Overexpression of calsequestrin in L6 myoblasts: formation of endoplasmic reticulum subdomains and their evolution into discrete vacuoles where aggregates of the protein are specifically accumulated.

Abstract

Monitoring Editor: Peter Walter Calsequestrin (CSQ), the major low-affinity Ca2+-binding glycoprotein of striated muscle fibers, is concentrated to yield aggregates that occupy the lumen of the terminal cisternae of the sarcoplasmic reticulum (SR). When infected or transfected into L6 myoblast, the protein is also concentrated, however, in dense vacuoles apparently separate from the endoplasmic reticulum (ER). CSQ-rich cells appear otherwise normal; in particular, neither other proteins involved in Ca2' homeostasis nor ER chaperones are increased. The CSQ dense vacuoles are shown herein to be specialized ER subdomains as demonstrated by 1) the endoglycosidase H sensitivity of their CSQ and 2) two markers, calreticulin and calnexin (but not others, protein disulfide isomerase and BiP), intermixed with the vacuole content. Their formation is shown to start with the aggregation of CSQ at discrete sites of the ER lumen. When cells were transfected with both CSQ and calreticulin, only the first gave rise to vacuoles; the second remained diffusely distributed within the ER lumen. The possibility that CSQ aggregation is an artifact of overexpression appears unlikely because 1) within dense vacuoles CSQ molecules are not disulfide cross-linked, 2) their turnover is relatively slow (t = 12 h), and 3) segregated CSQ is bound to large amounts of Ca2+. Transfection of a tagged CSQ into cells already overexpressing the protein revealed the continuous import of the newly synthesized protein into preassembled vacuoles. The tendency to aggregation appears, therefore, as a property contributing to the segregation of CSQ within the ER lumen and to its accumulation within specialized subdomains. The study of L6 cells expressing CSQ-rich vacuoles might thus ultimately help to unravel mechanisms by which the complexity of the sarcoplasmic reticulum is established in muscle fibers.

Role of Junctin Protein Interactions in Cellular Dynamics of Calsequestrin Polymer upon Calcium Perturbation

“…As CSQ interacts with JNT or TRN (4,5) and undergoes assembly into the RyR complex, JNT was cotransfected with CSQ. CSQ appeared as speckles in myoblasts, consistent with a previous finding (20). Ca 2ϩ perturbation was performed by treating cells with thapsigargin, which inhibits SERCA, resulting in SR Ca 2ϩ depletion within a few minutes and a corresponding transient Ca 2ϩ increase in the cytoplasm (21).…”

“…3), possibly due to abnormal SR Ca 2ϩ load and CSQ mislocalization, respectively. Pre- viously, CSQ condensation was observed in L6 myoblast and HeLa cells transfected with CSQ, which remained unchanged after depletion of endoplasmic reticulum Ca 2ϩ stores (20), possibly due to imbalance in the levels of CSQ relative to JNT. We replicated the necessity for JNT in CSQ depolymerization reactions in vitro in experiments using the purified proteins (Figs.…”

Correct targeting of dihydropyridine receptors and triadin in dyspedic mouse skeletal muscle in vivo

“…Thus domain formation in the SR is driven by a separation of a free SR protein, the calcium pump, and a junctional SR protein, triadin, even in the absence of the calcium release channels. This observation acquires further significance from the finding that calsequestrin also shows an apparently independent tendency towards segregating into specialized subdomains of the SR (Gatti et al, 1997). A remaining question is whether the distribution of calsequestrin drives that of triadin, or viceversa, or whether a third protein, perhaps the hypothetical protein responsible for SR-surface docking (Flucher and Franzini-Armstrong, 1996), drives both.…”

Neurosecretion Competence, an Independently Regulated Trait of the Neurosecretory Cell Phenotype

“…17. For electron microscopy the cells detached from the dishes and centrifuged were fixed with the aldehyde mixture followed by OsO 4 .…”