No description available.

Excessive release of neurotransmitter is a characteristic of epileptogenic cells. A number of lines of evidence implicate defects in the synaptic vesicle cycle as a cause of this excessive release. Synaptic vesicles are retrieved by more than one route in central nerve terminals. During mild stimulation the dominant synaptic vesicle retrieval pathway is classical clathrin mediated endocytosis. During elevated neuronal activity retrieval of synaptic vesicle membrane by bulk endocytosis is the predominant retrieval method. As it is triggered by strong stimulation, bulk endocytosis may be of importance in retrieval during epilepsy, however little is currently known about this pathway. In order to investigate the role of bulk endocytosis, we sought to establish a cell culture model of epilepsy, to develop an assay to distinguish retrieval by bulk endocytosis, and to use these tools to look at the molecular players controlling this form of endocytosis. Characterisation of bulk endocytosis through the development of tailored assay systems has revealed that bulk endocytosis is a fast event that is triggered during strong stimulation. Bulk endocytosis provides the nerve terminal with an appropriate mechanism to meet the demands of synaptic vesicle retrieval during periods of intense synaptic vesicle exocytosis. Inhibition of a dephosphorylation specific dynamin I-syndapin I interaction by competitive peptides inhibits activity dependent bulk endocytosis, implicating this interaction in a role in this method of synaptic vesicle retrieval. Having characterised the strength of stimulation needed to activate bulk endocytosis, and the speed at which it occurs, we also investigated the effects of known anti-epileptogenic drugs on bulk endocytosis in our central nerve terminal model system.

No description available.

<p>Signal transductions are essential processes for living cells to react to environmental stimuli adequately, and they need to be tightly regulated as they can affect cell survival and cell fate determination. Since many of these signaling events rely on the presence of receptors on the cell membrane, members of endocytic proteins play critical regulatory roles in signaling via changing the localization of the receptors. In particular, endocytic adaptors are the linkers that connect membrane cargo and other members of endocytic machinery to accomplish the process. We focused on the roles of the endocytic adaptors epsins and their cargoes in signaling, as both epsins’ transmembrane and cytosolic cargoes participate in signaling pathways.</p> <p>We investigated the molecular mechanism of how epsins recognize specific ubiquitinated membrane cargoes among other ubiquitinated membrane proteins. Through genetic, biochemical, and cell biological approaches, we identified the first yeast transmembrane cargo, Ena1, a P-type ATPase sodium pump. We report that the simultaneous presence of phosphorylation and ubiquitination on the Ena1 are required for epsin-specific recognition. We also demonstrated that post-translational modifications are Yck1/2 and Art3-Rsp5 dependent, and the spatial arrangement of the modifications is essential. </p> <p>In addition to the regulation of signaling pathways through internalizing transmembrane cargoes, epsins are also involved in the regulation of Rho GTPase signaling pathways. Through direct interaction, epsins inhibit activities of their cytosolic cargoes, Rho GTPase activating proteins (RhoGAPs). Ocrl1 is one of the epsin interacting RhoGAP domain-containing proteins. The deficiency of Ocrl1 leads to a lethal developmental disease called Lowe syndrome (LS). While the patients display developmental problems affecting the brain and eyes, they also suffer from kidney dysfunction that results in death. The pathological mechanism is currently obscure and no cure, partly due to the lack of an adequate cell model from the affected tissues. We generated the first iPSC model from fibroblasts of LS patients and normal individuals and further generated kidney cells from these iPSCs. Consistent with observations obtained from LS fibroblasts, the LS iPSC derived kidney cells from patient cells also have a deficiency in ciliogenesis.</p> Further, we discovered that Six2, a crucial transcriptional factor in kidney development, is mislocalized to the Golgi-apparatus in patient iPSC-derived kidney cells as well as in an <i>OC</i><i>RL1</i> K.O. proximal tubular cell line. Disproportional cell lineage differentiation is also observed in the patient group. The iPSC model provides an opportunity to investigate the differences between normal and disease cell differentiation in all the affected tissues, generate organoids, and develop cell replacement therapies.

Coprinopsis cinerea, 亦稀灰蓋鬼傘，是研究擔子菌發育過程的模範生物。它的生命週期短，容易培養，亦有已建立的遺傳和分子生物研究技術。最近，它的完整基因組序列亦被發表。C. cinerea 的子實體萌生和發展是快速而複雜的過程。它受多種因素影響，如交配基因，營養消耗，光照和溫度。然而，我們對於C. cinerea 出菇的基本機制和涉及的分子途徑仍不清楚。在這項研究中，我採用NimbleGen 微陣列，以了解13,320 個C. cinerea 基因模型的表達。此微陣列覆蓋不同發育階段，包括雙核菌絲體，子實體初體，第2階段原基體，年青和成熟子實體。11,815個預測，在至少一個發育階段表達。707個基因模型在出菇的萌生過程有差異表達。我發現一些可能參與子實體的萌生和發展的份子。它們可能在檢測養分、形態、信號轉導和應激反應方面，擔當重要角色。此外，我亦分析了轉錄因子 、蛋白激酶組和細胞色素P450的基因表達模式。 / C. cinerea 的子實體發展是與光暗週期同步。當子實體初體在沒有光的環境培植，使會形成dark stipe。101個基因在dark stipe 表達差異。它們可能參與原基體成熟的過程。此微陣列基因表達數據，對了解菇機制有價格的信息。 / 胞吞作用是真核細胞透過質膜內陷將外來物質攝取的過程。Rab5 和Rab7 分別控制早期和晚期的胞吞作用。C. cinerea 的胞吞作用是組織由110個基因組成。FM4-64 螢光顯示在C. cinerea 菌絲體的胞吞作用是依賴肌動蛋白和能源。從菌絲體到年青子實體，Cc. Rab5的表達維持相同水平，而Cc.Rab7 的表達則不斷增加，兩者在成熟子實體的表達都是最高，原位雜交體技術顯示 Cc.Rab5 和Cc.Rab7 的 mRNA在年青子實體的子實層以及整個子實層上菌摺表達。我在第2階段原基體進行RNA 乾擾，致使Cc.Rab5 和Cc.Rab7的基因表達敲落。這導致原基體的生長遲緩。及後形成的成熟子實體亦有異常形態。因此，我推測Cc. Rab5 和Cc.Rab7參與出菇過程，並影響擔孢子的形成。這些結果表明，胞吞作用在C.Cinerea 子實體發育過程中發揮定一定的作用。 / Coprinopsis cinerea, is a model organism for studying developmental processes in basidiomycetous fungi. It has a short life cycle, easy to be cultivated in laboratory and can be accessed by various genetic and molecular techniques. Recently, its complete genome sequence was released. The fruiting body development in C. cinerea is a rapid yet complicated process. It is under the regulation of various factors such as mating type genes, nutrients depletion, light and temperature. However, the underlying mechanism and molecular events involved during fruiting body initiation and development remains unclear. / In this study, fruiting body developmental stages including mycelium, fruiting initials, stage 2 primordium, young and mature fruiting body, were analyzed with a comprehensive NimbleGen microarray. 11,815 out of 13,320 predicted gene models were expressed in at least one of the stages. 707 genes were differentially expressed during fruiting body initiation. Potential players involved in nutrients sensing, morphogenesis, signaling pathways and stress response were identified. In particular, expression patterns of all transcription factors, kinome and cytochrome P450s were analyzed. / The fruiting body development of C. cinerea is synchronized with the light/dark cycle. Differentially expressed genes were found in dark stipe produced by keeping fruiting initials in complete darkness. 101 genes, which are likely to be involved in maturation of primordium were identified. / Endocytosis is an essential process in eukaryotes through which cells take up extracellular substrates by membrane invaginations. Rab5 and Rab7 control the early and late stage of endocytosis respectively. The C. cinerea endocytic machinery composed of 110 genes models. The endocytic pathway was traced by FM4-64 and was found to be actin- and energy-dependent. Temporal and spatial expressions of Cc.Rab5 and Cc.Rab7 during fruiting body development were studied. Cc.Rab5 expressed constitutively from mycelium to young fruiting body stage, and reached the highest in the mature fruiting body. The expression of Cc.Rab7 increased continually from mycelium to mature fruiting body stage. From the in situ RNA-RNA hybridization results, both transcripts were localized at the hymenium layer in the young fruiting body and throughout the gill tissue of the mature cap. Knock-down of Cc.Rab5 and Cc.Rab7 by siRNA resulted in retarded growth of the stage 2 primordium and abnormal mature fruiting body. Cc.Rab5 and Cc.Rab7 may be involved in the formation of basidiospores. Endocytosis may play some roles during fruiting body development in C. cinerea. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lee, Yung Yung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 156-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 論文摘要 --- p.iii / Abbreviations --- p.iv / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Importance of fruiting body (mushrooms) production --- p.1 / Chapter 1.2 --- Introduction on Coprinopsis cinerea --- p.1 / Chapter 1.2.1 --- General introduction --- p.1 / Chapter 1.2.2 --- Life cycle and morphology --- p.2 / Chapter 1.2.3 --- Growth conditions for C. cinerea --- p.4 / Chapter 1.3 --- Regulation of fruiting development in C. cinerea --- p.5 / Chapter 1.3.1 --- Regulation by mating types --- p.5 / Chapter 1.3.2 --- Regulation by light-dark cycle --- p.6 / Chapter 1.3.3 --- Regulation by physiological factors --- p.9 / Chapter 1.4 --- Fruiting-specific genes --- p.9 / Chapter 1.5 --- Genome project of C. cinerea --- p.10 / Chapter 1.6 --- Transformation and gene silencing in C. cinerea --- p.11 / Chapter 1.7 --- DNA microarray --- p.12 / Chapter 1.8 --- Endocytosis --- p.13 / Chapter 1.8.1 --- The endocytic pathway --- p.14 / Chapter 1.8.2 --- Rab GTPase --- p.17 / Chapter 1.8.2.1 --- Control of the active and inactive state of Rab proteins --- p.18 / Chapter 1.8.2.2 --- Functions of Rab GTPases in vesicular transport --- p.19 / Chapter 1.8.2.3 --- Rab5 and Rab7 GTPase --- p.20 / Chapter 1.8.3 --- Endocytosis in fungi --- p.21 / Chapter 1.9 --- Aims of project --- p.22 / Chapter Chapter 2 --- Whole genome expression analysis during fruiting body development --- p.25 / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- NimbleGen 12x135K gene expression microarray --- p.26 / Chapter 2.2.1.1 --- Strains and culture conditions --- p.26 / Chapter 2.2.1.2 --- RNA extraction --- p.27 / Chapter 2.2.1.3 --- Overall design of the NimbleGen custom Microarray --- p.28 / Chapter 2.2.1.4 --- Microarray hybridization, data extraction and normalization --- p.28 / Chapter 2.2.2 --- Microarray data analysis, clustering and GO assignment --- p.29 / Chapter 2.2.3 --- Validation of expression patterns of NimbleGen microarray and analysis of differentially expressed genes by quantitative real-time PCR --- p.29 / Chapter 2.2.3.1 --- cDNA synthesis --- p.29 / Chapter 2.2.3.2 --- Primer design and verification --- p.30 / Chapter 2.2.3.3 --- Real time PCR and data analysis --- p.32 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Whole-genome expression during fruiting body development --- p.33 / Chapter 2.3.2 --- Differentially expressed genes during fruiting body initiation --- p.48 / Chapter 2.3.2.1 --- Fruiting body initiation-specific genes --- p.52 / Chapter 2.3.3 --- Gene expression analysis during fruiting body development --- p.53 / Chapter 2.3.4 --- The C. cinerea kinome --- p.55 / Chapter 2.3.5 --- Transcription factors in C. cinerea --- p.60 / Chapter 2.3.6 --- The cytochrome P450 family in C. cinerea --- p.65 / Chapter 2.3.7 --- Validation of NimbleGen microarray data by quantitative real-time PCR --- p.68 / Chapter 2.4 --- Discussion --- p.77 / Chapter Chapter 3 --- Effect of light on gene expression of fruiting body development --- p.92 / Chapter 3.1 --- Introduction --- p.92 / Chapter 3.2 --- Materials and Methods --- p.93 / Chapter 3.2.1 --- NimbleGen 12x135K gene expression microarray --- p.93 / Chapter 3.2.2 --- Validation of expression patterns of NimbleGen microarray and analysis of differentially expressed genes by quantitative real-time PCR --- p.93 / Chapter 3.2.2.1 --- cDNA synthesis --- p.94 / Chapter 3.2.2.2 --- Primer design and verification --- p.94 / Chapter 3.3 --- Results --- p.95 / Chapter 3.3.1 --- Differentially expressed genes in dark stipes --- p.95 / Chapter 3.3.2 --- Validation of expression patterns of NimbleGen microarray by real-time PCR --- p.102 / Chapter 3.4 --- Discussion --- p.107 / Chapter Chapter 4 --- Endocytosis in C. cinerea and its role in fruiting body development --- p.111 / Chapter 4.1 --- Introduction --- p.112 / Chapter 4.2 --- Materials and Methods --- p.112 / Chapter 4.2.1 --- The endosomal machinery of C. cinerea --- p.112 / Chapter 4.2.2 --- Tracing the endocytic pathway sing FM-64 --- p.113 / Chapter 4.2.2.1 --- Strains and culture conditions --- p.113 / Chapter 4.2.2.2 --- FM4-64 internalization in mycelium of C. cinerea --- p.113 / Chapter 4.2.2.3 --- Drug treatment effect on the internalization of FM4-64 dye --- p.114 / Chapter 4.2.3 --- Temporal and spatial expression of Cc.Rab5 and Cc.Rab7 --- p.114 / Chapter 4.2.3.1 --- Cloning of Cc.Rab5 and Cc.Rab7 --- p.114 / Chapter 4.2.3.1.1 --- RNA extraction and cDNA synthesis --- p.114 / Chapter 4.2.3.1.2 --- TA cloning of amplification products and bacterial transformation --- p.115 / Chapter 4.2.3.1.3 --- PCR screening for positive transformants and sequencing --- p.115 / Chapter 4.2.3.2 --- Quantitative real-time PCR --- p.116 / Chapter 4.2.3.2.1 --- RNA extraction and cDNA synthesis --- p.116 / Chapter 4.2.3.2.2 --- Primer design and verification --- p.116 / Chapter 4.2.3.2.3 --- Real time PCR and data analysis --- p.117 / Chapter 4.2.3.3 --- In situ RNA-RNA hybridization --- p.117 / Chapter 4.2.3.3.1 --- Tissue preparation --- p.117 / Chapter 4.2.3.3.2 --- RNA probe synthesis --- p.117 / Chapter 4.2.3.3.3 --- Hybridization, signal development and image viewing --- p.118 / Chapter 4.2.4 --- Knock-down of endogenous Cc.Rab5 and Cc.Rab7 by siRNA --- p.119 / Chapter 4.2.4.1 --- Strains and culture conditions --- p.119 / Chapter 4.2.4.2 --- Production of dsRNA of Cc.Rab5 and Cc.Rab7 --- p.119 / Chapter 4.2.4.3 --- Digestion of dsRNA to give siRNA --- p.120 / Chapter 4.2.4.4 --- Effects of Cc.Rab5 and Cc.Rab7 siRNA on fruiting body development --- p.120 / Chapter 4.2.4.4.1 --- Application of siRNA to C. cinerea culture --- p.120 / Chapter 4.2.4.4.2 --- Validation of the knock-down efficacy by real-time PCR --- p.121 / Chapter 4.3 --- Results --- p.122 / Chapter 4.3.1 --- The endosomal machinery of C. cinerea --- p.122 / Chapter 4.3.2 --- The endocytic pathway of C. cinerea --- p.127 / Chapter 4.3.2.1 --- Time-course of FM4-64 internalization --- p.127 / Chapter 4.3.2.2 --- Validation of active transport of FM4-64 --- p.129 / Chapter 4.3.3 --- Cloning of Cc.Rab5 and Cc.Rab7 --- p.131 / Chapter 4.3.4 --- Temporal expression of Cc.Rab5 and Cc.Rab7 --- p.133 / Chapter 4.3.5 --- Spatial expression of Cc.Rab5 and Cc.Rab7 --- p.136 / Chapter 4.3.6 --- Effects of Cc.Rab5 and Cc.Rab7 knock-down by siRNA --- p.140 / Chapter 4.3.6.1 --- Observation of effect of siRNA on fruiting body development --- p.140 / Chapter 4.3.6.2 --- Validation of the efficacy of external application of siRNA --- p.143 / Chapter 4.4 --- Discussion --- p.146 / Chapter Chapter 5 --- Concluding remarks --- p.152 / References --- p.156 / Appendix --- p.180

SH2-containing inositol polyphosphate 5-phosphatases, SHIP2, has been established as a regulator of the insulin cascade, of cell adhesion and spreading, actin structures, remodelling and cytoskeletal organization. However, the molecular mechanisms underlying these processes still needed additional investigations. Among different regulatory mechanisms, protein-protein interaction play an essential role. To better understand the molecular mechanism of SHIP2 in signalling pathway as well as to reveal novel roles of SHIP2, a two-hybrid was performed to search for SHIP2 protein interactors. JNK-interacting protein 1 (JIP1) and intersectin 1 (ITSN1) were two of the newly identified protein partners of SHIP2. In this thesis, we characterized the associations of SHIP2 with JIP1 and ITSN1 in different aspects as identifying the interacting domain involved, biochemical function regulations and cellular biological roles. The JIP scaffold family of proteins associate with MAPK, MAPKK and MAPKKK creating functional signaling modules to control the specificity of signal transduction. JIP1 is characterized as a scaffold protein assembling JNK, MAPK kinase 7 (MKK7), mixed lineage kinase (MLK), dual leucine zipper-bearing kinase (DLK). It thus enhances the selectivity and effectiveness of kinase activation during JNK signaling. In this thesis, the SHIP2-JIP1 interaction has been confirmed both in overexpression system in COS-7 and CHO-IR cells, and in native cells of COS-7. Both the proline-rich (PR) domain (residues 359-487) and PTB domain of JIP1 participated in this interaction. Overexpression of SHIP2 in COS-7 cells up-regulated JIP1-mediated JNK activation and the tyrosine phosphorylations of both JIP1 and MLK3. These effects were independent of SHIP2 catalytic activity. By the use of kinase inhibitors, we showed that Abl and Src family tyrosine kinases might be implicated in the regulation of JIP1 tyrosine phosphorylation. The residue Y270 of JIP1, a potential target of Abl tyrosine kinase, was shown to be involved in SHIP2-increased JIP1 tyrosine phosphorylation. In an in vitro assay, JIP1 negatively regulated the catalytic activity of SHIP2. In addition, upon the stimulation of okadaic acid, the overexpression of SHIP2 caused less viability of COS-7 cells. These data provide a new molecular link between SHIP2 and JIP1-mediated JNK pathway, and may help explain the biochemical mechanisms of SHIP2 in cellular apoptosis, as well as in insulin pathway. Another protein partner, ITSN1, is a multi-domain protein which plays a role in endocytosis, MAPK signalling and actin cytoskeleton. The interaction between SHIP2 and ITSN1 was confirmed in overexpression systems in COS-7 cells, as well as at the physiological concentration with the endogenously expressed proteins in C2C12 and COS-7 cells. EGF stimulation did not modulate the association of SHIP2 and ITSN1. ITSN1-SH3D, A, C and E domains interacted with the C-terminal part of SHIP2 with the binding affinity as SH3D>SH3A>SH3C>SH3E. Upon the stimulation of EGF, the expression of SHIP2 may recruit ITSN1 short form (ITSN1-S) to cell membrane. The ITSN-mediated ERK1/2 and JNK activations in response to EGF were not modulated when SHIP2 or catalytic mutant of SHIP2 or TSHIP2 was overexpressed. The link between SHIP2 and ITSN may provide one of the molecular mechanisms used by SHIP2 to participate in receptor endocytosis regulation. In conclusion, our data of the associations of SHIP2 with JIP1 and ITSN1 provide evidence for potential novel biochemical mechanisms of SHIP2 to be implicated in JNK pathway as well as EGF receptor endocytosis. JIP1 and ITSN1, which are both implicated in the JNK pathway, may also have a link through the common protein partner SHIP2, giving rise to potential interesting study goal.

Thesis (M.S.)--Florida State University, 2004. / Advisor: Dr. Michael H. Peters, Florida State University, College of Engineering, Dept. of Chemical Engineering. Title and description from dissertation home page (viewed 6/15/04). Includes bibliographical references.

Cell-cell communication events are crucial to determine the fate of each cell during development. Notch signaling is involved in many different contexts in determining cell fate by mediating cell-cell communication. Furthermore, regulation of the Notch transduction pathway is critical for normal cellular function, which is implicated in various diseases, including cancers. At a certain developmental time point, intrinsic or extrinsic developmental cues induce biases in ligands and Notch receptors between neighboring cells. These initial biases are further amplified by various cellular factors which eventually dictate cell fates. In Drosophila, two Notch ligands, Delta and Serrate, trigger Notch receptor activation in nearby cells by virtue of numerous regulating factors. One important question in this area is how cells become Notch signal sending or receiving cells for cell fate decisions. I show evidence about a distinct mechanism for biasing the direction of Notch signaling that depends on a small GTPase, Ral, during Drosophila photoreceptor cell development. Investigations described here indicate that Fz signaling up-regulates Ral transcription in a signal sending fate cell, the R3 precursor, and Ral represses ligand-independent activation of Notch in the R3 precursor. This event ensures R3 to become a signaler and contributes to asymmetric Notch activation in the neighboring cell, R4. Ral is a small Ras-like GTPase that regulates membrane trafficking and signaling. Here, possible Ral effector pathways that are important for Notch regulation will be proposed. To trigger Notch activation in adjacent cells, Notch ligand endocytosis by the signaling cells is necessary. Recently, it was suggested that control of membrane trafficking is important not only for ligand signaling, but also for Notch receptor activation. Furthermore, Notch receptor trafficking regulates critical cellular functions, including proliferation, which is implicated in tumors. Therefore, another important question in Notch signaling is about the role of membrane trafficking in regulation of the Notch transduction pathway. Drosophila endocytic epsin, Liquid facets [Lqf], is a key component necessary for ligand endocytosis, thereby triggering Notch activation in adjacent cells. However, its function in signal receiving cells for Notch activation has not been studied. In this dissertation, I provide evidence that epsin is also required in signal receiving cells for Notch activation in developmental contexts. Furthermore, genetic and molecular evidence suggests that epsin regulates Notch receptor trafficking via Rab5-mediated endosomal sorting pathway for Notch activation. These studies support the idea that Notch activation at the plasma membrane is not the only way to transduce Notch signaling, but the Notch receptor must enter through an epsin-mediated endocytic pathway into subcellular compartments to be activated, at least in some contexts. / text

The transport of material across the plasma membrane and between subcellular compartments is a major trafficking event by which cells communicate, regulate developmental processes and maintain homeostasis. Clathrin-coated vesicles select and traffic proteins to specific compartments in eukaryotic cells. Recently a large number of proteins have been identified that serve as clathrin adaptors and accessory proteins. Information regarding the interaction between individual clathrin adaptors and accessory proteins during coated pit formation is currently lacking. Here we investigated the intracellular role of one clathrin adaptor, Dictyostelium discoideum Hip1r, and identified a functional relationship between Hip1r and a second clathrin adaptor, epsin. Hip1r is phosphorylated and localizes to punctae on the plasma membrane that also contain epsin. Moreover, expression of the NH₂-terminal ENTH domain of epsin alone was sufficient to restore both the phosphorylation and the restricted localization of Hip1r to the plasma membrane. Analysis of the individual Hip1r domains demonstrated the phosphorylation event is also dependent upon the expression of the central coiled-coil region of the Hip1r. During development, Hip1r null cells form mature fruiting bodies that yield defective spores. While the mutant spores contain both cellulose and at least one protein secreted from prespore vesicles, spore coats lack the organized fibrils typical of wild type spores. Moreover, Hip1r spores are round, rather than ovoid, and exhibit decreased viability. Domain analysis of Hip1r in conjunction with investigation of phenotypes associated with a Hip1r/epsin double mutant reveal a requirement for full length Hip1r in the production of robust spores. Results from this study suggest that the Hip1r protein functions with epsin during cellular events in both growing and developing Dictyostelium cells and reveals a previously unidentified interaction between two clathrin adaptors. / text

The goal of my graduate study is to understand the role of endocytosis for signaling receptor activation during development, especially ligand endocytosis for Notch activation. Notch is a transmembrane receptor which is conserved in metazoans. I am using the Drosophila model system. Notch is required in almost every developmental context and abnormality in Notch signaling components is related to many human diseases. Delta, one of the Notch ligands, is also a transmembrane protein. To activate Notch, endocytosis of Delta in the signaling cells is essential. However, the exact mechanism of how Delta endocytosis regulates Notch activation is not known. Liquid facets (Lqf) is an endocytic protein, called epsin in vertebrates, which is required only in the signaling cells for Delta endocytosis and Notch activation. Overexpression of Lqf in the eyes results in malformed eyes. Using this phenotype as a background, an EMS-mutagenesis screen was performed and auxilin mutants were isolated as enhancers of the eye phenotype. Auxilin is a J-domain protein involved in fission and uncoating of clathrin-coated vesicles. Mosaic clonal analysis showed that auxilin functions in Notch activation and that auxilin is required only in the signaling cells. The auxilin mutant phenotype was suppressed by addition of a clathrin heavy chain transgene. This result suggests that the auxilin phenotype is at least partly caused by clathrin depletion and that auxilin generates a pool of free clathrin which is required for Delta endocytosis. Auxilin is a multi-domain protein. Two C-terminal domains, the clathrin-binding and the J domains, are sufficient to function as auxilin in Drosophila. One of the popular models to explain why Delta endocytosis is required in the signaling cells is the 'recycling model' in which inactive Delta is endocytosed and recycled to the plasma membrane in active form. Rab11 is a small GTPase that regulates recycling. If the recycling model is correct, rab11 mutants may show a phenotype similar to auxilin, lqf and Delta mutants. The rab11 hypomorphs or expression of rab11 dominant negative result in fewer photoreceptor cells and less Delta protein in the eye. These phenotypes are the opposite of typical mutant phenotypes of Notch components. The rab11 mutant phenotype argues against the recycling model.