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Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

The actin cytoskeleton is the primary force-generating machinery in the cell, which can produce pushing (protrusive) forces using energy of actin polymerization and pulling (contractile) forces via sliding of bipolar filaments of myosin II along actin filaments, as well as perform other key functions. These functions are essential for whole cell migration, cell interaction with the environment, mechanical properties of the cell surface and other key aspects of cell physiology. The actin cytoskeleton is a highly complex and dynamic system of actin filaments organized into various superstructures by multiple accessory proteins. High resolution architecture of functionally distinct actin arrays provides key clues for understanding actin cytoskeleton functions. This review summarizes recent advance in our understanding of the actin cytoskeleton ultrastructure.

Tumor metastasis is responsible for ~90% of all cancer deaths. One of the key steps of tumor metastasis is tumor cell migration and invasion. Filopodia are cell surface extensions that are critical for tumor cell migration. Fascin protein is the main actin-bundling protein in filopodia. Small-molecule fascin inhibitors block tumor cell migration, invasion, and metastasis. Here we present the structural basis for the mechanism of action of these small-molecule fascin inhibitors. X-ray crystal structural analysis of a complex of fascin and a fascin inhibitor shows that binding of the fascin inhibitor to the hydrophobic cleft between the domains 1 and 2 of fascin induces a ~35o rotation of domain 1, leading to the distortion of both the actin-binding sites 1 and 2 on fascin. Furthermore, the crystal structures of an inhibitor alone indicate that the conformations of the small-molecule inhibitors are dynamic. Mutations of the inhibitor-interacting residues decrease the sensitivity of fascin to the inhibitors. Our studies provide structural insights into the molecular mechanism of fascin protein function as well as the action of small-molecule fascin inhibitors.

Prokaryotic microbes possess a variety of appendages on their cell surfaces. The most commonly known surface appendages of bacteria include flagella, pili, curli, and spinae. Although archaea have archaella (archaeal flagella) and various types of pili that resemble those in bacteria, cannulae, and hami are unique to archaea. Typically involved in cell motility, flagella, the thickest appendages, are 20-26 nm and 10-14 nm wide in bacteria and archaea, respectively. Bacterial and archaeal pili are distinguished by their thin, short, hair-like structures. Curli appear as coiled and aggregative thin fibers, whereas spinae are tubular structures 50-70 nm in diameter in bacteria. Cannulae are characterized by ∼25 nm-wide tubules that enter periplasmic spaces and connect neighboring archaeal cells. Hami are 1-3 μm in length and similar to barbed grappling hooks for attachment to bacteria. Recent advances in specimen preparation methods and image processing techniques have made cryo-transmission electron microscopy an essential tool for in situ structural analysis of microbes and their extracellular structures.

Macropinocytosis is a mechanism for the nonspecific bulk uptake and internalisation of extracellular fluid. This plays specific and distinct roles in diverse cell types such as macrophages, dendritic cells and neurons, by allowing cells to sample their environment, extract extracellular nutrients and regulate plasma membrane turnover. Macropinocytosis has recently been implicated in several diseases including cancer, neurodegenerative diseases and atherosclerosis. Uptake by macropinocytosis is also exploited by several intracellular pathogens to gain entry into host cells. Both capturing and subsequently processing large volumes of extracellular fluid poses a number of unique challenges for the cell. Macropinosome formation requires coordinated three-dimensional manipulation of the cytoskeleton to form shaped protrusions able to entrap extracellular fluid. The following maturation of these large vesicles then involves a complex series of membrane rearrangements to shrink and concentrate their contents, while delivering components required for digestion and recycling. Recognition of the diverse importance of macropinocytosis in physiology and disease has prompted a number of recent studies. In this article, we summarise advances in our understanding of both macropinosome formation and maturation, and seek to highlight the important unanswered questions.

Actin cortex controls cell migration Cell migration is mainly controlled by local actin polymerization–driven membrane protrusion. However, a second structural mechanism might also regulate membrane protrusions and directed migration: changes in the density of the attachment between the plasma membrane and the underlying F-actin cortex, a parameter related to membrane tension. Many types of attachment and signaling mechanisms are known to alter the density of membrane-proximal cortical actin. Bisaria et al. designed a membrane-proximal F-actin (MPA) reporter that could directly measure local changes in the density of MPA in living cells. Levels of MPA were surprisingly low toward the front of migrating cells despite an opposing high overall concentration of F-actin in the same front region. The researchers propose that MPA density can integrate different signaling processes to direct local membrane protrusions and stabilize cell polarity during cell migration. Science , this issue p. 1205

The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. With most studies failing to establish their structural identity and examine whether they are truly open-ended organelles, there is a need to study the anatomy of TNTs at the nanometer resolution. Here, we use correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structural organization of neuronal TNTs. Our data indicate that they are composed of a bundle of open-ended individual tunneling nanotubes (iTNTs) that are held together by threads labeled with anti-N-Cadherin antibodies. iTNTs are filled with parallel actin bundles on which different membrane-bound compartments and mitochondria appear to transfer. These results provide evidence that neuronal TNTs have distinct structural features compared to other cell protrusions., The architecture of functional TNTs is still under debate. Here, the authors combine correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structure of TNTs in neuronal cells, showing that they form structures that are distinct form other membrane protrusions.

Cellular motility is a fundamental process essential for embryonic development, wound healing, immune responses, and tissues development. Cells are mostly moving by crawling on external, or inside, substrates which can differ in their surface composition, geometry, and dimensionality. Cells can adopt different migration phenotypes, e.g., bleb-based and protrusion-based, depending on myosin contractility, surface adhesion, and cell confinement. In the few past decades, research on cell motility has focused on uncovering the major molecular players and their order of events. Despite major progresses, our ability to infer on the collective behavior from the molecular properties remains a major challenge, especially because cell migration integrates numerous chemical and mechanical processes that are coupled via feedbacks that span over large range of time and length scales. For this reason, reconstituted model systems were developed. These systems allow for full control of the molecular constituents and various system parameters, thereby providing insight into their individual roles and functions. In this review we describe the various reconstituted model systems that were developed in the past decades. Because of the multiple steps involved in cell motility and the complexity of the overall process, most of the model systems focus on very specific aspects of the individual steps of cell motility. Here we describe the main advancement in cell motility reconstitution and discuss the main challenges toward the realization of a synthetic motile cell.

Blebs are pressure-driven cell protrusions implicated in cellular functions such as cell division, apoptosis, and cell motility, including motility of protease-inhibited cancer cells. Because of their mechanical nature, blebs inform us about general cell-surface mechanics, including membrane dynamics, pressure propagation throughout the cytoplasm, and the architecture and dynamics of the actin cortex. Mathematical models including detailed fluid dynamics have previously been used to understand bleb expansion. Here, we develop mathematical models in two and three dimensions on longer timescales that recapitulate the full bleb life cycle, including both expansion and healing by cortex reformation, in terms of experimentally accessible biophysical parameters such as myosin contractility, osmotic pressure, and turnover of actin and ezrin. The model provides conditions under which blebbing occurs, and naturally gives rise to traveling blebs. The model predicts conditions under which blebs travel or remain stationary, as well as the bleb traveling velocity, a quantity that has remained elusive in previous models. As previous studies have used blebs as reporters of membrane tension and pressure dynamics within the cell, we have used our system to investigate various pressure equilibration models and dynamic, nonuniform membrane tension to account for the shape of a traveling bleb. We also find that traveling blebs tend to expand in all directions unless otherwise constrained. One possible constraint could be provided by spatial heterogeneity in, for example, adhesion density.

Blebs are pressure-driven protrusions that play an important role in cell migration, particularly in three-dimensional environments. A bleb is initiated when the cytoskeleton detaches from the cell membrane, resulting in the pressure-driven flow of cytosol toward the area of detachment and local expansion of the cell membrane. Recent experiments involving blebbing cells have led to conflicting hypotheses regarding the timescale of intracellular pressure propagation. The interpretation of one set of experiments supports a poroelastic model of the cytoplasm that leads to slow pressure equilibration when compared to the timescale of bleb expansion. A different study concludes that pressure equilibrates faster than the timescale of bleb expansion. To address this discrepancy, a dynamic computational model of the cell was developed that includes mechanics of and the interactions among the cytoplasm, the actin cortex, the cell membrane, and the cytoskeleton. The model results quantify the relationship among cytoplasmic rheology, pressure, and bleb expansion dynamics, and provide a more detailed picture of intracellular pressure dynamics. This study shows the elastic response of the cytoplasm relieves pressure and limits bleb size, and that both permeability and elasticity of the cytoplasm determine bleb expansion time. Our model with a poroelastic cytoplasm shows that pressure disturbances from bleb initiation propagate faster than the timescale of bleb expansion and that pressure equilibrates slower than the timescale of bleb expansion. The multiple timescales in intracellular pressure dynamics explain the apparent discrepancy in the interpretation of experimental results.

Although it is widely appreciated that cells migrate in a variety of diverse environments in vivo, we are only now beginning to use experimental workflows that yield images with sufficient spatiotemporal resolution to study the molecular processes governing cell migration in 3D environments. Since cell migration is a dynamic process, it is usually studied via microscopy, but 3D movies of 3D processes are difficult to interpret by visual inspection. In this review, we discuss the technologies required to study the diversity of 3D cell migration modes with a focus on the visualization and computational analysis tools needed to study cell migration quantitatively at a level comparable to the analyses performed today on cells crawling on flat substrates.

Female meiotic divisions are extremely asymmetric, producing large oocytes and small polar bodies (PBs). In mouse oocytes, the spindle relocates to the cortex before anaphase of meiosis I (MI). It is presumed that by displacing the future midzone, pre-anaphase spindle repositioning alone ensures asymmetry. But how subsequent anaphase events might contribute to asymmetric PB extrusion (PBE) is unknown. Here, we find that inactivation of cyclin-dependent kinase 1 (Cdk1) induces anaphase and simultaneously triggers cytoplasmic formin-mediated F-actin polymerisation that propels the spindle into the cortex causing it to protrude while anaphase progresses. Significantly, if post-anaphase-onset spindle migration fails, protrusion and asymmetry are severely threatened even with intact pre-anaphase migration. Conversely, post-anaphase migration can completely compensate for failed pre-anaphase migration. These data identify a cell-cycle-triggered phase of spindle displacement occurring after anaphase-onset, which, by inducing protrusion, is necessary for extreme asymmetry in mouse oocytes and uncover a pathway for maximising unequal division., Female meiotic divisions are asymmetric with the formation of large oocytes and small polar bodies, thought to result from cortical spindle placement before anaphase. The authors find that Cdk1 inactivation triggers F-actin dependent post-anaphase spindle migration, resulting in cortical protrusion.

Cell locomotion is essential for multicellular organism embryo development, organ homeostasis, tissue regeneration, immune responses, and tumor metastasis. Here we report that single mammalian cells can generate two extracellular membranous compartments: cytocapsulae and cytocapsular tubes. Cells migrate in cytocapsulae and engender cytocapsular tubes, which exhibit pleiotropic biological functions and provide tubular routes for directed cell transportation. Ultrastructural analysis by electron microscope revealed that nanoprotrusions surround and anchor cytocapsular tubes in place. Multiple cytocapsular tubes interconnect and form networks supporting directed cell transportation in diverse directions. Enhanced translation initiation factor eIF4E up-regulates translation of transcripts encoding proteins important for organelle development. Thus, this study proposes a mechanism of directed cell translocation in cytocapsular tubes, which may facilitate the management of diseases, including tumor metastasis.

Migrating cells sense variations of stiffness in connective tissue matrices but how cells detect and respond to stiffness orientation is not defined. We examined cell extension formation on collagen with underlying support (vertical stiffness gradient) or on collagen laterally supported by nylon (lateral stiffness gradient). At 6h after plating, cells plated on laterally-supported collagen exhibited >2-fold more abundant and ~2-fold longer cell extensions than cells plated on collagen with underlying support. We examined whether p21-activated kinase 1 (PAK1) influences extension formation that is dependent on the orientation of support. At 6h after plating on collagen with underlying support, wild-type cell extensions were 40% shorter than PAK1 knockdown cells. In contrast, on laterally-supported collagen, wild-type cell extensions were 2-fold longer than PAK1 knockdown cells. In cells plated on laterally-supported collagen, there were ~2-fold reductions of collagen fiber alignment and compaction in PAK1 knockdown cells compared with wild-type cells. PAK1 knockdown did not affect collagen fiber alignment or compaction by cells plated on collagen with underlying support. Wild-type cells with lateral support of collagen exhibited 3-fold increases of phospho-myosin staining at 6h, which was 2-fold lower in PAK1 knockdown cells. In contrast, cells on collagen with underlying support showed no increase of phospho-myosin staining at any times. PAK1 knockdown did not affect α2 or β1 integrin expression or function. We conclude that PAK1 is involved in the ability of cells to sense the orientation of stiffness in collagen substrates and generate contractile forces that affect cell extension formation.

The role of the actin-capping protein flightless I in collagen remodeling by mouse fibroblasts is examined. Flightless and nonmuscle myosin IIA cooperate to enable collagen phagocytosis., We examined the role of the actin-capping protein flightless I (FliI) in collagen remodeling by mouse fibroblasts. FliI-overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions that stained for myosin10 and fascin and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI-knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI-overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with nonmuscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1–6 capped actin filaments efficiently, whereas FliI GLD 2–6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1–6) with the rod domain of NMMIIA (kD = 0.146 μM), whereas FliI GLD 2–6 showed lower binding affinity (kD = 0.8584 μM). Cells expressing FliI GLD 2–6 exhibited fewer cell extensions, did not colocalize with NMMIIA, and showed reduced collagen uptake compared with cells expressing FliI GLD 1–6. We conclude that FliI interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling in fibroblasts.

High-resolution, real-time, three-dimensional fluorescence microscopy imaging shows the absence of clathrin-coated pits and vesicles at the ventral surfaces of lamellipodia and lamellae at the front of migrating cells. In addition, the data support the model invoking net membrane deposition at the cell front of migrating cells due to an imbalance between endocytic and exocytic membrane flow., Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In the experiments reported here, we used spinning-disk confocal and lattice light-sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2 tagged with enhanced green fluorescence (AP2-EGFP) and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene-edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium and at the approximate boundary between the lamellipodium and lamella and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane. We observed a similar dorsal/ventral asymmetry on membrane protrusions from SUM159 cells. Stationary coated pits formed and budded on the remainder of the dorsal and ventral surfaces of both types of cells. These observations support a previously proposed model that invokes net membrane deposition at the leading edge due to an imbalance between the endocytic and exocytic membrane flow at the front of a migrating cell.

Summary Polarization of cells into a protrusive front and a retracting cell body is the hallmark of mesenchymal-like cell migration. Many mRNAs are localized to protrusions, but it is unclear to what degree mRNA localization contributes toward protrusion formation. We performed global quantitative analysis of the distributions of mRNAs, proteins, and translation rates between protrusions and the cell body by RNA sequencing (RNA-seq) and quantitative proteomics. Our results reveal local translation as a key determinant of protein localization to protrusions. Accordingly, inhibition of local translation destabilizes protrusions and inhibits mesenchymal-like morphology. Interestingly, many mRNAs localized to protrusions are translationally repressed. Specific cis-regulatory elements within mRNA UTRs define whether mRNAs are locally translated or repressed. Finally, RNAi screening of RNA-binding proteins (RBPs) enriched in protrusions revealed trans-regulators of localized translation that are functionally important for protrusions. We propose that by deciphering the localized mRNA UTR code, these proteins regulate protrusion stability and mesenchymal-like morphology., Highlights • Local translation is a key regulator of protein localization to cell protrusions • Localization of mRNAs alone, however, does not determine local protein expression • Specific UTR elements and RBPs are associated with local translation in protrusions • Inhibition of local translation destabilizes protrusions, Establishment of front-back cell polarity is the first step in any form of active cell migration. Mardakheh et al. show that in mesenchymal-like cells, front-back asymmetry at the proteome level is maintained by differential local synthesis of cellular proteins.

Adaptation of cell shape and polarization through the formation and retraction of cellular protrusions requires balancing of endocytosis and exocytosis combined with fine-tuning of the local activity of small GTPases like Rab8. Here, we show that endocytic turnover of the plasma membrane at protrusions is directly coupled to surface removal and inactivation of Rab8. Removal is induced by reduced membrane tension and mediated by the GTPase regulator associated with focal adhesion kinase-1 (GRAF1, also known as ARHGAP26), a regulator of clathrin-independent endocytosis. GRAF1-depleted cells were deficient in multi-directional spreading and displayed elevated levels of GTP-loaded Rab8, which was accumulated at the tips of static protrusions. Furthermore, GRAF1 depletion impaired lumen formation and spindle orientation in a 3D cell culture system, indicating that GRAF1 activity regulates polarity establishment. Our data suggest that GRAF1-mediated removal of Rab8 from the cell surface restricts its activity during protrusion formation, thereby facilitating dynamic adjustment of the polarity axis., Vetenskapsrådet (Swedish Research Council) (dnr 811-2014-59), Stiftelsen fö r Strategisk Forskning (Swedish Foundation for Strategic Research) (ref no. FFL09-0181), the Kempestiftelserna (Kempe Foundation) (SMK-1348) and the Laboratory for Molecular Infection Medicine Sweden (MIMS) (ref no.316-706-10).

Tissue patterning, through the concerted activity of a small number of signaling pathways, is critical to embryonic development. While patterning can involve signaling between neighbouring cells, in other contexts signals act over greater distances by traversing complex cellular landscapes to instruct the fate of distant cells. In this review, we explore different strategies adopted by cells to modulate signaling molecule range to allow correct patterning. We describe mechanisms for restricting signaling range and highlight how such short-range signaling can be exploited to not only control the fate of adjacent cells, but also to generate graded signaling within a field of cells. Other strategies include modulation of signaling molecule action by tissue architectural properties and the use of cellular membranous structures, such as signaling filopodia and exosomes, to actively deliver signaling ligands to target cells. Signaling filopodia can also be deployed to reach out and collect particular signals, thereby precisely controlling their site of action.

Extracellular vesicles (exosomes and shedding vesicles) released by mesenchymal stem cells (MSCs) are regarded as a storable, cell-free alternative with comparable therapeutic potential to their parent cells. Shedding vesicles originate as bulges on the cell surface but little is known about their turnover or how their formation can be stimulated. We have used atomic force microscopy (AFM) to follow the formation dynamics of bulges in living adipose tissue-derived MSCs. AFM images showed that, in general, MSCs present hundreds of nanosized protrusions on their surface with life spans of 10–20 min. Scanning electron microscopy confirmed those images and showed that bulges are also formed on filamentous processes. Extracellular vesicles deposited on the culture surface have comparable sizes to those of bulges showing up on the cell surface. The amount of protrusions on cells treated with progesterone or PDGF-BB, two treatments that stimulate the secretion of extracellular vesicles in MSCs, was evaluated by AFM. Measurements of the cross-area at 50 nm over the cell surface provided estimates of the amount of protrusions and showed that these values increased with the stimulating treatments. Our study suggests that shedding vesicles constitute a large population of the extracellular vesicle pool.

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus-mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high-resolution quantitative live-cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili-associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl-β-cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria-induced plasma membrane reorganization is a rapid event driven by a direct cross-talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.

Arp2/3 is a protein complex that nucleates actin filament assembly in the lamellipodium in adherent cells crawling on planar 2-dimensional (2D) substrates. However, in physiopathological situations, cell migration typically occurs within a 3-dimensional (3D) environment, and little is known about the role of Arp2/3 and associated proteins in 3D cell migration. Using time resolved live-cell imaging and HT1080, a fibrosarcoma cell line commonly used to study cell migration, we find that the Arp2/3 complex and associated proteins N-WASP, WAVE1, cortactin, and Cdc42 regulate 3D cell migration. We report that this regulation is caused by formation of multigeneration dendritic protrusions, which mediate traction forces on the surrounding matrix and effective cell migration. The primary protrusions emanating directly from the cell body and prolonging the nucleus forms independent of Arp2/3 and dependent on focal adhesion proteins FAK, talin, and p130Cas. The Arp2/3 complex, N-WASP, WAVE1, cortactin, and Cdc42 regulate the secondary protrusions branching off from the primary protrusions. In 3D matrices, fibrosarcoma cells as well as migrating breast, pancreatic, and prostate cancer cells do not display lamellipodial structures. This study characterizes the unique topology of protrusions made by cells in a 3D matrix and show that these dendritic protrusions play a critical role in 3D cell motility and matrix deformation. The relative contribution of these proteins to 3D migration is significantly different from their role in 2D migration.—Giri, A., Bajpai, S., Trenton, N., Jayatilaka, H., Longmore, G. D., Wirtz, D. The Arp2/3 complex mediates multigeneration dendritic protrusions for efficient 3-dimensional cancer cell migration.

Recently, a consensus has emerged that cofilin severing activity can generate free actin filament ends that are accessible for F-actin polymerization and depolymerization without changing the rate of G-actin association and dissociation at either filament end. The structural basis of actin filament severing by cofilin is now better understood. These results have been integrated with recently discovered mechanisms for cofilin activation in migrating cells, which led to new models for cofilin function that provide insights into how cofilin regulation determines the temporal and spatial control of cell behaviour.

To Fuse or Not to Fuse? Cell-cell fusion is poorly understood, although we know that in the nematode, Caenorhabditis elegans , it involves cell-surface fusogens and in the fly, Drosophila , the actin cytoskeleton plays a role. Shilagardi et al. (p. 359 ) reconstituted a high-efficiency, inducible cell-fusion culture system, in which they found that the C. elegans fusogen, Eff-1, induced a low level of cell-cell fusion that was enhanced by coexpression with a cell adhesion molecule, Sticks and stones (Sns), required for myoblast fusion in Drosophila. Sns-enhanced cell-cell fusion was mediated by dynamic actin polymerization that generated invasive membrane protrusions at sites of fusion.

In this review, we summarized data on the formation and structure of the long and highly adhesive membrane tubulovesicular extensions (TVEs, membrane tethers or cytonemes) observed in human neutrophils and other mammalian cells, protozoan parasites and bacteria. We determined that TVEs are membrane protrusions characterized by a uniform diameter (130–250 nm for eukaryotic cells and 60–90 nm for bacteria) along the entire length, an outstanding length and high rate of development and a high degree of flexibility and capacity for shedding from the cells. This review represents TVEs as protrusions of the cellular secretory process, serving as intercellular adhesive organelles in eukaryotic cells and bacteria. An analysis of the physical and chemical approaches to induce TVEs formation revealed that disrupting the actin cytoskeleton and inhibiting glucose metabolism or vacuolar-type ATPase induces TVE formation in eukaryotic cells. Nitric oxide is represented as a physiological regulator of TVE formation.

Podosomes are cellular adhesion structures involved in matrix degradation and invasion that comprise an actin core and a ring of cytoskeletal adaptor proteins. They are most often identified by staining with phalloidin, which binds F-actin and therefore visualizes the core. However, not only podosomes, but also many other cytoskeletal structures contain actin, which makes podosome segmentation by automated image processing difficult. Here, we have developed a quantitative image analysis algorithm that is optimized to identify podosome cores within a typical sample stained with phalloidin. By sequential local and global thresholding, our analysis identifies up to 76% of podosome cores excluding other F-actin-based structures. Based on the overlap in podosome identifications and quantification of podosome numbers, our algorithm performs equally well compared to three experts. Using our algorithm we show effects of actin polymerization and myosin II inhibition on the actin intensity in both podosome core and associated actin network. Furthermore, by expanding the core segmentations, we reveal a previously unappreciated differential distribution of cytoskeletal adaptor proteins within the podosome ring. These applications illustrate that our algorithm is a valuable tool for rapid and accurate large-scale analysis of podosomes to increase our understanding of these characteristic adhesion structures.

Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In these cells, hundreds of podosomes are spatially organized in diversely shaped clusters. Although we and others established individual podosomes as micron-sized mechanosensing protrusive units, the exact scope and spatiotemporal organization of podosome clustering remain elusive. By integrating a newly developed extension of Spatiotemporal Image Correlation Spectroscopy with novel image analysis, we demonstrate that F-actin, vinculin and talin exhibit directional and correlated flow patterns throughout podosome clusters. Pattern formation and magnitude depend on the cluster actomyosin machinery. Indeed, nanoscopy reveals myosin IIA-decorated actin filaments interconnecting multiple proximal podosomes. Extending well-beyond podosome nearest neighbours, the actomyosin-dependent dynamic spatial patterns reveal a previously unappreciated mesoscale connectivity throughout the podosome clusters. This directional transport and continuous redistribution of podosome components provides a mechanistic explanation of how podosome clusters function as coordinated mechanosensory area., Podosomes are adhesive cytoskeletal structures found in several cell types, but whether or how they are interconnected is not known. Here the authors demonstrate mesoscale connectivity of podosome clusters by imaging directional flow patterns of podosome components vinculin, talin and F-actin.

© 2016. Published by The Company of Biologists Ltd. Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.

Cell migration within 3D interstitial microenvironments is sensitive to extracellular matrix (ECM) properties, but the mechanisms that regulate migration guidance by 3D matrix features remain unclear. To examine the mechanisms underlying the cell migration response to aligned ECM, which is prevalent at the tumor–stroma interface, we utilized time-lapse microscopy to compare the behavior of MDA-MB-231 breast adenocarcinoma cells within randomly organized and well-aligned 3D collagen ECM. We developed a novel experimental system in which cellular morphodynamics during initial 3D cell spreading served as a reductionist model for the complex process of matrix-directed 3D cell migration. Using this approach, we found that ECM alignment induced spatial anisotropy of cells' matrix probing by promoting protrusion frequency, persistence, and lengthening along the alignment axis and suppressing protrusion dynamics orthogonal to alignment. Preference for on-axis behaviors was dependent upon FAK and Rac1 signaling and translated across length and time scales such that cells within aligned ECM exhibited accelerated elongation, front-rear polarization, and migration relative to cells in random ECM. Together, these findings indicate that adhesive and protrusive signaling allow cells to respond to coordinated physical cues in the ECM, promoting migration efficiency and cell migration guidance by 3D matrix structure.

Within living systems, striking juxtapositions in symmetry and asymmetry can be observed and the superficial appearance of symmetric organization often gives way to cellular asymmetries at higher resolution. It is frequently asymmetry and polarity that fascinate and challenge developmental biologists. In multicellular eukaryotes, cell polarity and asymmetry are essential for diverse cellular, tissue, and organismal level function and physiology and are particularly crucial for developmental processes. In plants, where cells are surrounded by rigid cell walls, asymmetric cell divisions are the foundation of pattern formation and differential cell fate specification. Thus, cellular asymmetry is a key feature of plant biology and in the plant root the consequences of these asymmetries are elegantly displayed. Yet despite the frequency of asymmetric (formative) cell divisions, cell/tissue polarity and the proposed roles for directional signaling in these processes, polarly localized proteins, beyond those involved in auxin or nutrient transport, are exceedingly rare. Indeed, although half of the asymmetric cell divisions in root patterning are oriented parallel to the axis of growth, laterally localized proteins directly involved in patterning are largely missing in action. Here, various asymmetric cell divisions and cellular and structural polarities observed in roots are highlighted and discussed in the context of the proposed roles for positional and/or directional signaling in these processes. The importance of directional signaling and the weight given to polarity in the root-shoot axis is contrasted with how little we currently understand about laterally oriented asymmetry and polarity in the root.

Tunneling nanotubes (TNTs) are small membranous tubes of 50-1000 nm diameter observed to connect cells in culture. Transfer of subcellular organelles through TNTs was observed in vitro and in vivo, but the formation and significance of these structures is not well understood. A polydimethylsiloxane biochip-based coculture model was devised to constrain TNT orientation and explore both TNT-formation and TNT-mediated mitochondrial transfer. Two parallel microfluidic channels connected by an array of smaller microchannels enabled localization of stem cell and cardiomyocyte populations while allowing connections to form between them. Stem cells and cardiomyocytes were deposited in their respective microfluidic channels, and stem cell-cardiomyocyte pairs were formed via the microchannels. Formation of TNTs and transfer of stained mitochondria through TNTs was observed by 24 h real-time video recording. The data show that stem cells are 7.7 times more likely to initiate contact by initial extension of filopodia. By 24 h, 67% of nanotube connections through the microchannels are composed of cardiomyocyte membrane. Filopodial extension and retraction by stem cells draws an extension of TNTs from cardiomyocytes. MitoTracker staining shows that unidirectional transfer of mitochondria between stem cell-cardiomyocyte pairs invariably originates from stem cells. Control experiments with cardiac fibroblasts and cardiomyocytes show little nanotube formation between homotypic or mixed cell pairs and no mitochondrial transfer. These data identify a novel biological process, unidirectional mitochondrial transfer, mediated by heterotypic TNT connections. This suggests that the enhancement of cardiomyocyte function seen after stem-cell injection may be due to a bioenergetic stimulus provided by mitochondrial transfer.

Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over-expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over-expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over-expressing cells, Rab7 and LC3B-II co-localized and co-localization in large circular structures occurred only in Rab7 over-expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co-localization of Rab7 with cortactin and f-actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over-expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP-stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over-expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695-1708, 2016. © 2015 Wiley Periodicals, Inc.

Metastatic spread of cancer cells is the leading cause of mortality from cancer. Metastatic cancer cells must penetrate through several barriers to escape the primary tumor and gain entry into the bloodstream in order to spread to other tissues. It is believed that invasive cancer cells penetrate these barriers by forming specialized F-actin rich protrusions called invadopodia that localize matrix degrading activity to cell-substrate contact points. Invadopodia gain their protrusive ability by combining the physical force generated by actin polymerization with the chemical activity of matrix degradation. Accumulating data over the past few years have shed light on the molecular mechanisms as well as kinase signaling pathways that regulate the complex process of actin polymerization in invadopodia. Here we review some of these mechanisms, the signaling pathways that regulate this process, as well as the in vivo relevance of invadopodial structures. Understanding the mechanisms that govern invadopodia formation and function is an essential step in the prevention of cancer invasion and metastasis.

Protrusion formation is an essential step during cell migration. Cells migrating in three-dimensional environments and in vivo can form a wide variety of protrusion types, including actin polymerization-driven lamellipodia, and contractility-driven blebs. The ability to switch between different protrusions has been proposed to facilitate motility in complex environments and to promote cancer dissemination. However, plasticity in protrusion formation has so far mostly been investigated in the context of transitions between amoeboid and mesenchymal migration modes, which involve substantial changes in overall cell morphology. As a result, the minimal requirements of transitions between blebs and lamellipodia, as well as the time scales on which they occur, remain unknown. To address these questions, we investigated protrusion switching during cell migration at the single cell level. Using cells that can be induced to form either blebs or lamellipodia, we systematically assessed the mechanical requirements, as well as the dynamics, of switching between protrusion types. We demonstrate that shifting the balance between actin protrusivity and actomyosin contractility leads to immediate transitions between blebs and lamellipodia in migrating cells. Switching occurred without changes in global cell shape, polarity, or cell adhesion. Furthermore, rapid transitions between blebs and lamellipodia could also be triggered upon changes in substrate adhesion during migration on micropatterned surfaces. Together, our data reveal that the type of protrusion formed by migrating cells can be dynamically controlled independently of overall cell morphology, suggesting that protrusion formation is an autonomous module in the regulatory network that controls the plasticity of cell migration.

Frontal, cell body, and rear regions perform distinct functions in the complex process of cell migration. A low-capacity, directional mechanism in the front coupled to a high-capacity, nondirectional mechanism in the middle represents a highly appealing model for driving cell migration under high mechanical load., To understand the mechanism of cell migration, we cultured fibroblasts on micropatterned tracks to induce persistent migration with a highly elongated morphology and well-defined polarity, which allows microfluidic pharmacological manipulations of regional functions. The function of myosin II was probed by applying inhibitors either globally or locally. Of interest, although global inhibition of myosin II inhibited tail retraction and caused dramatic elongation of the posterior region, localized inhibition of the cell body inhibited nuclear translocation and caused elongation of the anterior region. In addition, local application of cytochalasin D at the tip inhibited frontal extension without inhibiting forward movement of the cell nucleus, whereas local treatment posterior to the nucleus caused reversal of nuclear movement. Imaging of cortical dynamics indicated that the region around the nucleus is a distinct compression zone where activities of anterior and posterior regions converge. These observations suggest a three-component model of cell migration in which a contractile middle section is responsible for the movement of a bulky cell body and the detachment/retraction of a resistive tail, thereby allowing these regions to undergo coordinated movement with a moving anterior region that carries little load.

SummaryCollective cell migration requires maintenance of adhesive contacts between adjacent cells, coordination of polarized cell protrusions, and generation of propulsive traction forces. We demonstrate that mechanical force applied locally to C-cadherins on single Xenopus mesendoderm cells is sufficient to induce polarized cell protrusion and persistent migration typical of individual cells within a collectively migrating tissue. Local tension on cadherin adhesions induces reorganization of the keratin intermediate filament network toward these stressed sites. Plakoglobin, a member of the catenin family, is localized to cadherin adhesions under tension and is required for both mechanoresponsive cell behavior and assembly of the keratin cytoskeleton at the rear of these cells. Local tugging forces on cadherins occur in vivo through interactions with neighboring cells, and these forces result in coordinate changes in cell protrusive behavior. Thus, cadherin-dependent force-inducible regulation of cell polarity in single mesendoderm cells represents an emergent property of the intact tissue.Video Abstract

Myo1c directly interacts with G-actin and is important for its transport to the leading edge during endothelial cell migration., Addition of actin monomer (G-actin) to growing actin filaments (F-actin) at the leading edge generates force for cell locomotion. The polymerization reaction and its regulation have been studied in depth. However, the mechanism responsible for transport of G-actin substrate to the cell front is largely unknown; random diffusion, facilitated transport via myosin II contraction, local synthesis as a result of messenger ribonucleic acid localization, or F-actin turnover all might contribute. By tracking a photoactivatable, nonpolymerizable actin mutant, we show vectorial transport of G-actin in live migrating endothelial cells (ECs). Mass spectrometric analysis identified Myo1c, an unconventional F-actin–binding motor protein, as a major G-actin–interacting protein. The cargo-binding tail domain of Myo1c interacted with G-actin, and the motor domain was required for the transport. Local microinjection of Myo1c promoted G-actin accumulation and plasma membrane ruffling, and Myo1c knockdown confirmed its contribution to G-actin delivery to the leading edge and for cell motility. In addition, there is no obvious requirement for myosin II contractile–based transport of G-actin in ECs. Thus, Myo1c-facilitated G-actin transport might be a critical node for control of cell polarity and motility.

We propose a new computation-based approach for elucidating how signaling molecules are decoded in cell migration. In this approach, we performed FRET time-lapse imaging of Rac1 and Cdc42, members of Rho GTPases which are responsible for cell motility and quantitatively identified the response functions that describe the conversion from the molecular activities to the morphological changes. Based on the identified response functions, we clarified the profiles of how the morphology spatiotemporally changes in response to local and transient activation of Rac1 and Cdc42 and found that Rac1 and Cdc42 activation triggers laterally propagating membrane protrusion. The response functions were also endowed with property of differentiator, which is beneficial for maintaining sensitivity under adaptation to the mean level of input. Using the response function, we could predict the morphological change from molecular activity and its predictive performance provides a new quantitative measure of how much the Rho GTPases participate in the cell migration. Interestingly, we discovered distinct predictive performance of Rac1 and Cdc42 depending on the migration modes, indicating that Rac1 and Cdc42 contribute to persistent and random migration, respectively. Thus, our proposed predictive approach enabled us to uncover the hidden information processing rules of Rho GTPases in the cell migration.

RhoA activated kinases (ROCKs) are potent effectors of RhoA signaling for regulation of the cytoskeleton. ROCKs have been shown to be localized to several different subcellular locations, suggesting that its localization is context specific and regulated. However, the signaling mechanisms that control ROCK localization have not been clearly described. In this study we measured ROCKII localization following stimulation with the chemokine CXCL12 or adhesion to collagen 1. Strikingly, each of these extracellular signals targeted ROCKII to membrane protrusions. We further determined that both RhoA and PI3-kinase signaling are required for these stimuli to induce efficient membrane localization. Furthermore, we used a mutational approach to show that two separate domains predicted to respond to these localization signals, the Rho Binding Domain (RBD) and the Pleckstrin Homology domain (PH). Unexpectedly, we found that these two domains work synergistically to lead to membrane localization. This suggests a novel mechanism for controlling ROCKII localization at the membrane, in which the ROCKII C-terminus acts as a coincidence detector for spatial regulatory signals. In other words, efficient membrane targeting requires the ROCKII RBD to receive the RhoA signal and the PH domain to receive the phospholipid signal.

Circular dorsal ruffles (CDRs) are transient actin-rich ring-like structures which form on the dorsal surface of growth-factor stimulated cells. However, the dynamics and mechanism of formation of CDRs are still unknown. It has been observed that CDR formation leads to stress fibers disappearing near the CDRs. Since stress fiber formation can be modified by substrate stiffness, we examined the effect of substrate stiffness on CDR formation by seeding NIH 3T3 fibroblasts on glass and polydimethylsiloxane (PDMS) substrates of varying stiffnesses from 20 kPa to 1800 kPa. We found that increasing substrate stiffness increased the lifetime of the CDRs. We developed a mathematical model of the signaling pathways involved in CDR formation to provide insight into this lifetime and size dependence which is linked to substrate stiffness via Rac-Rho antagonism. From the model, increasing stiffness raised mDia1-nucleated stress fiber formation due to Rho activation. The increased stress fibers present increased replenishment of the G-actin pool, therefore prolonging Arp2/3-nucleated CDR formation due to Rac activation. Negative feedback by WAVE-related RacGAP on Rac explained how CDR actin propagates as an excitable wave, much like wave propagation in other excitable medium, e.g., nerve signal transmission. Accepted version

Nano-spatial distribution of cell surface molecules on cell membrane fluctuations during T-cell activation has not been reported. In this study, we innovated application of near-field scanning optical microscopy (NSOM)/quantum dots (QD)-based nanotechnology through three-dimensional image fusion algorithm to merge the simultaneously-obtained dual-color fluorescence information and three-dimensional topography. This novel imaging system made it possible to visualize nano-spatial distribution and organization of early-activation molecules CD69 and late-activation molecules CD71 on cell-membrane fluctuations during T-cell activation. Interestingly, most CD69 molecules were clustered to form 250–500 nm nano-domains polarizing predominantly in the peak of the cell-membrane fluctuations. In contrast, although CD71 molecules were also clustered as 250–500 nm nano-domains, they polarized dominantly in the valley of the cell-membrane fluctuations. The peak-valley polarities of CD69 nano-domains and CD71 nano-domains implied their different functions. CD69 nano-domains polarizing on membrane-peak fluctuations might serve as transient platforms driving TCR/CD3-induced signaling and activation, whereas CD71 nano-domains distributing in the membrane-valley fluctuations appeared to facilitate iron uptake for increased metabolisms in T-cell activation. Importantly, this NSOM/QD-based fluorescence-topographic image fusion provides a powerful tool to visualize nano-spatial distribution of cell-surface molecules on cell-membrane fluctuations and enable better understanding of distribution-function relationship.

Exposure of live cells to shear flow induces major changes in cell shape, adhesion to the extracellular matrix, and migration. In the present study, we show that exposure of cultured multiple myeloma (MM) cells to shear flow of 4–36 dynes/cm2 triggers the extension of long tubular protrusions (denoted flow-induced protrusions, or FLIPs) in the direction of the flow. These FLIPs were found to be rich in actin, contain few or no microtubules and, apart from endoplasmic reticulum (ER)-like membranal structures, are devoid of organelles. Studying the dynamics of this process revealed that FLIPs elongate at their tips in a shear force-dependent manner, and retract at their bases. Examination of this force dependence revealed considerable heterogeneity in the mechanosensitivity of individual cells, most likely reflecting the diversity of the malignant B cell population. The mechanisms underlying FLIP formation following mechanical perturbation, and their relevance to the cellular trafficking of MM cells, are discussed. J. Cell. Physiol. 226: 3197–3207, 2011. © 2011 Wiley Periodicals, Inc.

Low pulling forces applied locally to cell surface membranes produce viscoelastic cell surface protrusions. As the force increases, the membrane can locally separate from the cytoskeleton and a tether forms. Tethers can grow to great lengths exceeding the cell diameter. The protrusion-to-tether transition is known as the crossover. Here we propose a unified approach to protrusions and tethers providing, to our knowledge, new insights into their biomechanics. We derive a necessary and sufficient condition for a crossover to occur, a formula for predicting the crossover time, conditions for a tether to establish a dynamic equilibrium (characterized by constant nonzero pulling force and tether extension rate), a general formula for the tether material after crossover, and a general modeling method for tether pulling experiments. We introduce two general protrusion parameters, the spring constant and effective viscosity, valid before and after crossover. Their first estimates for neutrophils are 50 pN μm−1 and 9 pN s μm−1, respectively. The tether elongation after crossover is described as elongation of a viscoelastic-like material with a nonlinearly decaying spring (NLDs-viscoelastic material). Our model correctly describes the results of the published protrusion and tether pulling experiments, suggesting that it is universally applicable to such experiments.

HIV-1 cell-to-cell transmission confers a strong advantage as it increases efficiency of transfer up to 100-fold compared with a cell-free route. Mechanisms of HIV-1 cell-to-cell transmission are still unclear and can in part be explained by the presence of actin-containing cellular protrusions. Such protrusions have been shown to facilitate cell-to-cell viral dissemination. Using fluorescence microscopy, electron tomography, and ion abrasion scanning electron microscopy we show that HIV-1 induces membrane extensions in immature dendritic cells through activation of Cdc42. We demonstrate that these extensions are induced after engagement of DC-SIGN by HIV-1env via a cascade that involves Src kinases, Cdc42, Pak1, and Wasp. Silencing of Cdc42 or treatment with a specific Cdc42 inhibitor, Secramine A, dramatically reduced the number of membrane protrusions visualized on the cell surface and decreased HIV-1 transfer via infectious synapses. Ion abrasion scanning electron microscopy of cell-cell contact regions showed that cellular extensions from immature dendritic cells that have the appearance of thin filopodia in thin section images are indeed extended membranous sheets with a narrow cross section. Our results demonstrate that HIV-1 binding on immature dendritic cells enhances the formation of membrane extensions that facilitate HIV-1 transfer to CD4+ T lymphocytes.

Arf6-dependent membrane dynamics concentrates active Cdc42 at the leading edge of migrating cells., Cell polarity is essential for cell division, cell differentiation, and most differentiated cell functions including cell migration. The small G protein Cdc42 controls cell polarity in a wide variety of cellular contexts. Although restricted localization of active Cdc42 seems to be important for its distinct functions, mechanisms responsible for the concentration of active Cdc42 at precise cortical sites are not fully understood. In this study, we show that during directed cell migration, Cdc42 accumulation at the cell leading edge relies on membrane traffic. Cdc42 and its exchange factor βPIX localize to intracytosplasmic vesicles. Inhibition of Arf6-dependent membrane trafficking alters the dynamics of Cdc42-positive vesicles and abolishes the polarized recruitment of Cdc42 and βPIX to the leading edge. Furthermore, we show that Arf6-dependent membrane dynamics is also required for polarized recruitment of Rac and the Par6–aPKC polarity complex and for cell polarization. Our results demonstrate influence of membrane dynamics on the localization and activation of Cdc42 and consequently on directed cell migration.

Invadopodia and podosomes have been intensively studied because of their involvement in the degradation of extracellular matrix. As both structures have been studied mostly on thin matrices, their commonly reported shapes and characteristics may differ from those in vivo. To assess the morphology of invadopodia in a complex 3D environment, we observed invadopodial formation in cells grown on a dense matrix based on cell-free dermis. We have found that invadopodia differ in morphology when cells grown on the dermis-based matrix and thin substrates are compared. The cells grown on the dermis-based matrix display invadopodia which are formed by a thick protruding base rich in F-actin, phospho-paxillin, phospho-cortactin and phosphotyrosine signal, from which numerous thin filaments protrude into the matrix. The protruding filaments are composed of an F-actin core and are free of phospho-paxillin and phospho-cortactin but capped by phosphotyrosine signal. Furthermore, we found that a matrix-degrading activity is localized to the base of invadopodia and not along the matrix-penetrating protrusions. Our description of invadopodial structures on a dermis-based matrix should greatly aid the development of new criteria for the identification of invadopodia in vivo, and opens up the possibility of studying the invadopodia-related signaling in a more physiological environment.

Cells remodel their plasma membrane and cytoskeleton during numerous physiological processes, including spreading and motility. Morphological changes require the cell to adjust its membrane tension on different timescales. While it is known that endo- and exocytosis regulate the cell membrane area in a timescale of 1 h, faster processes, such as abrupt cell detachment, require faster regulation of the plasma membrane tension. In this article, we demonstrate that cell blebbing plays a critical role in the global mechanical homeostasis of the cell through regulation of membrane tension. Abrupt cell detachment leads to pronounced blebbing (which slow detachment does not), and blebbing decreases with time in a dynamin-dependent fashion. Cells only start spreading after a lag period whose duration depends on the cell's blebbing activity. Our model quantitatively reproduces the monotonic decay of the blebbing activity and accounts for the lag phase in the spreading of blebbing cells.

It has long been speculated that cellular microdomains are important for many cellular processes, especially those involving Ca2+ signalling. Measurements of cytosolic Ca2+ report maximum concentrations of less than few micromolar, yet several cytosolic enzymes require concentrations of more than 20 microM Ca2+ to be activated. In this paper, we have resolved this apparent paradox by showing that the surface topology of cells represents an important and hitherto unrecognized feature for generating microdomains of high Ca2+ in cells. We show that whereas the standard modeling assumption of a smooth cell surface predicts only moderate localized effects, the more realistic "wrinkled" surface topology predicts that Ca2+ concentrations up to 80 microM can persist within the folds of membranes for significant times. This intra-wrinkle location may account for 5% of the total cell volume. Using different geometries of wrinkles, our simulations show that high Ca2+ microdomains will be generated most effectively by long narrow membrane wrinkles of similar dimensions to those found experimentally. This is a new concept which has not previously been considered, but which has ramifications as the intra-wrinkle location is also a strategic location at which Ca2+ acts as a regulator of the cortical cytoskeleton and plasma membrane expansion.

During yeast sporulation, a forespore membrane (FSM) initiates at each spindle-pole body and extends to form the spore envelope. We used Schizosaccharomyces pombe to investigate the role of septins during this process. During the prior conjugation of haploid cells, the four vegetatively expressed septins (Spn1, Spn2, Spn3, and Spn4) coassemble at the fusion site and are necessary for its normal morphogenesis. Sporulation involves a different set of four septins (Spn2, Spn5, Spn6, and the atypical Spn7) that does not include the core subunits of the vegetative septin complex. The four sporulation septins form a complex in vitro and colocalize interdependently to a ring-shaped structure along each FSM, and septin mutations result in disoriented FSM extension. The septins and the leading-edge proteins appear to function in parallel to orient FSM extension. Spn2 and Spn7 bind to phosphatidylinositol 4-phosphate [PtdIns(4)P] in vitro, and PtdIns(4)P is enriched in the FSMs, suggesting that septins bind to the FSMs via this lipid. Cells expressing a mutant Spn2 protein unable to bind PtdIns(4)P still form extended septin structures, but these structures fail to associate with the FSMs, which are frequently disoriented. Thus, septins appear to form a scaffold that helps to guide the oriented extension of the FSM.

Protein localization mechanisms dictate the functional and structural specialization of cells. Of the four polar surface organelles featured by the dimorphic bacterium Caulobacter crescentus , the stalk, a cylindrical extension of all cell envelope layers, is the least well characterized at the molecular level. Here we apply a powerful experimental scheme that integrates genetics with high-throughput localization to discover StpX, an uncharacterized bitopic membrane protein that modulates stalk elongation and is sequestered to the stalk. In stalkless mutants StpX is dispersed. Two populations of StpX were discernible within the stalk with different mobilities: an immobile one near the stalk base and a mobile one near the stalk tip. Molecular anatomy provides evidence that ( i ) the StpX transmembrane domain enables access to the stalk organelle, ( ii ) the N-terminal periplasmic domain mediates retention in the stalk, and ( iii ) the C-terminal cytoplasmic domain enhances diffusion within the stalk. Moreover, the accumulation of StpX and an N-terminally truncated isoform is differentially coordinated with the cell cycle. Thus, at the submicron scale the localization and the mobility of a protein are precisely regulated in space and time and are important for the correct organization of a subcellular compartment or organelle such as the stalk.