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Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped., In vivo, cells migrate across a diverse landscape of extracellular matrix containing gaps which present a challenge for cells to protrude across. Here, the authors show that T-Plastin strengthens protrusive actin networks to promote protrusion, extracellular matrix gap-bridging, and cell migration.

Cellular protrusion formation capacity is a key feature of developing neurons and many eukaryotic cells. However, the mechanisms underlying membrane growth in protrusion formation are largely unclear. In this study, photo-reactive unnatural amino acid 3-(3-methyl-3H-diazirin-3-yl)-propamino-carbonyl-Nε-l-lysine was incorporated by a genetic code expansion strategy into protrudin, a protein localized in acidic endosomes and in the endoplasmic reticulum, that induces cellular protrusion and neurite formation. The modified protrudin was used for covalent trapping of protrudin-interacting proteins in living cells. Fatty acid synthase (FASN), which synthesizes free fatty acids, was identified to transiently interact with protrudin. Further characterization revealed a unique cooperation mechanism in which protrudin cooperates with FASN to facilitate cellular protrusion formation. This work reveals a novel mechanism involved in protrusion formation that is dependent on transient interaction between FASN and protrudin, and establishes a creative strategy to investigate transient protein-protein interactions in mammalian cells.

This work is licensed under a Creative Commons Attribution 4.0 International License., Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion., This work has been funded by grants from Cancer Research UK (C7125/A9926; A13651) to GEJ and EM-V, the Medical Research Council (G0401026) to GEJ and from the Spanish Ministry of Economy and Competitiveness (SAF 2010-19152/SAF2013-46183R) and Community of Madrid (S2010/BMD-2359, SkinModel-CM) to MQ. EM-V is currently a recipient of a Postdoctoral Research Contract from Fundación Científica Asociación Española Contra el Cáncer (AECC).

Background: Myosin X (MYO10) was recently reported to promote tumour invasion by transporting integrins to filopodial tips in breast cancer. However, the role of MYO10 in tumours remains poorly defined. Here, we report that MYO10 is required in invadopodia to mediate invasive growth and extracellular matrix degradation, which depends on the binding of MYO10's pleckstrin homology domain to PtdIns(3,4,5)P3. Methods: The expression of MYO10 and its associations with clinicopathological and biological factors were examined in breast cancer cells and breast cancer specimens (n=120). Cell migration and invasion were investigated after the silencing of MYO10. The ability of cells to form invadopodia was studied using a fluorescein isothiocyanate-conjugated gelatin degradation assay. A mouse model was established to study tumour invasive growth and metastasis in vivo. Results: Elevated MYO10 levels were correlated with oestrogen receptor status, progesterone receptor status, poor differentiation, and lymph node metastasis. Silencing MYO10 reduced cell migration and invasion. Invadopodia were responsible for MYO10's role in promoting invasion. Furthermore, decreased invasive growth and lung metastasis were observed in the MYO10-silenced nude mouse model. Conclusions: Our findings suggest that elevated MYO10 expression increases the aggressiveness of breast cancer; this effect is dependent on the involvement of MYO10 in invadopodial formation.

Background: FSCN1 and matrix metalloproteinase 14 (MMP14) are both invadopodia-related proteins. We herein elucidate the tumourigenicity of these proteins and identify novel therapeutic agents in esophageal squamous cell carcinoma (ESCC). Methods: FSCN1 and MMP14 were evaluated by immunohistochemistry and quantitative PCR, and microRNA (miR)-133a was also evaluated by PCR in surgical ESCC specimens. The roles of FSCN1, MMP14 and miR-133a were established in ESCC cells. Results: The expression of FSCN1 or MMP14 was an independent poor prognostic factor according to a multivariate analysis of immunohistochemistry, and their co-expression correlated with the poorest overall survival (OS) out of all the examined factors. Additionally, their mRNAs significantly correlated and both inversely correlated with miR-133a in surgical specimens. Transfection of a miR-133a mimic decreased the mRNA and protein levels of both FSCN1 and MMP14 in ESCC cells. The knockdown of FSCN1 or MMP14 and transfection of a miR-133a mimic inhibited the proliferation and invasion of ESCC cells. Patients with a lower miR-133a expression have a significantly poorer OS than those with a higher expression. Conclusion: The combined expression of FSCN1 and MMP14 is associated with a poor prognosis, and miR-133a, which regulates their mRNAs, can serve as a strong tumour suppressor of ESCC.

Cell fate and differentiation in the Arabidopsis root epidermis are genetically defined but remain plastic to environmental signals such as limited availability of inorganic phosphate (Pi). Root hairs of Pi-deficient plants are more frequent and longer than those of plants grown under Pi-replete conditions. To dissect genes involved in Pi deficiency-induced root hair morphogenesis, we constructed a co-expression network of Pi-responsive genes against a customized database that was assembled from experiments in which differentially expressed genes that encode proteins with validated functions in root hair development were over-represented. To further filter out less relevant genes, we combined this procedure with a search for common cis-regulatory elements in the promoters of the selected genes. In addition to well-described players and processes such as auxin signalling and modifications of primary cell walls, we discovered several novel aspects in the biology of root hairs induced by Pi deficiency, including cell cycle control, putative plastid-to-nucleus signalling, pathogen defence, reprogramming of cell wall-related carbohydrate metabolism and chromatin remodelling. This approach allows the discovery of novel of aspects of a biological process from transcriptional profiles with high sensitivity and accuracy.

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.

During sexual contact, HIV-1 can enter the mucous membranes of the vagina, throat, or rectum. There, the virus encounters potential target cells, including antigen-presenting cells such as dermal dendritic cells (DC), Langerhans cells, and plasmacytoid DCs, which play pivotal roles in both innate and adaptive immunity. All DC subsets are susceptible to HIV-1 infection or internalization of intact virus, and appear to facilitate cell-mediated transmission of HIV-1 in the mucosa and lymphatic tissues. Once internalized, HIV-1 can be transmitted from DCs to T-cells at a specialized junction termed the infectious synapse (IS). Recent studies reveal that several actin-rich DC membrane extensions, including actin filopodia, membrane sheets, cytonemes, and nanotubes, establish direct contact between cells at the IS and facilitate HIV-1 transmission. Although the virus can infect T-cells directly (cis-infection), transmission of HIV-1 from DCs to T-cells at IS (trans-infection and de novo trans-infection) appears to be some 10 to 100 times more efficient than direct infection; therefore, cell-to-cell transmission is an attractive target for novel HIV therapeutics. We evaluated the effects of Slit2N, a ligand for the Roundabout (Robo) receptors, on the transmission of HIV-1 from DCs to T-cells. Slit2N inhibits HIV-1 transmission to T-cells and its replication. Binding of HIV-1 to DC-SIGN on the surface of DCs initiates signaling through cellular-Src (c-Src) and cell division control protein 42 (Cdc42), which modulates actin polymerization and filopodia formation via the actin-related protein 2/3 (Arp2/3) complex and mammalian Diaphanous 2 (mDia2). Slit2N inhibited HIV-1-induced signaling through both Arp2/3 and mDia2, decreased filopodial extensions on virus-infected DCs, and inhibited cell-to-cell transmission of HIV-1 at the IS in a Robo1-dependent manner. Of particular note, we identified Flightless-1 (Fli1) as a novel, Robo1-interacting protein, and demonstrated that it is required for efficient cell-to-cell transmission of HIV-1. These results suggest that targeting the Slit/Robo pathway involves the modulation of previously unrecognized cytoskeletal elements in HIV-1 transmission, and thus, offers an innovative strategy to limit viral infection in the host. Disclosures: No relevant conflicts of interest to declare.

Migration of endothelial cells is essential for wound healing and angiogenesis. Src kinase activity plays important roles at the protrusions of migrating endothelial cells. However, the spatiotemporal coordination between Src kinase activity and the protrusion of cell edge remains unclear. Therefore, we investigate these coordinated molecular events at the initiation of cell migration, by integrating microfabrication, fluorescence resonance energy transfer (FRET)-based biosensors and automated computational image analysis. We demonstrate that the physical release of restrictive micropattern triggered a significant decrease of Src activity at the protrusive edge of endothelial cells. Computational cross-correlation analysis reveals that the decrease of Src activity occurred earlier in time and was well-coordinated with the protrusion of cell edge in polarized cells, but not in non-polarized cells. These results suggest that the spatiotemporal control of Src kinase activity is well-coordinated with cell polarization and protrusion in endothelial cells upon the release of physical constraint, as that experienced by endothelial cells sprouting from stiff tumor micro-environment during angiogenesis. Therefore, our integrative approach enabled the discovery of a new model where Src is de-activated in coordination with membrane protrusion, providing important insights into the regulation of endothelial migration and angiogenesis.

Cellular protrusions are highly dynamic structures involved in fundamental processes,including cell migration and invasion. For a cell to migrate, its leading edge mustform protrusions and then adhere or retract. The spatial and temporal coordinationof protrusions and retraction is yet to be fully understood. The study of protrusiondynamics mainly relies on live-microscopy often coupled to fluorescent labeling.Here we report the use of an alternative, label-free, quantitative and rapid assayto analyze protrusion dynamics in a cell population based on the real-time recordingof cell activity by means of electronic sensors. Cells are seeded on a plate coveredwith electrodes and their shape changes map into measured impedance variations. Upongrowth factor stimulation the impedance increases due to protrusive activity anddecreases following retraction. Compared to microscopy-based methods, impedancemeasurements are suitable to high-throughput studies on different cell lines, growthfactors and chemical compounds. We present data indicating that this assay lendsitself to dissect the biochemical signaling pathways controlling adhesiveprotrusions. Indeed, we show that the protrusion phase is sustained by actinpolymerization, directly driven by growth factor stimulation. Contraction insteadmainly relies on myosin action, pointing at a pivotal role of myosin in lamellipodiaretraction.

Morphological polarization involving changes in cell shape and redistribution of cellular signaling machinery, initiate the migration of mammalian cells. Golgi complex typically localizes in front of the nucleus, and this frontwards polarization has been proposed to be involved in directional migration. However, the sequence of events remains unresolved. Does Golgi polarization precede directional migration or vice-versa? We address this question by constraining cells to specific areas and shapes then tracking their motile behavior and the spatio-temporal distribution of Golgi apparatus upon release. Results show that while the position of the Golgi complex depends on the cell geometry, the subcellular localization of the Golgi complex does not define the cell's leading edge. Cells constrained within elongated geometries exhibit polarized extension of lamellipodia and upon release, migrate preferentially along the long axis of the cell. Minimally constrained cells released from larger areas however, exhibit retarded migration regardless of lamellipodia protrusion activity.

The death receptor CD95 promotes apoptosis through well-defined signalling pathways. In colorectal cancer cells, CD95 primarily stimulates migration and invasion through pathways that are incompletely understood. Here, we identify a new CD95-activated tyrosine kinase pathway that is essential for CD95-stimulated tumour cell invasion. We show that CD95 promotes Tyr 783 phosphorylation of phospholipase C-gamma1 through the platelet-derived growth factor receptor-beta, resulting in ligand-stimulated phosphatidylinositol (4,5)-bisphosphate (PIP(2)) hydrolysis. PIP(2) hydrolysis liberates the actin-severing protein cofilin from the plasma membrane to initiate cortical actin remodelling. Cofilin activation is required for CD95-stimulated formation of membrane protrusions and increased tumour cell invasion.

COPII-coated vesicles form at the endoplasmic reticulum for cargo transport to the Golgi apparatus. We used in vitro reconstitution to examine the roles of the COPII scaffold in remodeling the shape of a lipid bilayer. Giant Unilamellar Vesicles were examined using fast confocal fluorescence and cryo-electron microscopy in order to avoid separation steps and minimize mechanical manipulation. COPII showed a preference for high curvature structures, but also sufficient flexibility for binding to low curvatures. The COPII proteins induced beads-on-a-string-like constricted tubules, similar to those previously observed in cells. We speculate about a mechanical pathway for vesicle fission from these multibudded COPII-coated tubules, considering the possibility that withdrawal of the Sar1 amphipathic helix upon GTP hydrolysis leads to lipid bilayer destabilization resulting in fission.

Expression of AMAP1 correlates well with the invasive phenotypes and malignancy of human primary breast carcinomas. AMAP1 recruits its binding proteins, such as cortactin and paxillin, to sites of Arf6 activation to form invadopodia. A mouse ortholog of AMAP1, ASAP1, is known to bind to CIN85, a binding partner of an E3 ligase, Cbl. Here, we found that CIN85 colocalizes with AMAP1 at invadopodia, and binding of AMAP1 with CIN85 is important for the invasive activities of breast cancer cells, including MDA-MB-231. siRNA-mediated silencing of CIN85, as well as Cbl, also inhibited the invasion. We moreover found that AMAP1 is monoubiquitinated, rather than polyubiquitinated, by virtue of Cbl and provide evidence that the ability of AMAP1 to be monoubiquitinated is important for its involvement in invasion. Our results indicate that CIN85, as well as Cbl, which is a well-known suppressor of growth factor receptor signaling, can be positively involved in tumor invasion, and suggest that a complex epigenetic process is involved in AMAP1 function in breast cancer cell invasion.

EMBO J 28, 248–260 (2009); published online 4 February 2009 [PMC free article] [PubMed] Axonal elongation involves the coordinated assembly and activation of molecular pathways in response to environmental signals. Neurons sense the environment at growth cones to decide on further growth, arborization or consolidation. The work by Ana Mingorance-Le Meur and Timothy P. O'Connor highlights the important role of neurite shaft dynamics in the consolidation process. The establishment of functional neuronal circuits requires a large number of precisely coordinated events, with the initial sprouting of neurites from the neuronal cell body being rather crucial. Neurites eventually develop into axons or dendrites, gaining the ability to convert chemical information into electrical signals. However, there is a transitional stage between these two events whereby the first neurites acquire some basic architectural and molecular features of axons or dendrites, yet their main aim is to grow to the territory where they will send or receive information, respectively. Studies on cortical development demonstrate that newborn neurons sprout two initial, very dynamic neurites, which guide their radial or tangential migration towards their final cortical layer position. Then, neurites need to acquire either axonal or dendritic identity, an event occurring early with the appearance of the first two neurites (Calderon de Anda et al, 2008), followed by a commitment phase, in which growth promoting molecules become restricted to one neurite only, the future axon (Wiggin et al, 2005). This period is critical in brain development, as it changes a neuron from a motile cell, with actively extending and retracting neurites into a stationary cell, in which its unique axon steadily grows towards its eventual target. A common feature of the advancing axon is the capacity to sense growth, positive or negative environmental cues and act accordingly, by advancing further, retracting or turning. Sensing is largely confined to the growth cones, which locally transduce those signals to the cytoskeleton, mitochondria and membrane reservoirs. In addition, at specific places along its trajectory, the axon can receive information for branching, which can occur through growth cone splitting or by the appearance of collateral branching spots along the shaft located at the rear of the growth cone (Dent et al, 2003). This brings into light that the neurite shaft must be, except for the peculiar sites of collateral branching, insensitive/irresponsive to the growth cues that elicit a growth response at the growth cone. Failure to do so would result in aberrant branching. In addition, the neurite shaft behind the advancing growth cone must contain all the machinery for the transformation of a dynamic bi-dimensional structure, the growth cone, into the more stable cylindrical neurite as growth proceeds and thus determines the consolidation of the growth. Consolidation may be seen as a simple distal–proximal passive mechanism, whereby the molecules supporting dynamic exploratory and advancing movement at the growth cone become gradually diluted out as one moves towards the cell body. This type of ‘default' mechanism may be sufficient to activate the machinery to prevent branching and to help making a cylinder. On the other hand, one cannot exclude an ‘active' mechanism for neurite consolidation. Despite the tremendous importance that neurite consolidation plays in the stabilization in axonal elongation and collateral branching, the different mechanisms governing these processes are far from complete. This confers great relevance to the work of Mingorance Le-Meur and O'Connor in this issue of The EMBO Journal. The main conclusion of the paper is that the restriction of the protease activity of calpain to the neurite shaft in developing neurons is a key event in the stabilization of these extensions. The election of calpains as potential regulators of consolidation was based on previous observations that calpains regulate actin dynamics and concentrate in the rear end of migrating cells, repressing the formation of protrusions. In agreement to their predictions, the activity of calpains, visualized using fodrin proteolysis as read-out, was restricted to the neurite shaft. Furthermore, inhibition of calpain activity increases the number of actin patches along the neurite shaft. These actin patches may represent sites of high actin turnover, required for localized growth, which is supported by life imaging experiments showing that the inhibition of calpain activity triggers filopodia and protrusion formation at the neurite shaft. The specificity of calpain activity on the process of consolidation/branching is suggested by the observation that calpain activity changes do not affect neurite length. Moreover, the overexpression of a protease dead mutant of calpain increased branching in contrast to the ectopic expression of the full-length calpain-2, which reduced branching capacity. Importantly, inhibition of calpain in vivo increased neurite branching in immature and mature neurons, adding biological significance to the in vitro data. Furthermore, exposing neurons to ligands known to induce branching inhibits calpain activity when added to neurons in vitro, which was blocked by excess of calpain-2. One mechanism by which calpains may prevent branching is through the proteolysis of cortactin, an actin-binding protein that activates the actin-nucleating complex of Arp2/3. Hence, it appears that neurite shaft calpain activity keeps cortactin at basal levels. As regulation of cortactin levels by basal proteolysis is energetically demanding and will need, eventually, to be turned off at the neurite-growth cone interphase, to allow advance, and along the shaft, to allow branching, consolidation needs to be seen as a key active process of axonal growth, required to keep neurites stable and ready to become dynamic at the same time. In all, the paper by Mingorance and O'Connor greatly contributes to the field of neuronal differentiation through the study of neurite stabilization. Their data in the context of basic aspects of axonal growth are illustrated in the accompanying figure (Figure 1). The current work leaves a number of important questions unanswered: how do activities regulating growth cone dynamics become repressed towards the neurite shaft? Is calpain activity in the shaft due to de-repression following the dilution of growth cone growing activities (retrograde) or is it because of a cell body-originated trafficking pathway (anterograde), unable to enter (or to be retained) at the growth cone? Do molecules with a presumed role in axonal growth or polarization (Wiggin et al, 2005) act in growth cones (pushing molecules) or in the shaft (consolidation molecules)? Clearly, the opening of venues of research is a natural consequence of novel data and therefore we envision that this work will serve as an inspiration to many, to finely dissect the molecular basis of neurite consolidation and how this contributes to axonal growth and branching, in health and pathology. The venues for the future, opened by this work are therefore multiple and exciting. Figure 1 Schematic representation of the balance between active growth and consolidation in extending neurites.