RESUMEN

Cell migration is largely determined by the type of protrusions formed by the cell. Mesenchymal migration is accomplished by formation of lamellipodia and/or filopodia, while amoeboid migration is based on bleb formation. Changing of migrational conditions can lead to alteration in the character of cell movement. For example, inhibition of the Arp2/3-dependent actin polymerization by the CK-666 inhibitor leads to transition from mesenchymal to amoeboid motility mode. Ability of the cells to switch from one type of motility to another is called migratory plasticity. Cellular mechanisms regulating migratory plasticity are poorly understood. One of the factors determining the possibility of migratory plasticity may be the presence and/or organization of vimentin intermediate filaments (VIFs). To investigate whether organization of the VIF network affects the ability of fibroblasts to form membrane blebs, we used rat embryo fibroblasts REF52 with normal VIF organization, fibroblasts with vimentin knockout (REF-/-), and fibroblasts with mutation inhibiting assembly of the full-length VIFs (REF117). Blebs formation was induced by treatment of cells with CK-666. Vimentin knockout did not lead to statistically significant increase in the number of cells with blebs. The fibroblasts with short fragments of vimentin demonstrate the significant increase in number of cells forming blebs both spontaneously and in the presence of CK-666. Disruption of the VIF organization did not lead to the significant changes in the microtubules network or the level of myosin light chain phosphorylation, but caused significant reduction in the focal contact system. The most pronounced and statistically significant decrease in both size and number of focal adhesions were observed in the REF117 cells. We believe that regulation of the membrane blebbing by VIFs is mediated by their effect on the focal adhesion system. Analysis of migration of fibroblasts with different organization of VIFs in a three-dimensional collagen gel showed that organization of VIFs determines the type of cell protrusions, which, in turn, determines the character of cell movement. A novel role of VIFs as a regulator of membrane blebbing, essential for manifestation of the migratory plasticity, is shown.

Asunto(s)

Adhesiones Focales , Filamentos Intermedios , Ratas , Animales , Filamentos Intermedios/metabolismo , Adhesiones Focales/metabolismo , Vimentina/genética , Vimentina/metabolismo , Vimentina/farmacología , Microtúbulos/metabolismo , Movimiento Celular , Extensiones de la Superficie Celular/metabolismo

RESUMEN

In the last few years, pulsed electric fields have emerged as promising clinical tools for tumor treatments. This study highlights the distinct impact of a specific pulsed electric field protocol, PEF-5 (0.3 MV/m, 40 µs, 5 pulses), on astrocytes (NHA) and medulloblastoma (D283) and glioblastoma (U87 NS) cancer stem-like cells (CSCs). We pursued this goal by performing ultrastructural analyses corroborated by molecular/omics approaches to understand the vulnerability or resistance mechanisms triggered by PEF-5 exposure in the different cell types. Electron microscopic analyses showed that, independently of exposed cells, the main targets of PEF-5 were the cell membrane and the cytoskeleton, causing membrane filopodium-like protrusion disappearance on the cell surface, here observed for the first time, accompanied by rapid cell swelling. PEF-5 induced different modifications in cell mitochondria. A complete mitochondrial dysfunction was demonstrated in D283, while a mild or negligible perturbation was observed in mitochondria of U87 NS cells and NHAs, respectively, not sufficient to impair their cell functions. Altogether, these results suggest the possibility of using PEF-based technology as a novel strategy to target selectively mitochondria of brain CSCs, preserving healthy cells.

Asunto(s)

Mitocondrias , Neoplasias , Mitocondrias/metabolismo , Membrana Celular/metabolismo , Electricidad , Citoesqueleto/metabolismo , Encéfalo/metabolismo , Neoplasias/metabolismo

RESUMEN

Chiral actin bundles have been shown to play an important role in cell dynamics, but our understanding of the molecular mechanisms which combine to generate chirality remains incomplete. To address this, we numerically simulate a crosslinked filopodial bundle under the actions of helical myosin motors and/or formins and examine the collective buckling and twisting of the actin bundle. We first show that a number of proposed mechanisms to buckle polymerizing actin bundles without motor activity fail under biologically-realistic parameters. We then demonstrate that a simplified model of myosin spinning action at the bundle base effectively "braids" the bundle, but cannot control compaction at the fiber tips. Finally, we show that formin-mediated polymerization and motor activity can act synergitically to compact filopodium bundles, as motor activity bends filaments into shapes that activate twist forces induced by formins. Stochastic fluctuations of actin polymerization rates and slower cross linking dynamics both increase buckling and decrease compaction. We discuss implications of our findings for mechanisms of cytoskeletal chirality.

Asunto(s)

Citoesqueleto de Actina , Actinas , Forminas , Citoesqueleto , Miosinas

RESUMEN

Brain development relies on dynamic morphogenesis and interactions of neurons. Filopodia are thin and highly dynamic membrane protrusions that are critically required for neuronal development and neuronal interactions with the environment. Filopodial interactions are typically characterized by non-deterministic dynamics, yet their involvement in developmental processes leads to stereotypic and robust outcomes. Here, we discuss recent advances in our understanding of how filopodial dynamics contribute to neuronal differentiation, migration, axonal and dendritic growth and synapse formation. Many of these advances are brought about by improved methods of live observation in intact developing brains. Recent findings integrate known and novel roles ranging from exploratory sensors and decision-making agents to pools for selection and mechanical functions. Different types of filopodial dynamics thereby reveal non-deterministic subcellular decision-making processes as part of genetically encoded brain development.

Asunto(s)

Neurogénesis , Seudópodos , Neurogénesis/fisiología , Neuronas , Morfogénesis , Encéfalo

RESUMEN

Filopodia are narrow cell extensions involved in various physiological processes. Integrins mediate filopodia adhesion and likely transmit adhesive force to regulate filopodia formation and functions, but the force is extremely weak to study and remains poorly understood. Using integrative tension sensor (ITS), we imaged filopodia adhesive force at the single molecular tension level and investigated the force dynamics and sources. Results show that filopodia integrin tension (FIT) is generated in discrete foci (force nodes) along single filopodia with a spacing of ∼1 µm. Inhibitions of actin polymerization or myosin II activity markedly reduced FIT signals in force nodes at filopodia tips and at filopodia bases, respectively, suggesting differential force sources of FIT in the distal force nodes and proximal ones in filopodia. Using two ITS constructs with different force thresholds for activation, we showed that the molecular force level of FIT is greater at filopodia bases than that at filopodia tips. We also tested the role of vinculin and myosin X in the FIT transmission. With vinculin knockout in cells, filopodia and associated force nodes were still formed normally, suggesting that vinculin is dispensable for the formation of filopodia and force nodes. However, vinculin is indeed required for the transmission of strong FIT (capable of rupturing DNA in a shear conformation), as the strong FIT vanished in filopodia with vinculin knockout. Co-imaging of FIT and myosin X shows no apparent co-localization, demonstrating that myosin X is not directly responsible for generating FIT, despite its prominent role in filopodium elongation.

Asunto(s)

Integrinas , Seudópodos , Seudópodos/metabolismo , Vinculina/metabolismo , Integrinas/metabolismo , Actinas/metabolismo , Adhesivos/metabolismo , Miosinas/metabolismo

RESUMEN

Post-therapeutic relapse remains the biggest challenge to breast cancer management. The re-initiation of proliferation of dormant tumor cells in either metastatic or primary tumor location marks the final rate-limiting step of malignancy and mortality. The underlying molecular mechanisms remain poorly understood. We have recently demonstrated that KLF8 promotes breast cancer metastasis via CXCR4 upregulation. Here we report a role and mechanisms for KLF8 in driving the recurrence-like tumor outgrowth in both secondary and primary sites in a CXCR4-dependent manner. Treatment of an MDA-MB-231 breast cancer cell variant with the CXCR4 ligand, CXCL12, induces formation of filopodia in monolayer culture and filopodium-like protrusions (FLPs) in 3D culture. The FLP+ cells proliferate significantly faster than FLP- cells in the 3D culture supplemented with CXCL12. Both the FLP formation and enhanced proliferation in the 3D culture can be prevented by silencing KLF8 expression in the cells. From this prevention, the cells can be rescued by overexpressing wild-type CXCR4 but not its inactive mutant form in the cells. Overexpression of KLF8 or CXCR4 in the cells dramatically enhances their invasive outgrowth and metastasis after being implanted into immunocompromised mice. Mechanistically, we found that the activated FAK was recruited to the nascent FLPs and that proliferation of the cells was completely prevented with a FAK-specific inhibitor. Taken together, these results shed new light on the role of KLF8 in promoting breast cancer recurrence, the fatal episode of the disease, by inducing CXCR4-dependent FLP formation.

RESUMEN

Branches are critical for neuron function, generating the morphological complexity required for functional networks. They emerge from different, well-described, cytoskeletal precursor structures that elongate to branches. While branches are thought to be maintained by shared cytoskeletal regulators, our data from mouse hippocampal neurons indicate that the precursor structures trigger alternative branch maintenance mechanisms with differing stabilities. Whereas branches originating from lamellipodia or growth cone splitting events collapse soon after formation, branches emerging from filopodia persist. Furthermore, compared to other developing neurites, axons stabilise all branches and preferentially initiate branches from filopodia. These differences explain the altered stability of branches we observe in neurons lacking the plasma membrane protein phospholipid phosphatase-related protein 3 (PLPPR3, also known as PRG2) and in neurons treated with netrin-1. Rather than altering branch stability directly, PLPPR3 and netrin-1 boost a 'filopodia branch programme' on axons, thereby indirectly initiating more long-lived branches. In summary, we propose that studies on branching should distinguish overall stabilising effects from effects on precursor types, ideally using multifactorial statistical models, as exemplified in this study.

Asunto(s)

Conos de Crecimiento , Neuronas , Animales , Axones , Células Cultivadas , Ratones , Neuritas

RESUMEN

BACKGROUND: Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post-translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis. METHODS: Immunoprecipitation and glutathione-S-transferase pull-down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP-associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF-mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live-cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry. RESULTS: Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti-AcK471-Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin-K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin-K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin-K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471-acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin-bundling activity. Clinically, high levels of AcK471-Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease-free survival of ESCC patients. CONCLUSIONS: Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin-K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin-bundling activity.

Asunto(s)

Proteínas Portadoras/metabolismo , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Proteínas de Microfilamentos/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Actinas , Humanos , Lisina/metabolismo , Procesamiento Proteico-Postraduccional

RESUMEN

Plasma membrane curvature is an important factor in the regulation of cellular phenotype and is critical for various cellular activities including the shedding of extracellular vesicles (EV). One of the most striking morphological features of cells is different plasma membrane-covered extensions supported by actin core such as filopodia and microvilli. Despite the various functions of these extensions are partially unexplained, they are known to facilitate many crucial cellular functions such as migration, adhesion, absorption, and secretion. Due to the rapid increase in the research activity of EVs, there is raising evidence that one of the general features of cellular plasma membrane protrusions is to act as specialized platforms for the budding of EVs. This review will focus on early observations and recent findings supporting this hypothesis, discuss the putative budding and shedding mechanisms of protrusion-derived EVs and their biological significance.

Asunto(s)

Membrana Celular/metabolismo , Vesículas Extracelulares/metabolismo , Microscopía Electrónica de Rastreo/métodos , Humanos

RESUMEN

The Arp2/3 complex generates branched actin filament networks operating in cell edge protrusion and vesicle trafficking. Here we employ a conditional knockout mouse model permitting tissue- or cell-type specific deletion of the murine Actr3 gene (encoding Arp3). A functional Actr3 gene appeared essential for fibroblast viability and growth. Thus, we developed cell lines for exploring the consequences of acute, tamoxifen-induced Actr3 deletion causing near-complete loss of functional Arp2/3 complex expression as well as abolished lamellipodia formation and membrane ruffling, as expected. Interestingly, Arp3-depleted cells displayed enhanced rather than reduced cell spreading, employing numerous filopodia, and showed little defects in the rates of random cell migration. However, both exploration of new space by individual cells and collective migration were clearly compromised by the incapability to efficiently maintain directionality of migration, while the principal ability to chemotax was only moderately affected. Examination of actin remodeling at the cell periphery revealed reduced actin turnover rates in Arp2/3-deficient cells, clearly deviating from previous sequestration approaches. Most surprisingly, induced removal of Arp2/3 complexes reproducibly increased FMNL formin expression, which correlated with the explosive induction of filopodia formation. Our results thus highlight both direct and indirect effects of acute Arp2/3 complex removal on actin cytoskeleton regulation.

RESUMEN

Cell migration frequently involves the formation of lamellipodial protrusions, the initiation of which requires Rac GTPases signalling to heteropentameric WAVE regulatory complex (WRC). While Rac-related RhoG and Cdc42 can potently stimulate lamellipodium formation, so far presumed to occur by upstream signalling to Rac activation, we show here that the latter can be bypassed by RhoG and Cdc42 given that WRC has been artificially activated. This evidence arises from generation of B16-F1 cells simultaneously lacking both Rac GTPases and WRC, followed by reconstitution of lamellipodia formation with specific Rho-GTPase and differentially active WRC variant combinations. We conclude that formation of canonical lamellipodia requires WRC activation through Rac, but can possibly be tuned, in addition, by WRC interactions with RhoG and Cdc42.

Asunto(s)

Seudópodos

RESUMEN

Predictive modelling of complex biological systems and biophysical interactions requires the inclusion of multiple nano- and micro-scale events. In many scenarios, however, numerical solutions alone do not necessarily enhance the understanding of the system. Instead, this work explores the use of an agent-based model with visualization capabilities to elucidate interactions between single cells. We present a model of juxtacrine signalling, using Cell Studio, an agent-based modelling system, based on gaming and three-dimensional visualization tools. The main advantages of the system are its ability to apply any cell geometry and to dynamically visualize the diffusion and interactions of the molecules within the cells in real time. These provide an excellent tool for obtaining insight about different biological scenarios, as the user may view the dynamics of a system and observe its emergent behaviour as it unfolds. The agent-based model was validated against the results of a mean-field model of Notch receptors and ligands in two neighbouring cells. The conversion to an agent-based model is described in detail. To demonstrate the advantages of the model, we further created a filopodium-mediated signalling model. Our model revealed that diffusion and endocytosis alone are insufficient to produce significant signalling in a filopodia scenario. This is due to the bottleneck at the cell-filopodium contact region and the long distance to the end of the filopodium. However, allowing active transport of ligands into filopodia enhances the signalling significantly compared with a face-to-face scenario. We conclude that the agent-based approach can provide insights into mechanisms underlying cell signalling. The open-source model can be found in the Internet hosting service GitHub.

Asunto(s)

Comunicación Celular , Imagenología Tridimensional , Modelos Biológicos , Seudópodos/metabolismo , Teoría del Juego

RESUMEN

Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Dictyostelium/metabolismo , Complejos Multiproteicos/metabolismo , Seudópodos/fisiología , Proteína de Unión al GTP rac1/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Movimiento Celular , Dictyostelium/citología , Dictyostelium/genética , Ratones , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/fisiología , Neuropéptidos/antagonistas & inhibidores , Neuropéptidos/metabolismo , Conformación Proteica , Células Tumorales Cultivadas , Familia de Proteínas del Síndrome de Wiskott-Aldrich/química , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína RCA2 de Unión a GTP

RESUMEN

Neuronal membrane glycoprotein M6a (Gpm6a) is a protein with four transmembrane regions and the N- and the C-ends facing the cytosol. It functions in processes of neuronal development, outgrowth of neurites, and formation of filopodia, spines, and synapsis. Molecular mechanisms by which Gpm6a acts in these processes are not fully comprehended. Structural similarities of Gpm6a with tetraspanins led us to hypothesize that, similarly to tetraspanins, the cytoplasmic tails function as connections with cytoskeletal and/or signaling proteins. Here, we demonstrate that the C- but not the N-terminal cytosolic end of Gpm6a is required for the formation of filopodia by Gpm6a in cultured neurons from rat hippocampus and in neuroblastoma cells N2a. Further immunofluorescence microcopy and flow cytometry analysis show that deletion of neither the N- nor the C-terminal intracellular domains interferes with the recognition of Gpm6a by the function-blocking antibody directed against the extracellular part of Gpm6a. Expression levels of both truncation mutants were not affected but we observed decrease in the amount of both truncated proteins on cell surface suggesting that the incapacity of the Gpm6a lacking C-terminus to induce filopodium formation is not due to the lower amount of Gpm6a on cell surface. Following colocalization assays shows that deletion of the C- but not the N-terminus diminishes the association of Gpm6a with clathrin implying involvement of clathrin-mediated trafficking events. Next, using comprehensive alanine scanning mutagenesis of the C-terminus we identify K250, K255, and E258 as the key residues for the formation of filopodia by Gpm6a. Substitution of these charged residues with alanine also diminishes the amount of Gpm6a on cell surface and in case of K255 and E258 leads to the lower amount of total expressed protein. Subsequent bioinformatic analysis of Gpm6a amino acid sequence reveals that highly conserved and functional residues cluster preferentially within the C- and not within the N-terminus and that K250, K255, and E258 are predicted as part of sorting signals of transmembrane proteins. Altogether, our results provide evidence that filopodium outgrowth induced by Gpm6a requires functionally critical residues within the C-terminal cytoplasmic tail.

RESUMEN

The ß-isoform of diacylglycerol kinase (DGK) localizes predominantly to neurons and induces neurite outgrowth and spine formation. However, the detailed molecular mechanisms underlying the functions of DGKß remain elusive. During the course of studies on other DGK isozymes, we unexpectedly found that the overexpression of wild-type DGKß in COS-7 cells markedly induced filopodium formation. Because filopodium formation is closely related to neurite outgrowth and spine formation, we constructed various DGKß mutants and compared their abilities to induce filopodium formation in order to elucidate the structure-function relationships of DGKß. We found that the C-terminal, C1 and catalytic domains and catalytic activity were indispensable for filopodium formation, but the recoverin homology domain and EF-hand motifs were not. Moreover, the extent of plasma membrane localization and F-actin colocalization were positively correlated with filopodium formation. Intriguingly, DGKß selectively interacted and colocalized at the plasma membrane with a Rac1-GTPase-activating protein, ß2-chimaerin, which is an inducer of filopodia; it also interacted, to lesser extent, with α2-chimaerin, but not with α1- or ß1-chimaerin. Moreover, DGKß enhanced the plasma membrane localization of ß2-chimaerin. These results suggest that DGKß plays an important role in neurite outgrowth and spine formation in neurons via its ability to induce filopodium formation.

Asunto(s)

Proteínas Activadoras de GTPasa/metabolismo , Lipoproteína Lipasa/metabolismo , Proteínas de Neoplasias/metabolismo , Seudópodos/fisiología , Animales , Células COS , Dominio Catalítico , Chlorocebus aethiops , Lipoproteína Lipasa/química , Lipoproteína Lipasa/genética , Mutación , Dominios Proteicos , Seudópodos/ultraestructura

Asunto(s)

Actinas , Movimiento Celular

RESUMEN

During cytoskeleton remodeling, cancer cells generate force at the plasma membrane that originates from chemical motors (e.g., actin). This force (pN) and its time course reflect the on and off-rates of the motors. We describe the design and calibration of a force-measuring device (i.e., optical tweezers) that is used to monitor this force and its time course at the edge of a cell, with particular emphasis on the temporal resolution of the instrument.

Asunto(s)

Movimiento Celular/fisiología , Pinzas Ópticas , Óptica y Fotónica/métodos , Algoritmos , Fenómenos Biomecánicos , Modelos Teóricos , Óptica y Fotónica/instrumentación , Relación Señal-Ruido , Temperatura

RESUMEN

Stress-responsive neuronal membrane glycoprotein M6a (Gpm6a) functions in neurite extension, filopodium and spine formation and synaptogenesis. The mechanisms of Gpm6a action in these processes are incompletely understood. Previously, we identified the actin regulator coronin-1a (Coro1a) as a putative Gpm6a interacting partner. Here, we used co-immunoprecipitation assays with the anti-Coro1a antibody to show that Coro1a associates with Gpm6a in rat hippocampal neurons. By immunofluorescence microscopy, we demonstrated that in hippocampal neurons Coro1a localizes in F-actin-enriched regions and some of Coro1a spots co-localize with Gpm6a labeling. Notably, the over-expression of a dominant-negative form of Coro1a as well as its down-regulation by siRNA interfered with Gpm6a-induced filopodium formation. Coro1a is known to regulate the plasma membrane translocation and activation of small GTPase Rac1. We show that Coro1a co-immunoprecipitates with Rac1 together with Gpm6a. Pharmacological inhibition of Rac1 resulted in a significant decrease in filopodium formation by Gpm6a. The same was observed upon the co-expression of Gpm6a with the inactive GDP-bound form of Rac1. In this case, the elevated membrane recruitment of GDP-bound Rac1 was detected as well. Moreover, the kinase activity of the p21-activated kinase 1 (Pak1), a main downstream effector of Rac1 that acts downstream of Coro1a, was required for Gpm6a-induced filopodium formation. Taken together, our results provide evidence that a signaling pathway including Coro1a, Rac1, and Pak1 facilitates Gpm6a-induced filopodium formation. Formation of filopodia by membrane glycoprotein M6a (Gpm6a) requires actin regulator coronin-1a (Coro1a), known to regulate plasma membrane localization and activation of Rac1 and its downstream effector Pak1. Coro1a associates with Gpm6a. Blockage of Coro1a, Rac1, or Pak1 interferes with Gpm6a-induced filopodium formation. Moreover, Gpm6a facilitates Rac1 membrane recruitment. Altogether, a mechanistic insight into the process of Gpm6a-induced neuronal filopodium formation is provided.

Asunto(s)

Glicoproteínas de Membrana/fisiología , Proteínas de Microfilamentos/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/ultraestructura , Seudópodos/fisiología , Quinasas p21 Activadas/fisiología , Proteína de Unión al GTP rac1/fisiología , Actinas/análisis , Animales , Células Cultivadas , Regulación hacia Abajo , Genes Reporteros , Hipocampo/citología , Proteínas de Microfilamentos/genética , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Biogénesis de Organelos , Cultivo Primario de Células , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteína de Unión al GTP rac1/antagonistas & inhibidores

RESUMEN

The assembly of a nervous system requires the extension of axons and dendrites to specific regions where they are matched with appropriate synaptic targets. Although the cues that guide long-range outgrowth have been characterized extensively, additional mechanisms are required to explain short-range guidance in neural development. Using a complementary combination of time-lapse imaging by fluorescence confocal microscopy and serial block-face electron microscopy, we identified a novel type of presynaptic projection that participates in the assembly of the vertebrate nervous system. Synapse formation by each hair cell of the zebrafish's lateral line occurs during a particular interval after the cell's birth. During the same period, projections emerge from the cellular soma, extending toward a specific subpopulation of mature hair cells and interacting with polarity-specific afferent nerve terminals. The terminals then extend along the projections to reach appropriately matched presynaptic sites, after which the projections recede. Our results suggest that presynaptic projections act as transient scaffolds for short-range partner matching, a mechanism that may occur elsewhere in the nervous system.

Asunto(s)

Diferenciación Celular , Sistema de la Línea Lateral/citología , Células Receptoras Sensoriales/citología , Sinapsis/fisiología , Pez Cebra/fisiología , Animales , Sistema de la Línea Lateral/crecimiento & desarrollo , Sistema de la Línea Lateral/ultraestructura , Microscopía Electrónica de Transmisión , Células Receptoras Sensoriales/ultraestructura , Pez Cebra/crecimiento & desarrollo

RESUMEN

The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been extensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime.

Asunto(s)

Actinas/fisiología , Movimiento Celular/fisiología , Modelos Biológicos , Animales , Fenómenos Biomecánicos/fisiología , Simulación por Computador , Fluorescencia , Cinética , Magnetismo , Polímeros , Conejos , Termodinámica