Browsing by Subject "Flagella"

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    A biochemical and genetic approach to understand the function of UNI2, a gene encoding a novel basal body protein in Chlamydomonas reinhardtii
    (2008-06) Piasecki, Brian Peter
    The unicellular green alga Chlamydomonas reinhardtii is typically biflagellate, but forward genetic screens have identified uniflagellar mutants. All uniflagellar mutants ( uni1, uni2, and uni3 ) contain ultrastructural defects in the basal body or transition zone and preferentially assemble a flagellum from the older basal body. The UNI2 gene encodes a novel coiled-coil protein with a potential homolog in the human genome. We rescued the uni2 mutant phenotype with an HA-epitope tagged gene construct. Immunoblot analysis demonstrated that the Uni2 protein migrates as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phophorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Immunofluorescence staining of the Uni2 protein in interphase cells demonstrated that it localizes to four distinct spots coinciding with the location of basal bodies and probasal bodies Immunogold labeling confirmed Uni2 protein localization on probasal bodies and the distal end of basal bodies at the precise point of triplet to doublet microtubule transition between the basal body and flagellum. Using the Uni2 protein as a marker of basal bodies during the cell cycle, we observed the sequential assembly of new probasal bodies beginning at prophase. Double mutant strains with uni1,uni2 or uni2,uni3 genotypes showed enhanced defects in flagellar assembly. Immunoblot analysis showed that phosphorylation of the Uni2 protein is significantly reduced in uni1 mutant cells but is similar to wild-type levels in uni3 mutant cells. Ultrastructural analysis demonstrated enhanced transition zone defects in the uni1,uni2 double mutant cells. Serial transverse sections through basal bodies in uni1 and uni2 single and double mutant cells revealed a previously undescribed defect in the transition from triplet to doublet microtubules between the basal body and flagellum. The transition defect was correlated with an inability to form axonemes. These mutants provide the first mechanistic insights into the pathway mediating the transition of triplet to doublet microtubules during flagellar assembly and suggest an overlap in the pathways mediating microtubule transition and basal body maturation.
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    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.
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    Swimming Despite Obstacles: Bacterial Swimming as an Evolution-selected Feature
    (2022-08) Kamdar, Shashank
    In 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.