UC Berkeley

One of the most exciting recent developments in cell biology is the recognition that the geometry, topography, and elasticity of the extracellular matrix (ECM) can provide signals to cells that can affect cell physiology via a process called tensional homeostasis – the ability of a cell to balance tractional forces against the ECM. Actomyosin stress fibers (SFs) directly connect the cell to its ECM via focal adhesions and are responsible for force generation and transmission. Despite the importance of these structures, we lack a clear understanding of how the geometry of SFs, particularly their length, regulates force generation. Furthermore, current techniques used to study SF mechanical properties consider SFs as isolated bodies that lack connections with the remaining cytoskeletal elements. As a result, we lack the understanding of how tension is distributed across an individual SF that is part of a two-dimensional (2D) network.

In this dissertation, we sought to understand SF viscoelasticity by considering an SF as part of a network by combining subcellular laser ablation (SLA) and single cell micropatterning. First, we show that longer SFs dissipate increased elastic energy after SLA. Second, we show that in addition to single SF geometry, the architecture of the connecting SF network also regulates the elastic energy dissipated by a single SF after SLA. We developed a computational model that takes in consideration the geometry of the surrounding SFs and can recapitulate SLA retraction kinetics. We then sought to understand the parameters that contribute to the observed differences in internal SF architectures and show that the initial cell binding position and spreading history on the micropattern may predict the final SF configuration. Finally, we expanded the theme of connectivity to study connections between cytoskeletal elements such as SFs and intermediate filaments. We show that vimentin interacts with central SFs and regulates SLA retraction kinetics. The work presented in this thesis provides a new framework of thinking about SFs as part of a 2D network both during cell spreading and during “steady state”.

Moreover, myosin in SFs can be phosphorylated via two parallel pathways: Rho Associated Kinase (ROCK) and Myosin Light Chain Kinase (MLCK). While each pathway regulates the formation of SFs in different cellular compartments, it is unclear how each kinase contributes to the viscoelastic properties of SFs found throughout the cell. The relationship between these pathways and SF formation have been explored through pharmacological inhibition making it hard to determine whether SF properties can be tuned according to the amount of myosin phosphorylation. To study these questions, we combine SLA with synthetic biology tools and show that gradient expression of each kinase results to gradient changes in viscoelastic properties of SFs within specific cellular compartments. Specifically, MLCK induces mono-phosphorylation of myosin which primarily localizes in peripheral SFs, altering their viscoelasticity whereas ROCK induces myosin di-phosphorylation which primarily localizes and controls the viscoelastic properties of central SFs.

Overall, this thesis represents a systematic attempt to combine single cell micropatterning and SLAtools to relate single SF geometry and architecture of the connecting SF network to single SF mechanics. Our findings enhance our understanding of how cells distribute tension across a network of SFs and other cytoskeletal structures, addressing a fundamental open question in cell biology. We also provide further insight into the mechanistic pathways of SF tension generation by ascribing the viscoelasticity of SFs found in different cellular compartments to the activity of specific kinases.