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

Abstract

Neurosecretion competence is intended as the ability of neurosecretory cells to express dense and clear vesicles discharged by regulated exocytosis (neurotransmitter release). Such a property, which so far has never been studied independently, is investigated here by a heterotypic cell fusion approach, using a clone of rat pheochromocytoma PC12 cells totally incompetent for neurosecretion that still largely maintains its typical molecular and cellular phenotype. When fused with wild-type partners of various species (rat, human) and specialization (PC12, neuroblastoma SH-SY5Y, HeLa), the defective cells reacquire their competence as revealed by the expression of their secretion-specific proteins. Fused wild-type cells therefore appear able to complement defective cells by providing them with factor(s) inducing the reactivation of their secretory program. The mechanism of action of these factors may consist not in a coordinate unblocking of transcription but in the prevention of a rapid post-transcriptional degradation of the mRNAs for secretion-specific genes.Regulated exocytosis, a fundamental activity of neurosecretory cells, is among the processes most intensely investigated. So far, however, the studies have focussed primarily on the identification of the proteins involved and on the mechanistic interactions that underly vesicle discharge (1-4). In contrast, the way by which neurosecretory cells become competent for neurosecretion has received much less attention. Competence acquisition requires the appearance of specific organelles, dense-core (DV) 1 and clear or synaptic-like (SLV) vesicles, an event that takes place during phenotype development. Because of this temporal coincidence, the first process is widely envisaged as a step of the second (5, 6).To investigate the relationships between competence and phenotype, we used clones isolated from a neurosecretory cell model, the rat pheochromocytoma PC12 line (7,8). Most PC12 clones, indicated here as wild-type clones, exhibit DVs from which exocytic release of secretory proteins and catecholamines occurs after appropriate stimulation. Moreover, Western blots, immunocytochemistry, and patch clamping document in these clones the expression and discharge of acetylcholine-containing SLVs (8 -10). An additional clone has been found to lack en block both types of secretory vesicles, i.e. to be neurosecretion incompetent, no matter whether analyzed at rest or after a variety of stimulatory treatments, while maintaining numerous markers typical of PC12 and other neurosecretory cells (7,8). These results suggest the lack in the defective clone of one or more factors that impart to the cells their neurosecretion competence, acting independently, at least in part, from the factors governing the rest of the phenotype (8). Here we report direct evidence supporting this hypothesis, showing that defective PC12 cells reacquire their competence when fused to various types of wild-type cells, regardless of their species of origin and of their specialization. Moreover, molecu...

Neurosecretory cells without neurosecretion: evidence of an independently regulated trait of the cell phenotype

“…The concomitant expression of at least one of the neurosecretion-specific proteins investigated, chromogranin B, was identified by speciesspecific antibodies against this protein. The hybrids were found to be positive for the two neurosecretion-specific markers tested, synaptotagmin and chromogranin B, with the latter being specifically recognized by an anti-rat but not by an anti-human antibody (Borgonovo et al 1998). We conclude therefore that complementation of the PC12_27 cell defect in the hybrids consists of the reactivation of their competence programme by factor(s) missing in the defective neurosecretory cells and provided by the fusion partners.…”

Neurosecretion Competence

“…In contrast to another reported cell model (12), it cannot be reverted by stimulatory treatments or by changes in growth conditions. However, the NI phenotype defect can be rescued by fusion of the NI cells with WT cells (regardless of the species or phenotype) and following transfection with a WT PC12 cDNA library (11,13). These data strongly suggest neurosecretion incompetence to reflect the lack of expression of a specific program.…”

“…In previous studies carried out by Northern and Western blotting, the degree of heterogeneity of the various PC12 clones had been underestimated. Indeed, of 29 gene products investigated in a single NI and a single WT clone, all the 11 mRNAs/proteins directly related to neurosecretion were found down-regulated, whereas the others (related to membranes, cytoskeleton, signaling, and endocytosis) appeared in contrast invariant (8,11,13). Neurosecretion competence had been therefore envisaged as a highly specific program, expressed independently of the other PC12 traits.…”

“…Previous NI cell fusion and cDNA library transfection experiments had suggested the involvement of one or more master genes 2 Giordano, T., Malosio, M. L., and Meldolesi, J., unpublished results. coding for specific suppressor(s) or enhancer(s) that in the NI clones could be up-or down-regulated, respectively (13,11). One or more master genes could control the coordinate expression of operative genes directly involved in the competence program.…”

“…To ensure the highest confidence to our analyses, we removed the bias due to single events by analyzing two independent NI PC12 clones (which most likely have the same genetic defect, because no complementation is observed after fusion between them, see Ref. 13), compared with two independent WT PC12 clones. Our analysis is focused on genes expressed at similar levels in both clone pairs (WT/WT and NI/NI) and at significantly different levels between pairs (WT/NI).…”