UVM Theses and Dissertations
Format:
Print
Author:
Stark, Benjamin C.
Dept./Program:
Cell and Molecular Biology Program
Year:
2013
Degree:
Ph. D.
Abstract:
Myosin motors compose a large family of proteins involved in numerous cellular activities including muscle contraction, vesicle transport, endocytosis, signal transduction, cell polarization, and cell division. Type-II myosins, the largest class of myosin motors, have been extensively studied in muscle physiology, yet members of this class of myosin are also found in non-muscle cells and their regulation is not well understood. Non-muscle myosin-II plays an active role in cellular division, with the actomyosin ring driving cleavage furrow formation. The machinery required for the generation of the cleavage furrow is conserved from yeast to humans, allowing yeast to serve as a model organism for the study of cytokinesis.
Tropomyosin is an actin-binding protein found throughout eukaryotes. In muscle cells, tropomyosin acts in conjunction with the troponin complex to regulate myosin-II binding to actin filaments in a calcium-dependent manner. In non-muscle cells, there exist over 40 alternatively spliced and differentially expressed isoforms of tropomyosin resulting in a much more complex regulatory mechanism. Fission yeast offer an excellent model system to study the regulation of the myosin-II motor by tropomyosin, as there is only one isoform (Cdc8p), which is essential for cytokinesis. Yet how Cdc8p and the type-II myosin, Myo2p, operate at the contractile ring remains to be fully understood. Changing the level of Myo2p influences contractile ring dynamics, whereas mutations to the motor domain or Cdc8p lead to delays in ring assembly. By doubling the amount of Myo2p in the cell, ring assembly defects associated with a cdc8 mutation were suppressed.
Experiments using purified proteins demonstrated the direct regulation of Myo2p by Cdc8p. Cdc8p-bound actin filaments increased the rate of Myo2p ATP hydrolysis. The Cdc8p-decorated filaments also showed a decrease in filament velocity while the efficiency of filament gliding was enhanced in the in vitro motility assay. By calculating the duty ratio for Myo2p with and without Cdc8p bound to actin, we found Cdc8p led to a two-fold increase in the amount of time Myo2p was strongly bound to actin filaments thus promoting more efficient actomyosin interactions.
DCS (Unc-45-/Crol-/She4-related) domain proteins serve as another form of myosin regulators. Their conserved DCS domain, a variable Central domain, and an N-terminal TPR domain characterize these proteins. The DCS domain has been shown to interact with myosin motors in numerous organisms. In higher eukaryotes the TPR domain has been shown to interact with the chaperone Hsp90, suggesting a role for the DCS proteins to act as a co-chaperone for proper myosin folding. Fungal DCS proteins lack a TPR domain suggesting they may function in a TPR-independent mechanism. In Schizosaccharomyces pombe, the DCS protein, Rng3p, is essential for cytokinesis. By examining contractile ring dynamics, we found the primary role of Rng3p lies in ring assembly, specifically in promoting Myo2p activity.
Purifying Myo2p in the absence of Rng3p function illustrated the DCS protein is not necessary for proper actin binding, but is essential to support actin filament gliding. Rng3p has also been proposed to function in the de novo folding of Myo2p as well as all other fission yeast myosin heavy chains. We found the type-I (Myo1p) and type-V (Myo52p) myosins do not require Rng3p function, suggesting its function is specific for Myo2p. Both regulation of the Myo2p motor by Cdc8p and Rng3p lead to efficient contractile ring assembly by promoting efficient actomyosin interactions and maintaining a local sub-population Qf active motors.
Tropomyosin is an actin-binding protein found throughout eukaryotes. In muscle cells, tropomyosin acts in conjunction with the troponin complex to regulate myosin-II binding to actin filaments in a calcium-dependent manner. In non-muscle cells, there exist over 40 alternatively spliced and differentially expressed isoforms of tropomyosin resulting in a much more complex regulatory mechanism. Fission yeast offer an excellent model system to study the regulation of the myosin-II motor by tropomyosin, as there is only one isoform (Cdc8p), which is essential for cytokinesis. Yet how Cdc8p and the type-II myosin, Myo2p, operate at the contractile ring remains to be fully understood. Changing the level of Myo2p influences contractile ring dynamics, whereas mutations to the motor domain or Cdc8p lead to delays in ring assembly. By doubling the amount of Myo2p in the cell, ring assembly defects associated with a cdc8 mutation were suppressed.
Experiments using purified proteins demonstrated the direct regulation of Myo2p by Cdc8p. Cdc8p-bound actin filaments increased the rate of Myo2p ATP hydrolysis. The Cdc8p-decorated filaments also showed a decrease in filament velocity while the efficiency of filament gliding was enhanced in the in vitro motility assay. By calculating the duty ratio for Myo2p with and without Cdc8p bound to actin, we found Cdc8p led to a two-fold increase in the amount of time Myo2p was strongly bound to actin filaments thus promoting more efficient actomyosin interactions.
DCS (Unc-45-/Crol-/She4-related) domain proteins serve as another form of myosin regulators. Their conserved DCS domain, a variable Central domain, and an N-terminal TPR domain characterize these proteins. The DCS domain has been shown to interact with myosin motors in numerous organisms. In higher eukaryotes the TPR domain has been shown to interact with the chaperone Hsp90, suggesting a role for the DCS proteins to act as a co-chaperone for proper myosin folding. Fungal DCS proteins lack a TPR domain suggesting they may function in a TPR-independent mechanism. In Schizosaccharomyces pombe, the DCS protein, Rng3p, is essential for cytokinesis. By examining contractile ring dynamics, we found the primary role of Rng3p lies in ring assembly, specifically in promoting Myo2p activity.
Purifying Myo2p in the absence of Rng3p function illustrated the DCS protein is not necessary for proper actin binding, but is essential to support actin filament gliding. Rng3p has also been proposed to function in the de novo folding of Myo2p as well as all other fission yeast myosin heavy chains. We found the type-I (Myo1p) and type-V (Myo52p) myosins do not require Rng3p function, suggesting its function is specific for Myo2p. Both regulation of the Myo2p motor by Cdc8p and Rng3p lead to efficient contractile ring assembly by promoting efficient actomyosin interactions and maintaining a local sub-population Qf active motors.