Cells divide to create two daughter cells through cytokinesis. Daughter cells of different sizes are created by shifting the position of the cleavage furrow. The cleavage furrow forms at the position of the metaphase plate so in asymmetric cytokinesis the spindle is shifted towards one pole. Unlike most systems, Drosophila neuroblasts have a centrally localized metaphase plate but divide asymmetrically. Drosophila neuroblasts divide asymmetrically due to the presence of a polarized myosin domain at the basal pole during mitosis. I investigated the mechanism by which the basal myosin domain produces asymmetric cytokinesis and the pathway regulating this domain. We tested several mechanisms by which the basal myosin domain could lead to asymmetric cytokinesis. Based on surface area and volume measurements, I demonstrated that asymmetric addition of new membrane is not involved. I determined that neuroblasts exhibit asymmetric cortical extension during anaphase with the apical pole extending 2-3 times more than the basal pole. Mutants that lose basal myosin extend equally at both poles supporting this model. Mutants that retain apical myosin exhibited symmetric cortical extension but still divided asymmetrically, demonstrating that asymmetric cortical extension is not required for asymmetric cytokinesis. Observations of the mitotic spindle show that the cleavage furrow forms at a position biased towards the basal pole when compared to the position of the metaphase plate even though this position is still equidistant between the centrosomes. I observed that midzone components shift basally in a basal domain dependent manner suggesting that contraction of the basal domain leads to new microtubule-cortex interactions at a position away from the spindle midzone. I demonstrated that the basal domain is regulated by the heterotrimeric G protein, Gβ13F, which is activated by Pins. In Gβ mutants, the localization of all basal components (myosin, anillin, and pavarotti) is lost and the cells divide symmetrically. Although the basal domain is contiguous with equatorial myosin, it is not regulated by the same pathway and photobleaching experiments indicate that they exhibit different behaviors during anaphase suggesting a difference in temporal regulation. This dissertation includes previously published coauthored material.

Fluorescence spectroscopy can be used to probe protein conformation and is recognized as a technique that provides very specific information. It has been applied/ in recent years/ to the study of tropomyosin (TM) and its role in regulation of contractile processes. In this thesis, two different approaches were used to further the understanding of the structure/function relationship in the two chain coiled coil of tropomyosin. The first involves a comparative study on TM and non-polymerizable TM (NPTM) (Mak, A.S., and Smillie, L.B. (1981) Biochim. Biophys. Res. Commun., 101, 208-214). Fluorescence involving pyrene (Py) and acrylodan (AD) bound at the only cysteine residue in the molecule (Cys-190), and circular dichroism (CD) studies led to the main conclusion that, while the two species, are very similar in stability, the COOH-terminus is required to hold the Cys-190 region in a specific conformation. This long-range structural effect may play a role in regulation of contraction. A species having one intact COOH-terminus, made by hybridizing TM and NPTM, was found to be non-polymerizable suggesting that one intact COOH-terminus is insufficient to permit overlap with the NH₂-terminus of a neighbouring TM under polymerizing conditions. Unlike the TM/NPTM hybrid, the hybrid of TM and platelet TM (P-TM) was difficult to make due to the sequence mismatches in the terminal regions, but small quantities could be detected by loss of excimer fluorescence from Py-P-TM on rapid cooling of a heated mixture of Py-P-TM and cardiac TM (C-TM). The second approach was to investigate the effect of actin-binding proteins on the structure and function of tropomyosin. DNase I depolymerizes F-actin and is known to interfere with the end-to-end polymerizability of tropomyosin (Payne, M.R., Baydoyannis, H., and Rudnick, S.E. (1986) Biochim. Biophys. Acta 883, 454-459). Results presented here from fluorescence studies suggest that this effect is caused by a localized loss of structure in the tropomyosin at the sites of labelling upon binding of DNase I. This result is supported by CD studies on labelled and unlabelled tropomyosins. Gelsolin is another actin-binding protein found in many cell types and in extracellular fluids. It is shown here to be able to depolymerize tropomyosin, but its mechanism of action is not the same as that of DNase I. The effect of interaction of gelsolin on the structure of tropomyosin, as determined from fluorescence studies, is negligible. / Science, Faculty of / Chemistry, Department of / Graduate

No description available.

Nonmuscle myosin from bovine thymus was purified, characterized , and phosphorylated with MLCK, H4PK, and Protein Kinase C. Phosphorylation occured exclusively on the myosin regulatory light chain. Phosphorylation by MLCK and H4PK resulted in the activation of the MgATPase activity as well as filament assembly of nonmuscle myosin.

No description available.

In this experiment, fluorescent N- (1-pyrenyl) iodoacetamide modified the two reactive thiols, SH1 (Cys 707) and SH2 (Cys 697) on myosin to detect SH1-SH2 a -helix melting. The excimer forming property of pyrene is well suited to monitor the dynamics of the SH1 and SH2 helix melting, since the excimer should only form during the melted state. Decreased anisotropy of the excimer relative to the monomeric pyrene fluorescence is consistent with the disordering of the melted SH1-SH2 region in the atomic model. Furthermore, nucleotide analogs induced changes in the anisotropy of the excimer, suggesting that the nucleotide site modulates the flexibility of SH1-SH2 region.

Toxoplasma gondii is an obligate intracellular parasite that can cause lifethreatening disease in immunocompromised individuals. Host cell invasion is therefore central to the pathology of the disease and parasite survival. Unlike many intracellular pathogens, T. gondii does not enter cells by manipulating the host’s phagocytic machinery; instead, the parasite enters the cell by a process of active penetration. Gliding motility and active penetration are driven by a complex of proteins termed the glideosome. The glideosome consists of four major proteins: TgMyoA, an unconventional myosin XIV, myosin light chain (TgMLC1) and glideosome-associated proteins 45 and 50 (TgGAP45, TgGAP50). TgMyoA has been shown to be essential for parasite motility, but the role of TgMLC1 in regulating myosin function remains unknown. Our lab has identified an inhibitor of T. gondii motility and invasion that results in a post-translational modification (PTM) to TgMLC1. Using molecular genetic and mass spectrometry methods we have shown cysteine 53 and cysteine 58 of TgMLC1 are essential for the modification to occur. To determine if the TgMLC1 PTM alters TgMyoA activity, glideosomes were isolated from DMSO- and 115556-treated parasites. Using an in vitro motility assay we have shown that the TgMyoA actin filament displacement velocities are decreased after 115556 treatment. This is the first evidence that TgMLC1 plays a role in regulating TgMyoA activity. The TgMLC1 PTM is responsible, at least in part, for the invasion and motility defects seen in the parasite after compound treatment. During the course of our investigations we have shown that TgMLC1 is dimethylated on lysine 95. This is an unusual modification for cytosolic proteins and has not been previously described for MLCs. Experiments using parasites expressing a non-methylatable form of TgMLC1 (TgMLC1-K95A) show that dimethylation is not necessary for TgMLC1 peripheral localization, TgMLC1 protein-protein interactions and is not required for TgMyoA activity in vitro. However, TgMLC1-K95A does not appear to be phosphoryalted indicating that TgMLC1 dimethylation is necessary for efficient phosphorylation of TgMLC1. These experiments will provide new insight into the ways in which TgMLC1 regulates this unconventional myosin motor complex.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "December 2000." Typescript. Vita. Includes bibliographical references (leaves 190-220). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 190-220). Also available on the Internet.

No description available.