published_or_final_version / Pathology / Master / Master of Philosophy

Kinesins are motor proteins responsible for the anterograde transport on microtubules. Kinesin-1 is the first characterized kinesin, and it consists of two heavy chains and two light chains. KIF5B is a form of Kinesin-1 heavy chains that is ubiquitously expressed in mammals. The head domain of KIF5B is responsible for ATP-dependent mechanical movement along microtubules, while the tail region is well-known for its interaction with cell specific cargos. Recent studies reveal a second microtubule binding site in the tail, suggesting special functions of KIF5B in microtubule sliding and bundling. To understand the role of KIF5B in long bone development, a conditional knockout mouse model was generated, in which Kif5b is deleted in early limb mesenchyme using Prx1-cre/LoxP mediated recombination. Unlike Col2a1-cre directed Kif5b knockout in chondrocytes, the expression of Prx1-cre in limb mesenchyme results in Kif5b knockout in both chondrocyte and osteoblast lineages. The Prx1-cre mediated Kif5b conditional knockout mice develop malformed long bones characterized by their bowed shape, shortened length and multiple fractures, which reflects a combination of defects in bone matrix and growth plate. The mutant mice demonstrate impaired bone matrix formation, as indicated by both collagen density reduction and collagen matrix disorganization. Also, the growth plate does not retain its normal organization, and the hypertrophic zone is absent. The KIF5B deficient chondrocytes not only lose planar cell polarity, but also undergo early apoptosis and fail in terminal differentiation. Interestingly, the binucleation rate is significantly increased in these chondrocytes, suggesting a severe cytokinesis defect. Besides, the intracellular retention of extracellular matrix (ECM) molecules and the uneven distribution of ECM in the cartilage imply both blockage and inappropriate direction of secretion. Cytokinetic defect in chondrocytes is closely associated with growth plate abnormality and growth retardation. In Kif5b knockout chondrocytes, cytokinetic defect is also one of the earliest and principal phenotypes. Therefore the underlying mechanism of cytokinetic defect was further investigated at cellular level. Since Kif5b knockout chondrocytes cannot survive in primary culture, RNA interference approach was adopted to generate a Kif5b-knockdown chondrogenic cell line. As expected, the Kif5b knockdown cells demonstrate cytokinetic defects characterized by increased binucleation rate and prolonged cytokinesis phase. In control cells, KIF5B becomes concentrated in the midbody during cytokinesis, and the midbody organization is disrupted in Kif5b knockdown cells. Furthermore, transient expression of full-length KIF5B significantly reduces the binucleation rate of these KIF5B deficient cells, whereas over-expression of a truncated KIF5B (without microtubule binding sites in tail region) cannot rescue the defect. Additionally, KIF5B is found to interact with midbody components PRC1 and Aurora B kinase by GST pull-down assay. This study demonstrates the multiple functions of KIF5B in long bone development and emphasizes its significant role as a key modulator in chondrocyte cytokinesis. More importantly, the study also brings new insights into the mechanisms of cytokinesis: we propose that KIF5B may participate in cytokinesis by regulating the midbody organization and stability via microtubule bundling and transporting or anchoring important components to the midbody. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The scaffolding protein anillin plays a crucial role during cytokinesis – the physical separation of daughter cells following chromosome segregation. Anillin binds filamentous F-actin, non-muscle myosin II and septins, and in cell culture models has been shown to restrict actomyosin contractility to the cleavage furrow. Whether anillin also serves this function during the incomplete cytokinesis that occurs in developing germ cells has remained unclear. Localization of anillin to several actin-rich structures in developing male germ cells also suggests potential roles for anillin outside of cytokinesis. In this study, I demonstrate that anillin is required for cytokinesis in dividing Drosophila spermatocytes. In addition, spermatid individualization is defective in anillin-depleted cells, although similarities to another cytokinesis mutant, four wheel drive, suggest this may be a secondary effect of failed cytokinesis. Anillin, septins and myosin II stably associate with the cleavage furrow in wild-type dividing spermatocytes. Anillin is necessary for recruitment of septins to the cleavage furrow, and for maintenance of Rho, F-actin and myosin II at the equator in late stages of cytokinesis. Membrane trafficking appears unaffected in anillin-depleted cells, although, unexpectedly, ectopic expression of one membrane trafficking marker, DE-cadherin-GFP, suppresses the cytokinesis defect. DE-cadherin-GFP recruits β-catenin (armadillo) and α-catenin to the cleavage furrow and stabilizes F-actin at the equator. Taken together, my results suggest that the anillin-septin and cadherin-catenin complexes can serve as alternative means to promote tight physical coupling of F-actin and myosin II to the cleavage furrow and successful completion of cytokinesis.

Thesis advisor: David R. Burgess / Cytokinesis is the final step in cell division, culminating in the formation of two daughter cells from a single mother cell. Previous studies from our lab have shown that lipid rafts are dynamic during cytokinesis in sea urchin embryos, migrating into the ingressing cleavage furrow then moving back outwards towards the poles prior to abscission. Here, I quantitated the mobility of GM1, a ganglioside enriched in lipid rafts, using cholera toxin subunit B (CTB). Despite previous observations of raft movement during cell division, I have found lipid rafts to be immobile throughout the cell cycle. Lipid raft stability is dependent on the activity of myosin light chain kinase (MLCK), most likely due to the dramatic reorganization of actin filaments upon MLCK inhibition. While further investigating the immobility of lipid rafts during cytokinesis using confocal microscopy, I have found that new membrane is added to the cell poles during anaphase, causing the plasma membrane to expand coincident with the constriction of the contractile ring. This membrane addition is dependent on actin and astral microtubules and occurs significantly earlier during mitosis than membrane addition at the furrow. The membrane that is added at the polar regions is compositionally distinct from the original cell membrane in that it is devoid of GM1, a component of lipid rafts. I also found that Rab11 vesicles are trafficked to the polar plasma membrane during the time of this new membrane event, suggesting that the growth of the plasma membrane at the cell poles during cell division is not due to stretching as previously thought, but due to the addition of new membrane through exocytosis. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Cell division is a universal cellular process responsible for the proliferation and differentiation of cells. After the chromosomes are faithfully segregated during mitosis, cells undergo cytokinesis, where one cell divides into two. Cytokinesis in many eukaryotes requires a structure known as the contractile ring, which contains actin, myosin and many other proteins assembled just beneath the plasma membrane. In this thesis, I present my studies on the function and organization of this ring. I used the powerful genetically tractable model organism the fission yeast Schizosaccharomyces pombe to study these processes in cytokinesis. First, I showed that one function of the cytokinetic ring is to regulate the assembly of the septum cell wall in a curvature dependent manner, suggesting a mechanosensitive mechanism. Second, I analyzed the substructure organization of the proteins within the ring, showing that ring proteins are arranged in clusters and in different layers. Finally, in a collaborative project, I studied the arrangement of chromosomes within the nucleus, and identified a protein required for linking centromeres to the spindle pole body at the nuclear envelope. In general, my thesis provides new insights into the spatial mechanisms of cytokinesis and chromosome organization.

The division of one cell to form two cells, or cytokinesis, is fundamental to the development of all known multi-cellular organisms, as well as the propagation of life between generations. The intracellular mechanisms that mediate the physical deformation of the cell membrane during division have proven to be remarkably robust, with multiple processes functioning together to achieve bisection. Here, I present my doctoral work, which seeks to illuminate the dynamic molecular interplay that coordinates and drives cytokinesis in the Caenorhabditis elegans single-cell zygote. In Chapter 1, I begin with an introduction on cytokinesis and the many proteins known to regulate cell division. Chapter 2 presents a detailed review of three intracellular signaling molecules that mediate the spatial control of cytokinesis, known as Rho family small GTPases. In Chapter 3, I present work in which we inactivated specific cytokinesis protein functions at precise stages of the division process, in order to map out the first “temporal atlas” of essential cytokinetic functions. In Chapter 4, I present evidence that the GTPase CDC-42 and the cortical polarity machinery sequester cytokinesis-inhibiting proteins away from the division plane and protect the fidelity of cytokinesis. Chapter 5 lays out preliminary evidence that another GTPase, RAC-1, is a suppresser of cytokinesis and must be inactivated in the division plane specifically by a spindle-associated regulatory protein. Through this body of work, I have attempted to elucidate the underpinnings of the complex intracellular orchestra that drives cytokinesis. This work provides valuable insight, not only into how this vital process occurs, but also how the disruption of its components could lead to the development of complex diseases like cancer.

Cytokinesis is the final separation of daughter cells after division. Membrane trafficking increases the surface area of dividing cells and may deliver cargo needed for division. The Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11 effector during this highly conserved process.

Cytokinesis is the final separation of daughter cells after division. Membrane trafficking increases the surface area of dividing cells and may deliver cargo needed for division. The Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11 effector during this highly conserved process.

Cytokinesis, the final step of cell division, is characterized by formation of a cleavage furrow that ingresses to separate the cell into two daughter cells. This process requires rearrangement of the cytoskeleton and addition of membrane to the growing furrow. The phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been implicated in regulating both actin dynamics and membrane trafficking and, thus, is uniquely poised to coordinate these different processes during cytokinesis. In this study, I show that PtdIns(4,5)P2 is involved in another aspect of cytokinesis: regulation of actomyosin contractility. Perturbing PtdIns(4,5)P2 levels in Drosophila spermatocytes caused constriction to fail and cleavage furrows to regress. Moreover, PtdIns(4,5)P2 hydrolysis is implicated in this process: inhibiting PLC or IP3R or chelating Ca2+ also caused defects in furrow ingression. In addition, I show that PLC and MLCK activities are required for myosin light chain phosphorylation and for proper myosin and actin localization to the cleavage furrow. Thus, I propose a model in which PtdIns(4,5)P2 hydrolysis-dependent Ca2+ release activates MLCK via Ca2+/calmodulin to maintain myosin filaments in the contractile ring and regulate cleavage furrow ingression. Furthermore, I show that PtdIns(4,5)P2 has a role in maintaining contractile ring components in the cleavage furrow that does not depend on PtdIns(4,5)P2 hydrolysis. I conclude that PtdIns(4,5)P2 regulates myosin contractility through a PLC-dependent pathway leading to myosin phosphorylation and is also involved in localizing contractile ring components to the furrow. Thus, PtdIns(4,5)P2 may coordinate signals leading to cytoskeleton rearrangement and actomyosin contractility during cytokinesis.

Cytokinesis, the final step of cell division, is characterized by formation of a cleavage furrow that ingresses to separate the cell into two daughter cells. This process requires rearrangement of the cytoskeleton and addition of membrane to the growing furrow. The phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been implicated in regulating both actin dynamics and membrane trafficking and, thus, is uniquely poised to coordinate these different processes during cytokinesis. In this study, I show that PtdIns(4,5)P2 is involved in another aspect of cytokinesis: regulation of actomyosin contractility. Perturbing PtdIns(4,5)P2 levels in Drosophila spermatocytes caused constriction to fail and cleavage furrows to regress. Moreover, PtdIns(4,5)P2 hydrolysis is implicated in this process: inhibiting PLC or IP3R or chelating Ca2+ also caused defects in furrow ingression. In addition, I show that PLC and MLCK activities are required for myosin light chain phosphorylation and for proper myosin and actin localization to the cleavage furrow. Thus, I propose a model in which PtdIns(4,5)P2 hydrolysis-dependent Ca2+ release activates MLCK via Ca2+/calmodulin to maintain myosin filaments in the contractile ring and regulate cleavage furrow ingression. Furthermore, I show that PtdIns(4,5)P2 has a role in maintaining contractile ring components in the cleavage furrow that does not depend on PtdIns(4,5)P2 hydrolysis. I conclude that PtdIns(4,5)P2 regulates myosin contractility through a PLC-dependent pathway leading to myosin phosphorylation and is also involved in localizing contractile ring components to the furrow. Thus, PtdIns(4,5)P2 may coordinate signals leading to cytoskeleton rearrangement and actomyosin contractility during cytokinesis.