Immunogold localization of inositol 1, 4, 5-trisphosphate (InsP3) receptor in mouse cerebellar Purkinje cells using three monoclonal antibodies.

Ultrastructural localization of InsP3 receptor in mouse cerebellar Purkinje cells was investigated by immunogold technique using three monoclonal antibodies (mab 10A6, 4C11 and 18A10). The epitopes of the three antibodies were numerously detected on the smooth endoplasmic reticulum (ER) (especially, on the stacks of flattened smooth ER, subsurface cisterns and spine apparatus), scantily on the rough ER and on the outer nuclear membrane, but were not detectable on either the plasmalemma, synaptic densities, mitochondria or Golgi apparatus. Not only mab 4C11 and 10A6 which bind to the N-terminal region of the receptor but also 18A10 which binds to the C-terminal region were localized on the cytoplasmic surface of the ER membranes. This indicates that the C terminus of InsP3 receptor is localized on the cytoplasmic surface of the ER. We noticed that gold particles are usually localized on the fuzzy structure of the cytoplasmic surface of smooth ER, which is suggested to correspond to the feet structure of the ryanodine receptor. In the Nissl body, gold particles were found not only on the ER membranes but also in the cytoplasmic matrix between the rough ER cisterns. We suggest that the peculiar structure of Nissl body, which is composed of parallel cisterns of rough ER, sandwiching a number of free polyribosomes between the cisternal elements, is due to the fact that the major proteins like InsP3 receptor are synthesized mostly on the free polyribosomes and become membrane bound only at the later stage of the biosynthesis.

The analysis of glycoproteins in cells and tissues by two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to identify and analyse subsets of proteins in cells and tissues. The combination of 2-D PAGE and [125I] concanavalin A overlay revealed an extraordinary complexity and diversity in the glycoprotein profiles of different cell types. However, the glycoproteins are not expressed idiosyncratically. Rather, their expression is closely linked to the state of differentiation of a particular cell type. Such glycoproteins can therefore be used to generate antibodies specific for differentiated cells. 2-D PAGE analyses of cellular glycoproteins also revealed a major common glycoprotein of 100 kDa. This was localised to the lumen of the endoplasmic reticulum and is referred to as endoplasmin. The combination of 2-D PAGE with electroblotting and 45Ca overlay revealed that endoplasmin and several other luminal endoplasmic reticulum proteins (reticuloplasmins) are high capacity, low affinity calcium binding proteins which could function as calcium storage proteins in the endoplasmic reticulum. One of these called calreticulin is also found in the sarcoplasmic reticulum. 2-D PAGE and 45Ca overlay has been used to demonstrate the presence of a calcium-binding protein (CP22/sorcin) in the cytosol of rodent multidrug resistant cells. Analyses of murine serum by 2-D PAGE revealed the presence of a novel stress protein serum amyloid P component. These studies illustrate the value of 2-D PAGE when used in combination with detection methods which select specific subsets of proteins such as glycoproteins.

A time course investigation of vitamin A level and lipid composition of the liver endoplasmic reticulum in rats following treatment with congeneric polychlorobiphenyls.

The drug metabolizing enzyme activities, the vitamin A content and the fatty acid composition in the endoplasmic reticulum membrane were studied in rat liver after a single injection of the polychlorobiphenyls (PCBs) 3,3',4,4'-tetrachlorobiphenyl [(3,4)2Cl] or 2,2',4,4',5,5'-hexachloro-biphenyl [(2,4,5)2Cl], 300 mumol/kg each. The microsomal vitamin A level was markedly lowered 3 days after treatment with (3,4)2Cl, a coplanar type inducer of cytochrome P-450. A marked increase in microsomal AHH and UDPGT activities occurred within 3 days after injection of (3,4)2Cl whereas (2,4,5,)2Cl treatment enhanced APDM activity only. Arachidonic, stearic and linoleic acid microsomal contents were enhanced by the two congeners. (3,4)2Cl caused the proportion of docosahexaenoic acid to decrease. No highly significant correlation was found between the vitamin A content and lipid components in the microsomal membrane. However, the vitamin A level was inversely related to the activities of drug metabolizing enzymes induced by coplanar compounds (cytochrome P-450 towards benzo[a]pyrene and UDP glucuronosyl transferase towards 4-nitrophenol).

Association of androgen binding sites with the endoplasmic reticulum of rat ventral prostate.

Microsomes from ventral prostate of 24-h castrated rats contain a single set of tissue-specific high-affinity, low-capacity androgen binding sites. These sites are indigenous to the endoplasmic reticulum, as shown by purification procedures associated with marker enzymes and electron microscopic analyses. When prostatic microsomal membranes are separated from plasma membranes using the nuclear or the mitochondrial pellets as the source of fractionation in sucrose gradients, the androgen binding activity is selectively associated with fractions rich in rough endoplasmic reticulum and ribosomes. Eighty-four percent of the total content of Na+/K+ adenosine triphosphatase (ATPase) and only 27% of the total binding capacity were concentrated in fractions rich in smooth-surfaced vesicular membranes, when nuclear suspensions constituted the membrane source. In contrast, the region of the same gradient when enriched in rough endoplasmic reticulum and deficient in plasma membrane content contained 73% of the androgen-binding capacity and only 14% of the ATPase. For fractions collected using mitochondrial suspensions as starting material, the ratio (total glucose-6-phosphatase/total binding capacity) was closer to 1.0 than similar ratios of ATPase/binding capacity, indicating co-sedimentation of binding sites with microsomal membranes and not with plasma membranes. Na+/K+ ATPase, but not 5' nucleotidase, is a valid plasma membrane marker for ventral prostate. Microsomal androgen receptors may constitute a new level of regulation of androgen action in target cells.

Albumin and collagen mRNA expression in normal and analbuminemic rodent liver: analysis by in situ hybridization using biotinylated probes.

We used in situ nucleic acid hybridization cytochemistry to examine cell types and subcellular sites expressing albumin (alb) or pro alpha 2 collagen (col) mRNA in livers from normal and analbuminemic rodents. Biotinylated cDNA or RNA probes were applied to aldehyde-fixed, non-frozen sections and the resulting DNA-RNA or RNA-RNA hybrids were subsequently visualized by enzymatic detection of either peroxidase or alkaline phosphatase conjugated to anti-biotin IgG or streptavidin. In normal rat liver, alb mRNA was expressed in all hepatocytes and was localized to discrete subcellular structures distributed as aggregates in the cytoplasm and in specific structures encircling the nucleus; these subcellular structures most likely represent the endoplasmic reticulum and nuclear envelope. In mouse liver, pro alpha 2 col mRNA was identified in a subpopulation of sinusoidal lining cells which have the morphological appearance of lipocytes. In liver from analbuminemic rats, a small number of hepatocytes, distributed throughout the hepatic lobule, expressed alb mRNA at high levels; the subcellular distribution of this alb mRNA was essentially identical to that observed in normal rat hepatocytes. Since non-radioactive in situ hybridization detected mRNA within the boundaries of individual cells and showed its precise subcellular location under conditions in which there was excellent preservation of tissue morphology, this procedure should be useful for a wide variety of histopathologic studies.

Characterization of high-capacity low-affinity calcium binding protein of liver endoplasmic reticulum: calsequestrin-like and divergent properties.

It had been previously demonstrated that endoplasmic reticulum membranes from rat hepatocytes contain a major calsequestrin-like protein, on account of electrophoretic and Stains All-staining properties (Damiani et al., J. Biol. Chem. 263, 340-343). Here we show that a Ca2+-binding protein sharing characteristics in size and biochemical properties with this protein is likewise present in the isolated endoplasmic reticulum from human liver. Human calsequestrin-like protein was characterized as 62 kDa, highly acidic protein (pl 4.5), using an extraction procedure from whole tissue, followed by DEAE-Cellulose chromatography, that was originally developed for purification of skeletal muscle and cardiac calsequestrin. Liver calsequestrin-like protein bound Ca2+ at low affinity (Kd = 4 mM) and in high amounts (Bmax = 1600 nmol Ca2+/mg of protein), as determined by equilibrium dialysis, but differed strikingly from skeletal muscle calsequestrin for the lack of binding to phenyl-Sepharose resin in the absence of Ca2+, and of changes in intrinsic fluorescence upon binding of Ca2+. Thus, these results suggest that liver 62 kDa protein, in spite of its calsequestrin-like Ca2+-binding properties, does not contain a Ca2+-regulated hydrophobic site, which is a specific structural feature of the calsequestrin-class of Ca2+-binding proteins.

A competition enzyme-linked immunosorbent assay (ELISA) for the measurement of pancreatic GP-2 glycoprotein.

A competition enzyme-linked immunosorbent assay has been developed for the quantitative detection of soluble and membrane-bound GP-2, a glycoprotein which is confined to the exocrine pancreas. Zymogen granule membranes fixed to microtiter plates with poly-L-lysine were used as the source of antigen. Detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS] was added to the assay in order to reveal all the antigens, more particularly in membranous samples. Presence of detergent at concentrations as high as 0.5% did not interfere with any particular steps of the ELISA. This competition ELISA can detect 10 ng of GP-2 and will be useful for measuring soluble as well as membrane GP-2 in order to elucidate its role in the secretory process of the pancreas as well as in certain pathologies such as cystic fibrosis.

Peripheral membrane proteins of sarcoplasmic and endoplasmic reticulum. Comparison of carboxyl-terminal amino acid sequences.

Peripheral endoplasmic reticulum membrane proteins residing in the lumen of the endoplasmic reticulum occupy the same space as other secreted proteins. The presence of a four amino acid salvage or retention signal (KDEL-COOH = Lys-Asp-Glu-Leu-COOH) at the carboxyl-terminal end of peripheral membrane proteins has been shown to represent a signal or an essential part of a signal for their retention within the endoplasmic reticulum membrane. In heart and skeletal muscle, a number of sarcoplasmic reticulum proteins have recently been identified which are peripheral membrane proteins. The high-affinity calcium-binding protein (55 kilodaltons (kDa] appears to conform to the above described mechanisms and contains the KDEL carboxyl-terminal tetrapeptide. Thyroid hormone binding protein is present in the sarcoplasmic reticulum, in addition to its endoplasmic reticulum location, and has a modified but related tetrapeptide sequence (RDEL = Arg-Asp-Glu-Leu), which also probably functions as the retention signal. Calsequestrin and a 53-kDa glycoprotein, two other peripheral membrane proteins residing in the lumen of the sarcoplasmic reticulum, do not contain the KDEL retention signal. The sarcoplasmic reticulum may have developed a unique retention mechanism(s) for these muscle-specific proteins.

Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum.

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.

Distribution of endoplasmic reticulum and calciosome markers in membrane fractions isolated from different regions of the canine brain.

Four regions of the canine brain (frontal lobe, parieto-occipital lobe, brainstem, and cerebellum) were each fractionated by differential centrifugation into a crude mitochondrial pellet (P2) and a crude microsomal pellet (P3). Markers of endoplasmic reticulum (glucose-6-phosphate phosphatase and rotenone-insensitive NADPH cytochrome c reductase) and markers of the 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store ([3H]IP3 binding and IP3-induced Ca2+ release) were measured. No correlation was found between the two classes of markers, which suggests that the IP3 receptor does not belong to the endoplasmic reticulum in canine brain. Cerebellum P2 and P3 fractions displayed levels of [3H]IP3 binding 10- to 30-fold higher, and rates of IP3-induced Ca2+ release greater than 15-fold faster than the homologous cerebrum and brainstem fractions. Actively accumulated Ca2+ was only partially released by IP3, both before and after saponin disruption of the plasma membrane compartment. The proportion of the IP3-sensitive Ca2+ store relative to that of the total (IP3-sensitive and IP3-insensitive) Ca2+ store was variable; i.e., it was larger in cerebellum P2 (approximately 90%) than in cerebrum fractions (less than 30%). Cerebellum fractions constitute the best source from which an IP3-sensitive Ca2+ storing organelle can be purified.

Immunocytochemical demonstration of amelogenins and enamelins secreted by ameloblasts during the secretory and maturation stages.

Rabbit polyclonal antibodies against bovine amelogenins and enamelins which did not show any cross-reaction were raised, and ultrathin sections of rat incisors were examined by the protein A-gold and ABC methods. The immunoreactivity of amelogenins was found in dense granules in the intercellular spaces between preameloblasts, and later over the fine- and coarse-textured material. The immunoreactivity was present over the cell organelles associated with the secretory pathway, as well as pale and dark lysosomes of the presecretory and secretory ameloblasts. Here the enamel was immunolabeled in the intercrystal spaces. The immunoreactivity in multivesicular bodies was stronger in preameloblasts than in secretory ameloblasts. In the region of second ruffle-ended ameloblasts at the maturation stage, the immunolabeling was intense in the ruffled-border, but in the rough endoplasmic reticulum and Golgi apparatus, the immunolabeling was much weaker than at the secretory stage. The immunolabeling for enamelins showed essentially the same intracellular topographical pattern as that for amelogenins by the secretory stage, but was weaker. The immunoreactivity was found mainly attached to the enamel crystals. Double immunostaining of amelogenins and enamelins revealed that both immunoreactivities were present over the same cell organelles associated with secretion and lysosomal systems. It is suggested that the presecretory and secretory ameloblasts are actively involved in the secretion, degradation and resorption of enamel proteins, and that multivesicular bodies and lysosomes in the cells take part in these processes. Ameloblasts are considered to be related to the synthesis of enamelins.

Bowringia milbraedii agglutinin. Specificity of binding to early processing intermediates of asparagine-linked oligosaccharide and use as a marker of endoplasmic reticulum glycoproteins.

We have purified a protein with hemagglutinating activity from the seeds of a West African legume, Bowringia milbraedii. The purified protein, designated BMA, has a native Mr = 38,000 on gel filtration and a subunit size of Mr = 16,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions. Hemagglutination was inhibited most effectively by Man alpha 1----2 linked sugars. Affinity chromatography of oligosaccharides on BMA-Sepharose showed that Man alpha 1----2Man alpha 1----2Man alpha 1----3Man beta 1----4GlcNAcol (where GlcNAcol is N-acetylglucosaminitol) and Man alpha 1----2Man alpha 1----3Man beta 1----4GlcNAcol were retarded on the column, whereas Man alpha 1----3Man beta 1----4GlcNAcol did not bind. Oligomannosidic-type glycans obtained by treatment of [3H] mannose-labeled baby hamster kidney cells with endo-beta-N-acetylglucosaminidase H bound more strongly to BMA-Sepharose and required 10 or 200 mM methyl-alpha-mannoside for elution. Oligosaccharides bearing the sequence Man alpha 1----2Man alpha 1----6Man alpha 1----6Man, i.e. Man9GlcNAc and certain isomers of Man8GlcNAc and Man7GlcNAc, bound more tightly than other Man8 GlcNAc and Man7GlcNAc isomers lacking this sequence. Man6GlcNAc and Man5GlcNAc were weakly bound. These results suggest that BMA binds preferentially to glycoproteins that are subjected to early steps of oligosaccharide processing in the endoplasmic reticulum but not to glycoproteins that are exposed to more extensive processing by Golgi mannosidases. Staining of permeabilized cells with BMA-chromophore conjugates revealed a reticular cytoplasmic pattern consistent with a preferential visualization of the endoplasmic reticulum. BMA staining was less evident in the juxtanuclear regions that were stained brightly with wheat germ agglutinin, a lectin that binds preferentially to sialylated glycoproteins located in Golgi compartments.

Intracellular distribution of Ca2+-Mg2+ adenosine triphosphatase (ATPase) in various tissues.

The cytochemical distribution of Ca2+-Mg2+-ATPase was studied ultrastructurally, using a lead capture method at pH 8.5 and compared in various tissues. In thymic, splenic and activated peripheral blood lymphocytes and in cultured HeLa cells activity was consistently localised on the nuclear envelope, endoplasmic reticulum, Golgi apparatus, mitochondria and weakly on centrioles, but not on the plasma membrane. Intracellular activity was similarly distributed in intestinal absorptive cells where activity was particularly strong in the Golgi apparatus, and in hepatocytes where, however, activity was generally weak. Intracellular activity was lacking in renal glomerular and tubular cells and in cerebellar neurons and neuroglia. Variable activity was present on the outer surface of the plasma membrane, particularly on the brush borders of intestinal and renal tubular absorptive cells, the basolateral invaginations of distal tubules and the bile canaliculi. Mitochondrial activity, when present, was inhibited by oligomycin. The localisation at different sites may represent biochemically different ATPases including endoplasmic reticular ATPase involved in intracellular calcium regulation, oligomycin-sensitive mitochondrial ATPase, dynein-like ATPase associated with centrioles and an ectoenzyme associated with cell surface specialisations.

Studies on the structure of the oligosaccharide chains of alpha 1-acid glycoprotein associated with rough membranes from rat liver.

The high mannose form of rat alpha 1-acid glycoprotein was isolated from rough membranes of rat liver using methods described previously. The high mannose glycopeptides were prepared by Pronase digestion, and oligosaccharides were isolated following digestion with endohexosaminidase-H. The structure of the carbohydrate chains of the high mannose glycopeptide and the oligosaccharides was examined by 300 MHz nuclear magnetic resonance spectroscopy. The glycopeptide contained a mixture of about equal amounts of AsnGlcNAc2Man9 and AsnGlcNAc2Man8. Analysis of the oligosaccharide fraction showed that it consisted of about equal amounts of GlcNAc Man9 and GlcNAc Man8; the GlcNAc Man8 fraction contained 85% of the "A" isomer (which was missing the terminal mannose from the middle antenna). The results suggested that mannose processing of alpha 1-acid glycoprotein in rough membranes of rat liver in vivo occurred only as far as the Man8 structure and that the "A" isomer was the main isomer formed.

Fluorescent labeling of endoplasmic reticulum.

The fluorescent molecule, DiOC6(3), can be used to label the ER in living cultured cells. The labeling procedures are simple and rapid, and in optimum conditions, the staining is bright and clear and bleaches slowly. The main disadvantage of the technique is toxicity. Photodynamic damage is probably the most serious of the toxic effects, because the damage can be relatively rapid and because the extent and nature of the damage during exposure to the light cannot be determined. To lessen the damage, the exposure of cells to light should be minimized. For many applications, it would be best to verify that cell function is normal during the experimental observations.

Immunocytochemistry of calciosomes in liver and pancreas.

Calciosomes are small cytoplasmic vacuoles identified in various nonmuscle cell types by their content of protein(s) similar to calsequestrin (CS), the Ca2+ storage protein of the muscle sarcoplasmic reticulum (SR). These entities have been interpreted as the "primitive" counterpart of the SR, and suggested to be the organelle target of inositol-1,4,5-triphosphate action (Volpe, P., K. H. Krause, S. Hashimoto, F. Zorzato, T. Pozzan, J. Meldolesi, and D. P. Lew. Proc. Natl. Acad. Sci. USA. 85:1091-1095). Immunoperoxidase and immunogold experiments carried out in both thick and ultrathin cryosections of rat hepatocytes and pancreatic acinar cells by using antimuscle CS antibodies revealed a specific labeling widely distributed in the entire cytoplasm, while nuclei were negative. Individual calciosomes appeared as small (105 nm) membrane-bound vacuoles intermingled with, and often apposed to ER cisternae and mitochondria. Other calciosomes were scattered in the Golgi area, in between zymogen granules and beneath the plasma membrane. The cumulative volume of the CS-positive organelles was measured to account for the 0.8 and 0.45% of the cytoplasm in liver and pancreas cells, respectively. The real total volume of the calciosome compartment is expected to be approximately twice as large. In hepatocytes, structures similar to CS-positive calciosomes were decorated by antibodies against the Ca2+ ATPase of muscle SR, while ER cisternae were not. By dual labeling, colocalization was revealed in 53.6% of the organelles, with 37.6% positive for the ATPase only. CS appeared preferentially confined to the content, and the Ca2+ ATPase to the contour of the organelle. The results suggested a partial segregation of the two antigens, reminiscent of their well-known segregation in muscle SR. Additional dual-label experiments demonstrated that hepatic calciosomes express neither two ER markers (cytochrome-P450 and NADH-cytochrome b5 reductase) nor the endolysosome marker, luminal acidity (revealed by 3-[2,4-dinitroanilino]-3'-amino-N-methyl dipropylamine). Calciosomes appear as unique cytological entities, ideally equipped to play a role in the rapid-scale control of the cytosolic-free Ca2+ in nonmuscle cells.

Localization of a renal kallikrein immunoreactive-like substance in rat ureter.

An antibody against rat kallikrein was produced in rabbits and its localization was studied in various organs of the rat to confirm its specificity. The distribution of immunoreactive kallikrein was studied in rat ureter by use of immunochemical techniques. Ureteral tissue was fixed in Zamboni's-glutaraldehyde fixative and immunostained with indirect immunofluorescence and the peroxidase-antiperoxidase (PAP) method for light and electron microscopy. Preabsorption of the primary polyclonal antiserum with purified rat urinary kallikrein and substitution with normal serum were used as controls. By light microscopy, kallikrein was localized in the lamina propria and in the adventitial connective tissue surrounding the entire ureter. Immunoelectron microscopy confirmed this immunolocalization. Immunoreactive kallikrein was concentrated in fibroblasts of connective tissue and was not present in collagen fibers. Immunoreactivity was associated with the Golgi complex, free polyribosomes, and rough endoplasmic reticulum. No immunostaining was observed in other subcellular components of fibroblasts.

Cytochemical analysis of the reconstitution of endoplasmic reticulum after microinjection of rat liver microsomes into Xenopus oocytes.

Fragments of rough and smooth endoplasmic reticulum purified from rat liver were injected into Xenopus oocyte cytoplasm. Light and electron microscopy, cytochemistry, immunocytochemistry, and enzyme assay were employed to determine the fate of heterologous membranes in the host cytoplasm. The in vivo-incubated microsomes disappeared in a time-dependent manner. Within 3 hr, rough microsomes were replaced by flattened ER cisternae and smooth microsomes were replaced by a network of anastomosing tubules. Polyclonal antibodies against rat liver microsomes and protein A-gold complexes were applied to glycol methacrylate sections of microinjected oocytes. Specific labeling was observed over discrete rough and smooth ER cisternae 3 hr after microinjection. Endogenous ER was not labeled by this technique, and label was not observed when sections were treated with pre-immune antibodies. Diaminobenzidene cytochemistry of microinjected rat lacrimal gland microsomes revealed enzyme activity in heterologous microsomes after 3 hr of in vivo incubation. Control injected microsomes (inactivated by heat denaturation) became associated with autophagic vacuoles, coincident with changes in lysosomal activity. Freshly isolated un-denatured microsomes did not provoke changes in lysosomal activity, and glucose-6-phosphatase activity associated with microinjected membranes could be detected 21 hr after in vivo incubation. Since rat liver microsomes reconstitute after in vivo incubation into cytoplasmic structures resembling those from which they were derived, we conclude that the microinjected membrane fragments act as templates for their own three-dimensional organization.