The endoplasmic reticulum of Purkinje neuron body and dendrites: molecular identity and specializations for Ca2+ transport.
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-triphosphate receptor and thus presumably specialized in Ca2+ transport (stacks of multiple smooth-surfaced cisternae; the dendritic spine apparatus) also exhibited labeling for BiP and p91, 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 Ca2+ 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, protein 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, p91, 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.
Villa
A
A
Department of Pharmacology, CNR Cytopharmacology and B. Ceccarelli Centers, S. Raffaele Scientific Institute, Milano, Italy.
Sharp
A H
AH
Racchetti
G
G
Podini
P
P
Bole
D G
DG
Dunn
W A
WA
Pozzan
T
T
Snyder
S H
SH
Meldolesi
J
J
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D003712:Dendrites / Q000378:metabolism* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008297:Male; D008861:Microsomes / Q000378:metabolism / Q000648:ultrastructure; D011689:Purkinje Cells / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D012433:Ryanodine / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure
D015220:Calcium Channels; D012433:Ryanodine; D002118:Calcium