# The assembly and function of cellular contractile systems

> **NIH NIH R35** · VANDERBILT UNIVERSITY · 2024 · $249,908

## Abstract

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 fo...

## Key facts

- **NIH application ID:** 11035467
- **Project number:** 3R35GM125028-07S1
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** Dylan Tyler Burnette
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $249,908
- **Award type:** 3
- **Project period:** 2017-08-01 → 2028-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11035467

## Citation

> US National Institutes of Health, RePORTER application 11035467, The assembly and function of cellular contractile systems (3R35GM125028-07S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/11035467. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*