Membrane traffic and turnover in TRP-ML1–deficient cells: a revised model for mucolipidosis type IV pathogenesis

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

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors

Pelvic organ prolapse and collagen-associated disorders

Contains fulltext : 109010.pdf (publisher's version ) (Open Access)INTRODUCTION AND HYPOTHESIS: Pelvic organ prolapse (POP) and other disorders, such as varicose veins and joint hypermobility, have been associated with changes in collagen strength and metabolism. We hypothesized that these various disorders were more prevalent in both POP patients and their family members. METHODS: In this study, the prevalence of various collagen-associated disorders, including POP, was compared between POP patients (n = 110) and control patients (n = 100) and their first and second degree family members. RESULTS: POP patients reported a higher prevalence of varicose veins, joint hypermobility and rectal prolapse and were more likely to have family members with POP as compared to the control group (p < 0.01). In contrast, the family members of the POP group did not report a higher prevalence of collagen-associated disorders compared to the family members of the control group (p = 0.82). CONCLUSIONS: POP and other collagen-associated disorders may have a common aetiology, originating at the molecular level of the collagens.1 maart 201

A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans

Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy

Activation of the Caenorhabditis elegans degenerin channel by shear stress requires the MEC-10 subunit

Mechanotransduction in Caenorhabditis elegans touch receptor neurons is mediated by an ion channel formed by MEC-4, MEC-10, and accessory proteins. To define the role of these subunits in the channel's response to mechanical force, we expressed degenerin channels comprising MEC-4 and MEC-10 in Xenopus oocytes and examined their response to laminar shear stress (LSS). Shear stress evoked a rapid increase in whole cell currents in oocytes expressing degenerin channels as well as channels with a MEC-4 degenerin mutation (MEC-4d), suggesting that C. elegans degenerin channels are sensitive to LSS. MEC-10 is required for a robust LSS response as the response was largely blunted in oocytes expressing homomeric MEC-4 or MEC-4d channels. We examined a series of MEC-10/MEC-4 chimeras to identify specific domains (amino terminus, first transmembrane domain, and extracellular domain) and sites (residues 130–132 and 134–137) within MEC-10 that are required for a robust response to shear stress. In addition, the LSS response was largely abolished by MEC-10 mutations encoded by a touch-insensitive mec-10 allele, providing a correlation between the channel's responses to two different mechanical forces. Our findings suggest that MEC-10 has an important role in the channel's response to mechanical forces

Control or TRP-ML1 siRNA–treated (5 d) HeLa cells were loaded with 3 mg/ml of FITC- and TMR-conjugated dextrans for 12 h

Lysosomal pH was determined by calculating the ratio of TMR/FITC fluorescence. Images were acquired as described in Materials and methods. Ratiometric data were converted to absolute values of pH using TMR/FITC ratios determined from permeabilized cells equilibrated with calibration solutions. Data from 20 random fields of cells were quantified, and the pH determined is presented as mean pH ± SEM. Similar results were obtained in four independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Membrane traffic and turnover in TRP-ML1–deficient cells: a revised model for mucolipidosis type IV pathogenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1477-1490.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413042.</p><p></p

(A) HeLa cells were transfected with either nonsilencing control or TRP-ML1–specific siRNA oligonucleotides

Cells were harvested for Western blot analysis after 24 h. Equal amounts of total protein were loaded for SDS-PAGE, as determined by protein assay. Samples were transferred to nitrocellulose and immunoblotted to detect endogenous levels of TRP-ML1 using an antibody directed against the first extracellular loop of the protein. The arrowhead denotes the migration of the cleaved form of TRP-ML1. (B) HeLa cells were transiently transfected with cDNA encoding HA epitope–tagged TRP-ML1 24 h after initial transfection with either control or TRP-ML1–specific siRNA duplexes. After an additional 24-h incubation, cells were solubilized and equal amounts of total protein were immunoprecipitated using anti-HA antibodies. After SDS-PAGE, proteins were transferred to nitrocellulose and probed using horseradish peroxidase–conjugated anti-HA antibody. 10% of the cell lysate was saved before immunoprecipitation and immunoblotted using antitubulin antibody as an additional loading control (bottom). (C) HeLa cells were transfected with either nonsilencing control or TRP-ML1–specific siRNA oligonucleotides and were harvested for Western blot analysis 1, 3, or 5 d after transfection (top), or were retransfected with siRNA duplexes after 2 d and were harvested for Western analysis at 3 or 5 d (bottom). Samples were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted to detect endogenous TRP-ML1. The migration of molecular mass markers (in kD) is noted on the left of the blots.<p><b>Copyright information:</b></p><p>Taken from "Membrane traffic and turnover in TRP-ML1–deficient cells: a revised model for mucolipidosis type IV pathogenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1477-1490.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413042.</p><p></p

Control or TRP-ML1 siRNA–treated (5 d) HeLa cells (A) or fibroblasts (B) were preloaded for 12 h with Alexa Fluor 647–conjugated dextran

Cells were incubated with DiI-LDL on ice for 60 min and were subsequently chased in prewarmed media at 37°C for an additional 30 or 120 min. At the indicated time points, cells were fixed and processed for immunofluorescence. Delivery of DiI-LDL to lysosomes in cells was measured by quantifying the percent overlap between DiI-LDL and the preloaded Alexa Fluor 647–dextran. Graphical representations of the quantifications are shown (A and B, right) and are expressed as the percent overlap ± SEM for cells under each condition ( = 20). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Membrane traffic and turnover in TRP-ML1–deficient cells: a revised model for mucolipidosis type IV pathogenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1477-1490.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413042.</p><p></p

(A and B) Electron micrographs of HeLa cells after either 1 or 5 d of TRP-ML1–specific or control siRNA oligonucleotide transfection

Bars: (A) 2 μm; (B, left) 200 nm; (B, right) 500 nm. (C) Quantitation of the effect of TRP-ML1 knockdown on the formation of storage inclusions. The number of inclusions was calculated using the automated particle counting function of ImageJ. Data are expressed as the number of inclusions per cell slice ± SEM (D) Western blot analysis of siRNA-treated cells expressing siRNA-resistant HA-ML1. HeLa cells were transfected with either control or TRP-ML1–specific siRNA, as indicated. 24 h after siRNA treatment, cells were cotransfected with plasmids encoding GFP and either HA-ML1 (non-siRNA resistant) or HA-ML1 (siRNA resistant). The next day, cells were sorted by FACS analysis to identify GFP-positive (transfected) cells and were subsequently retransfected with the appropriate siRNA and returned to culture for 48 h. Cells were harvested, and 30 μg of total protein was loaded for SDS-PAGE. Samples were transferred to nitrocellulose and immunoblotted to detect endogenous levels of TRP-ML1 (top), HA-ML1 or HA-ML1 (middle), or β-actin (bottom) as a loading control. (E) Quantitation of the effect of HA-ML1 expression on formation of storage inclusions in TRP-ML1 siRNA–treated cells. The number of inclusions was calculated as described in C. The migration of molecular mass markers (in kD) is noted on the left of the blots.<p><b>Copyright information:</b></p><p>Taken from "Membrane traffic and turnover in TRP-ML1–deficient cells: a revised model for mucolipidosis type IV pathogenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1477-1490.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413042.</p><p></p