Year: 2024
Type: dissertation
Abstract: Forces generated by the cell are critical to a number of cell biological processes including cell division, motility, embryogenesis, and wound healing. To generate force the cell utilizes a molecular toolkit known as the cytoskeleton. The cell cytoskeleton consists of filaments that interact via numerous accessory proteins like crosslinking and motor proteins. Interactions between filaments mediated by accessory proteins result in the production of cellular scale forces. The following work presents methods to quantitatively relate the behavior, motion, and abundance of cytoskeletal proteins to the generation of force at the scale of the cell. The asymmetric division of a mother cell into two differently sized daughter cells is one mechanism of cell fate specification. Forces mediated by microtubules, acting on the centrosomes, position the mitotic spindle asymmetrically within the zygote, a critical step of asymmetric division. In addition to spindle positioning, microtubule dependent force generation is critical to earlier events during mitosis in zygotes, like the separation of centrosomes and the migration of pro-nuclei. The microtubule mediated forces responsible for these mitotic events are generated via interactions between dynein, bound to the membrane via a cortical complex, and microtubules. The exact nature of the molecular interaction occurring between dynein and astral microtubules that results in force production is unknown. Determination of this molecular mechanism requires the ability to identify dynein in the presence of a microtubule and distinguish the interacting population from cortically located dynein in the absence of microtubules. I present a method to identify cortically located dynein interacting with microtubules from trajectories collected at the poles of the C. elegans zygote using high spatial-temporal resolution imaging. This work revealed that dynein microtubule interactions are short lived and numerous. The magnitude of the force acting on the centrosomes is regulated by astral microtubule number. In future work, this method will be built upon to determine the nature of the dynein microtubule interaction that results in force generation. The most well known actomyosin ring is the cytokinetic ring, which is responsible for the division of the mother cell into two daughter cells during cytokinesis. Actomyosin rings are found in other cell biological contexts like intra cellular and extra-cellular wound healing and as stable connections between germ cell compartments and the common cytoplasm within syncytia. Although actomyosin rings are found in diverse cellular contexts, they are composed of the same set of molecular components. The mechanism of actomyosin ring closure, and the extent of its conservation across cellular contexts is unknown. To sample the diversity of actomyosin rings, I imaged actomyosin ring closure across three subcellular contexts within a common cytoplasm. Using a novel active gel-based framework, measurements of ring component protein abundance and dynamics were related, which indicated that the role of non-muscle myosin II motoring varied across the subcellular contexts. In total, that work supported the conclusion that protein component density, distribution along F-actin, and myosin motoring, can be tuned via cellular context specific ques to regulate the contractility of actomyosin rings. Next, I analyze the novel active gel-based ring model to determine its dynamics and address a number of open questions related to cytokinesis in the C. elegans zygote. Models of actomyosin ring closure focus on either the microscopic or macroscopic aspects of the ring. The model evaluated here relates the forces generated between interacting filaments via accessory proteins to the material properties and resulting kinetics of the ring. In total, the model recapitulates a number of behaviors observed during cytokinesis in the first mitotic division of C. elegans and suggests mechanisms of actomyosin ring stability.
DOI: 10.17615/stx0-9x07
Author: Linehan, John
Institution: University of North Carolina at Chapel Hill
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W6963132199
Cites:
Cited by:
Related to: