Siga este enlace para ver otros tipos de publicaciones sobre el tema: Spindle (Cell division) Cell migration.
Tesis sobre el tema "Spindle (Cell division) Cell migration"
Crea una cita precisa en los estilos APA, MLA, Chicago, Harvard y otros
Consulte los 38 mejores tesis para su investigación sobre el tema "Spindle (Cell division) Cell migration".
Junto a cada fuente en la lista de referencias hay un botón "Agregar a la bibliografía". Pulsa este botón, y generaremos automáticamente la referencia bibliográfica para la obra elegida en el estilo de cita que necesites: APA, MLA, Harvard, Vancouver, Chicago, etc.
También puede descargar el texto completo de la publicación académica en formato pdf y leer en línea su resumen siempre que esté disponible en los metadatos.
Explore tesis sobre una amplia variedad de disciplinas y organice su bibliografía correctamente.
1
Nestor-Bergmann, Alexander. "Relating cell shape, mechanical stress and cell division in epithelial tissues." Thesis, University of Manchester, 2018. https://www.research.manchester.ac.uk/portal/en/theses/relating-cell-shape-mechanical-stress-and-cell-division-in-epithelial-tissues(ebf1bce8-ca35-4f5a-8be9-f2e19c96e20d).html.
Texto completoResumen
The development and maintenance of tissues and organs depend on the careful regulation and coordinated motion of large numbers of cells. There is substantial evidence that many complex tissue functions, such as cell division, collective cell migration and gene expression, are directly regulated by mechanical forces. However, relatively little is known about how mechanical stress is distributed within a tissue and how this may guide biochemical signalling. Working in the framework of a popular vertex-based model, we derive expressions for stress tensors at the cell and tissue level to build analytic relationships between cell shape and mechanical stress. The discrete vertex model is upscaled, providing exact expressions for the bulk and shear moduli of disordered cellular networks, which bridges the gap to traditional continuum-level descriptions of tissues. Combining this theoretical work with new experimental techniques for whole-tissue stretching of Xenopus laevis tissue, we separate the roles of mechanical stress and cell shape in orienting and cueing epithelial mitosis. We find that the orientation of division is best predicted by the shape of tricellular junctions, while there appears to be a more direct role for mechanical stress as a mitotic cue.
2
Chanasakulniyom, Mayuree. "Single cell devices for migration and division studies." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5072/.
Texto completoResumen
Microfluidic technologies and devices now provide powerful tools for many biological studies to gain knowledge and insight into cell behaviour because of their potential to control the local in vitro environment. This thesis aims to develop microfluidic devices for the single cell proliferation and migration studies that are fundamental in determining cell and tissue behaviour. There are two designs of microfluidic devices that have been used in this project. The first one is hydrodynamic single cell trap device having a bagatelle- like structure. The bagatelle-like devices were used to trap modified MCF7 cells expressing both mcherry-tubulin and GFP-actin and also to study the influences of the oestrogen hormone on MCF7 cells. It was found that the MCF7 cell proliferation could not be seen in the bagatelle-like devices either in the presence or absence of oestrogen. It was hypothesised that this might be due to cell stresses arising from being in a constrained area (trap) and subjected to strong fluid flow forces. The second, novel, device consists of three segregated layers and is termed a microhole device. It was specifically designed, fabricated, characterised and utilised in cancer cell proliferation and migration studies in this thesis. The microhole devices were designed to address the limitations of the bagatelle-like device. In each microhole device, the lower layer comprises of a network of submerged channels linked to an upper layer through cavity-like holes. The networks of submerged channels provide a route through which cells can migrate. The middle layer consists of an array of circular holes used to organise single cells into the cavities beneath. The top layer is a PDMS chamber for cell loading and culture medium perfusion. It was found that the recirculatory flow patterns inside the devices facilitate cell trapping, while also serving to separate high velocity flow in the top chamber from the middle and the bottom layer thereby protecting the cells from shear stress. MDA-MB-231 cells were used in this study. It was found that they can undergo cell cycling normally in the microhole devices, and migrate along an SDF-1α solution gradient produced inside the device, towards high SDF-1α concentration. To explore whether the cells were sensitive to SDF-1α on the surface to which they adhered (as opposed to solution gradients), the microhole devices were modified to have SDF-1α immobilised on selected interior surfaces. Despite each stage of the immobilisation process being verified using the appropriate fluorescence assays, relatively low levels of SDF-1α were detected in the completed devices. This may be due to fabrication processes that might deteriorate the immobilised SDF-1α functionality. It was found that unlike the situation when SDF-1α is in solution form, the MDA-MB-231 cells showed no migratory preference toward the immobilised SDF-1α. Taken together, the microhole devices developed in this thesis provide suitable environments for study cell migration toward stimuli under perfusion conditions. The geometry and the flow characteristics inside the array facilitate cell trapping and serve to protect cells from shear stress caused by high fluid flow. Further applications of the multilayer microhole devices can be found through modifying the different layers to accommodate different geometries for different cell types as well as more complex stimulation conditions, or in other application areas associated with droplet microfluidics and synthetic biology.
3
Stewart, Neil Padilla Pamela Ann Fox. "Identifying genetic interactions of the spindle checkpoint in Caenorhabditis elegans." [Denton, Tex.] : University of North Texas, 2009. http://digital.library.unt.edu/ark:/67531/metadc12203.
Texto completo
4
Golub, Ognjen. "Molecular Mechanisms Regulating Subcellular Localization and Function of Mitotic Spindle Orientation Determinants." Thesis, University of Oregon, 2016. http://hdl.handle.net/1794/20711.
Texto completoResumen
Proper orientation of the mitotic spindle is essential during animal development for the generation of cell diversity and organogenesis. To understand the molecular mechanisms regulating this process, genetic studies have implicated evolutionarily conserved proteins that function in diverse cell types to align the spindle along an intrinsic cellular polarity axis. This activity is achieved through physical contacts between astral microtubules of the spindle and a distinct domain of force generating proteins on the cell cortex. In this work, I shed light on how these proteins form distinct cortical domains, how their activity is coupled to their subcellular localization, and how they provide cytoskeletal and motor protein connections that are required to generate the forces necessary to position the mitotic spindle.
I first discuss the mechanisms by which Mushroom body defect (Mud; NuMA in mammals), provides spindle orientation cues from various subcellular locations. Aside from its known role at the cortex as an adapter for the Dynein motor, I reveal novel isoform-dependent Mud functions at the spindle poles during assembly of the mitotic spindle and astral microtubules, thus implicating Mud in spindle orientation pathways away from the cell cortex. Moreover, through collaborative efforts with former lab members, I describe molecular regulation and assembly of two ‘accessory’ pathways that activate cortical Mud-Dynein, one through the tumor suppressor protein Discs large (Dlg), and another through the signaling protein Dishevelled (Dsh).
I demonstrate that the Dlg pathway is spatially regulated by the polarity kinase atypical Protein Kinase C (aPKC) through direct phosphorylation of Dlg. This signal relieves Dlg autoinhibition to promote cortical recruitment of the Dlg-ligand Gukholder (Gukh), a novel microtubule-binding protein that provides an additional connection between astral microtubules and the cortex that is essential for activity of the Dlg pathway. Lastly, I determine that the Dsh accessory pathway provides an alternative cytoskeletal cue by recruiting Diaphanous (Dia), an actin nucleating protein. By demonstrating interchangeability between the two accessory pathways, we conclude that Mud-Dynein is activated by various cytoskeletal cues and that the mode of activation is cell-context dependent.
This dissertation includes unpublished and previously published co-authored material.<br>10000-01-01
5
Joslin, Elizabeth Jane. "Quantitative studies of EGFR autocrine induced cell signaling and migration." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39910.
Texto completoResumen
Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.<br>Includes bibliographical references.<br>Epidermal growth factor (EGF) receptor autocrine and/or paracrine signaling plays an important role in normal epithelial cell proliferation, survival, adhesion and migration. Aberrant expression of the EGF receptor and its cognate ligands have been implicated in various types of cancers, hence EGF receptor autocrine activation is thought to also be involved in tumorigenesis. EGF family ligands are synthesized as membrane-anchored proteins requiring proteolytic release to form the mature soluble, receptor-binding factor. Despite the pathophysiological importance of autocrine systems, how protease-mediated ligand release quantitatively influences receptor-mediated signaling and consequent cell behavior is poorly understood. Therefore, we explored the relationship between autocrine EGF release rate and receptor-mediated ERK activation and migration in human mammary epithelial cells. A quantitative spectrum of EGF release rates was achieved using chimeric EGF ligand precursors modulated by the addition of the metalloprotease inhibitor batimastat. We found that ERK activation increased with increasing ligand release rates despite concomitant EGF receptor downregulation.<br>(cont.) Cell migration speed depended linearly on the steady-state phospho-ERK level, but was much greater for autocrine compared to exogenous stimulation. In contrast, cell proliferation rates were constant across the various treatment conditions. In addition, we investigated an EGFR-mediated positive feedback through ERK that stimulated a 4-fold increase in release rate of our TGFa based construct. Thus, in these cells, ERK-mediated migration stimulated by EGF receptor signaling is most sensitively regulated by autocrine ligand control mechanisms.<br>by Elizabeth Jane Joslin.<br>Ph.D.
6
Chu, Calvin School of Biomedical Engineering UNSW. "Development of a semi-automatic method for cellular migration and division analysis." Awarded by:University of New South Wales. School of Biomedical Engineering, 2005. http://handle.unsw.edu.au/1959.4/20543.
Texto completoResumen
Binary image processing algorithms have been implemented in this study to create a background subtraction mask for the segmentation of cellular time lapse images. The complexity in the development of the background subtraction mask stems from the inherent difficulties in contrast resolution at the cellular boundaries. Coupling the background subtraction mask with the path reconstruction method via superposition of overlapping binary segmented objects in sequential time lapse images produces a semi-automatic method for cellular tracking. In addition to the traditional center of mass or centroid approximation, a novel quasi-center of mass (QCM) derived from the local maxima of the distance transformation (DT) has also been proposed in this study. Furthermore, image isolation and separation between spreading/motile and mitotic cells allows the extraction of both migratory and divisional cellular information. DT application to isolated mitotic cells permits the ability to identify distinct morphologic phases of cellular division. Application of standard bivariate statistics allows the characterization of cellular migration and growth. Determination of Hotelling???s confidence ellipse from cellular trajectory data elucidates the biased or unbiased migration of cellular populations. We investigated whether it was possible to describe the trajectory as a simple binomial process, where trajectory directions are classified into a sequence of (8) discrete states. A significant proportion of trajectories did not follow the binomial model. Additionally, a preliminary relationship between the image background area, approximate number of counted cells in an image frame, and imaging time is proposed from the segmentation of confluent monolayer cellular cultures.
7
Reschen, Richard Frederick. "The roles of Dgp71WD at the centrosome and spindle in Drosophila." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609808.
Texto completo
8
Stewart, Neil. "Identifying genetic interactions of the spindle checkpoint in Caenorhabditis elegans." Thesis, University of North Texas, 2009. https://digital.library.unt.edu/ark:/67531/metadc12203/.
Texto completoResumen
Faithful segregation of chromosomes is ensured by the spindle checkpoint. If a kinetochore does not correctly attach to a microtubule the spindle checkpoint stops cell cycle progression until all chromosomes are attached to microtubules or tension is experienced while pulling the chromosomes. The C. elegans gene, san-1, is required for spindle checkpoint function and anoxia survival. To further understand the role of san-1 in the spindle checkpoint, an RNAi screen was conducted to identify genetic interactions with san-1. The kinetochore gene hcp-1 identified in this screen, was known to have a genetic interaction with hcp-2. Interestingly, san-1(ok1580);hcp-2(ok1757) had embryonic and larval lethal phenotypes, but the phenotypes observed are less severe compared to the phenotypes of san-1(ok1580);hcp-1(RNAi) animals. Both san-1(ok1580);hcp-1(RNAi) and san-1(ok1580);hcp-2(RNAi) produce eggs that may hatch; but san-1(ok1580):hcp-1(RNAi) larvae do not survive to adulthood due to defects caused by aberrant chromosome segregations during development. Y54G9A.6 encodes the C. elegans homolog of bub-3, and has spindle checkpoint function. In C.elegans, bub-3 has genetic interactions with san-1 and mdf-2. An RNAi screen for genetic interactions with bub-3 identified that F31F6.3 may potentially have a genetic interaction with bub-3. This work provided genetic evidence that hcp-1, hcp-2 and F31F6.2 interact with spindle checkpoint genes.
9
Kosmin, Alan Simon. "Cell proliferation, apoptosis and migration within the human fetal retina." Thesis, University of Liverpool, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366488.
Texto completo
10
Hung, Hui-Fang. "Roles of the Mother Centriole Appendage Protein Cenexin in Microtubule Organization during Cell Migration and Cell Division: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/842.
Texto completoResumen
Epithelial cells are necessary building blocks of the organs they line. Their apicalbasolateral polarity, characterized by an asymmetric distribution of cell components along their apical-basal axis, is a requirement for normal organ function. Although the centrosome, also known as the microtubule organizing center, is important in establishing cell polarity the mechanisms through which it achieves this remain unclear. It has been suggested that the centrosome influences cell polarity through microtubule cytoskeleton organization and endosome trafficking. In the first chapter of this thesis, I summarize the current understanding of the mechanisms regulating cell polarity and review evidence for the role of centrosomes in this process.
In the second chapter, I examine the roles of the mother centriole appendages in cell polarity during cell migration and cell division. Interestingly, the subdistal appendages, but not the distal appendages, are essential in both processes, a role they achieve through organizing centrosomal microtubules. Depletion of subdistal appendages disrupts microtubule organization at the centrosome and hence, affects microtubule stability. These microtubule defects affect centrosome reorientation and spindle orientation during cell migration and division, respectively. In addition, depletion of subdistal appendages affects the localization and dynamics of apical polarity proteins in relation to microtubule stability and endosome recycling. Taken together, our results suggest the mother centriole subdistal appendages play an essential role in regulating cell polarity. A discussion of the significance of these results is included in chapter three.
11
Hung, Hui-Fang. "Roles of the Mother Centriole Appendage Protein Cenexin in Microtubule Organization during Cell Migration and Cell Division: A Dissertation." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/842.
Texto completoResumen
Epithelial cells are necessary building blocks of the organs they line. Their apicalbasolateral polarity, characterized by an asymmetric distribution of cell components along their apical-basal axis, is a requirement for normal organ function. Although the centrosome, also known as the microtubule organizing center, is important in establishing cell polarity the mechanisms through which it achieves this remain unclear. It has been suggested that the centrosome influences cell polarity through microtubule cytoskeleton organization and endosome trafficking. In the first chapter of this thesis, I summarize the current understanding of the mechanisms regulating cell polarity and review evidence for the role of centrosomes in this process.
In the second chapter, I examine the roles of the mother centriole appendages in cell polarity during cell migration and cell division. Interestingly, the subdistal appendages, but not the distal appendages, are essential in both processes, a role they achieve through organizing centrosomal microtubules. Depletion of subdistal appendages disrupts microtubule organization at the centrosome and hence, affects microtubule stability. These microtubule defects affect centrosome reorientation and spindle orientation during cell migration and division, respectively. In addition, depletion of subdistal appendages affects the localization and dynamics of apical polarity proteins in relation to microtubule stability and endosome recycling. Taken together, our results suggest the mother centriole subdistal appendages play an essential role in regulating cell polarity. A discussion of the significance of these results is included in chapter three.
12
Sze, Man-fong. "Characterization of mitotic checkpoint proteins, MAD1 and MAD2, in hepatocellular carcinoma." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36841286.
Texto completo
13
Smith, Nicholas Robert 1981. "Autoinhibition and ultrasensitivity in the Galphai-Pins-Mud spindle orientation pathway." Thesis, University of Oregon, 2010. http://hdl.handle.net/1794/11303.
Texto completoResumen
xiv, 81 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number.<br>Protein-protein interaction networks translate environmental inputs into specific physiological outputs. The signaling proteins in these networks require regulatory mechanisms to ensure proper molecular function. Two common regulatory features of signaling proteins are autoinhibition and ultrasensitivity. Autoinhibition locks the protein in an inactive state through cis interactions with a regulatory module until it is activated by a specific input signal. Ultrasensitivity, defined as steep activation after a threshold, allows cells to convert graded inputs into more switch-like outputs and can lead to complex decision making behaviors such as bistability. Although these mechanisms are common features of signaling proteins, their molecular origins are poorly understood. I used the Drosophila Pins protein, a regulator of spindle positioning in neuroblast cells, as a model to study the molecular origin and function of autoinhibition and ultrasensitivity.
Pins and its binding partners. Gαi and Mud, form a signaling pathway required for coordinating spindle positioning with cellular polarity in Drosophila neuroblasts. I found Pins switches from an autoinhibited to an activate state by modular allostery. Gαi binding to the third of three GoLoco (GL) domains allows Pins to interact with the microtubule binding protein Mud. The GL3 region is required for autoinhibitoon, as amino acids upstream and within GL3 constitute this regulatory behavior. This autoinhibitory module is conserved in LGN, the mammalian Pins orthologue.
I also demonstrated that Gαi activation of Pins is ultrasensitive. A Pins protein containing inactivating point mutations to GLs l and 2 exhibits non-ultrasensitive (graded) activation. Ultrasensitivity is required for Pins function in vivo as the graded Pins mutant fails to robustly orient the mitotic spindle. I considered two models for the source of ultrasensitivity in this pathway: cooperative or "decoy" Gai binding. I found ultrasensitivity arises from a decoy mechanism in which GLs 1 and 2 compete with the activating GL3 for the input, Gai. These findings suggest that molecular ultrasensitivity can be generated without cooperativity. This decoy mechanism is relatively simple, suggesting ultrasensitive responses can be evolved by the inclusion of domain repeats, a common feature observed in signaling proteins.
This dissertation includes previously published and unpublished co-authored material.<br>Committee in charge: Tom Stevens, Chairperson, Chemistry;
Kenneth Prehoda, Member, Chemistry;
Christopher Doe, Member, Biology;
Peter von Hippel, Member, Chemistry;
Karen Guillemin, Outside Member, Biology
14
Fish, Jennifer. "The evolution of neuronal progenitor cell division in mammals: The role of the abnormal spindle-like microcephaly associated (Aspm) protein and epithelial cell polarity." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2007. http://nbn-resolving.de/urn:nbn:de:swb:14-1184837029919-80275.
Texto completoResumen
Among mammals, primates are exceptional for their large brain size relative to body size. Relative brain size, or encephalization, is particularly striking among humans and their direct ancestors. Since the human-chimp split 5 to 7 million years ago, brain size has tripled in the human lineage (Wood &amp; Collard 1999). The focus of this doctoral work is to investigate some of the cell biological mechanisms responsible for this increase in relative brain size. In particular, the processes that regulate symmetric cell division (ultimately generating more progenitors), the constraints on progenitor proliferation, and how neural progenitors have overcome these constraints in the process of primate encephalization are the primary questions of interest. Both functionally analyses in the mouse model system and comparative neurobiology of rodents and primates are used here to address these questions. Using the mouse model system, the cell biological role of the Aspm (abnormal spindle-like microcephaly associated) protein in regulating brain size was investigated. Specifically, Aspm function in symmetric, proliferative divisions of neuroepithelial (NE) cells was analyzed. It was found that Aspm expression in the mouse neuroepithelium correlates in time and space with symmetric, proliferating divisions. The Aspm protein localizes to NE cell spindle poles during all phases of mitosis, and is down-regulated in cells that undergo asymmetric (neurogenic) cell divisions. Aspm RNAi alters the division plane in NE cells, increasing the likelihood of premature asymmetric division resulting in an increase in non-NE progeny. At least some of the non-NE progeny generated by Aspm RNAi migrate to the neuronal layer and express neuronal markers. Importantly, whatever the fate of the non-NE progeny, their generation comes at the expense of the expansion of the proliferative pool of NE progenitor cells. These data have contributed to the generation of an hypothesis regarding evolutionary changes in the regulation of spindle orientation in vertebrate and mammalian neural progenitors and their impact on brain size. Specifically, in contrast to invertebrates that regulate the switch from symmetric to asymmetric division through a rotation of the spindle (horizontal versus vertical cleavage), asymmetric NE cell division in vertebrates is accomplished by only a slight deviation in the cleavage plane away from the vertical, apical-basal axis. The requirement for the precise alignment of the spindle along the apical-basal axis in symmetric cell divisions may have contributed to selection on spindle “precision” proteins, thus increasing the number of symmetric NE cell division, and contributing to brain size increases during mammalian evolution. Previous comparative neurobiological analyses have revealed an increase in basally dividing NE cells in the brain regions of highest proliferation and in species with the largest brains (Smart 1972a,b; Martinez-Cerdeno et al. 2006). The cell biological characteristics of these basally dividing cells are still largely unknown. We found that primate basal progenitors, similar to rodent apical progenitors, are Pax6+. This suggests that primate basal progenitors may share other properties with rodent apical progenitors, such as maintenance of apical contact. Our previous finding that artificial alteration of cleavage plane in NE cells affects their ability to continue proliferating supports the hypothesis that the apical membrane and junctional complexes are cell fate determinants (Huttner &amp; Kosodo 2005). As such, the need to maintain apical membrane contact appears to be a constraint on proliferation (Smart 1972a,b; Smart et al. 2002). Together, these data favor the hypothesis that primate basally dividing cells maintain apical contact and are epithelial in nature.
15
Kim, Haein. "Temporal Coordination Of Mitotic Chromosome Alignment And Segregation: Structural And Functional Studies Of Kif18a." ScholarWorks @ UVM, 2018. https://scholarworks.uvm.edu/graddis/930.
Texto completoResumen
Chromosome alignment is highly conserved in all eukaryotic cell divisions. Microtubule (MT) -based forces generated by the mitotic spindle are integral for proper chromosome alignment and equal chromosome segregation. The kinetochore is a multi-subunit protein complex that assembles on centromeric regions of chromosomes. Kinetochores tether chromosomes to MTs (K fibers) that emanate from opposite poles, in a process called biorientation. This linkage translates K fiber dynamics into chromosome movements during alignment and segregation. Stable, high-affinity kinetochore attachments promote spindle assembly checkpoint (SAC) silencing, which is active when unattached kinetochores are present. During chromosome alignment, 1) K fiber plus-end dynamics decrease, confining chromosome movements near the spindle equator, and 2) electrostatic interactions between kinetochore proteins and MTs increase. Chromosome segregation occurs as soon as all chromosomes are stably attached to microtubules and the SAC has been silenced. SAC silencing and chromosome alignment are temporally coordinated during normal divisions, implying that the mechanisms regulating K fiber dynamics and kinetochore affinity must be linked. Interestingly, HeLa cells depleted of a kinesin-8 motor Kif18A, known for its role in promoting chromosome alignment, display a SAC-dependent mitotic delay due to kinetochore-MT attachment defects. This is puzzling, as Kif18A's function in chromosome alignment is to suppress MT growth by stably associating with MT plus-ends. Whether Kif18A is required for attachment in all cells and how it promotes kinetochore microtubule linkages are not understood.
The work presented in this dissertation supports a model in which Kif18A functions as a molecular link that coordinates chromosome alignment and anaphase onset. We find that Kif18A is required to stabilize kinetochore-MT attachments during mammalian germline development, as germline precursor cells in Kif18A mutant mice are unable to divide during embryogenesis due to an active SAC. However, while all cell types require functional Kif18A for chromosome alignment, mouse primary somatic cells can still divide with normal timing. This finding indicates a cell-type specific dependence on Kif18A for stabilizing kinetochore-MT attachments, and provides evidence that this function might be separate from Kif18A's known role in chromosome alignment. Consistent with this idea, we find that an evolutionarily conserved binding motif for protein phosphatase 1 (PP1) is required for Kif18A's novel role in regulating kinetochore microtubule attachments. Kif18A-PP1 interaction is required for Kif18A-mediated dephosphorylation of the kinetochore protein Hec1, which enhances attachment. However, Kif18A's interaction with PP1 is dispensable for chromosome alignment. Thus, point mutations that disrupt PP1 binding separate Kif18A's role in stabilizing kinetochore attachments from its function in promoting chromosome alignment. Additionally, through structure function studies of the motor domain, we identified a long surface loop (Loop2) that is required for Kif18A's unique MT plus-end binding activity, which is essential for its function in confining chromosome movements. Taken together, we find that Kif18A is molecularly tuned to provide temporal control of chromosome alignment and anaphase entry.
16
Allen, John C. "FGF4 Induced Wnt5a Gradient in the Limb Bud Mediates Mesenchymal Cell Directed Migration and Division." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/4309.
Texto completoResumen
The AER has a vital role in directing embryonic limb development. Several models have been developed that attempt to explain how the AER directs limb development, but none of them are fully supported by existing data. I provide evidence that FGFs secreted from the AER induce a gradient of Wnt5a. I also demonstrate that limb mesenchyme grows toward increasing concentrations of Wnt5a. We hypothesize that the changing shape of the AER is critical for patterning the limb along the proximal to distal axis. To better understand the pathway through which Wnt5a elicits its effects, we have performed various genetic studies. We demonstrate that Wnt5a does not signal via the Wnt/β-catenin pathway. However, we show that Wnt5a mutants share many common defects with Vangl2 mutants suggesting that Wnt5a signals through the Wnt/planar cell polarity (PCP) pathway.
17
Sze, Man-fong, and 施敏芳. "Characterization of mitotic checkpoint proteins, MAD1 and MAD2, in hepatocellular carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B38438550.
Texto completo
18
Müller-Reichert, Thomas. "Spindle organization in three dimensions." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1166107130476-22269.
Texto completoResumen
During cell division, chromosome segregation takes place on bipolar, microtubulebased spindles. Here, C. elegans is used to analyze spindle organization under both mitotic and meiotic conditions. First, the role of SAS-4 in organizing centrosome structure was analyzed. Partial depletion of SAS-4 in early embryos results in structurally defective centrioles. The study of this protein sheds light on the poorly understood role of the centrioles in dictating centrosome size. Second, the ultrastructure of wild-type mitotic spindle components was analyzed by electron tomography. This 3-D analysis reveals morphologically distinct microtubule end morphologies in the mitotic spindle pole. These results have structural implications for models of microtubule interactions with centrosomes Third, spindle assembly was studied in female meiosis. Specifically, the role of the microtubule severing complex katanin in spindle organization was analyzed. Electron tomography reveals fragmentation of spindle microtubules and suggests a novel katanin-dependent mechanism of meiotic spindle assembly. In this model, relatively long microtubules seen near the meiotic chromatin are converted into numerous short fragments, thus increasing the total number of polymers in an acentrosomal environment. Taken together, these results provide novel insights into the three-dimensional organization of microtubules during spindle assembly<br>Die Segregation der Chromosomen während der Zellteilung wird duch bipolare, von Microtubuli-aufgebauten Spindlen gewährleistet. In der vorliegenden Arbeit wird C. elegans zur Analyse der Spindelorganisation unter mitotischen und meiotischen Bedingungen herangezogen. Erstens wird die Rolle von SAS-4 in der Organisation von Zentrosomen untersucht. Die partielle Depletierung von SAS-4 in frühen Embryonen führt zu strukturell defekten Zentriolen und wirft somit Licht auf die wenig verstandene Rolle der Zentriolen in der Bestimmung der Zentrosomengröße. Zweitens wird die Ultrastruktur der mitotischen Spindelkomponenten im Wildtyp durch Elektronentomographie untersucht. Diese 3-D-Analyse zeigt, dass im mitotischen Spindlepol unterschiedliche Morphologien der Mikrotubulienden zu finden sind. Diese Ergebnisse haben strukturelle Implikationen für Modelle der Mikrotubuli-Zentrosomen-Interaktionen. Drittens wird der Aufbau der Spindel in der weiblichen Meiose, speziell die Rolle des Mikrotubuli-schneidenden Kataninkomplexes in der Spindelorganisation, untersucht. Die Elektronentomographie zeigt hier eine Fragmentierung der Spindelmikrotubuli. Basierend auf diesem Ergebnis wird ein neues Katanin-abhängiges Modell der Formierung der Meiosespindel entwickelt, in dem relativ lange Microtubuli in Nähe des meiotischen Chromatins in zahlreiche kurze Mikrotubuli “zerschnitten” werden. Dies erhöht die Anzahl der verfügbaren Polymere in dieser azentrosomalen Situation. Zusammenfassend bringen diese Ergebnisse neue Einsichten in die räumliche Organisation der Mikrotubuli während des Spindelaufbaus
19
Riche, Soizic. "Etude comparative du positionnement du fuseau mitotique dans les espèces de C.elegans et C. briggsae." Thesis, Lyon, École normale supérieure, 2015. http://www.theses.fr/2015ENSL1053/document.
Texto completoResumen
La division cellulaire asymétrique est un mécanisme fondamental qui assure la diversité cellulaire, le renouvellement des cellules souches et le maintien de l’identité cellulaire. Elle dépend du bon positionnement du fuseau mitotique car il dicte le plan de division des cellules. La première division des embryons de C. elegans, est asymétrique et génère deux cellules fille de taille et devenir différents. Elle consiste en deux étapes : la centration des pronoyaux en prophase puis le déplacement postérieur du fuseau mitotique en anaphase. Lors de l'anaphase le fuseau subit des oscillations transverses plus marquées au pôle postérieur qu’au pôle antérieur. Ces mouvements sont contrôlés par des forces de traction agissant sur les microtubules astraux. Les générateurs de force ont été moléculairement identifiés et sont évolutivement très conservés. Un complexe composé de protéines Gα, liées à GPR (protéine à domaine GoLoco, homologue de LGN/Pins), à LIN-5 (protéine à domaine super-enroulé, homologue de NuMA/Mud) et à la dynéine serait ancré au cortex et activé en début de mitose pour tirer le fuseau. En analysant la première division d’une espèce proche de C. elegans : C. briggsae, on observe des variations de trajectoire du fuseau. Les embryons de C. briggsae présentent un décalage antérieur des noyaux en prophase et les oscillations du fuseau sont réduites en anaphase. La combinaison de perturbations physiques et l'analyse de mutants dans ces espèces, ont montré que ces différences s’expliquent par des changements dans la régulation du complexe ternaire. Mais, nous avons découvert que dans les deux espèces 1) un switch positionnel conservé contrôle le démarrage des oscillations du fuseau, 2) la localisation postérieure de GPR détermine ce switch positionnel, et 3) l'amplitude maximum des oscillations est déterminée en partie par le temps passé dans la phase oscillatoire. Nous avons utilisés ces variants pour corréler les phénotypes, la localisation de GPR et la divergence de séquence entre espèces afin d’identifier les éléments de régulation de cette protéine. Nous avons alors échangé les protéines et construits des protéines chimères entre les deux espèces. Enfin, par optogénétique, nous avons essayé de contrôler la localisation temporelle de GPR et analyser les conséquences sur les mouvements des noyaux et du fuseau. En étudiant la microévolution d'un processus sous-cellulaire, nous avons identifié de nouveaux mécanismes qui contribuent à la compréhension du positionnement du fuseau<br>Asymmetric cell division is a fundamental mechanism essential in all organisms to assure cell diversity, stem cell renewal and cellular identity maintenance. It is relying on proper mitotic spindle positioning because it dictates the cell division plan. In C. elegans one-cell embryos, the first division is asymmetric and gives rise to two daughter cells of unequal size and fate. It occurs in two steps: pronuclei centration during prophase and spindle posterior displacement during anaphase. During anaphase, the mitotic spindle undergoes transverse oscillations that are more pronounced for the posterior than the anterior pole. These movements are controlled by pulling forces acting on astral microtubules. The force generators are identified and are evolutionary conserved. A complex made of Gα proteins, linked to GPR (a GoLoco containing protein, the LGN/Pins homologues), LIN-5 (a coiled-coil protein, the NuMA/Mud homologues) and dynein is thought to be anchored at the cortex and activated at the onset of mitosis to pull on the spindle. We identified variations in spindle trajectories by analyzing the outwardly similar one-cell stage embryo of a close relative of C. elegans, C. briggsae. Compared to C. elegans, C. briggsae embryos exhibit an anterior shifting of nuclei in prophase and reduced anaphase spindle oscillations. By combining physical perturbations and mutant analysis in both species, we show that differences can be explained by inter-species changes in the regulation of the cortical Gα/GPR/LIN-5 complex. However, we uncover that in both species 1) a conserved positional switch controls the onset of spindle oscillations, 2) GPR posterior localization may set this positional switch, and 3) the maximum amplitude of spindle oscillations is determined in part by the time spent in the oscillating phase. Interestingly, GPR is poorly conserved at the amino acid level between these species. We use these variants to correlate phenotypes, GPR localization and sequence divergence to identify GPR regulatory elements. To this end, we performed protein replacement between species, as well as analysis of protein chimeras. Finally we tried to use optogenetics in order to control GPR localisation temporally and analyze the consequences on pronuclei and spindle movements during the first division. By investigating microevolution of a subcellular process, we identified new mechanisms that are instrumental to decipher spindle positioning
20
Chen, Helen. "The non-motor protein RHAMM locates TPX2 to coordinate spindle assembly and balance motor forces needed to segregate chromosomes and complete cell division." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/60235.
Texto completoResumen
Cell division requires the assembly and organization of a microtubule-based mitotic spindle. Microtubule assembly at multiple sites is dependent on Aurora kinase A activity, which is promoted through a complex with TPX2 (targeting protein for XKlp2). Subsequent organization of these microtubules and progression into anaphase requires balance between forces orchestrated by antagonistic motor complexes. My studies show that the non-motor protein RHAMM (receptor for hyaluronan mediated motility) integrates structural and biochemical pathways to ensure the fidelity of cell division. Silencing RHAMM in HeLa cells delayed the kinetics of spindle assembly. I located RHAMM to centrosomes and non-centrosome sites for microtubule nucleation and found it necessary for TPX2 localization and Aurora A activity at kinetochores. The RHAMM-TPX2 complex requires a conserved leucine zipper motif in RHAMM and a domain that includes the nuclear localization signal in TPX2. These findings indicate RHAMM is needed for spatially-regulated activation of Aurora A by TPX2, which coordinates spindle assembly. I monitored mouse embryonic fibroblasts deficient for RHAMM through division and identified defects progressing through the spindle checkpoint. In RHAMM-silenced HeLa cells, I identified sustained activation of the checkpoint with unfocused spindles and unattached kinetochores, implicating unbalanced motor activities mediated by kinesins. In metaphase-delayed cells, the abundance or location of checkpoint proteins was not altered. Moreover, aberrant spindle orientation could not account for each delayed division. In RHAMM-silenced cells, I found that the reciprocal immunoprecipitation of Eg5-TPX2, an inhibitory complex, was reduced and that the concurrent inhibition of Eg5-generated force recovered division kinetics. I also observed a prolonged metaphase delay in a proportion of RHAMM-silenced cells, which resolved through cohesion fatigue. Together, my findings indicate that RHAMM-mediated attenuation of Eg5-dependent outward forces is needed to align chromosomes and progress through division. Lastly, I identified defects in spindle structure and function in redundant models for RHAMM over-expression. Collectively, my studies demonstrate that RHAMM coordinates Aurora A signaling and balances motor forces that are needed for cell division. These findings provide novel insights into processes that are essential for mammalian cell division and the maintenance of genome stability.<br>Medicine, Faculty of<br>Graduate
21
Deretic, Jovana. "Identifying new shared substrates of Aurora kinases at the mitotic apparatus." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31137.
Texto completoResumen
Aurora A and B are the major kinases that control key events in mitosis, such as centrosome function, spindle assembly, chromosome segregation and cytokinesis, through phosphorylation of multiple proteins. These kinases share identical consensus target motifs, so the substrate specificity is determined by distinctive sub-cellular localization of the Auroras. Many proteins have been identified as targets of either Aurora A, or Aurora B, or both kinases by mass spectrometry studies. However, only a few of the identified phosphorylation sites in these targets have a characterized function in vivo. Therefore, the molecular mechanisms underlying the regulation of certain mitotic events by Aurora kinases remain unclear. The objective of my work was to develop a tool for identifying new substrates of both Aurora kinases. More specifically, I aimed to identify the molecular targets of Aurora A at the kinetochores, and determine how Aurora A contributes to the error correction near spindle poles. I first demonstrated that the outer kinetochore protein HEC1/Ndc80, phosphorylated by Aurora B at kinetochores, can also be phosphorylated by Aurora A close to the centrosomes (Chapter 2). My finding showed that Aurora kinases can share substrates in the cells and revealed the mechanism by which Aurora A contributes to the error-correction near spindle poles. To identify and characterise novel substrates of Aurora kinases, I developed a bioinformatic approach in collaboration with the Centre Bioinformatician, Alastair Kerr. This bioinformatic method uses the Auroras’ shared consensus motifs combined with several parameters that control the substrate specificity of Aurora kinases. I tested the phosphorylation of the chosen candidates in vitro using radiolabelled kinase assays. In my study, five proteins were validated - SPICE1, TTLL4, AHCTF1, CLASP2 and an uncharacterized protein KIAA1468 - as in vitro substrates of Aurora A and Aurora B kinases (Chapter 3). I then focussed on the Aurora kinases-dependent regulation of spindle and centriole-associated protein, SPICE1, in cells (Chapter 4). Using either site-directed mutagenesis of SPICE1 or inhibition of Aurora kinases with small molecule inhibitors, I found that the predicted phosphorylation of the SPICE1 C terminus had the function in cells of directing the SPICE1 localization on the spindle MTs. My results demonstrate the high accuracy of this genome-wide bioinformatics approach. By complementing mass spectrometry studies, here lies a potential for the identification of other unknown substrates, which is important for the general understanding of how Aurora kinases regulate the mitotic apparatus.
22
Bauer, Nicola, Ana-Violeta Fonseca, Mareike Florek, et al. "New Insights into the Cell Biology of Hematopoietic Progenitors by Studying Prominin-1 (CD133)." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-136136.
Texto completoResumen
Prominin-1 (alias CD133) has received considerable interest because of its expression by several stem and progenitor cells originating from various sources, including the neural and hematopoietic systems. As a cell surface marker, prominin-1 is now used for somatic stem cell isolation. Its expression in cancer stem cells has broadened its clinical value, as it might be useful to outline new prospects for more effective cancer therapies by targeting tumor-initiating cells. Cell biological studies of this molecule have demonstrated that it is specifically concentrated in various membrane structures that protrude from the planar areas of the plasmalemma. Prominin-1 binds to the plasma membrane cholesterol and is associated with a particular membrane microdomain in a cholesterol-dependent manner. Although its physiological function is not yet determined, it is becoming clear that this cell surface protein, as a unique marker of both plasma membrane protrusions and membrane microdomains, might reveal new aspects of the cell biology of rare stem and cancer stem cells. The aim of this review is to outline the recent discoveries regarding the dynamic reorganization of the plasma membrane of rare CD133+ hematopoietic progenitor cells during cell migration and division<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
23
Bauer, Nicola, Ana-Violeta Fonseca, Mareike Florek, et al. "New Insights into the Cell Biology of Hematopoietic Progenitors by Studying Prominin-1 (CD133)." Karger, 2008. https://tud.qucosa.de/id/qucosa%3A27699.
Texto completoResumen
Prominin-1 (alias CD133) has received considerable interest because of its expression by several stem and progenitor cells originating from various sources, including the neural and hematopoietic systems. As a cell surface marker, prominin-1 is now used for somatic stem cell isolation. Its expression in cancer stem cells has broadened its clinical value, as it might be useful to outline new prospects for more effective cancer therapies by targeting tumor-initiating cells. Cell biological studies of this molecule have demonstrated that it is specifically concentrated in various membrane structures that protrude from the planar areas of the plasmalemma. Prominin-1 binds to the plasma membrane cholesterol and is associated with a particular membrane microdomain in a cholesterol-dependent manner. Although its physiological function is not yet determined, it is becoming clear that this cell surface protein, as a unique marker of both plasma membrane protrusions and membrane microdomains, might reveal new aspects of the cell biology of rare stem and cancer stem cells. The aim of this review is to outline the recent discoveries regarding the dynamic reorganization of the plasma membrane of rare CD133+ hematopoietic progenitor cells during cell migration and division.<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
24
Scrofani, Jacopo 1984. "Mechanism of RanGTP dependent microtubule assembly during mitosis." Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/289621.
Texto completoResumen
During mitosis, spindle assembly involves different sources of microtubules including centrosomes and chromosomes. While the role of centrosomes has been extensively studied, we still do not fully understand how chromosomes trigger microtubule assembly thereby contributing to the formation of the mitotic spindle. The chromosomal pathway is largely determined by a RanGTP gradient centered on the chromosomes that induces the local activation of spindle assembly factors. To get a better understanding on the RanGTP-dependent microtubule assembly during mitosis we aimed at:
i) Identifying new RanGTP regulated proteins involved in spindle assembly. Our results pointed to three novel proteins with a putative mitotic role in the RanGTP pathway.
ii) Understanding how the RanGTP pathway regulates microtubule nucleation during mitosis. We found that TPX2 together with Aurora-A and RHAMM are part of a RanGTP-dependent complex that binds and strongly stimulates the -TuRC nucleation activity.
iii) Investigating the contribution of the RanGTP pathway to spindle assembly. Our data provided novel evidences that MTs nucleated close to the chromosomes participate in the k-fibers assembly process.<br>Durante la mitosis, el proceso de ensamblaje del huso mitótico implica diferentes fuentes de microtúbulos incluyendo centrosomas y cromosomas. Mientras que el rol de los centrosomas ha sido extensamente estudiado, no se entiende en su totalidad como los cromosomas inducen la formación de microtúbulos contribuyendo de esta manera al ensamblaje del huso mitótico. La vía de los cromosomas está mayormente determinada por un gradiente de RanGTP, centrado en los cromosomas, que induce la activación local de factores mitóticos. Para entender el mecanismo que promueve el ensamblaje de los microtúbulos vía RanGTP durante la mitosis, este trabajo tuvo los siguientes objetivos:
i) La identificación de nuevas proteínas reguladas por RanGTP que participan en el ensamblaje del huso. Nuestros resultados apuntan a tres nuevas proteínas con un posible papel mitótico en la vía RanGTP de ensamblaje de los microtúbulos.
ii) El estudio de el mecanismo para el cual RanGTP regula la nucleación de microtúbulos durante la mitosis. Hemos descubierto que TPX2 junto con Aurora-A y RHAMM son parte de un complejo RanGTP dependiente que estimula la actividad de nucleación de el TuRC.
iii) El estudio del papel de la vía de RanGTP en el ensamblaje del huso. En particular, nuestros datos proporcionan nuevas evidencias sobre la participación de los MTs nucleados alrededor de los cromosomas en el proceso del ensamble de fibras cinetocóricas.
25
Zumdieck, Alexander. "Dynamics of Active Filament Systems." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1139849910030-68242.
Texto completoResumen
Aktive Filament-Systeme, wie zum Beispiel das Zellskelett, sind Beispiele einer interessanten Klasse neuartiger Materialien, die eine wichtige Rolle in der belebten Natur spielen. Viele wichtige Prozesse in lebenden Zellen wie zum Beispiel die Zellbewegung oder Zellteilung basieren auf dem Zellskelett. Das Zellskelett besteht aus Protein-Filamenten, molekularen Motoren und einer großen Zahl weiterer Proteine, die an die Filamente binden und diese zu einem Netz verbinden können. Die Filamente selber sind semifexible Polymere, typischerweise einige Mikrometer lang und bestehen aus einigen hundert bis tausend Untereinheiten, typischerweise Mono- oder Dimeren. Die Filamente sind strukturell polar, d.h. sie haben eine definierte Richtung, ähnlich einer Ratsche. Diese Polarität begründet unterschiedliche Polymerisierungs- und Depolymerisierungs-Eigenschaften der beiden Filamentenden und legt außerdem die Bewegungsrichtung molekularer Motoren fest. Die Polymerisation von Filamenten sowie Krafterzeugung und Bewegung molekularer Motoren sind aktive Prozesse, die kontinuierlich chemische Energie benötigen. Das Zellskelett ist somit ein aktives Gel, das sich fern vom thermodynamischen Gleichgewicht befindet. In dieser Arbeit präsentieren wir Beschreibungen solcher aktiven Filament-Systeme und wenden sie auf Strukturen an, die eine ähnliche Geometrie wie zellulare Strukturen haben. Beispiele solcher zellularer Strukturen sind Spannungsfasern, kontraktile Ringe oder mitotische Spindeln. Spannungsfasern sind für die Zellbewegung essentiell; sie können kontrahieren und so die Zelle vorwärts bewegen. Die mitotische Spindel trennt Kopien der Erbsubstanz DNS vor der eigentlichen Zellteilung. Der kontraktile Ring schließlich trennt die Zelle am Ende der Zellteilung. In unserer Theorie konzentrieren wir uns auf den Einfluß der Polymerisierung und Depolymerisierung von Filamenten auf die Dynamik dieser Strukturen. Wir zeigen, dass der kontinuierliche Umschlag (d.h. fortwährende Polymerisierung und Depolymerisierung) von Filamenten unabdingbar ist für die kontraktion eines Rings mit konstanter Geschwindigkeit, so wie in Experimenten mit Hefezellen beobachtet. Mit Hilfe einer mikroskopisch motivierten Beschreibung zeigen wir, wie &quot;filament treadmilling&quot;, also Filament Polymerisierung an einem Ende mit der gleichen Rate wie Depolymerisierung am anderen Ende, zur Spannung in Filament Bündeln und Ringen beitragen kann. Ein zentrales Ergebnis ist, dass die Depolymerisierung von Filamenten in Anwesenheit von filamentverbindenden Proteinen das Zusammenziehen dieser Bündel sogar in Abwesenheit molekulare Motoren herbeiführen kann. Ferner entwickeln wir eine generische Kontinuumsbeschreibung aktiver Filament-Systeme, die ausschließlich auf Symmetrien der Systeme beruht und von mikroskopischen Details unabhängig ist. Diese Theorie erlaubt uns eine komplementäre Sichtweise auf solche aktiven Filament-Systeme. Sie stellt ein wichtiges Werkzeug dar, um die physikalischen Mechanismen z.B. in Filamentbündeln aber auch bei der Bildung von Filamentringen im Zellkortex zu untersuchen. Schließlich entwickeln wir eine auf einem Kräftegleichgewicht basierende Beschreibung für bipolare Strukturen aktiver Filamente und wenden diese auf die mitotische Spindel an. Wir diskutieren Bedingungen für die Bildung und Stabilität von Spindeln<br>Active filament systems such as the cell cytoskeleton represent an intriguing class of novel materials that play an important role in nature. The cytoskeleton for example provides the mechanical basis for many central processes in living cells, such as cell locomotion or cell division. It consists of protein filaments, molecular motors and a host of related proteins that can bind to and cross-link the filaments. The filaments themselves are semiflexible polymers that are typically several micrometers long and made of several hundreds to thousands of subunits. The filaments are structurally polar, i.e. they possess a directionality. This polarity causes the two distinct filament ends to exhibit different properties regarding polymerization and depolymerization and also defines the direction of movement of molecular motors. Filament polymerization as well as force generation and motion of molecular motors are active processes, that constantly use chemical energy. The cytoskeleton is thus an active gel, far from equilibrium. We present theories of such active filament systems and apply them to geometries reminiscent of structures in living cells such as stress fibers, contractile rings or mitotic spindles. Stress fibers are involved in cell locomotion and propel the cell forward, the mitotic spindle mechanically separates the duplicated sets of chromosomes prior to cell division and the contractile ring cleaves the cell during the final stages of cell division. In our theory, we focus in particular on the role of filament polymerization and depolymerization for the dynamics of these structures. Using a mean field description of active filament systems that is based on the microscopic processes of filaments and motors, we show how filament polymerization and depolymerization contribute to the tension in filament bundles and rings. We especially study filament treadmilling, an ubiquitous process in cells, in which one filament end grows at the same rate as the other one shrinks. A key result is that depolymerization of filaments in the presence of linking proteins can induce bundle contraction even in the absence of molecular motors. We extend this description and apply it to the mitotic spindle. Starting from force balance considerations we discuss conditions for spindle formation and stability. We find that motor binding to filament ends is essential for spindle formation. Furthermore we develop a generic continuum description that is based on symmetry considerations and independent of microscopic details. This theory allows us to present a complementary view on filament bundles, as well as to investigate physical mechanisms behind cell cortex dynamics and ring formation in the two dimensional geometry of a cylinder surface. Finally we present a phenomenological description for the dynamics of contractile rings that is based on the balance of forces generated by active processes in the ring with forces necessary to deform the cell. We find that filament turnover is essential for ring contraction with constant velocities such as observed in experiments with fission yeast
26
Zumdieck, Alexander. "Dynamics of Active Filament Systems: The Role of Filament Polymerization and Depolymerization." Doctoral thesis, Technische Universität Dresden, 2005. https://tud.qucosa.de/id/qucosa%3A24642.
Texto completoResumen
Aktive Filament-Systeme, wie zum Beispiel das Zellskelett, sind Beispiele einer interessanten Klasse neuartiger Materialien, die eine wichtige Rolle in der belebten Natur spielen. Viele wichtige Prozesse in lebenden Zellen wie zum Beispiel die Zellbewegung oder Zellteilung basieren auf dem Zellskelett. Das Zellskelett besteht aus Protein-Filamenten, molekularen Motoren und einer großen Zahl weiterer Proteine, die an die Filamente binden und diese zu einem Netz verbinden können. Die Filamente selber sind semifexible Polymere, typischerweise einige Mikrometer lang und bestehen aus einigen hundert bis tausend Untereinheiten, typischerweise Mono- oder Dimeren. Die Filamente sind strukturell polar, d.h. sie haben eine definierte Richtung, ähnlich einer Ratsche. Diese Polarität begründet unterschiedliche Polymerisierungs- und Depolymerisierungs-Eigenschaften der beiden Filamentenden und legt außerdem die Bewegungsrichtung molekularer Motoren fest. Die Polymerisation von Filamenten sowie Krafterzeugung und Bewegung molekularer Motoren sind aktive Prozesse, die kontinuierlich chemische Energie benötigen. Das Zellskelett ist somit ein aktives Gel, das sich fern vom thermodynamischen Gleichgewicht befindet. In dieser Arbeit präsentieren wir Beschreibungen solcher aktiven Filament-Systeme und wenden sie auf Strukturen an, die eine ähnliche Geometrie wie zellulare Strukturen haben. Beispiele solcher zellularer Strukturen sind Spannungsfasern, kontraktile Ringe oder mitotische Spindeln. Spannungsfasern sind für die Zellbewegung essentiell; sie können kontrahieren und so die Zelle vorwärts bewegen. Die mitotische Spindel trennt Kopien der Erbsubstanz DNS vor der eigentlichen Zellteilung. Der kontraktile Ring schließlich trennt die Zelle am Ende der Zellteilung. In unserer Theorie konzentrieren wir uns auf den Einfluß der Polymerisierung und Depolymerisierung von Filamenten auf die Dynamik dieser Strukturen. Wir zeigen, dass der kontinuierliche Umschlag (d.h. fortwährende Polymerisierung und Depolymerisierung) von Filamenten unabdingbar ist für die kontraktion eines Rings mit konstanter Geschwindigkeit, so wie in Experimenten mit Hefezellen beobachtet. Mit Hilfe einer mikroskopisch motivierten Beschreibung zeigen wir, wie &quot;filament treadmilling&quot;, also Filament Polymerisierung an einem Ende mit der gleichen Rate wie Depolymerisierung am anderen Ende, zur Spannung in Filament Bündeln und Ringen beitragen kann. Ein zentrales Ergebnis ist, dass die Depolymerisierung von Filamenten in Anwesenheit von filamentverbindenden Proteinen das Zusammenziehen dieser Bündel sogar in Abwesenheit molekulare Motoren herbeiführen kann. Ferner entwickeln wir eine generische Kontinuumsbeschreibung aktiver Filament-Systeme, die ausschließlich auf Symmetrien der Systeme beruht und von mikroskopischen Details unabhängig ist. Diese Theorie erlaubt uns eine komplementäre Sichtweise auf solche aktiven Filament-Systeme. Sie stellt ein wichtiges Werkzeug dar, um die physikalischen Mechanismen z.B. in Filamentbündeln aber auch bei der Bildung von Filamentringen im Zellkortex zu untersuchen. Schließlich entwickeln wir eine auf einem Kräftegleichgewicht basierende Beschreibung für bipolare Strukturen aktiver Filamente und wenden diese auf die mitotische Spindel an. Wir diskutieren Bedingungen für die Bildung und Stabilität von Spindeln.<br>Active filament systems such as the cell cytoskeleton represent an intriguing class of novel materials that play an important role in nature. The cytoskeleton for example provides the mechanical basis for many central processes in living cells, such as cell locomotion or cell division. It consists of protein filaments, molecular motors and a host of related proteins that can bind to and cross-link the filaments. The filaments themselves are semiflexible polymers that are typically several micrometers long and made of several hundreds to thousands of subunits. The filaments are structurally polar, i.e. they possess a directionality. This polarity causes the two distinct filament ends to exhibit different properties regarding polymerization and depolymerization and also defines the direction of movement of molecular motors. Filament polymerization as well as force generation and motion of molecular motors are active processes, that constantly use chemical energy. The cytoskeleton is thus an active gel, far from equilibrium. We present theories of such active filament systems and apply them to geometries reminiscent of structures in living cells such as stress fibers, contractile rings or mitotic spindles. Stress fibers are involved in cell locomotion and propel the cell forward, the mitotic spindle mechanically separates the duplicated sets of chromosomes prior to cell division and the contractile ring cleaves the cell during the final stages of cell division. In our theory, we focus in particular on the role of filament polymerization and depolymerization for the dynamics of these structures. Using a mean field description of active filament systems that is based on the microscopic processes of filaments and motors, we show how filament polymerization and depolymerization contribute to the tension in filament bundles and rings. We especially study filament treadmilling, an ubiquitous process in cells, in which one filament end grows at the same rate as the other one shrinks. A key result is that depolymerization of filaments in the presence of linking proteins can induce bundle contraction even in the absence of molecular motors. We extend this description and apply it to the mitotic spindle. Starting from force balance considerations we discuss conditions for spindle formation and stability. We find that motor binding to filament ends is essential for spindle formation. Furthermore we develop a generic continuum description that is based on symmetry considerations and independent of microscopic details. This theory allows us to present a complementary view on filament bundles, as well as to investigate physical mechanisms behind cell cortex dynamics and ring formation in the two dimensional geometry of a cylinder surface. Finally we present a phenomenological description for the dynamics of contractile rings that is based on the balance of forces generated by active processes in the ring with forces necessary to deform the cell. We find that filament turnover is essential for ring contraction with constant velocities such as observed in experiments with fission yeast.
27
Shirasu-Hiza, Michele. "Mitotic microtubule depolymerization and XMAP215 /." 2004. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3109912.
Texto completo
28
Alsop, G. Bradley. "Dissecting induction of cell cleavage." Thesis, 2003. http://hdl.handle.net/1957/30497.
Texto completoResumen
Cytokinesis separates replicated chromosomes and cytoplasm into two
daughter cells. In animal cells, this is achieved by the formation of a cleavage
furrow that bisects the mitotic (or meiotic) spindle. It is known that the mitotic
apparatus defines the cell cleavage plane. However, it is not clear how the mitotic
apparatus initiates the cleavage furrow. Each part of the mitotic apparatus; namely
asters, central spindle (microtubule arrays and the spindle midzone), and
chromosomes, has been found capable of inducing a cleavage furrow in certain
cell types. Yet it is uncertain which part is the essential source of the signal and
whether all parts act in concert.
This thesis systematically examines in grasshopper spermatocytes 1) which
spindle constituent is the essential source of furrow signal; 2) the impact of
microtubules on distribution of actin filaments and positioning of cell cleavage
relative to spindle reorganization; 3) the independent role of the spindle midzone
relative to microtubules in furrow initiation and ingression. These examinations
combine micromanipulation with digital-enhanced polarization microscopy and
epifluorescence microscopy, in which mitotic spindles in living cells are
mechanically dissected and rearranged as desired as well as microfixed to evaluate
and propose models for cell cleavage.
This thesis has come to the conclusion that none of structural constituents
of the spindle apparatus is essential for cell cleavage induction except
microtubules. First, furrow induction occurs regardless of a particular spindle
constituent, so long as sufficient microtubules are present to form bipolar arrays.
Second, microtubules continuously dictate distribution of actin filaments and
positioning of cell cleavage. Asymmetric alterations of spindle microtubules
dynamically affect the location of the spindle midzone, distribution of actin
filaments, and ultimately position of the cleavage furrow in cells containing a
bipolar spindle, monopolar spindle, or half-spindle. Third, actin filaments are
distributed to the furrow region by microtubule-mediated transport, but organized
by the midzone, which is essential for furrow ingression, but not initiation. These
results suggest that during post-anaphase spindle assembly, actin filaments are
excluded by bipolar microtubule arrays to the equatorial cell cortex where they
bundle into a contractile ring with cytokinetic factors.<br>Graduation date: 2004
29
Tsai, Wan-Yu, and 蔡宛育. "Spindle Scaling and Orientation in Symmetric and Asymmetric Cell Division." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/67478467389797114286.
Texto completoResumen
碩士<br>國立臺灣大學<br>分子與細胞生物學研究所<br>104<br>During cell division, a microtubule-based macromolecular machine known as mitotic spindle plays an important role to segregate chromosomes to two daughter cells. Molecules locating at cell cortex and spindle equator regulate spindle architecture and orientation. In our study, mitotic spindles in symmetric and asymmetric cell division were investigated. Several human cancer cell lines from lung, breast and colon were chosen as models of symmetric division. By immunofluorescence, we found that the functionally selected highly invasive lung adenocarcinoma cell line, CL1-5, has lengthened steady-state metaphase spindle morphologically distinct from the original line, CL1-0. In addition, our recent study showed that in CL1-5, there was an up-regulation of kinesin-5, a motor protein involved in pole separation and chromosome segregation during mitosis. Therefore, we applied low dose kinesin-5 inhibitor to CL1-5 to reveal the relationship between kinesin-5 and spindle length scaling. As a result, dose-dependent change of spindle length was found. Similarly, an in vivo selected metastatic colon cancer cell line SW620 presented lengthened steady-state metaphase spindle compared to its in situ cell line SW480. These findings entitle spindle length scaling a candidate of cellular marker for metastatic cancer clone, and emphasize alternative benefits of kinesin-5 inhibitor in anti-cancer therapies. On the other hand, we analyzed spindle orientation in asymmetric cell division (ACD) by time-lapse imaging with mouse embryonic stem cells as our model. Microenvironment that promoted asymmetric cell division was built by localized Wnt3a beads. We have established live imaging platform and validated localized Wnt3a signals did orient cell division. We are currently investigating molecules locating at the cell cortex that mediate spindle orientation in asymmetric cell division.
30
Hirsch, Sophia Madeleine. "Spatial regulation of protein function in cell division and midbody assembly." Thesis, 2021. https://doi.org/10.7916/d8-rya4-0758.
Texto completoResumen
Cytokinesis is the physical division of one cell into two driven by an actomyosin contractile ring and positioned by signals from microtubules. This process is highly regulated spatially and temporally to ensure accurate division into two daughter cells. Here, I present work that builds upon our understanding of cytokinesis, focusing on the spatial requirements for protein function during cell division and midbody assembly. In Chapter 1, I present an introduction to cytokinesis and the cell and molecular mechanisms that govern the process. In Chapter 2, I present work I contributed to on the use of Upconverting nanoparticles for co-alignment of visible and infrared light on a light microscope. In Chapter 3, I present work developing a new microscopy technology called FLIRT (Fast Local Infrared Thermogenetics) that uses infrared light to inactivate fast-acting temperature sensitive protein function with subcellular precision and validate its use to study cytokinesis and cell fate signaling in the nematode Caenorhabditis elegans. In Chapter 4, I improve upon FLIRT technology by increasing its precision and demonstrate its use in studying the spatial regulation of key cytokinesis proteins including the actomyosin cytoskeleton in contractile ring constriction.
The central spindle is an array of antiparallel overlapping microtubules that forms between the separating chromosomes in anaphase and is thought to serve as a signaling hub for cytokinesis. The central spindle is thought to become compacted during contractile ring constriction to form the dense midbody at the end of cell division. In Chapter 5, I investigate the requirements for central spindle microtubules in assembling midbodies in the C. elegans one-cell embryo. I present evidence that the CENP-F-like protein HCP-1 plays a primary role relative to its paralog HCP-2 in assembling the central spindle, and that the midbody can form independently of central spindle assembly.
In Chapter 6, I discuss future directions for my work on both technology development and the mechanisms of cytokinesis. Through this work, I develop new technologies and hypotheses for how cytokinesis is spatially regulated within a cell, adding new complexity to our understanding of cell division.
31
Cui, Hong Ph D. "Role of the mitotic spindle in the equal segregation of an extrachromosomal element in Saccharomyces cerevisiae." 2008. http://hdl.handle.net/2152/17846.
Texto completoResumen
The Saccharomyces cerevisiae plasmid, 2 micron circle, resides in the yeast nucleus at a high copy number. It provides no apparent growth advantage to its host, nor imposes any significant growth disadvantage. The plasmid is an excellent paradigm for studying mechanisms utilized in the persistence of a eukaryotic selfish DNA element that is selectively neutral. The plasmid achieves stable propagation and copy number maintenance by combining a partitioning system and an amplification system. The partitioning proteins Rep1p and Rep2p promote the recruitment of the histone H3 variant Cse4p and the yeast cohesin complex to the partitioning locus STB during S phase, leading to the formation of a functional partitioning complex which segregates the plasmid equally during mitosis. The integrity of the mitotic spindle is a pre-requisite for the specific nuclear localization of the plasmid as well as for plasmid association with a subset of the partitioning proteins such as Cse4p and the cohesin complex. The work presented in this thesis reveals, using tools of molecular genetics and cell biology, the involvement and possible functions of a microtubule associated nuclear motor protein, Kip1p, in the 2 micron circle partitioning pathway. The plasmid missegregates in kip1[Delta] cells, but not in cells harboring deletions of genes coding for the other nuclear motors. Kip1p interacts with the plasmid partitioning system and promotes the association of Cse4p and the cohesin complex with STB. Lack of Kip1p function delocalizes the plasmid from its characteristic nuclear locale in close proximity to the spindle pole body. The distance between a reporter plasmid and the spindle pole body is nearly doubled in a kip1[Delta] host strain. We propose that, unlike the conventional roles played by nuclear motors in spindle function and chromosome segregation, the Kip1p motor assists the 2 micron circle in associating with the mitotic spindle and translocating to its ‘partitioning center’.<br>text
32
Enzor, Rikki S. "The Fanconi anemia signaling network regulates the mitotic spindle assembly checkpoint." Thesis, 2014. http://hdl.handle.net/1805/5904.
Texto completoResumen
Indiana University-Purdue University Indianapolis (IUPUI)<br>Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity.
33
Zimdahl, Bryan Jeffrey. "Requirement for Lis1 in Normal and Malignant Stem Cell Renewal." Diss., 2013. http://hdl.handle.net/10161/8042.
Texto completoResumen
<p>Stem cells are defined by their ability to make more stem cells, a property known as self-renewal and their ability to generate cells that enter differentiation. One mechanism by which fate decisions can be effectively controlled in stem cells is through asymmetric division and the correct partitioning and inheritance of cell fate determinants. While hematopoietic stem cells have the capacity to divide through asymmetric division, the molecular machinery that regulates this process is unknown and whether its activity is required in vivo remains unclear. Here we show that Lis1, a dynein-binding protein and regulator of asymmetric division, is critically required for blood development and for hematopoietic stem cell renewal in fetal and adult life. In particular, conditional deletion of Lis1 led to a severe bloodless phenotype and embryonic lethality in vivo. In both fetal and adult mice, loss of Lis1 led to a failure of normal self-renewal, which included impaired colony-forming ability in vitro and defects in long-term reconstitution ability following transplantation. As a possible mechanism, we find that the absence of Lis1 in hematopoietic cells, in part, accelerates differentiation linked to the incorrect inheritance of cell fate determinants. Furthermore, using a live cell imaging strategy, we find that the incorrect inheritance of cell fate determinants observed following the loss of Lis1 is due defects in spindle positioning and orientation. Finally, using two animal models of undifferentiated myeloid leukemia, we show that Lis1 is critical for the aberrant cell growth that occurs in cancer. Deletion of Lis1 both at the early and late stages of myeloid leukemia blocked its propagation in vivo and led to a marked improvement in survival. Together, these data identify Lis1 and the directed control of asymmetric division as key regulators of normal and malignant hematopoietic development.</p><br>Dissertation
34
Papaluca, Arturo. "Asymmetric cell division intersects with cell geometry : a method to extrapolate and quantify geometrical parameters of sensory organ precursors." Thèse, 2014. http://hdl.handle.net/1866/12060.
Texto completoResumen
La division cellulaire asymétrique (DCA) consiste en une division pendant laquelle des déterminants cellulaires sont distribués préférentiellement dans une des deux cellules filles. Par l’action de ces déterminants, la DCA générera donc deux cellules filles différentes. Ainsi, la DCA est importante pour générer la diversité cellulaire et pour maintenir l’homéostasie de certaines cellules souches. Pour induire une répartition asymétrique des déterminants cellulaires, le positionnement du fuseau mitotique doit être très bien contrôlé. Fréquemment ceci génère deux cellules filles de tailles différentes, car le fuseau mitotique n’est pas centré pendant la mitose, ce qui induit un positionnement asymétrique du sillon de clivage.
Bien qu’un complexe impliquant des GTPases hétérotrimériques et des protéines liant les microtubules au cortex ait été impliqué directement dans le positionnement du fuseau mitotique, le mécanisme exact induisant le positionnement asymétrique du fuseau durant la DCA n'est pas encore compris. Des études récentes suggèrent qu’une régulation asymétrique du cytosquelette d’actine pourrait être responsable de ce positionnement asymétrique du faisceau mitotique. Donc, nous émettons l'hypothèse que des contractions asymétriques d’actine pendant la division cellulaire pourraient déplacer le fuseau mitotique et le sillon de clivage pour créer une asymétrie cellulaire. Nos résultats préliminaires ont démontré que le blebbing cortical, qui est une indication de tension corticale et de contraction, se produit préférentiellement dans la moitié antérieure de cellule précurseur d’organes sensoriels (SOP) pendant le stage de télophase.
Nos données soutiennent l'idée que les petites GTPases de la famille Rho pourraient être impliqués dans la régulation du fuseau mitotique et ainsi contrôler la DCA des SOP. Les paramètres expérimentaux développés pour cette thèse, pour étudier la régulation de l’orientation et le positionnement du fuseau mitotique, ouvrirons de nouvelles avenues pour contrôler ce processus, ce qui pourrait être utile pour freiner la progression de cellules cancéreuses. Les résultats préliminaires de ce projet proposeront une manière dont les petites GTPases de la famille Rho peuvent être impliqués dans le contrôle de la division cellulaire asymétrique in vivo dans les SOP. Les modèles théoriques qui sont expliqués dans cette étude pourront servir à améliorer les méthodes quantitatives de biologie cellulaire de la DCA.<br>Asymmetric cell division (ACD) consists in a cellular division during which specific cell fate determinants are distributed preferentially in one daughter cell, which then differentiate from its sibling. Hence, ACD is important to generate cell diversity and is used to regulate stem cells homeostasis. For proper asymmetric distribution of cell fate determinants, the positioning of the mitotic spindle has to be tightly controlled. Frequently, this induces a cell size asymmetry, since the spindle is then not centered during mitosis, leading to an asymmetric positioning of the cleavage furrow.
Although small small GTPases have been shown to act directly on the spindle, the exact mechanism controlling spindle positioning during ACD is not understood. Recent studies suggest that an independent, yet uncharacterized pathway is involved in spindle positioning, which is likely to involve an asymmetric regulation of the actin cytoskeleton. Indeed, actin enables spindle anchoring to the cortex. Hence we hypothesize that asymmetric actin contractions during cytokinesis might displace the mitotic spindle and the cleavage furrow, leading to cell size asymmetry. Interestingly, from our preliminary results we observed that cortical blebbing, which is a read-out of cortical tension/contraction, preferentially occurs on the anterior side of the dividing sensory organ precursor (SOP) cells at telophase.
Our preliminary data support the idea that Rho small GTPases might be implicated in regulation of the mitotic spindle hence controlling asymmetric cell division of SOP cells. The experimental settings developed for this thesis, for studying regulation of the mitotic spindle orientation and positioning will serve as proof of concept of how geneticist and biochemist experts could design ways to control such process by different means in cancerous cells. The preliminary results from this project open novel insights on how the Rho small GTPases might be implicated in controlling asymmetric cell division hence their dynamics in vivo of such process during SOP development. Furthermore, the assays and the theoretical model developed in this study can be used as background that could serve to design improved quantitative experimental methods for cell biology synchronizing sub-networks of ACD mechanism.
35
Seldin, Lindsey. "The Role of Spindle Orientation in Epidermal Development and Homeostasis." Diss., 2015. http://hdl.handle.net/10161/9835.
Texto completoResumen
<p>Robust regulation of spindle orientation is essential for driving asymmetric cell divisions (ACDs), which generate cellular diversity within a tissue. During the development of the multilayered mammalian epidermis, mitotic spindle orientation in the proliferative basal cells is crucial not only for dictating daughter cell fate but also for initiating stratification of the entire tissue. A conserved protein complex, including LGN, Nuclear mitotic apparatus (NuMA) and dynein/dynactin, plays a key role in establishing proper spindle orientation during ACDs. Two of these proteins, NuMA and dynein, interact directly with astral microtubules (MTs) that emanate from the mitotic spindle. While the contribution of these MT-binding interactions to spindle orientation remains unclear, these implicate apical NuMA and dynein as strong candidates for the machinery required to transduce pulling forces onto the spindle to drive perpendicular spindle orientation. </p><p> In my work, I first investigated the requirements for the cortical recruitment of NuMA and dynein, which had never been thoroughly addressed. I revealed that NuMA is required to recruit the dynein/dynactin complex to the cell cortex of cultured epidermal cells. In addition, I found that interaction with LGN is necessary but not sufficient for cortical NuMA recruitment. This led me to examine the role of additional NuMA-interacting proteins in spindle orientation. Notably, I identified a role for the 4.1 protein family in stabilizing NuMA's association with the cell cortex using a FRAP (fluorescence recovery after photobleaching)-based approach. I also showed that NuMA's spindle orientation activity is perturbed in the absence of 4.1 interactions. This effect was demonstrated in culture using both a cortical NuMA/spindle alignment assay as well as a cell stretch assay. Interestingly, I also noted a significant increase in cortical NuMA localization as cells enter anaphase. I found that inhibition of Cdk1 or mutation of a single residue on NuMA mimics this effect. I also revealed that this anaphase localization is independent of LGN and 4.1 interactions, thus revealing two independent mechanisms responsible for NuMA cortical recruitment at different stages of mitosis. </p><p> After gaining a deeper understanding of how NuMA is recruited and stabilized at the cell cortex, I then sought to investigate how cortical NuMA functions during spindle orientation. NuMA contains binding domains in its N- and C-termini that facilitate its interactions with the molecular motor dynein and MTs, respectively. In addition to its known role in recruiting dynein, I was interested in determining whether NuMA's ability to interact directly with MTs was critical for its function in spindle orientation. Surprisingly, I revealed that direct interactions between NuMA and MTs are required for spindle orientation in cultured keratinocytes. I also discovered that NuMA can specifically interact with MT ends and remain attached to depolymerizing MTs. To test the role of NuMA/MT interactions in vivo, I generated mice with an epidermal-specific in-frame deletion of the NuMA MT-binding domain. I determined that this deletion causes randomization of spindle orientation in vivo, resulting in defective epidermal differentiation and barrier formation, as well as neonatal lethality. In addition, conditional deletion of the NuMA MT-binding domain in adult mice results in severe hair growth defects. I found that NuMA is required for proper spindle positioning in hair follicle matrix cells and that differentiation of matrix-derived progeny is disrupted when NuMA is mutated, thus revealing an essential role for spindle orientation in hair morphogenesis. Finally, I discovered hyperproliferative regions in the interfollicular epidermis of these adult mutant mice, which is consistent with a loss of ACDs and perturbed differentiation. Based on these data, I propose a novel mechanism for force generation during spindle positioning whereby cortically-tethered NuMA plays a critical dynein-independent role in coupling MT depolymerization energy with cortical tethering to promote robust spindle orientation accuracy. </p><p> Taken together, my work highlights the complexity of NuMA localization and demonstrates the importance of NuMA cortical stability for productive force generation during spindle orientation. In addition, my findings validate the direct role of NuMA in spindle positioning and reveal that spindle orientation is used reiteratively in multiple distinct cell populations during epidermal morphogenesis and homeostasis.</p><br>Dissertation
36
Fish, Jennifer [Verfasser]. "The evolution of neuronal progenitor cell division in mammals: the role of the abnormal spindle-like microcephaly associated (Aspm) protein and epithelial cell polarity / Jennifer Fish." 2007. http://d-nb.info/985849908/34.
Texto completo
37
Rieckhoff, Elisa Maria. "Hierarchical regulation of spindle size during early development." 2019. https://tud.qucosa.de/id/qucosa%3A74036.
Texto completoResumen
During embryogenesis, a single cell gives rise to a multi-cellular embryo through successive rounds of cell division. As cells become smaller, cellular organelles adapt their sizes accordingly. The size of the mitotic spindle—the microtubule-based structure controlling these divisions—is particularly important as it determines the distance over which chromosomes are segregated. To perform its function properly, spindle size scales with cell size. However, we still lack a mechanistic understanding of the underlying microtubule-based processes that regulate spindle scaling.
In this thesis, I combined quantitative microscopy and laser ablation in zebrafish embryos and Xenopus laevis egg extract encapsulated in oil droplets. My measurements revealed the influence of microtubule length dynamics, transport, and nucleation on cell size-dependent spindle scaling. Strikingly, I discovered a hierarchical regulation of spindle size. In large cells, microtubule nucleation exclusively scales spindle size relative to cell size by changing the number of microtubules within the spindle. In small cells, microtubule dynamics fine-tune spindle size by modulating microtubule length.
To understand the mechanism of spindle scaling, I proposed a theoretical model based on a limiting number of microtubule nucleators and microtubule-associated proteins that regulate microtubule length. The transition from nucleation- to dynamics-based scaling requires that microtubule number and the number of microtubule-associated proteins that promote microtubule growth scale differently with cell size. This can be achieved by sequestering an inhibitor of microtubule nucleation to the cell membrane, which is consistent with my measurements of microtubule nucleation. The differential regimes of spindle scaling modulated by microtubule nucleation and dynamics imply a gradual change in spindle architecture, which may ensure faithful chromosome segregation by spindles of all sizes.
38
Chee, Mark Kuan Leng. "B-cyclin/CDK Regulation of Mitotic Spindle Assembly through Phosphorylation of Kinesin-5 Motors in the Budding Yeast, Saccharomyces cerevisiae." Diss., 2012. http://hdl.handle.net/10161/5419.
Texto completoResumen
<p>Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCF<super>Cdc4</super> ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that in the budding yeast,<italic> Saccharomyces cerevisiae</italic>, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APC<super>Cdh1</super>). I have determined, however, that the Kip1 and Cin8 proteins are actually present at wild-type levels in yeast in the absence of Clb/Cdc28 kinase activity. Here, I show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28, and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's <italic>in vivo</italic> localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, I have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCF<super>Cdc4</super> target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, I propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors. </p><p>In addition to the positive regulation of kinesin-5 function in spindle assembly, I have also found evidence that suggests CDK phosphorylation of kinesin-5 motors at different sites negatively regulates kinesin-5 activity to prevent premature spindle pole separation. I have also begun to characterize a novel putative role for the kinesins-5 in mitochondrial genome inheritance in <italic>S. cerevisiae</italic> that may also be regulated by CDK phosphorylation. </p><p>In the course of my dissertation research, I encountered problems with several established molecular biology tools used by yeast researchers that I have tried to address. I have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in <italic>S. cerevisiae</italic> that can be propagated in the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes <italic>HIS2</italic> and <italic>ADE1</italic>, respectively. My new pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed in order to increase the availability of unique restriction sites within their polylinker regions. Hence, my pRSII plasmids are a complete set of integrating, centromere and 2 episomal plasmids with the biosynthetic marker genes <italic>ADE2</italic>, <italic>HIS3</italic>, <italic>TRP1</italic>, <italic>LEU2</italic>, <italic>URA3</italic>, <italic>HIS2</italic> and <italic>ADE1</italic> and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, I have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin and bialaphos. Finally, in the process of generating the new plasmids, I have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using updated plasmid sequences, I constructed pRS plasmid backbones with a unique restriction site for inserting new markers in order to facilitate future expansion of the pRS/pRSII series.</p><br>Dissertation