Browsing by Subject "Hemicentin"
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Item Open Access Basement Membranes Link Together and Stretch to Withstand Mechanical Forces(2022) Gianakas, ClaireBasement membranes (BMs) are thin, dense sheets of extracellular matrix that surround most animal tissues and provide structural support. While the role of BMs in the structural support of tissues is well established, how these matrices can structurally support tissues while accommodating dynamic tissue function is not well understood. Using C. elegans, a powerful model organism that allows for live imaging, genetic analysis, and rapid screening, I was able to utilize endogenous knock-in fluorescent proteins, conditional RNAi, optogenetics, and quantitative live imaging to investigate how BM components contribute to the BM’s ability to withstand mechanical load in various circumstances. In Chapter 1, I discuss the known roles of BM, introduce BM proteins of interest, explore gaps in our understanding of BM’s function in withstanding mechanical force, and expand upon the utility of C. elegans as a model system to investigate these questions. In Chapter 2, I show that BM-to-BM linkages can function to resist the mechanical forces involved in egg-laying. In Chapter 3, I explore how BM stretches to accommodate dynamic tissue movement. In Chapter 4, I discuss future directions and the implications of these findings and in Chapter 5 I summarize my conclusions.
Item Open Access Basement Membrane Dynamics During Anchor Cell Invasion(2015) Morrissey, Meghan AnnBasement membranes are a dense, sheet-like form of extracellular matrix that underlie epithelia and endothelia, and surround muscle, fat and Schwann cells. Basement membranes separate tissues and protect them from mechanical stresses. Although traditionally thought of as a static support structure, a growing body of evidence suggests that dynamic basement membrane deposition and modification instruct cell behavior and morphogenetic processes. In this thesis, I discuss how changes to basement membrane affect anchor cell (AC) invasion during C. elegans uterine vulval attachment. During AC invasion, the uterine AC breaches two juxtaposed basement membranes to contact the underlying vulval epithelium. Using live-cell imaging, genetics, molecular biology and electron microscopy I identify three modifications to the BM that affect AC invasion. In Chapter 2, I describe a system for linking juxtaposed basement membranes to stably align or connect adjacent tissues. This adhesion system promotes rapid AC invasion and also regulates a more long-term connection between the uterine tissue and the hypodermal seam cell in the adult worm. Chapter 3 elucidates how the BM component SPARC promotes cell invasion. As SPARC overexpression is correlated with cancer metastasis, this aims to understand how SPARC overexpression promote invasion in a pathological situation. In Chapter 4, I discuss preliminary data showing that the AC actively secretes laminin into the basement membrane targeted for invasion. I outline how future studies could elucidate the mechanism by which AC-derived laminin might promote cell invasion. Finally, Chapter 5 discusses conclusions and future directions for these studies.
Item Open Access Muscle contraction alters hemicentin dynamics at the B-LINK: a newly identified basement membrane adhesion system that connects tissues.(2017-05-11) Johnson, JamesBasement membranes (BMs) are thin, dense sheets of extracellular matrix found covering most tissues in multicellular organisms. In some instances, BMs of adjacent tissues can become linked and attach tissues together. A better understanding of BM-BM adhesion can help elucidate the mechanisms of conditions like Alport syndrome, a human pathology characterized by a loss of kidney function due to a failed BM-BM linkage. In order to further characterize the linkage of neighboring tissues through their BMs, I investigated how the Basement Membrane Linkage complex (B-LINK), a complex that mediates BM-BM adhesion at the uterine-hypodermal juncture in C. elegans, responds to biomechanical force. To accomplish this, I determined the necessity of specific BM proteins to B-LINK structural integrity by performing gene knockdown with RNA interference (RNAi) and scoring for vulval rupture, a phenotype that results from a disrupted B- LINK. Type IV collagen was the only common BM component to be identified as an important factor in BM adhesion due to high vulval rupture percentages when it was knocked down at the L1 (80.5%) and L4 (20.0%) larval stages (Fisher’s exact = 0.0001). Additionally, I used fluorescence recovery after photobleaching (FRAP) to measure the rate of the protein turnover of the B-LINK component hemicentin under different conditions. These FRAP experiments revealed that muscle contraction in animals significantly increases the hemicentin turnover rate when compared to immobilized worms over the same 15-minute time course (76.7% vs. 24.3%, p-value = 0.0041). These results provide a better understanding of which BM components are essential to B-LINK function and has revealed that muscle contraction influences B-LINK dynamics.