Clathrin-mediated endocytosis is the process whereby specific cargoes are internalized into coated vesicles from the plasma membrane. Numerous clathrin adaptors facilitate this process by linking the coat protein clathrin to the plasma membrane by associating with PI(4,5)P2 and binding to membrane-bound cargo. Here, I investigated the role of two clathrin adaptors, APβ1/2 and Hip1r, in clathrin-mediated endocytosis. I found that Dictyostelium APβ1/2 functions in both the AP1 and AP2 complexes, unlike vertebrates, which have distinct β subunits for each AP complex. I found that APβ1/2 function is required for several clathrin-dependent processes, including cytokinesis, development and osmoregulation. I also uncovered a role for APβ1/2 in the stability other subunits of the AP1 and AP2 complexes. Finally, phenotypic comparisons of APβ1/2 mutant cells with cells missing subunits that are specific to the AP1 or AP2 complex allowed me to distinguish between endocytic defects and endosomal trafficking defects in clathrin mutants. My investigation of Hip1r centered on the known requirement for Hip1r in actin dynamics during endocytosis and a possible role for Hip1r phosphorylation in regulating actin. To determine how phosphorylation contributes to Hip1r function, I identified a specific serine residue that serves as a Hip1r phosphorylation site. I also identified a novel role for the kinase PKB in Hip1r phosphorylation. I determined that phosphorylation is not required for Hip1r localization to the plasma membrane. Similar to Hip1r, PKB is required for proper actin dynamics during endocytosis. My results support a model in which epsin recruits Hip1r to the plasma membrane during formation of clathrin-coated vesicles. Here, Hip1r functions as both a clathrin adaptor and a negative regulator of actin polymerization. I propose that phosphorylation of Hip1r by PKB triggers a reduction in the affinity of Hip1r for clathrin, which may stimulate actin polymerization and tethering of clathrin-coated vesicles with the actin cytoskeleton. / text

AP2 complex protein is an essential clathrin adaptor protein during clathrin mediated endocytosis. However, this view has been challenged in simple organisms. To gain insight into this conflict, the role of AP2 in clathrin localization and other clathrin related processes were assessed in Dictyostelium discoideum. In Dictyostelium, deleting function AP2 caused mild phenotypes in clathrin membrane localization, cytokinesis, osmoregulation and cell development. This supported the idea that AP2 have significant roles in multicellular organisms but not in unicellular system. Clathrin mediated processes carries important function not only on the plasma membrane but also on some internal organelles. But clathrin coated vesicles on internal organelles are not as well studied as on the plasma membrane. To understand more of the clathrin coated vesicles on internal organelles, the clathrin coated vesicles on Dictyostelium discoideum contractile vacuole were studied. Contractile vacuole associated clathrin coated vesicles contained clathrin adaptor proteins AP2, AP180, and epsin but not Hip1r. The absence of AP180 or AP2 produced abnormal large vacuoles, but the absence of epsin did not cause any detectable contractile vacuole abnormality. The enlarged contractile vacuoles in AP180 minus cells were caused by excessive homotypic fusion among contractile vacuoles. Using both GST-pull down and immunostaining AP180 was identified as the possible adaptor protein for a contractile vacuole-associated SNARE protein, Vamp7B. Therefore recycling Vamp7B from contractile vacuole by AP180 through clathrin coated vesicles could be an efficient way to prevent excessive homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin coated vesicles on cell internal organelles. / text

Clathrin-mediated endocytosis (CME) plays a central role in the regulation of multiple cellular processes such as uptake of nutrients, recycling of housekeeping receptors and transporters, as well as for cell surface removal and downregulation of signaling receptors. Once endocytosed, cargo passes through early endosomes where sorting mechanisms traffic the cargo to the recycling pathway or to degradation in the lysosome. The general objectives of this doctoral research were to identify and characterize new players of the clathrin-mediated trafficking pathway to reveal differences between the abundant components of the trafficking machinery in two tissues, and to examine the mechanisms of endosomal sorting. / We used subcellular proteomics to reveal the differences in components of clathrin-coated vesicles (CCVs) isolated from brain and liver and to identify new molecules participating in clathrin trafficking. We demonstrated that the ratio between the clathrin adaptor proteins AP-1 and AP-2 is different in brain and liver, which indicates differential functions between the two tissues. We also discovered that clathrin-light chains, which have been proposed for many years to be regulatory proteins in the assembly of CCVs, were less abundant relative to clathrin-heavy chain in liver and in non-brain tissues compared to brain. / We identified a new DnaJ domain-containing protein, receptor-mediated endocytosis protein 8 (RME-8) that was detected in liver CCVs specifically. Further characterization revealed that the RME-8 DnaJ domain binds to the chaperone heat-shock cognate 70 (Hsc70) in an ATP-dependent manner. RME-8 is a ubiquitously expressed protein that tightly associates with endosomes, and its depletion causes intracellular trafficking defects. Moreover, we demonstrated that RME-8 depletion also leads to a decrease in levels of epidermal growth factor receptor (EGFR), as a result of an increase in EGFR degradation. RME-8 knock-down causes decreased EGFR levels even in cancer cells lines where EGFR is generally protected from degradation. / Globally this doctoral project revealed new insights on specialized functions for c1athrin-mediated trafficking in different tissues and allowed the identification and characterization of a novel protein implicated in sorting decisions occurring on endosomes.

Sterols are lipid components of eukaryotic membranes. Alterations of membrane sterol composition perturb the execution of cell division, which in diverse eukaryotes can have severe consequences for development of the organism. Partitioning of the cytoplasm during cell division occurs at the final stage of cell division named cytokinesis. In somatic plant cells, cytokinesis is initiated by fusion of membrane vesicles in the plane of cell division resulting in a transient compartment termed the cell plate. Cell plate maturation relies on temporal and spatial orchestration of membrane fusion and endocytosis. Impaired vesicle fusion or defects in endocytosis result in cytokinetic defects. In Arabidopsis thaliana, the KNOLLE and DYNAMIN-RELATED PROTEIN 1A (DRP1A) contribute to cytokinesis. KNOLLE mediates fusion of vesicles at the plane of cell division while DRP1A appears to be involved in cell plate maturation through its role in clathrin-mediated endocytosis. This thesis shows that KNOLLE is specifically restricted to the cell division plane through sterol-dependent endocytosis that involves a clathrin- and DRP1A-mediated mechanism. Sterols affect internalization of KNOLLE through their role in lateral membrane organization by keeping diffusion of KNOLLE to lateral membranes in check via its endocytic removal. It is shown that the cell plate represents a high-lipid-order membrane domain that depends on the correct composition and the right concentration of sterols. Accumulation of DRP1A at the cell plate requires correct sterol concentration and composition similar to high-lipid order. Conversely, high-lipid-order at the cell plate relies on DRP1A activity suggesting a feedback between DRP1A function and lipid order establishment. Finally, it is shown that sterols are also present at the tonoplast of dividing and elongated root cells. Taken together, the results reveal that formation of the cell plate in Arabidopsis thaliana depends on an intricate interplay between cytokinetic vesicle fusion, sterol-dependent lateral membrane and high-lipid-order domain organization as well as endocytic machinery function.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Eukaryotic cells are characterised by membranes with varied and dynamic compositions and shapes. Consequently, membrane-binding proteins are tuned to recognise and modify these membrane states to perform their functions. To study the curvature sensitivity of proteins, I have developed a single-particle assay using NanoSight technology that tracks the Brownian motion of particles to measure their size. I optimised this system to track fluorescently labelled lipid-binding domains bound to liposomes of different sizes moving freely in solution. The comparison of the size distribution of the total liposomes with the fluorescently labelled population allowed me to determine their curvature preferences. To validate the method I tested proteins from the Bin/Amphiphysin/Rvs (BAR) superfamily, which are inherently curved and have known curvature preferences. My method was capable of recapitulating the behaviour of BAR domains with different curvature preferences. I then expanded the range of targets and showed that this assay is also capable of detecting curvature preferences for a variety of other lipid-binding domain families. As such, I identified AKT PH domain as a new curvature-sensing domain. Finally, using the ENTH domain of Epsin1 that causes vesicle budding, I demonstrated that this method can also be used to study membrane remodelling. Trafficking involves generation and sensing of membrane curvature combined with recognition of specific cargo. Endophilin consists of a curvature-sensitive BAR domain followed by an SH3 (Src-homology 3) domain and has recently been identified in a clathrin-independent endocytosis pathway, FEME (fast endophilin-mediated endocytosis), involved in the uptake of cell surface receptors. Endophilin recognises ligands via its SH3 domain, binding G-protein coupled receptors (GPCRs) directly in their intracellular loop 3 and receptor tyrosine kinase (RTKs) via adaptor proteins. However, a specific recognition motif has not been identified yet. Here, using a combination of biophysical approaches and NMR spectroscopy, I characterised the Endophilin binding motif of ALIX (ALG-2-interacting protein X) adaptor protein and of the GPCR $\alpha$2A adrenergic receptor. Comparison of SH3-peptide models resulted in a putative Endophilin recognition site.

RALF é um peptídeo hormonal de aproximadamente 5kDa presente em diferentes espécies do reino vegetal regulando negativamente a expansão celular. AtRALF1 é uma isoforma específica de raiz das 37 presentes em Arabidopsis thaliana que regula negativamente o crescimento de raízes seguido de uma mobilização de Ca+2 intracelular e inibição na secreção de prótons (H+). Neste trabalho foi caraterizado a localização e tráfego intracelular do peptídeo AtRALF1. / RALF is a 5kDa peptide hormone ubiquitous in different species of the plant kingdom that regulates cell expansion. AtRALF1 is a root-specific isoform of 37 present in Arabidopsis thaliana that negatively regulates root growth by intracellular calcium mobilization and inhibition of proton secretion (H+). In this work was studied the localization and intracellular trafficking of the AtRALF1 peptide.

Endosomal trafficking can influence the composition of the plasma membrane and the ability of cells to polarize their membranes. Here, we examined whether trafficking through clathrin-independent endocytosis (CIE) affects the ability of T cells to form a cell-cell conjugate with antigen-presenting cells (APCs). We show that CIE occurs in both the Jurkat T cell line and primary human T cells. In Jurkat cells, the activities of two guanine nucleotide binding proteins, Arf6 and Rab22 (also known as Rab22a), influence CIE and conjugate formation. Expression of the constitutively active form of Arf6, Arf6Q67L, inhibits CIE and conjugate formation, and results in the accumulation of vacuoles containing lymphocyte function-associated antigen 1 (LFA-1) and CD4, molecules important for T cell interaction with the APC. Moreover, expression of the GTP-binding defective mutant of Rab22, Rab22S19N, inhibits CIE and conjugate formation, suggesting that Rab22 function is required for these activities. Furthermore, Jurkat cells expressing Rab22S19N were impaired in spreading onto coverslips coated with T cell receptor-activating antibodies. These observations support a role for CIE, Arf6 and Rab22 in conjugate formation between T cells and APCs.

Le PI(4,5)P2 est un des principaux régulateurs de la dynamique de l'actine. Entre autres, ce lipide active de multiples complexes impliqués directement dans la polymérisation de l'actine. Comprendre comment est régulée la concentration locale de PI(4,5)P2 dans le temps et l'espace est à cet égard un enjeu biologique important. L'hydrolyse du PI(4,5)P2 est cruciale pour le trafic endosomal et dépend essentiellement de la phosphatase OCRL dans les cellules non-neuronales. Cependant, le mécanisme qui permet le recrutement de cette enzyme sur les vésicules recouvertes de clathrine, précisément après leur scission de la membrane plasmique, est encore méconnu. Nous avons montré que la GTPase Rab35 recrute directement la phosphatase OCRL immédiatement après la scission de la vésicule recouverte de clathrine. De plus, la déplétion de Rab35 ou d'OCRL entraîne l'accumulation de récepteurs internalisés, tels que le CI-MPR, sur des endosomes à la périphérie de la cellule. Ces endosomes, alors anormalement associés à la clathrine, présentent également une accumulation de protéines liées au PI(4,5)P2 (telles que AP-2) ou à l'actine (telles que cortactine ou Arp2/3). Nous avons par ailleurs observé que le recrutement de Rab35 sur les vésicules recouvertes de clathrine suit rapidement le recrutement de DENND1A, la protéine GEF de Rab35 et la disparition d'EPI64B, la GAP de Rab35. Nos résultats montrent que l'activation spatiale et temporelle précise de la GTPase Rab35 déclenche le recrutement de la protéine OCRL sur les endosomes nouvellement formés pour contrôler les niveaux de PI(4,5)P2 et d'actine, et permettre un trafic intracellulaire normal à partir de ces endosomes. / PI(4,5)P2 is one of the major regulators of actin dynamics. This lipid activates numerous complexes that are directly involved in actin polymerisation. Therefore, it is a major biological issue to understand how the local concentration of PI(4,5)P2 is regulated in time and space. The hydrolysis of PI(4,5)P2 is crucial for endosomal trafficking and principally depends on the phosphatase OCRL in non-neuronal cells. However, this enzyme is recruited on clathrin-coated vesicles right after their separation from the plasma membrane but the underlying mechanism remained unanswered so far. During my thesis, I have shown that the GTPase Rab35 recruits a phosphatase, OCRL, straight after the separation of the clathrin-coated vesicle from the plasma membrane. The depletion of Rab35 or OCRL leads to an accumulation of internalised receptors such as CI-MPR on peripheral endosomes. These endosomes are abnormally associated with clathrin and also present an accumulation of proteins linked to PI(4,5)P2 (such as AP-2) or to actin (such as cortactin or Arp2/3). Moreover, we observed that the recruitment of Rab35 on clathrin-coated vesicles quickly follows the recruitment of DENND1A, the GEF of Rab35, and the disappearance of EPI64B, the GAP of Rab35. Our results reveal that the precise spatial and temporal activation of GTPase Rab35 triggers the recruitment of the protein OCRL on newly formed endosomes to control the levels of PI(4,5)P2 and actin thus enabling normal intracellular trafficking from these endosomes.

In schnell wachsenden Hyphen des filamentösen Pilzes Ashbya gossypii ist die Oberflächenvergrößerung bis zu 40-fach höher, als in den Knospen des nah verwandten Pilzes Saccharomyces cerevisiae. Um die Wachstumszonen auf die Hyphenspitzen zu begrenzen, müssen Polaritätsfaktoren wie Rezeptoren und Sensoren, sowie überschüssiges Membranmaterial in subapikalen Bereichen von der Zelloberfläche entfernt werden. Dies wird durch den Prozess der Endozytose erreicht. In S. cerevisiae ist der Hauptendozytoseweg die Clathrin- und Aktin-abhängige Endozytose und der Prozess ist bereits gut charakterisiert. A. gossypii besitzt Homologe zu fast allen Komponenten dieser endozytischen Maschinerie und ist daher besonders gut geeignet die Anpassung des endozytischen Prozesses an schnelles, polares Wachstum zu untersuchen. Um die Endozytose während des polaren Hyphenwachstums zu analysieren, wurden neun homologe Proteine des aus S. cerevisiae bekannten Endozytosemechanismus mittels „live cell imaging“ und TIRF-Mikroskopie sowohl in langsam, als auch in schnell wachsenden Hyphen untersucht. Hierbei zeigte sich, dass die Endozytoserate in den schnell wachsenden Hyphen in A. gossypii im Vergleich zu Hefe-Zellen deutlich erhöht ist. Dies wird sowohl durch die Beschleunigung des endozytischen Prozesses, als auch durch eine erhöhte Anzahl an endozytischen Ereignissen pro µm2 Zelloberfläche erreicht. Die fluoreszenzmikroskopischen Analysen zeigten zudem, dass sich die Endozytosezone bei hoher Wachstumsgeschwindigkeit um ca. 3 µm in den hinteren Hyphenbereich verlagert. Ein wesentlicher Unterschied des endozytischen Prozesses in A. gossypii im Vergleich zu S. cerevisiae ist die Funktion von Clathrin. Clathrin kolokalisierte mit keiner der getesteten endozytischen Komponenten und konnte ausschließlich an zellinternen Strukturen detektiert werden. Dies deutet darauf hin, dass Clathrin bei der Endozytose in A. gossypii keine Rolle spielt und seine Funktion auf interne Kompartimente wie die Endosomen oder das Golgi-Netzwerk beschränkt ist. Die Unterschiede in der Clathrin-Funktion zwischen S. cerevisiae und A. gossypii hängen vermutlich mit einer minimalen Abweichung im Genset endozytischer Komponenten in A. gossypii zusammen. So besitzt A. gossypii kein Homologes zu ScSla2, welches in Hefe sowohl mit Clc1, als auch mit dem Aktin-Zytoskelett interagiert. Der Sequenzvergleich der Clc1-Proteine aus S. cerevisiae und A. gossypii zeigt, dass in AgClc1 die Sla2-Bindedomäne fehlt. Mittels eines Komplementationstests konnte nachgewiesen werden, dass die Fusion dieser Bindedomäne an das AgCLC1-Gen ausreicht, um die endozytische Funktion von Clathrin in S. cerevisiae wieder herzustellen. In S. cerevisiae führt die Interaktion von Sla2 und Clc1 zu einer verminderten Aktin-Anlagerung an das entstehende Vesikel und dient als Regulationsmechanismus für die Membraneinstülpung. Das Fehlen dieses Mechanismus könnte in A. gossypii die Membraneinstülpung durch vermehrte Aktin-Anlagerung beschleunigen und auf diese Weise zur Anpassung an das schnelle Hyphenwachstum beitragen.