Project Summary/Abstract Spatial control of membrane traffic is essential for the morphogenesis and maintenance of polarized cell types such as epithelia and neurons, whose organization is integral to organ homeostasis and neurotransmission. Many advances have been made in understanding membrane traffic at points of origin (protein sorting, vesicle formation) and destination (vesicle docking/fusion). However, key challenges remain in understanding how long-range transport is spatially controlled en route to destination. The central hypothesis of our studies is that septins, a family of multimeric GTP-binding proteins, which associate with distinct subsets of microtubules and membrane domains, comprise a novel regulatory module for the spatial guidance of membrane traffic. Here, we request an administrative supplement for the acquisition of fast and super-resolution laser scanning confocal microscopy, which will improve the imaging capabilities, quality and data output (productivity) of our R35-funded projects. In investigating whether there is specificity between septins and distinct routes of polarized traffic (e.g., apical vs. basolateral), the new instrumentation will enable to image fixed and live epithelial sheets and organoids faster and with less photodamage as well as unprecedented resolution (100 nm lateral, 200 nm axial). In our studies of how lysosome trafficking and positioning is regulated by septins at steady state and in response to cellular cues, the new instrumentation will enable to track endolysosomes with unprecedented spatiotemporal resolution in 2D and 3D using detectors with superior quantum efficiency. Through the use of advanced modulations such as dynamic enhancement and adaptive image quality determination and reconstruction, we will be able to acquire high-quality time-lapse data, which currently is not possible with core confocal and spinning disk instrumentation that is over ten years old. Outcomes will shed valuable insights into the mechanisms that direct the transport of membrane vesicles and endolysosomes in response to morphogenetic and physiological signals. The proposed studies will also advance our understanding of septins as spatial regulators of intracellular organization, bearing significance on diseases triggered and/or exacerbated by abnormalities in septin expression.