Merck

Transitions of protein traffic from cardiac ER to junctional SR.

Journal of molecular and cellular cardiology (2015-02-03)
Naama H Sleiman, Timothy P McFarland, Larry R Jones, Steven E Cala
ABSTRACT

The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca(2+) release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies. Newly synthesized JCTdog and TRDdog appeared by 12-24h as bright fluorescent puncta close to the nuclear surface, decreasing in intensity with increasing radial distance. With increasing time (24-48h), fluorescent puncta appeared at further radial distances from the nuclear surface, eventually populating jSR similar to steady-state patterns. CSQ2-DsRed, a form of CSQ that polymerizes ectopically in rough ER, prevented anterograde traffic of newly made TRDdog and JCTdog, demonstrating common pathways of intracellular trafficking as well as in situ binding to CSQ2 in juxtanuclear rough ER. Reversal of CSQ-DsRed interactions occurred when a form of TRDdog was used in which CSQ2-binding sites are removed ((del)TRD). With increasing levels of expression, CSQ2-DsRed revealed a novel smooth ER network that surrounds nuclei and connects the nuclear axis. TRDdog was retained in smooth ER by binding to CSQ2-DsRed, but escaped to populate jSR puncta. TRDdog and (del)TRD were therefore able to elucidate areas of ER-SR transition. High levels of CSQ2-DsRed in the ER led to loss of jSR puncta labeling, suggesting a plasticity of ER-SR transition sites. We propose a model of ER and SR protein traffic along microtubules, with prominent transverse/radial ER trafficking of JCT and TRD along Z-lines to populate jSR, and an abundant longitudinal/axial smooth ER between and encircling myonuclei, from which jSR proteins traffic.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-谷氨酰胺, meets USP testing specifications, suitable for cell culture, 99.0-101.0%, from non-animal source
Sigma-Aldrich
丙烯酰胺, suitable for electrophoresis, ≥99%
Sigma-Aldrich
牛磺酸, ≥99%
Sigma-Aldrich
L-谷氨酰胺, ReagentPlus®, ≥99% (HPLC)
SAFC
L-谷氨酰胺
Sigma-Aldrich
丙烯酰胺, for molecular biology, ≥99% (HPLC)
Sigma-Aldrich
丙烯酰胺, suitable for electrophoresis, ≥99% (HPLC), powder
Sigma-Aldrich
一水肌酸, anhydrous
Sigma-Aldrich
抗-α微管蛋白抗体,小鼠单克隆抗体, clone B-5-1-2, purified from hybridoma cell culture
Sigma-Aldrich
丙烯酰胺 溶液, 40%, suitable for electrophoresis, sterile-filtered
Sigma-Aldrich
牛磺酸, suitable for cell culture, meets USP testing specifications
Supelco
丙烯酰胺, analytical standard
Sigma-Aldrich
丙烯酰胺, purum, ≥98.0% (GC)
Sigma-Aldrich
L-谷氨酰胺, BioUltra, ≥99.5% (NT)
Supelco
牛磺酸, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
L-谷氨酰胺, γ-irradiated, BioXtra, suitable for cell culture
Sigma-Aldrich
牛磺酸, BioUltra, ≥99.5% (T)
Supelco
丙烯酰胺, certified reference material, TraceCERT®
Supelco
L-谷氨酰胺, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
丙烯酰胺 溶液, 40% in H2O, for molecular biology
Sigma-Aldrich
丙烯酰胺, for Northern and Southern blotting, powder blend
SAFC
牛磺酸
Sigma-Aldrich
牛磺酸, ≥98%, FG
USP
牛磺酸, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
L-谷氨酰胺
Supelco
L-谷氨酰胺, certified reference material, TraceCERT®
Sigma-Aldrich
L-谷氨酰胺, Vetec, reagent grade, ≥99%