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As the interface between the cell and its environment, the cell cortex must be able to respond to a variety of external stimuli. This is made possible in part by cortical excitability, a behavior driven by coupled positive and negative feedback loops that generate propagating waves of actin assembly in the cell cortex. Cortical excitability is best known for promoting cell protrusion and allowing the interpretation of and response to chemoattractant gradients in migrating cells. It has recently become apparent, however, that cortical excitability is involved in the response of the cortex to internal signals from the cell-cycle regulatory machinery and the spindle during cell division. Two overlapping functions have been ascribed to cortical excitability in cell division: control of cell division plane placement, and amplification of the activity of the small GTPase Rho at the equatorial cortex during cytokinesis. Here, we propose that cortical excitability explains several important, yet poorly understood features of signaling during cell division. We also consider the potential advantages that arise from the use of cortical excitability as a signaling mechanism to regulate cortical dynamics in cell division.

Mechanical properties of the cell are important biomarkers for probing its architectural changes caused by cellular processes and/or pathologies. The development of microfluidic technologies has enabled measuring the cell’s mechanical properties at high throughput so that mechanical phenotyping can be applied to large samples in reasonable timescales. These studies typically measure the stiffness of the cell as the only mechanical biomarker and do not disentangle the rheological contributions of different structural components of the cell, including the cell cortex, the interior cytoplasm and its immersed cytoskeletal structures, and the nucleus. Recent advancements in high-speed fluorescent imaging have enabled probing the deformations of the cell cortex while also tracking different intracellular components in rates applicable to microfluidic platforms. We present a, to our knowledge, novel method to decouple the mechanics of the cell cortex and the cytoplasm by analyzing the correlation between the cortical deformations that are induced by external microfluidic flows and the nucleus displacements, induced by those cortical deformations, i.e., we use the nucleus as a high-throughput microrheological probe to study the rheology of the cytoplasm, independent of the cell cortex mechanics. To demonstrate the applicability of this method, we consider a proof-of-concept model consisting of a rigid spherical nucleus centered in a spherical cell. We obtain analytical expressions for the time-dependent nucleus velocity as a function of the cell deformations when the interior cytoplasm is modeled as a viscous, viscoelastic, porous, and poroelastic material and demonstrate how the nucleus velocity can be used to characterize the linear rheology of the cytoplasm over a wide range of forces and timescales/frequencies.

Septins are conserved guanine nucleotide-binding proteins that polymerize into filaments at the cell cortex or in association with other cytoskeletal proteins, such as actin or microtubules. As integral players in many morphogenic and signaling events, septins form scaffolds important for the recruitment of the cytokinetic machinery, organization of the plasma membrane, and orientation of cell polarity. Mutations in septins or their misregulation are associated with numerous diseases. Despite growing appreciation for the importance of septins in different aspects of cell biology and disease, septins remain relatively poorly understood compared with other cytoskeletal proteins. Here in this review, we highlight some of the recent developments of the last two years in the field of septin cell biology.

Cortical actin waves have emerged as a widely prevalent phenomena and brought pattern formation to many fields of cell biology. Cortical excitabilities, reminiscent of the electric excitability in neurons, are likely fundamental property of the cell cortex. Although they have been mostly considered to be biochemical in nature, accumulating evidence support the role of mechanics in the pattern formation process. Both pattern formation and mechanobiology approach biological phenomena at the collective level, either by looking at the mesoscale dynamical behavior of molecular networks or by using collective physical properties to characterize biological systems. As such they are very different from the traditional reductionist, bottom-up view of biology, which brings new challenges and potential opportunities. In this essay, we aim to provide our perspectives on what the proposed mechanochemical feedbacks are and open questions regarding their role in cortical excitable and oscillatory dynamics.

To segregate chromosomes in mitosis, cells assemble a mitotic spindle, a molecular machine with centrosomes at two opposing cell poles and chromosomes at the equator. Microtubules and molecular motors connect the poles to kinetochores, specialized protein assemblies on the centromere regions of the chromosomes. Bipolarity of the spindle is crucial for the proper cell division, and two centrosomes in animal cells naturally become two spindle poles. Cancer cells are often multicentrosomal, yet they are able to assemble bipolar spindles by clustering centrosomes into two spindle poles. Mechanisms of this clustering are debated. In this study, we computationally screen effective forces between 1) centrosomes, 2) centrosomes and kinetochores, 3) centrosomes and chromosome arms, and 4) centrosomes and cell cortex to understand mechanics that determines three-dimensional spindle architecture. To do this, we use the stochastic Monte Carlo search for stable mechanical equilibria in the effective energy landscape of the spindle. We find that the following conditions have to be met to robustly assemble the bipolar spindle in a multicentrosomal cell: 1) the strengths of centrosomes’ attraction to each other and to the cell cortex have to be proportional to each other and 2) the strengths of centrosomes’ attraction to kinetochores and repulsion from the chromosome arms have to be proportional to each other. We also find that three other spindle configurations emerge if these conditions are not met: 1) collapsed, 2) monopolar, and 3) multipolar spindles, and the computational screen reveals mechanical conditions for these abnormal spindles.

The actin cytoskeleton consists of structurally and biochemically different actin filament arrays. Among them, the actin cortex is thought to have key roles in cell mechanics, but remains a poorly characterized part of the actin cytoskeleton. The cell cortex is typically defined as a thin layer of actin meshwork that uniformly underlies the plasma membrane of the entire cell. However, this definition applies only to specific cases. In general, the cortex structure and subcellular distribution vary significantly across cell types and physiological states of the cell. In this review, I focus on our current knowledge of the structure and molecular composition of the cell cortex.

Proper mitotic spindle orientation requires that astral microtubules are connected to the cell cortex by the microtubule-binding protein NuMA, which is recruited from the cytoplasm. Cortical recruitment of NuMA is at least partially mediated via direct binding to the adaptor protein LGN. LGN normally adopts a closed conformation via an intramolecular interaction between its N-terminal NuMA-binding domain and its C-terminal region that contains four GoLoco (GL) motifs, each capable of binding to the membrane-anchored Gα(i) subunit of heterotrimeric G protein. Here we show that the intramolecular association with the N-terminal domain in LGN involves GL3, GL4, and a region between GL2 and GL3, whereas GL1 and GL2 do not play a major role. This conformation renders GL1 but not the other GL motifs in a state easily accessible to Gα(i). To interact with full-length LGN in a closed state, NuMA requires the presence of Gα(i); both NuMA and Gα(i) are essential for cortical recruitment of LGN in mitotic cells. In contrast, mInsc, a protein that competes with NuMA for binding to LGN and regulates mitotic spindle orientation in asymmetric cell division, efficiently binds to full-length LGN without Gα(i) and induces its conformational change, enhancing its association with Gα(i). In nonpolarized symmetrically dividing HeLa cells, disruption of the LGN–NuMA interaction by ectopic expression of mInsc results in a loss of cortical localization of NuMA during metaphase and anaphase and promotes mitotic spindle misorientation and a delayed anaphase progression. These findings highlight a specific role for LGN-mediated cell cortex recruitment of NuMA.

Background Improving stem cell (SC) deformability using pre-treatment strategies, or isolating more deformable sub-populations, may prevent non-specific entrapment of injected cells, maintain circulating numbers and thus increase the likelihood of capture by microvessels in injured organs. However, nothing is currently known about the basic mechanical properties of SCs, particularly with regards their elastic characteristics. This study therefore aimed to determine the mechanical characteristics of haematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) with comparisons made to neutrophils. Methods Micromanipulation and atomic force microscopy (AFM) were used to quantitate mechanical properties following large and small deformations respectively of neutrophils, MSCs and naive and stromal cell-derived factor-1α (SDF-1ɑ) or hydrogen peroxide (H2O2) pre-treated HSCs. Results Neutrophils and HSCs underwent rupture at ∼80% deformation. Nominal rupture stress (σR), nominal rupture tension (TR) and the Young's/elastic modulus at large deformations was significantly higher for neutrophils indicating they were stiffer and less deformable than HSCs. Surprisingly, MSCs did not rupture and were as deformable as HSCs despite their large size. Pre-treatment increased HSC deformability as indicated by lower rupture force, σR, TR and Young's modulus at large deformations. AFM demonstrated that pre-treatment increased the Young's modulus at smaller deformations indicating the HSC surface stiffened. This was accompanied by increased F-actin accumulation and its localisation in the cell cortex. Conclusion This is the first study to precisely demonstrate that mechanical distinctions exist amongst different therapeutic SCs with regards their deformability and rupture response to applied stress. This can potentially be utilized as label-free markers in microfluidic cell sorting systems to separate sub-populations of potentially more therapeutic SCs.

Global cytoskeleton reorganization is well-recognized when cells are exposed to distinct mechanical stimuli, but the localized responses at a specified region of a cell are still unclear. In this work, we mapped the cell-surface mechanical property of single cells in situ before and after static point loading these cells using atomic force microscopy in PeakForce-Quantitative Nano Mechanics mode. Cell-surface stiffness was elevated at a maximum of 1.35-fold at the vicinity of loading site, indicating an enhanced structural protection of the cortex to the cell. Mechanical modeling also elucidated the structural protection from the stiffened cell cortex, in which 9–15% and 10–19% decrease of maximum stress and strain of the nucleus were obtained. Furthermore, the flat-ended atomic force microscopy probes were used to capture cytoskeleton reorganization after point loading quantitatively, revealing that the larger the applied force and the longer the loading time are, the more pronounced cytoskeleton reorganization is. Also, point loading using a microneedle combined with real-time confocal microscopy uncovered the fast dynamics of actin cytoskeleton reorganization for actin-stained live cells after point loading (

Weibel-Palade bodies (WPBs) are specialized secretory organelles of endothelial cells that serve important functions in the response to inflammation and vascular injury. WPBs actively respond to different stimuli by regulated exocytosis leading to full or selective release of their contents. Cellular conditions and mechanisms that distinguish between these possibilities are only beginning to emerge. To address this we analyzed dynamic rearrangements of the actin cytoskeleton during histamine-stimulated, Ca2+-dependent WPB exocytosis. We show that most WPB fusion events are followed by a rapid release of von-Willebrand factor (VWF), the large WPB cargo, and that this occurs concomitant with a softening of the actin cortex by the recently described Ca2+-dependent actin reset (CaAR). However, a considerable fraction of WPB fusion events is characterized by a delayed release of VWF and observed after the CaAR reaction peak. These delayed VWF secretions are accompanied by an assembly of actin rings or coats around the WPB post-fusion structures and are also seen following direct elevation of intracellular Ca2+ by plasma membrane wounding. Actin ring/coat assembly at WPB post-fusion structures requires Rho GTPase activity and is significantly reduced upon expression of a dominant-active mutant of the formin INF2 that triggers a permanent CaAR peak-like sequestration of actin to the endoplasmic reticulum. These findings suggest that a rigid actin cortex correlates with a higher proportion of fused WPB which assemble actin rings/coats most likely required for efficient VWF expulsion and/or stabilization of a WPB post-fusion structure. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

The ability to divide is a fundamental property of a living cell. The 3D orientation of cell division is essential for embryogenesis, maintenance of tissue organization and architecture, as well as controlling cell fate. Much attention has been placed on the mitotic spindle’s role in placing itself along the cell’s longest axis, where a shape sensing mechanism between a population of microtubules extending from mitotic centrosomes to the cell cortex occurs. However, contractile forces at the cell cortex also likely play a decisive role in determining the final placement of daughter cells following division. In this review, we discuss recent literature that describes the role of these contractile forces and how these forces could be balanced by mitotic adhesion complexes.

The spatial organization of a cell is crucial for distribution of cell components and for cell morphogenesis in all organisms. Ustilago maydis, a basidiomycete fungus, has a yeast-like and a filamentous form. The former buds once per cell cycle at one of the cell poles, and can use the same site repeatedly or choose a new site at the same pole or opposite pole. The filamentous form consists of a long apical cell with short septate basal compartments lacking cytoplasm. It grows at the apex and can reverse growth forming a new growth zone at the basal end. We are interested in understanding how these different morphologies are generated. Here we present identification and characterization of U. maydis Tea1, a homologue of the fission yeast cell end marker Tea1. We demonstrate that UmTea1, a Kelch domain protein, interacts with itself and is an important determinant of the site of polarized growth: tea1 mutants bud simultaneously from both cell poles and form bifurcate buds. UmTea1 also regulates septum positioning, cell wall deposition, cell and neck width, coordination of nuclear division and cell separation, and localization of sterol-rich membrane domains. Some of these functions are shared with UmTea4, another cell end marker. We show that Tea1::GFP localizes to sites of polarized or potential polarized growth and to the septation site in the yeast-like form. Additionally, localization of Tea1::GFP as rings along the filament suggests that the filament undergoes septation. We hypothesize that Tea1 may act as a scaffold for the assembly of proteins that determine the site of polarized growth.

The orientation of the mitotic spindle is a crucial process that defines the axis of cell division, contributing to daughter cell positioning and fate, and hence to tissue morphogenesis and homeostasis. Pathological conditions associated with spindle orientation defects primarily include neuro-developmental disorders, while direct contributions to tumorigenesis are still under investigation. The trimeric NuMA/LGN/Gαi complex, the major determinant of spindle orientation, acts by anchoring to the cell cortex astral microtubules (MTs) and dynein motors, and exerting pulling forces on the spindle poles. Multiple mitotic kinases contribute to correct spindle orientation by regulating NuMA localization [7-9]. We and others have described a role for the Aurora-A centrosomal kinase in the targeting of NuMA to the cell cortex in metaphase. Aurora-A is frequently overexpressed in cancer, together with its major activator TPX2, raising the question as to whether spindle orientation is among the processes downstream the Aurora-A/TPX2 signaling axis that may be altered under pathological conditions. Here we have investigated the role of TPX2 in the Aurora-A-mediated regulation of NuMA and in spindle orientation. We show that in cultured human cells, the interaction with TPX2 is required for Aurora-A to exert these functions. We also show that Aurora-A, TPX2 and NuMA are part of a macromolecular assembly at spindle MTs, and that the Aurora-A/TPX2 complex favors the interaction between Aurora-A and NuMA, suggesting that TPX2 acts as a platform for Aurora-A regulation of NuMA at spindle MTs. Interestingly, we found that the excess of TPX2 does not influence NuMA localization, but induces a “super-alignment” of the spindle axis to the substrate, both in transformed and non-transformed cells. Conversely, an excess of Aurora-A induces spindle misorientation. We show that these opposite effects are both linked to altered MT dynamics. Overall our results highlight the importance of TPX2 for spindle orientation and suggest that distinct cellular components regulating spindle orientation are differentially sensitive to unbalanced levels of Aurora-A, TPX2 or the Aurora-A/TPX2 complex.

Actomyosin contractility regulates various biological processes, including cell migration and cytokinesis. The cell cortex underlying the membrane of eukaryote cells exhibits dynamic contractile behaviors facilitated by actomyosin contractility. Interestingly, the cell cortex shows reversible aggregation of actin and myosin called “pulsed contraction” in diverse cellular phenomena, such as embryogenesis and tissue morphogenesis. Although contractile behaviors of actomyosin machinery have been studied extensively in several in vitro experiments and computational studies, none of them successfully reproduced the pulsed contraction observed in vivo. Recent experiments have suggested the pulsed contraction is dependent upon the spatiotemporal expression of a small GTPase protein called RhoA. This only indicates the significance of biochemical signaling pathways during the pulsed contraction. In this study, we reproduced the pulsed contraction with only the mechanical and dynamic behaviors of cytoskeletal elements. First, we observed that small pulsed clusters or clusters with fluctuating sizes may appear when there is subtle balance between force generation from motors and force relaxation induced by actin turnover. However, the size and duration of these clusters differ from those of clusters observed during the cellular phenomena. We found that clusters with physiologically relevant size and duration can appear only with both actin turnover and angle-dependent F-actin severing resulting from buckling induced by motor activities. We showed how parameters governing F-actin severing events regulate the size and duration of pulsed clusters. Our study sheds light on the underestimated significance of F-actin severing for the pulsed contraction observed in physiological processes.

The actin cytoskeleton has two faces. One side provides the relatively stable scaffold to maintain the shape of cell cortex fit to the organs. The other side rapidly changes morphology in response to extracellular stimuli including chemical signal and physical strain. Our series of studies employing single-molecule speckle analysis of actin have revealed diverse F-actin lifetimes spanning a range of seconds to minutes in live cells. The dynamic part of the actin turnover is tightly coupled with actin nucleation activities of formin homology proteins (formins), which serve as rapid and efficient F-actin restoration mechanisms in cells under physical stress. More recently, our two studies revealed stabilization of F-actin either by actomyosin contractile force or by helical rotation of processively-actin polymerizing diaphanous-related formin mDia1. These findings quantitatively explain our proposed anti-mechanostress cascade in that G-actin released from F-actin upon loss of tension triggers frequent nucleation and subsequent fast elongation of F-actin by formins. This formin-restored F-actin may become specifically stabilized over long distance by helical polymerization-mediated filament untwisting. In this review, we discuss how and to what extent formins-mediated F-actin restoration might confer mechanostress resistance to the cell. We also give thought to the possible involvement of helical polymerization-mediated filament untwisting in the formation of diverse actin architectures including chirality control.

Protein polarization underlies directional cell growth, cell morphogenesis, cell division, fate specification and differentiation in plant development. Analysis of in vivo protein dynamics reveals differential mobility of polarized proteins in plant cells, which may arise from lateral diffusion, local protein-protein interactions, and is restricted by protein-membrane-cell wall connections. The asymmetric protein dynamics may provide a mechanism for the regulation of asymmetric cell division and cell differentiation. In light of recent evidence for preprophase band (PPB)-independent mechanisms for orienting division planes, polarity proteins and their dynamics might provide regulation on the PPB at the cell cortex to directly influence phragmoplast positioning or alternatively, impinge on cytoplasmic microtubule-organizing centers (MTOCs) for spindle alignment. Differentiation of specialized cell types is often associated with the spatial regulation of cell wall architecture. Here we discuss the mechanisms of polarized signaling underlying regional cell wall biosynthesis, degradation, and modification during the differentiation of root endodermal cells and leaf epidermal guard cells.

Actin bundling protein 34 (ABP34) is the one of 11 actin-crosslinking proteins identified in Dictyostelium discoideum, a novel model organism for the study of actin-associated neurodegenerative disorders such as Alzheimer's disease and Huntington's disease. ABP34 localizes at the leading and trailing edges of locomotory cells, i.e., at the cell cortex, filopodia, and pseudopodia. Functionally, it serves to stabilize membrane-associated actin at sites of cell-cell contact. In addition, this small crosslinking protein is involved in actin bundle formation, and its bundling activity is regulated by the concentration of calcium ion. Several studies have sought to determine the mechanism underlying the calcium-regulated actin bundling activity of ABP34, but it remains unclear. Using several mutational and structural analyses, we revealed that calcium binding to the EF2 motif disrupts the inter-domain interaction between the N- and C-domains, thereby inhibiting the actin bundling activity of ABP34. This finding provides clues about the pathogenesis of neurodegenerative disorders related to actin bundling.

The 14-3-3 family comprises a group of small proteins that are essential, ubiquitous, and highly conserved across eukaryotes. Overexpression of the 14-3-3 proteins σ, ϵ, ζ, and η correlates with high metastatic potential in multiple cancer types. In Dictyostelium, 14-3-3 promotes myosin II turnover in the cell cortex and modulates cortical tension, cell shape, and cytokinesis. In light of the important roles of 14-3-3 proteins across a broad range of eukaryotic species, we sought to determine how 14-3-3 proteins interact with myosin II. Here, conducting in vitro and in vivo studies of both Dictyostelium (one 14-3-3 and one myosin II) and human proteins (seven 14-3-3s and three nonmuscle myosin IIs), we investigated the mechanism by which 14-3-3 proteins regulate myosin II assembly. Using in vitro assembly assays with purified myosin II tail fragments and 14-3-3, we demonstrate that this interaction is direct and phosphorylation-independent. All seven human 14-3-3 proteins also altered assembly of at least one paralog of myosin II. Our findings indicate a mechanism of myosin II assembly regulation that is mechanistically conserved across a billion years of evolution from amebas to humans. We predict that altered 14-3-3 expression in humans inhibits the tumor suppressor myosin II, contributing to the changes in cell mechanics observed in many metastatic cancers.

Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a protein belonging to the formin family, which regulates, together with the small GTPase RhoA, the nucleation and the assembly of actin fibres through Wnt-Dishevelled PCP pathway. Its role has been investigated in essential biological processes, such as cell polarity, movement and adhesion during morphogenesis and organogenesis. In this work, we studied the expression of DAAM1 mRNA and protein by PCR and Western blot analyses and its co-localization with actin in adult mouse seminal vesicles by immunofluorescence. We show that both proteins are cytoplasmic: actin is evident at cell–cell junctions and at cell cortex; DAAM1 had a more diffused localization, but is also prominent at the apical plasmatic membrane of epithelial cells. These findings support our hypothesis of a role of DAAM1 in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular concerning actin filaments. We were also able to detect DAAM1 and actin association in the smooth muscle cells that surround the epithelium too. In this case, we could only speculate the possible involvement of this formin in muscular cells in the maintenance and the regulation of the contractile structures. The present results strongly suggest that DAAM1 could have a pivotal role in vesicle exocytosis and in the physiology of mouse seminal vesicles.

SummaryThe cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic reorganizations during a variety of essential biological processes including cell adhesion, cell migration, and cell division1,2. During cell division and cell locomotion, for example, waves of filamentous-actin (F-actin) assembly and disassembly develop in the cell cortex in a process termed “cortical excitability”3–7. In developing frog and starfish embryos, cortical excitability is generated through coupled positive and negative feedback, with rapid activation of Rho-mediated F-actin assembly followed in space and time by F-actin-dependent inhibition of Rho8,9. These feedback loops are proposed to serve as a mechanism for amplification of active Rho signaling at the cell equator to support furrowing during cytokinesis, while also maintaining flexibility for rapid error correction in response to movement of the mitotic spindle during chromosome segregation10. In this paper, we develop an artificial cortex based onXenopusegg extract and supported lipid bilayers (SLBs), to investigate cortical Rho and F-actin dynamics11. This reconstituted system spontaneously develops two distinct dynamic patterns: singular excitable Rho and F-actin waves and non-traveling oscillatory Rho and F-actin patches. Both types of dynamic patterns have properties and dependencies similar to the cortical excitability previously characterizedin vivo9. These findings directly support the longstanding speculation that the cell cortex is a self-organizing structure and present a novel approach for investigating mechanisms of Rho-GTPase-mediated cortical dynamics.HighlightsAn artificial cell cortex comprisingXenopusegg extract on a supported lipid bilayer self-organizes into complex, dynamic patterns of active Rho and F-actinWe identified two types of reconstituted cortical dynamics – excitable waves and coherent oscillationsReconstituted waves and oscillations require Rho activity and F-actin polymerization

Over the last decades, several techniques have been developed to study cell adhesion, however they present significant shortcomings. Such techniques mostly focus on strong adhesion related to specific protein-protein associations, such as ligand/receptor binding in focal adhesions. Therefore, weak adhesion, related to less specific or non-specific cell/substrate interactions, are rarely addressed. Hence, we propose in this paper a complete investigation of cell adhesion, from highly specific to non-specific adhesiveness, using variable-angle Total Internal Reflection Fluorescence (vaTIRF) nanoscopy. This technique allows us to map in real time, cell topography with a nanometric axial resolution, along with cell cortex refractive index. These two key parameters allow us to distinguish high and low adhesive cell/substrate contacts. Furthermore, vaTIRF provides cell/substrate binding energy; thus, revealing a correlation between cell contractility and cell/substrate binding energy. Here, we highlight the quantitative measurements achieved by vaTIRF on U87MG glioma cells expressing different amounts of α5 integrins, and distinct motility on fibronectin. Regarding integrins expression level, data extracted from vaTIRF measurements, such as the number and size of high adhesive contacts per cell, corroborate the adhesiveness of U87MG cells as intended. Interestingly enough, we found that cells overexpressing α5 integrins, present a higher contractility and lower adhesion energy.

Summary Cytokinesis, the process that partitions the mother cell into two daughter cells, requires the assembly and constriction of an equatorial actomyosin network. Different types of non-motor F-actin crosslinkers localize to the network, but their functional contribution remains poorly understood. Here, we describe a synergy between the small rigid crosslinker plastin and the large flexible crosslinker spectrin in the C. elegans one-cell embryo. In contrast to single inhibitions, co-inhibition of plastin and the βH-spectrin (SMA-1) results in cytokinesis failure due to progressive disorganization and eventual collapse of the equatorial actomyosin network. Cortical localization dynamics of non-muscle myosin II in co-inhibited embryos mimic those observed after drug-induced F-actin depolymerization, suggesting that the combined action of plastin and spectrin stabilizes F-actin in the contractile ring. An in silico model predicts that spectrin is more efficient than plastin at stabilizing the ring and that ring formation is relatively insensitive to βH-spectrin length, which is confirmed in vivo with a sma-1 mutant that lacks 11 of its 29 spectrin repeats. Our findings provide the first evidence that spectrin contributes to cytokinesis and highlight the importance of crosslinker interplay for actomyosin network integrity., Graphical abstract, Highlights • Single inhibitions of F-actin crosslinkers do not lead to cytokinesis failure • Cytokinesis fails upon double inhibition of the crosslinkers plastin and βH-spectrin • Their joint loss collapses the actomyosin network that forms the cytokinetic ring • Ring assembly with these two distinct crosslinker types is modeled in silico, Sobral, Chan et al. address how F-actin crosslinkers contribute to cytokinesis. Cooperation between the small/rigid crosslinker plastin and the large/flexible crosslinker βH-spectrin is shown to be essential for organization and stability of the equatorial actomyosin network that forms the cytokinetic ring.

The plant endoplasmic reticulum (ER) defines the biosynthetic site of lipids and proteins destined for secretion, but also contains important signal transduction and homeostasis components that regulate multiple hormonal and developmental responses. To achieve its various functions, the ER has a unique architecture, both reticulated and highly plastic, that facilitates the spatial-temporal segregation of biochemical reactions and the establishment of inter-organelle communication networks. At the cell cortex, the cortical ER (cER) anchors to and functionally couples with the PM through largely static structures known as ER-PM contact sites (EPCS). These spatially confined microdomains are emerging as critical regulators of the geometry of the cER network, and as highly specialized signalling hubs. In this review, we share recent insights into how EPCS regulate cER remodelling, and discuss the proposed roles for plant EPCS components in the integration of environmental and developmental signals at the cER-PM interface.

The Na/H exchange regulatory factor 1 or Ezrin-radixin-moesin-binding phosphoprotein 50 (NHERF1/EBP50) is an adaptor protein implicated in the stabilization of molecular complexes linking extracellular signals with the cytoskeleton machinery. NHERF1 expression at the cell cortex is associated with the maintenance of adherent junction integrity in polarized epithelia. The role of NHERF1 in cancer depends on its localization within the cell, acting, in most cases, as a tumor suppressor when localized at the cell membrane, and as an oncogene, when expressed in the cytoplasm or the nucleus of cancer cells. The distribution of NHERF1 in renal cell carcinoma (RCC) has not been yet investigated. In this study, NHERF1 expression was examined by immunohistochemistry in papillary and clear cell RCC. We observed membranous staining in papillary RCC, whereas NHERF1 expression was nuclear and membranous in clear cell RCC. In comparison, NHERF1 immunohistochemistry in clear cell carcinomas of the ovary showed mainly nuclear staining. Our finding of the specific NHERF1 nuclear expression in clear cell carcinomas may help to elucidate the molecular changes that regulate its nuclear accumulation and to better understand its role in this cell compartment.

Total internal reflection fluorescence microscopy (TIRFM) provides high optical sectioning capability and superb signal-to-noise ratio for imaging of cell cortex structures. The development of multi-angle (MA)-TIRFM permits high axial resolution imaging and reconstruction of cellular structures near the cell surface. Cytoskeleton is composed of a network of filaments, which are important for maintenance of cell function. The high-resolution imaging and quantitative analysis of filament organization would contribute to our understanding of cytoskeleton regulation in cell. Here, we used a custom-developed MA-TIRFM setup, together with stochastic photobleaching and single molecule localization method, to enhance the lateral resolution of TIRFM imaging to about 100 nm. In addition, we proposed novel methods to perform filament segmentation and 3D reconstruction from MA-TIRFM images. Furthermore, we applied these methods to study the 3D localization of cortical actin and microtubule structures in U373 cancer cells. Our results showed that cortical actins localize ∼ 27 nm closer to the plasma membrane when compared with microtubules. We found that treatment of cells with chemotherapy drugs nocodazole and cytochalasin B disassembles cytoskeletal network and induces the reorganization of filaments towards the cell periphery. In summary, this study provides feasible approaches for 3D imaging and analyzing cell surface distribution of cytoskeletal network. Our established microscopy platform and image analysis toolkits would facilitate the study of cytoskeletal network in cells.

Epithelial morphogenesis and homeostasis are essential for animal development and tissue regeneration, and epithelial disorganization is associated with developmental disorders and tumorigenesis. However, the molecular mechanisms that contribute to the morphogenesis and homeostasis of the epithelium remain elusive. Herein, we report a novel role for the cylindromatosis (CYLD) tumor suppressor in these events. Our results show that CYLD depletion disrupts epithelial organization in both Drosophila egg chambers and mouse skin and intestinal epithelia. Microscopic analysis of proliferating cells in mouse epithelial tissues and cultured organoids reveals that loss of CYLD synergizes with tumor-promoting agents to cause the misorientation of the mitotic spindle. Mechanistic studies show that CYLD accumulates at the cell cortex in epithelial tissues and cultured cells, where it promotes the formation of epithelial adherens junctions through the modulation of microtubule dynamics. These data suggest that CYLD controls epithelial morphogenesis and homeostasis by modulating the assembly of adherens junctions and ensuring proper orientation of the mitotic spindle. Our findings thus provide novel insight into the role of CYLD in development, tissue homeostasis, and tumorigenesis.

The plasma membrane forms the interface between the cell and its environment. It is composed essentially of a phospholipid matrix and many different types of protein molecules which may be embedded within the matrix (integral proteins) or more loosely associated with the cytoplasmic ‘face’ of the membrane (peripheral proteins). The passage of essential ions and molecules across the membrane is controlled by integral proteins acting as channels or transporters. Intercellular communication is mediated by protein receptors, which are activated by signalling molecules such as hormones and neurotransmitters. Physical contact between the cell and its environment (and between cells) is mediated by membrane adhesion proteins. The plasma membrane is a highly dynamic structure in terms of molecular composition and topological configuration. The linkage of the internal cytoskeleton to the plasma membrane (via peripheral and integral proteins) expedites cellular shape changes or amoeboid motion of some types of cell. The process of endocytosis enables the cell to internalize small volumes of extracellular fluid (pinocytosis) by invagination and formation of intracellular vesicles or to engulf entire cells by phagocytosis. Secretion of molecules, such as hormones, is accomplished by fusion of intracellular vesicles with the plasma membrane. This mechanism of exocytosis is mediated by another type of protein: the SNARE proteins.

Mechanical properties of cells are known to be influenced by the actin cytoskeleton. In this article, the action of drugs that interact with the actin cortex is investigated by tether extraction and rheology experiments using optical tweezers. The influences of Blebbistatin, Cytochalasin D and Jasplakinolide on the cell mechanical properties are evaluated. The results, in contradiction to current views for Jasplakinolide, show that all three drugs and treatments destabilize the actin cytoskeleton, decreasing the cell membrane tension. The cell membrane bending modulus increased when the actin cytoskeleton was disorganized by Cytochalasin D. This effect was not observed for Blebbistatin and Jasplakinolide. All drugs decreased by two-fold the cell viscoelastic moduli, but only Cytochalasin D was able to alter the actin network into a more fluid-like structure. The results can be interpreted as the interplay between the actin network and the distribution of myosins as actin cross-linkers in the cytoskeleton. This information may contribute to a better understanding of how the membrane and cytoskeleton are involved in cell mechanical properties, underlining the role that each one plays in these properties.

Classical cadherins are well known for their essential function in mediating cell-cell adhesion via their extra-cellular cadherin domains and intra-cellular connections to the actin cytoskeleton [1-3]. There is evidence, however, of adhesion-independent cadherin clusters existing outside of cell-cell junctions [4-6]. What function, if any, these clusters have is not known. HMR-1, the sole classical cadherin in Caenorhabditis elegans, plays essential roles during gastrulation, blastomere polarity establishment, and epidermal morphogenesis [7-11]. To elucidate the physiological roles of non-junctional cadherin, we analyzed HMR-1 in the C. elegans zygote, which is devoid of neighbors. We show that non-junctional clusters of HMR-1 form during the one-cell polarization stage and associate with F-actin at the cortex during episodes of cortical flow. Non-junctional HMR-1 clusters downregulate RHO-1 activity and inhibit accumulation of non-muscle myosin II (NMY-2) at the anterior cortex. We found that HMR-1 clusters impede cortical flows and play a role in preserving the integrity of the actomyosin cortex, preventing it from splitting in two. Importantly, we uncovered an inverse relationship between the amount of HMR-1 at the cell surface and the rate of cytokinesis. The effect of HMR-1 clusters on cytokinesis is independent of their effect on NMY-2 levels, and is also independent of their extra-cellular domains. Thus, in addition to their canonical role in inter-cellular adhesion, HMR-1 clusters regulate RHO-1 activity and NMY-2 level at the cell surface, reinforce the stability of the actomyosin cortex, and resist its movement to influence cell-shape dynamics.

Many cell-membrane-associated processes require transient spatiotemporal separation of components on scales ranging from a couple of molecules to micrometers in size. Understanding these processes mechanistically involves understanding how lipids and proteins self-organize and interact with the cell cortex. Here, we review recent advances in dissecting the mechanisms of cell membrane compartmentalization. We introduce the challenges in studying cell membrane organization, the current understanding of how complex membranes self-organize to form transient domains, and the role of protein scaffolds in membrane organization. We discuss the formation of signaling domains as an important example of transient membrane compartmentalization. We conclude by pointing to the current limitations of measuring membrane organization in living cells and the steps that are required to advance the field.

The early insect embryo develops as multinucleated cell distributing genomes uniformly to the cell cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing to date. Contemporary hypotheses propose actomyosin driven cytoplasmic movement transporting nuclei, or repulsion of neighbor nuclei driven by microtubule motors. Here, we show that microtubule crosslinking by Feo and Klp3A is essential for nuclear distribution and internuclear distance maintenance in Drosophila. RNAi knockdown in the germline causes irregular, less dense nuclear delivery to the embryo cortex and smaller distribution in ex vivo embryo explants. A minimal internuclear distance is maintained in explants from control embryos but not from Feo depleted embryos, following micromanipulation assisted repositioning. A dominant-negative Feo protein abolishes nuclear separation in embryo explants while the full-length protein rescues the genetic knockdown. We conclude that antiparallel microtubule overlap crosslinking by Feo and Klp3A generates a length-regulated mechanical link between neighboring microtubule asters. Enabled by a novel experimental approach, our study illuminates an essential process of embryonic multicellularity.

The construction of a low affinity Ca2+-probe that locates to the cell cortex and cell surface wrinkles, is described called. EPIC3 ( e zrin- p rotein i ndicator of C a2+). The novel probe is a fusion of CEPIA3 with ezrin, and is used in combination with a Ca2+-insensitive probe, ezrin-mCherry, both of which locate at the cell cortex. EPIC3 was used to monitor the effect of Ca2+ influx on intra-wrinkle Ca2+ in the macrophage cell line, RAW 264.7. During experimentally–induced Ca2+influx, EPIC3 reported Ca2+ concentrations at the cell cortex in the region of 30−50 μM, with peak locations towards the tips of wrinkles reaching 80 μM. These concentrations were associated with cleavage of ezrin (a substrate for the Ca2+ activated protease calpain-1) and released the C-terminal fluors. The cortical Ca2+ levels, restricted to near the site of phagocytic cup formation and pseudopodia extension during phagocytosis also reached high levels (50−80 μM) during phagocytosis. As phagocytosis was completed, hotspots of Ca2+ near the phagosome were also observed.

EpCAM has long been known as a cell surface protein highly expressed in carcinomas. It has since become one of the key cancer biomarkers. Despite its high fame, its actual role in cancer development is still controversial. Beyond a flurry of correlative studies, which point either to a positive or a negative link with tumour progression, there has been surprisingly few studies on the actual cellular mechanisms of EpCAM and on their functional consequences. Clearly, EpCAM plays multiple important roles, in cell proliferation as well as in cell adhesion and migration. The two latter functions, directly relevant for metastasis, are the focus of this review. We attempt here to bring together the available experimental data to build a global coherent view of EpCAM functions. We also include in this overview EpCAM2/Trop2, the close relative of EpCAM. At the core of EpCAM (and EpCAM2/Trop2) function stands the ability to repress contractility of the actomyosin cell cortex. This activity appears to involve direct inhibition by EpCAM of members of the novel PKC family and of a specific downstream PKD-Erk cascade. We will discuss how this activity can result in a variety of adhesive and migratory phenotypes, thus potentially explaining at least part of the apparent inconsistencies between different studies. The picture remains fragmented, and we will highlight some of the conflicting evidence and the many unsolved issues, starting with the controversy around its original description as a cell-cell adhesion molecule.

Actomyosin networks generate contractile force that changes cell and tissue shape. In muscle cells, actin filaments and myosin II appear in a polarized structure called a sarcomere, in which myosin II is localized in the center. Nonmuscle cortical actomyosin networks are thought to contract when nonmuscle myosin II (myosin) is activated throughout a mixed-polarity actin network. Here, we identified a mutant version of the myosin-activating kinase, ROCK, that localizes diffusely, rather than centrally, in epithelial cell apices. Surprisingly, this mutant inhibits constriction, suggesting that centrally localized apical ROCK/myosin activity promotes contraction. We determined actin cytoskeletal polarity by developing a barbed end incorporation assay for Drosophila embryos, which revealed barbed end enrichment at junctions. Our results demonstrate that epithelial cells contract with a spatially organized apical actomyosin cortex, involving a polarized actin cytoskeleton and centrally positioned myosin, with cell-scale order that resembles a muscle sarcomere., National Institutes of Health (U.S.) (GM06806), American Heart Association (Grant-in-aid 14GRNT1888005), American Cancer Society (grant 125792-RSG-14-039-01-CS), National Institutes of Health (U.S.) (Pre-Doctoral Training Grant T32GM00728)

The organization of filamentous actin and myosin II molecular motor contractility is known to modify the mechanical properties of the cell cortical actomyosin cytoskeleton. Here we describe a novel method, to our knowledge, for using force spectroscopy approach curves with tipless cantilevers to determine the actomyosin cortical tension, elastic modulus, and intracellular pressure of nonadherent cells. We validated the method by measuring the surface tension of water in oil microdrops deposited on a glass surface. We extracted an average tension of T ∼ 20.25 nN/μm, which agrees with macroscopic experimental methods. We then measured cortical mechanical properties in nonadherent human foreskin fibroblasts and THP-1 human monocytes before and after pharmacological perturbations of actomyosin activity. Our results show that myosin II activity and actin polymerization increase cortex tension and intracellular pressure, whereas branched actin networks decreased them. Interestingly, myosin II activity stiffens the cortex and branched actin networks soften it, but actin polymerization has no effect on cortex stiffness. Our method is capable of detecting changes in cell mechanical properties in response to perturbations of the cytoskeleton, allowing characterization with physically relevant parameters. Altogether, this simple method should be of broad application for deciphering the molecular regulation of cell cortical mechanical properties.

All 302 neurons in the C. elegans hermaphrodite arise through asymmetric division of neuroblasts. During embryogenesis, the C. elegans ham-1 gene is required for several asymmetric neuroblast divisions in lineages that generate both neural and apoptotic cells. By antibody staining, endogenous HAM-1 is found exclusively at the cell cortex in many cells during embryogenesis and is asymmetrically localized in dividing cells. Here we show that in transgenic embryos expressing a functional GFP::HAM-1 fusion protein, GFP expression is also detected in the nucleus, in addition to the cell cortex. Consistent with the nuclear localization is the presence of a putative DNA binding winged-helix domain within the N-terminus of HAM-1. Through a deletion analysis we determined that the C-terminus of the protein is required for nuclear localization and we identified two nuclear localization sequences (NLSs). A subcellular fractionation experiment from wild type embryos, followed by Western blotting, revealed that endogenous HAM-1 is primarily found in the nucleus. Our analysis also showed that the N-terminus is necessary for cortical localization. While ham-1 function is essential for asymmetric division in the lineage that generates the PLM mechanosensory neuron, we showed that cortical localization may not required. Thus, our results suggest that there is a nuclear function for HAM-1 in regulating asymmetric neuroblast division and that the requirement for cortical localization may be lineage dependent.

The roles of calmodulin (CaM), a multifunctional intracellular calcium receptor protein, as concerns selected morphological and functional characteristics of pure microglial cells derived from mixed primary cultures from embryonal forebrains of rats, were investigated through use of the CaM antagonists calmidazolium (CALMID) and trifluoperazine (TFP). The intracellular localization of the CaM protein relative to phalloidin, a bicyclic heptapeptide that binds only to filamentous actin, and the ionized calcium-binding adaptor molecule 1 (Iba1), a microglia-specific actin-binding protein, was determined by immunocytochemistry, with quantitative analysis by immunoblotting. In unchallenged and untreated (control) microglia, high concentrations of CaM protein were found mainly perinuclearly in ameboid microglia, while the cell cortex had a smaller CaM content that diminished progressively deeper into the branches in the ramified microglia. The amounts and intracellular distributions of both Iba1 and CaM proteins were altered after lipopolysaccharide (LPS) challenge in activated microglia. CALMID and TFP exerted different, sometimes opposing, effects on many morphological, cytoskeletal and functional characteristics of the microglial cells. They affected the CaM and Iba1 protein expressions and their intracellular localizations differently, inhibited cell proliferation, viability and fluid-phase phagocytosis to different degrees both in unchallenged and in LPS-treated (immunologically challenged) cells, and differentially affected the reorganization of the actin cytoskeleton in the microglial cell cortex, influencing lamellipodia, filopodia and podosome formation. In summary, these CaM antagonists altered different aspects of filamentous actin-based cell morphology and related functions with variable efficacy, which could be important in deciphering the roles of CaM in regulating microglial functions in health and disease.

Summary Symmetry breaking is an essential step in cell differentiation and early embryonic development. However, the molecular cues that trigger symmetry breaking remain largely unknown. Here, we show that mitochondrial H2O2 acts as a symmetry-breaking cue in the C. elegans zygote. We find that symmetry breaking is marked by a local H2O2 increase and coincides with a relocation of mitochondria to the cell cortex. Lowering endogenous H2O2 levels delays the onset of symmetry breaking, while artificially targeting mitochondria to the cellular cortex using a light-induced heterodimerization technique is sufficient to initiate symmetry breaking in a H2O2-dependent manner. In wild-type development, both sperm and maternal mitochondria contribute to symmetry breaking. Our findings reveal that mitochondrial H2O2-signaling promotes the onset of polarization, a fundamental process in development and cell differentiation, and this is achieved by both mitochondrial redistribution and differential H2O2-production.

Cell migration is orchestrated by dynamic interaction of microtubules with the plasma membrane cortex. However, the regulatory mechanisms underlying the cortical actin cytoskeleton and microtubule dynamics are less characterized. Our earlier study showed that small GTPase-activating proteins, IQGAPs, regulate polarized secretion in epithelial cells (1). Here, we show that IQGAP1 links dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein, SKAP. Biochemical characterizations revealed that IQGAP1 and SKAP form a cognate complex and that their binding interfaces map to the WWIQ motif and the C-terminal of SKAP, respectively. The WWIQ peptide disrupts the biochemical interaction between IQGAP1 and SKAP in vitro, and perturbation of the IQGAP1-SKAP interaction in vivo using a membrane-permeable TAT-WWIQ peptide results in inhibition of directional cell migration elicited by EGF. Mechanistically, the N-terminal of SKAP binds to EB1, and its C terminus binds to IQGAP1 in migrating cells. Thus, we reason that a novel IQGAP1 complex orchestrates directional cell migration via coupling dynamic microtubule plus-ends to the cell cortex.

Mitochondria are dynamic organelles that are highly motile and frequently fuse and divide. It has recently become clear that their complex behavior is governed to a large extent by interactions with other cellular structures. This review will focus on a mitochondria-plasma membrane tethering complex that was recently discovered and molecularly analyzed in budding yeast, the Num1/Mdm36 complex. This complex attaches mitochondria to the cell cortex and ensures that a portion of the organelles is retained in mother cells during cell division. At the same time, it supports mitochondrial division and integrates mitochondrial dynamics into cellular architecture. Recent evidence suggests that similar mechanisms might exist also in mammalian cells.

The zygote is the essential intermediate that allows interchange of nuclear, mitochondrial and cytosolic determinants between cells. Zygote formation in Saccharomyces cerevisiae is accomplished by mechanisms that are not characteristic of mitotic cells. These include shifting the axis of growth away from classical cortical landmarks, dramatically reorganizing the cell cortex, remodeling the cell wall in preparation for cell fusion, fusing with an adjacent partner, accomplishing nuclear fusion, orchestrating two steps of septin morphogenesis that account for a delay in fusion of mitochondria, and implementing new norms for bud site selection. This essay emphasizes the sequence of dependent relationships that account for this progression from cell encounters through zygote budding. It briefly summarizes classical studies of signal transduction and polarity specification and then focuses on downstream events.

The existence of membrane-rafts helps to conceptually understand the spatiotemporal organization of membrane-associated events (signaling, fusion, fission, etc.). However, as rafts themselves are nanoscopic, dynamic, and transient assemblies, they cannot be directly observed in a metabolizing cell by traditional microscopy. The observation of phase separation in giant plasma membrane-derived vesicles from live cells is a powerful tool for studying lateral heterogeneity in eukaryotic cell membranes, specifically in the context of membrane rafts. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. It remains unclear, however, if this lipid-driven process is tuneable in any way by interactions with proteins. Here, we demonstrate that MPP1, a member of the MAGUK family, can modulate membrane properties such as the fluidity and phase separation capability of giant plasma membrane-derived vesicles. Our data suggest that physicochemical domain properties of the membrane can be modulated, without major changes in lipid composition, through proteins such as MPP1.

In eukaryotic cells, an actin-based cortex lines the inner leaflet of the plasma membrane, endowing the cells with crucial mechanical and functional properties. Unfortunately, it has not been possible to study the structural dynamics of the actin cortex at high lateral resolution in living cells. Here, we performed atomic force microscopy time-lapse imaging and mechanical mapping of actin in the cortex of living cells at high lateral and temporal resolution. Cortical actin filaments adopted discernible arrangements, ranging from large parallel bundles with low connectivity to a tight meshwork of short filaments. Mixing of these architectures resulted in attuned cortex networks with specific connectivity, mechanical responses, and marked differences in their dynamic behavior.

SummaryThe mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt their migration mode to their environment. While the molecular pathways involved in this transition are well documented, the role of the microenvironment in the MAT is still poorly understood. Here, we investigated how confinement and adhesion affect this transition. We report that, in the absence of focal adhesions and under conditions of confinement, mesenchymal cells can spontaneously switch to a fast amoeboid migration phenotype. We identified two main types of fast migration—one involving a local protrusion and a second involving a myosin-II-dependent mechanical instability of the cell cortex that leads to a global cortical flow. Interestingly, transformed cells are more prone to adopt this fast migration mode. Finally, we propose a generic model that explains migration transitions and predicts a phase diagram of migration phenotypes based on three main control parameters: confinement, adhesion, and contractility.

Summary Many differentiated animal cells, and all higher plant cells, build interphase microtubule arrays of specific architectures without benefit of a central organizer, such as a centrosome, to control the location and geometry of microtubule nucleation. These acentrosomal arrays support essential cell functions such as morphogenesis [1, 2], but the mechanisms by which the new microtubules are positioned and oriented are poorly understood. In higher plants, nucleation of microtubules arises from distributed γ-tubulin ring complexes (γ-TuRCs) at the cell cortex that are associated primarily with existing microtubules [3–5] and from which new microtubules are nucleated in a geometrically bimodal fashion, either in parallel to the mother microtubule or as a branching event at a mean angle of approximately 40° to the mother microtubule. By imaging the dynamics of individual nucleation events in Arabidopsis , we found that a conserved peripheral protein of the γ-TuRC, GCP-WD/NEDD1 [6–8], associated with motile γ-TuRCs and localized to nucleation events. Knockdown of this essential protein resulted in reduction of γ-TuRC recruitment to cortical microtubules and total nucleation frequency, showing that GCP-WD controls γ-TuRC positioning and function in these interphase arrays. Further, we discovered an unexpected role for GCP-WD in determining the geometry of microtubule-dependent microtubule nucleation, where it acts to increase the likelihood of branching over parallel nucleation. Cells with normally complex patterns of cortical array organization constructed simpler arrays with cell-wide ordering, suggesting that control of nucleation frequency, positioning, and geometry by GCP-WD allows plant cells to build alternative cortical array architectures.

The physical properties of cells reflect how the structure and dynamics of the actomyosin cortex are coupled to the plasma membrane. In epithelia, adhesive E-cadherin clusters associate with the cell cortex to assemble the junctional actomyosin that participates in epithelial morphogenesis. E-cadherin is present not only at the apical zonula adherens (ZA), but also distributed throughout the lateral adherens junction (LAJ) below the ZA. However, the organizational dynamics of the actomyosin network at the LAJs remains elusive. To address this, we used quantitative real-time imaging to characterize the dynamics of actomyosin contractility at lateral cadherin contacts. Here, we report that contractility is coordinated into smaller actomyosin rings that link cadherin clusters together within the larger cortical network at the lateral junctions. We conclude that Myosin II activity determines the contractility of actomyosin cables between cadherin clusters to propagate pulsatility across lateral cell-cell contacts. Refereed/Peer-reviewed

Mechanics is at the heart of many cellular processes and its importance has received considerable attention during the last two decades. In particular, the tension of cell membranes, and more specifically of the cell cortex, is a key parameter that determines the mechanical behavior of the cell periphery. However, the measurement of tension remains challenging due to its dynamic nature. Here we show that a noninvasive interferometric technique can reveal time-resolved effective tension measurements by a high-accuracy determination of edge fluctuations in expanding cell blebs of filamin-deficient melanoma cells. The introduced technique shows that the bleb tension is ∼10–100 pN/μm and increases during bleb growth. Our results directly confirm that the subsequent stop of bleb growth is induced by an increase of measured tension, possibly mediated by the repolymerized actin cytoskeleton.

T cells are charged with surveying tissues for evidence of their cognate foreign antigens. Subsequently, they must navigate to effector sites, which they enter through the process of trans-endothelial migration (TEM). During interstitial migration, T cells migrate according to one of two modes that are distinguished by the strength and sequence of adhesions and the requirement for Myosin-IIA. In contrast during TEM, T cells require Myosin-IIA for the final process of pushing their nucleus through the endothelium. A generalized model emerges with dual roles for Myosin-IIA: This motor protein acts like a tensioning or expansion spring, transmitting force across the cell cortex to sites of surface contact and also optimizing the frictional coupling with substrata by modulating the surface area of the contact. The phosphorylation and deactivation of this motor following TCR engagement can allow T cells to rapidly alter the degree to which they adhere to surfaces and to switch to a mode of interaction with surfaces that is more conducive to forming a synapse with an antigen-presenting cell.

The endoplasmic reticulum (ER) in live cells is a highly mobile network whose structure dynamically changes on a number of timescales. The role of such drastic changes in any system is unclear, although there are correlations with ER function. A better understanding of the fundamental biophysical constraints on the system will allow biologists to determine the effects of molecular factors on ER dynamics. Previous studies have identified potential static elements that the ER may remodel around. Here, we use these structural elements to assess biophysical principles behind the network dynamics. By analyzing imaging data of tobacco leaf epidermal cells under two different conditions, i.e., native state (control) and latrunculin B (treated), we show that the geometric structure and dynamics of ER networks can be understood in terms of minimal networks. Our results show that the ER network is well modeled as a locally minimal-length network between the static elements that potentially anchor the ER to the cell cortex over longer timescales; this network is perturbed by a mixture of random and deterministic forces. The network need not have globally minimum length; we observe cases where the local topology may change dynamically between different Euclidean Steiner network topologies. The networks in the treated cells are easier to quantify, because they are less dynamic (the treatment suppresses actin dynamics), but the same general features are found in control cells. Using a Langevin approach, we model the dynamics of the nonpersistent nodes and use this to show that the images can be used to estimate both local viscoelastic behavior of the cytoplasm and filament tension in the ER network. This means we can explain several aspects of the ER geometry in terms of biophysical principles.

Vimentin intermediate filament expression is a hallmark of epithelial-to-mesenchymal transitions, and vimentin is involved in the maintenance of cell mechanical properties, cell motility, adhesion, and other signaling pathways. A common feature of vimentin-expressing cells is their routine exposure to mechanical stress. Intermediate filaments are unique among cytoskeletal polymers in resisting large deformations in vitro, yet vimentin's mechanical role in the cell is not clearly understood. We use atomic force microscopy to compare the viscoelastic properties of normal and vimentin-null (vim(-/-)) mouse embryo fibroblasts (mEFs) on substrates of different stiffnesses, spread to different areas, and subjected to different compression patterns. In minimally perturbed mEF, vimentin contributes little to the elastic modulus at any indentation depth in cells spread to average areas. On a hard substrate however, the elastic moduli of maximally spread mEFs are greater than those of vim(-/-)mEF. Comparison of the plastic deformation resulting from controlled compression of the cell cortex shows that vimentin's enhancement of elastic behavior increases with substrate stiffness. The elastic moduli of normal mEFs are more stable over time than those of vim(-/-)mEFs when cells are subject to ongoing oscillatory compression, particularly on a soft substrate. In contrast, increasing compressive strain over time shows a greater role for vimentin on a hard substrate. Under both conditions, vim(-/-)mEFs exhibit more variable responses, indicating a loss of regulation. Finally, normal mEFs are more contractile in three-dimensional collagen gels when seeded at low density, when cell-matrix contacts dominate, whereas contractility of vim(-/-)mEF is greater at higher densities when cell-cell contacts are abundant. Addition of fibronectin to gel constructs equalizes the contractility of the two cell types. These results show that the Young's moduli of normal and vim(-/-)mEFs are substrate stiffness dependent even when the spread area is similar, and that vimentin protects against compressive stress and preserves mechanical integrity by enhancing cell elastic behavior.