SCAR/WAVE is activated at mitosis and drives myosin-independent cytokinesis

Abstract

SummaryCell division requires the tight coordination of multiple cytoskeletal pathways. The best understood of these involves myosin-IIdependent constriction around the cell equator, but both Dictyostelium and mammalian cells also use a parallel, adhesion-dependent mechanism to generate furrows. We show that the actin nucleation factor SCAR/WAVE is strongly activated during Dictyostelium cytokinesis. This activation localises to large polar protrusions, driving separation of the daughter cells. This continues for 10 minutes after division before the daughter cells revert to normal random motility, indicating that this is a tightly regulated process. We demonstrate that SCAR activity is essential to drive myosin-II-independent cytokinesis, and stabilises the furrow, ensuring symmetrical division. SCAR is also responsible for the generation of MiDASes, mitosis-specific actin-rich adhesions. Loss of SCAR in both Dictyostelium and Drosophila leads to a similar mitotic phenotype, with severe mitotic blebbing, indicating conserved functionality. We also find that the microtubule end-binding protein EB1 is required to restrict SCAR localisation and direct migration. EB1-null cells also exhibit decreased adhesion during mitosis. Our data reveal a spindle-directed signalling pathway that regulates SCAR activity, migration and adhesion at mitosis.

Abi Is Required for Modulation and Stability but Not Localization or Activation of the SCAR/WAVE Complex

“…minally truncated Abi1 [⌬Ct 1 Abi]). The most distal C-terminal Abi2 sequence for which there are crystal structure data available (i.e., immediately before the start of the polyproline tail) was strikingly conserved (Fig.…”

“…For instance, during chemotaxis cells display an ability to detect and move along chemical gradients. Another example is during cytokinesis, where dividing cells establish opposing polarity and migrate apart from each other in a precisely coordinated manner so as to aid in the separation of the newly formed daughter cells (1). Directed migration requires the integrated activities of a multitude of proteins, ranging from sensory receptors and mediators of intracellular signaling to contractile actomyosin filaments, inducers of membrane curvature, and actin nucleators and their activators.…”

The use of Streptavidin Conjugates as Immunoblot Loading Controls and Mitochondrial Markers for use with Dictyostelium Discoideum

“…The single streptavidin-labeled band was extremely robust, and Dictyostelium cell pellets lysed with 150 mM NaCl, 10 mM Tris pH 7.5, 1 mM EGTA, 1 mM EDTA and either 1% NP40 (for SCAR blots) (4) or 0.1% SDS, 0.1% sodium deoxycholate, 50 mM DTT, and HALT protease inhibitors (Thermo-Scientific, IL, USA) (for cAR1 blots) gave identical results (Figure 1). Protein levels were then determined using Precision Red Advanced Protein Assay Reagent (Cytoskeleton, Inc., CO, USA) before boiling for 5 min in LDS loading buffer (Life Technologies), except for cAR1 samples, which were not boiled.…”

Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by single-molecule imaging

“…Imaging in other non-single-molecule modes shows that the SCAR/WAVE complex is recruited to the edge of the lamellipodium in fibroblasts (Stradal et al, 2001;Lai et al, 2008) and propagates in 'waves' of actin assembly at the leading edge of neutrophils and Dictyostelium (Weiner et al, 2007;King et al, 2010;Xiong et al, 2010). However, these imaging modes might obscure molecules whose dynamics differ from the global population.…”