WHIMP links the actin nucleation machinery to Src-family kinase signaling during protrusion and motility

ABSTRACTCell motility is governed by cooperation between the Arp2/3 complex and nucleation factors from the Wiskott-Aldrich Syndrome Protein (WASP) family, which together assemble actin filament networks to drive membrane protrusion. Here we identify WHIMP (WAVE Homology In Membrane Protrusions) as a new member of the WASP family. The Whimp gene is encoded on the X-chromosome of multiple animals, including mice. Murine WHIMP promotes Arp2/3-dependent actin assembly, but is less potent than other nucleation factors. Nevertheless, WHIMP-mediated Arp2/3 activation enhances both plasma membrane ruffling and wound healing migration, whereas WHIMP depletion impairs protrusion and slows motility. WHIMP expression also increases Src-family kinase activity, and WHIMP-induced ruffles contain the additional nucleation factors WAVE1, WAVE2, and N-WASP, but not JMY or WASH. Perturbing the function of Src-family kinases, WAVE proteins, or Arp2/3 complex inhibits WHIMP-driven ruffling. These results suggest that WHIMP-mediated actin assembly plays a direct role in membrane protrusion, but also results in feedback control of tyrosine kinase signaling to modulate the activation of multiple WASP-family proteins.AUTHOR SUMMARYThe actin cytoskeleton is a collection of protein polymers that assemble and disassemble within cells at specific times and locations. Sophisticated cytoskeletal regulators called nucleation factors ensure that actin polymerizes when and where it is needed, and most nucleation factors are members of the Wiskott-Aldrich Syndrome Protein (WASP) family. Several of the 8 known WASP-family proteins function in cell motility, but how the different factors collaborate with one another is not well understood. In this study, we identified WHIMP, a new WASP-family member which is encoded on the X chromosome of a variety of animals. In mouse cells, WHIMP enhances cell motility by assembling actin filaments that push the cell membrane forward. Unexpectedly, WHIMP also activates tyrosine kinase enzymes, proteins that stimulate multiple WASP-family members during motility. Our results open new avenues of research into how nucleation factors cooperate during movement and how the molecular activities that underlie motility differ in distinct cell types and organisms.

Genetic dissection of active forgetting in labile and consolidated memories in Drosophila

Different memory components are forgotten through distinct molecular mechanisms. In Drosophila, the activation of 2 Rho GTPases (Rac1 and Cdc42), respectively, underlies the forgetting of an early labile memory (anesthesia-sensitive memory, ASM) and a form of consolidated memory (anesthesia-resistant memory, ARM). Here, we dissected the molecular mechanisms that tie Rac1 and Cdc42 to the different types of memory forgetting. We found that 2 WASP family proteins, SCAR/WAVE and WASp, act downstream of Rac1 and Cdc42 separately to regulate ASM and ARM forgetting in mushroom body neurons. Arp2/3 complex, which organizes branched actin polymerization, is a canonical downstream effector of WASP family proteins. However, we found that Arp2/3 complex is required in Cdc42/WASp-mediated ARM forgetting but not in Rac1/SCAR-mediated ASM forgetting. Instead, we identified that Rac1/SCAR may function with formin Diaphanous (Dia), a nucleator that facilitates linear actin polymerization, in ASM forgetting. The present study, complementing the previously identified Rac1/cofilin pathway that regulates actin depolymerization, suggests that Rho GTPases regulate forgetting by recruiting both actin polymerization and depolymerization pathways. Moreover, Rac1 and Cdc42 may regulate different types of memory forgetting by tapping into different actin polymerization mechanisms.

Identification of Wiskott-Aldrich syndrome protein (WASP) binding sites on the branched actin filament nucleator Arp2/3 complex

Arp2/3 complex nucleates branched actin filaments important for cellular motility and endocytosis. WASP family proteins are Arp2/3 complex activators that play multiple roles in branching nucleation, but little is known about the structural bases of these WASP functions, owing to an incomplete understanding of how WASP binds Arp2/3 complex. Recent data show WASP binds two sites, and biochemical and structural studies led to models in which the WASP C segment engages the barbed ends of the Arp3 and Arp2 subunits while the WASP A segment binds the back side of the complex on Arp3. However, electron microscopy reconstructions showed density for WASP inconsistent with these models on the opposite (front) side of Arp2/3 complex. Here we use chemical cross-linking and mass spectrometry (XL-MS) along with computational docking and structure-based mutational analysis to map the two WASP binding sites on the complex. Our data corroborate the barbed end and back side binding models and show one WASP binding site on Arp3, on the back side of the complex, and a second site on the bottom of the complex, spanning Arp2 and ARPC1. The XL-MS-identified cross-links rule out the front side binding model and show that the A segment of WASP binds along the bottom side of the ARPC1 subunit, instead of at the Arp2/ARPC1 interface, as suggested by FRET experiments. The identified binding sites support the Arp3 tail release model to explain WASP-mediated activating conformational changes in Arp2/3 complex and provide insight into the roles of WASP in branching nucleation.

WASP family proteins and formins compete in pseudopod- and bleb-based migration

Actin pseudopods induced by SCAR/WAVE drive normal migration and chemotaxis in eukaryotic cells. Cells can also migrate using blebs, in which the edge is driven forward by hydrostatic pressure instead of actin. In Dictyostelium discoideum, loss of SCAR is compensated by WASP moving to the leading edge to generate morphologically normal pseudopods. Here we use an inducible double knockout to show that cells lacking both SCAR and WASP are unable to grow, make pseudopods or, unexpectedly, migrate using blebs. Remarkably, amounts and dynamics of actin polymerization are normal. Pseudopods are replaced in double SCAR/WASP mutants by aberrant filopods, induced by the formin dDia2. Further disruption of the gene for dDia2 restores cells’ ability to initiate blebs and thus migrate, though pseudopods are still lost. Triple knockout cells still contain near-normal F-actin levels. This work shows that SCAR, WASP, and dDia2 compete for actin. Loss of SCAR and WASP causes excessive dDia2 activity, maintaining F-actin levels but blocking pseudopod and bleb formation and migration.

Systematic analysis of the molecular architecture of endocytosis reveals a nanoscale actin nucleation template that drives efficient vesicle formation

Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially-ordered recruitment according to function. WASP family proteins form a circular nano-scale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template creates sufficient force for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.