Jan Faix

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-FP ₄ ligand interface that links EVH1-dependent targeting to dynamic control of actin assembly in adhesion, migration and pathogenesis.

The actin-rich cell cortex is a viscoelastic structure essential for processes such as cell migration and division, yet the full spectrum of actin assembly factors that build and regulate the cortex in mammalian cells remains incompletely defined. While the Arp2/3 complex generates actin networks mostly comprising short, branched filaments, formins like mDia1 and mDia3 assemble long, linear filaments capable of withstanding the high mechanical load produced by myosin-driven contraction at the cell rear. To obtain a complete inventory of formins that regulate mechanics of the contractile cortex in B16-F1 melanoma cells, we systematically screened Diaphanous-related formins (DRFs) for interactions with Rho-family GTPases regulating contractility. This screen led to the identification of Daam1/2 and FMNL2/3 as cortical formins, since active variants of these formins as well as the endogenous proteins prominently localized to the rear cortex of polarized B16-F1 cells. Yeast two-hybrid (Y2H) and GST pull-down assays revealed prominent interactions of Daam1/2 with active RhoA, RhoB, RhoC, RhoD, and Cdc42, and weaker binding to Rac1 and Rif. Pyrene-actin polymerization and single-filament TIRF assays further demonstrated that these GTPases effectively relieve autoinhibition of Daam-family formins, with Daam1 and 2 emerging as a potent elongators. In line with this, CRISPR/Cas9-generated Daam1/2 double-knockout cells exhibited multiple defects, including formation of multiple leading edges, impaired random 2D migration, reduced focal adhesion (FA) turnover and inefficient rear retraction. However, contrary to earlier work, we found no evidence that Daam1/2 regulate the formation of finger-like protrusions. By comparison, FMNL2 appears to act as a dual-role formin at both the cell front and rear, interacting with and being activated by several GTPases linked to contractility and protrusion. Analytical ultracentrifugation further clarified previous inconsistencies by showing that the affinity of FMNL2 for GTPases, particularly for Rac1, depends on the length of its N-terminal fragment and the presence of the full FH3 domain. Finally, we show that cells lacking Daam1/2 and mDia1/3 display pronounced cortical defects, including reduced rigidity, excessive blebbing, and delayed cytokinesis, demonstrating that these formins cooperate to preserve cortical architecture and function.

Cell migration frequently employs protrusions termed lamellipodia, constituting the prime model system for generation of branched actin filament networks. 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 Ena/VASP family polymerases in lamellipodial actin assembly. We show that Pfn counters Ena/VASP but promotes Arp2/3 complex activity, while Ena/VASP and CP mutually antagonize each other. While Pfn promotes Arp2/3 complex activity irrespective of Ena/VASP, sensitivity of CP to Pfn removal vanishes in the absence of Ena/VASP. Our findings establish Pfn as master regulator of Arp2/3 complex-dependent actin network formation, differentially regulating VASP and its antagonizer CP. Mathematical modeling of our data suggest Ena/VASP and CP to compete for recruitment to lamellipodial edges. Our work provides critical insights into the molecular logic of branched actin network assembly in protrusion and force generation.

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.

Tissue repair after myocardial infarction entails a vigorous angiogenic response that mitigates scarring and worsening of heart function. Angiogenesis in the infarct wound is guided by incompletely defined myeloid cell–endothelial cell interactions. Here, we identify the 75–amino acid microprotein BRICK1 (BRK1) as an indispensable driver of postinfarction angiogenesis in a mouse model of reperfused myocardial infarction. We show that BRK1 is preferentially expressed by myeloid cells and translocates to the extracellular space after myocardial infarction in mice and humans. As a subunit of the intracellular actin-regulatory WAVE complex, BRK1 was not previously known to function outside the cell. We find that BRK1 is not actively secreted but released during myeloid cell death. Cre- loxP –driven myeloid cell–selective genetic deletion of Brk1 or antibody-mediated neutralization of extracellular BRK1 impaired microvessel formation in the infarct border zone and resulted in severe postinfarction heart failure in mice. Conversely, treatment with recombinant BRK1 preserved heart function in infarcted mice. Mechanistically, BRK1 induced an angiogenic phenotype in human cardiac endothelial cells by signaling via the small GTPase Ras-related protein Rap-1 and mitogen-activated protein kinases 1 and 3 to promote retinoblastoma protein hyperphosphorylation and E2F transcription factor activation. BRK1 thus emerges as an angiogenic factor linking myeloid cell death to ischemic tissue repair, potentially enabling a protein-based therapy for myocardial infarction.

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.

Cortical formins, pivotal for the assembly of linear actin filaments beneath the membrane, exert only minor effects on unconfined cell migration of weakly and moderately adherent cells. However, their impact on migration and mechanostability of highly adherent cells remains poorly understood. Here, we demonstrate that loss of cortical actin filaments generated by the formins mDia1 and mDia3 drastically compromises cell migration and mechanics in highly adherent fibroblasts. Biophysical analysis of the mechanical properties of the mutant cells revealed a markedly softened cell cortex in the poorly adherent state. Unexpectedly, in the highly adherent state, associated with a hyperstretched morphology with exaggerated focal adhesions and prominent high-strain stress fibers, they exhibited even higher cortical tension compared to control. Notably, misguidance of intracellular forces, frequently accompanied by stress-fiber rupture, culminated in the formation of tension- and contractility-induced macroapertures, which was instantly followed by excessive lamellipodial protrusion at the periphery, providing critical insights into mechanotransduction of mechanically stressed and highly adherent cells.

In search of a more comprehensive structure-activity relationship (SAR) regarding the inhibitory effect of cytochalasin B (2) on actin polymerization, a virtual docking of 2 onto monomeric actin was conducted. This led to the identification of potentially important functional groups of 2 (i.e., the NH group of the isoindolone core (N-2) and the hydroxy groups at C-7 and C-20) involved in interactions with the residual amino acids of the binding pocket of actin. Chemical modifications of 2 at positions C-7, N-2, and C-20 led to derivatives 3-6, which were analyzed for their bioactivities. Compounds 3-5 exhibited reduced or no cytotoxicity in murine L929 fibroblasts compared to that of 2. Moreover, short- and long-term treatments of human osteosarcoma cells (U-2OS) with 3-6 affected the actin network to a variable extent, partially accompanied by the induction of multinucleation. Derivatives displaying acetylation at C-20 and N-2 were subjected to slow intracellular conversion to highly cytotoxic 2. Together, this study highlights the importance of the hydroxy group at C-7 and the NH function at N-2 for the potency of 2 on the inhibition of actin polymerization.

Summary: 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 β-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.

In search of a more comprehensive structure activity relationship (SAR) regarding the inhibitory effect of cytochalasin B (CB) on actin polymerization, a virtual docking of CB onto monomeric actin was conducted. This led to the identification of potentially important functional groups of CB (i.e. the NH group of the isoindolone core (N-2), and the hydroxyl groups at C-7 and C-20) involved in interactions with the residual amino acids of the binding pocket of actin. Chemical modifications of CB at positions C-7, N-2, and C-20 led to derivatives CB1-CB4, which were analyzed for their bioactivities. CB1-CB4 exhibited reduced or no cytotoxicity in murine L929 fibroblasts compared to CB. Moreover, short- and long-term treatments of human osteosarcoma cells (U-2OS) affected the actin network to variable extent, partially accompanied by the induction of multinucleation. Derivatives displaying acetylation at C-20 and N-2 were subject to slow intracellular conversion to highly cytotoxic CB. Together, this study highlights the importance of the hydroxy group at C-7 and the NH function at N-2 for CB potency on the inhibition of actin polymerization.