In accordance with previous observations , EGFP-WAVE, a representative type I NPF (Figure 2D), was localized to filopodia tips and moved along the lamellipodia edge (Movie S3). GST-N-WASP-VCA-activated Arp2/3 complex (single-color TIRFM experiments), Related to Figure 2J Movies show time-lapse images of polymerization of Alexa 488-actin (1.5 M) mixed with the Arp2/3 complex (20 nM) and GST-N-WASP-VCA (40 nM) in the absence (left) or presence (right) of GSK3145095 80 nM ACD-cross-linked actin oligomers. NIHMS958177-supplement-4.avi (19M) GUID:?43034F05-931E-4C93-B52D-450376308BF6 5: Movie S5. Directional movement of mDia1 formin in living cells is stalled at early points of ACD treatment, Related to Figure 3 A multi stack montage of time-lapse images of a peripheral region of an individual XTC cell expressing low levels of EGFP-mDia1-N3 and treated with either active ACD (upper row) or inactive ACD (lower row). NIHMS958177-supplement-5.avi (24M) GUID:?F5700436-47BC-49CB-B65A-199192A1487B 6: Movie S6. The leading edge dynamics in EGFP-VASP transfected cells is inhibited by ACD, Related to Figure 4B,C A multi stack montage of time-lapse images of a peripheral region of an individual XTC cell expressing low levels of EGFP-VASP and treated with either active ACD (upper row) or inactive ACD (lower row). NIHMS958177-supplement-6.avi (18M) GUID:?A26812F9-C61E-4E1F-B9C9-FAB73556DE77 7: Movie S7. Actin oligomers cause long pauses in elongation of Ena-bound, but not Ena-free filaments (single-molecule dual-color TIRFM experiments), Related to Figure 4FCI Movies show time-lapse images of polymerization of 1 1.5 M Alexa-488-actin (green) in the presence of 0.5 nM SNAP-EnaL (red), 3 M chickadee, and 5 nM actin oligomers. Open arrowheads denote unbound barbed ends, green filled arrowheads show growing Ena-bound ends (left panel), and a red filled arrowhead is for a stopped Ena-bound end (right panel). NIHMS958177-supplement-7.avi (3.6M) GUID:?22BE9D71-5CA5-4495-9013-615D786B9377 8: Movie S8. Dynamics of EGFP-Nt-Spire in living cells is impaired by ACD treatment. Related to Figure 5 A multi stack montage of time-lapse images of a peripheral region of an individual XTC cell expressing low levels of EGFP-Nt-Spire and treated with either active ACD (upper row) or inactive ACD (lower row). NIHMS958177-supplement-8.avi (11M) GUID:?A7291F4F-1E41-4317-A0BE-3E27CB44D1CD 9. NIHMS958177-supplement-9.pdf (1.0M) GUID:?994CB1B3-BFAB-4FAD-A927-A84FF883C6C1 SUMMARY Delivery of bacterial toxins to host cells is hindered by host protective barriers. This obstruction dictates a remarkable efficiency of toxins, a single copy of which may kill a host cell. Efficiency of actin-targeting toxins is further hampered by an overwhelming abundance of their target. The actin cross-linking domain (ACD) toxins of species and related bacterial genera catalyze the formation of GSK3145095 covalently cross-linked actin oligomers. Recently, we reported that the ACD toxicity can be amplified via a multivalent inhibitory association of actin oligomers with actin assembly factors formins, suggesting that the oligomers may act as secondary toxins. Importantly, many proteins involved in nucleation, elongation, severing, branching, and bundling of actin filaments contain G-actin-binding WASP homology motifs 2 (WH2) organized in tandems and, therefore, may act as a multivalent platform for high-affinity interaction with the ACD-cross-linked actin oligomers. Using live-cell single-molecule speckle (SiMS) microscopy, TIRF microscopy, Rabbit Polyclonal to DOCK1 and actin polymerization assays, we show that, in addition to formins, the oligomers bind with high affinity and potently inhibit several families of actin assembly factors: Ena/VASP, Spire, and the Arp2/3 complex, both and in live cells. As a result, ACD blocks the actin retrograde flow and membrane dynamics, and disrupts association of Ena/VASP with adhesion complexes. This study defines ACD as a universal inhibitor of tandem-organized G-actin binding proteins that overcomes the abundance of actin by redirecting the toxicity cascade towards less abundant targets and thus leading to profound disorganization of the actin cytoskeleton and disruption of actin-dependent cellular functions. eTOC Blurb The shared ability of actin assembly factors to bind several actin molecules aids actin filament nucleation and growth. Kudryashova, Heisler, et al. show that bacterial toxin ACD targets this common property by producing covalent actin oligomers, which potently inhibit many actin assembly factors leading GSK3145095 to disruption of cellular actin dynamics. INTRODUCTION The actin cytoskeleton plays numerous vital roles in innate and adaptive immune responses, and as such, represents a common and attractive target for microbial toxins. Despite the numerous toxins that target the actin cytoskeleton, few of them modify actin molecules directly . Due to the efficiency of immune barriers , delivery of protein toxins to host cells is heavily suppressed, which GSK3145095 applies a strong evolutionary pressure on toxin efficiency. Toxicity amplification is often achieved by targeting essential, low abundant host proteins in signaling.