Browsing by Subject "Motility"
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Item Flagellar proteins regulating motility, assembly and photobehavior in Chlamydomonas reinhardtii(2011-07) VanderWaal Mills, Kristyn E.Cilia and flagella are microtubule-based organelles that perform critical functions in human health and development. The I1 inner arm dynein and the IC138 subunit play a key role in the regulation of flagellar motility. To understand how the IC138 protein and its associated subunits modulate I1 activity, we characterized the molecular lesions and motility phenotypes of several bop5 alleles in Chlamydomonas reinhardtii. We first characterize a mutation (bop5-2) that disrupts an IC138 protein sub-complex located at the base of the I1 inner arm dynein. We found the bop5-2 deletion also affects the Tubby-1 (TBY1) gene. To characterize TBY1's activity, tagged versions of TBY1 were transformed into bop5-2. TBY1 protein localizes to a unique ring shaped structure found between the two contractile vacuoles and within the nucleo-flagellar apparatus. The bop5-3, bop5-4 and bop5-5 strains, like other I1 mutants, swim forwards with reduced swimming velocities and display an impaired reversal response during photoshock. However, unlike mutants lacking the entire I1 complex, bop5 strains exhibit normal phototaxis. Analysis of the bop5-3 flagellar waveform reveals that loss of the IC138 sub-complex reduces shear amplitude, sliding velocities, and the speed of bend propagation. The results indicate that the IC138 sub-complex is necessary to generate an efficient waveform for optimal forwards and backwards motility, but it is not essential for phototaxis. Assembly and maintenance of eukaryotic cilia and flagella requires the conserved, bidirectional movement of protein complexes along the length of the axoneme known as intraflagellar transport (IFT). We characterize the function of various components of the IFT complex responsible for the retrograde transport of particles towards the cell body. We quantify the defects in retrograde IFT and flagella assembly observed in a series of mutants of the retrograde complex subunit LIC. We also analyze the expression and distribution of retrograde IFT components in a family of flagellar assembly mutants known as fla, and attempt to correlate these patterns with defects in IFT parameters and other behavioral phenotypes. We provide new evidence that defects in IFT motors can alter photoshock and phototaxis behaviors.Item Swimming Despite Obstacles: Bacterial Swimming as an Evolution-selected Feature(2022-08) Kamdar, ShashankIn the 1670s, Leeuwenhoek used a single-lens microscope to bring the unfamiliar microscopic world of bacteria to human attention. In this research work, we use biophysical tools of quantitative microscopy and fluid dynamics to revisit the same world of microbes and shed light on the intricate yet fascinating motion of microbes. In particular, this thesis details two fundamentally significant problems related to microbial locomotion: 1) motility of microbes in complex fluids, and 2) impact of multiflagellarity on bacterial motility. Locomotion of flagellated microorganisms is of great importance for a wide range of biological processes from disease infection, to reproduction, and to ecosystem health. Bacterial swimming in simple Newtonian fluids is well understood; however, our understanding of their motion in their natural habitats comprising of microscopic particles and polymers is still far from complete. Even after six decades of research, whether bacteria show motility enhancement in polymer solutions and what is the origin of this enhancement remain under debate. We tackled this problem from a new perspective: we studied bacterial locomotion in dilute colloidal suspensions, which do not exhibit complex rheological behaviors such as shear thinning, thickening, etc. Surprisingly, we found that all the measurable swimming features of bacteria in colloidal suspensions are quantitatively the same as those in polymer solutions. This suggests a common origin of bacterial motility enhancement in all complex fluids and challenges all the existing theories which exclusively used polymer dynamics to explain this behavior. We subsequently developed a simple hydrodynamic model considering the colloidal nature of complex fluids, which predicted bacterial motility enhancement in both colloidal suspensions and polymer solutions. We also propose a new mechanism of bacterial wobbling that shows the enhancement and also reproduced bacterial helical trajectories with large pitches—another puzzling behavior of bacterial locomotion. Thus, our study combining experiments and theory unambiguously resolved the long-standing controversy of two problems at once, i.e., the origin of bacterial motility enhancement in complex fluids and the mechanism of bacterial wobbling in Newtonian fluids. Bacterial species also show variations in their flagellar architecture and adapt two common arrangements: monotrichous or uniflagellar bacteria possess a single flagellum at the pole of their body and peritrichous bacteria grow multiple flagella over their body, which form a helical rotating bundle propelling bacterial swimming. Although the cellular features of bacteria are under strong evolutionary selective pressures, extensive studies suggest that multiflagellarity confers no noticeable benefit to bacterial motility. These findings pose a long-standing question: why does multiflagellarity emerge in bacteria given the tremendous metabolic cost of flagellar synthesis? Here, contrary to common views that seek the answer beyond the basic function of flagella in motility, we show that multiflagellarity indeed provides a significant selective advantage in bacterial motility, allowing bacteria to maintain a constant swimming speed over a wide range of body sizes. Through experiments of immense sample sizes and detailed hydrodynamic modeling and simulations, we quantitatively reveal how bacteria utilize the increasing number of flagella to regulate the flagellar motor load, which leads to faster flagellar rotational speeds balancing the higher hydrodynamic drag on the bacterial body of larger sizes. Without such an elegant mechanism, the swimming speeds of uniflagellar bacteria decrease with increasing body sizes. This stark difference between the two swimming modes provides a novel fluid dynamic insight into the crucial role of multiflagellarity in maintaining optimum motility for navigation and survival in their native habitats. Beyond, the ecological implications, results, and insights from this thesis serve as guidelines for devising artificial swimmers that efficiently navigate complex biological environments for drug delivery.Item Visualizing CD8+ T cell responses to foreign and self-antigen(2017-08) Thompson, EmilyCD8+ T cells can recognize any infected cell of the body, making them essential in the immune response against intracellular pathogens. A critical function of CD8+ T cells is the directed killing of target cells through cytolysis. This mechanism is dependent on direct cell-cell contact, which makes the migratory capacity of CD8+ T cells paramount to their successful immune response. The small intestine (SI) is the biggest mucosal surface between the host and the environment. The immune system in this compartment must actively eliminate infection while maintaining tolerance to normal flora, self, and food-antigen. Using two-photon laser scanning microscopy, I evaluated foreign- and self-specific CD8+ T cell motility in the SI. I found that foreign-antigen specific CD8+ T cell behavior varied throughout infection, and was independent of the αE integrin. Interestingly, self-specific CD8+ T cells were initially reactive to self-antigen in vivo but this behavior was altered after further tolerance induction. These studies inform our understanding of the requirements for effective CD8+ T cell immunosurveillance in the SI. I also evaluated what characterizes and contributes to a self-specific CD8+ T cell response to protein in the SI. Using a heterologous prime-boost-boost (HPBB) approach, I generated functional self-specific CD8+ T cells. This response matured throughout boosting, showing the potential of self-specific CD8+ T cells. I also used HPBB to evaluate foreign-antigen specific CD8+ T memory development and showed that the time interval between each boost impacts CD8+ T cell memory longevity.