Cell motility is essential for many biological processes, including development and pathogenesis. Thus, themolecular mechanisms underlying this process have been intensively studied in many cell systems, forexample, leukocytes, amoeba and even bacteria. Intriguingly, bacteria are also able to move across solidsurfaces (gliding motility) like eukaryotic cells by a process that has remained largely mysterious. Theemergence of bacterial cell biology: the discovery that the bacterial cell also has a dynamic cytoskeleton andspecialized subcellular regions now provides new research angles to study the motility mechanism. Usingcell biology approaches, we previously suggested that the mechanism may be akin to acto-myosin-basedmotility in eukaryotic cells and proposed that bacterial focal adhesion complexes also power locomotion. Inthis project, we propose two complementary research axes to define both the mechanism and its spatialregulation in the cell at molecular resolution.Using the model motility bacterium Myxococcus xanthus, we first propose to develop a “toolbox” ofbiophysical and cell biology assays to analyze the motility process. Specifically, we will construct a TractionForce Microscopy assay designed to image the motility forces directly by live moving cells and usemicrofluidics to quantitate the secretion of a mucus that may participate directly in the motility process.These assays, combined with a newly developed laser trap system to visualize dynamic focal adhesions inthe cell envelope, will be instrumental not only to define new features of the motility process, but also tostudy the function of novel motility genes which may encode the components of the elusive motility engine.This way, we hope to establish the mechanism and structure function relationships within an entirely novelmotility machinery.In a second part, we propose to investigate the mechanism that controls a polarity switch, allowing M.xanthus cells to change their direction of movement. We have previously shown that dynamic motilityprotein pole-to-pole oscillations convert the initial leading cell pole into the lagging pole. Here, we proposethat like in a eukaryotic cells, a bacterial counterpart of small GTPases of the Ras superfamily, MglAcontrols the polarity cycle. To test this hypothesis, we will study both the MglA upstream regulation and theMglA downstream effectors. We thus hope to establish a model of dynamic polarity control in a bacterial