A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)-deficient cells are small and display a blunted response to nutrient and growth factor availability, we ...have hypothesized that mutant cell atrophy may be triggered by a change in global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the mammalian target of rapamycin (mTOR)/S6K pathway, here we evaluate the control of translational target phosphorylation and protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The phosphorylation of ribosomal protein S6 (rpS6), eukaryotic initiation factor-4B (eIF4B), and eukaryotic elongation factor-2 (eEF2) is predominantly regulated by mTOR in muscle cells. Conversely, in liver, the MAPK and phosphatidylinositol 3-kinase pathways also play an important role, suggesting a tissue-specific control. S6K deletion in muscle mimics the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2 phosphorylation. To gain insight on the functional consequences of these modifications, methionine incorporation and polysomal distribution were assessed in muscle cells. Rates and rapamycin sensitivity of global translation initiation are not altered in S6K-deficient muscle cells. In addition, two major pathways of protein degradation, autophagy and expression of the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our results do not support a role for global translational control in the growth defect due to S6K deletion, suggesting specific modes of growth control and translational target regulation downstream of mTOR.
The low‐density lipoprotein (LDL) receptor‐related protein (LRP) is a multiligand endocytic receptor and a member of the LDL receptor family. Here we show that sorting nexin 17 (Snx17) is part of the ...cellular sorting machinery that regulates cell surface levels of LRP by promoting its recycling. While the phox (PX) domain of Snx17 interacts with phosphatidylinositol‐3‐phosphate for membrane association, the FERM domain and the carboxyl‐terminal region participate in LRP binding. Immunoelectron microscopy shows that the membrane‐bound fraction of Snx17 is localized to the limiting membrane and recycling tubules of early endosomes. The NPxY motif, proximal to the plasma membrane in the LRP cytoplasmic tail, is identified as the Snx17‐binding motif. Functional mutation of this motif did not interfere with LRP endocytosis, but decreased LRP recycling from endosomes, resulting in increased lysosomal degradation. Similar effects are found after knockdown of endogenous Snx17 expression by short interfering RNA. We conclude that Snx17 binds to a motif in the LRP tail distinct from the endocytosis signals and promotes LRP sorting to the recycling pathway in the early endosomes.
A cisternal progression mode of intra-Golgi transport requires that Golgi resident proteins recycle by peri-Golgi vesicles, whereas the alternative model of vesicular transport predicts anterograde ...cargo proteins to be present in such vesicles. We have used quantitative immuno-EM on NRK cells to distinguish peri-Golgi vesicles from other vesicles in the Golgi region. We found significant levels of the Golgi resident enzyme mannosidase II and the transport machinery proteins giantin, KDEL-receptor, and rBet1 in coatomer protein I-coated cisternal rims and peri-Golgi vesicles. By contrast, when cells expressed vesicular stomatitis virus protein G this anterograde marker was largely absent from the peri-Golgi vesicles. These data suggest a role of peri-Golgi vesicles in recycling of Golgi residents, rather than an important role in anterograde transport.
The glycosylphosphatidylinositol (GPI)‐anchored proteoglycan Cripto binds Nodal and its type I receptor Alk4 to activate Smad2,3 transcription factors, but a role during Nodal precursor processing ...has not been described. We show that Cripto also binds the proprotein convertases Furin and PACE4 and localizes Nodal processing at the cell surface. When coexpressed as in early embryonic cells, Cripto and uncleaved Nodal already associated during secretion, and a Cripto‐interacting region in the Nodal propeptide potentiated the effect of proteolytic maturation on Nodal signalling. Disruption of the trans‐Golgi network (TGN) by brefeldin A blocked secretion, but export of Cripto and Nodal to the cell surface was not inhibited, indicating that Nodal is exposed to extracellular convertases before entering the TGN/endosomal system. Density fractionation and antibody uptake experiments showed that Cripto guides the Nodal precursor in detergent‐resistant membranes to endocytic microdomains marked by GFP–Flotillin. We conclude that Nodal processing and endocytosis are coupled in signal‐receiving cells.
Nemaline myopathies are heterogeneous congenital muscle disorders causing skeletal muscle weakness and, in some cases, death soon after birth. Mutations in nebulin, encoding a large sarcomeric ...protein required for thin filament function, are responsible for approximately 50% of nemaline myopathy cases. Despite the severity of the disease there is no effective treatment for nemaline myopathy with limited research to develop potential therapies. Several supplements, including L-tyrosine, have been suggested to be beneficial and consequently self-administered by nemaline myopathy patients without any knowledge of their efficacy. We have characterized a zebrafish model for nemaline myopathy caused by a mutation in nebulin. These fish form electron-dense nemaline bodies and display reduced muscle function akin to the phenotypes observed in nemaline myopathy patients. We have utilized our zebrafish model to test and evaluate four treatments currently self-administered by nemaline myopathy patients to determine their ability to increase skeletal muscle function. Analysis of muscle pathology and locomotion following treatment with L-tyrosine, L-carnitine, taurine, or creatine revealed no significant improvement in skeletal muscle function emphasizing the urgency to develop effective therapies for nemaline myopathy.
Many viruses modify cellular processes for their own benefit. The enterovirus 3A protein inhibits endoplasmic reticulum (ER)-to-Golgi transport, a function previously suggested to be important for ...viral suppression of immune responses. Here, we show that a virus carrying a 3A protein defective in inhibiting ER-to-Golgi transport is indeed less virulent in mice, and we unravel the mechanism by which 3A inhibits this trafficking step. Evidence is provided that 3A inhibits the activation of the GTPase ADP-ribosylation factor 1 (Arf1), which regulates the recruitment of the COP-I coat complex to membranes. 3A specifically inhibits the function of GBF1, a guanine nucleotide exchange factor for Arf1, by interacting with its N terminus. By specifically interfering with GBF1-mediated Arf1 activation, 3A may prove a valuable tool in dissecting the early steps of the secretory pathway.
Mutations in ATP8B1 cause familial intrahepatic cholestasis type 1, a spectrum of disorders characterized by intrahepatic cholestasis, reduced growth, deafness, and diarrhea. ATP8B1 belongs to the P4 ...P‐type adenosine triphosphatase (ATPase) family of putative aminophospholipid translocases, and loss of aminophospholipid asymmetry in the canalicular membranes of ATP8B1‐deficient liver cells has been proposed as the primary cause of impaired bile salt excretion. To explore the origin of the hepatic and extrahepatic symptoms associated with ATP8B1 deficiency, we investigated the impact of ATP8B1 depletion on the domain‐specific aminophospholipid translocase activities and polarized organization of polarized epithelial Caco‐2 cells. Caco‐2 cells were stably transfected with short hairpin RNA constructs to block ATP8B1 expression. Aminophospholipid translocase activity was assessed using spin‐labeled phospholipids. The polarized organization of these cells was determined by pulse‐chase analysis, cell‐fractionation, immunocytochemistry, and transmission electron microscopy. ATP8B1 was abundantly expressed in the apical membrane of Caco‐2 cells, and its expression was markedly induced during differentiation and polarization. Blocking ATP8B1 expression by RNA interference (RNAi) affected neither aminophospholipid transport nor the asymmetrical distribution of aminophospholipids across the apical bilayer. Nonetheless, ATP8B1‐depleted Caco‐2 cells displayed profound perturbations in apical membrane organization, including a disorganized apical actin cytoskeleton, a loss in microvilli, and a posttranscriptional defect in apical protein expression. Conclusion: Our findings point to a critical role of ATP8B1 in apical membrane organization that is unrelated to its presumed aminophospholipid translocase activity, yet potentially relevant for the development of cholestasis and the manifestation of extrahepatic features associated with ATP8B1 deficiency. (HEPATOLOGY 2010)
The RUN and FYVE domain proteins rabip4 and rabip4' are encoded by RUFY1 and differ in a 108 amino acid N-terminal extension in rabip4'. Their identical C terminus binds rab5 and rab4, but the ...function of rabip4s is incompletely understood. We here found that silencing RUFY1 gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4' yielded the adaptor protein complex AP-3, of which the hinge region in the β3 subunit bound directly to the FYVE domain of rabip4'. Rabip4' colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4' in regulating lysosome positioning through an interorganellar pathway.
Members of the Atg8 family of proteins are conjugated to autophagosomal membranes, where they have been proposed to drive autophagosome formation and selective sequestration of cargo. In mammals, the ...Atg8 family consists of six members divided into the LC3 and GABARAP subfamilies. To define Atg8 function, we used genome editing to generate knockouts of the LC3 and GABARAP subfamilies as well as all six Atg8 family members in HeLa cells. We show that Atg8s are dispensable for autophagosome formation and selective engulfment of mitochondria, but essential for autophagosome-lysosome fusion. We find that the GABARAP subfamily promotes PLEKHM1 recruitment and governs autophagosome-lysosome fusion, whereas the LC3 subfamily plays a less prominent role in these processes. Although neither GABARAPs nor LC3s are required for autophagosome biogenesis, loss of all Atg8s yields smaller autophagosomes and a slowed initial rate of autophagosome formation. Our results clarify the essential function of the Atg8 family and identify GABARAP subfamily members as primary contributors to PINK1/Parkin mitophagy and starvation autophagy.
The zebrafish is a powerful vertebrate system with great advantages for both forward and reverse genetic screens and as a model for human disease conditions. Light microscopy has been used ...extensively to study zebrafish development but less frequently have these studies been combined with ultrastructural information. Zebrafish embryos are ideal for electron microscopy (EM) with a single transverse section containing many different cell types and tissues. However, conventional methods of EM do not provide optimal preservation of all tissues and are usually incompatible with immunolabelling and visualisation of expressed fluorescently tagged proteins. Here we examine methods that overcome these problems. We summarise a range of methods, applicable to the ultrastructural analysis of zebrafish embryos, including methods for fast freezing and processing of zebrafish embryos. These methods preserve antigenicity, ultrastructure and GFP fluorescence even after embedding in resin. In addition, they are compatible with electron tomography. These methods provide a new set of research tools that provide an additional level of information, complementing current methods for study of this widely used model system.
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