Dissertations / Theses on the topic 'Pseudopodes'

La phagocytose est un mécanisme cellulaire essentiel de l’organisme. Elle joue un rôle à la fois dans le maintien de l’homéostasie tissulaire mais également dans le système immunitaire. Ce processus, réalisé par des cellules phagocytaires, telles que les cellules dendritiques, les polymorphonucléaires neutrophiles ou les macrophages, permet l’ingestion et l’élimination quotidienne de particules de grandes tailles (&gt;0,5 µm) : bactéries, champignons ou débris cellulaires. Il est induit par de nombreux récepteurs phagocytaires tels que les récepteurs aux fragments cristallisables des immunoglobulines (FcR) et les récepteurs au complément (CR3). Ceux-ci induisent des cascades de signalisation différentes mais aboutissant, toutes deux, à un remodelage du cytosquelette d’actine et de la membrane plasmique. Il y alors formation d’une coupe phagocytaire entourant et enfermant la particule à internaliser dans un compartiment clos appelé phagosome. Alors que de nombreuses études ont permis de disséquer l’organisation des coupes phagocytaires induites par les FcR, le mécanisme de fermeture des phagosomes n’était pas élucidé. Par ailleurs, les mécanismes moléculaires impliqués dans la formation des phagosomes suite à l’engagement des CR3 sont moins bien décrits. Au cours de ce travail, nous avons analysé le rôle de la dynamine 2, une GTPase impliquée dans les mécanismes de fission des vésicules d’endocytose, au cours de la formation et de la fermeture des phagosomes. Nous avons utilisé un système expérimental original utilisant la microscopie à ondes évanescentes pour montrer, que la dynamine 2 est recrutée avec l’actine dans les coupes phagocytaires en formation et qu’elle s’accumule au site de fermeture des phagosomes dans des macrophages vivants. L’inhibition de son activité GTPase induit une inhibition de l’efficacité de phagocytose et un défaut de la dynamique de l’actine lors de l’extension des coupes phagocytaires. De façon surprenante, la dépolymérisation de l’actine conduit à un défaut de recrutement de la dynamine 2 au site de la phagocytose mettant en évidence une régulation croisée entre la dynamine 2 et l’actine. Enfin cette étude a montré que la dynamine 2 joue un rôle critique dans la scission du phagosome. Dans un second temps, nous avons initié l’étude des mécanismes impliqués dans la régulation de l’activité du récepteur au complément CR3. L’activation de ce récepteur phagocytaire, qui fait partie de la famille des intégrines, requiert un ancrage à l’actine nécessaire à la signalisation vers la polymérisation d’actine et à la formation des coupes phagocytaires. L’ensemble de ces résultats contribue à une meilleure connaissance des mécanismes moléculaires fins impliqués dans la phagocytose<br>Phagocytosis is an important cellular mechanism. It plays a role in both the maintenance of tissue homeostasis and in the immune system. This process, performed by phagocytic cells, including dendritic cells, polymorphonuclear neutrophils or macrophages, enables daily ingestion and elimination of large particles (&gt; 0.5 microns) e.g. bacteria, fungi or cellular debris. It is induced by many phagocytic receptors such as the receptors for crystallizable fragments of immunoglobulins (FcR) and complement receptor (CR3). These receptors induce different signaling cascades but ultimately lead to a remodelling of the actin cytoskeleton and the plasma membrane. Next there is the formation of a phagocytic cup which surrounds and encloses the ingested particle in a closed compartment called the phagosome. While many studies have dissected the phagocytic cup organization induced by the FcR, the mechanism of phagosome closure was not understood. Furthermore, the molecular mechanisms involved in phagosome formation following CR3 engagement are less well described. In this work, we analyzed the role of dynamin 2, a GTPase involved in fission mechanisms of endocytosis vesicles, and in the formation and closure of phagosomes. We used an original experimental system using the total internal reflection fluorescence microscopy (TIRFM) to show that dynamin 2 is recruited with actin during phagocytic cup formation and accumulates at the site of phagosome closure in living macrophages. The inhibition of its GTPase activity induced an inhibition of phagocytosis and a defect in actin dynamics during pseudopod extension. Surprisingly, the depolymerization of actin lead to a defective recruitment of dynamin 2 at the phagocytic site showing there is a cross-regulation between dynamin 2 and actin. Finally, this study showed that dynamin 2 plays a critical role in the scission of the phagosome. Secondly, we initiated the study of the mechanisms involved in regulating the activity of the complement receptor CR3. Enabling this phagocytic receptor, part of the integrin family, requires anchoring actin which is necessary for signaling to the actin polymerization and the formation of phagocytic cups. All these results contribute to a better understanding of the molecular mechanisms involved in phagocytosis purposes

Le peptide antimicrobien LL-37 a été retrouvé surexprimé dans différents types de cancer et plus particulièrement dans le cancer du sein dans lequel il est associé au développement des métastases. Nous avons observé, in vitro, que la migration de trois lignées cancéreuses mammaires est augmentée par le peptide LL-37 et son énantiomère (D)-LL-37, excluant la fixation du peptide à un récepteur protéique. Sur les cellules cancéreuses mammaires MDA-MB-435s, le peptide se fixe à la membrane plasmique et diminue sa fluidité. La microscopie électronique localise LL-37 dans les cavéoles et à la surface de structures impliquées dans la migration cellulaire, les pseudopodes. LL-37 induit une entrée de calcium via le canal TRPV2 dont l’activité est augmentée par son recrutement dans les pseudopodes. Ce recrutement est dépendant de l’activation de la voie de signalisation PI3K/AKT induite par LL-37. L’entrée de calcium via TRPV2 est potentialisée par l’activation du canal potassique BKCa, localisé aussi dans les pseudopodes. Des ARN interférents contre TRPV2 inhibent à 70% la migration induite par LL-37, donnant un rôle prépondérant à ce canal dans les effets pro-migratoire du peptide. La fixation du peptide LL-37 aux membranes des cellules cancéreuses et l’activation de canaux ioniques constituent un nouvel axe de recherche pour comprendre le rôle du peptide dans la progression tumorale<br>The antimicrobial peptide LL-37 is overexpressed in several types of cancer, among which breast cancer were it is associated with metastasis development. Our experiments on three mammary cancer cell lines have shown that LL-37 increases cell migration. Both its natural (L)-form and its (D)-enantiomer are equally active, excluding a specific binding to a protein receptor. On the MDA-MB-435s cell line, LL-37 attaches to plasma membrane and reduces its fluidity. Electron microscopy localized LL-37 on the surface of pseudopodia, structures implicated in cell migration, and in caveolae. LL-37 induces calcium entry via the TRPV2 channel, which is recruited to pseudopodia. Recruitment depends on activation of PI3K/AKT signaling induced by LL-37. Calcium entry via TRPV2 is potentiated by activation of the BKCa potassium channel also located in pseudopodia. TRPV2 suppression by RNA interference results in 70% reduction of cell migration induced by LL-37, attributing a crucial role of this channel to the promigratory effects of the peptide. Binding of LL-37 to cancer cell membranes and in consequence the activation of ion channels constitutes a novel research field to understand its role in tumor progression

Les lymphocytes T sont les cibles privilégiées de la réplication du VIH-1. Ce virus détourne la machinerie cellulaire pour s'assurer l'infection efficace de son hôte. J'ai étudié les processus de réplication du VIH-1 et de sa transmission entre cellules T. J'ai observé simultanément différents modes de transmission virale directe entre cellules T comme la synapse virologique, le filopode ou la polysynapse, structure nouvellement caractérisée qui associe une cellule infectée à plusieurs cibles en même temps. J'ai quantifié précisément ces différentes modes de contact et leur importance. Mes observations aboutissent à proposer la synapse virale et la polysynapse comme principales voies de transmission du VIH. L'interface entre virus et cellules cibles est au cœur de mon travail de recherche. J'ai caractérisé les modifications du cytosquelette d'actine induites par le virus au niveau des cellules T. La protéine Nef se révèle ici comme un des inducteurs majeurs de ces réarrangements. Sous son impulsion, de profonds changements s'enclenchent : réduction de motilité cellulaire, diminution des replis membranaires de type « ruffles » et augmentation des filopodes. J'ai également étudie le rôle des Rhô GTPases Racl, 2 et Cdc42 ainsi que la GTPase dynamin 2 lors de la transmission virale directe de cellule à cellule et via des virions libres. Ces protéines responsables du remodelage du cytosquelette peuvent être considérées comme des cibles privilégiées du virus pour détourner des fonctions cellulaires majeures. Mes études révèlent et caractérisent quelques traits des interactions complexes entre le VIH et les cellules cibles<br>T lymphocytes are the main target cells for active HIV-1 replication. The virus subverts the cellular machineries to ensure the most efficient infection of the host. I studied the process of HIV-1 replication and intercellular spread in T cells. I visualized the simultaneous existence of different mechanisms of viral transmission between T lymphocytes, such as virological synapses (VS), filopodia, and by newly characterized polysynapses, structures formed between one infected cell and multiple adjacent recipients. I quantified further these diverse modes of contact and studied their relative importance in mediating new target infection. I observed that viral transfer mainly occurs across VS and through polysynapses. I investigated the interplay between the virus and its host cell upon infection. I showed that the virus modulates the physiology of T cells. This is, at least partially, due to the interactions of HIV with the cell cytoskeleton. The viral protein Nef appears as an important modulator of actin cytoskeleton remodeling. It mediates profound changes within the cells, reducing their motility and ability to undergo membrane ruffling, inducing at the same time the formation of filopodia. I also identified Rhô GTPases Racl, 2 and Cdc42 as well as GTPase dynamin2 as possible partners of HIV in mediating free viral infection and cell-cell spread. These regulators of actin cytoskeleton remodeling are likely targets for the virus to get the control over important cellular functions involving actin plasticity. The research presented here allowed to better characterize the means of HIV cell-to-cell spread and the modulation of the cellular biology by the viral infection

Le mouvement cellulaire est organisé par des processus moléculaires qui couplent la dynamique de la membrane plasmique à celle du cytosquelette d'actine, pour engendrer des protrusions cellulaires. Pour analyser le lien fonctionnel entre ces processus et le comportement motile qui en résulte, j'ai utilisé une approche biomimétique. J'ai mis au point une méthode rapide d'électrogonflement de liposomes géants unilamellaires, que j'ai fonctionnalisés avec la protéine N-WASP. Cette protéine catalyse la formation d'un réseau de filaments branchés par le complexe Arp2/3 au bord avant des cellules motiles. Les liposomes, placés dans un milieu reconstitué contenant l'actine, Arp2/3 et les protéines de régulation du treadmilling, sont déformés par la polymérisation insertionnelle de l'actine et se propulsent in vitro. J'ai montré que les liposomes adoptent un régime de propulsion soit continu, soit saltatoire périodique, la transition entre les deux régimes étant contrôlée par la concentration de Arp2/3. Les résultats établissent que le complexe Arp2/3 est le partenaire de N-WASP responsable de l'interaction entre la membrane et les filaments au cours de la réaction de branchement. Cette interaction est transitoire et détermine l'équilibre ségrégation-diffusion de N-WASP dans la bicouche lipidique et la formation d'un réseau cohésif de filaments branchés. Le modèle physique que nous proposons selon lequel l'équilibre ségrégation-diffusion de NWASP est contrôlé par les paramètres cinétiques du cycle catalytique de branchement, reproduit quantitativement les profils de densité de surface de NWASP observés expérimentalement.

Le cytosquelette d’actine est constitué de réseaux de filaments d’actine dont la dynamique d’assemblage et leur organisation sont contrôlées par de nombreuses protéines régulatrices. Parmi ces dernières, les formines sont des protéines possédant plusieurs domaines fonctionnels qui assurent le contrôle de la polymérisation de filaments longs et non-branchés dans la cellule. Les formines nucléent et accélèrent la vitesse d’allongement des filaments en suivant processivement les bouts barbés des filaments qui polymérisent. Grâce à des études in vitro à l’échelle du filament individuel, les détails moléculaires des mécanismes élémentaires de l’activité des formines en interaction avec les filaments sont mieux connus. Cependant, dans la cellule les filaments d’actine sont liés entre eux pour former des réseaux d’actine aux architectures variées, et l’impact de l’organisation spatiale des filaments sur l’activité des formines est mal connu.Nous avons reconstitué des faisceaux parallèles de filaments d’actine formés par l’action des fascines et avons étudié l’impact de ce type d’architecture sur l’activité des formines. Grâce à approches expérimentales combinant la microfluidique et la microscopie de fluorescence, nous montrons que l’ancrage des formines aux surfaces, qui contraignent ces dernières en rotation et/ou en translation, ainsi que les contraintes imposées aux filaments par la formation de faisceaux par les fascines, réduisent de manière importante la processivité des formines et la vitesse d’allongement des filaments d’actine<br>The actin cytoskeleton is a dynamic network of actin filaments whose polymerization, depolymerization, and remodeling are controlled by a large array of actin binding proteins. Formins are a family of multidomain actin binding proteins that promote the formation of long, unbranched filaments in cells. Formins achieve this by nucleating filaments and accelerating their elongation by processively tracking filament barbed ends as they polymerize. We now understand a great deal of elementary mechanisms by which formins interact with filaments and control their polymerization, thanks to a multitude of in vitro experiments that have been carried out using reconstituted, single actin filaments. However, in cells, actin filaments are crosslinked into different architectures, and to date, the impact of the network geometry on formin behavior has been poorly studied. In this work, we reconstituted parallel bundles of actin filaments using the actin crosslinking protein fascin, and we aimed to understand how the bundling geometry impacts formin behavior. Using a combination of microfluidics and fluorescence microscopy techniques, we shed light on how rotational constraints imposed on formin by filament crosslinking are an important factor governing formins’ ability to processively elongate filaments

In this thesis, I have applied a complementary and alternative understanding of melanoma chemotaxis by incorporating the role of LPA and through a pseudopod-centred approach. I have demonstrated that pseudopods are self organising actin entities that undergo mainly bifurcation (splitting) from preexisting protrusions rather than through synthesis of de novo pseudopods for melanoma cell chemotaxis. This observation was also extended to mouse melanoblast migration in vivo. These superior split pseudopods influence cell steering by alignment, size and lifetime regulation, and biases retraction. In non-metastatic cells, they are no longer able to form stable split pseudopods in response to external stimuli. LPA signaling is also established as vital for the stability of split pseudopods. Hence, in the event of LPAR1 perturbation, metastatic cells formed more actin protrusions but they adopted pseudopod morphologies similar to the non-metastatic lines. The role of Rac, Ras, Paxillin and Ezrin in split pseudopod regulation was also explored in this thesis. Finally, the increased stability of split pseudopods acting through LPA signalling could emerge as a signature of metastatic cells.

In a trajectory through 3 bodies of work the artist has attempted to define a lineage that discuses the act of creation. Starting with the procreative and the acknowledgement of the father/son relationship the discussion moves to the possibility of non pro-creative creativity. Quinn 's Da (completed in the first year of the doctorate) evolved through witnessing the demise of the artist's father. He acknowledges the line that passes to the eldest son and the putting down of the baton through choosing to be a non pro-creative unit. That this creates a void was the catalyst that led to the third body of work. Minor Wounds came about through a study of Major wounds. The artist had survived incestuous childhood abuse and had projected himself into the AIDS epidemic in New York from 1985 to 1996. These wounds had been dealt with in earlier work and there was a need to look at the other ways we fall apart, yet sit with dignity and even humour recognising our own mortality. This work is about the body's drive to renew itself. The wounds, unintentionally inflicted, were recorded and the body stands now as evidence of its recuperative powers. More Life celebrates continuum. Inspired by Jarry, Shakespeare, Goya, Rabelais, Roussel, Joyce, Cervantes , Goya and Duchamp, the artist plays with creative intuition, similitude, dreams and accidents to create a set of fictional selves that the self has bifurcated into. The creative bifurcators who have inspired him are considered and the possibility that we as a species might be evolving into bifurcatory, as opposed to sexual, reproducers, and that this would be falling in with the universal law of creativity. Pro meaning for would suggest that pro-creativity is (just) standing in for creativity. The artist's contribution comprises 92 large pseudo paintings on canvas, 21 of which are presently touring the USA. Like the Amoeba's pseudopodia the artist sends them out to feel their way and to create a space in which it might be safe to creatively bifurcate.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2005.<br>Includes bibliographical references (leaves 80-88).<br>Neutrophils traversing the pulmonary microcirculation are subjected to mechanical stimulation during their deformation into narrow capillaries. To better understand the time- dependant changes caused by this mechanical stimulus, in the first part of the thesis, neutrophils were caused to flow into a microchannel, which allowed simultaneous visualization of cell morphology, and passive rheological measurement by tracking the Brownian motion of endogenous granules. Above a threshold stimulus, mechanical deformation resulted in neutrophil activation with pseudopod projection. The activation time was inversely correlated to the rate of mechanical deformation experienced by the neutrophils. A reduction in shear moduli was observed within seconds after the onset of the mechanical stimulus, suggesting a sudden disruption of the neutrophil cytoskeleton when subjected to mechanical deformation. However, the magnitude of the reduction in moduli was independent of the degree of deformation. Recovery to nearly the initial values of viscoelastic moduli occurred within one minute. These observations confirm that mechanical deformation of neutrophils, similar to conditions encountered in the pulmonary capillaries is not a passive event; rather, it is capable of activating the neutrophils and enhancing their migratory tendencies. The second part of the thesis seeks to understand the changes in the cytoskeletal structure and the extent of biological activation as a result of this deformation process. Neutrophils were passed through narrow polycarbonate filter pores under physiological driving pressures, fixed and stained downstream in order to visualize the F-actin content and distribution.<br>(cont.) Below a threshold capillary size, the cell remodeled its cytoskeleton through initial F-actin depolymerization, followed by recovery and increase in F-actin content associated with formation of pseudopods. 'This rapid depolymerization and subsequent recovery of F-actin was consistent with our previous observation of an immediate reduction in moduli with eventual recovery when the cells were subjected to deformation. Results also show that neutrophils must be retained in their elongated shape for an extended period of time for pseudopod formation, suggesting that a combination of low driving pressures and small capillary diameters promotes cellular activation. These observations show that mechanical deformation of neutrophils into narrow pulmonary capillaries have the ability to influence cytoskeletal structure, the degree of cellular activation and migrational capabilities of the cells.<br>by Belinda Yap.<br>Ph.D.

Thesis (Ph. D.)--University of California, Riverside, 2009.<br>Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed March 16, 2010). Includes bibliographical references. Also issued in print.

La motilité cellulaire est un processus intégré essentiel à de nombreux phénomènes physiologiques tels que la formation du cône de croissance et la plasticité synaptique. Des dérégulations de la motilité cellulaire peuvent être à l’origine de la formation de métastases ou de pathologies neuropsychiatriques comme la schizophrénie et l'autisme. La compréhension des mécanismes régulant la migration cellulaire est donc un enjeu majeur. La motilité cellulaire repose sur la formation de diverses structures constituées de réseaux d’actine branchés telles que le lamellipode. La formation du lamellipode nécessite l’intervention de protéines régulatrices de l’actine telles que Rac1 et les complexes Wave et Arp2/3. Grâce à l’utilisation de suivi de protéine unique, nous avons pu comprendre comment la coordination spatio-temporelle de ces régulateurs contrôle la formation et la morphologie des lamellipodes de cellules migrantes. Nous avons ainsi découvert que l’activation et la localisation du complexe Wave étaient régulées de manière enzymatique mais également mécanique. Dans une première étude, nous avons montré que la RhoGTPase Rac1 active le complexe Wave spécifiquement à l’extrémité du lamellipode. Dans une seconde étude, nous avons révélé que la localisation du complexe Wave est régulée par la dynamique des filaments des réseaux branchés d’actine. Ces données soulignent l’importance du complexe Wave dans la formation du lamellipode et révèlent l’existence d’une régulation mécanique de la localisation du complexe Wave<br>Cell motility is an integrated process involved in critical phenomena such as axonal pathfinding and synaptic plasticity. Dysregulation of cell motility can induce metastasis and abnormal spine shapes observed in neuropsychiatric disorders like autism and schizophrenia. Therefore it is essential to understand how cell motility is regulated. Cell motility requires the formation of branched actin networks propelled by actin polymerization that lead to the formation of membrane protrusions such as the lamellipodium. Several actin regulatory proteins are involved in this process, such as Rac1 and the WAVE and ARP2/3 complexes. Using single protein tracking, we revealed key phenomena concerning the spatio-temporal regulation of lamellipodium formation by actin regulatory proteins. We found that the localization and activation of the WAVE complex was enzymatically regulated, but also mechanically. First, we showed that the Rac1 RhoGTPase activates the WAVE complex specifically at the tip of the lamellipodium. We also showed that WAVE complex localization is regulated by the dynamics of branched-network actin filaments. This study confirms the crucial role of the WAVE complex in lamellipodium formation and reveals the existence of a mechanical regulation of the localization of this complex in the cell

Thesis (M.S.)--University of California, San Diego, 2010.<br>Title from first page of PDF file (viewed July 16, 2010). Available via ProQuest Digital Dissertations. Includes bibliographical references (leaves 65-73).

Hypercholesterolemia is a dominant risk factor for a variety of cardiovascular diseases and involves a chronic inflammatory component in which neutrophil activity plays a critical role. Recently, fluid shear stress mechanotransduction has been established as a control mechanism that regulates the activity of neutrophils by reducing the formation of pseudopods and the surface expression of CD18 integrins, thereby rendering these cells rounded, deformable, and non-adhesive. This is critical for maintaining a healthy circulation, because chronically activated neutrophils not only release excess cytotoxic and degradative agents but also exhibit a reduced efficiency to pass through the small vessels of the microcirculation leading to increased microvascular resistance. We hypothesized that aberrant neutrophil mechanosensitivity to fluid shear stress due to the altered blood environment (i.e., excess plasma cholesterol) is a contributing factor for elevated hemodynamic resistance in the microcirculation associated with hypercholesterolemia. For this purpose, the present work firstly showed that the sensitivity of neutrophils to fluid shear stress depends on the cholesterol-dependent fluidity of the cell membrane, and that, in the face of hypercholesterolemia, the neutrophil mechanosensitivity highly correlated with the plasma levels of free cholesterol. The second part of this project demonstrated that, when subjected to shear stress fields, leukocyte suspensions exhibited transient (within 10 min of flow onset) time-dependent reductions in their apparent viscosity. Moreover, shear-induced changes in viscosity of cell suspensions were influenced by disturbances of membrane cholesterol and fluidity in a fashion similar to that for shear-induced pseudopod retraction. Finally, the third part of this work provided evidence that neutrophils played a role in hypercholesterolemia-related impairment of flow recovery response to transient ischemia. In conclusion, results of the current work provided the first evidence that cholesterol is an important component of the neutrophil mechanotransducing capacity and impaired neutrophil shear mechanotransduction may disturb the blood flow rheology, leading to elevations in the apparent viscosity as well as in the resistance. This cholesterol-linked perturbation may be a contributing factor for the pathologic microcirculation associated with hypercholesterolemia.

Poa L., is known as a highly diverse cosmopolitan genus with taxonomic difficulties that includes unknown species and species with uncertain affinities mainly in West Asia and North Africa. Poa also exhibits a close relationship with two West Asian genera, Eremopoa Roshev. and Oreopoa H. Scholz & Parolly. This study was conducted to: 1) fill the gap of information on the affinities between Poa species with an emphasis on West Asian Poa; 2) revise and evaluate the accuracy of traditional infrageneric classification of West Asian Poa; and 3) clarify the relationship between Poa and two allied genera of Poaceae Barnhart, Eremopoa and Oreopoa. DNA molecular evidence from present phylogenetic analyses of West Asian species of Poa, Eremopoa and Oreopoa, resulted in some great findings as follow: I) Poa caucasica Trin., which is currently assigned to subsection Nivicolae of section Poa from subgenus Poa resolved as a unique new distinct lineage within Poa. II), New treatments are suggested for Poa densa Troitsky, Poa masenderana Freyn & Sint., Poa cenisia All., Poa psychrophila Boiss. & Heldr. and Poa lipskyi. III) Three unclassified species of Poa pseudobulbosa, Poa diversifolia and Poa aitchisonii are assigned here to subgenus Poa and supersection Poa. IV), The present molecular evidence supports inclusion of Eremopoa in Poa and confirms reduction of Eremopoa to a level of subgenus of Poa. V) Present phylogenetic analyses also indicate that monotypic genus Oreopoa H. Scholz & Parolly is part of Poa. These findings require an urgent modification in subgeneric and sectional classification of the genus Poa.

The psuedopodial fraction of MSV-MDCK-INV cells was isolated and proteomic analysis by LS-MS/S revealed the enrichment of a variety of cytoskeletal and adhesion proteins, glycolytic enzymes, proteins required for translation, ubiquitin/proteasome associated proteins, RNA binding proteins and cell signaling proteins. The selective enrichment of these proteins indicated the potential existence of mRNA in the pseudopodial domain. Propidium iodide labeling demonstrated the presence of mRNA in the pseudopodia. The localization of mRNA in the pseudopodia could be inhibited by treatment with Rho-kinase inhibitor, Y27632. Further investigation of the involvement of Rho/OCK signaling in the pseudopodia demonstrated local activation of RhoA. This activation occurred preferentially to the activation of another RhoGTPase family member, Rac1 . However, Rac1 activation could be reversed with Y27632 treatment indicating communication between signaling components in the pseudopodia. Photobleaching of actin and mRNA in the pseudopodia in the presence of Y27632 showed a decreased recovery time for mRNA but not actin to the bleached regions. This supports a role for Rho-kinase in pseudopodial mRNA translocation. Microarray analysis of the pseudopodial fraction disclosed the presence of a number of upregulated mRNAs. In situ hybridization with anti-sense oligonucleotides to β-actin, RhoA, Shp-2, m-ras and Arp2/3 p41 subunit confirmed the presence of these mRNA in the pseudopodia. Active transport of mRNA via the cytoskeleton to intracellular domains has been documented in a number of cell types. It was shown here by treatment with nocodazole that mRNA localization to MSV-MDCK-INV cell pseudopodia occurs independently of microtubules. Therefore the MSV-MDCK-INV tumor cell model displaying distinct actin rich pseudopodial domains is characterized by distinct protein and mRNA complements. The regulation of these domains is mediated by local activation of a Rho/ROCK signaling pathway that contributes to mRNA localization in a microtubule independent manner.<br>Medicine, Faculty of<br>Graduate

This work identified an ortholog of Abp1 (actin binding protein 1) in Dictyostelium (Dabp1). In order to analyze the functions of Dabp1 in Dictyostelium, loss-of–function studies and gain-of-function studies were performed by generating cells that either deleted the Dabp1 gene from the genome or overexpressed the Dabp1 protein. In these mutants, most actin-based processes were intact. However, cell motility was altered during early development. During chemotactic streaming, more than 90% of wild type cells had a single leading pseudopodium and a single uropod, whereas more than 27% of Dabp1 null cells projected multiple pseudopodia. Similarly, ~ 90% of cells that overexpressed Dabp1 projected multiple pseudopodia during chemotactic streaming, and displayed reduced rates of cell movement. Expression of the SH3 domain of Dabp1 showed this domain to be an important determinant in regulating pseudopodium number. These results suggest that Abp1 controls pseudopodium number and motility in early stages of chemotactic aggregation in Dictyostelium. This work also revealed an interplay between Dabp1 and MyoB, one of the Myosin I proteins, in controlling pseudopodia formation in Dictyostelium. These two proteins colocalize partially at the cortex in growing cells. The peripheral localization of MyoB was dependent on Dabp1. Depletion of both Dabp1 and MyoB caused defects in organization of the actin cytoskeleton and actin related activities such as formation of small F-actin filled spikes on the cell cortex of growing cells, a higher percentage of multinucleated cells, and an increased number of pseudopodia branching extensively. When MyoB was overexpressed in Dabp1 null mutants, cells had similar phenotypes as Dabp1/MyoB double null mutants, and displayed an increased number of pseudopodia with many branches. Overexpression of Dabp1 in MyoB null mutants rescued the defects in pseudopodia formation. The SH3 of Dabp1 was shown to be important for the rescue of defects caused by depletion of MyoB. Collectively, these data suggest that MyoB and Dabp1 work cooperatively to regulate the uniformity and integrity of the actin extensions during chemotaxis. MyoB requires Dabp1 to function in this process. Dabp1 may function as a scaffold to recruit MyoB to the proper localization. These studies of Dabp1 in Dictyostelium raise broad question about functions of actinassociated proteins in pseudopodia formation and the importance of uniformity and integrity for actin structures in chemotaxis.<br>text

Transfer of the melanocyte-specific and lysosome-related organelle, the melanosome, from melanocytes to keratinocytes is crucial for the protection of the skin against harmful ultraviolet radiation (UVR)--our main physiological cutaneous stressor. However, this commonplace event remains a most enigmatic process despite several early hypotheses. Recently, we and others have proposed a role for filopodia in melanin transfer, although conclusive experimental proof remained elusive. Using known filopodial markers (MyoX/Cdc42) and the filopodial disrupter, low-dose cytochalasin-B, we demonstrate here a requirement for filopodia in melanosome transfer from melanocytes to keratinocytes and also, unexpectedly, between keratinocytes. Melanin distribution throughout the skin represents the key phenotypic event in skin pigmentation. Melanocyte filopodia were also necessary for UVR-stimulated melanosome transfer, as this was also inhibited by MyoX knockdown and low-dose cytochalasin-B. Knockdown of keratinocyte MyoX protein, in its capacity as a phagocytosis effector, resulted in the inhibition of melanin uptake by keratinocytes. This indicates a central role for phagocytosis by keratinocytes of melanocyte filopodia. In summary, we propose a new model for the regulation of pigmentation in human skin cells under both constitutive and facultative (post-UVR) conditions, which we call the "filopodial-phagocytosis model." This model also provides a unique and highly accessible way to study lysosome-related organelle movement between mammalian cells.

Bone morphogenetic proteins (BMPs) are a large family of multi-functional secreted signalling molecules. Previously BMP2/4 were shown to inhibit skin pigmentation by downregulating tyrosinase expression and activity in epidermal melanocytes. However, a possible role for other BMP family members and their antagonists in melanogenesis has not yet been explored. In this study we show that BMP4 and BMP6, from two different BMP subclasses, and their antagonists noggin and sclerostin were variably expressed in melanocytes and keratinocytes in human skin. We further examined their involvement in melanogenesis and melanin transfer using fully matched primary cultures of adult human melanocytes and keratinocytes. BMP6 markedly stimulated melanogenesis by upregulating tyrosinase expression and activity, and also stimulated the formation of filopodia and Myosin-X expression in melanocytes, which was associated with increased melanosome transfer from melanocytes to keratinocytes. BMP4, by contrast, inhibited melanin synthesis and transfer to below baseline levels. These findings were confirmed using siRNA knockdown of BMP receptors BMPR1A/1B or of Myosin-X, as well as by incubating cells with the antagonists noggin and sclerostin. While BMP6 was found to use the p38MAPK pathway to regulate melanogenesis in human melanocytes independently of the Smad pathway, p38MAPK, PI3-K and Smad pathways were all involved in BMP6-mediated melanin transfer. This suggests that pigment formation may be regulated independently of pigment transfer. These data reveal a complex involvement of regulation of different members of the BMP family, their antagonists and inhibitory Smads, in melanocytes behaviour.