Cytochrome-C localizes in secretory granules in pancreas and anterior pituitary.

We used quantitative immunogold electron microscopy to evaluate the subcellular distribution of cytochrome-c in normal rat tissues, employing a wide variety of monoclonal and polyclonal antibodies against mammalian cytochrome-c. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows highly specific labeling of mitochondria in all tissues examined, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen and thyroid. In pancreatic acinar cells and anterior pituitary, however, there was also strong cytochrome-c reactivity in zymogen granules and growth hormone granules, respectively. In the pancreas, strong immunoreactivity is also detected in condensing vacuoles and in the acinar lumen. Immunocytochemical controls included (i) use of monoclonal antibodies to horse cytochrome-c which recognize an epitope not present in rat cytochrome-c, (ii) preadsorption of antibodies with purified cytochrome-c, and (iii) omission of the primary antibody. The indicated presence of cytochrome-c outside mitochondria in certain tissues under normal physiological conditions raises interesting questions concerning translocation mechanisms and the cellular functions of cytochrome-c.

Localization of P32 protein (gC1q-R) in mitochondria and at specific extramitochondrial locations in normal tissues.

P32 protein, also known as the gC1q receptor for complement component C1q, is a binding protein for nuclear pre-mRNA splicing factor SF2/ASF and numerous other nuclear and cell surface proteins, yet is targeted to the mitochondrial matrix compartment where these proteins are not present. In the present study, we use immunogold electron microscopy to evaluate the subcellular distribution of P32 protein (gC1q-R) in cultured cell lines and in rat tissues embedded in the acrylic resin LR Gold. Immunogold labeling of Raji lymphoma, CHO, human fibroblasts, HeLa and B-SC-1 cells shows reactivity primarily within mitochondria. Highly specific labeling of mitochondria is also obtained in rat tissues, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen, testes and thyroid. However, strong P32 (gClq-R) reactivity is also present in (i) zymogen granules, condensing vacuoles, endoplasmic reticulum, and on the cell surface of pancreatic acinar cells, (ii) on the cell surface of microvascular endothelial cells in pancreas and kidney, (iii) on the cell surface and in nuclei of splenic lymphocytes, and (iv) in the acrosome of developing spermatids in testes. Western immunoblots show that the polyclonal antibody to P32 (gC1q-R) used in this study reacts specifically with a 32-kDa protein in both purified pancreatic zymogen granules and in mitochondria, and no other proteins are reactive. These results provide evidence that P32 (gC1q-R) is a mitochondrial protein that also localizes outside mitochondria in certain cells and tissues under normal physiological conditions.

Localization of mitochondrial 60-kD heat shock chaperonin protein (Hsp60) in pituitary growth hormone secretory granules and pancreatic zymogen granules.

We used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of Hsp60 in rat tissues. Western blot analysis, employing both monoclonal and polyclonal antibodies raised against mammalian Hsp60, shows that only a single 60-kD protein is reactive with the antibodies in brain, heart, kidney, liver, pancreas, pituitary, spleen, skeletal muscle, and adrenal gland. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows strong labeling of mitochondria in all tissues. However, in the anterior pitutary and in pancreatic acinar cells, Hsp60 also localizes in secretory granules. The labeled granules in the pituitary and pancreas were determined to be growth hormone granules and zymogen granules, respectively, using antibodies to growth hormone and carboxypeptidase A. Immunogold labeling of Hsp60 in all compartments was prevented by preadsorption of the antibodies with recombinant Hsp60. Biochemically purified zymogen granules free of mitochondrial contamination are shown by Western blot analysis to contain Hsp60, confirming the morphological localization results in pancreatic acinar cells. In kidney distal tubule cells, low Hsp60 reactivity is associated with infoldings of the basal plasma membrane. In comparison, the plasma membrane in kidney proximal tubule cells and in other tissues examined showed only background labeling. These findings raise interesting questions concerning translocation mechanisms and the cellular roles of Hsp60.

Mitochondrial proteins at unexpected cellular locations: export of proteins from mitochondria from an evolutionary perspective.

Researchers in a wide variety of unrelated areas studying functions of different proteins are unexpectedly finding that their proteins of interest are actually mitochondrial proteins, although functions would appear to be extramitochondrial. We review the leading current examples of mitochondrial macromolecules indicated to be also present outside of mitochondria that apparently exit from mitochondria to arrive at their destinations. Mitochondrial chaperones, which have been implicated in growth and development, autoimmune diseases, cell mortality, antigen presentation, apoptosis, and resistance to antimitotic drugs, provide some of the best studied examples pointing to roles for mitochondria and mitochondrial proteins in diverse cellular phenomena. To explain the observations, we propose that specific export mechanisms exist by which certain proteins exit mitochondria, allowing these proteins to have additional functions at specific extramitochondrial sites. Several possible mechanisms by which mitochondrial proteins could be exported are discussed. Gram-negative proteobacteria, from which mitochondria evolved, contain a number of different mechanisms for protein export. It is likely that mitochondria either retained or evolved export mechanisms for certain specific proteins.

Mitochondrial-matrix proteins at unexpected locations: are they exported?

Many proteins that were originally characterized on the basis of non-mitochondrial functions have unexpectedly been shown to be identical to mitochondrial-matrix proteins. Most of these proteins are encoded by single nuclear genes and are initially targeted to the mitochondrial matrix. We suggest that mitochondria, as organelles of bacterial origin, possess specific mechanisms for export of proteins to other compartments.

Cloning and some novel characteristics of mitochondrial Hsp70 from Chinese hamster cells.

The cDNA for Chinese hamster mitochondrial Hsp70 (mHsp70) was cloned and sequenced using a polymerase chain reaction probe based on conserved regions in the Hsp70 family of proteins. The encoded protein consists of 679 amino acids which includes a N-terminal mitochondrial targeting sequence of 46 amino acids. The mHsp70 protein contains several sequence signatures that are characteristics of prokaryotic and eukaryotic organellar Hsp70 homologs. In a phylogenetic tree based on Hsp70 sequences, it branches with the gram-negative proteobacteria, supporting the endosymbiotic origin of mitochondria from this group of prokaryotes. The mHsp70 cDNA was transcribed and translated in vitro and its import into isolated rat heart mitochondria was examined. The precursor mHsp70 was converted into a mature form of lower molecular mass (approximately 71 kDa) which became resistant to trypsin digestion. The import of mHsp70 into mitochondria was not observed in the presence of an uncoupler of energy metabolism or when the N-terminal presequence was lacking. The cDNA for mHsp70 was expressed in Escherichia coli and a polyclonal antibody to the purified recombinant protein was raised. The antibody shows no cross-reactivity to recombinant cytosolic Hsp70 protein and in 2-D gel blots it reacted specifically with the mHsp70 protein only. In immunofluorescence experiments, the antibody predominantly labeled mitochondria, and the observed labeling pattern was identical to that seen with a monoclonal antibody to the mitochondrial Hsp60 chaperonin. The affinity-purified antibody to mHsp70 was also employed to examine the subcellular distribution of the protein by cryoelectron microscopy and the immunogold-labeling technique. In these experiments, in addition to mitochondria, labeling with mitochondrial Hsp70 antibody was also observed on the plasma membrane and in unidentified cytoplasmic vesicles and granules. These studies raise the possibility that similar to the Hsp60 chaperonin and a number of other mitochondrial proteins, mHsp70 may have an extramitochondrial role.

Cell surface localization of the 60 kDa heat shock chaperonin protein (hsp60) in mammalian cells.

To investigate whether the 60-kDa heat shock chaperonin protein (hsp60) is present on the surface of mammalian cells, we used immunogold labeling of intact cells and backscattered electron imaging to image gold particles. Chinese hamster ovary cells and the human leukemic CD4-positive T-cell line CEM-SS on glass coverslips were labeled using affinity-purified monoclonal and polyclonal antibodies specific for hsp60 and 30 nm gold markers. Cells were imaged using the scanning mode of the conventional transmission electron microscope. Backscattered electron imaging provided definitive identification of the gold markers while secondary electron imaging gave information on surface architecture. Labeling intensity was 250-800 gold particles per cell in Chinese hamster ovary cells and 600-2000 in CEM-SS human lymphoblasts. The finding of hsp60 on the cell surface of mammalian cells may signify chaperone involvement in surface functions.

Identification of endoplasmic reticulum in the primitive eukaryote Giardia lamblia using cryoelectron microscopy and antibody to Bip.

Giardia lamblia trophozoites contain a complex endomembrane system as demonstrated by fluorescence and cryoelectron microscopy. The endomembrane system was weakly detected in live cells using the fluorescent membrane dye 3,3'-dihexyloxacarbocyanine iodide. The definitive identification of endoplasmic reticulum required the development of a molecular label. We expressed Giardial Bip in Escherichia coli and raised a polyclonal antibody to the purified protein. In western blots, the antibody was specific for Giardial Bip and did not react with human, monkey and rodent homologs. By immunofluorescence microscopy in methanol fixed cells the antibody visualized tubular structures and other subcellular components that required characterization by electron microscopy. Using cryotechniques we directly demonstrate the presence of a complex endomembrane system at the ultrastructural level. In conjunction with Bip immunogold labeling of cryosections we identify: (1) endoplasmic reticulum cisternae and tubules; (2) stacked perinuclear membranes; and (3) Bip presence in the nuclear envelope. Both the endoplasmic reticulum and nuclear envelope were found either with or without a cleft region suggesting each may contain common specialized sub-regions. In stacked perinuclear membranes, which may represent either multilamellar endoplasmic reticulum or a Golgi apparatus, Bip labeling was restricted to peripheral layers, also suggesting specialized sub-regions. Labeled endomembrane systems could be observed associated with microtubule structures, including axonemes and the adhesive disk. The presence of an extensive endomembrane system in Giardia lamblia, which represents one of the earliest diverging eukaryotic species, supports the view that both the nucleus and endomembrane system co-evolved in a common ancestor of eukaryotic cells.

Prokaryotic homolog of tubulin? Consideration of FtsZ and glyceraldehyde 3-phosphate dehydrogenase as probable candidates.

The bacterial FtsZ protein has recently been suggested as a probable prokaryotic homolog of the tubulin family of proteins (Cell 80, 1995, 367-370). We have compared the sequence similarity of tubulins to FtsZ and another protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Both these proteins exhibited similar levels of sequence identity to the tubulins, which in a few cases was indicated to be significant. We report that incubation of the GAPDH in microtubule assembly buffer causes its polymerization into filamentous structures. In eukaryotic cells, GAPDH is known to be associated with cytoskeletal structures and it binds specifically to both tubulins and colchicine. The latter is a distinctive characteristic of the tubulin family of proteins. These observations indicate that similar to the FtsZ proteins, GAPDH also exhibits a number of intriguing similarities to the tubulins. Whether any of these proteins truly represent the prokaryotic homolog of tubulin, however, is unclear at present.

Immunoelectron microscopic localization of the 60-kDa heat shock chaperonin protein (Hsp60) in mammalian cells.

The subcellular distribution of the 60-kDa heat shock protein (Hsp60) was examined in a variety of mammalian cells and tissues, including Chinese hamster ovary cells, human fibroblasts, B-SC-1 kidney cells, Daudi Burkitt's lymphoma cells, and rat liver, by immunogold electron microscopy employing six different monoclonal and polyclonal antibodies that are specific for Hsp60. In cryosections or LR Gold sections of different cultured cells, intense labeling of mitochondria was obtained, typically 200-500 gold particles per mitochondrion and accounting for 80-85% of the total gold particles. In addition, however, in all cell types and using all of the antibodies, about 15-20% of the labeling due to Hsp60 was seen at discrete extramitochondrial sites. Such sites included those in close proximity to mitochondrial outer membranes, foci on endoplasmic reticulum, on the cell surface, and in unidentified vesicles. In cryosections of rat liver, specific labeling due to Hsp60 antibodies was also observed within peroxisomes. Labeling of all cellular components by these antibodies could be prevented by preadsorption with purified recombinant mitochondrial Hsp60 indicating that the labeling is specific for Hsp60. Biotin labeling of cell surface proteins results in biotinylation of Hsp60 as analyzed by immunoprecipitation and Western blots, providing further evidence for Hsp60 presence on the plasma membrane. Immunoprecipitation experiments with Hsp60 antibodies show that under normal conditions no detectable precursor Hsp60 protein is present in cells. However, in cells treated with the potassium ionophore nonactin, which blocks mitochondrial import, only the precursor form of Hsp60 accumulates, providing evidence that at least partial mitochondrial import of Hsp60 is necessary for its maturation. These results also provide evidence that no other 60-kDa protein other than mitochondrial Hsp60 is recognized by the antibodies used for electron microscopy. These findings raise interesting questions concerning the possible role of Hsp60 at extramitochondrial sites.

Immunoelectron microscopy of Giardia lamblia cytoskeleton using antibody to acetylated alpha-tubulin.

Giardia lamblia trophozoites contain acetylated alpha-tubulin but lack detectable levels of tyrosinolated alpha-tubulin, as demonstrated in immunoblots with monoclonal antibodies specific for these tubulin forms. By immunofluorescence microscopy, acetylated alpha-tubulin is localized in axonemes, median bodies and in the adhesive disk. Post-embeddment immunogold labeling of thin sections of cells was used to evaluate acetylation at the level of individual microtubules by electron microscopy. Cells were fixed with glutaraldehyde and embedded in the acrylic resin LR Gold. Results indicate all microtubules in adhesive disk, axonemes, basal bodies, funis and the median bodies contain acetylated alpha-tubulin. Unlike immunofluorescence labeling, all microtubules of the adhesive disk and the funis could be gold labeled. No nonspecific labeling of the cytoplasm or of structures other than microtubules was observed. Acetylated microtubules in G. lamblia do not appear to be a subset of microtubules and acetylation appears uniform along the entire length of individual microtubules. Acetylation and the tyrosinolation state of microtubules in Giardia are discussed in the context of microtubule stability and crosslinked features of the cytoskeleton.

Presence and cellular distribution of a 60-kDa protein related to mitochondrial hsp60 in Giardia lamblia.

Giardia lamblia trophozoites contain a 60-kDa protein recognized in immunoblots by antibody to mammalian hsp60, a protein localized in mitochondria in eukaryotic cells. The cellular distribution of this protein is evaluated by immunofluorescence microscopy using monoclonal antibody to human hsp60, polyclonal antibody to rodent hsp60, and 2 monoclonal antibodies to mycobacterial 65-kDa antigen, a prokaryotic hsp60 homolog. All of these antibodies, except 1, which is specific for prokaryotic hsp60, give a punctate labeling pattern throughout the cytoplasm, indicating that the 60-kDa protein is concentrated at discrete sites in the cytoplasm. The polyclonal hsp60 antiserum reveals additional punctate labeling colocalized on axonemes of the anterior flagella. Postembedment immunogold labeling and electron microscopy confirm that the antigen is clustered at foci in the cytoplasm but show no evidence of association with a membranous organelle. The hsp60 reactivity is also observed on anterior axonemes within the cytoplasm and on the adhesive disc. Hoechst 33258 DNA staining as well as electron microscopy give no evidence of endosymbionts, so a false positive due to a prokaryotic hsp60 homolog is unlikely. The presence of an hsp60-related protein in G. lamblia raises interesting questions concerning its origin.

Changes in mitochondrial shape and distribution induced by ethacrynic acid and the transient formation of a mitochondrial reticulum.

We have examined the effect of ethacrynic acid on mitochondrial morphology and distribution as well as on cellular toxicity in cultured human fibroblasts, African Green Monkey B-SC-1 kidney cells, and Chinese hamster ovary cells. Treatment of the above cells with 66 microM ethacrynic acid causes no reduction in cell viability after 2 h but is cytotoxic upon prolonged (6-7 days) exposure. Ethacrynic acid treatment for up to 2 h is found to cause novel shape changes and redistribution of mitochondria, as assessed by immunofluorescence and electron microscopy. Early effects include the transient formation of a mitochondrial reticulum involving the majority of mitochondria, and these reticula are aligned along microtubules. At later times within 2 h, mitochondrial distributions become disoriented (show no association with microtubules), and an aggregation and final positioning of mitochondria around the nucleus is observed. Whole mount electron microscopy shows that mitochondria in treated cells increase in length and form junctions, indicating reticula result from mitochondrial fusion. Electron microscopy of sections through ethacrynic acid induced reticula demonstrates structural continuity in mitochondria at branch points and the presence of regular cristae. Staining of endoplasmic reticulum and mitochondria in intact cells with the cyanine dye 3,3'-dihexyloxacarbocyanine iodide provides evidence of concurrent aggregation of endoplasmic reticulum. Rhodamine 123 staining of living cells followed by immunofluorescent labeling of mitochondria in the same cells indicates that all mitochondria retain a transmembrane potential during the drug-induced shape changes and redistributions. The described effects of ethacrynic acid on mitochondrial morphology as well as on cellular toxicity are completely prevented by 0.5 mM dithiothreitol, indicating that ethacrynic acid is acting as a sulfhydryl reagent to produce the observed effects. The above observations also indicate that ethacrynic acid effects on mitochondrial morphology are an early event in the drug-induced cytotoxicity. The generation of varied mitochondrial morphologies by fusion and fission of mitochondria and its modulation by agents such as ethacrynic acid are discussed.

Interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules--a quadruple fluorescence labeling study.

To study the interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules, we have developed a quadruple fluorescence labeling procedure to visualize all four structures in the same cell. We applied this approach to study cellular organization in control cells and in cells treated with the microtubule drugs vinblastine or taxol. Endoplasmic reticulum was visualized by staining glutaraldehyde-fixed cells with the dye 3,3'-dihexyloxacarbocyanine iodide. After detergent permeabilization, triple immunofluorescence was carried out to specifically visualize mitochondria, vimentin intermediate filaments, and microtubules. Mitochondria in human fibroblasts were found to be highly elongated tubular structures (lengths up to greater than 50 microns), which in many cases were apparently fused to each other. Mitochondria were always observed to be associated with endoplasmic reticulum, although endoplasmic reticulum also existed independently. Intermediate filament distribution could not completely account for endoplasmic reticulum or mitochondrial distributions. Microtubules, however, always codistributed with these organelles. Microtubule depolymerization in vinblastine treated cells resulted in coaggregation of endoplasmic reticulum and mitochondria, and in the collapse of intermediate filaments. The spatial distributions of organelles compared with intermediate filaments were not identical, indicating that attachment of organelles to intermediate filaments was not responsible for organelle aggregation. Mitochondrial associations with endoplasmic reticulum, on the other hand, were retained, indicating this association was stable regardless of endoplasmic reticulum form or microtubules. In taxol-treated cells, endoplasmic reticulum, mitochondria, and intermediate filaments were all associated with taxol-stabilized microtubule bundles.

Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes.

Microtubule assembly in vivo was studied by hapten-mediated immunocytochemistry. Tubulin was derivatized with dichlorotriazinylaminofluorescein (DTAF) and microinjected into living, interphase mammalian cells. Sites of incorporation were determined at the level of individual microtubules by double-label immunofluorescence. The haptenized tubulin was localized by an anti-fluorescein antibody and a second antibody conjugated with fluorescein. Total microtubules were identified by anti-tubulin and a secondary antibody conjugated with rhodamine. Contrary to recent studies (Salmon, E. D., et al., 1984, J. Cell Biol., 99:2165-2174; Saxton, W. M., et al., 1984, J. Cell Biol., 99:2175-2186) which suggest that tubulin incorporates all along the length of microtubules in vivo, we found that microtubule assembly in interphase cells was in vivo, as in vitro, an end-mediated process. Microtubules that radiated out toward the cell periphery incorporated the DTAF-tubulin solely at their distal, that is, their plus ends. We also found that a proportion of the microtubules connected to the centrosomes incorporated the DTAF-tubulin along their entire length, which suggests that the centrosome can nucleate the formation of new microtubules.

Steroid modulation of human serum albumin binding properties. A spin label study.

The binding isotherm and unique electron spin resonance spectral characteristics of a monoanionic spin label (1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) and a dianionic spin label (1-glutamate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) are used to prove the steroid modulation of serum albumin binding properties. Effects of a selected number of steroids (progesterone, testosterone, estradiol, aldosterone, estriol, corticosterone, deoxycorticosterone, hydrocortisone, and cortisone) on the binding isotherm of the monoanionic spin label binding to serum albumin have been determined. At the steroid/albumin ratio of 0.5 to 1, progesterone, testosterone, and estradiol enhance binding of the spin label at all concentrations studied. However, the remaining steroids exert an inhibitory effect at low spin label/albumin ratios and an enhancement effect at high spin label/albumin ratios. Progesterone and cortisone effects on the resonance spectra of the spin label bound to serum albumin confirm the enhancement and displacement properties of these ligands. Thus, like fatty acids, steroids may bind to either the primary or secondary bilirubin binding sites and also allosterically perturb the binding properties of serum albumin. The in vivo importance of the steroid-albumin interaction is discussed.

Fatty acid enhancement of human serum albumin binding properties. A spin label study.

The introduction of a new spin-labeled anionic ligand, 1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene, is reported. Under the experimental conditions, the first molar equivalent of this ligand is 93% bound to human serum albumin. With the addition of palmitate, the free spin label concentration decreases greatly, by almost 80%, in the presence of a fatty acid:albumin ratio of 3:1 to 4:1. The spectral characteristics of the bound spin label are also affected. The changes seen in the intensity of and the splitting between the high and low field extrema are indicative of perturbations of the protein molecule. It is seen then that the binding of each molar equivalent of fatty acid effects the conformation state of albumin and allosterically affects albumin binding properties. Computer spectral subtractions, furthermore, suggest that the binding of the first molar equivalent of palmitate specifically increases the affinity of the first two 1-gamma-amino-butyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene binding sites. The present results indicate that fluctuations in serum free fatty acid levels within the physiological range may have a major modulatory effect on the free serum levels of certain drugs and/or physiological substances that bind to albumin.