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  • br Results br Discussion The actin cytoskeleton is a

    2024-07-09


    Results
    Discussion The adenosine kinase cytoskeleton is a highly attractive target for many bacterial toxins, owing to its role in activation and locomotion of immune cells, secretion of humoral response factors, and maintenance of protective barriers at the cellular (sub-membrane cytoskeleton) and organ (cell-cell and cell-matrix contacts) levels. Furthermore, due to the low homology between eukaryotic actin and its bacterial counterparts, actin-targeting toxins are highly specific against hosts but benign for pathogens. On the other hand, the actin cytoskeleton would not appear to be an easy target due to actin’s high abundance and the cells’ ability to promptly adjust its levels in response to environmental [28] and pathogenic cues [29]. In this study, we combined the precision and predictive power of biochemical assays, substantiated by mathematical modeling, with the quantitative analysis of the actin cytoskeleton dynamics in living cells achieved via SiMS microscopy to characterize the pathological mechanisms of the ACD family of toxins. These approaches allowed us to discover that the distortion of the actin cytoskeleton by the ACD-produced oligomers is more profound and multifaceted than previously appreciated. Particularly, the dynamics of mDia1 formin (Figure 3) was severely inhibited at concentrations of the oligomers undetectable by western blotting (i.e., estimated to be below 1% of total actin; Figure 1B) within 30 min of toxin addition to the medium, long before any morphological changes in the affected cells can be detected (Figure 1A). In addition to formins, several families of actin assembly factors containing WH2 domains were also inhibited by the nanomolar range of oligomer concentrations. The emergence of cellular effects for particular proteins correlated well with the in vitro data from TIRFM and bulk actin polymerization assays. Thus, cellular dynamics of mDia1 formin and Ena/VASP, which showed the highest apparent affinities for oligomers in functional assays (appKd 2–5 nM for formins [13] and 3–6 nM for Ena/VASP; Figure 4), were inhibited at earlier stages than that of the Arp2/3 complex, activation of which by N-WASP was inhibited in vitro with lower efficiencies (appKd in 10–23 nM range; Figure 2O). This lower potency likely stems from a fewer (than in formins and Ena/VASP) number of G-actin binding domains and, possibly, a less favorable geometry for oligomer binding to NPFs. Of note, depending on the involvement in a particular cellular process (i.e., remodeling of cytoplasmic [30], endosomal [31], autophagosomal [32, 33], Golgi, and endoplasmic reticulum [ER] membranes [34, 35] or contribution to cytoplasmic streaming [36], etc.), the Arp2/3 complex can be activated by different NPFs, of which only two were tested in the present study: VCA fragment of N-WASP in the in vitro assays (Figures 2G–2O) and WAVE in the cellular context (Figures 2D–2F). Given that the oligomers interact with NPFs rather than with the Arp2/3 complex (Figure 2H), the inhibition efficiency of the complex-controlled actin dynamics would likely vary by NPF, proportional to the number of G-actin binding domains they comprise. Thus, junction-mediating and regulatory protein (JMY), an NPF that regulates transcription in the nucleus and vesicle trafficking between the ER and trans-Golgi network [37, 38, 39], with its three WH2 domains, is likely to be inhibited more efficiently than N-WASP and WASP homolog associated with actin, Golgi membranes, and microtubules (WHAMM) (each containing two WH2 domains), which regulate endocytosis [40] and autophagosome formation [32], respectively. Furthermore, all these NPFs are likely to be more efficiently inhibited than single-WH2-domain NPFs WASP, WAVE, and WASP homolog complex (WASH), which are involved in endocytosis, cell migration, and endosomal trafficking (recently reviewed in [30]). Notably, inhibition of many of these processes would be beneficial for pathogenic bacteria to interrupt migration, phagocytosis, and activation of immune cells; disrupt epithelial contacts; inhibit Golgi-dependent maturation of granules in immune effector cells and their release; and inhibit autophagy.