Abstract

The assembly of actin, the essential cytoskeleton protein, into filaments and bundles/networks is important in various cellular processes including cell movement and morphogenesis. Actin bundle formation occurs in crowded intracellular environments with the aid of actin-crosslinking proteins. The role of actin-crosslinking proteins such as fascin and a-actinin in bundle formation has been investigated, however, how intracellular environments affect actin crosslinker-induced bundle formation is unknown. In the first two parts of this dissertation, we explore the effects of macromolecular crowding and cation interactions on the organization and mechanics of actin crosslinker-induced bundles. To determine how changing environmental conditions modulate actin bundling, we utilize total internal reflection fluorescence microscopy, atomic force microscopy, and transmission electron microscopy. Through biophysical analysis on actin bundles we show crowding and crosslinking proteins competitively involve in bundle formation. Cations shift actin organization from networks to bundles through destabilization of binding interactions. In addition, molecular dynamics simulations support the counteractive interactions between crowding/cations and actin-crosslinking proteins during bundle formation. Similarly, cellular environments can further be affected by external factors such as nanomaterials that are used in various biomedical applications. Graphene is a widely used nanomaterial that potentially affects the dynamics of actin cytoskeleton. However, its influence on actin assembly dynamics at the molecular level is not well understood. In the third part of this dissertation, we investigate the effect of graphene on actin assembly by localized fluorescence microscopy and real-time pyrene assembly kinetics. Both direct visualization of individual filaments growth and bulk actin polymerization demonstrate that graphene accelerates the actin assembly kinetics. The extent of graphene effects on actin assembly depends on the nanomaterial structural properties including excluded area effects and hydrophobicity. Taken together, these studies provide fundamental insights into how the actin organization, mechanics, and assembly are modulated by intracellular and external factors.

Notes

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Graduation Date

2021

Semester

Fall

Advisor

Kang, Ellen

Degree

Doctor of Philosophy (Ph.D.)

College

College of Engineering and Computer Science

Department

Materials Science and Engineering

Degree Program

Materials Science and Engineering

Format

application/pdf

Identifier

CFE0008883; DP0026162

Language

English

Release Date

December 2022

Length of Campus-only Access

1 year

Access Status

Doctoral Dissertation (Campus-only Access)

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