James B. Reinecke

Convergence of linkage, gene expression and association data demonstrates the influence of the RAR-related orphan receptor alpha (RORA) gene on neovascular AMD: A systems biology based approach

To identify novel genes and pathways associated with AMD, we performed microarray gene expression and linkage analysis which implicated the candidate gene, retinoic acid receptor-related orphan receptor alpha (RORA, 15q). Subsequent genotyping of 159 RORA single nucleotide polymorphisms (SNPs) in a family-based cohort, followed by replication in an unrelated case-control cohort, demonstrated that SNPs and haplotypes located in intron 1 were significantly associated with neovascular AMD risk in both cohorts. This is the first report demonstrating a possible role for RORA, a receptor for cholesterol, in the pathophysiology of AMD. Moreover, we found a significant interaction between RORA and the ARMS2/HTRA1 locus suggesting a novel pathway underlying AMD pathophysiology.

Rabankyrin-5 Interacts with EHD1 and Vps26 to Regulate Endocytic Trafficking and Retromer Function

Rabankyrin‐5 (Rank‐5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank‐5 as an interaction partner for the recycling regulatory protein, Eps15 homology domain 1 (EHD1). We have demonstrated this interaction by glutathione S‐transferase‐pulldown, yeast two‐hybrid assay, isothermal calorimetry and co‐immunoprecipitation, and found that the binding occurs between the EH domain of EHD1 and the NPFED motif of Rank‐5. Similar to EHD1, we found that Rank‐5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps26), suggesting a role for Rank‐5 in retromer‐based transport. Indeed, depletion of Rank‐5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6‐phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank‐5 is required for normal retromer distribution, as overexpression of a wild‐type Rank‐5‐small interfering RNA‐resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank‐5 or EHD1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank‐5 and EHD1, and novel endocytic regulatory roles that include retromer‐based transport and secretion.

Endocytosis and the Src family of non-receptor tyrosine kinases

Abstract The regulated intracellular transport of nutrient, adhesion, and growth factor receptors is crucial for maintaining cell and tissue homeostasis. Endocytosis, or endocytic membrane trafficking, involves the steps of intracellular transport that include, but are not limited to, internalization from the plasma membrane, sorting in early endosomes, transport to late endosomes/lysosomes followed by degradation, and/or recycling back to the plasma membrane through tubular recycling endosomes. In addition to regulating the localization of transmembrane receptor proteins, the endocytic pathway also controls the localization of non-receptor molecules. The non-receptor tyrosine kinase c-Src (Src) and its closely related family members Yes and Fyn represent three proteins whose localization and signaling activities are tightly regulated by endocytic trafficking. Here, we provide a brief overview of endocytosis, Src function and its biochemical regulation. We will then concentrate on recent advances in understanding how Src intracellular localization is regulated and how its subcellular localization ultimately dictates downstream functioning. As Src kinases are hyperactive in many cancers, it is essential to decipher the spatiotemporal regulation of this important family of tyrosine kinases.

Novel Functions for the Endocytic Regulatory Proteins MICAL-L1 AND EHD1 in Mitosis

During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like‐1 (MICAL‐L1) and C‐terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL‐L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi‐nucleated cells. We provide evidence that bi‐nucleation in MICAL‐L1‐ and EHD1‐depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL‐L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1‐independent function for MICAL‐L1 earlier in mitosis. Moreover, we provide evidence that MICAL‐L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL‐L1 and EHD1 during the cell cycle.

The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome

The endocytic recycling compartment maintains segregation of cargo after sorting in peripheral endosomes, and cargo is recycled by distinct pathways to the plasma membrane. In addition, tubular recycling endosomes can be generated from sorting endosomes and preferentially traffic clathrin-independent cargo.

Control of mitochondrial homeostasis by endocytic regulatory proteins

ABSTRACT Mitochondria play essential roles in cellular energy processes, including ATP production, control of reactive oxygen species (ROS) and apoptosis. While mitochondrial function is regulated by the dynamics of fusion and fission, mitochondrial homeostasis remains incompletely understood. Recent studies implicate dynamin-2 and dynamin-related protein-1 (Drp1, also known as DNM1L), as GTPases involved in mitochondrial fission. Here, we identify the ATPase and endocytic protein EHD1 as a novel regulator of mitochondrial fission. EHD1 depletion induces a static and elongated network of mitochondria in the cell. However, unlike dynamin-2 and Drp1, whose depletion protects cells from staurosporine-induced mitochondrial fragmentation, EHD1-depleted cells remain sensitive to staurosporine, suggesting a different mechanism for EHD1 function. Recent studies have demonstrated that VPS35 and the retromer complex influence mitochondrial homeostasis either by Mul1-mediated ubiquitylation and degradation of the fusion protein Mfn2, or by removal of inactive Drp1 from the mitochondrial membrane. We demonstrate that EHD1 and its interaction partner rabankyrin-5 interact with the retromer complex to influence mitochondrial dynamics, likely by inducing VPS35-mediated removal of inactive Drp1 from mitochondrial membranes. Our study sheds light on mitochondrial dynamics, expanding a new paradigm of endocytic protein regulation of mitochondrial homeostasis. Highlighted Article: Mitochondrial homeostasis is maintained by fusion and fission, and is essential for healthy cells. We identify a novel endocytic protein, EHD1, that, together with its interaction partner rabankyrin-5, indirectly regulates mitochondrial fission.

Regulation of Src trafficking and activation by the endocytic regulatory proteins MICAL-L1 and EHD1

ABSTRACT Localization of the non-receptor tyrosine kinase Src to the cell periphery is required for its activation and to mediate focal adhesion turnover, cell spreading and migration. Inactive Src localizes to a perinuclear compartment and the movement of Src to the plasma membrane is mediated by endocytic transport. However, the precise pathways and regulatory proteins that are responsible for SRC transport are incompletely understood. Here, we demonstrate that Src partially colocalizes with the endocytic regulatory protein MICAL-L1 (molecule interacting with CasL-like protein 1) in mammalian cells. Furthermore, MICAL-L1 is required for growth-factor- and integrin-induced Src activation and transport to the cell periphery in HeLa cells and human fibroblasts. Accordingly, MICAL-L1 depletion impairs focal adhesion turnover, cell spreading and cell migration. Interestingly, we find that the MICAL-L1 interaction partner EHD1 (EH domain-containing protein 1) is also required for Src activation and transport. Moreover, the MICAL-L1-mediated recruitment of EHD1 to Src-containing recycling endosomes is required for the release of Src from the perinuclear endocytic recycling compartment in response to growth factor stimulation. Our study sheds new light on the mechanism by which Src is transported to the plasma membrane and activated, and provides a new function for MICAL-L1 and EHD1 in the regulation of intracellular non-receptor tyrosine kinases.

A zebrafish model for uremic toxicity: role of the complement pathway.

Many organic solutes accumulate in end-stage renal disease (ESRD) and some are poorly removed with urea-based prescriptions for hemodialysis. However, their toxicities have been difficult to assess. We have employed an animal model, the zebrafish embryo, to test the toxicity of uremic serum compared to control. Serum was obtained from stable ESRD patients predialysis or from normal subjects. Zebrafish embryos 24 h postfertilization were exposed to experimental media at a water:human serum ratio of 3:1. Those exposed to serum from uremic subjects had significantly reduced survival at 8 h (19 ± 18 vs. 94 ± 6%, p < 0.05, uremic serum vs. control, respectively). Embryos exposed to serum from ESRD subjects fractionated at 50 kDa showed significantly greater toxicity with the larger molecular weight fraction (83 ± 11 vs. 7 ± 17% survival, p < 0.05, <50 vs. >50 kDa, respectively). Heating serum abrogated its toxicity. EDTA, a potent inhibitor of complement by virtue of calcium chelation, reduced the toxicity of uremic serum compared to untreated uremic serum (96 ± 5 vs. 28 ± 20% survival, p < 0.016, chelated vs. nonchelated serum, respectively). Anti-factor B, a specific inhibitor of the alternative complement pathway, reduced the toxicity of uremic serum, compared to untreated uremic serum (98 ± 6 vs. 3 ± 9% survival, p < 0.016, anti-factor B treated vs. nontreated, respectively). Uremic serum is thus more toxic to zebrafish embryos than normal serum. Furthermore, this toxicity is associated with a fraction of large size, is inactivated by heat, and is reduced by both specific and nonspecific inhibitors of complement activation. Together these data lend support to the hypothesis that at least some uremic toxicities may be mediated by complement.

Contents Vol. 35, 2013

230 Selected Abstracts from the 31th International Vicenza Course on Critical Care Nephrology Vicenza, June 11–14, 2013 (available online only)