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Leep1 interacts with PIP3 and the Scar/WAVE complex to regulate cell migration and macropinocytosis.

ABSTRACT: Polarity is essential for diverse functions in many cell types. Establishing polarity requires targeting a network of specific signaling and cytoskeleton molecules to different subregions of the cell, yet the full complement of polarity regulators and how their activities are integrated over space and time to form morphologically and functionally distinct domains remain to be uncovered. Here, by using the model system Dictyostelium and exploiting the characteristic chemoattractant-stimulated translocation of polarly distributed molecules, we developed a proteomic screening approach, through which we identified a leucine-rich repeat domain-containing protein we named Leep1 as a novel polarity regulator. We combined imaging, biochemical, and phenotypic analyses to demonstrate that Leep1 localizes selectively at the leading edge of cells by binding to PIP3, where it modulates pseudopod and macropinocytic cup dynamics by negatively regulating the Scar/WAVE complex. The spatiotemporal coordination of PIP3 signaling, Leep1, and the Scar/WAVE complex provides a cellular mechanism for organizing protrusive structures at the leading edge.

SUBMITTER: Yang Y

PROVIDER: S-EPMC8127007 | biostudies-literature |

REPOSITORIES: biostudies-literature

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AKT and SGK kinases regulate cell migration by altering Scar/WAVE complex activation and Arp2/3 complex recruitment.

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Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo.

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Extracellular Signalling Modulates Scar/WAVE Complex Activity through Abi Phosphorylation.

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Project description:The WAVE/SCAR complex promotes actin nucleation through the Arp2/3 complex, in response to Rac signaling. We show that loss of WVE-1/GEX-1, the only C. elegans WAVE/SCAR homolog, by genetic mutation or by RNAi, has the same phenotype as loss of GEX-2/Sra1/p140/PIR121, GEX-3/NAP1/HEM2/KETTE, or ABI-1/ABI, the three other components of the C. elegans WAVE/SCAR complex. We find that the entire WAVE/SCAR complex promotes actin-dependent events at different times and in different tissues during development. During C. elegans embryogenesis loss of CED-10/Rac1, WAVE/SCAR complex components, or Arp2/3 blocks epidermal cell migrations despite correct epidermal cell differentiation. 4D movies show that this failure occurs due to decreased membrane dynamics in specific epidermal cells. Unlike myoblasts in Drosophila, epidermal cell fusions in C. elegans can occur in the absence of WAVE/SCAR or Arp2/3. Instead we find that subcellular enrichment of F-actin in epithelial tissues requires the Rac-WAVE/SCAR-Arp2/3 pathway. Intriguingly, we find that at the same stage of development both F-actin and WAVE/SCAR proteins are enriched apically in one epithelial tissue and basolaterally in another. We propose that temporally and spatially regulated actin nucleation by the Rac-WAVE/SCAR-Arp2/3 pathway is required for epithelial cell organization and movements during morphogenesis.

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High-resolution X-ray structure of the trimeric Scar/WAVE-complex precursor Brk1.

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Structural Basis of CYRI-B Direct Competition with Scar/WAVE Complex for Rac1.

Project description:Rac1 is a major regulator of actin dynamics, with GTP-bound Rac1 promoting actin assembly via the Scar/WAVE complex. CYRI competes with Scar/WAVE for interaction with Rac1 in a feedback loop regulating actin dynamics. Here, we reveal the nature of the CYRI-Rac1 interaction, through crystal structures of CYRI-B lacking the N-terminal helix (CYRI-BΔN) and the CYRI-BΔN:Rac1Q61L complex, providing the molecular basis for CYRI-B regulation of the Scar/WAVE complex. We reveal CYRI-B as having two subdomains - an N-terminal Rac1 binding subdomain with a unique Rac1-effector interface and a C-terminal Ratchet subdomain that undergoes conformational changes induced by Rac1 binding. Finally, we show that the CYRI protein family, CYRI-A and CYRI-B can produce an autoinhibited hetero- or homodimers, adding an additional layer of regulation to Rac1 signaling.

| S-EPMC7955166 | biostudies-literature

Endogenous ARF6 interacts with Rac1 upon angiotensin II stimulation to regulate membrane ruffling and cell migration.

Project description:ARF6 and Rac1 are small GTPases known to regulate remodelling of the actin cytoskeleton. Here, we demonstrate that these monomeric G proteins are sequentially activated when HEK 293 cells expressing the angiotensin type 1 receptor (AT(1)R) are stimulated with angiotensin II (Ang II). After receptor activation, ARF6 and Rac1 transiently form a complex. Their association is, at least in part, direct and dependent on the nature of the nucleotide bound to both small G proteins. ARF6-GTP preferentially interacts with Rac1-GDP. AT(1)R expressing HEK293 cells ruffle, form membrane protrusions, and migrate in response to agonist treatment. ARF6, but not ARF1, depletion using small interfering RNAs recapitulates the ruffling and migratory phenotype observed after Ang II treatment. These results suggest that ARF6 depletion or Ang II treatment are functionally equivalent and point to a role for endogenous ARF6 as an inhibitor of Rac1 activity. Taken together, our findings reveal a novel function of endogenously expressed ARF6 and demonstrate that by interacting with Rac1, this small GTPase is a central regulator of the signaling pathways leading to actin remodeling.

| S-EPMC1783798 | biostudies-literature

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