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International audience; Talks at the Minisymposium on “Intermediate Filaments from Cytoplasm to Nucleus” demonstrated the structural and functional diversity of intermediate filaments (IFs) and their multiple binding partners and highlighted their importance in both cellular mechanics and gene regulation.

Talks in the “Organelle Homeostasis and Turnover” Minisymposium covered a broad range of topics, including the formation of bacterial magnetosomes, a new understanding of endoplasmic reticulum (ER) protein translocation, the mechanisms for mitochondrial turnover, and phase transition in amyotrophic lateral sclerosis (ALS) protein aggregates.

Talks at the “New Technologies and Immuno-Signaling” Minisymposium featured the engineering and development of new technologies for cell signaling studies. Specifically, proteins have been engineered to be controlled by chemical stimulation or photostimulation for the manipulation of signaling transduction in live cells. Experimental and computational approaches have also been combined to systematically identify phosphatase substrates. Hui Wang from Klaus Hahn’s lab at the University of North Carolina described a novel and versatile set of new reagents termed LOVTRAP for reversible light-induced dissociation of tightly bound protein pairs. Proteins of interest are fused to a small reagent derived from high-throughput screening that binds to the LOV domain. The protein of interest is reversibly released upon irradiation of LOV, and different versions of the system are used for rapid kinetic control or long-term activation requiring only brief periodic irradiation. LOV was anchored to mitochondria, where it bound and sequestered proteins of interest in the dark. Light caused release of proteins from the mitochondria, leading to action at the plasma membrane or in the nucleus. Wang used LOVTRAP to induce precisely timed oscillations of Rho-family signaling circuits. This light-controlled activation of molecular functions can be used to regulate signaling pathways and control cellular phenotypes in a broad spectrum of biological and biomedical applications. Patrick O’Neill from the Gautam lab at the Washington University School of Medicine presented two complementary optogenetic approaches based on the engineering of different photosensitive proteins to reveal the dynamic contributions of G proteins to immune cell migration. The first used light-induced membrane recruitment of an RGS protein to inhibit heterotrimeric G protein signaling, and the second used light-induced membrane recruitment of a guanine nucleotide exchange factor to activate Cdc42. In cells treated with uniform chemoattractant, optically triggered inhibition of endogenous heterotrimeric G proteins created signaling gradients capable of guiding cell motility. In the absence of chemoattractant, subcellular activation of Cdc42 generated a leading edge and a myosin-dependent signaling sufficient to retract the cell rear. The molecular engineering of synthetic proteins integrated with photoactivation can allow the control of Cdc42 activation sufficient to initiate, direct, and reverse cell migration. Andrei Karginov of the Department of Pharmacology at the University of Illinois, Chicago, reported on technologies for interrogation of phosphorylation-mediated signaling. Karginov’s lab has developed a new method that enables transient activation of a specific protein kinase in living cells. Activation of a kinase is achieved using a rapamycin-regulated (RapR) method (Karginov et al., 2010 ) that uses the insertion of an engineered allosteric switch, the iFKBP domain, at a specific site within the catalytic domain of a kinase. Treatment with rapamycin or its nonimmunosuppressive analogues induces interaction between iFKBP and a coexpressed FRB domain, leading to kinase activation. Inactivation of the engineered kinase can be achieved by introducing a previously reported mutation into the catalytic domain of a kinase that makes kinase sensitive to inhibition by an analogue of PP1 compound, 1NA-PP1 (Bishop et al., 1998 ). Using this strategy, Karginov’s lab successfully generated an engineered Src tyrosine kinase, RapR-Src-as2, that can be transiently activated in living cells for a defined period of time. Application of this method revealed that transient activation of Src induces PI3K/Akt signaling that continues after Src inactivation and stimulates cell spreading independently of Src. This method has also been used to regulate protein tyrosine phosphatases. A specific site within the catalytic domain of Shp2 was identified where insertion of iFKBP enables rapamycin-mediated activation of phosphatase. RapR analogues of Shp2, PTP-PEST, and PTP1B were then generated. Analysis of RapR-Shp2 activity in living cells reveals that it can stimulate endogenous Erk1/2 kinases, demonstrating that it functions similarly to wild-type Shp2. Through conjugation of FRB to a selected protein, phosphatase activation was further restricted to a complex with a specific downstream target and/or specific subcellular location. In fact, RapR-Shp2 was engineered in complex with focal adhesion kinase to down-regulate its signaling. These methods successfully provide specific and efficient control of kinase and phosphatase activities in living cells. Jagoree Roy from Martha Cyert’s lab in the Department of Biology at Stanford University reported a novel approach combining experimental and computational methods to systematically identify substrate peptide sequences of phosphatase calcineurin (CN). Systems-level analyses of phosphorylation-based signaling networks have transformed the understanding of kinase function, but knowledge of phosphatase signaling is limited. CN is ubiquitously expressed and critically regulates Ca2+-dependent processes in the human immune system, heart, and brain (Roy and Cyert, 2009 ). CN acts on phosphosites with little primary sequence similarity; thus specificity is not encoded within regions contiguous to the phosphosite. Instead, the enzyme binds to short linear motifs (SLiMs) PxIxIT and LxVP, which can occur hundreds of residues away from dephosphorylation sites (Grigoriu et al., 2013 ). In fact, SLiMs are a growing class of sequences that localize within intrinsically disordered regions, that is, flexible protein domains that lack a defined structure. SLiMs mediate the majority of protein–protein interactions in cells and evolve rapidly to rewire signaling networks, including that of CN (Goldman et al., 2014 ). However, degenerate sequences and low-affinity interactions make SLiMs challenging to identify. Roy and Cyert combined experimental and computational approaches to identify CN-binding SLiMs systematically in the human proteome. Structure-based approaches were first used for PxIxIT-site prediction, incorporating the structural features of experimentally verified PxIxIT motifs, including intrinsic disorder, hydrophobicity, secondary structure content, and a newly derived PxIxIT score (PS) reflecting the binding energy of any 6-mer peptide for the PxIxIT docking pocket, as defined by published CN:PxIxIT structures (Li et al., 2007 , 2012 ; Grigoriu et al., 2013 ). This method successfully identified nine of 12 verified PxIxIT motifs and also predicted new PxIxIT fragments in eight other proteins that were previously identified CN interactors. Two of these novel sequences, found in the CN substrates KSR2 and amphiphysin, were confirmed to bind CN in vitro. This computational strategy has been applied to the entire human proteome to identify the spectrum of human CN-interacting proteins that contain PxIxIT-like sequences. In parallel, proteome peptide phage display (ProP-PD) was used to experimentally select CN-interacting sequences from all predicted disordered regions of the human proteome (Ivarsson et al., 2014 ). Many of the novel sequences identified by ProP-PD were confirmed to bind CN in vitro. Sequences that were identified both in the Pro-PD screen and by the computational approach constitute strong candidates for new CN substrates. Altogether, 50 novel CN targets, including ion channels, kinases, and receptors, were identified. These methods not only can be used to expand our understanding of phosphatase substrates and signaling networks, but they also can be applied to systematically identify any SLiM-mediated interaction network.

At this year's ASCB meeting, the dynamic genome took center stage. Three Minisymposia carried this thread from start to finish, revealing the impacts of cell biology on all aspects of the genome. New concepts about the physical nature and organization of chromatin were presented, highlighting cutting-edge techniques such as high-throughput chromosome conformation capture (Hi-C) and biophysical approaches to assess the mechanical properties of chromosomes. The intimate connection between transcription and the building of nuclear bodies took on new importance as both cause and consequence of cellular transformation. Several presentations focused on how the physical state of chromatin can be transduced into biochemical signals to support homeostasis, an emerging theme in nuclear cell biology. Four of the talks are highlighted here. Pol I and Pol III in nuclear architecture and cancer Sui Huang (Northwestern University) presented evidence of a functional connection between perinucleolar compartment (PNC) formation and cancer metastasis. Using loss of the PNC as a readout to screen chemical compounds, Huang's team identified a lead molecule that remarkably inhibited metastasis in an animal model without displaying cytotoxicity. The compound altered nucleolar structure and selectively decreased Pol I and Pol III, but not Pol II, activity. The success of this chemical biology approach highlights a new way to probe the mechanistic connection between nuclear architecture and cancer. Together, insulator proteins hold all the ACEs In addition to their transcriptional roles, the transcription factor TFIIIC and pol III have been implicated in contributing to chromatin boundary activity. Kevin Van Bortle (Victor Corces’ laboratory, Emory University) presented evidence that topologically associated domains (TADs) are defined by architectural clustering elements (ACEs)—sites associated with multiple insulator proteins, including TFIIIC, CTCF, PRDM5, and the SMC complexes cohesin and condensin (see Figure 1). Thus, these protein complexes likely carry out both unique functions at distinct, nonboundary sites while working cooperatively at common sites to define boundaries, thereby separating TADs. FIGURE 1: Combinatorial binding of insulator proteins at ACEs shapes TAD structure and regulatory function. Chromatin immunoprecipitation followed by high-throughput sequencing reveals that insulators form dense clusters and that TAD border strengths identified ... SMC proteins stiffen mitotic chromosomes Mingxuan Sun (John Marko's laboratory, Northwestern University) presented work demonstrating that condensins do double duty by contributing to the physical properties of metaphase chromatin. After individual mitotic chromosomes were mechanically removed from cells, glass pipettes were used to probe the mechanical properties of the chromosomes. While the DNA polymer makes the largest contribution to chromosome structure (digestion with nucleases can “liquefy” the chromosomes), both condensin I and more prominently condensin II contribute to chromosome stiffness. Provocatively, threads of DNA between individual chromosomes were also detected using this approach, raising interesting questions about the biological context for these connections and the mechanisms of their resolution. Tel1 tells short telomeres to get replicating Investigating how telomere length can be sensed and communicated to the replication machinery, Akila Sridhar (Anne Donaldson's laboratory, University of Aberdeen) uncovered an essential role for the Tel1 kinase (the budding yeast ATM homologue) in shifting the timing of telomere replication earlier in response to a “short telomere” signal. Normally, the telomere-binding factor Rif1 attenuates this Tel1 cascade, but Tel1 can override this blockade at short telomeres. Sridhar found that Rif1 becomes a substrate for Tel1 in response to short telomeres, suggesting that Rif1 phosphorylation may contribute to this change in state. However, the minor effect of mutating the Rif1 phosphorylation sites on replication timing suggests additional inputs remain to be uncovered.

The “Applications of Cell Biology in the Real World” Minisymposium comprised two full sessions. Although the topic was overwhelmingly broad, several central themes emerged. Disease mechanisms and therapeutics permeated many of the talks, with an array of different, and often unexpected, experimental systems, new analytical tools, and model organisms also discussed. Cancer was a focus of a number of presentations, with new approaches addressing long-standing problems. Lisa Belmont (Genentech) discussed strategies for targeting the tumor suppressor BRG1, an ATP-dependent helicase frequently mutated in cancer, which is part of the BAF complex that remodels chromatin. Based on synthetic lethal interaction identifications, the data she discussed suggest that inactivating related helicases might be a promising strategy. Bert Gough (University of Pittsburgh) described methods for exploiting (rather than bemoaning) the broad heterogeneity among different cell types to facilitate drug discovery, focusing on investigations of signaling heterogeneity in the IL-6-activated STAT3 pathway and describing novel tools such as a “heterogeneity browser.” Hirofumi Matsui (University of Tsukubu) discussed the development of optical cell separation and culture systems that use photodegradable hydrogels, photoirradiation, and cell picking to separate cells based on morphological criteria, along with the development of automated systems useful for the study of cancer cells. Another overarching theme encompassed cell death, aging, and neurodegeneration, with numerous new tools and approaches described here as well. Vlad Denic (Harvard University) described his studies of the essential protein heat shock factor 1 (Hsf1) in yeast. Because Hsf1 inactivation causes protein aggregation, he used an “anchor-away” approach to acutely deplete Hsf1 in the presence of rapamycin and found that heat shock protein family members, in particular Hsp70 and Hsp90, were necessary and sufficient to allow cells to survive in the absence of Hsf1. Marc Hammarlund (Yale University) spoke about axon regeneration, using pulsed-laser axotomy in Caenorhabditis elegans as an in vivo model and emphasizing the critical role of inhibiting poly(ADP-ribosylation) in stimulating regeneration. Jonny Nixon-Abell (University College London and National Institutes of Health) used emerging superresolution imaging approaches to clarify the distinct morphologies and dynamics of peripheral ER tubules and noted that important disorders such as hereditary spastic paraplegia are linked to proteins involved in ER morphology. Grazing incidence illumination (GI)-SIM and lattice light sheet-point accumulation for imaging in nanoscale topography (LLS-PAINT) were used to reveal novel, ultrafast dynamism in the peripheral ER and further indicated that many structures classically considered peripheral sheets are instead dense tubular matrices. Christopher Medina (University of New Mexico) spoke about kinesin-1 deficiency and imaging in living mouse brain, presenting techniques such as tracing circuitry in vivo using magnetic resonance imaging after focal manganese injection. These techniques were able to show altered axonal transport in vivo in hippocampal-to–basal forebrain memory circuits, pathogenically implicating decreased synaptic vesicle replacement in active synapses. Moving to injury repair, Virginia Ayres (Michigan State University) identified nanoscale cues for regenerative neural cell systems, specifically for polyamide nanofiber scaffolds used in spinal cord injury repair, using specially adapted atomic force microscopy for the cues and superresolution imaging for reactive astrocyte protein responses. A variety of neurodegenerative disorders also took center stage. Aditya Venkatesh (University of Massachusetts) spoke about retinitis pigmentosa (RP), an inherited photoreceptor degenerative disorder (with many known mutated genes in rod genes) that results in blindness from secondary loss of retinal cones. Cone survival depends on mTORC1, which has an essential role in clearance of autophagic aggregates. Activating mTORC1 by reducing TSC1 promotes long-term cone survival, prefiguring therapeutic potential to prolong vision in RP. Alzheimer’s disease was the topic of several talks. Rylie Walsh (Brandeis University) investigated Drosophila neuromuscular junctions to describe how perturbations in the retromer protein complex cause changes in amyloid precursor protein (APP)–positive exosome levels. Neuronal retromer was able to rescue APP accumulation in a retromer mutant. Natalya Gertsik (Weill Cornell Medical College) discussed how γ-secretase inhibitors and modulators might be useful for Alzheimer’s disease treatment via their inducement of distinct conformational changes within the active sites of γ-secretase and signal peptide peptidase that she identified by photophore walking. Risa Broyer (University of California, San Diego) leveraged the cell biology of metabolic enzymes to uncover new insights into orphan genetic diseases affecting metabolic pathways, emphasizing studies of PRPP synthase, where filament formation may be involved in pathogenesis. Infections and vascular disorders are also prominent themes in medicine, and cell biology is enlightening these areas. Meron Mengistu (University of Maryland) spoke about HIV vaccine development, using three-dimensional dSTORM microscopic visualization of vulnerable sites exposed on cell-bound HIV to inform better therapy. Robert Cooper (University of California, San Diego) described how neighbor killing via the type VI secretion system enables high-efficiency, cross-species acquisition of antibiotic resistance via “gene snatching” in competent Acinetobacter bacteria—a strain of serious threat to hospitalized patients. Vascular disease is always of interest, and Akira Sawaguchi (University of Miyazaki) spoke about the dynamics of thrombus formation in mouse testicular surface vein using a new vascular mapping method for correlative light and electron microscopy (CLEM) in vivo, revealing detailed structure of the thrombus. Larry Lemanski (Texas A&M University) described how cardiac-inducing RNAs help differentiation of nonmuscle cells from muscle cells, which might play a role in helping those with myocardial infarction recover function. Sometimes extreme examples can teach us fundamental biology, relevant beyond human disease. Thomas Boothby (University of North Carolina) introduced many participants to tardigrades, one of the most resilient survivors in existence, which tolerate a very broad range of temperatures, even that of outer space, and desiccation. Specific genes are up-regulated and required for survival when desiccated. The proteins encoded by these genes can form bioglasses that stabilize proteins upon desiccation. Understanding such mechanisms may lead to creation of drought-resistant crops and heat-stable vaccines. Finally, Shiva Razavi (Johns Hopkins University) spoke about reconstitution of chemotaxis in giant unilamellar vesicles, employing a rapamycin-induced dimerization system (FKBP and FRB). Cell biology studies affect a wide range of diseases and other basic cellular questions, with new tools leading the way. Cells are the basic unit of life, and the presentations in this minisymposium highlight how broad and fundamental cellular insights are for understanding and improving life.

The Minisymposium on Nuclear Structure and Function featured new strategies and approaches for understanding how the vast amount of information in the nucleus is parsed out in individual cells. The field faces the problem of deducing the structure of a dynamic polymer (chromatin) in a living cell. Classical structural biology approaches have not been as forthcoming for dissecting the structure of this highly flexible and dynamic polymer. From a statistical mechanics perspective, the number of states that chromatin can adopt provides a rich source of conformational energy. Extrapolating to nuclear domains, we appreciate these are statistical definitions for regions within the nucleus that are on average differentiated from adjacent regions. Nuclear domains are not confined by membranes; rather, they are more like eddies in a highly viscous environment that provide chemistry favoring one chromatin state over another. The question is how we probe subtle features in such a fragile environment as the cell. Domain targeting Jason Brickner (Northwestern University) has cracked the code for clustering of active genes at the nuclear periphery in yeast. Gene promoters contain “DNA zip codes” that target genes to nuclear pores as one mechanism to enhance expression. The positional information is epigenetically propagated through several cell cycles, providing a mechanism of transcriptional memory. Aspects of this mechanism are conserved to humans, suggesting that the interaction of genes with nuclear pore proteins plays an important, conserved role in regulating transcription and chromatin structure. Gatekeeping function of the nuclear pore Weidong Yang (Temple University) is interested in the mechanism of RNA transport through the nuclear pore and has developed a new method called SPEED (single-point edge excitation subdiffusion) microscopy. SPEED microscopy enabled him to beat the loss of spatial resolution in z and visualize single RNA molecules as they traverse a nuclear pore with an unprecedented spatiotemporal superaccuracy of 8 nm and 2 ms. The three-dimensional mapping of export routes indicates that mRNAs primarily interact with the periphery on the nucleoplasmic side and in the center of the NPC (nuclear pore complex) without entering the central conduit utilized for passive diffusion of small molecules. These are important findings that enable discrimination of various models in the field. Gene and chromosome pairing Megan Bodnar (David Spector's laboratory, Cold Spring Harbor Laboratory) investigated the organization of pluripotency genes during early embryonic stem cell differentiation. She reported that a DNA element mediates allelic pairing at the Oct4 gene locus during the onset of Oct4 gene repression. Thus, just as active genes may be corralled for maximum expression, inactive genes may also rely on positional cues. On a larger scale, Ofer Rog (Abby Dernberg's laboratory, University of California–Berkeley) showed us chromosome synapsis in live meiosis. These remarkable images revealed a rapid and processive event that initiates from previously identified pairing sites and elongates the length of the chromosome. This study opens up a new understanding of the intimate contacts and exchange that occur between homologous chromosomes. Finally, we heard two talks on mechanisms of chromosome segregation. Ajit Joglekar (University of Michigan) updated his nanometer localization mapping of kinetochore protein position in live cells to address mechanisms of force transduction. Using a sensitive fluorescence resonance energy transfer (FRET) assay and statistical analysis of the data, Joglekar demonstrated that the microtubule (mt)-binding complex (Ndc80) is responsible for attachment to shortening mt-plus ends, while the homologue of XMAP215 (Stu2) seems to be required for corralling growing mt-plus ends. Kerry Bloom (University of North Carolina–Chapel Hill) discussed the structure of the chromatin spring that is required for tension sensing and distribution of forces between sister kinetochores. The spring is most likely a cross-linked polymer network. The cluster of 16 kinetochores in budding yeast is analogous in structure to one mammalian kinetochore that makes multiple microtubule attachments in mitosis.

The Minisymposium on “Cell Migration in Tissues” focused on analysis of dynamic collective cell migration in several model systems and was cochaired by Alissa M. Weaver (Vanderbilt University Medical Center) and Celeste M. Nelson (Princeton University). The program highlighted both the physical forces exerted during migration and the signaling pathways involved in the process. Celeste Nelson (Princeton University) presented results suggesting that cells migrate collectively through fibrous extracellular matrix (ECM) by exerting tensile forces at the leading edge of the cohort. Three-dimensional traction force microscopy was used to quantify the physical forces exerted by normal and malignant mammary epithelial cells undergoing collective migration through collagen gels (Gjorevski et al., 2015 ). The forces and displacements were surprisingly dynamic, with the cohort periodically relaxing as it migrated forward. Jessica Konen (Marcus laboratory, Emory University) described a new technique called spatiotemporal genomic analysis, which was used to select rare populations of leader cells from three-dimensional (3D) lung cancer spheroids and compare their gene expression patterns to that of follower cells. Leader cells were found to express higher levels of genes related to cell adhesion and vascular sprouting. These gene expression changes persisted in purified cultures. Eliah Shamir (Ewald laboratory, Johns Hopkins University) showed that in 3D culture of mammary epithelial organoids, the myoepithelial layer was required to prevent invasion and dissemination of Twist1-expressing luminal epithelial cells. Surprisingly, Twist1-induced invasion required the expression of E-cadherin, and knocking down expression of this cell–cell adhesion protein prevented dissemination downstream of Twist1 (Shamir et al., 2014 ). Joshua Bloomekatz (Yelon laboratory, University of California, San Diego) showed a critical role for platelet-derived growth factor (PDGF) in the movement of cardiomyocytes during cardiac fusion. A forward genetic screen was used to identify a mutation called refuse-to-fuse, which causes inhibition of cardiac fusion and the subsequent formation of a bifurcated ventricle. This mutation was found to disrupt splicing of pdgfra, leading to expression of a truncated form of the PDGF receptor. Expression of PDGF ligands in relatively medial locations suggested a model in which PDGF signaling coordinates the movement of cardiomyocytes toward the midline during cardiac fusion. Bong Hwan Sung (Weaver laboratory, Vanderbilt University) showed that directionally persistent migration of cancer cells is promoted by autocrine secretion of exosomes, late endosome–derived extracellular vesicles (Sung et al., 2015 ). Exosomes were shown to contain ECM proteins as cargo and to be secreted at sites of nascent adhesions. Accordingly, exosome secretion promoted adhesion assembly, migrational polarity, and the stability of lamellipodia. David Matus (Stony Brook University) used an RNA interference screen to identify a transcriptional program that maintains invasive anchor cells in a cell cycle–arrested state during uterine-vulval attachment in Caenorhabditis elegans. Nuclear hormone receptor nhr-67 (TLX) expression leads to up-regulation of cyclin-dependent kinase inhibitor cki-1, causing cell cycle arrest in G1 (Matus et al., 2015 ). Cells that lack nhr-67 are proliferative and noninvasive, failing to express matrix or form invadopodia. The Minisymposium concluded with three lightning talks. Micah Webster (Fan laboratory, Carnegie Institution for Science) showed a role for “ghost fibers,” ECM remnants of damaged muscle fibers, in organizing skeletal muscle progenitor migration, division, and fusion during regeneration in mice (Webster et al., 2016 ). Rebecca Green (Oegema laboratory, University of California, San Diego) developed a four-dimensional high-content imaging approach to screen for developmentally important genes within C. elegans embryos. Yutaka Matsubayashi (Stramer laboratory, King’s College London) showed that formation of the basement membrane in the Drosophila embryo requires rapid anterior–posterior dispersal of macrophages (hemocytes), which first appear in the head of the embryo.

The Minisymposium “Cell Migration and Motility” was attended by approximately 500 visitors and covered a broad range of questions in the field using diverse model systems. Topics comprised actin dynamics, cell polarity, force transduction, signal transduction, barrier transmigration, and chemotactic guidance. The session started with the 2010 American Society for Cell Biology Early Career Life Science awardee, Anna Kashina (University of Pennsylvania), who reported on the role of arginylation in the dynamics of lamellipodial actin. In contrast to γ-actin, β-actin preferentially localizes to the leading edge of migrating cells and is arginylated. Surprisingly, the reason for this differential arginylation is not inferred by differences at the amino acid level but by different mRNA codon usage that results in faster β-actin translation and protein folding kinetics, which in turn affect arginylation, protein stability, and turnover rates. The talk was followed by a presentation on the chemotropic response of budding yeast—the basis of mate recognition. Jayme Johnson (Duke University) showed data supporting a new model in which the “polarity patch,” a polarity complex that is condensed by positive feedback and directs polarized growth, searches the inner membrane for active receptors. Local, receptor-mediated signaling slows patch motility and thereby biases the growth response toward the pheromone gradient. Cochair Carole Parent (National Cancer Institute, National Institutes of Health) then reported on a new chemotactic signalling pathway in neutrophil granulocytes that shows partial homology with attractant sensing in Dictyostelium. The pathway involves a mammalian target of rapamycin complex 2, protein kinase C, and cyclic adenosine monophosphate–dependent signalling cascade that regulates myosin II–dependent contractility at the trailing edge of the cell and thereby balances leading-edge protrusion with trailing-edge retraction during directed motility. Daria Siekhaus (Skirball Institute, New York University) followed with a presentation on Drosophila immune cell distribution during early embryogenesis. The path of these cells involves the penetration of an epithelial layer, and a genetic screen revealed that, in analogy to the leukocyte extravasation process in vertebrates, this step involves inside-out signalling of integrins, while migration through the rest of the interstitium is independent of these receptors. Integrin activation is mediated by the guanosine 5′-triphosphate–binding proteins Rap1 and RhoL, and the weakening of epithelial junctions is sufficient to rescue the immune cell autonomous penetration defects. In the next presentation cochair Michael Sixt (Institute of Science and Technology, Austria) presented data supporting the idea that the presentation of guidance cues determines the migratory mode of leukocytes. When dendritic cells are exposed to soluble chemokines, they migrate in a nonadhesive manner, and consequently the extracellular substrate does not play instructive roles for cell guidance. When the chemokine is immobilized to surfaces, integrin activation is triggered, the cells become adhesive, and they migrate along the chemokine-decorated matrix structure. The session concluded with a talk by Matthew Paszek (University of California, San Francisco), who proposed a new role of the glycocalyx in cell adhesion. A combination of immunofluorescence and fluorescence interference contrast microscopy demonstrated that at sites of high glycoprotein density the plasma membrane of adherent cells is elevated from the substrate, while the spatially separated adhesion sites are in close proximity to the substrate. Consequently, enhanced glycoprotein expression causes irregular membrane topography and imposes a vertical pull on integrin receptors, causing enhanced force coupling and adhesion site maturation.

How does the nucleus move within the cell? How is the nucleus compartmentalized? How is nuclear size maintained? How is chromatin organized within the nucleus? These are a few of the questions related to nuclear cell biology addressed by our Minisymposium. Dan Starr led off with a description of how nuclei migrate in Caenorhabditis elegans. A KASH protein in the outer nuclear membrane recruits microtubule motors to the nucleus. Live imaging of nuclear migration and microtubules suggests that kinesin-1 provides the major force to move nuclei. To resolve large cellular roadblocks, dynein mediates backward movements or dramatic nuclear rolling events (Fridolfsson and Starr, 2010 ). G. W. Gant Luxton discussed mechanisms of nuclear migration in wounded cultured fibroblasts. Nesprin2G, SUN2, lamin, and actin cables associate in transmembrane actin-associated nuclear (TAN) lines to move nuclei (Luxton et al., 2010 ). The AAA+ ATPase TorsinA disease gene regulates the formation of TAN lines and nuclear migration. TorsinA knockdown reduced the localization and mobility of Nesprin2G and disrupted nuclear migration, suggesting that TorsinA regulates SUN-KASH interactions in the nuclear envelope. Levels of lamin-A/-C are known to increase during differentiation of embryonic stem cells (Constantinescu et al., 2006 ). Joe Swift described new mass spectrometry methods to accurately measure the ratios of lamin-A/-C to lamin-B isoforms, which had not been directly measured previously. The findings are just beginning to enhance our understanding of lamin changes throughout development and aging. Dan Levy described using Xenopus laevis and tropicalis as model organisms with similar DNA content but differently sized nuclei to understand nuclear scaling. Sperm nuclear assembly in tropicalis and laevis egg extracts mixed to different proportions directly correlated with nuclear size, suggesting size control by an egg cytoplasmic factor (Levy and Heald, 2010 ). Amounts of importin-α, Ntf2, and lamin were all involved in nuclear scaling. Future analyses will identify additional nuclear size factors and show how changes in size affect genome function. Jason Brickner described mechanisms of targeting a gene to the nuclear periphery in yeast. Genes were localized by LacO repeats and GFP-LacI. Mutational analyses revealed DNA elements that function as “DNA zip codes” to promote targeting to the nuclear periphery through association with the nuclear pore complex (Ahmed et al., 2010 ). A different DNA zip code controls posttranscriptional intranuclear targeting of the INO1 gene. This “memory” zip code is necessary and sufficient for H2A.Z incorporation and priming for reactivation (Light et al., 2010 ). Specific zip codes may also promote long-range interchromatin interactions between genomic regions sharing the same elements. Finally, Ana Pombo described how changes in chromatin positioning accompany gene activation, using the human uPA gene (Ferrai et al., 2010 ). Prior to induction, uPA alleles are found at the interior of their chromosome territory, associated with poised transcription factories characterized by RNA polymerase II phosphorylation on Serine5, but not Serine2. Gene activation with phorbol esters induced uPA gene repositioning out of its chromosome territory. Investigation of a looping requirement for transcriptional activation showed that gene looping is not required for activity upon induction, but that the internal chromosome territory position prior to activation is important for reinforcing the silent state. The Minisymposium on Nuclear Cell Biology covered the breadth of nuclear research, from events that control nuclear positioning within cells, to nuclear size control, to chromatin positioning and gene regulation. Future studies will focus on how nuclear position and size influence genome function, how nuclear envelope components might regulate the peripheral localization of individual loci, and how gene activity is regulated in different areas of the nucleus. We therefore look forward to future ASCB programs highlighting nuclear cell biology.

Cells must thrive in a variety of stressful environments. Moreover, a significant number of proteins fold slowly or inefficiently, so there is a constant threat that misfolded proteins might accumulate. An unmitigated response to these stresses can result in cell death. To offset the catastrophic effects of cellular stress, proteins are subject to quality control checkpoints that target aberrant polypeptides for degradation or for refolding via the action of molecular chaperones. In addition, defective or superfluous proteins, lipids, and organelles can be selected for destruction, or are inherited asymmetrically during cell division. Finally, inducible transcriptional programs facilitate protein triage pathways. The “Organelle and Proteome Quality Control Mechanisms” session at the 2013 ASCB Annual Meeting focused on each of these events. Protein quality control and the cytoplasmic–nuclear nexus The first talk in this session was delivered by Thibault Mayor (University of British Columbia), who highlighted the mechanism that leads to the degradation of ubiquitinated, cytoplasmic proteins in heat-stressed yeast cells. After incubation at elevated temperatures, damaged cytosolic proteins are ubiquitinated and routed to the proteasome. By screening for mutants in which this phenomenon was compromised, Mayor and colleagues identified Hul5 as the ubiquitin ligase that modifies most of these substrates (Fang et al., 2011 ). Hul5 resides in the nucleus, but, upon heat stress, a significant population migrates to the cytoplasm. Consistent with this model, yeast expressing a form of Hul5 that remains trapped in the nucleus are unable to fully recover from heat shock and exhibit decreased levels of ubiquitinated cytoplasmic proteins. Current efforts are defining how heat-damaged proteins are selected for ubiquitination, assessing whether Hul5 acts as a bona fide E3 ligase or as a ubiquitin extension enzyme (i.e., an E4), and identifying other ligases that function with Hul5. Chris Guerriero from the Brodsky Laboratory (University of Pittsburgh) described new chimeric proteins that can be used to investigate specific questions underlying the selection of substrates for endoplasmic reticulum–associated degradation (ERAD) and cytoplasmic quality control. Each chimera contains a single “degron,” which derives from a well-characterized yeast ERAD substrate, Ste6p*. When expressed in the cytosol, the degron undergoes Hsp70- and proteasome-dependent degradation (Guerriero et al., 2013 ). Examining substrate ubiquitination in a new in vitro assay provided evidence to suggest that Hsp70 acts both before and after substrate ubiquitination. Surprisingly, degron ubiquitination and degradation utilized a nuclear resident E3 ligase, San1. This result is in contrast to the E3 requirements for the degradation of a degron-containing protein tethered to the ER. In this case, proteolysis via the ERAD pathway is mediated by different E3 ligases, even though degradation remains Hsp70 dependent. A presentation by Shengyun Fang (University of Maryland School of Medicine) provided another example of how misfolded proteins are selected and routed to different compartments for degradation. Fang and colleagues discovered that the chemical entrapment of a mammalian nuclear export factor, Crm1, led to the aggregation of ERAD substrates in the nucleus. Some substrates also accumulated in this compartment when p62, an autophagy delivery factor, was silenced. Therefore, a shuttle escorts misfolded proteins from the nucleus to the cytosol and, in some cases, to the autophagic machinery. Ongoing work is exploring the substrate selectivity of Crm1 and p62, and identifying how these factors facilitate the delivery of ubiquitinated and nonubiquitinated proteins from the nucleus. Combined with the results from Mayor and Guerriero, these data emphasize how cellular compartments communicate to mediate protein quality control. Modulating substrate interactions Kevin Morano (University of Texas Health Science Center at Houston Medical School) addressed Hsp70 regulation. Hsp70s interact dynamically with substrates through an ATPase cycle (Zuiderweg et al., 2013 ). Three families of nucleotide exchange factors (NEFs) regulate Hsp70 nucleotide release: Hsp110, HspBP1, and Bag. The yeast NEFs were shown to maintain protein homeostasis, with Hsp110 promoting protein folding on its own and degradation of a misfolded substrate in coordination with HspBP1. Reflecting these roles, loss of HspBP1 alone or in combination with Hsp110 induces a stress response and chaperone elevation, presumably due to the accumulation of misfolded proteins. Yet, the NEFs were dispensable for recovery after heat shock, suggesting that nucleotide exchange may not be a limiting event once Hsp70 levels are elevated. Tricia Serio (University of Arizona) next discussed how the presence of one chaperone substrate promotes the refolding of another. The yeast prion Sup35 assembles into amyloid aggregates in vivo, which are amplified through fragmentation by the AAA+ATPase Hsp104 (Tuite and Serio, 2010 ). While the amyloid is normally stable, it is efficiently lost at elevated temperature (Newnam et al., 2011 ). This loss depends not only on the elevation of chaperone expression but also on the formation of heat-induced aggregates of other proteins. It was shown that Hsp104 engagement with heat-induced aggregates increases its levels in a subset of cells, which promotes amyloid disassembly. Thus, substrate interactions can promote chaperone accumulation beyond expression-level changes. Organelle quality control The final presentations described emerging work on quality control at the whole-organelle level. Damaged mitochondria contribute to aging and neurodegeneration, and proteins that target damaged mitochondria have emerged (PINK1, Parkin, Nix, Atg 32). Nevertheless, the mechanisms of selectivity are incompletely understood. Hagai Abeliovich (Hebrew University) described a dramatic autophagic turnover of mitochondria (mitophagy) when yeast cells are cultured under stationary-phase conditions. Turnover requires both mitochondrial fission and fusion. Proteomic analyses revealed subpopulations of mitochondrial proteins degraded at divergent rates, again dependent on fission and fusion. Abeliovich et al. (2013) speculate that protein subsets are retained or turned over through phase transition–like condensations (Brangwynne, 2013 ). Alex Merz (University of Washington) described links between the SNARE disassembly proteins Sec17 and Sec18 (NSF and α-SNAP) and SM proteins essential for SNARE-mediated fusion. SM function was needed to withstand Sec17 and Sec18 overproduction, and SMs directly protected SNARE complexes from disassembly. Unexpectedly, Sec17 stimulated formation of 1:3:1 SNARE–Sec17–SM cocomplexes. Merz et al. propose that SMs stimulate fusion by augmenting SNARE function and protecting incipient complexes from premature disassembly. The system taken as a whole has properties consistent with a kinetic proofreading system that accelerates on-pathway membrane traffic while eliminating complexes that lead to incorrect fusions or irreversible organelle aggregation.

The actomyosin cytoskeleton is at the heart of many of the most dramatic cell and tissue shape changes, but linking molecular-scale organization and activity to cellular shape changes remains a substantial challenge. The dazzling array of force-generating structures assembled through the interplay of actin-binding proteins was on show in the Minisymposium on actin dynamics and structure in studies examining actin network functions in tissues, isolated cells, and in vitro. Myosin contractility is the best-studied mechanism for force production in isolated cells. However, until recently, it was widely believed that myosins only generate steady contraction. New evidence presented by Michelle Baird (Waterman lab) suggests that myosin IIA can also generate pulsatile contractions. Downstream of contractility, the resulting traction forces exerted on the cell matrix or an adjacent cell depend on the extent of coupling between adhesion molecules and the actin cytoskeleton. Kenneth Buck (Forscher lab) presented evidence that localized Arp2/3-dependent actin assembly can shield adhesion molecules from the forces generated by actin retrograde flow and provide a pushing force in the anterograde direction. The role of actin polymerization in generating protrusive forces is well established in the lamellipodium, a protruding sheet of membrane at the leading edge of migrating cells. Lamellipodia have been most studied in cells crawling on planar substrates, but cells in vivo can also migrate along fibrils of matrix. Charlotte Guetta-Terrier (Gauthier and Ladoux labs) analyzed cell motility on fabricated nanofibers coated with extracellular matrix to mimic this type of cell movement. Under these conditions, cells assemble an Arp2/3-dependent, fin-like structure that emanates from the cell body and travels along the fiber at a constant rate for up to several hundred micrometers. By studying how cells from the inner progenitor layer of the frog epidermis can move to the outer, mature epithelium, Jakub Sedzinski (Wallingford lab) revealed that actin polymerization also plays a role in apical surface expansion. The force necessary for this “apical emergence” is generated primarily by the emerging cell rather than by the neighboring cells. Formin-mediated actin assembly in the apical cortex of the emerging cell provides a pushing force sufficient to expand the apical membrane. Cortical actin was also the topic of a presentation by Guillaume Charras, who presented his group’s work on how this actin network is generated. Cortical actin lies just beneath the plasma membrane, where it plays important roles in cell mechanics and morphogenesis. Charras and colleagues showed that blebs shorn from cells can reform the cortical actin network. This enabled determination of cortical composition and identified Arp2/3 and mDia1 as the only two actin nucleation factors present in the cortex. Matt Smith (Paluch lab) presented new computational approaches devised to understand how tension is controlled by the properties and organization of actin filaments in the cortex. He showed that actin filament length and arrangement can modulate cortical tension, providing a new level of regulation for cell surface tension in addition to myosin contractility. Josh Winkelman (Kovar lab) presented work showing that differences in filament spacing arising from distinct cross-linkers may represent a sorting mechanism for localization of subsets of actin-binding proteins to different structures, such as myosin motors for the generation of contractility. Although it is widely recognized that actin polymerization can generate mechanical forces, a potential role for depolymerization has been invoked in theoretical work but is poorly documented experimentally. Philippe Bun (Lenart lab) described an actin network that forms in the nuclear region encompassing chromosomes during the first meiotic division in starfish oocytes. This network collects chromosomes under the plasma membrane to ensure a successful division of these very large cells. Experiments and modeling suggested that chromosome movement is driven by asymmetric assembly and disassembly of the actin network to create a pressure gradient that pushes the chromosomes toward the plasma membrane. Part of the reason that forces generated by depolymerization have rarely been documented is that the molecular mechanisms of depolymerization remain poorly understood. Vivian Tang (Tang and Brieher labs) presented recent progress on understanding the mechanism of action of cofilin, coronin, and Aip1 in depolymerizing actin filaments. This revealed a new mode of catastrophic depolymerization of filaments mediated by Aip1 where long stretches of F-actin rapidly dissociated into monomers.

Some exciting new findings were reported at the Minisymposium “The Cell Biology of Genetic Information” held at the 2015 annual meeting of the American Society for Cell Biology. G. V. Shivashankar (Mechanobiology Institute, Singapore) spoke about how geometric constraints, such as extracellular matrix stiffness, can regulate nuclear organization in mammalian cells, from nuclear shape to chromatin dynamics and gene expression. The regulation is mediated by a pathway that includes focal adhesion, perinuclear actin, and chromatin modulations. Reduction of adhesion was found to reduce perinuclear actin filaments and increase the turnover of core histones (Toh et al., 2015 ), suggesting that gene expression and chromatin state tightly couple with changes of extracellular conditions. In another system, K. Weis (ETH Zurich Institute of Biochemistry, Switzerland) showed that nutritional or pH conditions could affect nuclear stiffness and chromatin dynamics in yeast. Genome organization and expression can also be regulated by nucleolar function (S. Huang, Northwestern University) and by differentiation (A. Wood, Northwestern University). Chromatin state, in turn, was shown to play a key role in shaping physical properties of the nucleus (A. D. Stephens, Northwestern University). A more euchromatic genome associates with a more elastic nucleus. These reports demonstrate dynamic functional coordination between the organization of the genetic material in the nucleus and cytoplasmic and extracellular function in response to environmental changes. The MCM helicase was reported to play important roles in preventing early replication stress– and loss-of-function–induced mitotic missegregation and genome rearrangement in fission yeast, resembling micronuclei in cancer cells (S. L. Forsburg, University of Southern California; Sabatinos et al., 2015 ). Micronucleolus formation, on the other hand, was shown by A. Spektor (Dana-Farber Cancer Institute) to be one of the causes of chromothripsis common in cancer cells (Zhang et al., 2015 ). Y. Kim (University of California, Berkeley) showed the activity of CHK-2 to be part of a conserved feedback mechanism that is important for meiosis and is facilitated by HORMA-domain proteins within the chromosome axis in C. elegans (Kim et al., 2015 ). Single RNA tracking in live cells defined the kinetics of mRNA export and demonstrated that mRNA remained associated with the outside of the nuclear envelope for an extended time upon exiting the nucleus in budding yeast (A. Lari, University of Alberta, Canada; Smith et al., 2015 ). Influenza virus mRNAs were shown by A. Mor (University of Texas Southwestern Medical Center) to be spliced and exported through nuclear speckles—nuclear organelles highly enriched with factors for pre-mRNA splicing. The reports on development of innovative methods provided exciting and more precise tools for future studies of nuclear functions. R. E. Kleiner (Rockefeller University) showed a novel chemical proteomics approach for quantitative profiling of direct binders to phosphorylated γ-H2AX (Kleiner et al., 2015 ) or other proteins in the future. Y. Chen (University of Illinois, Urbana–Champaign) spoke of a specific cross-link method, TSA-Seq, for measuring cytological distance in micrometers between genes and specific nuclear organelles, which can also be used to identify DNAs associated with specific nuclear organelles. D. S. Nelles (University of California, San Diego) showed that clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology can be suited for tracking RNA in living cells without modifying the targeted RNA (Nelles et al., 2015 ). These findings provided glances at current studies and future research trajectories. There remain many black boxes regarding nuclear structure, function, and coordination between the nucleus, cytoplasm, and extracellular environment in normal and diseased conditions. The increasingly sophisticated tools make it an exciting time to explore these mysteries.

Recent advances in fluorescence and electron microscopy (FM and EM) are closing the resolution gap between the two technologies, allowing cellular structures to be studied across the scales between structural and cell biology. FM and EM not only cover different scales, they provide complementary information. FM provides dynamic information, allows the imaging of large volumes, and is specific for the fluorescently labeled protein. EM, on the other hand, while imaging static and smaller volumes, offers contextual information, because the nonlabeled components of the cell are also seen. This working group presented methods and applications that bridge the gaps in resolution and integrate information from different FM and EM imaging modalities. John Briggs discussed motivations and methods for correlating FM and EM. He introduced sample preparation for transmission EM, comparing high-contrast overviews available after plastic-embedding with low-contrast, high-resolution structural views from cryo-EM. When correlative FM/EM is required, FM can be carried out before or after EM sample preparation. FM before sample preparation allows live imaging, but movements or distortions may occur between the FM and EM images. FM after sample preparation requires preservation of the FM signal during preparation. Methods and applications from different laboratories were presented (e.g., Muller-Reichert et al., 2007 ; Nixon et al., 2009 ; van Driel et al., 2009 ; Jun et al., 2011 ), as was the application of a sensitive, precise correlative FM–EM method (Kukulski et al., 2011 ) to study spatiotemporal dynamics of membrane bending during yeast endocytosis. Melike Lakadamyali presented an overview of superresolution FM techniques, such as stimulated emission depletion microscopy (STED), stochastic optical reconstruction microscopy (STORM), and photoactivation localization microscopy (PALM), and their extension to three-dimensional, multicolor, and live-cell imaging (Hell, 2009 ; Huang et al., 2010 ; Patterson et al., 2010 ). She also discussed their limitations, particularly in imaging living cells (e.g., trade-off between temporal and spatial resolution). Finally, she gave two examples of biological applications of superresolution: 1) using three-dimensional, multicolor STORM provides improved accuracy in neuronal tracing in the context of connectomics (Lakadamyali et al., 2012 ), and 2) correlating fast organelle dynamics with superresolution images of the cytoskeleton can reveal the biophysical mechanisms of organelle transport (Balint and Lakadamyali, unpublished). Stephen Smith of Stanford University's Department of Molecular and Cellular Physiology highlighted array tomography, a versatile method that can potentially reveal the synaptic connectivity of neurons at the light and electron microscopic scale (Micheva and Smith, 2007 ). Cutting ultrathin tissue sections and immunostaining them with multiple rounds of antibodies allow imaging of a large number of synaptic molecules. Using array tomography, the Smith laboratory is getting the first glimpse into the rich molecular diversity of synapses and searching for principles that govern the complex molecular architectures of synapses and synaptic circuits (Micheva et al., 2010 ). Array tomography is also compatible with EM and superresolution FM and allows correlating molecular and ultrastructural information across different scales. Finally, Erik Jorgensen of the Biology Department at the University of Utah gave an example of how molecular content of cells captured by superresolution FM can be correlated with ultrastructural information derived using scanning EM. Having previously correlated STED-based methods with EM, he described the correlation of PALM-based superresolution methods with scanning EM (Watanabe et al., 2011 ). This method allowed the imaging of specific ultrastructural features of synapses within Caenorhabditis elegans. He also introduced a three-dimensional PALM-based system capable of generating images with resolution of 20 nm in the plane of the image and 50 nm in the axial dimension.

Talks at the “Cell Biology at the Host–Microbe Interface” Minisymposium highlighted mechanisms used by viral, bacterial, and eukaryotic pathogens to hijack host-cell functions and establish a replicative niche. Manipulation of membrane dynamics Nihal Altan-Bonnet (National Institutes of Health) described positive-sense RNA enteroviruses that manipulate the host to generate phosphatidylinositol 4-phosphate (PI4P) and cholesterol-rich membrane platforms for efficient viral replication. The enzyme PI4KIIIβ is hijacked by the enteroviral protein 3A to generate new pools of PI4P at these membrane platforms and to recruit RNA-dependent polymerases that are important for viral replication. Indeed, inhibition of PI4K activity led to decreased viral replication, which might provide an antiviral strategy for positive-strand RNA viruses. Altan-Bonnet also described how enteroviruses stimulate endocytic uptake of cholesterol, which is re-rerouted to Rab11-positive recycling endosomes that eventually fuse with viral replication organelles. Increased cholesterol levels may facilitate viral replication by decreasing the fluidity of replication organelle membranes. This reprogramming of membrane trafficking is not unique to viruses. Charles Larson (Heinzen Laboratory, Rocky Mountain National Labs) described the manipulation of endocytosis by a secreted protein from the bacterial pathogen Coxiella burnetti, the causative agent of Q fever. In infected cells, Coxiella resides in a vacuole that shares many of the features of classical lysosomes. For the pathogen to survive in this organelle, it delivers specific “effector” proteins, including Coxiella vacuolar protein A (CvpA), which Larson described as being essential for bacterial replication in mammalian cells. CvpA localizes to the pathogenic vacuole and interacts with adaptor complex 2 (AP2). Depletion of cellular AP2 or clathrin with small interfering RNA impaired Coxiella replication, suggesting regulation of AP2–clathrin membrane transport events contributes to pathogen growth. Xing Liu (Yao Laboratory, Morehouse) provided an example of a bacterial toxin that reprograms proton secretion in the stomach. Normally, gastric acid secretion involves translocation of proton pumps by ACAP4 and associated proteins to the apical membrane, so protons can be released into lumen of gastric glands. VacA from the bacterium Helicobacter pylori, a leading cause of gastric ulcers and cancers, inhibits the dephosphorylation of ACAP4, resulting in the translocation of ACAP4 and the proton pump to the basolateral membrane, and low acid secretion to the gastric lumen. Altering the normal function of host organelles Anju Sreelatha (Orth Laboratory, University of Texas Southwestern) presented an example of a pathogen protein that modifies the function of organelles to drastically impact the health of the cell. Sreelatha presented data indicating that the protein VopQ from Vibrio parahemolyticus, a marine bacterium that causes food poisoning, makes a gated channel in lysosomal membranes. The mechanism of VopQ action appears to be a disruption in the turnover of autophagosomes, which causes lowered autophagic flux. Sreelatha and coworkers demonstrated that VopQ forms a channel in liposomes that permits passive diffusion of small molecules. They presented a model wherein VopQ blocks lysosomal function by dissipating the proton gradient, leading to the accumulation of autophagosomes and death of cells. Rewiring of cellular processes by obligate intracellular pathogens Malina Bakowski (Troemel Laboratory, University of California, San Diego) described infection of the nematode Caenorhabditis elegans by its natural pathogen Nematocida parisii, which belongs to the Microsporidia phylum of fungal-related pathogens. N. parisii infection induces expression of genes involved in the C. elegans ubiquitin proteasome system (UPS). This pathway appears to be an important arm of the worm's innate immune system, as interfering with the UPS system limits the ability of the worm to control microsporidia replication and viral replication. A subset of intracellular microsporidia is decorated with conjugated ubiquitin, which requires a host E3 ubiquitin ligase. Interestingly, lowered UPS capacity induces expression of ubiquitin ligases, indicating that the host monitors UPS function to regulate defense responses to intracellular infection. Yi-Shan Chen (Valdivia Laboratory, Duke University) described a new effector delivered by Chlamydia trachomatis, an obligate intracellular bacterium that is a leading cause of sexually transmitted infections. This protein, named TepP, is rapidly phosphorylated at tyrosine residues during bacterial entry. The host scaffolding protein CrK is subsequently recruited to the early Chlamydia vacuole in a process that is entirely dependent on TepP, as Chen described that a mutant strain defective for this factor—a first for this formerly “genetically intractable” pathogen—no longer recruited Crk. These mutants are defective for global changes in tyrosine phosphorylation patterns in infected cells, altered induction of immunity-related genes, and the disruption of cell–cell junctions in polarized endocervical epithelial cells.

mbc.E13-11-0671 Molecular Biology of the Cell Volume 25 Page 731 MBoC is pleased to publish this summary of the Minisymposium “Cell−Cell/Cell−Matrix Interactions and Intracellular Signaling” held at the American Society for Cell Biology 2013 Annual Meeting, New Orleans, LA, December 15, 2013.

mbc.E13-11-0670 Molecular Biology of the Cell Volume 25 Page 730 MBoC is pleased to publish this summary of the Minisymposium “Cells Shaping Tissues: Mechanisms Underlying Cell Polarity, Fate Specification, and Morphogenesis” held at the American Society for Cell Biology 2013 Annual Meeting, New Orleans, LA, December 15, 2013.

The Minisymposium ”Stem Cells and Their Niche in Homeostasis/Regeneration and Disease” was aimed at highlighting the importance of cell biology aspects in understanding stem cell biology. Tudorita (Doina) Tumbar (Cornell University) started the session by summarizing two models of stem cell behavior during normal homeostasis. Stem cells can divide asymmetrically to generate a stem cell and a more differentiated cell. Conversely, she showed that hair follicle stem cells behave as a population: some differentiate and eventually die, while others self-renew by symmetric divisions. During hair follicle stem cell G0 quiescence, the transcription factor Runx1 drives a reversible step of differentiation to progenitors. The latter succumb irreversibly to rapid proliferation and terminal differentiation upon activation of tissue signals. Pantelis Rompolas (Valentica Greco's Lab, Yale University) reported evidence for a direct link between a specific niche location and cell fate, using in vivo imaging to trace single hair-follicle stem cells long term in live mice. Furthermore, by utilizing laser ablation, he showed that hair follicle stem cells may be dispensable and that other epithelial populations can be reprogrammed to support hair regeneration by entering the niche. Gage Crump (University of Southern California) presented evidence for a transfating of chondrocytes to the osteoblasts that rebuild bone during jawbone regeneration in zebrafish. Such transfating is not observed during development of the same bone, challenging the notion that adult regeneration of an organ simply recapitulates its development. He also presented evidence for a novel role of Ihh signaling in this process and for a similar process in mammalian bone regeneration. Heike Kroeger (Jonathan Lin's Lab, University of California, San Diego) presented a study that links unfolded protein response signaling to hESC differentiation and maturation via the function of ATF6. hESC activates the ATF6 signaling during hESC differentiation through the FGF2 signaling cascade. ATF6 signaling leads to dramatic expansion of the endoplasmic reticulum (ER) and enhances hESC differentiation. These findings provide a new mechanism underlying hESC differentiation that involves ATF6’s induction and activation of ER functions. Daniela Malide (National Institutes of Health) reported the use of combinatorial 5 fluorescent proteins to mark hematopoietic stem and progenitor cells that allowed in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allowed noninvasive fate mapping of multicolored hematopoietic stem/progenitor cell–derived cells in intact nonhematopoietic tissues for extensive periods of time following transplantation, demonstrating clearly delineated clones progressing from multicolored to monochromatic over time. Abby Gerhold (Paul Maddox's Lab, University of North Carolina, Chapel Hill) reported in vivo live imaging of Caenorhabditis elegans germ-line stem cells to investigate how mitotic progression is influenced by the physiological context. She provided evidence that 1) the duration of chromosome congression is reduced during developmental expansion of the stem cell pool, and 2) dietary restriction induces a spindle assembly checkpoint–dependent delay in anaphase onset, suggesting the basic mitotic process is affected by physiological factors. Yukiko Yamashita (University of Michigan, Ann Arbor) discussed her group's recent identification of Klp10A kinesin as the first stem cell–specific centrosomal component in the Drosophila male germ line. Klp10A seems to specifically regulate the length of stem cells’ mother centrosomes. She presented additional data suggesting Klp10A regulates stem cell behavior through the regulation of nanos mRNA.

The central dogma uniquely positions RNA as the key molecular player for translating genetic software into protein hardware. But it has become increasingly clear that RNA also plays an essential regulatory role within cells. MicroRNAs in particular have emerged as ubiquitous and still poorly understood molecules involved in the control of a wide variety of biological processes. RNA has also recently been shown to have important roles in structural assembly within cells. A diverse set of speakers at the Minisymposium on Micro- and Coding RNA highlighted some of these new and often unexpected roles of RNA across a range of length scales. Tracy Johnson (University of California–San Diego) discussed her lab's work on cotranscriptional mRNA splicing. Her talk underscored the importance of ATP-dependent RNA helicases in rearranging RNA secondary structure and the integral role for chromatin modification in coordinating RNA rearrangements with transcription in space and time. Albert Rosana (Owttrim lab, University of Alberta) also discussed RNA helicases within the fascinating context of temperature fluctuations of cyanobacteria. He described his work on the altered expression of the oligomeric RNA helicase CrhR as a temperature stress response. His work provides new insights into RNA regulation, suggesting CrhR autoregulates its expression through a combination of RNA processing and RNA–protein stabilization mechanisms, which in turn leads to temperature-dependent regulation of small regulatory RNAs. Several presentations explored how RNAs play a role in the assembly of large-scale intracellular structures. Magdalena Strzelecka (Heald lab, University of California–Berkeley) discussed her work on RNAs in the assembly of the mitotic spindle. She utilized Xenopus egg extracts and next-generation sequencing approaches to demonstrate a surprising role for splicing factors in mitosis. This suggests the intriguing possibility that RNA processing plays a regulatory role in cell division. Moving to even larger scales, Cliff Brangwynne (Princeton University) discussed his lab's work on the biophysics of ribonucleoprotein (RNP) bodies—large, non–membrane bound assemblies that behave as liquid-phase droplets of RNA and protein. This work exploits the nucleus of Xenopus oocytes to study the nucleolus, an RNP droplet involved in ribosome biogenesis. The presentation revealed some unexpected biophysical consequences of the large length scales involved, with important implications for nuclear RNP organization and function. The nucleolus was also the central theme of a talk by Thoru Pederson (University of Massachusetts Medical School), who summarized his lab's recently published work on a subset of microRNAs localized in the nucleolus of rat myoblasts. He also reported new results on the nucleolar presence of certain mRNAs in these cells, proposing there might be microRNA–mRNA interactions in the nucleolus to pre-set the translational status of exported mRNAs. The remaining talk of the session, by Rebecca Holmes (Tollervey lab, University of Edinburgh), focused on the yeast protein Npl3, a member of the large SR family of RNA-binding proteins with diverse functions in pre-mRNA processing. She described experiments utilizing in vivo cross-linking and deep sequencing, which revealed the binding of Npl3 to a surprising variety of RNA species. Her data suggest an unexpected role for Npl3 in regulating noncoding RNAs (ncRNAs), with consequences for the expression of surrounding genes. These results raise the possibility that other previously described functions of Npl3 may, in fact, be consequences of Npl3 regulation of ncRNAs.

Visualization of cellular and molecular processes is an indispensable tool for cell biologists, and innovations in microscopy methods unfailingly lead to new biological discoveries. Today, light microscopy (LM) provides ever-higher spatial and temporal resolution and visualization of biological process over enormous ranges. Electron microscopy (EM) is moving into the atomic resolution regime and allowing cellular analyses that are more physiological and sophisticated in scope. Importantly, much is being gained by combining multiple approaches, (e.g., LM and EM) to take advantage of their complementary strengths. The advent of high-throughput microscopies has led to a common need for sophisticated computational methods to quantitatively analyze huge amounts of data and translate images into new biological insights. In vivo imaging requires carefully balancing conflicting parameters to achieve high imaging speed, low photobleaching and phototoxicity, good three-dimensional resolution, high signal-to-noise ratio, and excellent physical coverage. Light-sheet microscopy provides outstanding performance in all of these categories and has become a key imaging method for the life sciences. Philipp Keller (Janelia Farm) showed how entire Drosophila embryos can be imaged throughout embryogenesis at a spatiotemporal resolution that enables comprehensive cell tracking. The use of automated approaches for computational image analysis enables systematical reconstruction of cell lineage information from such recordings in real time. Progress in the light-sheet microscopy field is faster than ever, and further improvements in temporal and spatial resolution are expected in the near future. These capabilities will directly synergize with the rapid progress in related fields, such as the development of advanced fluorescent reporter strategies, powerful approaches to high-throughput data analysis, and computational tools for biophysical modeling, opening up exciting new opportunities for microscopy-based research in the life sciences. Superresolution imaging is enabling the visualization of intracellular relationships unobtainable using traditional fluorescent microscopy. However, different superresolution techniques are based on distinct physical principles to break conventional light microscopy limitations, and these principles determine the apparent size of the biological structure being imaged. The 25-nm-microtubule diameter appears to be between 30 and 120 nm, depending on the technique used. These specific principles also define the acquisition speed of each method, resulting in a family of techniques, with each technique optimized for both size (e.g., organelles vs. transmembrane receptors) and dynamics (e.g., slow transport vs. diffusion). Recent advances that combine multiple approaches to address a biological question show great promise for overcoming these limitations. Catherine Galbraith (National Institutes of Health) showed how live-cell superresolution imaging of single molecules can be combined with computer-vision tracking and conventional microscopy. This integration provides a dense functional dynamics map that can be used to correlate the behavior of multiple proteins to that of the entire cell, while visualizing biochemistry at the single-molecule level. Because biology is the complex integration of multiple systems, the combination of multiple imaging approaches presents the greatest opportunity for making meaningful discoveries. Cellular transmission EM has traditionally suffered from the need to section the sample. Sectioning of resin-embedded material is relatively easy but can suffer from poor sample preservation, while cryosectioning of frozen-hydrated samples is inefficient and hampered by cutting and compression artifacts. A recent breakthrough is the use of focus ion beam (FIB) methodology to carve out cellular slices, thus opening a window into deep regions of the eukaryotic cell without displaying the shortcomings of previous methods. Although EM methodologies cannot provide live imaging, they can produce snapshots that allow inference of biological transitions. The resolution and number of snapshots have been increasing with the high-throughput automated data collection and analysis. Eva Nogales (University of California–Berkeley, Howard Hughes Medical Institute, and Lawrence Berkeley National Laboratory) reported the structure of GMPCPP and GDP microtubules at 5-A resolution, showing how hydrolysis of GTP results in an accordion-like deformation of protofilaments that is overcome by the presence of Taxol. New developments (silicon-based cameras for direct electron detection, several phase-plate implementations for in-focus EM imaging, and innovative image analysis algorithms) will extend the scope of molecular and cellular EM studies, opening the door for higher-quality structures and quantitative conformational descriptions.

Talks at the Organelle Structure and Vesicle Formation Minisymposium featured new ways to look at vesicle formation, described molecular links between coat components, and uncovered the unexpected ways that organelles connect with one another.

In this year's annual ASCB Meeting Minisymposium on Cell–Cell and Cell–Matrix Interactions, new data were presented on several aspects of the biology of cell adhesions. The role of cell adhesions in cancer was the topic of three of the talks, two of which were focused on identifying how invasion is affected by cell–matrix and cell–cell adhesions. Yasmin Moshfegh from Louis Hodgson's lab (Albert Einstein College of Medicine) presented data showing a role for Rac1 in invadopodia formation, one of the initial steps in tumor cell invasion. The data showed that Rac1 suppresses invadopodia formation, and by using a fluorescence resonance energy transfer (FRET)-based Rac biosensor, the researchers showed that Rac1 activity is suppressed at sites of invadopodia formation. They further linked this to Rac1-mediated activation of PAK1, which in turn led to cortactin phosphorylation and invadopodia disassembly. Thomas Marshall (winner of the MBoC Paper of the Year Award) from Jody Rosenblatt's lab (Huntsman Cancer Institute) presented data on how APC (adenomatous polyposis coli) protein regulates the directionality of cell extrusion and how this could contribute to tumor cell metastasis in colon cancer, in which APC is frequently mutated. Cells with normal APC mainly extrude to the apical side of the tissue, whereas mutant APC reverses this directionality. The researchers also showed that the C-terminal domain of APC regulated this directionality. APC lacking this domain led to basal protrusion of the cancer cells, perhaps contributing to tumor cell invasion to basal layers. Taru Muranen from Joan Brugge's lab (Harvard Medical School) presented data on how extracellular matrix contact mediates drug resistance in tumor cells, wherein only matrix-attached cancer cells up-regulate a protective response (including anti-apoptotic Bcl-2 family proteins) in response to drug treatment, leading to cancer cell survival. In the other half of the Minisymposium, the speakers took a more mechanistic approach toward research on cell–matrix interactions. Viola Vogel (ETH Zurich) presented recent work on probes and new assays that demonstrate how cells exploit fibronectin fibrils as mechanochemical signal converters. She also presented proteomic data–derived evidence that extracellular phosphorylation and phosphorylated proteins are physiologically far more important than previously thought and are associated particularly with diseased tissues, such as those found in cancer. Michael Rubashkin from Valerie Weaver's lab (University of California–San Francisco) presented recent work on the development of a novel microscopy technique—scanning angle interference microscopy (SAIM)—that allows the study of focal adhesion structures in live cells at the nanoscale. SAIM was used to study how the relative position of vinculin changes in response to traction forces and how myosin contractility affects this localization. The researchers utilized vinculin activity mutants to study this and showed that active vinculin positions itself closer to the cell surface. This localization was also reflected in the structure of focal adhesions, suggesting that the relative position of vinculin corresponds to different adhesions. Caitlin Collins from Ellie Tzima's lab (University of North Carolina at Chapel Hill) reported on the role of the mechanosensory molecule PECAM-1 in endothelial mechanotransduction. The approach the researchers took was to use magnetic tweezers to apply tensional forces on PECAM-1 magnetic beads. On application of mechanical force on PECAM-1, they observed integrin and phosphatidylinositol 3-kinase pathway activation, both of which were required for adaptive stiffening of the endothelial cells. They further showed that this pathway led to global activation of RhoA and global formation of focal adhesions in endothelial cells. This pathway could have implications in vivo for atherosclerotic plaque formation, in which there is stiffening of the endothelial cells, and PECAM-1 could be employed to elicit some of the early events leading to plaque formation.

mbc.E13-01-0002 Molecular Biology of the Cell Volume 24 Page 678 MBoC is pleased to publish this summary of the Minisymposium “Cell Growth and Cell Cycle Control” held at the American Society for Cell Biology 2012 Annual Meeting, San Francisco, CA, December 19, 2012.

To deliver the right amount of the right protein to the right place at the right time is a fundamental cell biology event underlying diverse cellular, physiological, and pathological processes. As cochairs of the Minisymposium on Intracellular Sorting and Trafficking we reviewed a large number of deserving abstracts, but we could choose only six speakers, ranging from junior to senior investigators. The talks covered major cellular organelles involved in sorting and trafficking, including the endoplasmic reticulum (ER), Golgi, endocytic compartments, the plasma membrane, and the primary cilia. We were thrilled to have Gia Voeltz from the University of Colorado at Boulder in our session, and we congratulated her on her Early Career Life Scientist Award. She described her original work in defining the molecular basis governing the organization and dynamics of the different subdomains and connectivity of the ER using in vitro systems, biochemical approaches, and in vivo cell biological validation. The small GTPase Rab10 was identified as a major player. A part of the story was just published (English and Voeltz, 2012 ). Moving to the Golgi apparatus, Wanjin Hong from the Institute of Molecular and Cell Biology in Singapore talked about an approach of data-browsing the Human Protein Atlas (www.proteinatlas.org) to identify several new potential Golgi membrane proteins and focused on the novel Golgi protein TMEM115. TMEM115 is an evolutionarily conserved protein with four predicted transmembrane domains and a C-terminus facing the cytoplasm. Functionally, TMEM115 may regulate retrograde trafficking from the Golgi back to the ER through interaction with the conserved oligomeric Golgi complex. Moving from the Golgi to the cell's surface, Thierry Galli from the Institut Jacques Monod in France focused on Ti-VAMP/VAMP7 and its interacting network in regulating the migration of vesicles from the cell center to the cell periphery via the microtubules to mediate exocytosis. A multiprotein interacting network for Ti-VAMP was described, including the Rab21 guanine nucleotide exchange factor Varp, MACF1, GolginA4, and the kinesin 1 Kif5A. Part of the work has appeared recently (Burgo et al., 2012 ). Also at the cell surface, Ludger Johannes from the Institut Curie in France talked about his exciting discovery underlying the molecular aspects of clathrin-independent endocytosis. His group has identified an endogenous protein that drives the biogenesis of clathrin-independent carriers for the uptake of transmembrane cargo proteins, such as CD44. In experiments on cell and model membranes, they found that glycosphingolipids were key to the formation of endocytic membrane invaginations. These finding were condensed into the first mechanistic model based on specific protein machinery to describe how the uptake of certain endogenous cargoes is initiated without the help of the cytosolic clathrin machinery. Moving to later stages in endocytosis, Anne Spang from the University of Basel in Switzerland described a systematic study to address coordination of various processes, such as membrane fusion, acidification, and intralumenal vesicle formation underlying the transition from the early to the late endosome, which occurs during endosome maturation (Poteryaev et al., 2010 ). Novel roles and the action of the HOPS (homotypic fusion and vacuole protein sorting) and the CORVET (class C core vacuole/endosome tethering) tethering complexes in conjunction with SAND-1/Mon1 and RABX-5 on endosome maturation in the model organism Caenorhabditis elegans were presented. Projecting from the cell surface are primary cilia, which are receiving increased attention due to the growing number of human diseases found to be related to the biogenesis, trafficking, and maintenance of the cilia. Importantly, primary cilia appear to be key signaling platforms. Peter Jackson from Genentech in San Francisco discussed many molecules and interacting networks important for cilia biogenesis and trafficking. A key aspect is the role of the small GTPase Arl3 in targeting myristoylated and prenylated proteins, such as NPHP3, to the primary cilium via UNC119 and PDE6D effectors. Some of this work has already been published (Wright et al., 2011 ). We thank all the speakers for their participation and contri­butions and the discussion participants for making this Minisymposium such a great event.

Cellular signaling can be complex and dynamic. In the past several decades, it has become clear that many signal transduction pathways are not simple one-way transmissions of upstream stimuli, but are mediated through extremely complicated networks. Our next challenge is to move beyond the static biochemical description of networks into developing a functional quantitative understanding of their behavior. In this Minisymposium, we presented diverse approaches for beginning to achieve this goal. Galit Lahav (Harvard Medical School) opened the session and demonstrated how the temporal dynamics of the tumor suppressor protein p53 in single cells affect cell fate decisions. Cells that naturally show a series of p53 pulses in response to irradiation and recover from the damage were perturbed to instead show sustained p53. This perturbation led to activation of different target genes and pushed cells toward permanent cell cycle arrest. p53 dynamics therefore contribute to transferring information in cells and provide a new axis for pushing cells toward a specific cellular outcome. Andrei V. Karginov (University of Illinois at Chicago) focused on the spatiotemporal regulation of the Src signaling pathway by engineering a chimera of the Src kinase domain and an insertable FKBP12 protein, iFKBP. By fusing the FKBP12-rapamycin binding domain (FRB) to the specific downstream effectors, he restricted the Src activation to the complex it forms with FRB-bearing downstream targets. He presented evidence that a specific complex of Src and FAK leads to focal adhesion, while a complex of Src and p130Cas leads to filopodia formation. Robin E. Lee (Dana-Farber Cancer Institute) demonstrated that, upon tumor necrosis factor-α (TNF-α) stimulation, the early-response genes of NF-κB use memory to assess NF-κB activation. He analyzed the nuclear translocation dynamics of p65, a major NF-κB transcription factor, and then the number of mRNA transcripts for target genes in the same cells. The gene transcriptions of three early-response genes, IL8, A20, and IκB α, were regulated by the fold changes of nuclear NF-κB. His results, supported by computational modeling, suggest that competitive transcription factor–DNA interactions can provide memory of pre-ligand NF-κB states, allowing fold-change signal detection. The NF-κB signaling pathway has been also shown to involve ubiquitin modification of important molecules. Fumiyo Ikeda (Institute of Molecular Biotechnology) reported that an atypical type of ubiquitin chain, linearly linked chains, plays a critical role in the regulation of a Sharpin-dependent, anti-apoptotic pathway downstream of TNF. She presented a critical apoptosis signaling molecule, FADD, as a novel substrate of the Sharpin-containing E3 ligase that plays a role in the regulation of the apoptotic cascade. Jeffery A. Nickerson (University of Massachusetts Medical School) focused on the regulation of mRNA export by phosphatidylinositol 3-kinase/protein kinase B (PI3 kinase/Akt) signal transduction. His group implemented a fluorescence recovery after photobleaching system for screening signal transduction pathways that regulate mRNA nuclear export and the binding of the exon junction complex core proteins. By combining the analysis of mRNA export to the cytoplasm, they showed that the PI3 kinase/AKT pathway regulates not only mRNA export complex formation, but also the rate of mRNA nuclear export. Anna Podgornaia (Massachusetts Institute of Technology) tackled a challenging question to understand the transient protein–protein interaction surface that determines protein pairing by using a systematic mutagenesis approach. She focused on the histidine kinase PhoQ and its cognate response regulator PhoP, which together regulate bacterial signaling. Out of 160,000 PhoQ variants completely randomized at four residues, 4600 variants were signal-responsive. She is exploring why all these 4600 sequences are not used by extant PhoQ orthologues, which only have 260 different interface sequences.

Properly regulated actin organization and dynamics are crucial for cellular processes such as cell migration, cell division, and maintenance of cell–cell junctions. Speakers in the “Actin Organization and Dynamics” Minisymposium at the ASCB 2012 meeting highlighted new and interesting mechanisms that underlie the regulation of actin dynamics. The session was broad in scope, ranging from in vitro biochemical and biophysical studies with purified components to cellular- and tissue-level studies. There was an emphasis on severing mechanisms and mechanics, as well as the complexities of regulatory pathways in cells. Hyeran Kang (Yale University), a postdoc with Enrique M. De La Cruz, presented work on how cations affect filament mechanics and severing via the regulatory protein, cofilin. Kang identified, using structural bioinformatics, two potential filament-specific cation-binding sites and classified them as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent assembly and bending mechanics, respectively. Cofilin enhances actin filament bending and twisting compliance, and it is hypothesized that local mechanical discontinuities in partially decorated filaments promote severing at boundaries of bare and cofilin-decorated segments. Using native and engineered yeast actin mutants, Kang showed that dissociation of stiffness site cations by human cofilin drives changes in filament bending mechanics and that this is required for severing. Pinar Gurel (Geisel School of Medicine at Dartmouth), a graduate student with Harry Higgs, described research investigating the severing activity of INF2, a formin linked to two human genetic diseases: Charcot-Marie-Tooth disease and focal segmental glomerulosclerosis. Using a combination of classic biochemical experiments and total internal reflection fluorescence (TIRF) microscopy, Gurel showed that, in addition to the normal formin binding site at the barbed end, INF2 binds the sides of actin filaments stoichiometrically (one INF2 per subunit) and with submicromolar affinity. INF2-catalyzed filament severing occurs within INF2-decorated regions of partially and fully decorated filaments. This property, along with INF2’s ability to bind ADP-Pi filaments, distinguishes INF2-mediated filament severing from that of cofilin. Dennis Breitsprecher (Brandeis University), a postdoc with Bruce Goode, presented recent work demonstrating a new role for Srv2/cyclase-associated protein. Breitsprecher observed, using dual-color TIRF microscopy, that the N-terminal half of Srv2 (N-Srv2) increases the severing activity of cofilin without appreciably affecting cofilin binding to filaments. Electron microscopy and single-particle analysis revealed that N-Srv2 self-associates into hexameric ring structures. N-terminal truncations weaken N-Srv2 oligomerization, compromise enhanced severing activity, and yield actin organization and cell polarity defects in cells. Peter Bieling (University of California, San Francisco), a postdoc with Dyche Mullins and Dan Fletcher, evaluated how reconstituted dendritic actin network growth responds to external loads. Dendritic actin networks assembled in vitro from micron-sized patterns of surface-immobilized nucleation-promoting factor were challenged with force applied via an atomic force microscope (AFM) cantilever, and actin assembly was monitored using multicolor TIRF microscopy and AFM. Bieling demonstrated that the dendritic actin networks dynamically adapt to external forces; the actin network growth velocity decreases with load, while actin density in the force-generating region of the network increases with load. Barbed-end capping is force-dependent and slows with increasing load. Colleen G. Bilancia (University of North Carolina, Chapel Hill), a postdoc with Mark Peifer, presented work on how Enabled (Ena) and Diaphanous (Dia) coordinate to regulate actin filament barbed-end polymerization. Overexpressing active Dia in Drosophila D16 cells promotes long, stable filopodial protrusions, while overexpressing Ena leads to dynamic, fan-like protrusions. Coexpression of Dia and Ena promotes protrusions distinct from those produced with expression of either regulator independently. Bilancia noted that filopodia retract when active Dia and Ena colocalize, suggesting that Ena negatively regulates Dia, possibly through direct binding interactions. Indeed, Bilancia showed that the Ena EVH1 domain directly binds the Dia FH1 domain, and the EVH1 domain inhibits Dia-mediated in vitro pyrene actin assembly and filopodia formation in cells. Ann Miller (University of Michigan) described her lab's finding that the cytokinesis regulator anillin plays a novel role in regulating epithelial cell–cell junctions. Using Xenopus embryos as a model system, Miller showed that a population of anillin localizes at cell–cell junctions in both dividing and nondividing cells. Further, anillin functionally regulates junctional integrity. Both tight junctions and adherens junctions are disrupted when anillin is knocked down. Anillin binds actin filaments and the small GTPase RhoA, both of which regulate cell–cell junction structure and function. Miller's group tested the effects of anillin expression on RhoA activation and found that anillin knockdown results in increased spontaneous flares of active RhoA that are prominent at cell–cell junctions in both dividing cells and nondividing regions of the epithelium.

The broad-ranging topic of this Minisymposium attracted four presentations describing in vivo and in vitro experimental work and two presentations that primarily addressed theoretical aspects of chromatin behavior over long and short length scales. Overall, the focus was very much on chromosomes, with presentations addressing chromosome ends (telomeres), centromeres, chromosome segregation, heterochromatin, and specific gene regions. In two complementary papers from the Andrew Spakowitz laboratory, Peter Mulligan and Elena Koslover (Stanford University), addressed theoretical models of DNA mechanics across the very different length scales in chromosomes and cooperative binding during heterochromatin formation. The theme of nucleosome structure and packing was maintained by Abbas Padeganeh (Universite de Montreal), who used total internal reflection fluorescence–coupled, photobleaching-assisted copy-number counting of single nucleosomes obtained from cultured cells to provide evidence to support the view that the histone H3 variant CENP-A, which is critical for centromere identity and function, forms octameric nucleosomes containing dimers of CENP-A. Geoff Lovely (Caltech, Pasadena) addressed the molecular mechanism that generates antigenic variation by using a single-molecule in vitro assay to capture and characterize pairing intermediates in V(D)J recombination mediated by RAG1-2 and HMGB1. David Sherratt (Oxford University) described in vivo single-molecule biochemistry experiments addressing the architecture of the Escherichia coli SMC (Structural Maintenance of Chromosomes) complexes, MukBEF, and its role in chromosome segregation. Clusters of relatively immobile dimer-of-dimer MukBEF complexes associate with the replication origin region to apparently position it and facilitate the positioning of newly replicated sisters. Finally, Daniela Rhodes (Nanyang Technological University) presented the first three-dimensional structure of active, full-length human telomerase determined using single-particle electron microscopy. Telomerase is medically important, as telomere maintenance in the majority of cancer cells involves the activation of telomerase. Telomerase consists of a large RNA subunit TER and a protein catalytic subunit TERT. The structural information revealed that telomerase has a bilobal architecture in which the two monomers in the dimer have “open” and “closed” conformations, linked by a flexible dimer interface. Fitting of the atomic structure of the beetle TERT subunit into the electron microscopy density revealed the spatial relationship between RNA and protein subunits, providing novel insights into the architecture of the telomerase enzyme. Furthermore, Rhodes presented data showing that catalytic activity requires both TERT active sites to be functional, providing unambiguous evidence that human telomerase functions as a dimer.

Following a successful panel discussion at last years' American Society for Cell Biology Annual Meeting, the first minisymposium in Synthetic Cell Biology was held at this years' annual meeting in Denver. This is an exciting first for the society, and the attendance clearly supported the interest of cell biologists in applying the concepts of synthetic biology to the understanding and engineering of cells. Pamela Silver opened the session with a brief overview of the rapidly growing field of synthetic biology. She began by defining the goal of synthetic biology as making the building of biological systems faster, cheaper, and more predictable. She anticipated that the engineering of biology will be the defining technology of this century. Over 50 years of molecular and cell biology have brought us to a point where biology is much in the space where organic chemistry was 30 years ago—we understand many of the pieces and the fundamental reactions, and we are well situated to start building with biology. Indeed, the modular nature of much of cell biology offers an important starting point for the logical engineering of a wide range of useful biological systems. And DNA—the basic building material for biology—is getting increasingly cheaper to synthesize, thereby making it possible to consider creating large pieces of reprogrammed chromosomes and in some cases, whole genomes. But how do we start, and what should we build? The minisymposium offered some useful visions and guidance. For example, Silver discussed the modular nature of the control of carbon fixation in photosynthetic bacteria. She presented results from Dave Savage that indicate that it is possible to reassemble carbon-fixing units (carboxysomes in nonphotosynthetic bacteria), a first step in moving carbon fixation from one organism to another. In another series of experiments, she demonstrated the artificial assembly of RNA-based scaffolds engineered to carry out biosynthetic reactions more efficiently inside cells. Together these results represent important steps forward in programming new behaviors into cells using the modularity of biology. Ron Weiss, consistent with his background as a computer scientist turned synthetic biologist, went on to argue that the cell is a highly programmable entity. One of his goals is to create gene programs that control tissue patterning through the engineering of artificial homeostasis and timing of differentiation. One strategy to accomplish this involves a biocompiler designed to take a high-level cell behavior, abstract it into a regulatory network, and expand it into a logical operation in terms of a gene network that then instructs a robot to assemble the necessary DNA. These talks were followed by four short talks from younger investigators. Brian Goodman from Sam Reck-Peterson's lab discussed using DNA as a scaffold on which to position molecular motors for directional transport. Karmella Haynes, a new assistant professor at Arizona State University, talked about designing a collection of modules from chromatin to guide a cell to differentiation in programmable ways. Takanari Inoue presented novel intracellular logic gates that could be chemically controlled. And Clifford Wong built on the engineering parallels to analyze gene dosage and expression by analogy to band-pass filters. This session engaged many who were unfamiliar with synthetic biology and stimulated cell biologists to think about new joint applications. Our deep understanding from the years of cell biological research will play a major role in the future of synthetic biology.

The Minisymposium on Bioengineering and Mechanobiology focused on engineering approaches to investigate the mechanical signaling of cells and tissues and was cochaired by Adam J. Engler (University of California, San Diego) and Celeste M. Nelson (Princeton University). The program moved thematically from the mechanics of the nucleus to those of the cell cortex and finally to the mechanical behaviors of tissues. Jan Lammerding (Cornell University) described work probing the role of the nuclear envelope proteins, lamin A and C, in the structure and mechanical properties of the nuclei of individual cells and whole tissues. Fibroblasts isolated from patients harboring mutations in the LMNA gene that cause muscular dystrophies had softer nuclei than controls. Expressing these mutated forms of the LMNA gene in otherwise lamin A/C–deficient mouse embryonic fibroblasts also yielded softer nuclei and defects in coupling of the nucleus to the cytoskeleton. Importantly, applying strain to the body wall musculature of genetically modified Drosophila melanogaster showed that lamin mutations altered nuclear mechanics within intact muscle tissues. Ben Fabry (University of Erlangen–Nuremberg) demonstrated that the binding between the focal adhesion proteins p130Cas and vinculin is important for mechanotransduction. Mechanical stress is coupled to p130Cas through vinculin and initiates the activation of downstream signaling through pathways such as the extracellular signal–regulated kinase (ERK1/2) pathway. Phosphorylation of p130Cas on residue Y12 or mutation of this residue to a phosphomimicking glutamate prevents vinculin binding, reduces the localization of p130Cas to focal adhesion sites, and suppresses ERK1/2 signaling in response to mechanical stretch. Yee Seir Kee (Robinson and Iglesias Laboratories, Johns Hopkins University School of Medicine) used micropipette aspiration and compression approaches to investigate how assembly of the cleavage furrow responds to mechanical stress in Dictyostelium. During cytokinesis, cell shape is controlled by a mechanosensory system that includes the myosin II motor protein and the actin cross-linker, cortexillin I. Accumulation of myosin II at the cleavage furrow is governed by a regulatory network composed of IQGAP1, IQGAP2, kinesin-6, and INCENP. Feedback loops present within this network are responsible for regulating the levels of myosin II at the cleavage furrow. Celeste Nelson (Princeton University) presented results suggesting that cells respond to the mechanical properties of their surrounding microenvironment, in part by regulating the subcellular localization of small GTPases and the assembly and activation of NADPH oxidase. Mammary epithelial cells underwent epithelial–mesenchymal transition (EMT) on stiff, but not soft, substrata. Cells on stiff substrata apportioned Rac1b, a splice variant of Rac1, to the plasma membrane, at which it formed an activated complex with NADPH oxidase and permitted the generation of reactive oxygen species. The complex did not form on soft substrata, and blocking membrane localization of Rac1b prevented EMT on stiffer microenvironments. Dylan Tyler Burnette (Lippincott–Schwartz Laboratory, National Institutes of Health) was the recipient of the Merton Bernfield Memorial Award, which is given every year to one graduate student or postdoctoral fellow in recognition of his or her meritorious achievements in cell biology research. Burnette discussed his efforts in expanding the imaging toolbox to the nanometer scale using conventional fluorophores, in an approach dubbed bleaching/blinking-assisted localization microscopy (BaLM; Burnette et al., 2011 ). Point localization–based superresolution images with a resolution on the scale of tens of nanometers can be produced from commonly used fluorescent molecules by taking advantage of their intrinsic bleaching and blinking. Four-color images can be generated via BaLM with standard electron-multiplying charge-coupled device cameras by using multiple fluorophores in a single cell and can resolve individual motor proteins along intact cytoskeletal networks. Adam Engler (University of California, San Diego) reported on the differences in differentiation response between adipose-derived and bone marrow–derived stem cells. Both types of stem cells are sensitive to the stiffness of their surrounding microenvironment and differentiate down a muscle lineage when cultured on extracellular matrix of a specific compliance mimicking that of muscle in vivo. The adipose-derived stem cells (ASCs), however, express markers of muscle differentiation at 10-fold higher levels than do the bone marrow–derived stem cells. Furthermore, a fraction of ASCs cultured under these conditions form multinucleated myotubes, which could be maintained when replated on non­permissive substrata.

In the Chemical Biology Minisymposium, cochaired by Alice Ting and Lisa Belmont, the first three talks focused on the development of novel probes for cell biology, while the last three presentations highlighted inhibitors of proteins previously believed to be “undruggable.” Daniel Hochbaum (Cohen lab, Harvard University) described an optical probe for imaging single action potentials using the fluorescence of a rhodopsin protein, Archaerhodopsin 3 (Arch), expressed in cultured rat hippocampal neurons. This voltage indicator exhibited a 10-fold improvement in sensitivity and speed over existing protein-based voltage indicators, with a twofold increase in brightness between −150 mV and +150 mV and a submillisecond response time. Arch detected single electrically triggered action potentials with a signal-to-noise ratio > 10. The mutant ArchD95N lacked endogenous proton pumping and showed 50% greater sensitivity than wild-type. Although it had a slower response (41 ms), ArchD95N resolved individual action potentials. Alice Ting (MIT) presented a novel approach to determining the proteomic composition of subcellular compartments by targeting a promiscuous biotin-conjugating enzyme to subcellular regions. The labeled proteins were enriched and identified by mass spectrometry. This approach was used to determine the proteomes of mitochondria and the endoplasmic reticulum of live mammalian cells without using subcellular fractionation. Katie White, a graduate student in Ting's lab, then described improvements to in vivo fluorophore labeling using mutants of lipoic acid ligase (LplA). She engineered LplA to accept a blue coumarin fluorophore and then used yeast display evolution to evolve LplA into a probe ligase with high activity in the secretory pathway. The LplA variants allowed imaging of intra- or intercellular protein–protein contacts. To expand the scope of bioluminescence imaging, Stephen Miller (University of Massachusetts Medical School) developed new aminoluciferin substrates for firefly luciferase that emit light at longer wavelengths than d-luciferin. Although these substrates were initially limited by product inhibition, this could be ameliorated by mutation of luciferase. Moreover, mutant luciferases were identified that displayed selectivity for these synthetic substrates over d-luciferin. This system has two advantages: it is potentially better suited for in vivo imaging, because tissue is more transparent to light at longer wavelengths, and orthogonal luciferase–luciferin pairs could allow multiplexed bioluminescence imaging. An NMR-based fragment screen for inhibitors of the Ras oncoprotein presented by Guowei Fang (Genentech) identified 25 compounds that inhibit Ras with two distinct mechanisms of action. One class of compounds binds to a small pocket between Switch I and Switch II that expands to accommodate the inhibitor. These compounds competitively inhibit nucleotide exchange by blocking the interaction of RasGDP with its nucleotide exchange factor, SOS. The second class of compounds binds in a pocket created by the interface of the Ras–SOS complex and acts by accelerating nucleotide release. Corey Nislow (University of Toronto) highlighted the power of yeast genetics by screening cell-active, structurally diverse compounds against 1100 heterozygous strains (haploid for essential genes) and 5000 homozygous deletion strains. He identified 55 novel drug targets, including Sec14 and septin. Sec14 coordinates lipid biosynthesis with signaling pathways and was thought to be undruggable. He then showed how chemical genetic profiling revealed that elesclomol, a compound demonstrating efficacy in metastatic melanoma, inhibits the electron transport chain. These approaches will continue to bear fruit as additional targets are confirmed and similar technology is applied to Candida albicans and other organisms. Lisa Belmont described a model for synergy between paclitaxel and navitoclax, a Bcl-2/Bcl-xL inhibitor, in which cells in mitotic arrest slowly degrade Mcl-1, while navitoclax causes acute inhibition of Bcl-xL. Across 50 cancer cell lines, cells with high levels of Bcl-xL relative to Mcl-1 exhibited lowered paclitaxel response and higher paclitaxel/navitoclax synergy. The cell line synergy translated to xenograft studies, and analysis of ovarian cancer tissue from paclitaxel-treated patients demonstrated that high Bcl-xL predicted poor response to paclitaxel. Taken together, the data suggest the paclitaxel–navitoclax combination might be effective in cancers expressing high levels of Bcl-xL.

In recent years, definitions of the nuclear periphery have become increasingly blurred with the realization that nuclear envelope (NE) and nuclear pore complex (NPC) components interact with and influence the behavior of both nuclear and cytoplasmic structures. The Minisymposium on the Nuclear Periphery reflected this evolving outlook by bringing together an eclectic group of researchers working on a variety of problems related to the biology of the NE/NPC. A. Szymborska (Ellenberg laboratory, European Molecular Biology Laboratory, Heidelberg) described how emerging techniques in superresolution light microscopy can be used to define the disposition of NPC components with precision in the range of nanometers. This approach permitted the first visualization of the radial substructure of the NPC by light microscopy and was able to resolve distinct members of an NPC subcomplex (the Nup107-160/Y complex). This has provided new insights into the orientation of this complex within the NPC. L. Lu (Kirchhausen laboratory, Harvard Medical School, Boston) raised the controversial issue of how NPCs become incorporated into the reforming NE at the end of mitosis. Using a combination of three-dimensional live-cell imaging and electron microscope tomography, he showed that newly segregated chromatids become coated by planar membrane sheets derived from the peripheral endoplasmic reticulum prior to NPC assembly. This suggests that mitotic and interphase NPC assembly may follow similar pathways (Lu et al., 2011 ). D. Osorio (Gomes laboratory, Universite Pierre et Marie Curie, Paris) described a small interfering RNA (siRNA)-based screen that revealed a role for Ndc1, an NPC membrane protein, in the positioning of the nucleus relative to the centrosome during fibroblast migration. His data indicate that the interaction of Ndc1 with Sun2, a constituent of LINC (LInker of the Nucleoskeleton and Cytoskeleton) complexes might regulate nuclear positioning and cell migration. D. Hu (Vallee laboratory, Columbia University, New York) focused on the cell cycle–dependent nuclear oscillation in neural progenitor cells. This process, termed interkinetic nuclear migration (INM), involves dynein-driven migration of the nucleus toward the apical surface of the neocortex in G2 (Tsai et al., 2010 ). Interestingly, in vivo siRNA-mediated depletion of factors previously implicated in dynein recruitment to the NE during G2 in nonneuronal cells, namely BicD2 (Splinter et al., 2010 ) and Nup133 (Bolhy et al., 2011 ), successively impaired INM in G2. These findings strongly support a role for dynein recruitment to the NE in apical nuclear migration during INM and provide insight into the mechanism of cell cycle control. H. Horn (Stewart laboratory, Institute of Medical Biology, Singapore) reported on the role of nesprin-4 (Nesp4), an outer nuclear membrane protein and LINC complex component. Nesp4 functions as an adaptor for kinesin-1 and is expressed in certain epithelial cells, including hair cells of the inner ear (Roux et al., 2009 ). Derivation of Nesp4-deficient mice reveals that this protein is required for the basal positioning of nuclei in outer hair cells. In the absence of Nesp4, outer hair cells are lost through apoptosis, resulting in progressive hearing loss. Y. Kim (Zheng laboratory, Carnegie Institute for Science, Baltimore) unexpectedly reported that lamin B1− and B2− double-knockout embryonic stem cells (ESCs), despite expressing no other type of lamin (A/C or B3), are indistinguishable from wild-type cells and are fully capable of differentiating toward the trophectoderm lineage. Furthermore, gene-expression analyses revealed that B-type lamins are not required for the silencing of their bound genes that are required for lineage specification in differentiating ESCs. Remarkably, lamin B double-knockout mice develop to term, albeit with defects in multiple organs (Kim et al., 2011 ). Clearly, this study is going to change our view of nuclear lamina function in both health and disease. Taken together, the studies described in this minisymposium impact a number of areas of cell and developmental biology. There is little doubt that they will provide the foundation for future work in this rapidly evolving field. We look forward to seeing the fruits of this work at future ASCB meetings.

Cell–cell and cell–extracellular matrix (ECM) interactions are fundamental to the complexity of multicellularity achieved in the metazoa. These interactions share many conceptual similarities, the most prominent being a dependence on transmembrane adhesion receptors, binding of adhesion receptors to specific extracellular ligand partners, and the linkage of receptor cytoplasmic domains to intracellular cytoskeletal systems via protein complexes. In both cases, these protein complexes also integrate intracellular signaling, cytoskeletal organization, and regulation of multiple cellular functions. The 2011 Minisymposium on Cell–Cell and Cell–Matrix Interactions brought together current topics and model systems within this broad area of cell biology.

A comparative or evolutionary approach is a powerful addition to the cell biologist's armory. It can provide context for observations in more classical model systems; it can elucidate the forces shaping the morphology, organization, and complexity of the cell; and it can identify new phenomena that may eventually be recognized as crucial to how cells work. Over the past 15 years, genome sequencing has facilitated comparative work in microbial eukaryotes, while advances in cellular imaging technologies have opened up prokaryotes as models for the study of cell biology. At the 2011 ASCB meeting, the Minisymposium entitled “Cell Biology of Micro-organisms and the Evolution of the Eukaryotic Cell” highlighted mechanisms that underpin the evolution of complexity in cells, described new and unexpected microbial cellular phenomena, and reported the development of technologies that will allow us to explore new avenues in the study of microbial cells. The session began with the theme of eukaryotic cell evolution and emergent complexity. Using a combination of comparative genomics and structural modeling Fred Mast (University of Alberta) described an evolutionary model for how multiple organellar cargoes compete for transport by myosin V (Mast et al., 2011 ). Aaron Turkewitz (University of Chicago) extended this theme by discussing evidence that evolutionary diversification of the Rab family of small GTPases scales with increasing complexity of cellular membrane trafficking and compartmentalization (Bright et al., 2010 ). Mark Slabodnick (University of California, San Francisco) concluded the session's presentations on unicellular eukaroytes with his progress report on the development of new molecular tools to study cell division and regeneration in the giant ciliate, Stentor. The second half of the minisymposium centered on the biology of bacterial cells, and began with Alex Valm (Marine Biological Laboratory–Woods Hole), who described an exciting new fluorescence imaging method. Combinatorial labeling and spectral imaging fluorescence in situ hybridization (CLASI-FISH) uses dozens of combinations of fluorescent probes that provide an unprecedented look at the spatial composition of complex bacterial cell communities (Valm et al., 2011 ). Joel Kralj (Harvard University) reported the development of a voltage-sensitive fluorescent protein known as the proteorhodopsin optical proton sensor (PROPS). Expression of PROPS in Escherichia coli revealed unexpected, high-frequency electrical spiking (Kralj et al., 2011 ) that coincided with efflux of a small fluorophore from the cell. The results of these studies suggest that some bacterial cell efflux processes may be electrically regulated. Finally, Sean Crosson (University of Chicago) described recent data on molecular and genetic determinants of cell cycle control and cell differentiation in the bacterium Caulobacter crescentus. These studies have defined how the broadly conserved regulatory molecules guanosine tetraphosphate and inorganic polyphosphate link nutrient limitation to cell differentiation at the G1–S cell cycle transition in a bacterial cell (Boutte et al., 2012 ). All of these talks make us rethink our ideas about “normal” cell biology and awakened us to the “endless forms most beautiful and most wonderful” into which the cell has evolved.