Alphée Michelot

Clathrin-mediated endocytosis is a conserved eukaryotic trafficking process where an Arp2/3 complex–nucleated branched actin network provides force for vesicle formation. The mechanisms that initiate endocytic actin assembly are incompletely understood. In the fission yeast, Schizosaccharomyces pombe, actin assembly is initiated by Dip1, an Arp2/3 complex activator. In the budding yeast, Saccharomyces cerevisiae, the initiation of actin assembly has remained a mystery. Here, we show that S. cerevisiae Ldb17, the homolog of Dip1, functions as an on-switch for endocytic actin assembly. Unexpectedly, the regulation of Ldb17 is more complicated than that of constitutively active Dip1. Ldb17 is controlled by a coat protein, Sla1, via separate recruitment and activation steps. This regulation was likely lost in the S. pombe lineage, and this simplification may be related to other changes in actin assembly between these species. Our findings add a key missing piece in the understanding of endocytosis in S. cerevisiae and reveal an intriguing evolutionary tinkering of the actin on-switch.

In Myxococcus xanthus, a predatory soil bacterium, cell polarity and motility are regulated by MglA (a small GTPase) and its regulators, MglB (a potential GAP) and RomRX (a previously proposed GEF), but their precise roles remain unclear. Using fluorescent nucleotides suitable for fluorescence anisotropy measurements, we show that RomRX does not function as a GEF but instead acts as an effector binding MglA_GTP. We further find, using an enzymatic model, that MglB exhibits both GAP and GEF properties depending on the local MglA_GDP concentration. Additionally, RomRX, specifically RomR, effectively dissociates the MglA-MglB complex by sequestering MglB. This low-affinity interaction permits GAP activity while still partitioning the proteins in vivo, as shown by a biomimetic oil-emulsion droplet assay in a cell-sized system. A minimal mathematical model incorporating these interactions and previous findings recapitulates how dynamic interactions between these three players form an invertible polarity axis in the M. xanthus cell.

A dynamic actin cytoskeleton, characterized by rapid filament turnover, is essential for driving intracellular transport and cellular movement. Despite extensive study, the contributions of many actin-binding proteins (ABPs) that regulate the different steps of the actin turnover cycle (filament assembly, disassembly and recycling into polymerizable actin monomers) remain ill-defined. Here, we introduce novel sensitive in vitro assays to quantitatively assess how ABPs catalyze actin turnover. By accurately measuring nucleotide exchange dynamics and ATP consumption, these assays enable robust characterization of ABP activity across broad concentration ranges. Using these methods and modeling of these reactions, we systematically examined the contributions of five conserved regulators, both individually and in combination, and identified conditions that maximize turnover efficiency. We also determined that increasing F-actin concentration to cellular levels affects ABP activity. Finally, we demonstrated that rapid actin turnover is preserved during encapsulation in cell-sized vesicles using the cDICE method. Together, these advances provide versatile tools and new insights into actin cytoskeletal dynamics under physiologically relevant conditions.

Clathrin-mediated endocytosis is a conserved eukaryotic trafficking process where an Arp2/3 complex nucleated branched actin network provides force for vesicle formation. The mechanisms that initiate endocytic actin assembly are incompletely understood. In the fission yeast, Schizosaccharomyces pombe , actin assembly is initiated by Dip1, an Arp2/3 activator. In the budding yeast, Saccharomyces cerevisiae, the initiation of actin assembly has remained a mystery. Here we show that S. cerevisiae Ldb17, the homolog of Dip1, functions as an on-switch for endocytic actin assembly. Unexpectedly, the regulation of Ldb17 is more complicated than that of constitutively active Dip1. Ldb17 is controlled by a coat protein, Sla1, via separate recruitment and activation steps. This regulation was likely lost in the S. pombe lineage and this simplification may be related to other changes in actin assembly between these species. Our findings add a key missing piece in the understanding of endocytosis in S. cerevisiae and reveal an intriguing evolutionary tinkering of the actin on-switch.

In Myxococcus xanthus , cell polarity and motility are regulated by the small GTPase MglA and its associated regulators, MglB, a potential GTPase Activating Protein and the RomRX complex, a potential Guanine nucleotide Exchange Factor. However, recent results have questioned the exact function of these regulators. Using a new type of fluorescent nucleotides for fluorescence-based anisotropy, we first demonstrate that RomRX does not function as a GEF but as an effector that binds strictly to MglA _GTP . Secondly, based on an enzymology model, we found that MglB functions as a weak catalyst, unlike typical GAPs, and requires additional factors to dissociate from MglA. We demonstrate that RomRX, and specifically RomR, effectively competes with MglA by sequestering MglB. This low affinity interaction is still permissive for GAP activity but sufficient to partition these proteins in vivo as indicated by a biomimetic assay in oil emulsion droplets reconstituting these interactions in a cell-sized environment. These results suggest a new model for cell polarity where RomRX exerts a dual function, targeting MglA _GTP to the pole via its effector function and preventing its accumulation at the lagging pole by destabilizing the MglA-MglB complex.

In a recent study, Wang et al. (https://doi.org/10.1083/jcb.202206074) demonstrate that subtle differences between two ADF/cofilin isoforms allow fine spatial regulation of the actin cytoskeleton in pollen tubes. This article illustrates how two similar proteins have progressively evolved to adapt their localization and activity according to the cellular environment.

Correction for ‘Non-linear elastic properties of actin patches to partially rescue yeast endocytosis efficiency in the absence of the cross-linker Sac6’ by Belbahri Reda et al., Soft Matter, 2022, 18, 1479–1488, https://doi.org/10.1039/D1SM01437D.

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