Date

2015

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Physics

First Adviser

Vavylonis, Dimitrios

Other advisers/committee members

Gunton, James D.; Huang, Xiaolei; Mittal, Jeetain; Ou-yang, Daniel

Abstract

The cellular actin cytoskeleton is an intricate system of actin filaments that supports cell morphology and is crucial for numerous cell functions including cell growth and cell division. Among the most important actin cytoskeleton structures are actin cables and cytokinetic ring, which are bundles of actin filaments. The actin cables span the cell and serve as tracks for vesicle transport while the actin cytokinetic ring forms in the middle and constricts to divide the cell. The focus of my work is to gain a quantitative understanding of how such cables and ring are formed, what are the essential components, how overexpression and underexpression of certain proteins will affect the structure and dynamics. I built a 3D computational model that starts out from the basic physical and mechanical properties of actin filaments and accounts for known interactions with other proteins, to reproduce the experimental observations of the actin cytoskeleton in different cell systems and further make testable predictions for cell mutants.First, I modeled individual actin filament as a semiflexible worm like chain. I coarse grained the filamentous actin segment using a bead-spring model with spring, bending and thermal forces. This model represents of the actin filament's spatial and dynamical properties. I tested that the model reproduces the correct persistence length, relaxation dynamics and equipartition of energy.Second, to obtain a quantitative understanding of these actin structures and dynamics in fission yeast, I extended the individual actin filament model and added actin-interacting factors. Polymerization out of formin cortical sites, bundling by cross-linkers, pulling by type V myosin, and severing by cofilin, are simulated as growth, cross-linking, pulling and turnover of the semiflexible polymers. With the above mechanisms my quantitative model generated actin cable structures and dynamics similar to those observed in live cell experiments. The simulations reproduced the particular actin cable structures in myoVΔ cells and predicted the effect of increased myosin V pulling. I found that increasing cross-linking parameters generated thicker actin cables and led to anti-parallel and parallel phases with straight or curved cables. I further analyzed the cable number, curvature and loop occurrences of experimental images of cells overexpressing crosslinkers and cell treated with drugs that depolymerize actin patches, provided by our collaborator Damien Laporte. Our predictions are in quantitative agreement with the experiments. Furthermore, the model predicts that clustering of formins at cell tips promotes actin cable formation.Third, I adapted the actin cable model to budding yeast, another well-studied model organism. Budding yeast differs from fission yeast in that it has a more complex geometry and different types of interacting proteins. I refined the previous fission yeast actin cable model by considering a more accurate model of orientation-dependent crosslinking by fimbrin and a more accurate aging mechanism for turnover. It also included polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosins stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which I suggest as a model for the assembly of actin filaments to the contractile ring.Finally, my colleague Dr. Tamara Bidone and I further extended the model to simulate the actin contractile ring. We showed that the ring formation region in parameter space lies close to regions leading to clumps, meshworks or double rings, and stars/cables, which are consistent with prior experiments with mutations that alter the morphology of the condensing network. We also quantified tension along actin filaments and forces on nodes during ring assembly and showed that the mechanisms describing ring assembly can also drive ring constriction once the ring is formed.In summary, this work provides a numerical way to study the morphology and dynamics of the actin cytoskeleton in model cell organisms. Combining simulated, analytical and experimental results, the proposed model with minimal set of interactions successfully reproduced experimental observations and made predictions for further studies.

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