Funding is requested for the purchase of a Zeiss Airyscan detector with FAST capabilities for imaging of Drosophila larval brain neuroblast divisions. The detector will be used primarily by my research group and secondarily by my colleague Brad Nolen who has submitted a cross- referenced application. In R35GM127092, the parent grant for this supplement application, my laboratory proposes to uncover the mechanism of animal cell polarization using the neuroblast as a model system. Neuroblast polarization is a dynamic process, with the central Par complex transitioning from the cytoplasm in interphase to the apical membrane during mitosis. We would like to observe the dynamics of neuroblast polarization so that we can understand how upstream regulators such as the actin cytoskeleton and the Rho GTPase Cdc42 in?uence di?erent steps of the process. However, because the transition from cytoplasmic to apical Par localization occurs over a short time period, it is di?cult to obtain the required temporal resolution and sensitivity. We recently tested the Airyscan detector, which foregoes the typical confocal pinhole (which rejects a signi?cant amount of light) and replaces it with a high sensitivity area detector. This detector allowed us to perform live imaging of the process with unprecedented sensitivity such that we were able to visualize both the Par complex and its substrates at frame rates su?cient to follow polarization. If we are able to acquire the detector, we will closely examine which steps in the transition from cytoplasmic to polarized localization are in?uenced by known polarity regulators, using F-actin depolymerizing drugs and known Drosophila mutant lines.
! This administrative supplement requests funds to purchase a Zeiss Airyscan detector to allow us to monitor the dynamics of neural stem cell divisions. We would like to use this detector to monitor the localization of fate determinant proteins during these divisions as they become polarized and segregated into di?erent daughter cells. Our previous detector allowed us to monitor the end state of the polarization process, but not the steps that precede it. By shedding light on the multistep nature of the neural stem cell polarization, we will better be able to determine how upstream components regulate polarity.!
