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Mechanismy ustavení a udržení polarity PIN přenašečů v Arabidopsis / Mechanisms of establishment and maintenance of PIN polarity in ArabidopsisGlanc, Matouš January 2019 (has links)
Cell polarity is a key concept in plant biology. The subcellular localization of Pin- formed (PIN) auxin efflux carriers in the root of "#$%&'()*&* is remarkably asymmetrical, making PINs prominent markers to study cell polarity. In spite of its developmental importance and two decades of research, the molecular basis of PIN polarity remains largely unknown. In this thesis, I employed advanced transgenic and fluorescence microscopy approaches to gain insight into several aspects of PIN polarity regulation. I participated in establishing a novel genetically encoded inhibitor of endocytosis, an invaluable tool for the study of the importance of endocytosis for various cellular processes, including PIN polarity. I demonstrated that apical polarity of PIN2 needs to be re-established after cell division and that this process depends on endocytosis, '+!,(-( protein secretion and the action of WAG1 and related protein kinases, but not transcytosis, cell-cell signaling or intact cytoskeleton. Finally, I identified the previously unknown role of MAB4/MEL proteins in PIN polarity, which lies in the ability of MAB4/MELs to reduce PIN lateral diffusion and thus contribute to PIN polarity maintenance. My results, besides broadening current understanding of PIN polarity regulation, identify mechanisms that...
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The involvement of ARF6 in rapid membrane recycling during Drosophila spermatocyte cytokinesisFoster, Naomi 14 February 2007 (has links)
Cytokinesis involves constriction of the cell at the equator. Without decreasing in volume, a spherical cell requires a net increase in the surface area during this constriction. The constriction is driven by formation of an actomyosin contractile ring, and the surface increase by addition of membrane during the formation of the cleavage furrow. Both events depend on the central spindle microtubules at the midzone of the spindle and, in particular, on the centralspindlin protein complex. The communication between the central spindle microtubules and the actomyosin ring involves binding of a GAP and a GEF for RhoA to the centralspindlin kinesin Pavarotti/MKLP1. However, it is still unclear which molecular machinery connects the mitotic spindle to membrane trafficking during cleavage furrow ingression. ARF6 is a member of the ARF family of small GTPases, and previous studies suggest that it is an important regulator of membrane trafficking through the endocytic pathway, and cortical Actin remodelling. I generated an arf6 null mutant in Drosophila. arf6 null mutants survive to adulthood without obvious morphological defects, indicating that ARF6 is not required for Drosophila somatic development. However, ARF6 is required for cytokinesis in Drosophila spermatocytes. The centralspindlin kinesin Pavarotti, identified as an ARF6 interactor in a Yeast-2-Hybrid assay, binds ARF6 in GST pulldowns, and interacts genetically with the arf6 mutant. ARF6 localizes to the plasma membrane and a population of early and recycling endosomes. During cytokinesis, ARF6 is enriched on recycling endosomes at the central spindle. arf6 mutants form a cleavage furrow during cytokinesis, which later regresses. Cytokinesis in arf6 mutant spermatocytes lacks the rapid plasma membrane expansion observed during normal divisions. The results of this study suggest that ARF6 might promote rapid recycling of endosomal membrane stores at the central spindle to the plasma membrane during cytokinesis. ARF6 might be recruited to the central spindle via its interaction with Pavarotti, and act as part of the molecular link between the central spindle cytoskeleton and the rapid plasma membrane addition necessary for cytokinesis. Für die Ansicht der quick-time-Movies mit der Endung "avi" ist die Installation des "Apple QuickTime-Players" erforderlich.
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Insights into the Role of Oncogenic BRAF in Tetraploidy and Melanoma InitiationDarp, Revati A. 09 March 2021 (has links)
Melanoma, the most lethal form of skin cancer, arises from altered cells in the melanocyte lineage, but the mechanisms by which these cells progress to melanoma are unknown. To understand the early cellular events that contribute to melanoma formation, we examined melanocytes in melanoma-prone zebrafish strains expressing BRAFV600E, the most common oncogenic form of the BRAF kinase that is mutated in nearly 50% of human melanomas. We found that, unlike wild-type melanocytes, melanocytes in transgenic BRAFV600Eanimals were binucleate and tetraploid. Furthermore, melanocytes in p53-deficient transgenic BRAFV600Eanimals exhibited 8N and greater DNA content, suggesting bypass of a p53-dependent arrest that stops cell cycle progression of tetraploid melanocytes. These data implicate tetraploids generated by increased BRAF pathway activity as contributors to melanoma initiation. Previous studies have used artificial means of generating tetraploids, raising the question of how these cells arise during actual tumor development. To gain insight into the mechanism by which BRAFV600E generates binucleate, tetraploid cells, we established an in vitro model by which such cells are generated following BRAFV600E expression. We demonstrate thatBRAFV600E-generated tetraploids arise via cytokinesis failure during mitosis due to reduced activity of the small GTPase RhoA. We also establish that oncogene-induced centrosome amplification in the G1/S phase of the cell cycle and subsequent increase in the activity of the small GTPase Rac1, partially contribute to this phenotype. These data are of significance as recent studies have shown that aneuploid progeny of tetraploid cells can be intermediates in tumor development, and deep sequencing data suggest that at least one third of melanomas and other solid tumors have undergone a whole genome doubling event during their progression. Taken together, our melanoma-prone zebrafish model and in vitro data suggest a role for BRAFV600E-inducedtetraploidy in the genesis of melanomas. To our knowledge, this is the first in vivo model showing spontaneous rise of tetraploid cells that can give rise to tumors. This novel role of the BRAF oncogene may contribute to tumorigenesis in a broader context.
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Understanding Regulation of the Cytoskeleton during Cell Cycle Transitions through Examination of Crosstalk between Homologous Fission Yeast Pathways, Septation Initiation Network and Morphogenesis ORB6 Network: A DissertationGupta, Sneha 10 December 2013 (has links)
The fission yeast Schizosaccharomyces pombe has become a powerful model system for studying cytokinesis, a process of cytoplasmic division by which one cell divides into two identical daughter cells. Like mammalian cells, S. pombe divides through the use of an actomyosin contractile ring, which is composed of a set of highly conserved cytoskeletal proteins. Cytokinesis in S. pombe is primarily regulated by the SIN pathway, which is activated in late mitosis and is required for actomyosin contractile ring and septum assembly, and also plays a role in spindle checkpoint inactivation, and telophase nuclear positioning. The various functions of the SIN are carried out by the terminal kinase in the pathway called Sid2. The lack of information in the downstream targets of Sid2 has limited our understanding of the different functions of the SIN. We recently showed that, in addition to its other functions, the SIN promotes cytokinesis through inhibition the MOR signaling pathway, which normally drives cell separation and initiation of polarized growth following completion of cytokinesis (Ray et al, 2010). The molecular details of this inhibition and the physiological significance of inhibiting MOR during cytokinesis was unclear. The results presented in Chapter II describe our approach to identify Sid2 substrates, particularly focusing on Nak1 and Sog2 that function in the MOR signaling cascade. We identified and characterized Sid2 phosphorylation sites on the Nak1 and Sog2 proteins. Chapter III explores how post translational modification of MOR proteins by Sid2 regulates polarized growth during cytokinesis. This includes delineating the effect of Sid2 mediated phosphorylation of Nak1 and Sog2 on protein-protein interactions in the MOR pathway as well as on the regulation of their localization during late mitosis. Finally, results in Chapter IV demonstrate that failure to inhibit MOR signaling is lethal because cells initiate septum degradation/cell separation before completing cytokinesis thereby emphasizing the importance of cross-regulation between the two pathways to prevent initiation of the interphase polarity program during cytokinesis.
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Mechanismy ustavení a udržení polarity PIN přenašečů v Arabidopsis / Mechanisms of establishment and maintenance of PIN polarity in ArabidopsisGlanc, Matouš January 2019 (has links)
Cell polarity is a key concept in plant biology. The subcellular localization of Pin- formed (PIN) auxin efflux carriers in the root of "#$%&'()*&* is remarkably asymmetrical, making PINs prominent markers to study cell polarity. In spite of its developmental importance and two decades of research, the molecular basis of PIN polarity remains largely unknown. In this thesis, I employed advanced transgenic and fluorescence microscopy approaches to gain insight into several aspects of PIN polarity regulation. I participated in establishing a novel genetically encoded inhibitor of endocytosis, an invaluable tool for the study of the importance of endocytosis for various cellular processes, including PIN polarity. I demonstrated that apical polarity of PIN2 needs to be re-established after cell division and that this process depends on endocytosis, '+!,(-( protein secretion and the action of WAG1 and related protein kinases, but not transcytosis, cell-cell signaling or intact cytoskeleton. Finally, I identified the previously unknown role of MAB4/MEL proteins in PIN polarity, which lies in the ability of MAB4/MELs to reduce PIN lateral diffusion and thus contribute to PIN polarity maintenance. My results, besides broadening current understanding of PIN polarity regulation, identify mechanisms that...
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Coordination des réseaux cytosquelettiques dans la cytokinèseChambaud, Guillaume 12 1900 (has links)
La cytokinèse est un processus minutieusement régulé par une structure corticale appelée
l'anneau contractile d'actomyosine, sous le contrôle de la petite GTPase RhoA. La protéine
d'échafaudage Anilline est un effecteur de RhoA et organise les différents éléments de l'anneau
permettant sa fermeture. Via son N-terminus, l'Anilline interagit avec la Citron kinase Sticky, la
myosine II et l'actine-F ce qui permet la stabilisation de l'anneau contractile et sa maturation en
anneau du corps central ou «midbody». Via son domaine C-terminal, l'Anilline interagit avec la
RhoA-GTP et les septines pour ancrer l'anneau à la membrane. De précédentes études du
laboratoire ont montré que Sticky et les septines ont des actions opposées sur l'Anilline. Nous
avons donc défini plusieurs ensembles d'interactions entre l'Anilline et les cytosquelettes
d'actomyosine et de septines, qui possèdent des fonctions différentes voire opposées dans la
cytokinèse: ce sont des réseaux cytosquelettiques. L'Anilline est ainsi impliquée dans la
coordination de ces réseaux opposés et RhoA-dépendants. Nous devons encore déterminer si ces
interactions de l'Anilline en N-ter et C-ter peuvent se produire en même temps ou si elles sont
mutuellement exclusives pour coordonner les différents éléments cytosquelettiques pendant la
fermeture de l'anneau contractile. Les cellules S2 de drosophile ont été utilisées pour déterminer
comment l'Anilline coordonnait les réseaux cytosqulettiques de l'anneau contractile. Deux
modèles ont été proposés : l'un où une seule molécule d'Anilline se lie simultanément aux réseaux
en N-ter et C-ter; l'autre modèle suggère qu'une ou plusieurs populations d'Anilline coordonnent
les réseaux de façon mutuellement exclusive. Pour distinguer entre ces deux modèles, des allèles
de séparation de fonction de l'Anilline ont été testés : l'AnillinΔ1-5 qui n'interagit plus avec
Sticky, ainsi que l'AnillinRBD* qui n'interagit plus avec Rho1-GTP et qui ne recrute plus les
septines à l'anneau contractile. Des expériences de sauvetage suite à la déplétion de l'Anilline
endogène ont été réalisées et les tentatives de division ont été captées par microscopie en temps
réel. L'expression de chaque mutant individuellement menait à une fermeture de l’anneau
décalée, ralentie et souvent incomplète. En revanche, la co-expression de l'AnillinΔ1-5 et
AnillinRBD* en trans dans les mêmes cellules a restauré la cinétique normale de la fermeture de
l'anneau. Ce résultat supporte le modèle des populations multiples d'Anilline. Cette étude avance
significativement nos connaissances de l'organisation de l'anneau contractile qui gère la division
de toutes cellules animales. / Cytokinesis is a process thoroughly regulated by a cortical structure called the
actomyosine contractile ring, under the control of the RhoA GTPase. The scaffolding protein
Anillin is a RhoA effector organizing the several elements of the ring, thus permitting its closure.
The AnillinN-terminus interacts with the Citron kinase Sticky, Myosin II and F-actin to stabilize
the contractile ring and drive its maturation to the midbody ring. The AnillinC-terminus interacts
with the RhoA GTPase and the septins to anchor the ring to the membrane. Previous works
revealed that Sticky retains Anillin while the septins shed Anillin from the ring. Therefore,
Anillin is involved in opposed RhoA-dependent cytoskeletal sub-networks to generate or reduce
the tension at the membrane, and their balance is necessary to improve the ring closure. This
study aims to decipher the coordination between these opposed sub-networks. We proposed two
models : either sub-networks on AnillinN-ter and AnillinC-ter are simultaneously organized by
the same molecule of Anillin, or several pools of Anillin coordinate separately the opposed subnetworks.
We generated and expressed several inducible Anillin mutants in drosophila S2 cells :
AnillinΔ1-5 prevents the interaction with Sticky; AnillinRBD* does not interact with RhoA and
perturbs the Anillo-septin assembly. The expression of each mutant individually delayed, slowed
down and failed the ring closure. However, co-expression of single mutants in trans rescued the
ring closure. Moreover, Sticky over-expression improved AnillinRBD* recruitment in the ring.
These results support the multiple pools of Anillin model. This study improves our knowledge on
the contractile ring organization, necessary to succeed cytokinesis in animal cells.
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A Comprehensive Model of the Structure and Function of the FtsZ Ring of Escherichia coliRedfearn, James C. 21 April 2016 (has links)
No description available.
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Actomyosin mechanics at the cell levelErzberger, Anna 29 February 2016 (has links) (PDF)
Almost all animal cells maintain a thin layer of actin filaments and associated proteins underneath the cell membrane. The actomyosin cortex is subject to internal stress patterns which result from the spatiotemporally regulated activity of non-muscle myosin II motors in the actin network. We study how these active stresses drive changes in cell shape and flows within the cortical layer, and how these cytoskeletal deformations and flows govern processes such as cell migration, cell division and organelle transport. Following a continuum mechanics approach, we develop theoretical descriptions for three different cellular processes, to obtain - in collaboration with experimental groups - a detailed and quantitative understanding of the underlying cytoskeletal mechanics.
We investigate the forces and cortex flows involved in adhesion-independent cell migration in confinement. Many types of cell migration rely on the extension of protrusions at the leading edge, where the cells attach to the substrate with specific focal adhesions, and pull themselves forward, exerting stresses in the kPa range. In confined environments however, cells exhibit migration modes which are independent of specific adhesions. Combining hydrodynamic theory, microfluidics and quantitative imaging of motile, non-adherent carcinosarcoma cells, we analyze the mechanical behavior of cells during adhesion-independent migration. We find that the accumulation of active myosin motors in the rear part of these cells results in a retrograde cortical flow as well as the contraction of the cell body in the rear and expansion in the front, and we describe how both processes contribute to the translocation of the cells, depending on the geometric and mechanical parameters of the system. Importantly, we find that the involved propulsive forces are several orders of magnitude lower than during adhesive motility while the achieved migration velocities are similar. Moreover, the distribution of forces on the substrate during non-adhesive migration is fundamentally different, giving rise to a positive force dipole. In contrast to adhesive migration modes, non-adhesive cells move by exerting pushing forces at the rear, acting to expand rather than contract their substrate as they move. These differences may strongly affect hydrodynamic and/or deformational interactions between collectively migrating cells.
In addition to the work outlined above, we study contractile ring formation in the actin cytoskeleton before and during cell division. While in disordered actin networks, myosin motor activity gives rise to isotropic stresses, the alignment of actin filaments in the cortex during cell division introduces a preferred direction for motor-filament interactions, resulting in anisotropies in the cortical stress. Actin filaments align in myosin-dependent shear flows, resulting in possible feedback between motor activity, cortical flows and actin organization. We investigate how the mechanical interplay of these different cortical properties gives rise to the formation of a cleavage furrow during cell division, describing the level of actin filament alignment at different points on the cortex with a nematic order parameter, in analogy to liquid crystal physics. We show that cortical anisotropies arising from shear-flow induced alignment patterns are sufficient to drive the ingression of cellular furrows, even in the absence of localized biochemical myosin up-regulation. This mechanism explains the characteristic appearance of pseudocleavage furrows in polarizing cells.
Finally, we study the characteristic nuclear movements in pseudostratified epithelia during development. These tissues consist of highly proliferative, tightly packed and elongated cells, with nuclei actively travelling to the apical side of the epithelium before each cell division. We explore how cytoskeletal properties act together with the mechanics of the surrounding tissue to control the shape of single cells embedded in the epithelium, and investigate potential mechanisms underlying the observed nuclear movements. These findings form a theoretical basis for a more detailed characterization of processes in pseudostratified epithelia.
Taken together, we present a continuum mechanics description of the actomyosin cell cortex, and successfully apply it to several different cell biological processes. Combining our theory with experimental work from collaborating groups, we provide new insights into different aspects of cell mechanics.
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Caractérisation du rôle de Citron Kinase durant la cytokinèseEl-Amine, Nour 12 1900 (has links)
La cytokinèse est un processus dont le but est une séparation de deux cellules soeurs en deux entités suite à une mitose. La cytokinèse nécessite la formation d’un anneau contractile (AC) qui va conduire un sillon de clivage vers une ingression à l’équateur de la cellule. L’une des étapes critiques de ce processus est la transition d’un AC dynamique vers une structure stable surnommée l’anneau du midbody (AM), organelle qui va guider la cellule vers l’abscision. La compréhension des mécanismes moléculaires impliqués dans cette transition nous permettrait de mieux comprendre les complexes protéiques impliqués autant au niveau de l’initiation qu’à la terminaison de la cytokinèse. Des défauts ayant lieu lors de cette transition mènent à la formation de cellules binucléées tétraploïdes qui sont observées dans plusieurs pathologies comme le cancer. Afin d’approfondir nos connaissances à ce sujet j’ai utilisé un modèle d’imagerie optique en temps réel dans un modèle cellulaire de Drosophila melanogaster : les cellules S2 de Schneider. Ces études ont mis l’emphase sur un nouveau mécanisme de maturation de la transition AC/AM. Nous avons pu démontrer que la kinase Citron, Sticky, et la septine, Peanut, agissent de manière opposée sur la protéine Anillin pour retenir ou éliminer, respectivement, la membrane plasmique lors de la transition AC/AM. En effet, la diminution d’expression de Sticky par ARNi engendre une perte de contrôle de rétention membranaire de l’AM. À l’inverse, la diminution d’expression de Peanut inhibe la maturation par excrétion membranaire de l’AM. La diminution d’expression simultanée de Sticky et de Peanut conduit l’AC vers des mouvements oscillatoires typiques d’une instabilité de l’AC suite à la perte de fonction de l’Anillin. Sticky est une protéine corticale lors de la cytokinèse dont le rôle et les partenaires d’interaction restent controversés. Pour approfondie nos connaissance de ce sujet, nous avons effectué une étude structurelle et fonctionnelle de Sticky. Cette étude démontre que Sticky possède deux mécanismes de localisation corticale. Le premier dépend de l’Anillin et le deuxième dépend de la petite GTPase Rho1, le régulateur maître de la cytokinèse. Sticky est capable de se localiser à l’AC en présence de l’un ou l’autre de ces deux mécanismes, mais chacun semble être essentiel pour la réussite de la cytokinèse. Le domaine minimal d’interaction entre la Sticky et l’Anillin a été identifié. Une version d’Anillin qui manque le site de liaison à la Sticky est incapable de supporter l’achèvement de la cytokinèse, et les cellules échouent la cytokinèse d’une manière semblable aux cellules dont l’expression de Sticky est diminuée. Similairement, les cellules exprimant une protéine Sticky mutée au site d’interaction avec Rho1-GTP, sont incapables de compléter la cytokinèse lorsque les niveaux endogènes de Sticky sont diminués par ARNi. Ceci suggère que Sticky agit avec Anillin et Rho1 au niveau du cortex pour guider la transition d’un AC dynamique vers un AM stable. Par la mise en évidence et la caractérisation d’un nouveau mécanisme moléculaire essentiel à la cytokinèse, cette thèse constitue des avancements importants au niveau de la cytokinèse. / Cytokinesis is a multistep process that allows two sister cells to undergo complete separation following mitosis. Cytokinesis requires the formation of a contractile ring (CR) that will drive cleavage furrow ingression at the equator of the cell. One of the crucial steps in this process is the transition from a dynamic CR to a more stable structure named the midbody ring (MR), which directs the final separation or abscission. Our knowledge of the molecular mechanisms involved in the CR-to-MR transition would presumably improve our understanding of the molecular complexes involved throughout cytokinesis from initiation to abscission. Defects that occur during this transition can lead to the formation of bi-nucleate tetraploid cells that are often observed in pathological conditions such as cancer. I have used Drosophila melanogaster Schneider’s S2 cells to study the CR-to-MR transition. My findings have highlighted a previously uncharacterized maturation process essential for the transition. More specifically, I demonstrate that the Citron Kinase, Sticky, and the Septin, Peanut, have opposing actions on the scaffold protein Anillin to either retain or extrude, respectively, membrane-positive proteins during the CR-to-MR transition. Indeed, Sticky depletion by RNAi led to uncontrolled loss of membrane-associated Anillin at the MR. Conversely, Peanut depletion led to inhibition of MR maturation by membrane extrusion. Co-depletion of Sticky and Peanut led to oscillatory movements of the CR, typical of Anillin depletion. Sticky is a cortical protein during cytokinesis whose role and interacting partners are controversial. I have performed a structure/function analysis of Sticky to better define its role and regulation during cytokinesis. My work shows that Sticky has two mechanisms of cortical localization. The first is through an Anillin interaction and the second is through the small GTPase Rho1, a master regulator of cytokinesis. Sticky can localize to the cortex in the absence of either one of these mechanisms. However, loss of both inhibits its localization. Following the identification of the minimal interaction sites of Anillin and Sticky, I expressed an Anillin mutant that lacked part of this site and found that cells failed cytokinesis in a similar manner to cells depleted of Sticky. Mutation of the Rho1 binding site on Sticky produced similar cytokinesis failures. Altogether, the results suggest that Sticky interacts with Anillin and Rho1 at the cortex to guide the transition from dynamic CR to stable MR. This thesis advances our understanding of cytokinesis by highlighting a previously uncharacterized process of MR maturation and by defining the importance and regulation of Citron Kinase during this process.
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Spatiotemporal dynamics of cytoskeletal and chemosensory proteins in the bacterium Rhodobacter sphaeroidesChiu, Sheng-Wen January 2014 (has links)
The discovery of the prokaryotic cytoskeleton has revolutionized our thinking about spatial organisation in prokaryotes. However, the roles different bacterial cytoskeletal proteins play in the localisations of diverse biomolecules are controversial. Bacterial chemotaxis depends on signalling through large protein clusters and each cell must inherit a cluster on cytokinesis. In Escherichia coli the membrane chemosensory clusters are polar and new static clusters form at pre-cytokinetic sites, ensuring positioning at new poles after cytokinesis and suggesting a role for the bacterial FtsZ and MreB cytoskeletons. Rhodobacter sphaeroides has both polar, membrane-associated and cytoplasmic, chromosome-associated chemosensory clusters. This study sought to investigate the roles of FtsZ and MreB in the partitioning of the two chemosensory clusters in R. sphaeroides. The relative positioning between the two chemosensory systems, FtsZ and MreB in R. sphaeroides cells during the cell cycle was monitored using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which gradients of FtsZ extend circumferentially to form the Z-ring. The proposed node-precursor model might represent a common mechanism for the formation of cytokinetic rings. The MreB cytoskeleton continuously reorganizes between patchy and filamentous structures, and colocalises with FtsZ at midcell. Membrane chemosensory proteins form individual dynamic unit-clusters with mature clusters containing about 1000 CheW<sub>3</sub> proteins. These unit-clusters diffuse randomly within the membrane but have a higher propensity for curved regions like cell poles. Membrane clusters do not colocalise with FtsZ and MreB and appear excluded from the Z-ring vicinity. The bipolar localisation of membrane clusters is established after cell division via random diffusion and polar trapping of clusters. The cytoplasmic chemosensory clusters colocalise with FtsZ at midcell in new-born cells. Before cytokinesis one cluster moves to a daughter cell, followed by the second moving to the other cell. FtsZ and MreB do not participate in the positioning of cytoplasmic clusters. Therefore the two homologous chemosensory clusters use different mechanisms to ensure partitioning, and neither system utilizes FtsZ or MreB for positioning.
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