Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 164-183.

Intraflagellar transport (IFT), the bi-directional movement of particles along the length of flagella, is required for flagellar assembly. The IFT particles are moved by kinesin II from the base to the tip of the flagellum, where flagellar assembly occurs. The IFT particles are then moved in the retrograde direction by cytoplasmic dynein 1b/2 to the base of the flagellum. The IFT particles of Chlamydomonas are composed of ~16 proteins, organized into complexes A and B. Alhough IFT is believed to transport cargoes into flagella, few cargoes have been identified and little is known about how the cargos are transported. To study the mechanism of IFT and how IFT is involved in flagellar assembly, this thesis focuses on two questions. 1) In addition to a heavy chain, DHC1b, and a light chain, LC8, what other proteins are responsible for the retrograde movement of IFT particles? 2) What is the specific function of an individual IFT-particle protein? To address these two questions, I screened for Chlamydomonas mutants either defective in retrograde IFT by immunofluorescence microscopy, or defective in IFT-particle proteins and D1bLIC, a dynein light intermediate chain possibly involved in retrograde IFT, by Southern blotting. I identified several mutants defective in retrograde IFT and one of them is defective in the D1bLIC gene. I also identified several mutants defective in several IFT-particle protein genes. I then focused on the mutant defective in D1bLIC and the one defective in IFT46, which was briefly reported as an IFT complex B protein. My results show that as a subunit of the retrograde IFT motor, D1bLIC is required for the stability of DHC1b and is involved in the attachment of IFT particles to the retrograde motor. The P-loop in D1bLIC is not necessary for the function of D1bLIC in retrograde IFT. My results also show that as a complex B protein, IFT46 is necessary for complex B stability and is required for the transport of outer dynein arms into flagella. IFT46 is phosphorylated in vivo and the phosphorylation is not critical for IFT46’s function in flagellar assembly.

Orientador: Jorg Kobarg / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T03:42:39Z (GMT). No. of bitstreams: 1 Migueleti_DeividLucasdosSantos_M.pdf: 8057948 bytes, checksum: ef239a4533686a90e511e97afd0f97ba (MD5) Previous issue date: 2012 / Resumo: A proteína UNC-76 foi identificada como necessária para a fasciculação e elongação de axônios do verme Caenorhabditis elegans durante o desenvolvimento do sistema nervoso. A homóloga de mamíferos FEZ1 apresenta altos níveis de expressão em tecidos neuronais e camundongos knockout para o gene FEZ1 apresentam desvios de comportamento que remetem a desordens neurológicas. O papel de FEZ1 no desenvolvimento do sistema nervoso parece residir na sua associação com elementos do citoesqueleto e vias de sinalização (e.g., PKC?, E4B, DISC1) que conduzem o crescimento axonal e a polarização celular. Trabalhos do grupo mostram que FEZ1 é uma proteína multifuncional (hub), capaz de interagir com mais de 50 parceiros através de seus domínios coiled-coil. Além disso, a superexpressão de FEZ1 em células HEK293 provoca o aparecimento de núcleos multilobulados, um fenótipo comum em alguns tipos de leucemia. Nesse trabalho foi investigado o papel de FEZ1 nos mecanismos causadores dos núcleos multilobulados e as consequências funcionais de sua superexpressão na viabilidade celular, tentando extrapolar esse modelo para leucemias. Análises in silico de diversas leucemias mostraram que FEZ1 está superexpressa em LMAs e que isso pode se relacionar à ocorrência da fusão 11q23/MLL. A expressão de FEZ1 na linhagem leucêmica THP-1 foi detectada por Western blotting, mas, a expressão em PBMCs de pacientes ainda permanece sem provas empíricas. Para avaliar as consequências funcionais da superexpressão, uma linhagem com expressão estável e indutível foi obtida e utilizada em ensaios de proliferação e resistência a quimioterápicos. Porém, não foram observadas diferenças entre as linhagens expressando a fusão FLAG-FEZ1 e as que expressavam o FLAG tag apenas. Em um ensaio de IP-MS utilizando tais linhagens, foram identificadas proteínas cuja interação com FEZ1 pode ser modulada pela atividade de PKCs. Finalmente, a cotransfecção de FEZ1 inteira com coiled-coils C-terminais diminui a formação de núcleos multilobulados em quase 40%. A transfecção com o mutante FEZ1 nocys contendo 5 cisteínas mutadas não teve o mesmo efeito, mas, novos experimentos são necessários para determinar o potencial de sinergismo que esses dois componentes podem ter sobre a ocorrência desse fenômeno / Abstract: The protein UNC-76 was identified as necessary for fasciculation and elongation of axons of the worm Caenorhabditis elegans during development of the nervous system. The mammalian homologue FEZ1 is mostly expressed in neuronal tissues and FEZ1 knockout mice present behavior abnormalities that resemble neurological disorders. The role of FEZ1 in the development of the nervous system seems to lie in its association with cytoskeletal elements and signaling pathways (e.g., PKC?, E4B, DISC1) regulating axon outgrowth and cell polarization. The studies of our group have shown that FEZ1 is a hub, able to interact with more than 50 partners through its coiled-coil domains. Furthermore, overexpression of FEZ1 in HEK293 cells causes the appearance of flower-like nuclei, a common phenotype to certain types of leukemia. In this work the role of FEZ1 in the mechanisms of flower-like nuclei formation and functional consequences of its overexpression on cell viability were investigated, attempting to extrapolate this model for leukemias. In silico analysis of several leukemias showed that FEZ1 is overexpressed in AML patients and that this may relate to the occurrence of 11q23/MLL genetic fusion. FEZ1 expression in leukemic THP-1 cells was detected by Western blotting, but the expression in PBMCs of leukemic patients still lacks empirical evidence. To assess the functional consequences of overexpression, cell lineage with stable and inducible expression of FEZ1 was obtained and used in proliferative assays. However, it was not observed any differences between lineages expressing FLAG-FEZ1 fusion protein or FLAG tag alone. IP-MS assay using these lineages identified proteins whose interaction with FEZ1 could be modulated by the activity of PKCs. Finally, cotransfection of C-terminal coiled-coils and FEZ1 full-length decreases flower-like nuclei formation to nearly 40%. Transfection with FEZ1nocys mutant containing five substituted cysteines did not play the same, but further experiments are needed to determine the potential synergism these two components may have on this phenomenon / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Abstract Functional defects in the muscle specific chloride channel ClC-1 result in reduced chloride conductance and electrical hyperexcitability, which in turn impairs muscle relaxation and leads to myotonia. The gene CLCN 1 codes for ClC-1 in humans, and mutations in CLCN 1 cause the disease known as myotonia congenita. Worldwide over 80 mutations in CLCN1 have been described, but only three were found in patients in Northern Finland. These included two missense mutations and a nonsense mutation. The behavior and localization of the normal and mutated ClC-1 mRNA and protein were analyzed in muscle cell cultures. In intact muscle the ClC-1 protein was seen in the sarcolemma, but after myofiber isolation the protein was located intracellularly. Sarcolemmal localization was restored when myofibers were electrically stimulated or treated with a protein kinase C inhibitor. When mutated ClC-1 proteins were examined in a myofiber cell culture system, retardation in the ER was observed with the two missense mutations. The nonsense mutation did not have an effect on the transport from the ER to the Golgi elements, but the mutated ClC-1 was degraded more rapidly than the wild type ClC-1, at least in myotubes. Both retardation and degradation of the mutated ClC-1 are likely to result in too few channels present at the plasma membrane of the muscle cell to maintain normal physiological function. A very strict quality control in muscle cells was observed. The behavior and survival of multinuclear skeletal muscle cells is dependent on innervation and muscle activity, and the balance between the phosphorylation and dephosphorylation pathways modulates the function of muscle chloride channels. / Tiivistelmä Lihasspesifisen kloridikanavan ClC-1:n toiminnalliset virheet johtavat alentuneeseen kloridin johtumiseen solukalvon läpi ja lihassolun ylieksitoitumiseen. Tämän seurauksena lihaksen rentoutuminen vaikeutuu ja havaitaan myotoniaa, lihasjäykkyyttä. Pohjoissuomalaisesta potilasmateriaalista tautiin johtavia geenimutaatioita löytyi kolme erilaista. Poikkeuksellista havainnoissa on erilaisten mutaatioiden vähyys, mikä on tyypillistä suomalaiselle tautiperinnölle. Yhteensä tämän kloridikanavan mutaatioita on julkaistu yli 80 erilaista. Tutkiessamme normaalin ja mutatoidun ClC-1 lRNA:n ja proteiinin käyttäytymistä ja sijaintia lihassoluviljelmissä. Havaitsimme eron lihasleikkeiden ja eristettyjen myofiibereiden välillä. Lihasleikkeissä ClC-1 paikantui solun pinnalle sarkolemmalle, mutta eristetyissä myofiibereissä lähinnä solun sisälle. Stimuloimalla eristettyjä myofiibereitä sähkövirralla tai käsittelemällä proteiini kinaasi C inhibiittorilla, saimme kloridikanava-proteiinin siirtymään takaisin solun pinnalle. Proteiinitasolla kuljetuksessa on havaittavissa eroja. Aminohappomuutokseen johtavat pistemutaatiot aiheuttivat proteiinin jäämisen endoplasmiseen kalvostoon, kun taas ennenaikaisen stop-kodonin johdosta lyhentynyt proteiini kuljetetaan eteenpäin Golgin laitteeseen. Myotuubeissa tämä lyhentynyt proteiini kuitenkin hajotettiin nopeammin kuin normaali kloridikanavaproteiini. Sekä kuljetuksen hidastuminen että nopeampi hajotus johtavat tilanteeseen, jossa lihassolun solukalvolla on liian vähän kloridikanavia ylläpitämään lihaksen normaalia fysiologista toimintaa. Monitumaisten lihassolujen laaduntarkkailu havaittiin vielä monitahoisemmaksi kuin yksitumaisilla. Monitumainen lihassolu on riippuvainen hermoärsytyksestä ja lihasaktiivisuudesta. Lisäksi fosforylaatioon liittyvä signalointi on tärkeää ClC-1 proteiinin oikealle paikantumiselle lihassolussa.

No description available.

Cotranslational translocation is initiated by targeting of a ribosome-bound nascent polypeptide chain (RNC) to the endoplasmic reticulum (ER) membrane. The targeting reaction is coordinated by the signal recognition particle (SRP) through its interaction with the RNC and the membrane-bound SRP receptor (SR). A vacant translocon is a prerequisite for the subsequent nascent chain release from SRP-SR-RNC complex. It has been proposed that the protease-accessible cytosolic domains of the Sec61p complex play an important role in posttargeting steps by providing the binding site for the ribosome or interacting with the SR to initiate the signal sequence releasing. In this study, we have investigated the detailed mechanism that allows transfer of the ribosome-nascent chain (RNC) from the SRP-SR complex to the translocon using yeast S. cerevisiaeas the model system. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in translocation of nascent polypeptides that utilize the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome binding activity, indicating that the L6 and L8 sec61mutants impact different steps in the cotranslational translocation pathway. An interaction between the signal recognition particle receptor (SR) and the Sec61 complex has been proposed to facilitate transfer of the ribosome-nascent chain (RNC) complex to an unoccupied translocon. The slow growth and cotranslational translocation defects caused by deletion of the transmembrane span of yeast SRβ (srp102pΔTMD) are exaggerated upon disruption of the SSH1 gene, which encodes the pore subunit of a cotranslational translocation channel. Disruption of the SBH2 gene, which encodes the β-subunit of the Ssh1p complex, likewise causes a synthetic growth defect when combined with srp102pΔTMD. The in vivo kinetics of translocon gating by RNCs were slow and inefficient in the ssh1Δ srp102pΔTMD mutant. A critical role for translocon β-subunits in SR recognition is supported by the observation that deletion of both translocon β-subunits causes a block in the cotranslational targeting pathway that resembles elimination of either subunit of the SR, and could be partially suppressed by expression of carboxy-terminal Sbh2p fragments.

in English Formation of mitochondria by the conversion of a bacterial endosymbiont is the fundamental moment in the evolution of eukaryotes. An integral part of the organelle genesis was the displacement of the endosymbiont genes to host nucleus and simultaneous creation of new pathways for delivery of proteins synthesized now in the host cytoplasm. Resulting protein translocases are complexes combining original bacterial components and eukaryote-specific proteins. In addition to these novel protein import machines, some components of the original bacterial secretory pathways have remained in the organelle. While the function of a widely distributed mitochondrial homolog of YidC, Oxa1, is well understood, the role of infrequent components of Sec or Tat translocases has not yet been elucidated. So far, more attention has been paid to their abundant plastid homologs, which assemble photosynthetic complexes in the thylakoid membrane. In the thesis, the structure and function of prokaryotic YidC, Sec and Tat machineries and their eukaryotic homologs are described. By comparing both organelles of the endosymbiotic origin, the hypothesis is drawn on why these translocases have been more "evolutionary successful" in plastids than in mitochondria.

The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7 and TM8 in Sec61p) to expose the signal sequence-binding (SSB) site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in channel gating. The translocation defect caused by L7 mutations could be suppressed by the prl class of sec61 alleles that reduce the fidelity of signal sequence recognition. The prl mutants are proposed to act by destabilizing the closed conformation of the translocation channel. Our results indicate that the equilibrium between the open and closed conformations of the protein translocation channel maintains a balance between translocation activity and signal sequence recognition fidelity. In the opening of the translocation channel, both the lateral and lumenal gate must open in a coordinated fashion for efficient protein translocation to occur. The lumenal gate is composed of a short helix of the loop preceding the second TM span, referred to as the plug helix, and six hydrophobic pore ring residues which form the constriction ring in the center of the channel. We identified three lateral gate polar residues and three hydrophobic residues from the plug domain that affect channel gating. Mutagenesis of the lateral gate polar cluster residues yields either a gain of function (prl phenotype) or a loss of function (translocation defect) phenotype. The combination of polar cluster mutations with each other or with plug domain mutations which cause a prl phenotype resulted in the mutually suppressive or additive phenotypes in double mutant strains. Cooperation between these residues is made possible through a structural link which connects the two translocation channel gates at their interface. The structural link provides a mechanism for the channel to coordinate the movement of multiple domains in the channel gating conformational change. Translocation assays demonstrated that this mechanism of gating regulation is particularly important for efficient protein translocation of substrates using the posttranslational translocation pathway. Our results indicate that residues from the plug and lateral gate domain form a regulatory cluster of residues responsible for efficient translocation channel gating.

Protruding from the apical surface of nearly every cell in our body lies a specialized sensory organelle—the primary cilium. Eukaryotic cells use these ubiquitous structures to monitor the extracellular environment, defects in which result in an ever-growing list of human maladies termed ciliopathies including obesity, retinal degeneration and polycystic kidney disease. The sensory functions of primary cilia rely on the unique complement of receptors concentrated within the ciliary membrane. Vital to the proper functioning of the cilium is the cell's ability to target specific proteins to the ciliary membrane yet little is known how a cell achieves this highly polarized distribution. IFT20, a subunit of the intraflagellar transport particle is localized to the Golgi complex that is hypothesized to sort proteins to the ciliary membrane. We show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP-210 and mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction and heart defects. Cilia on GMAP210 mutant cells have reduced amounts of the membrane protein polycystin-2 localized to them suggesting IFT20 and GMAP-210 function together in the sorting or transport of proteins to the ciliary membrane. To better understand the mechanism of ciliary protein trafficking, we identify a ciliary targeting sequence (CTS) contained within fibrocystin, the gene mutated in autosomal recessive polycystic kidney disease, and investigate a series of proteins required for the delivery of this sequence to the primary cilium. We demonstrate the small G protein Rab8 interacts with the CTS of fibrocystin and controls the ciliary levels of the CTS. Arf4 is another small G protein deemed a key regulator of ciliary protein trafficking. We show Arf4 binds the CTS of fibrocystin but is not absolutely required for trafficking of the fibrocystin CTS to cilia. Arf4 mutant mice are embryonic lethal and die at mid-gestation likely due to defects in the non-ciliated visceral endoderm, where the lack of Arf4 caused defects in cell structure and apical protein localization. This suggests Arf4 is not only important for the efficient transport of fibrocystin to cilia, but also plays critical roles in non-ciliary processes. Together this work aims to elucidate the mechanisms of protein targeting to the ciliary membrane.

The primary cilium is an immotile, microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Critically, the transport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, this work employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. Using SPEED microscopy and single molecule tracking, we mapped the movement of membrane and soluble proteins at the base of the primary cilium. In addition to the well-known intraflagellar transport (IFT) route, we identified two new pathways within the lumen of the primary cilium - passive diffusional and vesicle transport routes - that are adopted by proteins for cytoplasmic-cilium transport in live cells. Independent of the IFT path, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins. While the 3D transport pathways in this work are always replicated multiple times with a high degree of consistency, several experimental parameters directly affect the 3D transport routes’ error, such as single molecule localization precision and the number of single molecule localizations. In fact, if these experimental parameters do not meet a minimum threshold, the resultant 3D transport pathways may not have significant resolution to determine any biological details. To estimate the 3D transport routes’ error, this work will explain in detail the component of SPEED microscopy that estimates 3D sub-diffraction-limited structural or dynamic information in rotationally symmetric bio-structures, such as the primary cilium. This component is a post-localization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability distributions based on prior known structural knowledge. This analysis is ideal in cases where the ultrastructure of a cellular structure is known but the sub-structural localization of a particular protein is not. This work will demonstrate how the 2D-to-3D component of SPEED microscopy can be successfully applied to achieve 3D structural and functional sub-diffraction-limited information for 25-300 nm subcellular organelles that meet the rotational symmetry requirement, such as the primary cilium and microtubules. Furthermore, this work will provide comprehensive analyses of this method by using computational simulations which investigate the role of various experimental parameters on the 3D transport pathway error. Lastly, this work will demonstrate that this method can distinguish different types of 3D transport pathway distributions in addition to their locations. / Biology