Browsing by Subject "Endocytosis"
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Item APβ1/2 and Hip1r : insights into early and late stage clathrin adaptors in Dictyostelium discoideum(2012-05) Sosa, Ramiro Thomas; O'Halloran, Theresa; Gross, Jeffrey M.; Johnson, Arlen W.; Mehdy, Mona; Morgan, Jennifer R.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.Item Characterization of the epsin homolog EpnA in Dictyostelium discoideum(2008-05) Brady, Rebecca Jane, 1980-; O'Halloran, TheresaClathrin-coated pits on the plasma membrane invaginate into coated vesicles to internalize receptors and membrane. The clathrin adaptor epsin contains an aminoterminal ENTH domain that binds PI(4,5)P₂ and a carboxy-terminal domain that binds clathrin, and accessory proteins such as AP2. Here, we assessed how inter- and intramolecular factors affect the contribution of epsin to coated-pit function in living cells. We found Dictyostelium epsin was not required for global clathrin function, but plays an essential role in spore development. We demonstrated that clathrin, but not AP2, was critical for epsin to associate with clathrin-coated pits. We found that the carboxy-terminal region of epsin was essential, but not sufficient, for targeting epsin within clathrin-coated pits on the plasma membrane. In addition to targeting epsin to the membrane, the amino-terminal ENTH domain regulates the interaction between epsin and clathrin, an essential property that cannot be replaced by an alternate PI(4,5)P₂ binding domain. Moreover, the ENTH domain facilitates the functional interaction between clathrin and actin during late stages of endocytosis, possibly by regulating the activity of the adaptor Hip1r. Both the ability to bind PI(4,5)P₂ and another function mediated by residue T107 are critical for the activity of the ENTH domain. Our results support a model where the ENTH domain coordinates with the clathrin-binding C-terminal domain to allow a dynamic interaction of epsin with coated pits. Furthermore, we propose that the ENTH domain of epsin facilitates the membrane recruitment and phosphorylation of Hip1r, which in turn mediates the productive interaction of clathrin with the actin cytoskeleton at the plasma membrane.Item Function and regulation of Drosophila Epsin in notch signaling(2011-12) Xie, Xuanhua; Fischer, Janice Ann; Macdonald, Paul M.; O'Halloran, Theresa J.; Morgan, Jennifer R.; Thompson, Wesley J.Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. How Epsin promotes ligand endocytosis and thus Notch signaling is unclear. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, I tested each of the five protein or lipid interaction modules of Epsin for a role in Notch activation by each of the two Drosophila ligands, Serrate and Delta. here are five main results of this work that impact present thinking about endocytic machinery/Epsin, Epsin/ligand, or ligand/receptor interactions at the plasma membrane. First, I discovered that deletion or mutation of both UIMs destroys Epsin’s function in Notch signaling and has a greater negative effect on Epsin’s ability to function than removal of any other module type. Second, only one of the two UIMs of Epsin is essential. Third, the lipid-binding function of the ENTH domain is required for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins are necessary collectively for Epsin activity, their functions are highly redundant. Finally, I detected no ligand-specific requirements for Epsin modules. Most unexpected was the finding that Epsin’s Clathrin binding motifs were dispensable. All of these observations are consistent with a model where Epsin’s essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins.Item Functional analysis of the clathrin assembly protein, AP180, in Dictyostelium discoideum(2006) Stavrou, Irene; O'Halloran, Theresa J.AP180, an important coat component of clathrin-coated vesicles, is known to assemble clathrin triskelia into cages of uniform size. To gain insight into the relationship between AP180 and clathrin, the gene that encodes AP180 in Dictyostelium discoideum was cloned and a mutant strain carrying a deletion in this gene was constructed using homologous recombination. Unlike clathrin mutants, AP180 null cells displayed normal pinocytosis, cytokinesis and development into fruiting bodies. However, AP180 mutant cells were osmosensitive, a phenotype also exhibited by clathrin light chain and clathrin heavy chain mutants. The contractile vacuole in AP180 mutant cells became abnormally large in a hypotonic environment and the cycle of expansion and discharge of the vacuole took twice as long compared to that of wild-type cells. Expression of GFP tagged AP180 showed that it localized to punctae at the plasma membrane, cytoplasm and perinuclear area and that it associated extensively with clathrin at these sites. AP180 also localized to the contractile vacuole and in the absence of AP180 more contractile vacuoles were labeled with clathrin. The association of AP180 with the contractile vacuole was affected in the absence of clathrin light chain and the internalization of AP180 into cytoplasmic punctae required the presence of clathrin heavy chain. This work also investigated the dynamics between clathrin, AP180 and AP-2, which is another clathrin assembly protein. A double mutant strain was constructed that had the genes for both AP180 and AP-2 deleted. Of all the clathrin mediated processes examined only osmoregulation was more severe than in the AP180 or AP-2 single mutant cells. The osmosensitivity of the double mutant was an indication that clathrin events at the plasma membrane and the contractile vacuole are linked to some extent. In the absence of both adaptor proteins, the membrane association of clathrin was decreased but not completely abolished whereas the presence of clathrin on the contractile vacuole was markedly decreased. These results present a functional relationship between clathrin, AP180 and AP-2 and suggest that endocytic events mediated by clathrin, AP180 and AP- 2 are important in the normal function of the contractile vacuole.Item The Functions of Auxilin and Rab11 in Drosophila Suggest That the Fundamental Role of Ligand Endocytosis in Notch Signaling Cells Is Not Recycling(Public Library of Science, 2011-03-23) Banks, Susan M. L.; Cho, Bomsoo; Eun, Suk Ho; Lee, Ji-Hoon; Windler, Sarah L.; Xie, Xuanhua; Bilder, David; Fischer, Janice A.Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling.Item Overcoming the plasma membrane barrier to improve the efficiency of therapeutic delivery to the cellular cytoplasm(2017-06-19) Gadok, Avinash Kaur; Stachowiak, Jeanne Casstevens; Smyth, Hugh D. C.; Zoldan, Janet; O'Halloran, Theresa; Yeh, Hsin-ChihDifficulties in controlling endocytosis limit the success of many nanoparticle-based drug delivery strategies. Therefore, there is a need to both (i) introduce new mechanisms of therapeutic delivery that overcome the limitations of endocytic uptake, and (ii) gain better control of endocytosis by understanding its underlying mechanisms at a molecular level. Towards achieving efficient therapeutic delivery independently of endocytosis, I first report the development of targeted Connectosomes, cell-derived lipid vesicle materials that contain embedded connexons and are capable of forming functional gap junctions with cells. These materials encapsulated diverse molecular cargo, including dyes and drugs. These materials achieved efficient delivery of molecular cargo directly into the cytoplasm of specific populations of target cells, through interactions of embedded multi-functional, multi-domain transmembrane targeting proteins that target cell-specific receptors. By opening direct routes to the cytoplasm, targeted Connectosomes reduced the therapeutically effective dose (LD50) of doxorubicin for target cells by more than an order of magnitude in comparison to the unencapsulated drug, and by several orders of magnitude in comparison to conventional liposomal doxorubicin. These data illustrate the therapeutic importance of direct access to the cell cytoplasm, and highlight the potential of gap junction-mediated cytoplasmic delivery to increase the effectiveness of diverse therapeutics. Towards furthering our basic biophysical understanding of the mechanisms that drive clathrin-mediated endocytosis, I then investigated the curvature sensing abilities of clathrin, a critical question limiting our understanding of how nanoparticles and other molecular cargo are internalized. In particular, my findings demonstrate that clathrin binds preferentially to highly curved membranes, suggesting a possible new explanation for clathrin’s early participation in endocytic vesicle formation. In sum, this work represents key steps towards improving the success of nanoparticle-based drug delivery strategies from both applied and fundamental standpoints.Item The role of auxilin and endocytosis in delta signaling(2012-05) Banks, Susan Marie-Louise; Fischer, Janice Ann; Huibregtse, Jon; Macdonald, Paul; Morgan, Jennifer; O'Halloran, TheresaNotch signaling is important for cell-cell signaling during development. Notch signaling is highly conserved across all metazoans and failure in Notch signaling is causative in many human diseases. In the Drosophila eye, activation of the Notch pathway requires Lqf (Drosophila Epsin)-dependent and Clathrin-dependent internalization of the Notch receptor ligands, Delta or Serrate, by the signal-sending cells. However, it is unclear why ligand must be internalized into the signal-sending cells to activate Notch signaling in the signal-receiving cells. Evidence suggests that in addition to Clathrin and Epsin, Auxilin is essential for signaling and is indirectly required for internalization of the Notch receptor ligand Delta. Auxilin functions in uncoating Clathrin-coated vesicles to maintain a pool of free Clathrin and Epsin in the cell. auxilin mutants were used as an entryway to identify previously unknown components of the Notch signaling pathway. An F1, FLP/FRT, EMS screen was performed and enhancers of an auxilin mutant rough eye defect were isolated. The enhancers ultimately formed one complementation group on the 2nd chromosome and fourteen complementation groups on the 3rd chromosome. Three of the 3rd chromosome complementation groups were each identified as Delta, lqf, or hsc70. A single allele was identified as faf. Delta and Epsin have known roles in signaling cells to activate Notch as described above. Hsc70 is an ATPase that functions with Auxilin to uncoat Clathrin-coated vesicles and Faf is a deubiquitinating enzyme that maintains levels of active Epsin in the cell. These results suggest I have isolated mutations in genes closely tied to Notch signaling or functioning directly with Auxilin. Mutations in two genes previously undescribed in Notch signaling in the developing Drosophila eye were also isolated from the screen and identified. The second chromosome complementation group was identified as α-adaptin. α-Adaptin is a subunit of the heterotetrameric Clathrin adaptor protein AP-2. One of the third chromosome complementation groups was identified as crumbs. Crumbs is an integral membrane protein that functions at adherens junctions and in establishing apical/basal polarity in cells. Characterizing roles for α-Adaptin and Crumbs during Notch signaling may elucidate the purpose for Delta internalization to activate Notch signaling.Item The role of bone morphogenetic proteins in the development of the vertebrate midbrain(2010-12) Eom, Dae Seok; Agarwala, Seema; Wallingford, John; Gross, Jeffrey; Thompson, Wesley; De Lozanne, ArturoThe purpose of the thesis is to explore the role of BMP signaling in developing vertebrate midbrain. BMP signaling plays important roles in various tissues and stages of neural development to regulate cell fate, proliferation, differentiation, morphogenesis and more. We observed that several major BMPs are expressed not only at the roof plate but also the floor plate of the midbrain. This has led us to ask the role of BMP signaling in dorsal and ventral midbrain patterning. Despite ventral experiments, we found that BMP signaling does not regulate ventral cell fate specification in the midbrain. Instead BMPs profoundly influence the shape and early morphogenesis of the midbrain neural plate as it closes into a neural tube. During neural tube closure, one of the early events occurring at the ventral midline is median hinge point (MHP) formation. Failure to form MHP leads to neural tube closure defects, the 2nd most common birth defects in humans. However, the molecular mechanisms underlying MHP formation are not well known. We found that the lowest BMP signaling occurs at the MHP during early neurulation and BMP blockade is necessary and sufficient for MHP formation. Interestingly, we also demonstrated that BMP blockade directs MHP formation by regulating the apicobasal polarity pathway and this regulation may be mediated by biochemical interactions between pSMAD5 and the apical protein, PAR3. Additionally, our time-lapse data suggest that BMP blockade slows cell cycle progression by increasing duration of G1 to S transition and S phase which leads cell nuclei stay at the basal location longer. This mimics basal nuclear migration seen at the MHP where low BMP signaling occurs. Thus, we conclude that BMP signaling regulates neural tube closure via the apicobasal polarity pathway and in a cell cycle dependent manner at the ventral midline. We observed that BMP signaling is necessary and sufficient for the dorsal cell fate specification in a context-dependent manner and ventral BMP signaling affects dorsal cell fates. Taken together, we propose the idea that BMP signaling has distinct roles in different contexts. BMPs regulate tissue morphogenesis in the ventral midbrain and dorsally cell fate specification.Item The role of Dropsophila auxilin in Notch signaling(2007-12) Eun, Suk Ho, 1973-; Fischer, Janice AnnThe 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.Item Study of Hip1r: insights from a Dictyostelium discoideum clathrin adaptor(2007) Repass, Shannon Lea, 1970-; O'Halloran, TheresaThe 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.Item Transmembrane stem cell factor protein therapeutics enhance revascularization in ischemia without mast cell activation(2020-05-07) Takematsu, Eri; Baker, Aaron Blair; Dunn, Andrew K.; Terreson, David W.; Stachowiak , Jeanne C.; Suggs, Laura J.Diabetes mellitus affects approximately 350 million people worldwide, leading to the death of about 4.6 million people per year. As a complication of diabetes, 30-40 percent of patients age 50 and older develop peripheral artery disease (PAD). The current standard of care treatments for PAD includes surgical revascularization with bypass grafting or percutaneous interventions. However, these interventions cannot be performed in a significant portion of patients, and many do not respond to these therapies. An alternative approach for treating PAD is to use proteins to stimulate the body to create new vasculature, thus restoring blood flow through its own regenerative processes. Stem cell factor (SCF) is a cytokine that acts through the receptor tyrosine kinase c-Kit to regulate hematopoiesis and has been a candidate protein for treating PAD. Clinical use of soluble SCF would be highly beneficial but has been limited due to toxicity related to mast cell activation. SCF also exists in a transmembrane form that has differential activity from soluble SCF and has not been explored as a therapeutic agent. To explore the transmembrane SCF (tmSCF) as a therapeutic we created formulations of tmSCF embedded in proteoliposomes or in lipid nanodiscs. Mouse models of anaphylaxis and ischemia revealed the tmSCF-based therapies did not activate mast cells and were effective in improving the recovery from ischemia in both wild type and diabetic mice. We also found that the formulation of the lipid nanocarrier to deliver tmSCF altered the biological response and trophism of the tmSCF-based treatments. Proteoliposomal tmSCF preferentially acted on mature endothelial cells to induce angiogenesis while tmSCF nanodiscs had greater activity in inducing stem cell mobilization from the bone marrow and recruitment to ischemic sites. A mechanistic analysis of the effects of the treatments on mast cells, mature endothelial cells and endothelial progenitor cells, revealed that the nanocarriers altered the relative utilization of clathrin- versus caveolin-mediated uptake pathways of c-Kit in response to the treatments. Overall, our studies support tmSCF-based therapies can provide therapeutic benefits without off-target effects on mast cells and that lipid nanocarriers can be used to tailor the properties of membrane protein-based therapeutics.