Transitions of protein traffic from cardiac ER to junctional SR

Abstract

The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca2+ 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 12-24 h as bright fluorescent puncta close to the nuclear surface, decreasing in intensity with increasing radial distance. With increasing time (24-48 h), 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 CSQD-sRed interactions occurred when a form of TRDdog was used in which CSQ2-binding sites are removed (delTRD). 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.

Phospholamban regulates nuclear Ca2+ stores and inositol 1,4,5-trisphosphate mediated nuclear Ca2+ cycling in cardiomyocytes

“…The use of animals in the study was approved by the IACUC of Indiana University School of Medicine and the Methodist Research Institute, Indianapolis, Indiana and conformed to the NIH Guide for the care and use of laboratory animals. CM isolation from adult C57BL/6 mice and PLB-KO mice (2 to 6 month old) using protocols modified from our previously reported [26, 28]. In brief, CMs were isolated with 15μg/ml liberase (Roche) stored in normal Tyrode’s solution containing (in mM/L): 138 NaCl, 5.33 KCl, 0.33 NaH 2 PO 4 , 1.18 MgCl 2 , 10 HEPES, 10 taurine, and 10 glucose, pH 7.4 (NaOH).…”

Heart failure leads to altered β2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain

“…which is reminiscent of overexpressed jSR proteins retained in the biosynthetic pathway29 . Since optical microscopy techniques with its resolution close to half of the wavelength of light do not allow to properly resolve T-tubular membrane from jSR which are just 10-30 nm apart, we cannot conclude on the localization of the biosensor pool or even a part of it in the T-tubular membrane.…”

The relationship between form and function throughout the history of excitation–contraction coupling

“…Three regions of Trsk 95 needed for targeting and retention at couplons have been identified ( Rossi et al, 2014 ). Trafficking of junctin, triadin, and CASQ follows a linked path from rough ER to jSR in cardiac muscle ( Sleiman et al, 2015 ), where also it was shown that CASQ joins peripheral couplings before RYRs ( Tijskens et al, 2003 ).…”

“…Transformation of the generic ER of differentiating muscle cells into the highly dedicated SR and particularly the sorting of components into two distinct but continuous regions, the free SR and jSR, is quite complex, involving membrane rearrangements and independent as well as concerted migrations of specific components ( Cusimano et al, 2009 ). The question has already been approached ( Barone et al, 2015 ; Sleiman et al, 2015 ), and it is worth pursuing in view of the known interactions of many components.…”