Neuroscience Vol. 49, No. 2, pp. 467471, 1992 Printed in Great Britain 0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd IBRO zyxwvu THE ENDOPLASMIC RETICULUM OF PURKINJE NEURON BODY AND DENDRITES: MOLECULAR IDENTITY AND SPECIALIZATIONS FOR Ca2+ TRANSPORT A. VILLA,* A. H. SHARP,? G. RACCHETTI,* P. PODINI,* D. G. BoLE,~: W. A. DUNN,$ T. POZZAN,]~S. H. SNYDER? and J. MELDOLPSI*~ *Department of Pharmacology, CNR Cytopharmacology and B. Ceccarelli Centers, S. Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy tDe.partments of Neuroscience, Pharmacology and Molecular Sciences, Psychiatry and Behavioral Sciences, Johns Hopkins University, School of Medicine, Baltimore, MD, U.S.A. IHoward Hughes Medical Institute Research Laboratory, The University of Michigan Medical Center, Ann Arbor, MI, U.S.A. §Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainsville, FL, U.S.A. IIInstitute of General Pathology, CNR Biomembrane Center, University of Padova, Italy Abstract-Immunofluorescence and immunogold labeling, together with sucrose gradient separation and Western blot analysis of microsomal subfractions, were employed in parallel to probe the endoplasmic reticulum in the cell body and dendrites of rat cerebellar Purkinje neurons. Two markers, previously investigated in non-nerve cells, the membrane protein p91 (calnexin) and the lumenal protein BiP, were found to be highly expressed and widely distributed to the various endoplasmic reticulum sections of Purkinje neurons, from the cell body to dendrites and dendritic spines. An antibody (denominated anti-rough-surfaced endoplasmic reticulum), which recognized two membrane proteins, p14 and p40, revealed a similar immunogold labeling pattern. However, centrifugation results consistent with a widespread distribution were obtained for p14 only, while p40 was concentrated in the rough microsome- enriched subfractions. The areas enriched in the inositol 1,4,5&phosphate receptor and thus presumably specialized in Ca*+ transport (stacks of multiple smooth-surfaced cisternae; the dendritic spine apparatus) also exhibited labeling for BiP and ~91, and were positive for the anti-rough-surfaced endoplasmic reticulum antibody (presumably via the p14 antigen). Additional antibodies, that yielded inadequate immunocytochemical signals, were employed only by Western blotting of the microsomal subfractions, while the ryanodine receptor was studied by specific binding. The latter receptor and the Ca*+ ATPase, known in other species to be concentrated in Purkinje neurons, exhibited bimodal distributions with a peak in the light and another in the heavy subfractions. A similar distribution was also observed with another lumenal protein, proteine disulfide isomerase. Taken as a whole, the results that we have obtained suggest the existence in the endoplasmic reticulum of Purkinje neurons of two levels of organization; the first identified by widespread, probably general markers (BiP, ~91, possibly p14 and others), the second by specialization markers, such as the inositol 1,4,5-triphosphate receptor and, possibly, p40, which appear restricted to areas where specific functions appear to be localized. Knowledge about neuron endoplasmic reticulum (ER) is still incomplete. Up until recently, most studies concentrated on the axonal reticulum, investi- gated because of its possible involvement in axonal transport29s49,M and neurotransmitter release (see Ref. 11). In other regions of the neuron, only few studies have been carried out. Except for experiments reveal- ing the distribution of glucose-6-phosphatase activity TTo whom correspondence should be addressed. Abbreoiarions: BSA, bovine serum albumin; EGTA, ethyleneglycolbis(aminoethylether)tetra-acetate; (R)ER, (rough-surfaced) endoplasmic reticulum; HEPES, N-2- hydroxyethylpiperaxine-N’-2-ethanesulfonic acid; Ins-P, and Ins-P,R, inositol 1,4,5_trisphosphate and its receptor; PBS, phosphate-buffered saline; PDI, protein disulfide isomerase; PMSF, phenylsulfonylfluoride; RyR, ryanodine receptor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. in the entire ER,6 the widespread distribution of concanavalin A and the absence of other lectin binding,‘3v67*68 the information was limited to con- ventional electron microscopy. The latter revealed in most neurons the co-existence and occasional lumenal continuities of the rough- and smooth- surfaced sections of the ER in the cell body up to the dendrite stalk(s). Moreover, the smooth ER was shown to be distributed along the dendrites, up to the tips and (where present) the spines, giving rise to a complex tridimensional network of tubules and longi- tudinal cistemae. Whether at all these sites the ER expresses common molecular markers or includes highly specialized areas, designed to match the struc- tural and functional specializations of the various regions of the cell, is still largely unknown. Recently, interest in these problems has greatly increased in view of the possible key involvement of NSC 49,2--H 461