Mechanisms of cancer cell motility in vivo.

This thesis describes investigations into mechanisms responsible for cancer cell motility in vivo. Chapters 1 and 2 provide a review of current literature in this field and also describe the techniques used to generate the following the results. Chapter 3 describes a candidate-based approach to investigate whether ROCK1 might be regulated by phosphorylation. Mutagenesis of ROCK1 was carried out at 3 chosen sites (T233 T380 T398) in the activation loop and the hydrophobic domain and the phenotypes of the mutants were analysed. Chapter 4 describes a parallel approach finding phosphorylation sites in ROCK1 by mass spectrometry. From these results T518 was chosen for further investigation and its possible function is investigated. Chapter 5 describes an siRNA screen designed to identify novel regulators of the cortical acto-myosin cytoskeleton. The read-out for this was based on the disruption of rounded blebbing morphology of A375 cells cultured on 3D gel matrices. The rounded morphology is similar to that observed in amoeboid cancer cell motility in vivo, therefore we hypothesised that genes required for contracted, rounded morphology might also be required for motility. Results identified PDK1 amongst other genes as a potential regulator of contractile forces in A375 cells and the role of PDK1 was investigated further. It was found that PDK1 was required both in vitro and in vivo for amoeboid cell motility. Chapters 6,7 and 8 detail the investigations into the mechanism of how PDK1 regulates the cytoskeleton and amoeboid cell motility. It was shown that PDK1 was responsible for the localisation of ROCK1 but not ROCK2 at the plasma membrane. This regulation was achieved by the direct binding of ROCK1 to PDK1. It was further found that PDK1 was able to compete with and prevent RhoE, a negative regulator of ROCK1, from binding. Chapter 9 investigates the relationship between cell morphology, motility and pigment production. It was found that it was possible to image melanin containing vesicles using multiphoton excitation, and using this technique, the motile behaviour of pigmented melanoma cells was observed in vivo. It was found that motile invasive cells tended to contain less melanin than non-motile cells suggesting that they were less well differentiated. This chapter details investigations into what differences in signalling could be responsible for a switch to a de-differentiated, more invasive/metastatic phenotype. The final chapter discusses the findings contained within this thesis and the possible implications

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Development of techniques for time-lapse imaging of the dynamics of glial-axonal interactions in the central nervous system

Background: Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualise the various stages of myelination. Methodology/Principal findings: Two approaches have been taken to follow myelination over time i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. The data demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, evidence from time-lapse fluorescence microscopy and confocal microscopy suggest that the oligodendrocytes’ cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs which then extend longitudinally along the axons. From this model it is predicted that these spiral cuffs must extend over each other first before extending to form internodes of myelin. Conclusion: These experiments show the feasibility of visualising the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualisation of an entire internodal length of myelin and the ex vivo approach validating the in vitro data

PKA-Mediated Regulation of Profilin-1 - Implication in Sprouting Angiogenesis

Angiogenesis is a process of vessel outgrowth from a pre-existing capillary that is implicated in many physiological and pathological conditions. Profilin-1 (Pfn1), a ubiquitously expressed actin-binding protein, has been previously shown to be up-regulated in vascular endothelial cells during capillary morphogenesis and required for endothelial cell migration, morphogenesis and invasion in vitro. In the first part of this study, we demonstrated that depletion of Pfn1 interferes with sprouting angiogenesis in vitro and ex vivo. In the second part, we further explored how Pfn1 might be biochemically regulated. Our studies suggested that a significant fraction of Pfn1 could exist in a number of phosphorylated states in cells. We showed that Pfn1 can be phosphorylated by Protein Kinase A (PKA) in vitro and in a PKA-dependent manner in vivo. By mass-spectrometry, we identified several potential PKA phosphorylation sites of Pfn1, one of which, T89, at least also appeared to be a bona fide modification site of Pfn1 in vivo. We performed biochemical and in silico analyses to determine the potential consequence of this phosphorylation on the properties of Pfn1. Finally, we showed that activating the PKA pathway affects ligand binding of Pfn1 in cells and negatively impacts both endothelial cell migration and sprouting angiogenesis. Collectively, these observations implicate a possible role for Pfn1 post-translational modification in the PKA-mediated regulation of sprouting angiogenesis

Molecular mechanisms of cell migration in amoeboid cells

Chemotaxis is critical during development, tissue repair, immune response and cancer metastasis. The excitable network hypothesis can account for recent observations of propagating waves of signal transduction and cytoskeleton events as well as behaviors of migrating cells. However, the molecular feedback loops involved in these networks that bring about excitability are poorly understood. We show that during random migration and in response to chemoattractants, cells maintain complementary spatial and temporal distributions of Ras activity and PI(3,4)P2 in Dictyostelium cells. In addition, depletion of PI(3,4)P2 leads to elevated Ras activity, cell spreading and altered migratory behavior. Furthermore, RasGAP2 and RapGAP3 bind to PI(3,4)P2 and the phenotypes of cells lacking these genes mimic those with low PI(3,4)P2 levels, providing a molecular mechanism. These findings suggest that Ras activity drives PI(3,4)P2 down causing the PI(3,4)P2-binding GAPs to dissociate from the membrane, further activating Ras, completing a positive feedback loop essential for excitability. Furthermore, we demonstrated that there is an ongoing flow of vesicular PI(3,4)P2 through the cell and a compensatory forward flow along the membrane, which establishes a back-to-front gradient of PI(3,4)P2. Specifically, first, we show that PI(3,4)P2 localized to the lagging edge of Dictyostelium and neutrophils. Surprisingly, this lagging edge component also localizes to retracting leading edge protrusions and nascent macropinosomes, even in the absence of PIP3. Second, PI(3,4)P2 is internalized on macropinosomes and transported on microtubules into the cytosol. Once internalized, the macropinosomes break up into smaller PI(3,4)P2-enriched vesicles, which dock and fuse to the plasma membrane at the cell rear. Third, we determined that the PI(3,4)P2 molecules incorporated at the back diffuse along the membrane towards the front, where they are degraded. Last, a stochastic mathematical model confirmed that this cycle brings about a stable back-to-front gradient. This reverse fountain flow of PI(3,4)P2 in establishing the back-to-front gradient could be essential for polarity in cell migration. Taken together, our findings uncovered a mutually inhibitory Ras-PI(3,4)P2 mechanism essential for excitability, and shed light on the dynamics and role of PI(3,4)P2 in regulating polarity in cell migration. Our work provides novel frameworks to control cell migration in many physiological processes. Primary Reader and Adviser: Dr. Peter Devreotes Secondary Reader: Dr. Erin Gole

Rho GTPase Dynamics in the Regulation of Cellular Signaling and Migration

Cell migration is critical to the development and maintenance of higher organisms, and is required for the patterning of the nervous system, for the development of organs, and for responses to wounds or sites of inflammation. Because cell migration is so widely utilized, it must be very tightly controlled, as is apparent when the process goes awry, such as in cancer cell metastasis or chronic inflammation. Growth factors and other cues mediate the activation of a variety of pathways that induce cell migration. Despite these many pathways, a family of proteins called Rho GTPases are universally engaged to cause changes in the cellular cytoskeleton, leading to cell migration. Because Rho GTPases are so critical to cell migration, yet can be used to mediate many different types of cellular responses, they must be precisely controlled. In most cases, it is the timing and placement of Rho GTPase activity that defines cellular behaviors in response to specific signals. However, tools to investigate the spatiotemporal dynamics of Rho GTPase activity in live cells have only recently been developed. I this work, I characterize the spatial and temporal dynamics of the Rho GTPases in cell migration through the development of sensors for Rho GTPase activity for live cell imaging. I establish the spatial and temporal dynamics of RhoA, Rac1, and Cdc42 at the leading edge of migrating cells, and the role of RhoG in its ability to precisely position and activate Rac1 at the leading edge of cells. This work provides the first thorough characterization of the roles of these GTPases at the leading edge relative to one another and the mechanisms by which they are regulated. This work also demonstrates preliminary studies on the roles of these GTPases during leukocyte transendothelial migration. It is critical to gain an understanding of the mechanisms by which cells control cell migration via the Rho GTPases. Aberrant signaling through the GTPases leads to a variety of disease processes. Thus, a better understanding of normal Rho GTPase signaling will provide a framework for understanding how cell migration goes awry and how it can be potentially treated.Doctor of Philosoph

Combining Smart Material Platforms and New Computational Tools to Investigate Cell Motility Behavior and Control

Cell-extracellular matrix (ECM) interactions play a critical role in regulating important biological phenomena, including morphogenesis, tissue repair, and disease states. In vivo, cells are subjected to various mechanical, chemical, and electrical cues to collectively guide their functionality within a specific microenvironment. To better understand the mechanisms regulating cell adhesive, differentiation, and motility dynamics, researchers have developed in vitro platforms to synthetically mimic native tissue responses. While important information about cell-ECM interactions have been revealed using these systems, a knowledge gap currently exists regarding how cell responses in static environments relate to the dynamic cell-ECM interaction behaviors observed in vivo. Advances at the intersection of materials science, biophysics, and cell biology have recently enabled the production of dynamic ECM mimics where cells can be exposed to controlled mechanical, electrical or chemical cues to directly decouple cell-ECM related behaviors from cell-cell or cell-environmental factors. Utilization of these dynamic synthetic biomaterials will enable discovery of novel mechanisms fundamental in tissue development, homeostasis, repair, and disease. In this dissertation, the primary goal was to evaluate how mechanical changes in the ECM regulate cell motility and polarization responses. This was accomplished through two major aims: 1) by developing a modular image processing tool that could be applied in complex synthetic in vitro microenvironments to asses cell motility dynamics, and 2) to utilize that tool to advance understanding of mechanobiology and mechanotransduction processes associated with development, wound healing, and disease progression. Therefore, the first portion of this thesis (Chapters 2 and 3) dealt with proof of concept for our newly developed automated cell tracking system, termed ACTIVE (automated contour-based tracking for in vitro environments), while the second portion of this thesis (Chapter 4-7) addressed applying this system in multiple experimental designs to synthesize new knowledge regarding cell-ECM or cell-cell interactions. In Chapter 1, we introduced why cell-ECM interactions are essential for in vivo processes and highlighted the current state of the literature. In Chapter 2, we demonstrated that ACTIVE could achieve greater than 95% segmentation accuracy at multiple cell densities, while improving two-body cell-cell interaction error by up to 43%. In Chapter 3 we showed that ACTIVE could be applied to reveal subtle differences in fibroblast motility atop static wrinkled or static non-wrinkled surfaces at multiple cell densities. In Chapters 4 and 5, we characterized fibroblast motility and intracellular reorganization atop a dynamic shape memory polymer biomaterial, focusing on the role of the Rho-mediated pathway in the observed responses. We then utilized ACTIVE to identify differences in subpopulation dynamics of monoculture versus co-culture endothelial and smooth muscle cells (Chapter 6). In Chapter 7, we applied ACTIVE to investigate E. coli biofilm formation atop poly(dimethylsiloxane) surfaces with varying stiffness and line patterns. Finally, we presented a summary and future work in Chapter 8. Collectively, this work highlights the capabilities of the newly developed ACTIVE tracking system and demonstrates how to synthesize new information about mechanobiology and mechanotransduction processes using dynamic biomaterial platforms