Location of endoplasmic reticulum relative to sarcoplasmic reticulum in cultured chick myotubes.

Abstract

Calcium plays a major role as a second messenger modulating a wide range of cellular processes (1). Endoplasmic and sarcoplasmic reticulum calcium pumps (SERCAs) play a major role in controlling the levels of cytoplasmic calcium but the way in which these pumps are targeted to and maintained in specific cellular compartments is unclear. In skeletal muscle cells it has been assumed that either proteins destined for the sarcoplasmic reticulum (SR) are synthesised on the endoplasmic reticulum (ER) and that the SR forms by extension of the ER membrane or that regions of ER develop into SR (2). However, it is not known what signals result in either proteins moving from ER to SR or in proteins being synthesised on newly forming SR. A similar targeting problem may exist in non-muscle cells where it has been suggested that there is more than one discernible calcium store and that there am. specialised regions of ER involved in calcium uptake and release, i.e.. the calciosomes (3). If there is more than one type of store then there must be mechanisms for targeting calcium pumps to these stores. In order to study this process more closely it is first necessary to determine whether it is possible to discriminate between ER and SR. This has been achieved using antibodies against proteins normally associated with ER and SR in conjunction with laser confocal microscopy. Antibodies against, the ER resident transmembranous protein calnexin (4). the ER retention signal KDEL (5) and the calcium pump (SERCAla) (6) have been used with cultured chick myotubes. Primary cultures of myotubes were prepared from 11-day old embryonic chick breast muscle (modified from (7)). Chopped muscle tissue from 4 birds was dispersed in 10 ml HBSS (calciummagnesium free) supplemented with 0.25% Trypsin-EDTA and stirred for 20 minutes at stirred for 20 minutes at 37C.Undisrupted material was removed following sedimentation and the digestion was stopped by the addition of 10 ml HBSS (calcium-magnesium free) supplemented with soya bean trypsin inhibitor (80 pg ml'), DNAase