161 research outputs found
Singularities of the curve shrinking flow for space curves
No full textAltschuler, Steven J.. (1991). Singularities of the curve shrinking flow for space curves. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/4439
Shortening space curves and flow through singularities
No full textAltschuler, Steven J.; Grayson, Matthew A.. (1991). Shortening space curves and flow through singularities. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/4606
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Role of cell identity in growth cone extension during Drosophila visual circuit development
No full textThe formation of repeated patterns is a recurring theme during tissue development. Well-studied models include the pigmentation of fish skins, the spacing and orientation of hair follicles, and the connection of sensory neurons. How do seemingly complex, yet highly organized patterns emerge? What rules underlie the development of these cellular patterns? A major challenge is to uncover rules in the face of “biological noise”, arising from stochastic variability of individual cells, local warping of the developing tissue, and specimen-to-specimen differences. Here, I investigated rules governing neuronal pattern formation in the context of Drosophila visual circuit development, where the outer photoreceptors, R1-R6, resort in the lamina layer of the brain to form the stereotyped neural superposition (NSP) circuit. To hunt for signals within the biological noise, I developed a data-driven, standardized coordinate system to characterize the ensemble behaviors of photoreceptors. With this, I was able to identify rules that govern cell-type-specific extension velocities of the photoreceptor pair R3/R4, which uniquely exhibit asymmetric targeting. Specifically, I found that the extension speeds of the R3 and R4 growth cones are inherent to their cell identities. Further, to showcase the predictive power of the coordinate system, I made a computational prediction that extension angles can be explained by a weighted nearest-neighbor repulsion model. My work provides a new case study of how pattern formation rules — hidden within phenotypic variability — can be inferred by quantitative analysis during the development of living organisms
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Non-genetic factors influencing drug persistence and survival in cancer cells
No full textDespite the development of new, promising therapies for treating cancer, the emergence of cancer cells that do not respond to treatment remains a major barrier to cure. These emerging populations of cancer cells can result from secondary mutations that give rise to inherited drug tolerance or resistance, but in many cases there is no clear genetic basis for improved drug survival. In fact, prior work has shown that non-genetic mechanisms (e.g. chromatin remodeling) can enable longer-term persistence of cells in drug (Sharma et al., 2010, Hinohara et al., 2018), and we and others have demonstrated that these “persister” cells are an important reservoir for the emergence of drug-resistant mutants (Ramirez et al., 2016, Hata et al., 2016). Here through a combination of data analysis, mathematical modeling, and borrowed insight from microbiology, we further our understanding of non-genetic factors that alter cancer cell response to drug treatment. In the first chapter, I summarize literature describing the persister state in both antibiotic-treated bacteria and drug-treated cancer cells, and I highlight shared features of these persisters (such as slow growth and distinct metabolic activity). Of particular relevance to this dissertation, I discuss epigenetic and metabolic changes that influence cancer progression and drug survival. In the second chapter, I pair a close study of cancer cells differing only by expression of the epigenetic modifier TET2 with mathematical modeling to demonstrate how TET2 loss can confer a fitness advantage in drug by altering the dynamics of cell-state switching. Finally, in the third chapter, I present unpublished work focused on metabolic changes and vulnerabilities associated with TET2 loss. Through these studies, I describe several non-genetic mechanisms that affect persistence and tolerance of cancer cells in drug and highlight the challenges in pharmacologically targeting these complex and ill-defined modes of drug survival
Single-Cell Image Analysis Enables High-Throughput Phenotypic Drug Screen and Elucidates Cell-Fate Decision Principles
No full textCellular phenotypes encode information that can be used to infer the external signals cells experience. Here we applied quantitative image analysis to enable a one-pass multi-class phenotypic drug screen. Our combined experimental and computational approach can functionally annotate large compound libraries across diverse drug classes in a single-pass screen with high prediction accuracy confirmed via orthogonal, secondary validation assays. We further investigated how heterogeneity arose from an isogenic population. We monitored the dynamics of p21 to understand the proliferation-senescence cell-fate decision in single cells under non-lethal dose of chemotherapy via time-lapse microscopy before, during and days after treatment. Surprisingly, while high p21 is associated with senescence at late times, we find the opposite at early times during drug treatment: most senescence-fated cells have low p21 levels, while proliferation-fated cells have much higher p21 expression. Further, we identify a p21 "Goldilocks zone" for proliferation, in which increasing p21 levels has the undesirable effect of increasing proliferative outcomes. Our study identifies a counter-intuitive role for early p21 dynamics in cell-fate decision and pinpoints the source of proliferative cancer cells that emerge after exposure to non-lethal doses of chemotherapy
Spatiotemporal Control of Actin Cytoskeletal Machinery in Cells: Multivalency Mediated Protein Clustering at the Plasma Membrane and Optogenetic Tool Development
No full textPrecise regulation of actin cytoskeleton dynamics is crucial for numerous cellular processes. My thesis work has demonstrated two different mechanisms that cells use to achieve spatiotemporal control of actin assembly -- multivalency mediated phase separation and subcellular activation of actin polymerization machinery.
The plasma membrane of the living cell is believed to function as a dynamic platform for signal transduction, rather than existing simply as a static barrier between intracellular and extracellular environments. Moreover the membrane is organized into distinct lipid and protein microdomains. Recent work has shown the importance of protein assembly at the membrane in the formation of these domains. However, the mechanisms by which proteins organize at the membrane are largely unknown. Here I demonstrate that multivalent interactions among proteins in the Nephrin/Nck/N-WASP actin-regulatory pathway can drive clustering of the components at the plasma membrane in cells. Generation of the micronsized domains is dependent on the phosphorylation-mediated interaction between the transmembrane protein, Nephrin, and its effectors, Nck and N-WASP, in the cytoplasm. These structures are dynamic and associated with increased actin polymerization activity, suggesting the importance of clustering in local rearrangements of the cytoskeleton. My studies suggest that multivalent interactions between the signaling proteins can, in general, contribute to spatiotemporal regulation of cellular processes by clustering of molecules at the plasma membrane.
Precise spatiotemporal regulation of protein activity is a key feature of cellular signaling pathways. While contemporary tools in biology have limited control in space and time, recent developments in optogenetics have demonstrated that light can be used as a general tool to investigate protein function in vivo. Many optogenetic tools, however, indirectly regulate protein activity by modulating expression level or cellular localization. Other optogenetic tools with direct control of protein activity have limited applications. Here I demonstrate a method based on fragment complementation that can be generally applicable to a protein of interest based on the light-dependent interaction of Phytochrome B (PhyB) and phytochrome interacting factor 3 (Pif3). My method allows subcellular control of protein function via light-induced reconstitution of a split protein. In particular, I show that light-dependent activation of SopE, a bacterial GEF protein, induces membrane ruffling in a living cell. Furthermore, as an application to inter-molecular regulation between two different molecules, I induced light-mediated interactions between Rho-GTPase Cdc42 and its effector Wiskott-Aldrich Syndrome Protein (WASp) to promote filopodia formation in HeLa cells. Using these optogenetic tools, I demonstrate a general method that can be used to study a broad range of proteins and protein-protein interactions
Investigating Roles for Cellular Heterogeneity in Cancer
No full textCell populations, even those derived from a single clone, can exhibit a high degree of phenotypic variability. However, most biological studies take measurements as averages of entire populations without consideration for the underlying distribution of cellular phenotypes. Though there is growing evidence that variability within cellular populations has some functional consequences, the significance of cell to cell heterogeneity is still poorly understood. Here, we present an analytical platform that represents heterogeneity of cell populations as mixtures of distinct cell phenotypes, or subpopulations, based on immunofluorescent images. These "subpopulation profiles" make the heterogeneity of cell populations more tractable and comparable. We go on to demonstrate that subpopulation profiles can be predictive of clonal populations' drug responses. This separation is shown to be independent of the population's cell-cycle distribution. The subpopulation profiles are then shown to be robust population readouts and used to classify diverse cell lines. We show that, in diverse panels of cell populations, the relationship between basal state heterogeneity and drug response tends to break down. We also show, however, that the subpopulation profiles of diverse cell lines can be useful for identifying independently informative biomarkers. Taken together, these results demonstrate that a subpopulation level reduction of heterogeneity can be a useful readout of cell populations with many potential applications
Mapping the Landscape of Acquired Vulnerabilities in Ovarian Cancer
No full textRecent undertakings to identify the genetic lesions associated with ovarian cancer have noted the striking diversity of mutations occurring in this disease. This genetic diversity has complicated the search for novel therapies. However, recent data has suggested that one commonality of ovarian tumors might be ablation of miRNA biogenesis. Here I conducted a broad-scale gain-of-function microRNA (miRNA) screen in 16 ovarian cancer cell lines to annotate the functional landscape present in such a chaotic genetic background. miRNAs function as multigenic perturbations allowing for interrogation of maximal gene space with few experiments. This screen identified multiple miRNAs reducing cell viability with the majority of hits being toxic in only one or two lines screened. This surprising finding reflected the commonality of altered miRNA function in ovarian tumors while also suggesting that specifics of this alteration in function are unique to each tumor. To investigate more public vulnerabilities, I focused mechanistic studies on miRNAs displaying penetrance in greater than 5 cell lines. miR-517a reduced cell viability in over 30% of the panel and also reduced tumor burden in vivo. Functional analysis of the predicted targets of miR-517a revealed that expression of this miRNA reduced protein levels of ARCN1, a member of the coatamer complex, and that knockdown of ARCN1 reduced cell viability similar to miR-517a. Another penetrant miRNA, miR-124a, reduced cell viability in 37.5% of the panel and functional analysis of this miRNA revealed it promoted a cell differentiation program. Analysis of predicted targets revealed that expression of miR-124a reduced expression the homeodomain transcription factor SIX4, resulting in increased signaling along the tumor suppressive AMPK pathway and epithelial differentiation. Furthermore, SIX4 displayed increased expression in ovarian tumors and depletion of SIX4 expression reduced tumor cell viability in vitro and in vivo. Therefore, SIX4 overexpression might function to deflect cell differentiation in tumors. Thus, the common loss of miRNA function observed in ovarian tumors might serve to maintain an undifferentiated state, and engagement of cell fate determination programs via re-expression of miRNAs can result in catastrophic consequences for cancer cell viability
Quantitative Single-Cell Imaging Reveals Insulation of Morphogenic Signal Transduction
No full textHow cells integrate external cues in order to make behavioral decisions is a central problem of cell biology. In development and in tissue-homeostasis, cell-fate decisions are made by the integration of multiple morphogenic signals, but how cells convert such combinations of signals into distinct behaviors is not well understood. A major complication is our incomplete knowledge of which signal properties encode the information that cells use for decision-making. A further complication is that the static networks we use to describe cellular signaling pathways are likely to be overly-complex; the true signaling network, in a given cellular context and at a particular point in time, may be much simpler. Using a rigorous and quantitative single-cell imaging approach, I find that such simplicity is present in the integration between Wnt and Transforming Growth Factor Beta (TGFB), which are key developmental pathways. Surprisingly, this insulation extends to the integration of signals within the TGFB superfamily, which are expected to compete for shared components and so interfere with one another during signal transduction. My results thus add clarity to and simplify our understanding of how cells integrate information from the Wnt and TGFB pathways, and further suggest that insulation of signal transduction may be a common feature of morphogenic pathways
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