The synapse, the minimal element required for interneuronal communication in the nervous sytems, is a structure with a great deal of plasticity, capable of undergoing changes that alter transmission strength, and even forming new connections. This property has great implications for a number of processes, including circuit formation and learning and memory. However, the proteins behind this synaptic plasticity are still not fully understood. To uncover and characterize the proteins that regulate the plastic nature of the synapse, I turned to the Drosophilalarval neuromuscular junction (NMJ), a powerful and accessible model system. I began by examining synaptic cell adhesion, as Cell Adhesion Molecules (CAMs) have long been implicated in synaptic outgrowth as well as learning and memory. CAMs have traditionally been thought of as molecules that mediate cell adhesion between the pre- and postsynaptic membrane. However, through the course of the studies presented here I demonstrate a CAM function that goes beyond simple cell adhesion, acting as a receptor that transduces adhesive signals to the intracellular space. In particular, I have demonstrated a role for the Drosophila CAM, Fasciclin II(FasII), in a signaling complex involving the Amyloid Precursor Protein-Like (APPL) and the Drosophila homolog of X11/MINT/Lin-10 (dX11). Further results show that deletion of either APPL or dX11 inhibits the FasII mediated outgrowth. These studies show that during NMJ expansion the transinteraction between FasII molecules in the pre- and postsynaptic membrane results in the recruitment of APPL and dX11 to the presynaptic cell surface, and the initiation of a signaling cascade that leads to bouton outgrowth. The next question addressed here was regarding the cytoskeletal changes that must occur during synapse remodeling. In particular I centered on the evolutionarily conserved cell polarity complex aPKC-Par3-Par6, which is know to regulate axon growth, the cell cytoskeleton during polarized cell division, and learning and memory. To understand the role of the cytoskeleton during NMJ expansion, I examined the organization of microtubules and actin during this process. Further, I identified atypical protein kinase C (aPKC) as a regulator of microtubule dynamics. I found that aPKC is required for regulating the degree of stabilization of synaptic microtubules. This stabilization requires the Microtubule Associated Protein-1B (MAP1B) homolog Futsch, which I demonstrated was required for aPKC to associate with and stabilize the microtubule cytoskeleton. The process of synaptic expansion not only requires modifications to the presynapse, but to the postsynapse as well. Previous work demonstrates that levels of the scaffolding proteins DrosophilaMembrane Associated Guanlyate Kinase (MAGUK) protein Discs-large (DLG), as well as the vertebrate homolog Postsynaptic Density-95 (PSD-95), which are concentrated at synapses, determine the size of postsynaptic membranes. To identify the underlying mechanisms of the regulation of postsynaptic size, we performed a yeast two hybrid screen, searching for DLG interacting proteins. We found a novel interaction between DLG, and a t-SNARE, GUK-interacting Syntaxin (Gtaxin; GTX), and went on to demonstrate that this interaction is required for proper postsynaptic membrane addition. Strong hypomorphic mutations in either dlg or gtx show a dramatic reduction in postsynaptic expansion. Overexpression of DLG produces an increase of synaptic GTX, as well as an increase in postsynaptic size, and an increased formation of GTX positive SNARE complexes. Taken together, these observations suggest that the MAGUK DLG regulates postsynaptic membrane addition by modulating the formation of a SNARE complex of the t-SNARE Gtaxin, and by targeting GTX to sites of postsynaptic membrane addition. In summary, the studies performed in this thesis probe a trans-synaptic adhesion based signaling complex required for presynaptic expansion, a specific pathway for dynamic microtubule stabilization required for pre- and postsynaptic expansion, and how a scaffolding protein regulates postsynaptic membrane expansion. These processes are all interconnected to maintain the efficacy of the synapse. The studies conducted revealed important information about how these processes are accomplished, and constitute an important step to elucidate the mechanisms by which synapse plasticity occurs at the level of single synaptic terminals.

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.

No description available.

No description available.

No description available.

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...