Coordinated assembly and disassembly of branched actin networks by Arp2/3 complex and its regulators - admin supplement to purchase Zeiss Airyscan detector upgrade

University Of Oregon, Eugene OR

Investigators

Linked publications, trials & patents

Abstract

Project abstract Here we request funding to purchase a Zeiss Airyscan detector upgrade for a Zeiss ELYRA SIM/LSM 880 microscope. This detector will be used predominantly by my laboratory, and by the laboratory of Ken Prehoda, who has also submitted an administrative supplement requesting funds for this upgrade. In R01-GM092917, the parent grant for this supplement, my laboratory proposes to determine how WDS family proteins and WASP family proteins coordinately regulate Arp2/3 complex to assemble branched actin filament networks. A critical aspect of these studies is to test biochemical mechanisms of regulation of WDS and WASP family in cells. To accomplish this, we proposed to use spinning disk confocal microscopy to visualize how perturbations in either Arp2/3 complex or its regulators influence endocytic actin network assembly. However, we recently demoed a Zeiss LSM with Airyscan detection, and found that this instrument is superior to our current spinning disk confocal microscope and even state-of-the-art spinning disk confocal microscopes. The improved resolution of this instrument makes it possible to observe endocytic actin networks with unprecedented detail, revealing aspects of their morphology and dynamics that allow us to better connect our mechanistic and structural insights to in vivo function. Likewise, the increased sensitivity of this instrument would allow us to track fluorescently tagged proteins at endocytic sites that are expressed at low levels or suffer from high cytoplasmic background, such as the fission yeast WDS protein, Dip1. Our ability to visualize Dip1 and mutants of Dip1 at endocytic sites with the Airyscan detector will allow us to conduct more sophisticated experiments to address the regulation and localization of Dip1 (Aim 3 in R01-GM092917). Further, we note that the Airyscan detector will also greatly benefit our recently funded R01, GM127440, which focuses on understanding WASP-mediated activation of branching nucleation by Arp2/3 complex. Specifically, we will be able to determine how perturbations at each biochemical step in WASP-mediated Arp2/3 complex activation influence actin assembly at endocytic sites. As with our experiments on Dip1, we anticipate the increased resolution and sensitivity of the Airyscan detector will reveal morphological and dynamic features of the actin network and its regulators not detectable with our spinning disk confocal instrument. Therefore, we are certain that this instrument will substantially increase the immediate impact of our work and have a significant and sustained positive impact on our research program and the field.