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Ultrastructure of the Spermatozoid of Lycopodium Obscurum (Lycopodiaceae)Maden, Angel R., Renzaglia, Karen Sue, Whittier, Dean P. 01 January 1996 (has links)
Ultrastructural observations reveal that the spermatozoid of Lycopodium obscurum is crescent shaped and contains two posteriorly directed flagella that are inserted at the front of the cell. The nucleus is broad and elongated with a narrow posterior projection or nuclear diverticulum. Spline microtubules (MTs) number 180 at their maximum and provide the framework for the cell. These MTs extend from the anterior of the locomotory apparatus and along the outermost surface of the nucleus, with a central shank of 14-17 MTs encircling the cell for at least one-third gyre beyond the nucleus. The two basal bodies are slightly staggered and positioned at the front of the cell over a highly elongated multilayered structure (MLS). The MLS extends laterally around the cell anterior and curves posteriorly over the nucleus. One large anterior mitochondrion is situated subjacent to the MLS, while numerous small mitochondria are scattered near or among the lobes of the single plastid. The plastid rests on the inner nuclear surface and contains numerous large starch grains. This cell differs from that of L. cernuum, the only other species of Lycopodium examined to date, in that it is more elongated and has an anterior-posterior orientation of the nucleus, basal bodies, MLS, and spline. Comparisons with coiled gametes of bryophytes and Selaginella suggest that some degree of coiling and cell streamlining may be ancestral in archegoniate spermatozoids.
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Functions of conserved centriole proteins in African trypanosomesScheumann, Nicole January 2012 (has links)
Centriole and basal bodies are related nine-fold symmetric microtubule-based eukaryotic organelles central to the organisation of cilia/flagella and centrosomes. Mechanisms of eukaryotic centriole and basal body assembly are mainly based on studies in animal systems. To understand which centriolar proteins are the universally important ones in the assembly across eukaryotes, a bioinformatic survey presented here investigates the distribution of centriolar and cilia-associated proteins across a diverse range of eukaryotes. This analysis showed also that the basal body function is ancestral to eukaryotes, whereas centrosomal components are specific to Holozoa (which include animals). It also suggested that the ancestor of all eukaryotes possessed a cilium/cilia not only with motility function but also with a sensory role. The most frequently conserved proteins in extant ciliated eukaryotes found in this analysis included SAS-6, SAS-4 and WDR16. To test whether these proteins are also important for basal body assembly in distantly-related species to metazoan and other model organisms where the proteins have been studied to date, the proteins were investigated in Trypanosoma brucei. I used a combination of genetic tools and microscopy techniques to demonstrate that SAS-6 but not SAS-4 is essential for basal body assembly in T. brucei. I showed that WDR16 is a stably integrated component of the transition zone and axoneme but not the basal body. Furthermore, I identified a novel SAS-6 like protein which localises to a position consistent with the basal plate and has the capacity to form into filaments. This thesis provides new insights into the evolution of centrioles and basal bodies, and into the function of conserved centriole proteins in T. brucei, a distantly-related organism to animals.
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The centriole in evolution : from motility to mitosisSmith, Amy Elisabeth January 2013 (has links)
Centrioles and basal bodies with their characteristic 9+2 structure are found in all major eukaryotic lineages. The correlation between the occurrence of centrioles and the presence of cilia/flagella, but not centrosome-like structures, suggests that the ciliogenesis function of centrioles is ancestral. Here, it is demonstrated that the centriole domain of centrosomes emerged within the Metazoa from an ancestral state of possessing a centriole with basal body function but no functional association with a centrosome. Centrosome structures involving a centriole are metazoan innovations. When an axoneme is still present but no longer fully functional, such as the sensory cilia of Caenorhabditis elegans or, as depicted here, the flagellum of the intracellular amastigote stage of the Leishmania mexicana parasite, the basal body structure is less constrained and can depart from the canonical structure. A general view has emerged that classifies axonemes into canonical motile 9+2 and noncanonical, sensory 9+0 structures. This study reveals this view to be overly simplistic, and additional axonemal architectures associated with potential sensory structures should be incorporated into prevailing models. Here, a striking similarity between the axoneme structure of Leishmania amastigotes and vertebrate primary cilia is revealed. This striking conservation of ciliary structure, despite the evolutionary distance between Leishmania and mammalian cells, suggests a sensory function for the amastigote flagellum. Adding weight to a sensory hypothesis, close examination of Leishmania positioning inside the parasitophorous vacuole revealed frequent contact between the flagellum tip and the vacuole membrane. A sensory function could also explain the retention of a flagellum in Trypanosoma cruzi amastigotes, an intracellular stage that, as shown in this study, emerged independently to the Leishmania amastigote. Basal body appendages, such as pro-basal bodies and microtubule rootlets, also vary widely in their structure. Choanoflagellates, a sister group to the Metazoa, posses an extensive microtubule rootlet system that provides support for their characteristic collar tentacles. This atypical structure is reflected in the underlying molecular components of the choanoflagellate basal body. The importance of choanoflagellates as the closest known relative of metazoans was first revealed by their similarity to choanocytes, the feeding cells of sponges. Although phylogenetic analyses leave little doubt that choanoflagellates are a sister group of animals, comparisons of molecular and structural components of appendages associated with the collar tentacles highlight significant differences and questions the extent to which the collar structures of choanoflagellates and choanocytes can be assumed to be homologous. Finally, the confinement of a centriole-based centrosome to the Metazoa provides little support for the flagellar synthesis constraint as an explanation for the origin of multicellularity. There is, indeed, an apparent constraint; no flagellated or ciliated metazoan cell ever divides. This constraint, however, did not arise until after the incorporation of centrioles into the centrosome in the metazoan lineage and the co-option of centrioles as a structural and functional component of the centrosome. The flagellar synthesis constraint is therefore not an explanation for the origin of multicellularity but a consequence of it.
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Etude des protéines VFL3 et OFD1 dans le mécanisme d'ancrage des corps basaux chez la paramécie / Roles of VFL3 and OFD1 in basal body anchoring process in ParameciumBengueddach, Hakim 14 December 2016 (has links)
Les cils sont des organites conservés au cours de l’évolution émanant de corps basaux et qui, motiles ou non, jouent des rôles essentiels dans de nombreux processus physiologiques. Leur formation est conditionnée par le positionnement et l’ancrage correct des corps basaux à la surface cellulaire. Chez la paramécie, trois protéines conservées, FOR20, Centrine 2 et Centrine 3 recrutées séquentiellement jouent un rôle dans ce processus d’ancrage. J’ai réalisé l’analyse fonctionnelle de deux autres protéines évolutivement conservées OFD1 et VFL3 susceptibles d’être impliquées dans cet ancrage. L’analyse d’OFD1 a été également dictée par le fait que sa fonction dans l’assemblage des cils motiles demeurait peu étudiée. Dans l’espèce Paramecium tetraurelia, qui a subi au moins trois duplications globales de son génome au cours de l’évolution, un seul gène code la protéine OFD1 tandis que deux familles VFL3-A et VFL3-B coexistent. La déplétion des protéines de la famille VFL3-B n’ayant pas produit d’effet je n’ai pas pu leur attribuer une fonction mais une de ses isoformes se localise au niveau des corps basaux. Bien qu’OFD1 et les protéines VFL3-A soient impliquées dans le positionnement et l’ancrage des corps basaux, les mécanismes dans lesquels elles interviennent sont différents. Pour OFD1 les défauts d’ancrage étaient associés à des anomalies de formation de la partie distale des corps basaux, ce qui est en accord avec la fonction connue de cette protéine dans l’assemblage des appendices distaux des corps basaux des cils primaires. Elle se localise au niveau de la zone de transition entre les doublets de microtubules et la membrane ciliaire. Les recrutements d’OFD1 et FOR20 au sein des corps basaux sont interdépendants alors qu’il n’y a pas de relation entre le recrutement d’OFD1 et celui de la Centrine 2. Ces observations démontrent une conservation fonctionnelle de la protéine OFD1 dans les mécanismes d’ancrage des cils motiles et précisent ses relations avec FOR20 et Centrine 2. Outre les défauts d’ancrage, la déplétion des deux isoformes VFL3-A induit une distribution anarchique des racines striées qui constituent des marqueurs de leur polarité rotationnelle. Ceci suggère que ces protéines sont impliquées dans l’établissement de cette polarité. Cette polarité étant indispensable au positionnement correct des différents appendices qui guident le mouvement des corps basaux néoformés vers la surface cellulaire, son altération pourrait expliquer les défauts d’ancrage observés. Une isoforme de VFL3-A se localise transitoirement à l’extrémité proximale des corps basaux pères à un stade précoce de leur duplication entre la racine striée et les microtubules auxquels elles sont associées. Cette protéine pourrait donc constituer un facteur extrinsèque contrôlant la polarité du corps basal. L’ensemble de ces résultats souligne la complexité du mécanisme d’ancrage des corps basaux chez cet organisme qui est conditionné non seulement par un assemblage correct de leur extrémité distale mais également par celui de ses structures associées en partie proximale. / Cilia are evolutionary conserved organelles developing from basal bodies and which play essential roles in many physiological processes. Their development depends upon a correct anchoring of basal bodies at the cell surface. In Paramecium, three conserved proteins, FOR20, Centrin 2 and Centrin 3, sequentially recruited are required for the anchoring process. I analyzed the function of two others conserved proteins, OFD1 and VFL3, likely involved in the anchoring process. In particular, the role of OFD1 in motile cilia biogenesis had not been really studied yet. In P. tetraurelia, which has undergone at least three global genome duplications, a single gene encodes OFD1, while two families VFL3-A and VFL3-B coexist. Depletion of the VFL3-B proteins produced no effect, but VFL3-3 was localized at the basal bodies. Although OFD1 and the VFL3-A proteins are both involved in the positioning and anchoring of the basal bodies, they participate in different mechanisms. Concerning OFD1, the anchoring defects reflected defects in basal body distal part assembly, in agreement with its known role in the assembly of the distal appendages of primary cilia. It localizes in the transition zone, between the microtubule doublets and the ciliary membrane. The recruitment of OFD1 and FOR20 to the basal bodies is interdependent, while OFD1 and Centrin2 were not. These observations demonstrate the conserved role of OFD1 in the anchoring mechanisms in motile cilia and clarify its relations with FOR20 and Centrin 2. In addition to the anchoring defects, depletion of the two VFL3-A isoforms causes an anarchic distribution of the striated rootlets which mark the rotational polarity of basal bodies. This suggests that these proteins are involved in the establishment of this polarity, required for the correct positioning of the different appendages which guide the neoformed basal bodies towards the cell surface. One isoform of VFL3-A is transiently localizes at the proximal tip of the mother basal body, at an early stage of its assembly, between the striated rootlet and the microtubules to which they are associated. VFL3-1 might then be an extrinsic polarity factor for the basal body. Altogether, these results underscore the complexity of the anchoring process which requires not only the correct assembly of the distal part but also of the proximal appendages in Paramecium.
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The little engine that could: Characterization of noncanonical components in the speed-variable flagellar motor of the symbiotic soil bacterium Sinorhizobium melilotiSobe, Richard Charles 07 June 2022 (has links)
The bacterial flagellum is a fascinating corkscrew-shaped macromolecular rotary machine used primarily to propel bacterial cells through their environment via the conversion of chemical potential energy into rotational power and thrust. Flagella are the principal targets of complex chemotaxis systems, which allow microbes to navigate their habitats to locate favorable conditions and avoid harmful ones by continuous sampling of environmental compounds and cues. Flagella serve as surface and temperature sensors, mediators of host cell adherence by bacterial pathogens and symbionts alike, and important virulence factors for disease-causing microbes. They play several essential roles in accelerating the foundational stages of biofilm formation, during which bacteria build highly intricate microbial communities with increased resistance to predation and environmental assaults. Flagellum-mediated chemotaxis has broad and impactful implications in fields of bioremediation, targeted drug delivery, bacterial-mediated cancer therapy and diagnostics, and cross-kingdom horizontal gene transfer.
While the core structural and functional components of flagella have been well characterized in the closely related enteric bacteria, Escherichia coli and Salmonella typhimurium, major departures from this paradigm have been identified in other diverse species that merit further investigation. Many bacteria employ additional reinforcement modules to surround and stabilize their more powerful flagellar motors and provide increased contact points in the inner membrane, the peptidoglycan sacculus, and, in Gram-negative bacteria, the outer membrane. Additionally, the soil-dwelling bacterium Sinorhizobium meliloti exhibits marked distinctions in the regulation, structure, and function of its navigation systems. S. meliloti is a nitrogen-fixing symbiont of the agronomically valuable leguminous plant, Medicago sativa Lucerne, and uses its coupled chemotaxis and flagellar motility systems to search for host plant roots to colonize. Following root colonization, the bacterium converts to a nitrogen-fixing factory for the plant and the combined influences of this symbiosis can quadruple the yields of the host.
This dissertation is aimed at delivering a thorough representative overview of the processes facilitating bacterial flagellum-mediated chemotaxis and motility. Chapter 1 describes the interplay between chemotaxis and flagellar motility pathways as well as the structure, function, and regulation of these systems in several model bacteria. Particular emphasis is placed on the comparison of flagellar systems from the soil-dwelling legume symbiont, Sinorhizobium meliloti with other model systems, and a brief introduction is provided for its primary counterpart, the agronomically valuable legume, Medicago sativa, more commonly referred to as alfalfa.
Chapter 2 embodies the first report of a flagellar system to require two copies of a protein known as FliL for its function. FliL is found in all bacterial flagellar systems reported to date but is only essential for some to drive motility. The more conserved copy of the protein has retained the title of FliL and several experiments to assay the proficiency of flagellar motor function revealed that in the absence of FliL swimming is essentially abolished as is the presence of flagella on the cell body. Flagellar motor activity and swimming proficiency of mutants lacking the FliL-paralog MotF was nearly as abysmal as those without FliL but flagellation was essentially normal indicating distinct roles for the two proteins. FliL is implicated in initial stator recruitment to the motor while MotF was found to serve as a power or speed modulator. A model to accommodate and explain the roles of these proteins in the flagellar motor of S. meliloti is provided.
Chapter 3 links a never-before characterized flagellar protein, currently named Orf23, to a role in promoting maximum swimming velocity and perhaps stator alignment with the rotor in a peptidoglycan-dependent manner. The loss of LdtR, a transcriptional regulator of peptidoglycan-modification genes, caused defects in swimming motility that are restored only by removal of Orf23 or by replacing a nonpolar glycine with a polar serine in the periphery of stator units. Bioinformatics analyses, immunoblotting, and membrane topology reporter assays revealed that Orf23 is likely embedded in the inner membrane and that the remainder of the protein extends into the periplasm. Building on findings from Chapter 2, Orf23 is anticipated to influence stator positioning through interactions with MotF, FliL, and/or stator units directly. The chapter is concluded with the description of future experiments aimed to more thoroughly characterize Orf23.
Altogether, this work increases the depth and breadth of knowledge regarding the composition and function of the speed-variable bacterial flagellar motor. We have identified several components required for stator incorporation and function, as well as an accessory component that improves stator performance. A wise society will draw inspiration from these fascinating and powerful machines to inform new technologies to achieve modern goals including targeted drug delivery, bioremediation, and perhaps one day our own exploration. / Doctor of Philosophy / Bacteria are small autonomous single-celled organisms capable of existing and thriving in highly diverse environments. Motility is achieved by these organisms in various ways, but the most common approach is to produce one or more corkscrew-shaped propeller systems known as flagella that are constructed upon and anchored within the wall of the bacterial cell. Rotation of these propellers relies on power converters known as stators to transform the flow of ions down self-produced gradients into useful rotational energy. This process can be likened to the way that the stored energy of water behind a dam can be harnessed and used to power hydroelectric generators. While the core components of flagellar motors are well conserved and understood among distantly related bacteria, billions of years of evolution and refinement of additional structures have allowed bacteria to accommodate swimming in diverse habitats with e.g. low nutrient availability or high viscosity.
Here we describe the discovery and characterization of additional components in the flagellar motor system of the soil-dwelling bacterium Sinorhizobium meliloti to navigate soil environments. We report the first identification of a flagellar motor that requires two copies of a pervasive flagellar motor protein known as FliL and have named the more distinct version of the protein MotF. We found that FliL is required for the power converter components to install into the motor and that MotF is necessary to activate them. Next, we identify another motor component, Orf23, that is dispensible for motility but appears to be required to achieve maximum swimming velocity and may serve to shift the motor into a "higher gear". We find that disruption of a regulator of cell wall modification systems leads to defects in motility that are only restored when Orf23 is removed or when the power converter is modified. Ideas are proposed for how FliL, MotF, and Orf23 are integrated into the motor and may contribute to stator function.
An advanced understanding of the mechanisms governing flagellar motor structure and function will provide avenues for the improvement of bacteria-based agricultural improvements, development of optimized bacteria-mediated drug delivery systems, bioremediation techniques, and more.
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The evolution of eukaryotic ciliaHodges, Matthew Edmiston January 2011 (has links)
Eukaryotic cilia are complex, highly conserved microtubule-based organelles with a broad phylogenetic distribution. Cilia were present in the last eukaryotic common ancestor and many proteins involved in cilia function have been conserved through eukaryotic diversification. The evolution of these ciliary functions may be inferred from the distribution of the molecular components from which these organelles are composed. By linking protein distribution in 45 diverse eukaryotes with organismal biology, I define an ancestral ciliary inventory. Analysis of these core proteins allows the inference that the cenancestor of the eukaryotes possessed a cilium for motility and sensory function. I show that the centriolar basal body function is ancestral, whereas the centrosome is specific to the Holozoa, and I use this information to predict a number of roles for proteins based on their phylogenetic profile. I also show that while remarkably conserved, significant divergence in ciliary protein composition has occurred in many lineages, such as the unusual centriole of Caenorhabditis elegans and the transitional changes throughout the land plants. I exemplify this divergence through ultrastructural studies of the fern Ceratopteris richardii and the liverwort Marchantia polymorpha both of which have cilia that exhibit a number of distinctive morphological features, the most conspicuous of which is a general breakdown of canonical microtubule arrangements. Cilia have also been lost multiple times in different lineages: at least twice within the land plants. During these evolutionary transitions proteins with ancestral ciliary functions may be lost or co-opted into different functions. I have interrogated genomic data to identify proteins that I predict had an ancestral ciliary role, but which have been maintained in non-ciliated land plants. I demonstrate that several of these proteins have a flagellar localisation in protozoan trypanosomes and I use expression data correlation to predict potential non-ciliary plant roles.
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