Abstract
Expression of dense-core granules, a typical exocytic organelle, is widely believed to be controlled by coordinate gene expression mechanisms specific to neurones and neurosecretory cells. Recent studies in PC12 cells, however, have suggested the number of granules/cells depends on the levels of only one of their cargo proteins, chromogranin A, regulating the metabolism of the other proteins, and thus the composition of the organelles, by an on/off switch mechanism. In addition, transfection of chromogranin A was reported to induce appearance of dense-core granules in the non-neurosecretory fibroblasts of the CV-1 line. Here the role of chromogranin A has been reinvestigated using not the heterogeneous PC12 line but several clones isolated therefrom. In these clones, investigated as such or after transfection with chromogranin A antisense sequences, the ratio between chromogranin A and its secretory protein mate, chromogranin B, was not constant but highly and apparently randomly variable. Variability of the chromogranin A/chromogranin B ratio was seen by confocal immunofluorescence also among the cells of single clones and subclones and among the granules of single cells. Moreover, stable and transient transfections of chromogranin A in a PC12 clone characterised by a low number of dense-core granules (one fifth of the reference clone) failed to modify significantly the number of the organelles, despite the several-fold increase of the granin. Finally, in three types of non-neurosecretory cells (CV-1, adenocarcinoma TS/A and a clone of PC12 incompetent for secretion) the transfected chromogranin A accumulated mostly in the Golgi/transGolgi area and was released rapidly from resting cells (constitutive secretion) as revealed by both immunofluorescence during cycloheximide treatment and pulse-chase experiments. Only a minor fraction was sorted to discrete organelles that were not dense-core granules, but primarily lysosomes because they contained no chromogranin B, and were largely positive for the late endosomal-lysosomal markers, lamp1 and lamp3. Dense-core granules are therefore true hallmarks of neurones and neurosecretory cells. Their number/cell appears independent of chromogranin A and their composition does not appear to be constant; in particular, they exhibit considerable, and so far unexplained variability in the chromogranin A/chromogranin B ratio.
Highlights
Regulated secretion, i.e. the stimulation-induced, exocytic release of cargo molecules segregated within the lumen of membrane-bound organelles, has classically been envisaged as a property of secretory granules and vesicles, taking place only in professional secretory cells: exocrine, endocrine and neurones
Recent studies in PC12 cells, have suggested the number of granules/cells depends on the levels of only one of their cargo proteins, chromogranin A, regulating the metabolism of the other proteins, and the composition of the organelles, by an on/off switch mechanism
Variability of the chromogranin A/chromogranin B ratio was seen by confocal immunofluorescence among the cells of single clones and subclones and among the granules of single cells
Summary
I.e. the stimulation-induced, exocytic release of cargo molecules segregated within the lumen of membrane-bound organelles, has classically been envisaged as a property of secretory granules and vesicles, taking place only in professional secretory cells: exocrine, endocrine and neurones. Regulated exocytosis of lysosomes, believed for a long time to be specific for a subclass of these organelles, the secretory lysosomes expressed in blood leukocytes and a few other cell types, was shown to be ubiquitous (Jaiswal et al, 2002; Reddy et al, 2001), possibly involved in membrane repair. Another widely expressed family of vesicles, competent for regulated exocytosis, was identified (Borgonovo et al, 2002). A fall in CgA levels was reported to induce the rapid turnover of other proteins of DCGs, with ensuing reduction of the number and preservation of the composition of the organelles (Kim et al, 2001)
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