Project Summary/Abstract Proper mitotic spindle positioning and orientation of the cell division plane is imperative for tissue morphogenesis, development, and determination of cell fate. When the spindle is mislocalized or the division plane is askew, defects impacting the genetic content of daughter cells, tissue architecture, and development of tumors may arise. In vertebrates, cytoplasmic dynein and its cortically localized regulators, such as Leu/Gly/Asn-repeat containing protein (LGN) anchor the spindle and provide the mechanical force needed to move the spindle into its proper position and orientation. The location of this spindle positioning machinery is, in turn, controlled by a variety of factors including polarity proteins, cell-cell junctions, or both, depending on the cellular context. In addition, studies going back more than a century have identified the so-called “default” spindle positioning mechanism, wherein the spindle spontaneously positions itself parallel to the longest axis of the cell and perpendicular to its shortest axis. In the absence of any other information, this default mode of spindle placement ensures formation of two equally-sized daughter cells. The small GTPase, Cdc42, has been repeatedly implicated as an upstream participant in spindle orientation in a variety of organisms and tissues, based largely on knockdown or knockout approaches. However, the means by which Cdc42 controls spindle positioning is poorly understood, in part because it can impact many processes known to be involved in spindle positioning including cell polarity, cell-cell junction formation, and cell-substrate adhesion formation. Further, almost nothing is known about Cdc42 dynamics during spindle positioning. Our preliminary data show that Cdc42 is present in propagating cortical waves during spindle positioning in Xenopus embryos, and that experimental suppression of these Cdc42 waves results in inappropriately positioned cleavage planes, a hallmark of mispositioned or misoriented spindles. I will use a combination of imaging, global and optogenetic manipulation of Cdc42, and knockdown approaches in intact Xenopus embryos and dissociated blastomeres to test the hypotheses that Cdc42 controls proper spindle positioning and to test whether it acts on the spindle positioning machinery directly or whether it acts through upstream control mechanisms such as cell polarization. In Aim 1, I will assess the role of Cdc42 in spindle positioning in intact embryos and dissociated blastomeres via both global and optogenetically controlled Cdc42 wave modulation. This will allow me to determine where and when in the spindle positioning hierarchy Cdc42 acts. In Aim 2, I will identify the Cdc42 activator, GEF, and inactivator, GAP, responsible for regulating Cdc42 waves. This will allow me to understand the molecular regulation of cortical Cdc42 dynamics during spindle positioning, and provide a complementary approach for wave manipulation. Collectively, ...