Le produit naturel salinomycine (SAL) est largement utilisé comme médicament anticoccidien et maintenant de plus en plus reconnu comme un agent destiné à réduire la proportion de population CD44⁺ / CD24⁻ cellules souches du cancer du sein. Ce facteur est important et intervient lors des rechutes des tumeurs du sein. Pour la première fois, nous avons décrit que l'action n’était pas ionophorique mais que le proton dit "éponge" de la salinomycine ciblait particulièrement la population des cellules souches du cancer. De plus, un analogue alcyne-amine synthétisé de la salinomycine a une action similaire à cette dernière sur la population CD44⁺ / CD24⁻ mais à une concentration inférieure : 30 nM pour 500 nM pour la salinomycine. En utilisant la méthode de clic-imagerie, nous avons observé le composé incolore dans les lysosomes et les auto-lysosomes. En augmentant le pH des vésicules acides, la salinomycine et ses analogues inhibent les activités des cathepsines B, L et D empêchant ainsi l'autophagie. Cette autophagie joue un rôle important dans la survie des cellules souches du cancer conduisant à une augmentation du facteur ROS et à une mort cellulaire par apoptose. Notre étude donne un aperçu du mécanisme par lequel la salinomycine élimine les cellules souches cancéreuses et propose des stratégies pour le traitement de cancers résistants. / A natural product Salinomycin (SAL) is widely used as an anticoccidial drug now being increasingly recognized as an agent for reducing the proportion of CD44⁺/CD24⁻ breast cancer stem cell which is perceived as important factor for breast tumor relapse. We first time report that not ionophoric action but the proton “sponge” of SAL is responsible for distinguishingly targeting cancer stem cell population. In addition, one SAL-analog alkyne-amine performed the similar action with SAL on CD44⁺/CD24⁻ population but at much lower concentration than SAL, at 30 nM compare to 500 nM of SAL. Using click-imaging method we visually observed the colorless compound saturated in lysosomes and autolysosomes. By raising pH of acidic vesicles, SAL and its analogs inhibit cathepsin B, L, D activity preventing the autophagy which plays an important role in cancer stem cell maintain and survival thus lead to cell death via increasing ROS and apoptosis. Our study provides the insight mechanism how SAL actually eradicates cancer stem cells and suggests sharpened strategies for treating resistant cancers.

Lysosomes are acidic, hydrolase-rich vesicles capable of degrading most biological macromolecules. During the past several decades, much has been learned about different aspects of lysosome biogenesis. The selective phosphorylation of mannose residues on lysosomal enzymes, in conjunction with specific receptors for the mannose-6-phosphate recognition marker, has been found to be largely responsible for the targeting of newly synthesized lysosomal enzymes to lyzosomes. It is known that lysosomes receive input from both the endocytotic and biosynthetic pathways. Nevertheless the exact molecular mechanisms responsible for sorting of the biosynthetic imput involved in the lysosome biogenesis is still a matter of debate. Because osteoclast precursors do not secrete their lysosomal enzymes and osteoclasts do, the observation of modifications occuring during osteoclastogenesis is a good model to observe mechanisms responsible for lysosomal enzymes traffic. Osteoclasts are bone-degrading cells. To perform this specific task they have to reorganise the sorting of their lysosomal enzymes to be able to target them toward the bone surface in mature cells. Since few years, the differentiation of osteoclasts in vitro did help to study these cells. Osteoclast morphology has been therefore already well studied, and the nature of their specific membrane domains is now established. Sensing the proximity of a bone-like surface the cell reorganises its cytoskeleton, and creates specific membrane domains: an actin-rich ring-like zone (named actin ring) surrounded by highly ruffled membrane (named the ruffled border) where enzymes are secreted, while subsequent bone degradation products are endocytosed. Endocytosed material is then transported through the cell inside transcytotic vesicles and released at the top of the cell in an area named the functional secretory domain. Several molecular machineries are thought to control these different phenomena. The main purpose of this thesis was to identify the major regulators of lysosomal enzymes secretion and therefore to identify the molecular switches responsible for such a membrane traffic re-organisation.

<p>The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. While these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a, and H+-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role for notochord vacuoles in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.</p> / Dissertation

Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The orthologue of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, PHI-33, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppress cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Les astrocytes sont capables de sécréter des gliotransmetteurs en réponse à une stimulation qui engendre l'augmentation de la concentration calcique intra-cellulaire. Différents mécanismes de sécrétion ont été proposés, parmi lesquels l'exocytose régulée. Mais les expériences menées dans le but d'observer la fusion d'organelles individuels dans des astrocytes en culture ont conduit à des résultats contradictoires, notamment en terme d'identité des vésicules libérables. Nos expériences préliminaires nous ont convaincus que les conflits sur l'identité des organelles libérables sont dus à de fausses colocalisations à cause du recouvrement spectral des marqueurs fluorescents utilisés et de la présence d'autofluorescence dans les astrocytes en culture. Nous avons donc adapté la décomposition spectrale à l'identification rigoureuse d'organelles individuels et au suivi de leur exocytose. La décomposition spectrale permet la séparation de sources de fluorescence mal séparées et ainsi l'étude de l'expression et de la colocalisation de protéines fluorescentes, même en présence d'autofluorescence. Nous avons à cette occasion introduit un intervalle de confiance du résultat de l'estimation des quantités de colorants. Appliquée au marquage des organelles astrocytaires avec la EGFP et l'acridine orange, cette méthode a montré que l'apparente colocalisation entre ces marqueurs reflète en fait la présence d'acridine orange plus intense que la EGFP et coexistant dans les mêmes organelles sous deux formes verte et rouge. A l'aide de la décomposition spectrale et de la microscopie à onde évanescente, nous avons ensuite montré que les organelles autofluorescents dans les astrocytes sont en majorité des lysosomes capables de fusionner lors d'une stimu\-lation qui engendre l'augmentation du calcium intra-cellulaire. Ces lysosomes sont peut-être les organelles majoritairement responsables de l'exocytose dans les astrocytes en culture.

The vesicle-mediated degradation of low-density lipoprotein (LDL) is an essential cellular function due to its role in cellular biosynthesis of membranes and steroids. Using multi-color single particle tracking fluorescence microscopy, the intracellular degradation of LDL was probed in live, intact cells. Unique to these experiments is the direct observation of LDL degradation using an LDL-based probe that increases fluorescence intensity upon degradation. Specifically, individual LDL particles were labeled with multiple fluorophores resulting in a quenched fluorescent signal. The characteristics of the vesicle responsible for degradation were determined and the vesicle dynamics involved in LDL degradation were quantified. Visualization of early endosomes, late endosomes and lysosomes was accomplished by fluorescently labeling vesicles with variants of GFP. Transient colocalization of LDL with specific vesicles and the intensity of the LDL particle were measured simultaneously. These studies, which are the first to directly observe the degradation of LDL within a cell, strive to completely describe the endo-lysosomal pathway and quantify the dynamics of LDL degradation in cells.

Traumatic brain injury (TBI) is a relatively unknown disease considering the tens of millions of people affected around the world each year. Many TBI patients die from their injuries and survivors often suffer from life-long disabilities. The primary injury initiates a variety of cellular and molecular processes that are both beneficial and detrimental for the brain, but that are not fully understood. The focus of this thesis has been to study the role of astrocytes in clearance of dead cells after TBI and to identify injury specific proteins that may function as biomarkers, by using cell cultures, animal models and in cerebrospinal fluid (CSF) from TBI patients. The result demonstrates a new function in that astrocytes, the most numerous cell type in the brain, engulf dead cells after injury both in cell cultures and in adult mice and thereby save neurons from contact-induced apoptosis. Astrocytes are effective phagocytes, but degrade the ingested dead cells very slowly. Moreover, astrocytes express the lysosome-alkalizing proteins Rab27a and Nox2 as well as major histocompatibility complex class II, the receptors on which antigens are being presented. By lowering the pH of the lysosomes with acidic nanoparticles, the degradation increases, but the astrocytes still remained less effective than macrophages. Taken together, the data indicates that the low acidification in astrocytes can preserve antigens and that astrocytes may be able to activate T cells. The expression and secretion of injury-specific proteins was studied in a cell culture model of TBI by separate mass spectrometry analysis of cells and medium. Interestingly, close to 30 % of the injury-specific proteins in medium are linked to actin, for example ezrin of the ezrin/radixin/moesin (ERM) protein family. Ezrin, but none of the other ERM proteins or actin, is actively secreted after injury. Extracellular ezrin also increases in CSF in response to experimental TBI in rats and is present in CSF from TBI patients, indicating that ezrin is a potential biomarker for TBI.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

<p>Due to its close proximity to the gastrointestinal tract, the human urinary tract is</p><p>subjected to constant barrage by gut-­associated bacteria. However, for the most part, this tract has resisted infection by various microbes. The impregnability of the urinary tract to microbial colonization is attributable to the ability of the bladder to promptly sense and mount robust responses to microbial challenge. A powerful mechanism for the elimination of invading bacteria was recently described in bladder epithelial cells, involving non-­lytic ejection of intracellular bacteria back into the extracellular milieu. In spite of the effectiveness of this defense strategy, much of the underlying mechanisms surrounding how this powerful cellular defense activity detects intracellular UPEC and shuttles them from their intracellular location to the plasma membrane of BECs to be exported remains largely a mystery.</p><p> Here, we describe uropathogenic E.coli (UPEC) expelled from infected bladder</p><p>epithelium cells (BECs) within membrane-­bound vesicles as a distinct cellular defense</p><p>response. Examination of the intracellular UPEC revealed that intracellular bacteria were</p><p>initially processed via autophagy, the conventional degradative pathway, then delivered</p><p>into multivesicular bodies (MVBs) and encapsulated in nascent intraluminal vesicle membrane. We further show the bacterial expulsion is triggered when intracellular UPEC follow the natural degradative trafficking pathway and reach lysosomes and attempt to neutralize its pH to avoid degradation. This pathogen-­mediated activity is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized on lysosomes, which spontaneously initiates lysosome exocytosis resulting in expulsion of exosome-­encased bacteria. These studies reveal a cellular default system for lysosome homeostasis and also, how it is coopted by the autonomous defense program to clear recalcitrant pathogens.</p> / Dissertation