Arf GTPase activates the WAVE regulatory complex through a distinct binding site.

Cross-talk between Rho- and Arf-family guanosine triphosphatases (GTPases) plays an important role in linking the actin cytoskeleton to membrane protrusions, organelle morphology, and vesicle trafficking. The central actin regulator, WAVE regulatory complex (WRC), integrates Rac1 (a Rho-family GTPase) and Arf signaling to promote Arp2/3-mediated actin polymerization in many processes, but how WRC senses Arf signaling is unknown. Here, we have reconstituted a direct interaction between Arf and WRC. This interaction is greatly enhanced by Rac1 binding to the D site of WRC. Arf1 binds to a previously unidentified, conserved surface on the Sra1 subunit of WRC, which, in turn, drives WRC activation using a mechanism distinct from that of Rac1. Mutating the Arf binding site abolishes Arf1-WRC interaction, disrupts Arf1-mediated WRC activation, and impairs lamellipodia formation and cell migration. This work uncovers a new mechanism underlying WRC activation and provides a mechanistic foundation for studying how WRC-mediated actin polymerization links Arf and Rac signaling in cells.

Lamellipodia-like actin networks in cells lacking WAVE regulatory complex.

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases activating WAVE regulatory complex (WRC) to drive Arp2/3 complex-dependent actin assembly. Previous genome editing studies in B16-F1 melanoma cells solidified the view of an essential, linear pathway employing the aforementioned components. Here, disruption of the WRC subunit Nap1 (encoded by Nckap1) and its paralog Hem1 (encoded by Nckap1l) followed by serum and growth factor stimulation, or active GTPase expression, revealed a pathway to formation of Arp2/3 complex-dependent lamellipodia-like structures (LLS) that requires both Rac and Cdc42 GTPases, but not WRC. These phenotypes were independent of the WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from Ena/VASP proteins, LLS contained all lamellipodial regulators tested, including cortactin (also known as CTTN), the Ena/VASP ligand lamellipodin (also known as RAPH1) and FMNL subfamily formins. Rac-dependent but WRC-independent actin remodeling could also be triggered in NIH 3T3 fibroblasts by growth factor (HGF) treatment or by gram-positive Listeria monocytogenes usurping HGF receptor signaling for host cell invasion. Taken together, our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery that operates without WRC and Ena/VASP.

Spire1 and Myosin Vc promote Ca2+-evoked externalization of von Willebrand factor in endothelial cells.

Weibel-Palade bodies (WPB) are endothelial cell-specific storage granules that regulate vascular hemostasis by releasing the platelet adhesion receptor von Willebrand factor (VWF) following stimulation. Fusion of WPB with the plasma membrane is accompanied by the formation of actin rings or coats that support the expulsion of large multimeric VWF fibers. However, factor(s) organizing these actin ring structures have remained elusive. We now identify the actin-binding proteins Spire1 and Myosin Vc (MyoVc) as cytosolic factors that associate with WPB and are involved in actin ring formation at WPB-plasma membrane fusion sites. We show that both, Spire1 and MyoVc localize only to mature WPB and that upon Ca2+ evoked exocytosis of WPB, Spire1 and MyoVc together with F-actin concentrate in ring-like structures at the fusion sites. Depletion of Spire1 or MyoVc reduces the number of these actin rings and decreases the amount of VWF externalized to the cell surface after histamine stimulation.

EPLIN-α and -ß Isoforms Modulate Endothelial Cell Dynamics through a Spatiotemporally Differentiated Interaction with Actin.

Actin-binding proteins are essential for linear and branched actin filament dynamics that control shape change, cell migration, and cell junction remodeling in vascular endothelium (endothelial cells [ECs]). The epithelial protein lost in neoplasm (EPLIN) is an actin-binding protein, expressed as EPLIN-α and EPLIN-ß by alternative promoters; however, the isoform-specific functions are not yet understood. Aortic compared to cava vein ECs and shear stress-exposed cultured ECs express increased EPLIN-ß levels that stabilize stress fibers. In contrast, EPLIN-α expression is increased in growing and migrating ECs, is targeted to membrane protrusions, and terminates their growth via interaction with the Arp2/3 complex. The data indicate that EPLIN-α controls protrusion dynamics while EPLIN-ß has an actin filament stabilizing role, which is consistent with FRAP analyses demonstrating a lower EPLIN-ß turnover rate compared to EPLIN-α. Together, EPLIN isoforms differentially control actin dynamics in ECs, essential in shear stress responses, cell migration, and barrier function.

Actin dynamics during Ca2+-dependent exocytosis of endothelial Weibel-Palade bodies.

Weibel-Palade bodies (WPBs) are specialized secretory organelles of endothelial cells that serve important functions in the response to inflammation and vascular injury. WPBs actively respond to different stimuli by regulated exocytosis leading to full or selective release of their contents. Cellular conditions and mechanisms that distinguish between these possibilities are only beginning to emerge. To address this we analyzed dynamic rearrangements of the actin cytoskeleton during histamine-stimulated, Ca2+-dependent WPB exocytosis. We show that most WPB fusion events are followed by a rapid release of von-Willebrand factor (VWF), the large WPB cargo, and that this occurs concomitant with a softening of the actin cortex by the recently described Ca2+-dependent actin reset (CaAR). However, a considerable fraction of WPB fusion events is characterized by a delayed release of VWF and observed after the CaAR reaction peak. These delayed VWF secretions are accompanied by an assembly of actin rings or coats around the WPB post-fusion structures and are also seen following direct elevation of intracellular Ca2+ by plasma membrane wounding. Actin ring/coat assembly at WPB post-fusion structures requires Rho GTPase activity and is significantly reduced upon expression of a dominant-active mutant of the formin INF2 that triggers a permanent CaAR peak-like sequestration of actin to the endoplasmic reticulum. These findings suggest that a rigid actin cortex correlates with a higher proportion of fused WPB which assemble actin rings/coats most likely required for efficient VWF expulsion and/or stabilization of a WPB post-fusion structure. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Endocytic Adaptor Protein Tollip Inhibits Canonical Wnt Signaling.

Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of ß-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of ß-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of ß-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive ß-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.