Biology subjects

Faix, J.

Publications and source records attributed to Faix, J..

10 recordsLinked to original sources

bioRxiv2026

Conserved tyrosine Y16 acts as a phosphorylation switch controlling Ena/VASP recruitment to sites of actin assembly

Ena/VASP proteins are powerful actin polymerases that accumulate at sites of active actin assembly, including lamellipodial and microspike tips, focal adhesions and pathogen surfaces, where they drive processive filament elongation. However, the mechanisms controlling their recruitment remain elusive. Here, we used cells lacking all Ena/VASP-family members to systematically test phosphomimetic and nonphosphorylatable substitutions at conserved tyrosine and serine residues implicated in subcellular targeting. Combining cell-based reconstitution with biochemical assays, we identify phosphorylation of Y16 in vertebrate Ena/VASP proteins as the decisive inhibitory modification controlling subcellular Ena/VASP recruitment, in contrast to previously proposed roles for Y39 and S157. Corresponding phosphomimetic substitutions in the C. elegans orthologue UNC-34 phenocopied uncoordinated locomotion of UNC-34 null worms, supporting the evolutionary conservation of this regulatory mechanism. Thus, our findings identify a conserved phosphorylation-sensitive switch at the EVH1-FP4 ligand interface that links EVH1-dependent targeting to dynamic control of actin assembly in adhesion, migration and pathogenesis.

cell biology↗

bioRxiv2026

IqgD is a Rac1-interacting IQGAP required for efficient growth of Dictyostelium discoideum on bacterial lawns

IQGAPs are large multidomain scaffold proteins that interact with the Rho family GTPases Cdc42 and Rac1, functioning both as their effectors and as regulators by stabilizing their active GTP-bound state. In this study, we analyzed the function of IqgD, an IQGAP-related protein from the professional phagocyte Dictyostelium discoideum. IqgD contains a calponin homology domain (CHD), a GAP-related domain (GRD), and a RasGAP C-terminal (RGCt) domain. We show that the CHD is essential for F-actin binding and cortical localization, whereas the GRD and RGCt domains mediate interactions with Rac1 GTPases and the actin-bundling proteins cortexillins. Moreover, similar to mammalian IQGAPs, IqgD maintains Rac1 in its active conformation. IqgD is enriched in macropinocytic and phagocytic cups and co-localizes with F-actin and active Rac1 in the ring-like structure that forms around surface-bound particles at the cell bottom. Loss of IqgD results in markedly reduced growth on bacterial lawns and significantly smaller cell size. While mutant cells internalize bacteria from suspension as efficiently as wild-type cells, they display a strong defect in phagocytosis of surface-bound particles, accompanied by decreased adhesion to the cell substrate. Together, our data show that although IqgD localizes to macroendocytic cups, it is dispensable for macropinocytosis and phagocytosis of suspended particles. Instead, IqgD is specifically required for efficient phagocytosis of surface-bound particles, likely by facilitating robust F-actin polymerization at the cell bottom to generate the force necessary for detachment of surface-bound bacteria. Significance StatementPhagocytosis of surface-bound microbes is essential for host defense and environmental feeding strategies, yet its underlying mechanisms remain poorly understood. We identify the IQGAP-related protein IqgD in D. discoideum as a key factor required for efficient uptake of bacteria attached to solid surfaces. IqgD localizes to an F-actin- and Rac1-rich circular structure analogous to the phagocytic adhesion ring (PAR) recently described in mammalian macrophages, suggesting that this mode of force-driven particle detachment is evolutionarily conserved. Our findings provide mechanistic insight into substrate-dependent phagocytosis and establish IqgD as a central regulator of this process.

cell biology↗

bioRxiv2025

The fibrocystin C-terminal domain inhibits Src/STAT3 signal induced cystogenesis of kidney epithelia.

Autosomal recessive polycystic kidney disease (ARPKD) is caused by impaired function of fibrocystin/polyductin (FPC) in collecting duct epithelia resulting in cyst formation. We hypothesized that the membrane-bound C-terminal FPC domain (FPCct) is necessary to suppress cystogensis and facilitate epithelial homeostasis. In ARPKD, cystic kidney epithelia are characterized by a secretory phenotype associated with high intracellular cAMP levels and enhanced STAT3-dependent transcription. Moreover, impaired FPC function may lead to enhanced activation of Src tyrosine kinase, thereby activating STAT3 signaling and its downstream transcriptional activity. To investigate the effects of FPC loss on the cystic epithelial cell phenotype, we used an established principal-like MDCK cell line (pl-MDCK) and studied monolayers in both two and three-dimensional culture. In this in vitro model of collecting duct epithelia, FPC-deficient cells showed two-fold elevated basal cAMP levels and enhanced apical secretion leading to three-fold higher luminal pressure. Forskolin-stimulated elevation of cAMP levels triggered enhanced Src-dependent activation of STAT3 resulting in a pronounced cystic phenotype. Notably, expression of wildtype FPCct reduced both STAT3-dependent transcription and the secretory phenotype in knockout epithelial cells. Our data suggest that FPCct interacts with Src kinase at the plasma membrane, thereby reducing Src-mediated STAT3 phosphorylation and limiting STAT3-dependent transcription. Thus, FPCct appears to act like a physiological suppressor of cystogenic signaling, as found in healthy kidney epithelia, that is essential for maintaining epithelial homeostasis. Protein constructs that restore FPC C-terminal function may offer a therapeutic lead to mitigate epithelial dysfunction and slow disease progression in ARPKD.

cell biology↗

bioRxiv2025

Profilin promotes lamellipodium protrusion by tuning the antagonistic activities of CP and VASP

Cell migration on rigid surfaces employs flat protrusions termed lamellipodia, which constitute the prime model system for branched actin filament networks that generate pushing forces for membrane movement. Branched actin filaments are nucleated by the Arp2/3 complex and play vital roles in various cell biological processes, such as organelle trafficking, phagocytosis and autophagy. Here we utilize genome editing to explore the functional connections between the actin monomer-binding protein profilin (Pfn), the filament nucleating Arp2/3 complex, its co-factor heterodimeric capping protein (CP) and the Ena/VASP family of actin filament polymerases in lamellipodial actin assembly. Individual and combinations of knockouts show that Pfn counters Ena/VASP but promotes Arp2/3 complex activity, while Ena/VASP and CP mutually antagonize each other. Notably, while Pfn is important for Arp2/3 complex activity irrespective of Ena/VASP, sensitivity of CP to Pfn removal is lost in the absence of Ena/VASP. Our findings establish Pfn as master regulator of Arp2/3 complex-dependent actin network formation, which differentially regulates VASP and its antagonizer CP. Finally, mathematical modeling of our data suggest that Ena/VASP and CP compete for binding at the lamellipodial edge, likely contributing to their functional antagonism at this subcellular site. This work provides critical insights into the molecular logic of branched actin network assembly in membrane protrusion and cellular force generation.

cell biology↗

bioRxiv2024

Differential interference with actin-binding protein function by acute Cytochalasin B

Dynamic actin filament remodeling is crucial for a plethora of fundamental cell biological processes, ranging from cell division and migration to cell communication, intracellular trafficking or tissue development. Cytochalasin B and -D are fungal secondary metabolites frequently used for interference with such processes. Although generally assumed to block actin filament polymerization at their rapidly growing barbed ends and compete with regulators at these sites, our molecular understanding of their precise effects in dynamic actin structures is scarce. Here we combine live cell imaging and analysis of fluorescent actin-binding protein dynamics with acute treatment of lamellipodia in migrating cells with cytochalasin B. Our results show that in spite of an abrupt halt of lamellipodium protrusion, cytochalasin B affects various actin filament barbed end-binding proteins in a differential fashion. Cytochalasin B enhances instead of diminishes the accumulation of prominent barbed end-binding factors such as Ena/VASP family proteins and heterodimeric capping protein (CP) in the lamellipodium. Similar results were obtained with cytochalasin D. All these effects are highly specific, as cytochalasin-induced VASP accumulation requires the presence of CP, but not vice versa, and coincides with abrogation of both actin and VASP turnover. Cytochalasin B can also increase apparent barbed end interactions with the actin-binding {beta}-tentacle of CP and partially mimic its Arp2/3 complex-promoting activity in the lamellipodium. In conclusion, our results reveal a new spectrum of cytochalasin activities on barbed end-binding factors, with important implications for the interpretation of their effects on dynamic actin structures.

cell biology↗

bioRxiv2022

Convergence of Ras- and Rac-regulated formin pathways is pivotal for phagosome formation and particle uptake in Dictyostelium

Macroendocytosis comprising phagocytosis and macropinocytosis are actin-driven processes regulated by small GTPases that depend on the dynamic reorganization of the membrane that protrudes and internalizes extracellular material by cup-shaped structures. To effectively capture, enwrap, and internalize their targets, these cups are arranged into a peripheral ring or ruffle of protruding actin sheets emerging from an actin-rich, non-protrusive zone at its base. Despite extensive knowledge of the mechanism driving actin assembly of the branched network at the protrusive cup edge, which is initiated by the actin-related protein (Arp) 2/3 complex downstream of Rac signaling, our understanding of actin assembly in the base is still incomplete. In the Dictyostelium model system, the Ras-regulated formin ForG was previously shown to specifically contribute to actin assembly at the cup base. Loss of ForG is associated with a strongly impaired macroendocytosis and a 50% reduction of F-actin content at the base of phagocytic cups, in turn indicating the presence of additional factors that specifically contribute to actin formation at the base. Here, we show that ForG synergizes with the Rac-regulated formin ForB to form the bulk of linear filaments at the cup base. Consistently, combined loss of both formins virtually abolishes cup formation and leads to severe defects of macroendocytosis, emphasizing the relevance of converging Ras- and Rac-regulated formin pathways in assembly of linear filaments in the cup base, which apparently provide mechanical support to the entire structure. Remarkably, we finally show that active ForB, unlike ForG, additionally drives phagosome rocketing to aid particle internalization. Significance StatementCup formation in macroendocytosis is a decisive, actin-dependent process that relies on distinct actin assembly factors generating the necessary mechanical forces to drive rearrangements of the plasma membrane and engulfment of extracellular material. Hitherto, in Dictyostelium the Arp2/3 complex and VASP were shown to promote actin assembly at the protrusive rim of phagocytic cups, while the Ras-regulated formin ForG generates about half of the actin filament mass at the base. Here, we show that ForG synergizes with the Rac-regulated formin ForB to form the bulk of filaments at the cup base. Loss of both formins virtually abolishes cup formation and leads to dramatic defects in macroendocytosis, illustrating the relevance of converging Ras- and Rac-regulated signaling pathways in this process.

cell biology↗

bioRxiv2022

Ena/VASP clustering at microspike tips involves Lamellipodin but not I-BAR proteins, and absolutely requires unconventional Myosin-X

Sheet-like membrane protrusions at the leading edge, termed lamellipodia, drive 2D-cell migration using active actin polymerization. Microspikes comprise actin-filament bundles embedded within lamellipodia, but the molecular mechanisms driving their formation and their potential functional relevance have remained elusive. Microspike formation requires the specific activity of clustered Ena/VASP proteins at their tips to enable processive actin assembly in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering are poorly understood. Systematic analyses of B16-F1 melanoma mutants lacking potential candidate proteins revealed that neither inverse BAR-domain proteins, nor lamellipodin or Abi are essential for clustering, although they differentially contribute to lamellipodial VASP accumulation. In contrast, unconventional myosin-X (MyoX) identified here as proximal to VASP was obligatory for Ena/VASP clustering and microspike formation. Interestingly, and despite the invariable distribution of other relevant marker proteins, the width of lamellipodia in MyoX-KO mutants was significantly reduced as compared to B16-F1 control, suggesting that microspikes contribute to lamellipodium stability. Consistently, MyoX removal caused marked defects in protrusion and random 2D-cell migration. Strikingly, Ena/VASP-deficiency also uncoupled MyoX cluster dynamics from actin assembly in lamellipodia, establishing their tight functional association in microspike formation. Significance StatementUnlike filopodia that protrude well beyond the cell periphery and are implicated in sensing, morphogenesis and cell-to-cell communication, the function of microspikes consisting of actin-filament bundles fully embedded within lamellipodia is less clear. Microspike formation involves specific clustering of Ena/VASP family members at filament-barbed ends to enable processive actin polymerization in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering have remained unknown. Here, we systematically analyzed these processes in genetic knockout mutants derived from B16-F1 cells and show that Ena/VASP clustering at microspike tips involves Lamellipodin, but not inverse BAR-domain proteins, and strictly requires unconventional Myosin-X. Complete loss of microspikes was confirmed with CRISPR/Cas9-mediated MyoX knockout in Rat2 fibroblasts, excluding cell type-specific effects.

cell biology↗

bioRxiv2020

Loss of Hem1 disrupts macrophage function and impacts on migration, phagocytosis and integrin-mediated adhesion

The hematopoietic-specific protein 1 (Hem1) comprises an essential subunit of the WAVE Regulatory Complex (WRC) in immune cells. WRC has a fundamental role in Arp2/3 complex activation and the protrusion of branched actin networks in motile cells. Hem1 deficiency leads to suppression of the entire WRC in immune cells. Defective WRC function in macrophages results in loss of lamellipodia and migration defects. Moreover, phagocytosis, commonly accompanied by lamellipodium protrusion during cup formation, is altered in Hem1 null cells concerning frequency and efficacy. When analyzing cell spreading, adhesion and podosome formation, we found that Hem1 null cells are capable, in principle, of podosome formation and consequently, do not show any quantitative differences in extracellular matrix degradation. Their adhesive behavior, however, was significantly altered. Specifically, adhesion as well as de-adhesion of Hem1 null cells was strongly compromised, likely contributing to the observed reduced efficiency of phagocytosis. In line with this, phosphorylation of the prominent adhesion component paxillin was diminished. Non-hematopoietic somatic cells disrupted in expression for both Hem1 and its ubiquitous orthologue Nck-associated protein 1 (Nap1) or the essential WRC components Sra-1/PIR121 did not only confirm defective paxillin phosphorylation, but also revealed that paxillin turnover in focal adhesions is accelerated in the absence of WRC. Finally, adhesion assays using platelets lacking functional WRC as model system unmasked radically decreased IIb{beta}3 integrin activation. Our results thus demonstrate that WRC-driven actin networks impact on integrin-dependent processes controlling formation and dismantling of different types of cell-substratum adhesion. One sentence summaryInterference of Hem1 function in mice and cells uncovers a hitherto unrecognized role in integrin-mediated cell adhesion that is crucial for macrophage function and connects to recently discovered immunodeficiencies in patients carrying Hem1 mutations.

cell biology↗

bioRxiv2020

Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion

Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel, cellular Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.

cell biology↗

bioRxiv2019

Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation

Efficient migration on adhesive surfaces involves the protrusion of lamellipodial actin networks and their subsequent stabilization by nascent adhesions. The actin binding protein lamellipodin (Lpd) is thought to play a critical role in lamellipodium protrusion, by delivering Ena/VASP proteins onto the growing plus ends of actin filaments and by interacting with the WAVE regulatory complex (WRC), an activator of the Arp2/3 complex, at the leading edge. Using B16-F1 melanoma cell lines, we demonstrate that genetic ablation of Lpd compromises protrusion efficiency and coincident cell migration without altering essential parameters of lamellipodia, including their maximal rate of forward advancement and actin polymerization. We also confirmed lamellipodia and migration phenotypes with CRISPR/Cas9-mediated Lpd knockout Rat2 fibroblasts, excluding cell type-specific effects. Moreover, computer-aided analysis of cell edge morphodynamics on B16-F1 cell lamellipodia revealed that loss of Lpd correlates with reduced temporal protrusion maintenance as a prerequisite of nascent adhesion formation. We conclude that Lpd optimizes protrusion and nascent adhesion formation by counteracting frequent, chaotic retraction and membrane ruffling. Summary statementWe describe how genetic ablation of the prominent actin- and VASP-binding protein lamellipodin combined with software-aided protrusion analysis uncovers mechanistic insights into its cellular function during cell migration.

cell biology↗