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 Alsin and Its Role in IGF-1-Mediated Neuronal Survival(2005-04-29) Topp, Justin David; Horazdovsky, BruceThe transport of proteins between organelles is a highly regulated and complex process that is crucial for many of the functions required for cellular homeostasis. Many distinct proteins are involved in each trafficking step with roles in vesicle formation, budding, movement, and fusion. One class of proteins, the Rab GTPases, is required for docking and fusion of transport vesicles with their target membrane. These proteins are regulated by their state of nucleotide binding, with GTP-bound Rabs thought to provide specificity to transport steps via their interactions with specific effector proteins. While much work has been focused on proteins downstream of Rab GTPases, little is known as to how the activation of these proteins is controlled. This is particularly true of Rab5, the Rab protein required for vesicle fusion at the endosome. Endocytosis of plasma membrane proteins requires Rab5(GTP, and humans possess at least seven proteins (Vps9 family) that are expected to activate Rab5. An intriguing aspect of the Vps9 family of proteins is that they appear to link signal transduction to receptor trafficking via the specific coupling of particular receptors to Rab5-mediated endocytosis. Cell biological, biochemical, and immunohistochemical techniques were employed to characterize one of the Vps9 family proteins named Alsin. Alsin is required for motor neuron maintenance and/or survival, as loss-of-Alsin function results in multiple juvenile-onset neurodegenerative disorders (ALS2, JPLS, IAHSP). It was found here that Alsin is an endosomal protein that activates both Rac1 and Rab5. This protein is present in all of the tissues associated with the aforementioned diseases and intriguingly is upregulated in the cerebellum, an unknown site of pathology for this class of disorders. Alsin was found to couple Rab5 activation specifically to the IGF-1 signal transduction pathway via its regulation of IGF-1 receptor endocytosis. This function of Alsin was shown to be essential for IGF-1-mediated cell survival. These results provide the first characterization of Alsin and identify a novel cause for neurodegeneration.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 How to Mend a Broken Heart: Massive Endocytosis and the Role of Lipidic Forces in Membrane Trafficking(2012-07-20) Fine, Michael Jon; Hilgemann, Donald W.Novel forms of membrane internalization defined as massive endocytosis (MEND) were characterized for mechanism and physiological significance in isolated cells and intact cardiac tissue. These non-canonical forms of membrane trafficking are related to perturbations within the outer lipid monolayer of the plasmalemma or through coupled calcium-mediated fusion and phosphatidylinositol 4,5-bisphosphate (PIP2) signaling. Both forms of MEND do not rely on classical endocytic proteins such as clathrin or dynamin and appear independent of the cytoskeletal or extracellular matrix. A hypothesis emerged implicating lipidic driven processes as the mechanistic basis of MEND. One explanation involves lipid domain formation with excessive inward curvature from rapid accumulation of PIP2 within the inner monolayer subsequent to large calcium transients. Alternatively, perturbation of the outer monolayer through amphipathic detergents or modulation of lipid content also promotes inward curvature of the membrane leading to massive endocytosis. Electrophysiological and optical methods support MEND preferentially internalizing membrane of liquid-ordered (Lo) domains leading to the potential selection of certain proteins and markers due to physiological segregation into their respective energetically favorable domains. MEND demonstrates that lipidic reorganization of the membrane may be sufficient for rapid and selective internalization of the plasma membrane. The implications of such a novel form of endocytosis could dramatically impact numerous physiological and cellular activities. In cardiac pathologies, short-term changes in the surface membrane expression of vital ionic transporters have been implicated in hypertrophy, atrial defibrillation, and damage post myocardial ischemia. It is possible that the properties underlying MEND mechanics may be responsible for some of these acute cardiac changes. Protocols that induce massive endocytosis were performed on isolated cardiac myocytes with similar results described in the fibroblast cell lines. MEND occurs in cardiomyocytes with calcium transients. MEND also occurs upon isolation of myocytes without stimulation. While this indicates that MEND does occur in primary cells, protocols to mimic MEND in intact cardiac tissue have remained inconclusive. Cellular processes during the isolation procedures cause dramatic changes in the membrane of cardiac myocytes. Subsequently, it is likely isolated cardiomyocytes have distinct differences from unstressed intact tissue. MEND may still occur in cardiac tissue as well as other physiological systems and newer protocols to monitor membrane movements in intact tissues are currently being investigated. [Keywords: endocytosis, Triton X-100, lipid raft, cardiac remodeling, transporters, NCX1, membrand trafficking, cytoplasmic calcium, lipid domains, phase separation]Item Rab5 Activation by the Vps9 Domain(2007-05-21) Carney, Darren Scott; Horazdovsky, BruceThe movement of proteins through the endocytic pathway is a complex and highly regulated process. Not only is this pathway used to internalize cellular nutrients, but it is also used to modulate a cell's response to extracellular stimuli. Internalization and subsequent trafficking of transmembrane receptor proteins that receive these signals from the external milieu play an essential role in establishing and maintaining cellular homeostasis. As key regulators of the early stages the endocytic pathway, the small GTPases of the Rab5 family serve an essential function in integrating intracellular protein traffic and these cell signaling events. Rab5 proteins exert their influence on protein trafficking only when bound to GTP. A large family of proteins containing a conserved domain (Vps9) activate Rab5 by promoting the release of GDP and reloading of GTP. These nucleotide exchange factors contain additional domains which link them to specific cellular locations or signaling cascades. The multiplicity of these Rab5 proteins and exchange factors raises the question of how these proteins specifically interact to regulate individual trafficking events. To investigate this specificity, the three Rab5 proteins of Saccharomyces cerevisiae, Vps21, Ypt52 and Ypt53, and the two yeast Vps9 domain-containing proteins, Vps9 and Muk1, were analyzed. This analysis identified previously unappreciated roles for Ypt53 and Muk1 in a relatively late stage of endocytosis. A mutational analysis of Vps9 identified several residues important for Vps9 domain function and shed light on a possible intramolecular regulation of this domain by the carboxy-terminal ubiquitin-binding CUE domain. Finally, structural studies of the Rab5/Vps9 domain complex were initiated to gain a better understanding of the molecular mechanisms by which Rab5 proteins interact with and are activated by the Vps9 domain.Item Regulation of Endocytic Recycling by FGD4, A CDC42 GEF(2012-07-10) Cardenas, Jessica A.; White, Michael A.The family of Receptor Tyrosine Kinases (RTKs) are a group of cell surface receptors with the capability of activating, through phosphorylation, multiple kinase cascades in response to activation by an extracellular ligand. This allows a cell to respond to its environment and induce a range of cellular processes such as proliferation, differentiation, migration, and apoptosis. Unsurprisingly, these powerful transducers of extracellular signaling are often found mutated in human disease, such as cancer. Therefore, learning how these receptors are downregulated and processed once they have been activated may provide novel avenues of therapeutic intervention. How receptors are processed after internalization and fusion into the sorting endosome (also known as the early endosome) still largely remains unknown. Here, we discovered a Cdc42 GEF, FGD4, that may be important for shuttling ErbB receptors to the recycling endosome via a microtubule dependent mechanism. Through protein depletion studies we show that FGD4 is important for mitosis, microtubule stability, migration and endocytic trafficking of EGF, an ErbB1 ligand. Dynamic microtubule regulation are critical in these cell biological process, therefore we hypothesize that FGD4 may be regulating these diverse cell functions via a microtubule dependent mechanism. [Keywords: FGD4, Cdc42, endocytic recycling, microtubules, ErbB receptors]Item Regulation of Erythropoietin Receptor Endocytosis and Signaling(2009-01-14) Sulahian, Rita; Huang, LilyErythropoietin (Epo) and its receptor (EpoR) play an essential role in the survival, proliferation and differentiation of precursor red blood cells into mature erythrocytes. EpoR lacks intrinsic enzymatic activity essential to mediate downstream signaling cascades, instead, it associates with Janus tyrosine kinase 2 (JAK2), which upon Epo binding is auto-phosphorylated and activated. Activated JAK2 phosphorylates many of the tyrosines in the EpoR which recruit SH2-domain containing proteins that will carry on the signaling event. EpoR -/- and JAK2 -/- mice die during embryogenesis due to the absence of definitive erythropoiesis. Once activated, the EpoR is internalized and degraded through unidentified mechanisms. Sustained EpoR signaling and aberrant JAK2 activation are observed in hematological malignancies. Here we show that cell-surface EpoR is internalized via clathrin-mediated endocytosis. Ligand-dependent EpoR internalization requires both JAK2 kinase activity and EpoR cytoplasmic tyrosines. In addition, phosphorylated Y429, Y431, and Y479 in the EpoR cytoplasmic domain binds p85 subunit of phosphoinositide 3-kinase (PI3-kinase) upon Epo stimulation and individually is sufficient to mediate Epo-dependent EpoR internalization. Knockdown of p85 alpha and p85 beta but not inhibition of PI3-kinase activity dramatically impaired EpoR internalization, indicating that p85 alpha and p85 beta may recruit proteins in the endocytic machinery upon Epo stimulation. We carried on a candidate siRNA screen for endocytic proteins involved in EpoR endocytosis. c-Cbl, a E3 ubiquitin ligase associated with p85, was identified to be essential in mediating EpoR internalization. Ligase-deficient c-Cbl mutants dramatically diminished ligand-induced EpoR internalization. Consistent with this finding, c-Cbl knockout animals exhibit erythroid hyperplasia. We further demonstrate that ubiquitination of the EpoR itself is not required for internalization but may be important for endosomal sorting and degradation, and K428 in the EpoR cytoplasmic domain may be the primary ubiquitination site. Further studies will uncover the role of c-Cbl in EpoR down-regulation. These results provide the first characterization of EpoR downregulation mechanisms.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.