Regulation of focal adhesion complex assembly during the adventurous motility of Myxococcus xanthus

Les adhérences focales sont des assemblages couvrant l'enveloppe cellulaire qui connectent le substrat sous-jacent au cytosquelette intracellulaire, contribuant à la migration cellulaire. Chez les bactéries, l'assemblage de ces complexes d'adhésion focale (bFA) est contrôlée par la machinerie de motilité Agl-Glt connectée aux protéines de la plateforme cytoplasmique, MglA et AglZ, associées au cytosquelette bactérien. Chez Myxococcus xanthus, le fonctionnement de cette machinerie reste inexpliqué. Ici, nous révélons que la protéine membranaire GltJ du complexe Agl-Glt sert de lien entre la machinerie de motilité et la plateforme cytoplasmique pour son activation. En combinant la spectroscopie RMN pour les études de structure et d'interaction aux tests de motilité et au suivi de la dynamique des bFA par microscopie TIRF, nous montrons que la région N-terminale de GltJ interagit avec MglA et AglZ, entraînant ainsi l'assemblage des bFA. De plus, nous révélons que GltJ agit comme un switch moléculaire basé sur l'interaction intramoléculaire du domaine ZnR de GltJ avec la région désordonnée de GltJ. Cette interaction ne peut être libérée que lors de la liaison à MglA au niveau de l'assemblage du bFA au pôle avant de la cellule. Ensuite, il a été démontré que le domaine ZnR de GltJ libéré recrute MglB au pôle arrière de la cellule, déclenchant ainsi le désassemblage du bFA. Les domaines N-terminaux de GltJ émergent ainsi comme une nouvelle classe de commutateurs moléculaires qui agissent de concert avec les GTPases pour contrôler la dynamique des bFA.Focal adhesions (FA) are assemblies spanning the cell envelope to connect the underlying substrate to the cytoskeleton, transmitting forces on cytoskeleton into forces contributing to cell migration in a highly regulated manner. In bacteria, FAs assembly is mediated by the Agl-Glt motility machinery, however how this machinery is connected to the cytoplasmic platform proteins, MglA and AglZ, associated to the bacterial cytoskeleton remains unknown. Here, we identify the GltJ membrane protein as the connecting link between the motility machinery and the cytoplasmic platform. Combining NMR spectroscopy for structure and interaction studies to motility assays and bacterial FA (bFA) dynamics monitoring by TIRF microscopy, we show that GltJ N-terminal region interacts with MglA and AglZ, thus driving bFA assembly. Moreover, we show that GltJ acts as a molecular switch based on intramolecular interaction of ZnR-GltJ domain with the disordered linker-GltJ region, interaction that can be released upon MglA binding at the leading cell pole promoting bFA assembly. Then, released ZnR-GltJ is shown to recruit MglB at the lagging cell pole thus triggering bFA disassembly. GltJ N-terminal domains thus emerge as a new class of molecular switches that act in concert with GTPases to control bFA dynamics

FrzS acts as a polar beacon to recruit SgmX , a central activator of type IV pili during Myxococcus xanthus motility

International audienceIn rod-shaped bacteria, type IV pili (Tfp) promote twitching motility by assembling and retracting at the cell pole. In Myxococcus xanthus, a bacterium that moves in highly coordinated cell groups, Tfp are activated by a polar activator protein, SgmX. However, while it is known that the Ras-like protein MglA is required for unipolar targeting, how SgmX accesses the cell pole to activate Tfp is unknown. Here, we demonstrate that a polar beacon protein, FrzS, recruits SgmX at the cell pole. We identified two main functional domains, including a Tfp-activating domain and a polarbinding domain. Within the latter, we show that the direct binding of MglA-GTP unveils a hidden motif that binds directly to the FrzS N-terminal response regulator (CheY). Structural analyses reveal that this binding occurs through a novel binding interface for response regulator domains. In conclusion, the findings unveil the protein interaction network leading to the spatial activation of Tfp at the cell pole. This tripartite system is at the root of complex collective behaviours in this predatory bacterium

1H, 13C and 15N chemical shift assignments of the ZnR and GYF cytoplasmic domains of the GltJ protein from Myxococcus xanthus

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A molecular switch controls assembly of bacterial focal adhesions

Cell motility universally relies on spatial regulation of focal adhesion complexes (FAs) connecting the substrate to cellular motors. In bacterial FAs, the Adventurous gliding motility machinery (Agl-Glt) assembles at the leading cell pole following a Mutual gliding-motility protein (MglA)-guanosine 5'-triphosphate (GTP) gradient along the cell axis. Here, we show that GltJ, a machinery membrane protein, contains cytosolic motifs binding MglA-GTP and AglZ and recruiting the MreB cytoskeleton to initiate movement toward the lagging cell pole. In addition, MglA-GTP binding triggers a conformational shift in an adjacent GltJ zinc-finger domain, facilitating MglB recruitment near the lagging pole. This prompts GTP hydrolysis by MglA, leading to complex disassembly. The GltJ switch thus serves as a sensor for the MglA-GTP gradient, controlling FA activity spatially