Structure and Dynamics of Microtentacles

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Ory, Eleanor Claire-higgins
Losert, Wolfgang
Martin, Stuart
While modern cancer diagnostics and treatments are often interpreted through a biomolecular perspective, cancer abounds with many mechanically interesting characteristics and questions. Metastasis, the process by which a primary tumor spreads and forms a second tumor in a distant site is currently responsible for 90% of cancer fatalities [1–3]. One of the key limiting steps in metastasis is extravasa- tion; the process by which a circulating tumor cell (CTC) moves from the blood- stream into surrounding tissue. So far, most in vitro studies in metastasis focus on cell migration and invasiveness with few focused on reattachment of cells to a blood vessel wall, and extravasation. One possible attachment mechanism involves tubulin-based structures called microtentacles, which have been observed to poke into crevices between cells that line blood vessels. Based on biomolecular assays, the current hypothesis is that microtentacles are formed as the result of unbalanced, mechanical interactions between microtubules and actin, allowing microtubules to push the plasma membrane beyond the cell body. The focus of my thesis is to gain insights into the dynamics and mechanical properties of microtentacles and evaluate how microtentacles may be altered by cytoskeletal drugs. In this thesis, I will measure changes in microtubule dynamics using cytoskele- tal drugs to the actomyosin cortex and microtubules. The first study presented examines how drug treatments targeting the actomyosin cortex impact microtubule dynamics for attached cells. The results of the first study demonstrate that weaken- ing the actomyosin cortex allows microtubule end-binding-protein-1 (EB1) to move beyond the cell body boundary. Weakening the actomyosin cortex also results in changes to the speed and straightness of microtubule growth. In the second study, an image analysis framework is presented to quantify microtentacles as well as an eval- uation of the dynamics of microtubules in suspended cells. The study demonstrates a successful image analysis technique that can evaluate microtentacle phenotype for both free-floating and tethered cells as well as dynamics for tethered cells. This second study shows that while microtubule stabilizing drug treatment with Taxol increases total microtentacle phenotype, it also reduces microtentacle dynamics. On the other hand, while microtubule destabilizing drug treatment Colchicine decreases total microtentacle phenotype, Colchicine also reduces microtentacle dynamics. As a summary and outlook, I present a mechanical framework and present hypotheses for 4 different genetic modifications spanning a spectrum of different cytoskeletal states. I also show preliminary, qualitative results for 3 out of the 4 different cell lines. Critical to evaluating microtentacles within this physical framework is a direct mechanical assay; here, I show preliminary work taken at the University of Leipzig on an optical stretcher. Given that microtentacles have demonstrated to be a sufficient prerequisite for reattachment, better understanding of what circumstances lead to microtentacles is a critical basic research question. My work applies a physical perspective to the bal- ance between the actomyosin cortex and microtubules and demonstrates changes in microtubule dynamics. Such work contributes towards the possibility of identifying morphological and dynamics signatures of CTCs with higher metastatic potential.