Y. Rbaibi - Academia.edu (original) (raw)

Papers by Y. Rbaibi

Research paper thumbnail of Lysosomal deficiencies and cell death in mucolipidosis type IV

Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology, 2009

Lysosomal storage diseases are a set of genetic diseases that occur due to dysfunction or misloca... more Lysosomal storage diseases are a set of genetic diseases that occur due to dysfunction or mislocalization of proteins responsible for degradation of the endocytosed material. As a result, the cells accumulate storage bodies containing undigested proteins, lipids and other high-molecular compounds. A majority of storage diseases is associated with developmental delays, blindness and premature death. One of such diseases, mucolipidosis type IV (MLIV), is caused by mutation in a gene MCOLN1 . MCOLN1 codes for a novel channel mucolipin 1 (TRPML1), which is localized in lysosomes. The two main models that attempt to explain the function of this ion channel suggest that it regulates: 1) membrane traffic within the lower portions of the endocytic pathway or 2) ionic conditions in the lysosomes and, therefore, the activity of lysosomal digestive enzymes. Identifying the role of TRPML1 in the endocytic pathway will define the direction of the future search for MLIV treatments.

Research paper thumbnail of PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

PLoS ONE, 2013

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] at clathrin coated... more Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P 2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (a, b or c). PIP5KIb localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIb whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P 2 -containing liposomes of the PtdIns(4,5)P 2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P 2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIb have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P 2 mediated by PIP5KIb is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P 2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis.

Research paper thumbnail of Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization

Molecular Biology of the Cell, 2013

The sorting signals that direct proteins to the apical surface of polarized epithelial cells are ... more The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N-and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proximal stalk, but how this domain mediates targeting is unknown. Protein oligomerization or clustering has been suggested as a common step in the segregation of all apical proteins. Like many apical proteins, p75 forms dimers, and we hypothesized that formation of higher-order clusters mediated by p75 dimerization and interactions of the stalk facilitate its apical sorting. Using fluorescence fluctuation techniques (photon-counting histogram and number and brightness analyses) to study p75 oligomerization status in vivo, we found that wild-type p75-green fluorescent protein forms clusters in the trans-Golgi network (TGN) but not at the plasma membrane. Disruption of either the dimerization motif or the stalk domain impaired both clustering and polarized delivery. Manipulation of O-glycan processing or depletion of multiple galectins expressed in Madin-Darby canine kidney cells had no effect on p75 sorting, suggesting that the stalk domain functions as a structural prop to position other determinants in the lumenal domain of p75 for oligomerization. Additionally, a p75 mutant with intact dimerization and stalk motifs but with a dominant basolateral sorting determinant (Δ250 mutant) did not form oligomers, consistent with a requirement for clustering in apical sorting. Artificially enhancing dimerization restored clustering to the Δ250 mutant but was insufficient to reroute this mutant to the apical surface. Together these studies demonstrate that clustering in the TGN is required for normal biosynthetic apical sorting of p75 but is not by itself sufficient to reroute a protein to the apical surface in the presence of a strong basolateral sorting determinant. Our studies shed new light on the hierarchy of polarized sorting signals and on the mechanisms by which newly synthesized proteins are segregated in the TGN for eventual apical delivery.

Research paper thumbnail of Membrane traffic and turnover in TRP-ML1-deficient cells: a revised model for mucolipidosis type IV pathogenesis

Journal of Experimental Medicine, 2008

Research paper thumbnail of Mitochondrial Aberrations in Mucolipidosis Type IV

Journal of Biological Chemistry, 2006

Mucolipidosis type IV is a genetic lysosomal storage disease associated with degenerative process... more Mucolipidosis type IV is a genetic lysosomal storage disease associated with degenerative processes in the brain, eye, and other tissues. Mucolipidosis type IV results from mutations in the gene MCOLN1, which codes for the TRP family ion channel, mucolipin 1. The connection between lysosomal dysfunction and degenerative processes in mucolipidosis type IV is unclear. Here we report that mucolipidosis type IV and several unrelated lysosomal storage diseases are associated with significant mitochondrial fragmentation and decreased mitochondrial Ca 2؉ buffering efficiency. The mitochondrial alterations observed in these lysosomal storage diseases are reproduced in control cells by treatment with lysosomal inhibitors and with the autophagy inhibitor 3-methyladenine. This suggests that inefficient autophagolysosomal recycling of mitochondria generates fragmented, effete mitochondria in mucolipidosis. Mitochondria accumulate that cannot properly buffer calcium fluxes in the cell. A decrease in mitochondrial Ca 2؉ buffering capacity in cells affected by these lysosomal storage diseases is associated with increased sensitivity to apoptosis induced by Ca 2؉ -mobilizing agonists and executed via a caspase-8-dependent pathway. Deficient Ca 2؉ homeostasis may represent a common mechanism of degenerative cell death in several lysosomal storage diseases.

Research paper thumbnail of TRP-ML1 Is a Lysosomal Monovalent Cation Channel That Undergoes Proteolytic Cleavage

Journal of Biological Chemistry, 2005

Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel... more Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel TRP-ML1 lead to the lipid storage disorder mucolipidosis type IV (MLIV). The function and role of TRP-ML1 are not well understood. We report here that TRP-ML1 is a lysosomal monovalent cation channel. Both native and recombinant TRP-ML1 are cleaved resulting in two products.

Research paper thumbnail of TRP_2, a Lipid/Trafficking Domain That Mediates Diacylglycerol-induced Vesicle Fusion

Journal of Biological Chemistry, 2008

Physics Arch. Quant. Methods arXiv:0806.2394v1). Herein we test our functional predictions for th... more Physics Arch. Quant. Methods arXiv:0806.2394v1). Herein we test our functional predictions for the TRP_2 domain of TRPC3; a domain of unknown function that is conserved in all TRPC channels. Our functional models of this domain identify both lipid binding and trafficking activities. In this study, we reveal: (i) a novel structural determinant of ion channel sensitivity to lipids, (ii) a molecular mechanism for the difference between diacylglycerol (DAG)-sensitive and DAG-insensitive TRPC subfamilies, and (iii) evidence that TRPC3 can comprise part of the vesicle fusion machinery. Indeed, the TRPC3 TRP_2 domain mediates channel trafficking to the plasma membrane and binds to plasma membrane lipids. Further, mutations in TRP_2, which alter lipid binding, also disrupt the DAG-mediated fusion of TRPC3-containing vesicles with the plasma membrane without disrupting SNARE interactions. Importantly, these data agree with the known role of DAG in membrane destabilization, which facilitates SNARE-dependent synaptic vesicle fusion (Villar, A. V., Goni, F. M., and Alonso, A. (2001) FEBS Lett. 494, 117-120 and Goni, F. M., and Alonso, A. (1999) Prog. Lipid Res. 38, 1-48). Taken together, functional models generated by GDDA-BLAST provide a computational platform for deriving domain functionality, which can have in vivo and mechanistic relevance.

Research paper thumbnail of TRP-ML1 Regulates Lysosomal pH and Acidic Lysosomal Lipid Hydrolytic Activity

Journal of Biological Chemistry, 2006

which was hypothesized to be caused by the altered membrane fusion and fission events. How mutati... more which was hypothesized to be caused by the altered membrane fusion and fission events. How mutations in TRP-ML1 lead to aberrant lipolysis is not known. Here we present evidence that MLIV is a metabolic disorder that is not associated with aberrant membrane fusion/fission events. Thus, measurement of lysosomal pH revealed that the lysosomes in TRP-ML1 ؊/؊ cells obtained from the patients with MLIV are over-acidified. TRP-ML1 can function as a H ؉ channel, and the increased lysosomal acidification in TRP-ML1 ؊/؊ cells is likely caused by the loss of TRP-ML1-mediated H ؉ leak. Measurement of lipase activity using several substrates revealed a marked reduction in lipid hydrolysis in TRP-ML1 ؊/؊ cells, which was rescued by the expression of TRP-ML1. Cell fractionation indicated specific loss of acidic lipase activity in TRP-ML1 ؊/؊ cells. Furthermore, dissipation of the acidic lysosomal pH of TRP-ML1 ؊/؊ cells by nigericin or chloroquine reversed the lysosomal storage disease phenotype. These findings provide a new mechanism to account for the pathogenesis of MLIV.

Research paper thumbnail of Evidence for core 2 to core 1 O-glycan remodeling during the recycling of MUC1

Research paper thumbnail of Mitotic slippage in non-cancer cells induced by a microtubule disruptor, disorazole C1

BMC Chemical Biology, 2010

Background: Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation brot... more Background: Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C 1 was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C 1 in both non-cancer and cancer cells and compared our results to vinblastine and taxol. Results: In non-cancer cells, disorazole C 1 induced a prolonged mitotic arrest, followed by mitotic slippage, as confirmed by live cell imaging and cell cycle analysis. This mitotic slippage was associated with cyclin B degradation, but did not require p53. Four assays for apoptosis, including western blotting for poly(ADP-ribose) polymerase cleavage, microscopic analyses for cytochrome C release and annexin V staining, and gel electrophoresis examination for DNA laddering, were conducted and demonstrated little induction of apoptosis in non-cancer cells treated with disorazole C 1 . On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C 1 . Conclusion: Our studies demonstrate that non-cancer cells undergo mitotic slippage in a cyclin B-dependent and p53-independent manner after prolonged mitotic arrest caused by disorazole C 1 . In contrast, cancer cells induce the apoptotic pathway after disorazole C 1 treatment, indicating a possibly significant therapeutic window for this compound.

Research paper thumbnail of Galectin-7 modulates the length of the primary cilia and wound repair in polarized kidney epithelial cells

AJP: Renal Physiology, 2011

Galectins (Gal) are β-galactoside-binding proteins that function in epithelial development and ho... more Galectins (Gal) are β-galactoside-binding proteins that function in epithelial development and homeostasis. An overlapping role for Gal-3 and Gal-7 in wound repair was reported in stratified epithelia. Although Gal-7 was thought absent in simple epithelia, it was reported in a proteomic analysis of cilia isolated from cultured human airway, and we recently identified Gal-7 transcripts in Madin-Darby canine kidney (MDCK) cells (Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP. J Biol Chem 286: 6780-6790, 2011). We now report that Gal-7 is localized exclusively on the primary cilium of MDCK, LLC-PK(1) (pig kidney), and mpkCCD(c14) (mouse kidney) cells as well as on cilia in the rat renal proximal tubule. Gal-7 is also present on most cilia of multiciliated cells in human airway epithelia primary cultures. Interestingly, exogenous glutathione S-transferase (GST)-Gal-7 bound the MDCK apical plasma membrane as well as the cilium, while the lectin Ulex europeaus agglutinin, with glycan preferences similar to Gal-7, bound the basolateral plasma membrane as well as the cilium. In pull-down assays, β1-integrin isolated from either the basolateral or apical/cilia membranes of MDCK cells was similarly bound by GST-Gal-7. Selective localization of Gal-7 to cilia despite the presence of binding sites on all cell surfaces suggests that intracellular Gal-7 is specifically delivered to cilia rather than simply binding to surface glycoconjugates after generalized secretion. Moreover, depletion of Gal-7 using tetracycline-induced short-hairpin RNA in mpkCCD(c14) cells significantly reduced cilia length and slowed wound healing in a scratch assay. We conclude that Gal-7 is selectively targeted to cilia and plays a key role in surface stabilization of glycoconjugates responsible for integrating cilia function with epithelial repair.

Research paper thumbnail of Rab11a-positive compartments in proximal tubule cells sort fluid-phase and membrane cargo

AJP: Cell Physiology, 2014

The proximal tubule (PT) reabsorbs the majority of sodium, bicarbonate, and chloride ions, phosph... more The proximal tubule (PT) reabsorbs the majority of sodium, bicarbonate, and chloride ions, phosphate, glucose, water, and plasma proteins from the glomerular filtrate. Despite the critical importance of endocytosis for PT cell (PTC) function, the organization of the endocytic pathway in these cells remains poorly understood. We have used immunofluorescence and live-cell imaging to dissect the itinerary of apically internalized fluid and membrane cargo in polarized primary cultures of PTCs isolated from mouse kidney cortex. Cells from the S1 segment could be distinguished from those from more distal PT segments by their robust uptake of albumin and comparatively low expression of γ-glutamyltranspeptidase. Rab11a in these cells is localized to variously sized spherical compartments that resemble the apical vacuoles observed by electron microscopy analysis of PTCs in vivo. These Rab11a-positive structures are highly dynamic and receive membrane and fluid-phase cargo. In contrast, fluid-phase cargoes are largely excluded from Rab11a-positive compartments in immortalized kidney cell lines. The unusual morphology and sorting capacity of Rab11a compartments in primary PTCs may reflect a unique specialization of these cells to accommodate the functional demands of handling a high endocytic load.

Research paper thumbnail of Shear stress-dependent regulation of apical endocytosis in renal proximal tubule cells mediated by primary cilia

Proceedings of the National Academy of Sciences, 2014

The kidney has an extraordinary ability to maintain stable fractional solute and fluid reabsorpti... more The kidney has an extraordinary ability to maintain stable fractional solute and fluid reabsorption over a wide range of glomerular filtration rates (GFRs). Internalization of filtered low molecular weight proteins, vitamins, hormones, and other small molecules is mediated by the proximal tubule (PT) multiligand receptors megalin and cubilin. Changes in GFR and the accompanying fluid shear stress (FSS) modulate acute changes in PT ion transport thought to be mediated by microvillar bending. We found that FSS also affects apical endocytosis in PT cells. Exposure of immortalized PT cell lines to physiologically relevant levels of FSS led to dramatically increased internalization of the megalin-cubilin ligand albumin as well as the fluid phase marker dextran. FSS-stimulated apical endocytosis was initiated between 15 and 30 min postinduction of FSS, occurred via a clathrin- and dynamin-dependent pathway, and was rapidly reversed upon removing the FSS. Exposure to FSS also caused a rapid elevation in intracellular Ca(2+) [Ca(2+)]i, which was not observed in deciliated cells, upon treatment with BAPTA-AM, or upon inclusion of apyrase in the perfusion medium. Strikingly, deciliation, BAPTA-AM, and apyrase also blocked the flow-dependent increase in endocytosis. Moreover, addition of ATP bypassed the need for FSS in enhancing endocytic capacity. Our studies suggest that increased [Ca(2+)]i and purinergic signaling in response to FSS-dependent ciliary bending triggers a rapid and reversible increase in apical endocytosis that contributes to the efficient retrieval of filtered proteins in the PT.

Research paper thumbnail of Lysosomal deficiencies and cell death in mucolipidosis type IV

Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology, 2009

Lysosomal storage diseases are a set of genetic diseases that occur due to dysfunction or misloca... more Lysosomal storage diseases are a set of genetic diseases that occur due to dysfunction or mislocalization of proteins responsible for degradation of the endocytosed material. As a result, the cells accumulate storage bodies containing undigested proteins, lipids and other high-molecular compounds. A majority of storage diseases is associated with developmental delays, blindness and premature death. One of such diseases, mucolipidosis type IV (MLIV), is caused by mutation in a gene MCOLN1 . MCOLN1 codes for a novel channel mucolipin 1 (TRPML1), which is localized in lysosomes. The two main models that attempt to explain the function of this ion channel suggest that it regulates: 1) membrane traffic within the lower portions of the endocytic pathway or 2) ionic conditions in the lysosomes and, therefore, the activity of lysosomal digestive enzymes. Identifying the role of TRPML1 in the endocytic pathway will define the direction of the future search for MLIV treatments.

Research paper thumbnail of PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

PLoS ONE, 2013

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] at clathrin coated... more Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P 2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (a, b or c). PIP5KIb localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIb whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P 2 -containing liposomes of the PtdIns(4,5)P 2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P 2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIb have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P 2 mediated by PIP5KIb is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P 2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis.

Research paper thumbnail of Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization

Molecular Biology of the Cell, 2013

The sorting signals that direct proteins to the apical surface of polarized epithelial cells are ... more The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N-and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proximal stalk, but how this domain mediates targeting is unknown. Protein oligomerization or clustering has been suggested as a common step in the segregation of all apical proteins. Like many apical proteins, p75 forms dimers, and we hypothesized that formation of higher-order clusters mediated by p75 dimerization and interactions of the stalk facilitate its apical sorting. Using fluorescence fluctuation techniques (photon-counting histogram and number and brightness analyses) to study p75 oligomerization status in vivo, we found that wild-type p75-green fluorescent protein forms clusters in the trans-Golgi network (TGN) but not at the plasma membrane. Disruption of either the dimerization motif or the stalk domain impaired both clustering and polarized delivery. Manipulation of O-glycan processing or depletion of multiple galectins expressed in Madin-Darby canine kidney cells had no effect on p75 sorting, suggesting that the stalk domain functions as a structural prop to position other determinants in the lumenal domain of p75 for oligomerization. Additionally, a p75 mutant with intact dimerization and stalk motifs but with a dominant basolateral sorting determinant (Δ250 mutant) did not form oligomers, consistent with a requirement for clustering in apical sorting. Artificially enhancing dimerization restored clustering to the Δ250 mutant but was insufficient to reroute this mutant to the apical surface. Together these studies demonstrate that clustering in the TGN is required for normal biosynthetic apical sorting of p75 but is not by itself sufficient to reroute a protein to the apical surface in the presence of a strong basolateral sorting determinant. Our studies shed new light on the hierarchy of polarized sorting signals and on the mechanisms by which newly synthesized proteins are segregated in the TGN for eventual apical delivery.

Research paper thumbnail of Membrane traffic and turnover in TRP-ML1-deficient cells: a revised model for mucolipidosis type IV pathogenesis

Journal of Experimental Medicine, 2008

Research paper thumbnail of Mitochondrial Aberrations in Mucolipidosis Type IV

Journal of Biological Chemistry, 2006

Mucolipidosis type IV is a genetic lysosomal storage disease associated with degenerative process... more Mucolipidosis type IV is a genetic lysosomal storage disease associated with degenerative processes in the brain, eye, and other tissues. Mucolipidosis type IV results from mutations in the gene MCOLN1, which codes for the TRP family ion channel, mucolipin 1. The connection between lysosomal dysfunction and degenerative processes in mucolipidosis type IV is unclear. Here we report that mucolipidosis type IV and several unrelated lysosomal storage diseases are associated with significant mitochondrial fragmentation and decreased mitochondrial Ca 2؉ buffering efficiency. The mitochondrial alterations observed in these lysosomal storage diseases are reproduced in control cells by treatment with lysosomal inhibitors and with the autophagy inhibitor 3-methyladenine. This suggests that inefficient autophagolysosomal recycling of mitochondria generates fragmented, effete mitochondria in mucolipidosis. Mitochondria accumulate that cannot properly buffer calcium fluxes in the cell. A decrease in mitochondrial Ca 2؉ buffering capacity in cells affected by these lysosomal storage diseases is associated with increased sensitivity to apoptosis induced by Ca 2؉ -mobilizing agonists and executed via a caspase-8-dependent pathway. Deficient Ca 2؉ homeostasis may represent a common mechanism of degenerative cell death in several lysosomal storage diseases.

Research paper thumbnail of TRP-ML1 Is a Lysosomal Monovalent Cation Channel That Undergoes Proteolytic Cleavage

Journal of Biological Chemistry, 2005

Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel... more Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel TRP-ML1 lead to the lipid storage disorder mucolipidosis type IV (MLIV). The function and role of TRP-ML1 are not well understood. We report here that TRP-ML1 is a lysosomal monovalent cation channel. Both native and recombinant TRP-ML1 are cleaved resulting in two products.

Research paper thumbnail of TRP_2, a Lipid/Trafficking Domain That Mediates Diacylglycerol-induced Vesicle Fusion

Journal of Biological Chemistry, 2008

Physics Arch. Quant. Methods arXiv:0806.2394v1). Herein we test our functional predictions for th... more Physics Arch. Quant. Methods arXiv:0806.2394v1). Herein we test our functional predictions for the TRP_2 domain of TRPC3; a domain of unknown function that is conserved in all TRPC channels. Our functional models of this domain identify both lipid binding and trafficking activities. In this study, we reveal: (i) a novel structural determinant of ion channel sensitivity to lipids, (ii) a molecular mechanism for the difference between diacylglycerol (DAG)-sensitive and DAG-insensitive TRPC subfamilies, and (iii) evidence that TRPC3 can comprise part of the vesicle fusion machinery. Indeed, the TRPC3 TRP_2 domain mediates channel trafficking to the plasma membrane and binds to plasma membrane lipids. Further, mutations in TRP_2, which alter lipid binding, also disrupt the DAG-mediated fusion of TRPC3-containing vesicles with the plasma membrane without disrupting SNARE interactions. Importantly, these data agree with the known role of DAG in membrane destabilization, which facilitates SNARE-dependent synaptic vesicle fusion (Villar, A. V., Goni, F. M., and Alonso, A. (2001) FEBS Lett. 494, 117-120 and Goni, F. M., and Alonso, A. (1999) Prog. Lipid Res. 38, 1-48). Taken together, functional models generated by GDDA-BLAST provide a computational platform for deriving domain functionality, which can have in vivo and mechanistic relevance.

Research paper thumbnail of TRP-ML1 Regulates Lysosomal pH and Acidic Lysosomal Lipid Hydrolytic Activity

Journal of Biological Chemistry, 2006

which was hypothesized to be caused by the altered membrane fusion and fission events. How mutati... more which was hypothesized to be caused by the altered membrane fusion and fission events. How mutations in TRP-ML1 lead to aberrant lipolysis is not known. Here we present evidence that MLIV is a metabolic disorder that is not associated with aberrant membrane fusion/fission events. Thus, measurement of lysosomal pH revealed that the lysosomes in TRP-ML1 ؊/؊ cells obtained from the patients with MLIV are over-acidified. TRP-ML1 can function as a H ؉ channel, and the increased lysosomal acidification in TRP-ML1 ؊/؊ cells is likely caused by the loss of TRP-ML1-mediated H ؉ leak. Measurement of lipase activity using several substrates revealed a marked reduction in lipid hydrolysis in TRP-ML1 ؊/؊ cells, which was rescued by the expression of TRP-ML1. Cell fractionation indicated specific loss of acidic lipase activity in TRP-ML1 ؊/؊ cells. Furthermore, dissipation of the acidic lysosomal pH of TRP-ML1 ؊/؊ cells by nigericin or chloroquine reversed the lysosomal storage disease phenotype. These findings provide a new mechanism to account for the pathogenesis of MLIV.

Research paper thumbnail of Evidence for core 2 to core 1 O-glycan remodeling during the recycling of MUC1

Research paper thumbnail of Mitotic slippage in non-cancer cells induced by a microtubule disruptor, disorazole C1

BMC Chemical Biology, 2010

Background: Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation brot... more Background: Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C 1 was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C 1 in both non-cancer and cancer cells and compared our results to vinblastine and taxol. Results: In non-cancer cells, disorazole C 1 induced a prolonged mitotic arrest, followed by mitotic slippage, as confirmed by live cell imaging and cell cycle analysis. This mitotic slippage was associated with cyclin B degradation, but did not require p53. Four assays for apoptosis, including western blotting for poly(ADP-ribose) polymerase cleavage, microscopic analyses for cytochrome C release and annexin V staining, and gel electrophoresis examination for DNA laddering, were conducted and demonstrated little induction of apoptosis in non-cancer cells treated with disorazole C 1 . On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C 1 . Conclusion: Our studies demonstrate that non-cancer cells undergo mitotic slippage in a cyclin B-dependent and p53-independent manner after prolonged mitotic arrest caused by disorazole C 1 . In contrast, cancer cells induce the apoptotic pathway after disorazole C 1 treatment, indicating a possibly significant therapeutic window for this compound.

Research paper thumbnail of Galectin-7 modulates the length of the primary cilia and wound repair in polarized kidney epithelial cells

AJP: Renal Physiology, 2011

Galectins (Gal) are β-galactoside-binding proteins that function in epithelial development and ho... more Galectins (Gal) are β-galactoside-binding proteins that function in epithelial development and homeostasis. An overlapping role for Gal-3 and Gal-7 in wound repair was reported in stratified epithelia. Although Gal-7 was thought absent in simple epithelia, it was reported in a proteomic analysis of cilia isolated from cultured human airway, and we recently identified Gal-7 transcripts in Madin-Darby canine kidney (MDCK) cells (Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP. J Biol Chem 286: 6780-6790, 2011). We now report that Gal-7 is localized exclusively on the primary cilium of MDCK, LLC-PK(1) (pig kidney), and mpkCCD(c14) (mouse kidney) cells as well as on cilia in the rat renal proximal tubule. Gal-7 is also present on most cilia of multiciliated cells in human airway epithelia primary cultures. Interestingly, exogenous glutathione S-transferase (GST)-Gal-7 bound the MDCK apical plasma membrane as well as the cilium, while the lectin Ulex europeaus agglutinin, with glycan preferences similar to Gal-7, bound the basolateral plasma membrane as well as the cilium. In pull-down assays, β1-integrin isolated from either the basolateral or apical/cilia membranes of MDCK cells was similarly bound by GST-Gal-7. Selective localization of Gal-7 to cilia despite the presence of binding sites on all cell surfaces suggests that intracellular Gal-7 is specifically delivered to cilia rather than simply binding to surface glycoconjugates after generalized secretion. Moreover, depletion of Gal-7 using tetracycline-induced short-hairpin RNA in mpkCCD(c14) cells significantly reduced cilia length and slowed wound healing in a scratch assay. We conclude that Gal-7 is selectively targeted to cilia and plays a key role in surface stabilization of glycoconjugates responsible for integrating cilia function with epithelial repair.

Research paper thumbnail of Rab11a-positive compartments in proximal tubule cells sort fluid-phase and membrane cargo

AJP: Cell Physiology, 2014

The proximal tubule (PT) reabsorbs the majority of sodium, bicarbonate, and chloride ions, phosph... more The proximal tubule (PT) reabsorbs the majority of sodium, bicarbonate, and chloride ions, phosphate, glucose, water, and plasma proteins from the glomerular filtrate. Despite the critical importance of endocytosis for PT cell (PTC) function, the organization of the endocytic pathway in these cells remains poorly understood. We have used immunofluorescence and live-cell imaging to dissect the itinerary of apically internalized fluid and membrane cargo in polarized primary cultures of PTCs isolated from mouse kidney cortex. Cells from the S1 segment could be distinguished from those from more distal PT segments by their robust uptake of albumin and comparatively low expression of γ-glutamyltranspeptidase. Rab11a in these cells is localized to variously sized spherical compartments that resemble the apical vacuoles observed by electron microscopy analysis of PTCs in vivo. These Rab11a-positive structures are highly dynamic and receive membrane and fluid-phase cargo. In contrast, fluid-phase cargoes are largely excluded from Rab11a-positive compartments in immortalized kidney cell lines. The unusual morphology and sorting capacity of Rab11a compartments in primary PTCs may reflect a unique specialization of these cells to accommodate the functional demands of handling a high endocytic load.

Research paper thumbnail of Shear stress-dependent regulation of apical endocytosis in renal proximal tubule cells mediated by primary cilia

Proceedings of the National Academy of Sciences, 2014

The kidney has an extraordinary ability to maintain stable fractional solute and fluid reabsorpti... more The kidney has an extraordinary ability to maintain stable fractional solute and fluid reabsorption over a wide range of glomerular filtration rates (GFRs). Internalization of filtered low molecular weight proteins, vitamins, hormones, and other small molecules is mediated by the proximal tubule (PT) multiligand receptors megalin and cubilin. Changes in GFR and the accompanying fluid shear stress (FSS) modulate acute changes in PT ion transport thought to be mediated by microvillar bending. We found that FSS also affects apical endocytosis in PT cells. Exposure of immortalized PT cell lines to physiologically relevant levels of FSS led to dramatically increased internalization of the megalin-cubilin ligand albumin as well as the fluid phase marker dextran. FSS-stimulated apical endocytosis was initiated between 15 and 30 min postinduction of FSS, occurred via a clathrin- and dynamin-dependent pathway, and was rapidly reversed upon removing the FSS. Exposure to FSS also caused a rapid elevation in intracellular Ca(2+) [Ca(2+)]i, which was not observed in deciliated cells, upon treatment with BAPTA-AM, or upon inclusion of apyrase in the perfusion medium. Strikingly, deciliation, BAPTA-AM, and apyrase also blocked the flow-dependent increase in endocytosis. Moreover, addition of ATP bypassed the need for FSS in enhancing endocytic capacity. Our studies suggest that increased [Ca(2+)]i and purinergic signaling in response to FSS-dependent ciliary bending triggers a rapid and reversible increase in apical endocytosis that contributes to the efficient retrieval of filtered proteins in the PT.