Animals, fungi, and amoebas require an actomyosin contractile ring at the division site to perform cytokinesis. The contractile ring initiates and guides the invagination of the plasma membrane as it forms new barriers between the nuclei at the cell equator. Defects in the contractile ring can result in misdirected, delayed, or premature cytokinesis, which leads to abnormal chromosome numbers. Aneuploidies resulting from failed cytokinesis sometimes lead to aggressive forms of cancer. This dissertation was motivated by the goal of better understanding the properties of the contractile ring and how it drives cytokinesis. Actomyosin is initially recruited to the cell equator through the coordination of scaffolding factors, actin-binding proteins, and signaling cascades. Subsequently, the sliding of actin filaments by myosin reshapes the resulting meshwork into a compact ring. Once fully assembled, the contractile ring establishes tension, which leads the plasma membrane inward. The primary motor proteins in the contractile ring of animal cells are class-II nonmuscle myosins, which typically function as bipolar filaments. Filament assembly is activated by phosphorylation and plays a central role in myosin function during cytokinesis. However, many underlying processes that regulate contractile ring function are poorly understood. Current models of cytokinesis have been based on mechanistic insights provided by two decades of work in the fission yeast system Schizosaccharomyces pombe. In fission yeast, the class-II myosin Myo2 provides the major source of motor activity in the contractile ring. Myo2 is two-headed and has a rod-like tail, which is consistent with other class-II myosins. Yet, it was unknown whether Myo2 assembles into filaments, or how phosphorylation affects its activity. To investigate these features, recombinant Myo2 was purified from the baculovirus/Sf9 insect cell expression system. Hydrodynamic measurements were used to examine whether Myo2 forms filaments. These sedimentation velocity data gave no indication that Myo2 self-assembles under the typical physiological salt concentrations, which suggests that Myo2 is unlike any class-II myosin known to date. Myo2 was also treated in vitro with its native kinase Pak1. Phosphorylation of Myo2 molecules had no effect on self-assembly, however it reduced actin-binding in motility assays and increased steady-state ATPase rates by two fold. Our results imply that the function and regulation of fission yeast Myo2 during cytokinesis depends on a specific scaffolding scheme at the plasma membrane, which has not been observed in other eukaryotes. Another interest of this dissertation was how the contractile ring is regulated during cytokinesis. We examined one cytokinesis protein, Cyk3, believed to mediate between the ring and extracellular processes. Genetics and live cell imaging analyses indicated that Cyk3 functions through a catalytically-inactive enzyme domain, which implicated Cyk3's involvement in one of the primary cytokinesis signaling pathways. This dissertation sheds new light on core aspects of how fission yeast undergo cytokinesis, especially with respect to the mechanism of Myo2 activity in the contractile ring. Characterizing the physical and enzymatic properties of an essential myosin in a simple organism should provide insights into cytokinesis in higher organisms.

1. ABSTRACT Myosins are proteins that convert chemical energy stored in ATP into mechanical force that is applied on an actin filament. Nuclear myosin 1 (NM1) was the first myosin detected in the cell nucleus. Together with nuclear actin they were shown to play important roles in DNA transcription and chromatin remodeling. However, the molecular details of the NM1 functions are largely unknown. To expand our knowledge about this molecular motor we studied tissue expression, mechanism of nuclear localization and molecular interactions of this myosin motor. In the first part we examined the expression pattern of NM1 in various mouse tissues. We demonstrated that NM1 is present in cell nuclei of all mouse tissues examined except for cells in terminal stages of spermatogenesis. Quantitative PCR and western blots demonstrated that the expression of NM1 in tissues varies, with the highest levels in the lungs. NM1 is a nuclear isoform of earlier identified myosin 1c (Myo1c), which was described initially as a cytosolic, and plasma membrane associated protein. The only known difference between these two proteins was the presence of additional 16 amino acids at the N-terminus of NM1. Next we focused on the influence of NM1 domains, including the N-terminus, on the subcellular localization of this protein. We found...

(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids. Their inositol head can be phosphorylated at three positions generating seven differently phosphorylated species. Cytoplasmic phosphoinositides regulate membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generate second messengers. In the nucleus, PIs are implicated in pre-mRNA processing, DNA transcription and chromatin remodelling. However, their nuclear functions are still poorly understood. Here we focus on nuclear phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). We describe their localization and interaction with proteins involved in regulation of DNA transcription. PI(4)P localizes to nuclear membrane, nuclear speckles and nucleoplasm. The majority of nuclear PI(4)P is associated with chromatin and colocalizes with H3K4me2. PI(4,5)P2 localizes to nucleoli and nuclear speckles. Besides, 30 % of nuclear PI(4,5)P2 forms small nucleoplasmic PI(4,5)P2 islets. They have carbon rich core, which is probably formed by lipids, and are surrounded by proteins and nucleic acids. The active form of RNA polymerase II associates with PI(4,5)P2 islets and DNA is actively transcribed in the vicinity of PI(4,5)P2 islets. Moreover,...

O objetivo deste trabalho é estudar a afinidade e especificidade de sítios EF-hand, e correlacionar estas propriedades com a estrutura primária do sítio, com as interações entre aminoácidos nas posições de coordenação, e com prováveis características da estrutura terciária da proteína. Os efeitos de três mutações no sítio EF-hand da cadeia leve regulatória de miosina (RLC) foram estudados: D5S, em que o aspartato presente na posição 5 do sítio foi substituído por uma serina; D9E, substituindo o aspartato da posição 9 por um glutamato, e D12E, substituindo o aspartato da posição 12 por um glutamato. Todas as combinações destas três mutações foram produzidas. Os mutantes simples D5S e D9E e o duplo mutante D5S/D9E têm baixa afinidade por cálcio. Todos os mutantes contendo a mutação D12E são específicos para cálcio, com afinidades maiores que RLC tipo selvagem. Todos os mutantes estudados possuem menor afinidade por magnésio que RLC tipo selvagem. As mudanças na energia livre de ligação e as energias de acoplamento sugerem que há interações inespecíficas entre todas as posições, e uma interação específica entre uma serina na posição 5 e um glutamato na posição 9. Esta interação ocorre somente na presença de magnésio, e quando há um aspartato na posição 12. O glutamato na posição 9 pode ser capaz de coordenar a ligação de magnésio diretamente no duplo mutante D5S/D9E. Embora um aminoácido ou um certo arranjo deles possa determinar características específicas do sítio EF-hand, o conjunto de propriedades depende da estrutura terciária, uma vez que sítios homólogos podem possuir afinidades e especificidades bastante diferentes. / The aim of this thesis was to study affinity and specificity in EF-hand sites, and how these properties are related to the site primary structure, interactions between amino acids in coordinating positions, and probable tertiary structure properties. The effects of three mutations on the EF-hand Ca2+/Mg2+ binding site of smooth muscle myosin regulatory light chain (RLC) were studied: D5S, in which an aspartate is replaced by a serine in position 5 of the loop; D9E, in which an aspartate is replaced by a glutamate in position 9, and D12E, in which the aspartate in position 12 is replaced by a glutamate. All possible combinations of the three mutations were produced. The single mutants D5S and D9E and the double mutant D5S/D9E have low affinity for Ca2+. All the mutants containing mutation D12E are Ca2+-specific and have higher affinities than wild type, even when containing mutations D5S or D9E. All the mutants studied have lower affinity for Mg2+ than wild type RLC. Coupling energies and changes in binding free energy suggest that all positions interact in a non-specific way, and a specific interaction occurs between a serine in position 5 and a glutamate in position 9. This interaction can be seen only in the presence of magnesium, and with an apartate in position 12. Glutamate in position 9 may be able to coordinate Mg2+ directly in the double mutant D5S/D9E. Even though an amino acid or a few amino acids in certain positions can determine specific characteristics for an EF-hand site, the site properties depend on the tertiary structure, since homologue sites can have very different affinities and specificities.

Thesis advisor: David R. Burgess / Polarity established by the first cleavages in sea urchin embryos was investigated in this thesis revealing precocious embryonic polarity. Studies of embryonic polarity have focused on protostomes such as <italics>C. elegans</italics>, and those on deuterostomes have focused on later developmental stages. I find asymmetries in the sea urchin membrane cell cortex as early as the first division after fertilization as a result of new membrane addition in the cleavage furrow. Membrane domains and the polarity determinants Par6, aPKC, and Cdc42 are polarized to the apical, or free, cell surface, while the cell-cell contact site remains distinct. Using immunofluorescence, fluorescence recovery after photobleaching (FRAP), and specific inhibitor treatments, myosin filaments were identified as the major regulator of membrane cortex polarity. However, membrane domains and cortical polarity determinants are differentially regulated with respect to blastomere dissociation. These asymmetries are required for proper spindle alignment and cleavage plane determination and are responsible for polarized fluid phase endocytosis. The work in this thesis and future studies addressing the connection between the membrane cortex and myosin filaments has and will lead to a greater understanding of the maintenance of embryonic polarity in cleavage stage sea urchin embryos. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Das Ziel dieser Studie war es, die Beteiligung der nichtmuskulären schweren Myosinketten an der Kontraktion der glatten Muskeln unter physiologischen Bedingungen zu untersuchen. Als Versuchsmodell wurde die Harnblase von neugeborenen Wildtyp und transgenen Mäusen verwendet, bei denen das Gen für die glattmuskelspezifischen schweren Myosinketten durch "Gene Targeting" funktionell eliminiert wurde (Knock-Out). Das Fehlen der Expression der glattmuskelspezifischen schweren Myosinketten wurde durch Elektrophorese und Immunfärbung bestätigt. Im Gegensatz dazu blieb die Expression der nichtmuskulären schweren Myosinketten unverändert. Die mechanische Analyse des glatten Muskels wurde mit intakten Muskelpräparaten aus der Harnblase durchgeführt. Das Muskelpräparat wurde in KCl-Lösung oder mit Phorbolester stimuliert. Die Aktivierung mittels depolarisierender KCl-Lösung führte bei neugeborenen Wildtyp Mäusen zuerst zu einer transienten Kontraktion (Phase 1) mit hoher Kraftentwicklung und maximaler Verkürzungsgeschwindigkeit, und danach zu einer tonischen Kontraktion (Phase 2) mit niedrigerer Kraftentwicklung und maximaler Verkürzungsgeschwindigkeit. Blasenpräparate neugeborener Knock-Out Mäuse dagegen zeigten keine Phase 1, sondern nur eine tonische Kontraktion, die mit Wildtyp Mäusen vergleichbar war. Daher scheint nichtmuskuläres Myosin an der tonischen Kontraktion des glatten Muskels beteiligt zu sein. Durch Stimulierung mit Phorbolester waren ähnliche tonische Muskelkontraktionen der Blasenpräparate sowohl bei Wildtyp als auch bei Knock-Out Mäusen zu beobachten. Vermutlich wird also das nichtmuskuläre Myosin durch Stimulierung mit Phorbolester aktiviert. Intrazelluläre Filamente wurden durch Immunfluoreszenz mit einem spezifischen Antikörper gegen nichtmuskuläre schwere Myosinketten in kultivierten primären glatten Muskelzellen untersucht. Dabei zeigten die Muskelzellen sowohl von Wildtyp als auch von Knock-Out Mäusen intrazelluläre dicke Myosinfilamente, was für die Beteiligung des nichtmuskulären Myosins an der glatten Muskelkontraktion spricht. Entsprechend wurden intrazelluläre Filamente mit einem Antikörper gegen glattmuskelspezifische schwere Myosinketten in kultivierten primären glatten Muskelzellen untersucht. Wie erwartet, konnten nur in glatten Muskelzellen von Wildtyp Mäusen intrazelluläre Filamente nachgewiesen werden, nicht aber in denen von Knock-Out Mäusen. In dieser Arbeit konnte zum ersten Mal gezeigt werden, dass nichtmuskuläres Myosin zumindest an der tonischen Kontraktion glatter Muskelzellen beteiligt sein kann. / The aim of the present study was to investigate the involvement of non-muscle myosin heavy chain in smooth muscle contraction under physiological conditions. As an experimental model urinary bladder from neonatal wild-type mice as well as from neonatal mice with disrupted smooth muscle myosin heavy chain expression was used. This animal model was established through gene targeting technology, resulting in complete elimination of the expression of smooth muscle myosin heavy chains. The lack of expression of smooth muscle myosin heavy chains was confirmed by electrophoresis and immunoblotting. On the other hand, non-muscle myosin heavy chain expression remained normal, as verified by Western blot analysis. The mechanical analysis of smooth muscle was performed with intact urinary bladder preparations, stimulated using prolonged KCl depolarization or with phorbol ester. Prolonged activation by KCl depolarization of intact bladder preparations from wild-type neonatal mice produced an initial transient state (phase 1) of high force generation and maximal shortening velocity, followed by a sustained state (phase 2) with lower force generation and maximal shortening velocity. In contrast, bladder preparations from homozygous knockout neonatal mice did not exhibit phase 1, but phase 2 could be observed, i.e. a similar isometric force and maximal shortening velocity, compared to wild-type phase 2. Thus, non-muscle myosin appears to be recruited in the sustained phase of smooth muscle contraction during prolonged KCl depolarization in the animal model used. Upon stimulation with phorbol ester a similar sustained contraction was observed in both wild-type and knockout smooth muscle preparations. Therefore, non-muscle myosin may also be recruited during phorbol ester stimulation in both wild-type and knockout muscle preparations. The participation of non-muscle myosin in smooth muscle contraction was further supported by the finding of longitudinally arranged intracellular filaments in cultivated smooth muscle cells from both wild-type and knockout mice by immunofluorescence microscopy, using a specific antibody raised against non-muscle myosin heavy chain. In a similar way, smooth muscle myosin heavy chain structures were investigated in cultivated smooth muscle cells. As expected, longitudinally arranged intracellular filamentous structures of smooth muscle myosin were observed in wild-type smooth muscle cells, but not in smooth muscle cells from knockout mice. In conclusion, in neonatal smooth muscle the initial phase of contraction elicited by KCl depolarization is generated by smooth muscle myosin heavy chain recruitment. Upon prolonged KCl depolarization non-muscle myosin is recruited in the sustained phase of contraction, as well as upon stimulation with phorbol ester. Thus, it was possible, for the first time, to verify the involvement of the non-muscle myosin in smooth muscle contraction in vivo. The results of the present study contribute to the understanding of the regulatory mechanisms of smooth muscle contraction.

O objetivo deste trabalho é estudar a afinidade e especificidade de sítios EF-hand, e correlacionar estas propriedades com a estrutura primária do sítio, com as interações entre aminoácidos nas posições de coordenação, e com prováveis características da estrutura terciária da proteína. Os efeitos de três mutações no sítio EF-hand da cadeia leve regulatória de miosina (RLC) foram estudados: D5S, em que o aspartato presente na posição 5 do sítio foi substituído por uma serina; D9E, substituindo o aspartato da posição 9 por um glutamato, e D12E, substituindo o aspartato da posição 12 por um glutamato. Todas as combinações destas três mutações foram produzidas. Os mutantes simples D5S e D9E e o duplo mutante D5S/D9E têm baixa afinidade por cálcio. Todos os mutantes contendo a mutação D12E são específicos para cálcio, com afinidades maiores que RLC tipo selvagem. Todos os mutantes estudados possuem menor afinidade por magnésio que RLC tipo selvagem. As mudanças na energia livre de ligação e as energias de acoplamento sugerem que há interações inespecíficas entre todas as posições, e uma interação específica entre uma serina na posição 5 e um glutamato na posição 9. Esta interação ocorre somente na presença de magnésio, e quando há um aspartato na posição 12. O glutamato na posição 9 pode ser capaz de coordenar a ligação de magnésio diretamente no duplo mutante D5S/D9E. Embora um aminoácido ou um certo arranjo deles possa determinar características específicas do sítio EF-hand, o conjunto de propriedades depende da estrutura terciária, uma vez que sítios homólogos podem possuir afinidades e especificidades bastante diferentes. / The aim of this thesis was to study affinity and specificity in EF-hand sites, and how these properties are related to the site primary structure, interactions between amino acids in coordinating positions, and probable tertiary structure properties. The effects of three mutations on the EF-hand Ca2+/Mg2+ binding site of smooth muscle myosin regulatory light chain (RLC) were studied: D5S, in which an aspartate is replaced by a serine in position 5 of the loop; D9E, in which an aspartate is replaced by a glutamate in position 9, and D12E, in which the aspartate in position 12 is replaced by a glutamate. All possible combinations of the three mutations were produced. The single mutants D5S and D9E and the double mutant D5S/D9E have low affinity for Ca2+. All the mutants containing mutation D12E are Ca2+-specific and have higher affinities than wild type, even when containing mutations D5S or D9E. All the mutants studied have lower affinity for Mg2+ than wild type RLC. Coupling energies and changes in binding free energy suggest that all positions interact in a non-specific way, and a specific interaction occurs between a serine in position 5 and a glutamate in position 9. This interaction can be seen only in the presence of magnesium, and with an apartate in position 12. Glutamate in position 9 may be able to coordinate Mg2+ directly in the double mutant D5S/D9E. Even though an amino acid or a few amino acids in certain positions can determine specific characteristics for an EF-hand site, the site properties depend on the tertiary structure, since homologue sites can have very different affinities and specificities.

A yeast two-hybrid assay suggested the possibility of an interaction between Cdc4p, a small EF-hand protein essential for cytokinesis, and Pik1p in S. pombe. This interaction was unexpected, as one function of Cdc4p is that of an essential light chain, bound to the first IQ-motif of type II myosins, whereas Pik1p is a phosphatidylinositol 4-kinase. The objective of this work was to analyze the effects of Pik1p lipid kinase activity on the cell cycle of S. pombe. Another goal of this study was to evaluate the functional significance of the interaction between Cdc4p and Pik1p. This was performed by generating two mutants of pik1: one that abolished lipid kinase activity (pik1-D709A) and one that abolished Pik1p Cdc4p-binding activity (pik1-R838A). Pik1p has a conserved IQ-motif in its C-terminal region. A mutation in this site (R838A), homologous to a residue which was mutated in myosin and abrogated the interaction with Cdc4p, prevented the interaction with Cdc4p in a yeast two-hybrid assay and ELISA. An increase in lipid kinase activity was observed in cell extracts upon ectopic expression of pik1-wt from an episome, which was abolished by a mutation in the lipid kinase domain of Pik1p (D709A), but not by the R838A mutation. However, little to no increase in lipid kinase activity was observed upon ectopic expression of pik1-wt and pik1-R838A in a strain carrying a conditionally lethal allele of cdc4 (cdc4-G107S). This mutation in Cdc4p was shown previously to prevent the interaction with Pik1p in yeast two-hybrid assays. Ectopic expression of pik1-wt suppressed cell proliferation, with disruption of actin cytoskeletal structures and contractile ring formation. These results were not observed with the ectopic expression of the pik1-R838A mutant or when pik1-wt was expressed in the cdc4-G107S strain. Ectopic expression of pik1-R838A resulted in cell shortening, likely through inhibition of growth, and many of the short cells showed an accumulation of the expressed Pik1p protein at the cell tips. Formation of the contractile ring appeared unaffected in cells with ectopic expression of the pik1-D709A mutant, but many of these cells had thick or more than one septum, characteristic of a septation defect. The ectopic expression phenotypes were dosage dependent since lower levels of expression greatly reduced the severity of the ectopic phenotypes. Pik1p lipid kinase activity is essential and, based on ectopic expression studies, is required for septation. There is a physical and functional interaction between Cdc4p and Pik1p which is not essential for cell viability, but suggests a role for Cdc4p in phosphoinositide metabolism.

Background and aims: It is known that smooth muscle possesses substantial mechanical plasticity in that it is able to adapt to large changes in length without compromising its ability to generate force. It is believed that structural malleability of the contractile apparatus underlies this plasticity. There is strong evidence suggesting that myosin thick filaments of the muscle are relatively labile and their length in vivo is determined by the equilibrium between monomeric and filamentous myosin. The equilibrium in turn is governed by the state of phosphorylation of the 20-kD regulatory myosin light chain (MLC20, or RLC). It is known that phosphorylation of the myosin light chain favors formation of the filaments; it is not known how the light chain phosphorylation affects the lability of the filaments. The major aim of this thesis was to measure the mechanical integrity of the filaments formed from purified myosin molecules from bovine airway smooth muscle, and to determine whether the integrity was influenced by phosphorylation of the myosin light chain. Methods: Myosin was purified from bovine trachealis to form filaments, in ATP containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of RLC within 20-40 s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy. Results: RLC phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated. Conclusion: Our results suggest that RLC phosphorylation is a key regulatory step in modifying the structural properties of myosin filaments in smooth muscle, where formation and dissolution of the filaments are required in the cells’ adaptation to different cell length.

A yeast two-hybrid assay suggested the possibility of an interaction between Cdc4p, a small EF-hand protein essential for cytokinesis, and Pik1p in S. pombe. This interaction was unexpected, as one function of Cdc4p is that of an essential light chain, bound to the first IQ-motif of type II myosins, whereas Pik1p is a phosphatidylinositol 4-kinase. The objective of this work was to analyze the effects of Pik1p lipid kinase activity on the cell cycle of S. pombe. Another goal of this study was to evaluate the functional significance of the interaction between Cdc4p and Pik1p. This was performed by generating two mutants of pik1: one that abolished lipid kinase activity (pik1-D709A) and one that abolished Pik1p Cdc4p-binding activity (pik1-R838A). Pik1p has a conserved IQ-motif in its C-terminal region. A mutation in this site (R838A), homologous to a residue which was mutated in myosin and abrogated the interaction with Cdc4p, prevented the interaction with Cdc4p in a yeast two-hybrid assay and ELISA. An increase in lipid kinase activity was observed in cell extracts upon ectopic expression of pik1-wt from an episome, which was abolished by a mutation in the lipid kinase domain of Pik1p (D709A), but not by the R838A mutation. However, little to no increase in lipid kinase activity was observed upon ectopic expression of pik1-wt and pik1-R838A in a strain carrying a conditionally lethal allele of cdc4 (cdc4-G107S). This mutation in Cdc4p was shown previously to prevent the interaction with Pik1p in yeast two-hybrid assays. Ectopic expression of pik1-wt suppressed cell proliferation, with disruption of actin cytoskeletal structures and contractile ring formation. These results were not observed with the ectopic expression of the pik1-R838A mutant or when pik1-wt was expressed in the cdc4-G107S strain. Ectopic expression of pik1-R838A resulted in cell shortening, likely through inhibition of growth, and many of the short cells showed an accumulation of the expressed Pik1p protein at the cell tips. Formation of the contractile ring appeared unaffected in cells with ectopic expression of the pik1-D709A mutant, but many of these cells had thick or more than one septum, characteristic of a septation defect. The ectopic expression phenotypes were dosage dependent since lower levels of expression greatly reduced the severity of the ectopic phenotypes. Pik1p lipid kinase activity is essential and, based on ectopic expression studies, is required for septation. There is a physical and functional interaction between Cdc4p and Pik1p which is not essential for cell viability, but suggests a role for Cdc4p in phosphoinositide metabolism.