Browsing by Subject "fluorescent protein"
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Item Embargo Experimental and Modeling Approaches to Investigate Molecular-Scale Mechanosensitive Processes in Collective Cell Migration(2024) Shoyer, Timothy CurtisThe coordinated movement of groups of cells, called collective cell migration (CCM), plays important roles in many developmental, physiological, and pathological processes. During CCM, cells remain mechanically coupled to their neighbors, which enables both long-range coordination and local rearrangements. This coupling requires the ability of cell adhesions to transmit and adapt to mechanical forces. However, the molecular mechanisms that underly these mechanosensitive processes remain poorly understood, hindering efforts to manipulate CCM for therapeutic or engineering purposes. To address this gap, this dissertation develops and applies a combination of experimental and modeling approaches to investigate molecular scale mechanosensitive processes. In the first part of this dissertation, we asked how mechanical forces and biochemical regulation interact to control mechanical coupling during CCM. We focused on the mechanical linker protein vinculin, which is known to mediate adhesion strengthening. Using a set of Förster resonance energy transfer (FRET)-based biosensors, we probed the mechanical function and biochemical regulation of vinculin, elucidating a switch that toggles both the activation and molecular loading of vinculin at cell adhesions. We found that the vinculin switch controlled both the speed and coordination of CCM, resulting in a covariation of these variables that suggested changes in adhesion-based friction. To bridge molecular and cellular measurements, we developed molecularly specific models of frictional forces at cell adhesions based on the force-sensitive bond dynamics of key proteins. In these models, increases in vinculin activation and loading produced increases in friction at adhesion structures, and this was due to the engagement of vinculin-actin catch bonding. Together, this work reveals how the biochemical regulation of a linker protein (vinculin) affects a cell-level mechanical property (adhesion-based friction) to control a multicellular behavior (CCM).
In the second part of this dissertation, we focused on how cells sense mechanical forces at the molecular scale. This is thought to occur by force-induced changes in the structure/function of proteins. However, how forces affect protein function inside cells remains poorly understood due to a lack of tools to probe this inside cells. Motivated by in vitro work showing that the mechanical loading of fluorescent proteins (FPs) causes a reversible switching of their fluorescence, we investigated if this phenomenon could be detected inside cells to directly visualize force-sensitive protein function. Using a mathematical model of FP mechanical switching, we developed a framework to detect it inside FRET-based biosensors. Applying this framework, we observed FP mechanical switching in two sensors, a synthetic actin-crosslinker and the linker protein vinculin, and we found that mechanical switching was altered by manipulations to cellular forces on the sensor as well as force-dependent bond dynamics of the sensor. Together, this work develops a new framework for assessing the mechanical stability of FPs and enables visualizing the effect of forces on protein function inside cells.
Overall, the work in this dissertation advances our basic understanding of mechanosensitive processes, addressing knowledge gaps in CCM and mechanobiology. The frameworks we have developed for integrating molecular- and cellular-level experiments with mathematical models will facilitate new mechanistic studies into mechanosensitive processes involving other proteins and biological contexts.
Item Open Access Investigating the Structure of FtsZ to Understand its Functional Role in Bacterial Cell Division(2016) Moore, Desmond AntoineFtsZ, a bacterial tubulin homologue, is a cytoskeleton protein that plays key roles in cytokinesis of almost all prokaryotes. FtsZ assembles into protofilaments (pfs), one subunit thick, and these pfs assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane, and also serves as a scaffold to recruit cell-wall remodeling proteins for complete cell division in vivo. FtsZ can be subdivided into 3 main functional regions: globular domain, C terminal (Ct) linker, and Ct peptide. The globular domain binds GTP to assembles the pfs. The extreme Ct peptide binds membrane proteins to allow cytoplasmic FtsZ to function at the inner membrane. The Ct linker connects the globular domain and Ct peptide. In the present studies, we used genetic and structural approaches to investigate the function of Escherichia coli (E. coli) FtsZ. We sought to examine three questions: (1) Are lateral bonds between pfs essential for the Z ring? (2) Can we improve direct visualization of FtsZ in vivo by engineering an FtsZ-FP fusion that can function as the sole source of FtsZ for cell division? (3) Is the divergent Ct linker of FtsZ an intrinsically disordered peptide (IDP)?
One model of the Z ring proposes that pfs associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of E. coli FtsZ by inserting either small peptides or whole FPs. Of the four lateral surfaces on FtsZ pfs, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174 located on the left and right surfaces, completely blocked function, and were identified as possible sites for essential lateral interactions. Another goal was to find a location within FtsZ that supported fusion of FP reporter proteins, while allowing the FtsZ-FP to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by super-resolution techniques.
The Ct linker is the most divergent region of FtsZ in both sequence and length. In E. coli FtsZ the Ct linker is 50 amino acids (aa), but for other FtsZ it can be as short as 37 aa or as long as 250 aa. The Ct linker has been hypothesized to be an IDP. In the present study, circular dichroism confirmed that isolated Ct linkers of E. coli (50 aa) and C. crescentus (175 aa) are IDPs. Limited trypsin proteolysis followed by mass spectrometry (LC-MS/MS) confirmed Ct linkers of E. coli (50 aa) and B. subtilis (47 aa) as IDPs even when still attached to the globular domain. In addition, we made chimeras, swapping the E. coli Ct linker for other peptides and proteins. Most chimeras allowed for normal cell division in E. coli, suggesting that IDPs with a length of 43 to 95 aa are tolerated, sequence has little importance, and electrostatic charge is unimportant. Several chimeras were purified to confirm the effect they had on pf assembly. We concluded that the Ct linker functions as a flexible tether allowing for force to be transferred from the FtsZ pf to the membrane to constrict the septum for division.