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1.  Evidence for core 2 to core 1 O-glycan remodeling during the recycling of MUC1 
Glycobiology  2013;23(8):935-945.
The apical transmembrane glycoprotein MUC1 is endocytosed to recycle through the trans-Golgi network (TGN) or Golgi complex to the plasma membrane. We followed the hypothesis that not only the known follow-up sialylation of MUC1 in the TGN is associated with this process, but also a remodeling of O-glycan core structures, which would explain the previously described differential core 2- vs core 1-based O-glycosylation of secreted, single Golgi passage and recycling membrane MUC1 isoforms (Engelmann K, Kinlough CL, Müller S, Razawi H, Baldus SE, Hughey RP, Hanisch F-G. 2005. Glycobiology. 15:1111–1124). Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles. To address this novel observation, we used recombinant epitope-tagged MUC1 (MUC1-M) and mutant forms with abrogated clathrin-mediated endocytosis (MUC1-M-Y20,60N) or blocked recycling (palmitoylation-defective MUC1-M-CQC/AQA). We show that the CQC/AQA mutant transits the TGN at significantly lower levels, concomitant with a strongly reduced shedding from the plasma membrane and its accumulation in endosomal compartments. Intriguingly, the O-glycosylation of the shed MUC1 ectodomain subunit changes from preponderant sialylated core 1 (MUC1-M) to core 2 glycans on the non-recycling CQC/AQA mutant. The O-glycoprofile of the non-recycling CQC/AQA mutant resembles the core 2 glycoprofile on a secretory MUC1 probe that transits the Golgi complex only once. In contrast, the MUC1-M-Y20,60N mutant recycles via flotillin-dependent pathways and shows the wild-type phenotype with dominant core 1 expression. Differential radiolabeling of protein with [35S]Met/Cys or glycans with [3H]GlcNH2 in pulse-chase experiments of surface biotinylated MUC1 revealed a significantly shorter half-life of [3H]MUC1 when compared with [35S]MUC1, whereas the same ratio for the CQC/AQA mutant was close to one. This finding further supports the novel possibility of a recycling-associated O-glycan processing from Gal1-4GlcNAc1-6(Gal1-3)GalNAc (core 2) to Gal1-3GalNAc (core 1).
doi:10.1093/glycob/cwt030
PMCID: PMC3695752  PMID: 23640779
glycan processing; membrane trafficking; MUC1; O-glycosylation; recycling
2.  Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization 
Molecular Biology of the Cell  2013;24(12):1996-2007.
The mechanisms that regulate the apical sorting of proteins are unclear, but clustering may play an important role. A role for dimerization and higher-order oligomerization in the biosynthetic transport of the model O-glycosylated protein p75 has been identified. This study also suggests that the O-glycans of p75 have a structural role in apical sorting.
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.
doi:10.1091/mbc.E13-02-0078
PMCID: PMC3681702  PMID: 23637462
3.  OCRL1 Modulates Cilia Length in Renal Epithelial Cells 
Traffic (Copenhagen, Denmark)  2012;13(9):1295-1305.
Lowe syndrome is an X-linked disorder characterized by cataracts at birth, mental retardation and progressive renal malfunction that results from loss of function of the OCRL1 (oculocerebrorenal syndrome of Lowe) protein. OCRL1 is a lipid phosphatase that converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The renal pathogenesis of Lowe syndrome patients has been suggested to result from alterations in membrane trafficking, but this cannot fully explain the disease progression. We found that knockdown of OCRL1 in zebrafish caused developmental defects consistent with disruption of ciliary function, including body axis curvature, pericardial edema, hydrocephaly and impaired renal clearance. In addition, cilia in the proximal tubule of the zebrafish pronephric kidney were longer in ocrl morphant embryos. We also found that knockdown of OCRL1 in polarized renal epithelial cells caused elongation of the primary cilium and disrupted formation of cysts in three-dimensional cultures. Calcium release in response to ATP was blunted in OCRL1 knockdown cells, suggesting changes in signaling that could lead to altered cell function. Our results suggest a new role for OCRL1 in renal epithelial cell function that could contribute to the pathogenesis of Lowe syndrome.
doi:10.1111/j.1600-0854.2012.01387.x
PMCID: PMC3652247  PMID: 22680056
kidney; Lowe syndrome; MDCK; oculocerebrorenal syndrome of Lowe; phosphatidylinositol 4,5-bisphosphate; zebrafish
4.  PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells 
PLoS ONE  2013;8(1):e53790.
Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] 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)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ 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 PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ 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)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis.
doi:10.1371/journal.pone.0053790
PMCID: PMC3547069  PMID: 23342003
5.  Mitotic slippage in non-cancer cells induced by a microtubule disruptor, disorazole C1 
BMC Chemical Biology  2010;10:1.
Background
Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C1 was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C1 in both non-cancer and cancer cells and compared our results to vinblastine and taxol.
Results
In non-cancer cells, disorazole C1 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 C1. On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C1.
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 C1. In contrast, cancer cells induce the apoptotic pathway after disorazole C1 treatment, indicating a possibly significant therapeutic window for this compound.
doi:10.1186/1472-6769-10-1
PMCID: PMC2834648  PMID: 20181182
6.  Autophagy, mitochondria and cell death in lysosomal storage diseases 
Autophagy  2007;3(3):259-262.
Lysosomal storage diseases (LSDs) are debilitating genetic conditions that frequently manifest as neurodegenerative disorders. They severely affect eye, motor and cognitive functions and, in most cases, abbreviate the lifespan. Postmitotic cells such as neurons and mononuclear phagocytes rich in lysosomes are most often affected by the accumulation of undegraded material. Cell death is well documented in parts of the brain and in other cells of LSD patients and animal models, although little is known about mechanisms by which death pathways are activated in these diseases, and not all cells exhibiting increased storage material are affected by cell death. Lysosomes are essential for maturation and completion of autophagy-initiated protein and organelle degradation. Moreover, accumulation of effete mitochondria has been documented in postmitotic cells whose lysosomal function is suppressed or in aging cells with lipofuscin accumulation. Based upon observations in the literature and our own data showing similar mitochondrial abnormalities in several LSDs, we propose a new model of cell death in LSDs. We suggest that the lysosomal deficiencies in LSDs inhibit autophagic maturation, leading to a condition of autophagic stress. The resulting accumulation of dysfunctional mitochondria showing impaired Ca2+ buffering increases the vulnerability of the cells to pro-apoptotic signals.
PMCID: PMC2777544  PMID: 17329960
autophagy; mitochondrial homeostasis; lysosome; calcium; caspase; programmed cell death; mucolipidosis; neurodegeneration; Niemann-Pick; neuronal ceroid lipofuscinosis
7.  Membrane traffic and turnover in TRP-ML1–deficient cells: a revised model for mucolipidosis type IV pathogenesis 
The Journal of Experimental Medicine  2008;205(6):1477-1490.
The lysosomal storage disorder mucolipidosis type IV (MLIV) is caused by mutations in the transient receptor potential–mucolipin-1 (TRP-ML1) ion channel. The “biogenesis” model for MLIV pathogenesis suggests that TRP-ML1 modulates postendocytic delivery to lysosomes by regulating interactions between late endosomes and lysosomes. This model is based on observed lipid trafficking delays in MLIV patient fibroblasts. Because membrane traffic aberrations may be secondary to lipid buildup in chronically TRP-ML1–deficient cells, we depleted TRP-ML1 in HeLa cells using small interfering RNA and examined the effects on cell morphology and postendocytic traffic. TRP-ML1 knockdown induced gradual accumulation of membranous inclusions and, thus, represents a good model in which to examine the direct effects of acute TRP-ML1 deficiency on membrane traffic. Ratiometric imaging revealed decreased lysosomal pH in TRP-ML1–deficient cells, suggesting a disruption in lysosomal function. Nevertheless, we found no effect of TRP-ML1 knockdown on the kinetics of protein or lipid delivery to lysosomes. In contrast, by comparing degradation kinetics of low density lipoprotein constituents, we confirmed a selective defect in cholesterol but not apolipoprotein B hydrolysis in MLIV fibroblasts. We hypothesize that the effects of TRP-ML1 loss on hydrolytic activity have a cumulative effect on lysosome function, resulting in a lag between TRP-ML1 loss and full manifestation of MLIV.
doi:10.1084/jem.20072194
PMCID: PMC2413042  PMID: 18504305

Results 1-7 (7)