Thursday, November 21, 2024
11/21/2024
Summary We request funds to purchase a Spinning Disk Microscope for fast multi-color 3D imaging. This equipment is needed to achieve goals of the parent grant #R35 GM125028. Summary of the parent funded grant. A) We will continue elucidating how specific molecular components of myosin II filaments, as well as those of other contractile system proteins like a-actinin and formins, regulate cellular force production. B) We will determine the roles, and specific differences in function, of the non-muscle and muscle paralogs of myosin II, a-actinin, and potentially other sarcomeric proteins during both sarcomere formation and mitosis/cytokinesis (we have found that some muscle paralogs re-localize from the sarcomere to the cortex during cell division). C) Finally, we will use zebrafish embryos to test the hypotheses that come out of our in vitro experiments in vivo. Techniques used in the parent project. 1) Instant Structured Illumination Microscopy (iSIM) is employed to discern macromolecular complexes within cells, specifically to distinguish the motor-groups within myosin II filaments. 2) Spinning disk confocal microscopy is utilized to observe the dynamic changes in cellular processes, such as retrograde actin flow and sarcomere assembly, influenced by the genetic or pharmacological manipulation of contractile system components in zebrafish embryos. Scientific justification of the request. The spinning disk confocal microscope has been instrumental in the study of cytokinesis and cell motility, favored over iSIM for its lower light intensity requirements that prevent cell cycle arrest. In addition to dividing cells, we are also studying a novel class of large extracellular vesicles we call "blebbisomes" that have their own myosin II-based contractile systems and undergo continuous blebbing. Blebbisomes are studied using spinning disk confocal to avoid phototoxicity, as they stop blebbing and moving during super-resolution imaging. These blebbisomes are autonomous in motion, capable of secreting and internalizing vesicles, housing functional mitochondria and organelles for independent function, and can even initiate apoptosis, with their dynamics investigated in cell culture and zebrafish embryos to align findings across in vitro and in vivo model systems. Existing Equipment. The Burnette lab's Visitech iSIM microscope, while capable of fixed and live cell imaging, often cannot image dim samples or those with rapid movements due to its high photon requirement, leading to photobleaching and phototoxicity. As such, we are reliant on a heavily used Yokogawa CSU-X1 spinning disk confocal in Vanderbilt’s microscopy core for its rapid, multi-channel 3D volume acquisition with minimal photo-damage, as evidenced by its extensive use the lab’s MIRA-funded projects. Requested equipment. The proposal requests a Nikon spinning disk confocal microscope system with automatic focus, DIC, a Yokogawa CSU-X1 spinning disk, a Hamamatsu ORCA-Fusion CMOS camera, and four solid-state lasers to facilitate high-quality, long-term time- lapse imaging with minimal photo-damage.
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