To better understand the pathophysiology of ASDs we

To better understand the pathophysiology of ASDs, we would need comprehensive information on a) the functions of ASD-associated proteins in the brain, b) how mutations affect the expression level and function of these proteins, c) how mutations affect their function in neurons, and d) how changed neuronal function impacts the circuits and behavior. We currently have a good overview of the different pathways affected in ASD and the next step will be to characterize the proteins encoded by these genes, as well as the effects of mutations. The recent CAMK2A study (Stephenson et al., 2017) provides a good example on how we should proceed with each protein and mutation.
Authors contributions
Funding M. Joensuu is supported by The Academy of Finland Postdoctoral Research Fellowship (298124) and P. Hotulainen by Instrumentarium Foundation senior researcher fellowship.
Introduction The tbtu 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 [1]. Due to the efficiency of immune barriers [2], delivery of protein toxins to host cells is heavily suppressed, which applies a strong evolutionary pressure on toxin efficiency. Toxicity amplification is often achieved by targeting essential, low abundant host proteins in signaling or neurotransmission cascades [3, 4, 5, 6], whereas actin, a highly abundant structural protein, is a rare exception to this rule. The actin cross-linking domain (ACD), produced by gram-negative Vibrio, Aeromonas, and other species, is one of the toxins that utilize actin monomers as a substrate [7]. ACD is delivered to the host cytoplasm as one of several effectors of the multifunctional auto-processing repeats-in-toxin (MARTX) toxins [8] or as a single effector domain fused to valine–glycine repeat protein G1 (VgrG1) toxin of the type VI secretion system [9]. Once inside a host cell, ACD catalyzes the covalent cross-linking of actin monomers into various-length oligomers through a formation of amide bonds between the side chains of lysine-50 and glutamate-270 [10, 11]. The oligomers fail to polymerize, and their bulk accumulation eventually leads to cell rounding [12]. However, to be effective, this toxicity mechanism would require high doses of the toxin, reducing its value for the micro-organisms. Instead, an unusual “gain-of-function” mechanism of toxicity amplification was recently proposed whereby the actin oligomers, while showing negligible effects on spontaneous actin dynamics, act as potent secondary toxins that bind to and directly inhibit formins [13]. Formins function as homodimers that nucleate actin filaments and accelerate elongation of filament barbed ends while also protecting them from the inhibitory activity of capping proteins [14]. Formins contain conserved formin-homology domains 1 and 2 (FH1 and FH2) capable of simultaneously interacting with actin filament ends and several actin-profilin complexes. Recently, we demonstrated that, by providing a multivalent platform for high-affinity interaction with FH1 and FH2 domains, the ACD-cross-linked actin oligomers potently block nucleation and elongation activities of formins in vitro, which correlated with a distortion of the host cytoskeleton in living cells [13]. Notably, a diverse pool of actin-organizing proteins involved in nucleation, elongation, severing, branching, and bundling of actin filaments contain G-actin-binding Wiskott-Aldrich syndrome homology 2 (WH2) motifs [15] that are arranged in tandem or closely positioned upon functional oligomerization, i.e., they share the property targeted by the ACD-cross-linked oligomers in case of formins. We employed single-molecule speckle (SiMS) live-cell microscopy to demonstrate that low doses of the oligomers effectively shut down the dynamics of formins and several families of WH2-containing proteins. Specifically, the oligomers strongly inhibit the dynamics of mDia1, Ena/vasodilator-stimulated phosphoprotein (VASP), Spire, and the Arp2/3 complex nucleation-promoting factors (NPFs) in lamellipodia and filopodia, leading to disorganization of adhesion contacts and arrest of actin dynamics at the leading edge of affected cells. Total internal reflection fluorescence microscopy (TIRFM), bulk actin polymerization assays, and modeling of actin polymerization with sets of ordinary differential equations supported the experimental data and revealed additional details on the proposed mechanisms of oligomer-induced inhibition of the Arp2/3 complex, Ena/VASP, and Spire. Therefore, we report that ACD converts actin into toxic covalent oligomers that are universally poisonous toward numerous tandem and oligomeric actin organizers.