The outer dynein arm-docking complex (ODA-DC) specifies the outer dynein arm-binding site on the flagellar axoneme. The ODA-DC of Chlamydomonas contains equimolar amounts of three proteins termed DC1, DC2, and DC3 (Takada et al., 2002). DC1 and DC2 are predicted to be coiled-coil proteins, and are encoded by ODA3 and ODA1, respectively (Koutoulis et al., 1997; Takada et al., 2002). Prior to this work, nothing was known about DC3. To fully understand the function(s) of the ODA-DC, a detailed analysis of each of its component parts is necessary. To that end, this dissertation describes the characterization of the smallest subunit, DC3. In Chapter II, I report the isolation and sequencing of genomic and full-length cDNA clones encoding DC3. The sequence predicts a 21,341 D protein with four EF hands that is a member of the CTER (Calmodulin, Troponin C, Essential and Regulatory myosin light chains) group and is most closely related to a predicted protein from Plasmodium. The DC3 gene, termed ODA14, is intronless. Chlamydomonas mutants that lack DC3 exhibit slow, jerky swimming due to loss of some but not all, outer dynein arms. Some outer doublet microtubules without arms had a "partial" docking complex, indicating that DC1 and DC2 can assemble in the absence of DC3. In contrast, DC3 cannot assemble in the absence of DC1 or DC2. Transformation of a DC3-deletion strain with the wild-type DC3 gene rescued both the motility phenotype and the structural defect, whereas a mutated DC3 gene was incompetent to rescue. The results indicate that DC3 is important for both outer arm and ODA-DC assembly. As mentioned above, DC3 has four EF-hands: two fit the consensus pattern for calcium binding and one contains two cysteine residues within its binding loop. To determine if the consensus EF-hands are functional, I purified bacterially expressed wild-type DC3 and analyzed its calcium-binding potential in the presence and absence of DTT and Mg2+. As reported in Chapter III, the protein bound one calcium ion with an affinity (Kd) of ~1 x 10-5 M. Calcium binding was observed only in the presence of DTT and thus is redox sensitive. DC3 also bound Mg2+ at physiological concentrations, but with a much lower affinity. Changing the essential glutamate to glutamine in both EF-hands eliminated the calcium-binding activity of the bacterially expressed protein. To investigate the role of the EF hands in vivo, I transformed the modified DC3 gene into a Chlamydomonas insertional mutant lacking DC3. The transformed strain swam normally, assembled a normal number of outer arms, and had a normal photoshock response, indicating that the E to Q mutations did not affect ODA-DC assembly, outer arm assembly, or Ca2+-mediated outer arm activity. Thus, DC3 is a true calcium-binding protein, but the function of this activity remains obscure. In Chapter IV, I report the initial characterization of a DC3 insertional mutant having a phenotype intermediate between that of the DC3-deletion strain and wild type. Furthermore, I suggest future experiments that may help elucidate the specific role of DC3 in outer arm assembly and ODA-DC function. Lastly, I speculate that the ODA-DC may play a role in flagellar regeneration.

No description available.

Regulation of size and growth is a fundamental problem in biology and often closely related to functionality and fitness. A prominent example is the mitotic spindle, whose size needs to be perfectly tuned to ensure proper chromosome segregation during cell division. It is known that spindle size generally scales with cell volume, most likely as a result of limiting components. However, this relation breaks down in very large cells where spindles have a maximum size. How the size and microtubule mass are set and why spindles show an upper size limit in large cells is still not understood. Spindles mainly consist of highly dynamic short microtubules that turn over very quickly in comparison to the lifetime of the entire structure. Thus, microtubules need to be constantly created throughout the spindle, a process called nucleation. Understanding the role of microtubule nucleation in setting the size of spindles is limited by the fact that little is known about the rate, distribution, and regulation of microtubule nucleation in these structures. This is partly due to the lack of methods to measure microtubule nucleation in spindles. During this work, I developed an assay based on laser ablation to probe microtubule nucleation in monopolar spindles assembled in Xenopus laevis egg extract. Using this new method in combination with quantitative microscopy, I found that microtubule nucleation in these structures is spatially regulated. Furthermore, I observed that nucleation is stimulated by pre-existing microtubules leading to new microtubule growth in their physical proximity. Combining my experimental results on nucleation with theory and further biochemical perturbations, I show that this autocatalytic nucleation mechanism is limited by the availability of active nucleators. In spindles, the amount of active nucleators decreases with distance from the chromosomes. Thus, this mechanism provides an upper limit to spindle size even when resources are not limiting.

No description available.

Aggregation of microtubule-associated protein tau is involved in the pathology of several neurodegenerative diseases, including Alzheimer’s disease. The affibody TP4 has been shown to inhibit this aggregation process, and its target-binding positions were previously grafted onto a dimeric affibody scaffold, creating the sequestrin seqTP4. This project constitutes a part of the affinity maturation process of seqTP4, using two different bacterial display methods. An error-prone PCR library was first expressed on Staphylococcus carnosus cells for selection of variants with improved tau-binding properties, resulting in a library of 1.4×107 transformants. Flow cytometric tests indicated difficulties in the setup due to nonspecific interactions, and whereas several different approaches to alleviate the problems were investigated, two cell sorting attempts were ultimately unsuccessful. Subcloning of seqTP4 and the library to an Escherichia coli surface display vector resulted in functional surface expression of seqTP4 on E. coli JK321 and BL21 cells, and a BL21 library size of 1.6×109 transformants. An initial flow cytometric test of this library indicates the presence of improved tau-binding variants, paving the way for future cell sorting. / Aggregering av mikrotubuli-associerat protein tau är involverad i patologin av flera neurodegenerativa sjukdomar, däribland Alzheimers sjukdom. Affibodymolekylen TP4 har visat sig inhibera denna aggregeringsprocess, och överföring av dess målbindande positioner till ett dimeriskt affibodyprotein har tidigare gett upphov till seqTP4, en så kallad sequestrin. Detta projekt utgör ett led i processen att affinitetsmaturera seqTP4, med hjälp av två olika metoder för presentation av proteiner på ytan av bakterieceller. Ett error-prone PCR-bibliotek uttrycktes först på ytan av Staphylococcus carnosus-celler för selektion av varianter med ökad affinitet för tau, vilket resulterade i ett bibliotek av 1.4×107 transformanter. Flödescytometriska tester tydde på svårigheter i detta upplägg på grund av ospecifika interaktioner, och emedan flera olika angreppssätt för att förmildra dessa problem undersöktes, misslyckades slutligen två cellsorteringsförsök. Omkloning av seqTP4 och biblioteket till en vektor för ytpresentation på Escherichia coli resulterade i funktionellt ytuttryck av seqTP4 på E. coli JK321- och BL21-celler, och ett BL21-bibliotek bestående av 1.6×109 transformanter. Ett första flödescytometriskt test av detta bibliotek tyder på närvaron av varianter med förbättrad förmåga att binda tau, och vägen ligger nu relativt öppen för cellsortering.

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No description available.

No description available.

During embryogenesis, a single cell gives rise to a multi-cellular embryo through successive rounds of cell division. As cells become smaller, cellular organelles adapt their sizes accordingly. The size of the mitotic spindle—the microtubule-based structure controlling these divisions—is particularly important as it determines the distance over which chromosomes are segregated. To perform its function properly, spindle size scales with cell size. However, we still lack a mechanistic understanding of the underlying microtubule-based processes that regulate spindle scaling. In this thesis, I combined quantitative microscopy and laser ablation in zebrafish embryos and Xenopus laevis egg extract encapsulated in oil droplets. My measurements revealed the influence of microtubule length dynamics, transport, and nucleation on cell size-dependent spindle scaling. Strikingly, I discovered a hierarchical regulation of spindle size. In large cells, microtubule nucleation exclusively scales spindle size relative to cell size by changing the number of microtubules within the spindle. In small cells, microtubule dynamics fine-tune spindle size by modulating microtubule length. To understand the mechanism of spindle scaling, I proposed a theoretical model based on a limiting number of microtubule nucleators and microtubule-associated proteins that regulate microtubule length. The transition from nucleation- to dynamics-based scaling requires that microtubule number and the number of microtubule-associated proteins that promote microtubule growth scale differently with cell size. This can be achieved by sequestering an inhibitor of microtubule nucleation to the cell membrane, which is consistent with my measurements of microtubule nucleation. The differential regimes of spindle scaling modulated by microtubule nucleation and dynamics imply a gradual change in spindle architecture, which may ensure faithful chromosome segregation by spindles of all sizes.

MT nucleation from γ-tubulin complexes, located at centrosome, is an essential step in the formation of MT cytoskeleton. In mammalian cells, -tubulin is encoded by two genes. We functionally characterized two γ-tubulin proteins and have found that both are functionally equivalent. γ-Tubulin 2 is able to substitute for γ-tubulin 1 in MT nucleation. However, we revealed that unlike TUBG1, TUBG2 expression is downregulated in mouse preimplantation development. Mast cells represent effectors of the allergy reaction. Their activation by antigen induces number of cellular processes such as degranulation, proliferation and cytoskeleton rearrangements. The regulatory mechanisms of MT reorganization during mast cell activation are unknown. We identified new signaling proteins, GIT1 and PIX that interact with - tubulin. Depletion of GIT1 or PIX leads to changes in MT nucleation. GIT1 is phosphorylated on tyrosine and associates with γ-tubulin in a Ca2+ -dependent manner. Our data suggested a novel signaling pathway for MT rearrangement in mast cells where tyrosine kinase-activated GIT1 and βPIX work in concert with Ca2+ signaling to regulate MT nucleation. We tested the capability of GIT1 and PIX to influence -tubulin function in more cell types. We found out that GIT1/βPIX signaling proteins together...