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(Pro)Insulin Associates with Golgi Membranes of Pancreatic B Cells (Golgi Complex/Conversion/Sorting Out/Secretory Po!Ypeptides/Immunocytochemistry) L

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(Pro)Insulin Associates with Golgi Membranes of Pancreatic B Cells (Golgi Complex/Conversion/Sorting Out/Secretory Po!Ypeptides/Immunocytochemistry) L Proc. Natl. Acad. Sci. USA Vol. 81, pp. 6743-6746, November 1984 Cell Biology (Pro)insulin associates with Golgi membranes of pancreatic B cells (Golgi complex/conversion/sorting out/secretory po!ypeptides/immunocytochemistry) L. ORCI, M. RAVAZZOLA, AND A. PERRELET Institute of Histology and Embryology, University of Geneva Medical School, 1211 Geneva 4, Switzerland Communicated by A. Frey-Wyssling, July 11, 1984 ABSTRACT The immunocytochemical demonstration of lowing incubation, sections were washed in Pi/NaCl, further (pro)insulin on intracellular membrane compartments of the incubated for 30 min in the protein A/gold solution (9), pancreatic B cell reveals that the immunolabeling detected by washed again, stained with uranyl acetate, and embedded in the protein A/gold methQd is associated, at the level of the methylcellulose (8) to be observed in a Philips 300 electron Golgi apparatus, with the inner aspect of the cisternal mem- microscope. Gold particles of small ("'8 nm) diameter were brapes; on the secretory granules, by contrast, insulin immu- prepared according to ref. 10. Controls of specificity of the noreactive sites predominate over the granule core, and very immunolabeling comprised the use of the antiserum ad- little immunoreactivity is associated with the granule mem- sorbed with native insulin (4 units/ml) or of antisera-e.g., brane. The localization of (pro)insulin immunoreactivity on anti-glucagon or anti-somatostatin-unrelated to insulin. No Golgi membranes is compatible with the presence, at this level, labeling above background was observed in these condi- of specific binding sites for (pro)insulin, which could be relat- tions. As detailed in ref. 11, all anti-insulin antisera show a ed to the proteolytic processing or sorting out (or both) of these certain degree of cross-reactivity with proinsulin. The quan- peptides on their way from the rough endoplasmic reticulum titation of the immunolabeling was carried out as described to the storage secretory granules. (12). During their transit from the rough endoplasmic reticulum RESULTS (RER) to the storage secretory granules of the pancreatic B cell, proinsulin polypeptides undergo an important process- The typical distribution of gold particles on Golgi and secre- ing step at the level of the Golgi apparatus. This step com- tory granule compartments of B cells following immuno- prises both conversion of proinsulin to insulin (1, 2) and the staining is shown in Figs. 1A and 2 A-D. At the level of the sorting out of insulin polypeptides from other (e.g., lysosom- Golgi apparatus, gold particles appeared associated prefer- al enzymes) RER-derived proteins. From the RER-related entially with the periphery of the Golgi cisternae, whereas on pole (or cis pole) of the Golgi apparatus to the secretory the secretory granules, immunolabeling predominated over granule-related pole (or trans pole) (3), the membranes limit- the dense secretory granule core. A close 'examination of ing the Golgi cisternae show, with morphological markers, a these compartments indicated that their respective limiting decrease in their content of proteins (4) and an increase in membrane was preserved and that in the Golgi cisternae the sterols (5), changes which are further enhanced on the secre- immunolabeling was close to or over these membranes (Fig. tory granule membrane derived from the trans Golgi cister- 1B). On a given cisterna, segments of labeled membrane al- nae (6). This suggests a correlation between the maturation ternated with unlabeled segments (Fig. 2 A, B, and D). The of the proinsulin polypeptides and the composition/organi- cryoultramicrotomy process induced a moderate degree of zation of the membrane limiting the respective intracellular swelling of Golgi cisternae, which helped to localize and compartments involved. With immunocytochemistry on quantify the immunoreactivity on the membrane (Table 1). plastic embedded tissue, we showed previously a preferen- In the Golgi area, 88% of the gold particles were associated tial association of insulin immunoreactive sites with the pe- with the inner aspect of the cisternal membrane; on secre- riphery of Golgi cisternae (7). Using an anti-insulin antise- tory granules, by contrast, 94% of the particles were over the rum revealed by the protein A/gold method on cryoultrami- dense granule core (Fig. 2C). A marked asymmetry was ob- crotomy sections that minimize antigen loss, we have now served between the Golgi cisternae without or with condens- quantified the localization of (pro)insulin polypeptides in the ing secretory material (Fig. 2 B and D). The latter, situated at Golgi apparatus and observed that most immunoreactivity the trans pole of the Golgi, presented a secretory granule- appeared associated with the membrane of Golgi cisternae. type distribution of the immunoreactivity-i.e., a concentra- On the contrary, the membrane limiting the secretory gran- tion of gold particles over the dense condensing material but ule showed very little immunoreactivity, but this was con- very little immunoreactivity on the cisternal membrane itself centrated over the secretory granule core. (1 gold particle per ,um of length cisternae versus 12 particles per Am of cisternae without condensing secretory material). A degree of membrane-associated labeling comparable to the MATERIAL AND METHODS latter value was observed among cisternae without condens- Rat pancreata were perfusion fixed with 1% phosphate-buff- ing material. In relationship with Golgi stacks, profiles re- ered glutaraldehyde and islets were isolated under the dis- sembling "rigid cisternae" and considered as elements of the secting microscope. Microdissected islets were cryoprotect- Golgi-associated endoplasmic reticulum from which lyso- ed in 2.3 M sucrose and directly thin sectioned by cryoultra- somes form (GERL) (14) appeared devoid of immunolabel- microtomy (8). Thin sections were picked on copper grids ing. and incubated at room temperature for 1 hr on a drop of anti- The number of clearly identifiable RER profiles and trans- insulin antiserum (P. H. Wright, Indianapolis), which was fer vesicles (15) on the cryosections studied was not suffi- diluted 1:800 with phosphate-buffered saline (Pj/NaCl). Fol- cient to allow a quantification of the gold particles per um' of each compartment. Furthermore, the narrow width of the The publication costs of this article were defrayed in part by page charge identifiable RER cisternae and the small diameter of transfer payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: RER, rough endoplasmic reticulum. 6743 Downloaded by guest on October 2, 2021 6744 Cell Biology: Orci et aL Proc. NatL. Acad. Sci. USA 81 (1984) 1A .:r * * =;*W; .* * Ajr G ..P4 'tG. tz'$Sf.lm.. *s ivii.,;, |.. i as.,w. .............................'Aa r.¢ i . t 4* '-' iiSG4i"¢ ' u at i.. At. I" Yl'. 3:. 7 ..* Uw *.0 . 0 .- G 9 .99/ 1. .: jig'. Be :4-:. 4 FIG. 1. (A) Overview of a B-cell cytoplasm in cryosection following immunostaining with anti-insulin antiserum. Two Golgi complexes (G) are apparent as well as numerous secretory granules (SG). Even at this low magnification, the immunolabeling over the Golgi complex appears related to the periphery ofthe cisternae, while it is concentrated over the dense secretory granule core. Part of the nucleus is visible in the lower left corner. Mitochondria (m) appear unlabeled. (x 19,000.) (B) Higher magnification of part of the Golgi complex delimited by arrowheads in A. The trilaminar structure of the membrane limiting individual Golgi cisternae is revealed (arrows) and the association of individual gold particles with the inner aspect of cisternal membrane is apparent. (x69,000.) Approximately 15-nm gold particles in A and B were prepared according to ref. 9. vesicles prevented determination of whether the immunore- (pro)insulin membrane association on secretory granule frac- activity was membrane bound or cisternal. Therefore, the tions (16). These results were obtained by assessing the bind- cytoplasmic area containing RER and transfer vesicles was ing both of endogenous, biosynthetically labeled proinsulin evaluated as a whole. The low degree of labeling obtained and of exogenously added marked hormone, and the differ- with the anti-insulin antiserum adsorbed with its antigen is ence, for secretory granules, between these and our data shown in Fig. 2E. As mentioned previously, the known cannot be simply explained at present. The specificity of the cross-reactivity of the anti-insulin antiserum with proinsulin immunocytochemical labeling, as evidenced by the lack of (11) does not distinguish, at the level of the Golgi mem- staining with adsorbed anti-insulin antiserum or with antise- branes, between insulin or proinsulin antigenic sites (the ki- ra directed against islet hormones other than insulin, speaks netics of conversion suggest the presence of both polypep- against the detected immunoreactivity being due to polypep- tides at this level) (1, 2, 6). It is clear, however, that the gold tides not related to proinsulin; the fact that rigid cistemae, particles over the dense secretory granule core represent elements of the GERL (14), were also unreactive can be in- mostly insulin since the conversion of proinsulin has mark- terpreted similarly. Furthermore, the processing protocol in- edly progressed at this stage (1, 2, 6). volving glutaraldehyde fixation alone prior to cryosectioning should have minimized proinsulin/insulin
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