Student Theses and Dissertations

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Kapoor Laboratory

Abstract

Chemical probes are valuable tools for investigating rapid cellular processes, such as anaphase in cell division, by allowing for the modulation of protein activity over the course of minutes. In addition, resistance to chemical probes by mutation can provide insight into inhibitor binding interactions and may provide morphological pharmacodynamic markers that allow for the identification of drug resistance or sensitivity. Here, I present a two-part thesis in which I (i) explore the role of spastin during late anaphase and nuclear envelope reformation using our recently developed spastin inhibitor and (ii) use inhibitor resistance to identify morphological markers of drug resistance via high-content microscopy. In the first part of my thesis, I use chemical probes to examine spastin’s role in cell division. Spastin is a AAA (ATPases associated with diverse cellular activities) protein that is recruited in anaphase by the endosomal sorting complex required for transport (ESCRT) to the reforming nuclear envelope, where spastin is proposed to sever stable microtubules. However, we currently lack an understanding of spastin’s dynamics during anaphase, particularly the roles of microtubules and ESCRT proteins in said dynamics. Using live cell imaging and chemical probes that inhibited spastin’s ATPase activity (spastazoline) or affected microtubules (nocodazole, taxol, or monastrol), I quantified spastin dynamics during anaphase. Spastin foci accumulated on the periphery of chromosomes and were similar on the spindle pole-facing and midzone-facing sides of chromosomes. However, foci that colocalized with microtubules persisted longer than microtubule-free foci. Inhibiting spastin with spastazoline resulted in more stable microtubule-proximal foci compared to microtubule-free foci. Spastazoline had no measurable effect on the accumulation of the ESCRT-III protein, charged multivesicular body protein 4B (CHMP4B). Together, these data suggest that spastin dynamics during late anaphase are decoupled from ESCRT dynamics and are instead modulated by the presence of microtubules and spastin’s ATPase activity. In the second part of my thesis, I use chemical probes to examine morphological markers of drug resistance. Drug resistance is a confounding factor in the treatment of many diseases, particularly cancers, where uncontrolled and error-prone cell divisions can give rise to resistance-conferring mutations. While identifying drug-resistant cell populations in advance could help personalize therapeutics and prevent delays in administering effective treatments, we currently lack rapid methods for broadly analyzing drug resistance. Here, I help generate drug-resistant cell lines and image these cells using Cell Painting, a high-content microscopy method, to characterize morphological markers of resistance. Working in collaboration with colleagues at the Broad Institute, we found that our data support the identification of resistant cell lines using profiles generated from high-content microscopy. This work suggests that image-based cell profiling may be a valuable method of identifying drug-resistant or -sensitive cells and could be a useful tool to guide therapeutic strategies.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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