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Spontaneous mutations in the human LIS1 gene are responsible for Type I lissencephaly ("smooth brain"). The distribution of neurons within the cerebral cortex of lissencephalic children appears randomized, probably owing to a defect in neuronal migration during early development. LIS1 has been implicated in the dynein pathway by genetic analyses in fungi. We previously reported that the vertebrate LIS1 co-localized with dynein at prometaphase kinetochores, and interference with LIS1 function at kinetochore caused misalignment of chromosomes onto the metaphase plate. This leads to a hypothesis that LIS1 might regulate kinetochore protein targeting. In order to test this hypothesis, I created dominant inhibitory constructs of LIS1. After removal of the endogenous LIS1 from the kinetochore by overexpression of the N-terminal self-association domain of LIS1, dynein and dynactin remained at the kinetochores. This result indicated that LIS1 is not required for dynein to localize at the kinetochore. Next, CLIP-170 was displaced from the kinetochores in the LIS1 full-length and the C-terminal WD-repeat overexpressers, suggesting a role for LIS1 in targeting CLIP-170 onto kinetochores. LIS1 was co-immunoprecipitated with dynein and dynactin. Its association with kinetochores was mediated by dynein and dynactin, suggesting LIS1 might interact directly with subunits of dynein and/or dynactin complexes. I found that LIS1 interacted with the heavy and intermediate chains (HC and IC) of dynein complex, and the dynamitin subunit of dynactin complex. In addition to kinetochore targeting, the LIS1 C-terminal WD-repeat domain was responsible for interactions with dynein and dynactin. Interestingly, LIS 1 interacted with two distinct sites on HC: one in the stem region containing the subunit-binding domain, and the other in the first AAA motif of the motor domain, which is indispensable for the ATPase function of the motor protein. This LIS1-dynein motor domain interaction suggests a role for LIS1 in regulating dynein motor activity. To test this hypothesis, changes of dynein ATPase activity was measured in the presence of LIS1 protein. The ATPase activity of dynein was stimulated by the addition of a recombinant LIS1 protein. Besides kinetochores, others and we have found LIS1 also localized at microtubule plus ends. LIS1 may mediate dynein and dynactin mitotic functions at these ends by interacting with astral microtubules at cortex, and associating with the spindle microtubules at kinetochores. Overexpression of LIS1 displaced dynein and dynactin from the microtubule plus ends, and mitotic progression was severely perturbed in LIS1 overexpressers. These results suggested that the role for LIS1 at microtubule plus ends is to regulate dynein and dynactin interactions with various subcellular structures. Results from my thesis research clearly favored the conclusion that LIS1 activates dynein ATPase activity through its interaction with the motor domain, and this activation is important to establish an interaction between dynein and microtubule plus ends during mitosis. I believe that my thesis work not only has provided ample implications regarding dynein dysfunction in disease formation, but also has laid a significant groundwork for more future studies in regulations of the increasing array of dynein functions.

Diss. (sammanfattning) Umeå : Univ., 2009. / Härtill 4 uppsatser.

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.