PHYSICAL FACTORS IN B CELL ACTIN DYNAMICS AND ACTIVATION

Cells adapt to their changing world by sensing environmental cues and responding appropriately. This is made possible by complex cascades of biochemical signals that originate at the cell membrane. In the last decade it has become apparent that the origin of these signals can also arise from physical cues in the environment. Our motivation is to investigate the role of physical factors in the cellular response of the B lymphocyte. B cells patrol the body for signs of invading pathogens in the form of antigen on the surface of antigen presenting cells. Binding of antigen with surface proteins initiates biochemical signaling essential to the immune response. Once contact is made, the B cell spreads on the surface of the antigen presenting cell in order to gather as much antigen as possible. The physical mechanisms that govern this process are unexplored. In this research, we examine the role of the physical parameters of antigen mobility and cell surface topography on B cell spreading and activation. Both physical parameters are biologically relevant as immunogens for vaccine design, which can provide laterally mobile and immobile antigens and topographical surfaces. Another physical parameter that influences B cell response and the formation of the cell-cell junction is surface topography. This is biologically relevant as antigen presenting cells have highly convoluted membranes, resulting in variable topography. We found that B cell activation required the formation of antigen-receptor clusters and their translocation within the attachment plane. We showed that cells which failed to achieve these mobile clusters due to prohibited ligand mobility were much less activation competent. To investigate the effect of topography, we use nano- and micro-patterned substrates, on which B cells were allowed to spread and become activated. We found that B cell spreading, actin dynamics, B cell receptor distribution and calcium signaling are dependent on the topographical patterning of the substrate. A quantitative understanding of cellular response to physical parameters is essential to uncover the fundamental mechanisms that drive B cell activation. The results of this research are highly applicable to the field of vaccine development and therapies for autoimmune diseases. Our studies of the physical aspects of lymphocyte activation will reveal the role these factors play in immunity, thus enabling their optimization for biological function and potentially enabling the production of more effective vaccines.

THE ROLE OF THE MECHANICAL ENVIRONMENT ON CANCER CELL TRANSMIGRATION AND MRNA LOCALIZATION

Most cancer-related deaths are due to metastasis formation, the ability of cancer cells to break away from the primary tumor site, transmigrate through the endothelium, and form secondary tumors in distant areas. Many studies have identified links between the mechanical properties of the cellular microenvironment and the behavior of cancer cells. Cells may experience heterogeneous microenvironments of varying stiffness during tumor progression, transmigration, and invasion into the basement membrane. In addition to mechanical factors, the localization of RNAs to lamellipodial regions has been proposed to play an important part in metastasis. This dissertation provides a quantitative evaluation of the biophysical effects on cancer cell transmigration and RNA localization. In the first part of this dissertation, we sought to compare cancer cell and leukocyte transmigration and investigate the impact of matrix stiffness on transmigration process. We found that cancer cell transmigration includes an additional step, ‘incorporation’, into the endothelial cell (EC) monolayer. During this phase, cancer cells physically displace ECs and spread into the monolayer. Furthermore, the effects of subendothelial matrix stiffness and endothelial activation on cancer cell incorporation are cell-specific, a notable difference from the process by which leukocytes transmigrate. Collectively, our results provide mechanistic insights into tumor cell extravasation and demonstrate that incorporation into the endothelium is one of the earliest steps. In the next part of this work, we investigated how matrix stiffness impacts RNA localization and its relevance to cancer metastasis. In migrating cells, the tumor suppressor protein, adenomatous polyposis coli (APC) targets RNAs to cellular protrusions. We observed that increasing stiffness promotes the peripheral localization of these APC-dependent RNAs and that cellular contractility plays a role in regulating this pathway. We next investigated the mechanism underlying the effect of substrate stiffness and cellular contractility. We found that contractility drives localization of RNAs to protrusions through modulation of detyrosinated microtubules, a network of modified microtubules that associate with, and are required for localization of APC-dependent RNAs. These results raise the possibility that as the matrix environment becomes stiffer during tumor progression, it promotes the localization of RNAs and ultimately induces a metastatic phenotype.

REGULATION OF SHAPE DYNAMICS AND ACTIN POLYMERIZATION DURING COLLECTIVE CELL MIGRATION

This thesis aims to understand how cells coordinate their motion during collective migration. As previously shown, the motion of individually migrating cells is governed by wave-like cell shape dynamics. The mechanisms that regulate these dynamic behaviors in response to extracellular environment remain largely unclear. I applied shape dynamics analysis to Dictyostelium cells migrating in pairs and in multicellular streams and found that wave-like membrane protrusions are highly coupled between touching cells. I further characterized cell motion by using principle component analysis (PCA) to decompose complex cell shape changes into a serial shape change modes, from which I found that streaming cells exhibit localized anterior protrusion, termed front narrowing, to facilitate cell-cell coupling. I next explored cytoskeleton-based mechanisms of cell-cell coupling by measuring the dynamics of actin polymerization. Actin polymerization waves observed in individual cells were significantly suppressed in multicellular streams. Streaming cells exclusively produced F-actin at cell-cell contact regions, especially at cell fronts. I demonstrated that such restricted actin polymerization is associated with cell-cell coupling, as reducing actin polymerization with Latrunculin A leads to the assembly of F-actin at the side of streams, the decrease of front narrowing, and the decoupling of protrusion waves. My studies also suggest that collective migration is guided by cell-surface interactions. I examined the aggregation of Dictyostelim cells under distinct conditions and found that both chemical compositions of surfaces and surface-adhesion defects in cells result in altered collective migration patterns. I also investigated the shape dynamics of cells suspended on PEG-coated surfaces, which showed that coupling of protrusion waves disappears on touching suspended cells. These observations indicate that collective migration requires a balance between cell-cell and cell-surface adhesions. I hypothesized such a balance is reached via the regulation of cytoskeleton. Indeed, I found cells actively regulate cytoskeleton to retain optimal cell-surface adhesions on varying surfaces, and cells lacking the link between actin and surfaces (talin A) could not retain the optimal adhesions. On the other hand, suspended cells exhibited enhanced actin filament assembly on the periphery of cell groups instead of in cell-cell contact regions, which facilitates their aggregation in a clumping fashion.