1,721,015 research outputs found
Vesicle budding from endoplasmic reticulum is involved in calsequestrin routing to sarcoplasmic reticulum of skeletal muscles
Full text linkCS (calsequestrin) is an acidic glycoprotein of the SR (sarcoplasmic reticulum) lumen and plays a crucial role in the storage of Ca2+ and in excitation-contraction coupling of skeletal muscles. CS is synthesized in the ER (endoplasmic reticulum) and is targeted to the TC (terminal cisternae) of SR via mechanisms still largely unknown, but probably involving vesicle transport through the Golgi complex. In the present study, two mutant forms of Sari and ARF1 (ADP-ribosylation factor 1) were used to disrupt cargo exit from ER-exit sites and intra-Golgi trafficking in skeletal-muscle fibres respectively. Co-expression of Sar1-H79G (His79 → Gly) and recombinant, epitope-tagged CS, CSHA1 (where HA1 stands for nine-amino-acid epitope of the viral haemagglutinin 1), barred segregation of CSHA1 to TC. On the other hand, expression of ARF1-N126I altered the subcellular localization of GM130, a cis-medial Golgi protein in skeletal-muscle fibres and myotubes, without interfering with CSHA1 targeti..
Retention of skeletal muscle calsequestrin in the terminal cisternae upon deletion of the 2nd or 3rd domain.
No full textMultiple/heterogeneous Ca2+ stores in cerebellum Purkinje neurons.
No full text1. The rapid and transient redistribution of Ca2+ from intracellular membrane-bound compartments (stores) is a key event of cell activation. 2. The cytological nature and molecular composition of such Ca2+ stores have been the object of intense investigation in recent years. 3. Here we review: (a) the current knowledge on intracellular Ca2+ stores of Purkinje neurons at the functional, biochemical, molecular, morphological and ultrastructural level; and discuss: (b) the relationship between Ca2+ stores and the endoplasmic reticulum, and (c) the occurrence of multiple/heterogeneous Ca2+ stores
Phosphorylation of the triadin cytoplasmic domain by CaM protein kinase in rabbit fast-twitch muscle sarcoplasmic reticulum
No full textSkeletal muscle triadin is a sarcoplasmic reticulum (SR) membrane protein that had been shown to interact structurally and functionally at the cytoplasmic domain (amino acid residues 1-47) with the ryanodine receptor (RyR1), and to undergo phosphorylation by endogenous calmodulin protein kinase (CaM K II) in isolated terminal cisternae from rabbit fast-twitch muscle. Here we show that triadin cytoplasmic domain expressed as glutathione-S-transferase fusion protein, is a substrate of the protein kinase. This finding is corroborated by identification of a specific consensus sequence in the deduced amino sequence between residue 34 and 37 of triadin. Confirming the regulatory features of CaM K II, we show the phosphorylation of triadin cytoplasmic segment by the kinase when converted to the autonomous form. We propose that triadin modulates RyR1 in a phosphorylation-dependent manner
Targeting of calsequestrin to the sarcoplasmic reticulum of skeletal muscle upon deletion of its glycosylation site
Full text linkThe glycoprotein calsequestrin (CS) is segregated to the junctional sarcoplasmic reticulum (jSR) and is responsible for intraluminal Ca(2+) binding. A chimeric CS-hemoagglutinin 1 (HA1), obtained by adding the nine amino acid viral epitope hemoagglutinin to the carboxy terminal of CS and shown to be correctly segregated to skeletal muscle jSR [A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe (1997). Chimeric calsequestrin and its targeting to the junctional sarcoplasmic reticulum of skeletal muscle. Am. J. Physiol. 272, C1420-C1428] lends itself as a molecular tool to investigate the targeting domains of CS. A putative targeting mechanism of CS to jSR implies glycosylation-dependent steps in the endoplasmic reticulum (ER) and Golgi complex. To test this hypothesis, CS-HA1DeltaGly, a mutant in which the unique N-glycosylation site Asn316 was changed to Ile, was engineered by site-directed mutagenesis. The mutant cDNA was transiently transfected in either HeLa cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of CS-HA1DeltaGly was studied by double-labeling epifluorescence by means of antibodies against either CS, HA1, or the ryanodine receptor calcium release channel. CS-HA1DeltaGly was expressed and retained to ER and ER/sarcoplasmic reticulum of HeLa cells and myotubes, respectively, and expressed, sorted, and correctly segregated to jSR of regenerating soleus muscle fibers. Thus, the targeting mechanism of CS in vivo appears not to be affected by glycosylation-that is, the sorting, docking, and segregation of CS are independent of cotranslational and posttranslational glycosylation or glycosylation
Targeting of calsequestrin to the sarcoplasmic reticulum of skeletal muscle following deletion of its carboxy-terminal acidic tail
No full textCalsequestrin (CS) is the Ca(2+) binding protein of the junctional sarcoplasmic reticulum (jSR) lumen. Recently, a chimeric CS-HA1, obtained by adding the nine-amino-acid viral epitope hemagglutinin (HA1) to the COOH terminus of CS, was shown to be correctly segregated to the sarcoplasmic reticulum [A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe. Am. J. Physiol. 272 (Cell Physiol. 41): C1420-C1428, 1997]. A putative targeting mechanism of CS to jSR implies electrostatic interactions between negative charges on CS and positive charges on intraluminal domains of jSR integral proteins, such as triadin and junctin. To test this hypothesis, 2 deletion mutants of chimeric CS were engineered: CS-HA1DeltaGlu-Asp, in which the 14 acidic residues [-Glu-(Asp)(5)-Glu-(Asp)(7)-] of the COOH-terminal tail were removed, and CS-HA1Delta49(COOH), in which the last, mostly acidic, 49 residues of the COOH terminus were removed. Both mutant cDNAs were transiently transfected in HeLa cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of CS-HA1 mutants were studied by epifluorescence microscopy with use of antibodies against CS or HA1. CS-HA1 mutants were shown to be expressed, sorted, and correctly segregated to jSR. Thus short or long deletions of the COOH-terminal acidic tail do not influence the targeting mechanism of C
INTRACELLULAR CA2+ STORES OF RAT CEREBELLUM - HETEROGENEITY WITHIN AND DISTINCTION FROM ENDOPLASMIC-RETICULUM
No full textRat cerebellum microsomes were subfractionated on isopycnic linear sucrose (20-42%)-density gradients. The distribution of endoplasmic reticulum (ER) markers (RNA, signal-sequence receptor alpha, calnexin, calreticulin, the immunoglobulin-binding protein Bip) and markers of intracellular rapidly exchanging Ca2+ stores [Ca2+ channels sensitive to either Ins(1,4,5)P3 or ryanodine) was investigated biochemically and immunologically. The comparison indicates that: (a) vesicles bearing the InsP3 receptor were separated from those bearing the ryanodine receptor; (b) ER markers, i.e. Bip, calnexin, signal-sequence receptor alpha, RNA, did not sediment as either InsP3 or ryanodine receptors did; (c) calreticulin, an intralumenal low-affinity high-capacity Ca(2+)-binding protein, had a widespread distribution, similar to that of Bip and calnexin, and was present in Purkinje, granule, Golgi and stellate neurons, as indicated by immunofluorescent labelling of cerebellum cortex cryosections. The present results show that the ER is not a homogeneous entity, and that Ca2+ stores are heterogeneous insofar as InsP3 receptors and ryanodine receptors are segregated, either to discrete intracellular organelles or to specialized ER subcompartment
Site-directed mutagenesis and deletion of three phosphorylation sites of calsequestrin of skeletal muscle sarcoplasmic reticulum: Effects on intracellular targeting
No full textCalsequestrin (CS) is segregated to the junctional sarcoplasmic reticulum (jSR) of skeletal muscle fibers and is responsible for intraluminal Ca(2+) binding. A chimeric CS-HA1, obtained by adding the nine-amino-acid viral epitope hemagglutinin (HA1) to the carboxy-terminal of CS and shown to be correctly segregated to skeletal muscle jSR in vivo (A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe, 1997, Am. J. Physiol. 272, C1420-C1428), is mutagenized in order to identify domains of CS involved in targeting. Since a putative targeting mechanism of CS implies phosphorylation-dependent steps in the endoplasmic reticulum (ER) and/or Golgi complex, five CS-HA1 mutants disrupting the three phosphorylation sites of CS (Thr(189), Thr(229), and Thr(353)) were engineered by either site-directed mutagenesis or deletion: CS-HA1DeltaP1 (Thr(189) --> Ile); CS-HA1DeltaP2 (Thr(229) --> Asn); CS-HA1DeltaP1,2; in which Thr(189) and Thr(229) were changed to Ile and Asn, respectively; and CS-HA1Delta14(COOH) and CS-HA1Delta49 (COOH), in which 14 residues (Glu(354)-Asp(367)) and 49 residues (Asp(319)-Asp(367)), respectively, were deleted at the carboxy-terminal. Mutant cDNAs were transiently transfected in either HeLa cells, cultured myoblasts of rat skeletal muscle, or regenerating soleus muscle fibers of adult rats. Each CS-HA1 mutant was identified by Western blot as a single polypeptide of the predicted molecular weight. The intracellular localization of CS-HA1 mutants was studied by immunofluorescence using specific antibodies against either CS or HA1. CS-HA1 mutants colocalized with ER markers, e.g., calreticulin, and partially overlapped with Golgi complex markers, e.g., alpha-mannosidase II, in HeLa cells and myotubes. CS-HA1 mutants were expressed and retained in ER and ER/SR of HeLa cells and myotubes, respectively, and correctly segregated to jSR of regenerating soleus muscle fibers. Thus, the targeting mechanism of CS in vivo is not affected by phosphorylation(s); i.e., sorting and segregation of CS appear to be independent of posttranslational phosphorylation(s)
Electrotransfer in differentiated myotubes: a novel, efficient procedure for functional gene transfer
Full text linkDevelopment of reliable techniques for experimental manipulation of gene expression in multinucleated skeletal muscle fibers is critical for understanding molecular mechanisms involved in both physiology and pathophysiology. At present, viral vectors represent the only method to obtain efficient gene transfer in terminally differentiated myotubes. Here we present an in vitro procedure that relies on the application of a pulsed electric field for transferring naked DNA into differentiated myotubes seeded on coverslips. Compared with standard transfection methods, electroporation was at least 1000 times more efficient, as judged by quantitative determination of luciferase content. Percentage of transfected myotubes averaged around 45%. Moreover, we were successful in transfecting a dominant-negative ADP ribosylation factor 1 (ARF1) mutant, i.e., ARF1N126I, in myotubes, thus interfering with endoplasmic reticulum-Golgi traffic, as indicated by alterations of subcellular distribution of GM130, a cis/medial-Golgi marker. Co-transfection experiments with beta-galactosidase also showed that the ARF1 mutant appeared to inhibit myoblast fusion and could not be used before myotube formation. The present work validates the use of electroporation as a highly efficient approach for gene transfer in fully differentiated myotubes
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