Biology subjects

Pokrant, T.

Publications and source records attributed to Pokrant, T..

3 recordsLinked to original sources

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↗

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 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↗