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PubMed1991

Compartmentalization and actin binding properties of ABP-50: the elongation factor-1 alpha of Dictyostelium.

ABP-50 is the elongation factor-1 alpha (EF-1 alpha) of Dictyostelium discoideum (Yang et al.: Nature 347:494-496, 1990). ABP-50 is also an actin filament binding and bundling protein (Demma et al.: J. Biol. Chem. 265:2286-2291, 1990). In the present study we have investigated the compartmentalization of ABP-50 in both resting and stimulated cells. Immunofluorescence microscopy shows that in addition to being colocalized with F-actin in surface extensions in unstimulated cells, ABP-50 exhibits a diffuse distribution throughout the cytosol. Upon addition of cAMP, a chemoattractant, ABP-50 becomes localized in the filopodia that are extended as a response to stimulation. Quantification of ABP-50 in Triton-insoluble and -soluble fractions of resting cells indicates that 10% of the total ABP-50 is recovered in the Triton cytoskeleton, while the remainder is in the soluble cytosolic fraction. Stimulation with cAMP increases the incorporation of ABP-50 into the Triton cytoskeleton. The peak of incorporation of ABP-50 at 90 sec is concomitant with filopod extension. Immunoprecipitation of the cytosolic ABP-50 from unstimulated cells using affinity-purified polyclonal anti ABP-50 results in the coprecipitation of non-filamentous actin with ABP-50. Purified ABP-50 binds to G-actin with a Kd of approximately 0.09 microM. The interaction between ABP-50 and G-actin is inhibited by GTP but not by GDP, while the bundling of F-actin by ABP-50 is unaffected by guanine nucleotides. We conclude that a significant amount of ABP-50 is bound to either G- or F-actin in vivo and that the interaction between ABP-50 and F-actin in the cytoskeleton is regulated by chemotactic stimulation.

Actins↗
PubMed2006

Glutamate leakage from a compartmentalized intracellular metabolic pool and activation of the lipoxygenase pathway mediate oxidative astrocyte death by reversed glutamate transport.

Astrocytes have essential roles for neuron survival and function, so that their demise in neurodegenerative insults, such as ischemia, deserves attention. A major event of the cell death cascade in ischemia is the reversed operation of excitatory amino acid transporters (EAAT), releasing glutamate. Cytotoxicity is conventionally attributed to extracellular glutamate accumulation. We previously reported that mimicking such dysfunction by EAAT substrate inhibitors, whose uptake induces glutamate release by heteroexchange, triggers glutathione (GSH) depletion and oxidative death of differentiated astrocytes in culture. Here we demonstrate that astrocyte death, although correlated with glutamate release, is not resulting from high extracellular glutamate-mediated toxicity. L-glutamate per se was gliotoxic only at concentrations much higher than the maximum reached with the potent EAAT substrate inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), and toxicity was lower. Moreover, high glutamate concentrations offered protection against PDC. Protection was also provided by L-aspartate, which is both transported by EAAT and metabolized into glutamate, and by inhibiting glutamine synthetase, which uses transported glutamate to synthesize glutamine. Neither D-aspartate, a metabolically inert EAAT substrate, nor compounds that can provide glutamate intracellularly but are not EAAT substrates offered protection. Interestingly, only the compounds providing protection prevented PDC-induced GSH depletion. These data strongly suggest that reversed uptake-mediated astrocyte death results from the leakage of glutamate from a compartmentalized intracellular metabolic pool specifically fuelled by EAAT, crucial for preserving GSH contents. In addition, we provide evidence for a minor contribution of the cystine-glutamate antiporter x(c) (-) but a major role of the 5-lipoxygenase pathway in this death mechanism.

Amino Acid Transport System X-AG↗
PubMed1993

Differential intracellular compartmentalization of phosphotyrosine phosphatases in a glial cell line: TC-PTP versus PTP-1B.

Intracellular levels of protein-phosphotyrosine are regulated by the protein-tyrosine phosphatase (PTP) family. Cellular compartmentalization may play an important role in modulating the function of these enzymes. The recent demonstration that PTP-1B is localized to the endoplasmic reticulum (Frangioni et al: Cell 68:545, 1992) is consistent with this proposition. In this study we have examined the intracellular distribution of TC-PTP in a glial cell line (C6). Using indirect immunofluorescence we have shown that this enzyme is distributed differently from PTP-1B and is mainly concentrated in the perinuclear region of these cells.

Amino Acid Sequence↗
PubMed1980

Compartmentalized ATP pools produced from adenosine are nuclear pools.

Incubation of African green monkey kidney (BS-C-1) cells and mouse fibroblasts (3T6) in the presence of adenosine for 4 hours resulted in increases in the nuclear compartment pools of adenosine 5'-triphosphate (ATP) and nuclear ATP/adenosine 5'-diphosphate (ADP) ratios. Adenine and inosine, which yield increases in total cellular ATP pools and ATP/ADP ratios similar to those promoted by adenosine, do not produce similar increases in the nuclear compartment. Adenosine-promoted increases in nuclear ATP pools were higher in the untransformed, serially propagated, BS-C-1 cells than in the spontaneously transformed 3T6 cells. Adenosine-promoted compartmentalized ATP pools in primary chick embryo fibroblasts were reduced upon transformation of these cells with Rous sarcoma virus, resulting in free mixing of all of the ATP pools synthesized from various salvage precursors. The growth regulatory properties of the nuclear compartment pools of adenine nucleotides is suggested by the big increases in nuclear ATPase and adenosine 5'-monophosphate (AMP) deaminase activities upon the entry of 3T6 cells into the S phase of their cycle. These enzymatic activities would tend to lower the nuclear ATP/ADP ratios and reduce the total adenine nucleotide pools in these nuclei respectively--conditions which were shown by earlier in vitro studies to be favorable to DNA replication.

Adenosine↗
PubMed2001

Intracellular compartmentation of pyruvate in primary cultures of cortical neurons as detected by (13)C NMR spectroscopy with multiple (13)C labels.

The intracellular compartmentation of pyruvate in primary cultures of cortical neurons was investigated by high resolution (13)C NMR using mixtures of different pyruvate precursors conveniently labeled with (13)C or unlabeled. Cells were incubated with 1-5 mM (1-(13)C, 1,2-(13)C(2) or U-(13)C(6)) glucose only or with mixtures containing 1.5 mM (1-(13)C or U-(13)C(6)) glucose, 0.25-2.5 mM (2-(13)C or 3-(13)C) pyruvate and 1 mM malate. Extracts from cells and incubation media were analyzed by (13)C NMR to determine the relative contributions of the different precursors to the intracellular pyruvate pool. When ((13)C) glucose was used as the sole substrate fractional (13)C enrichments and (13)C isotopomer populations in lactate and glutamate carbons were compatible with a unique intracellular pool of pyruvate. When mixtures of ((13)C) glucose, ((13)C) pyruvate and malate were used, however, the fractional (13)C enrichments of the C2 and C3 carbons of lactate were higher than those of the C2 and C3 carbons of alanine and depicted a different (13)C isotopomer distribution. Moreover, neurons incubated with 1 mM (1,2-(13)C(2)) glucose and 0.25-5 mM (3-(13)C) pyruvate produced exclusively (3-(13)C) lactate, revealing that extracellular pyruvate is the unique precursor of lactate under these conditions. These results reveal the presence of two different pools of intracellular pyruvate; one derived from extracellular pyruvate, used mainly for lactate and alanine production and one derived from glucose used primarily for oxidation. A red-ox switch using the cytosolic NAD(+)/NADH ratio is proposed to modulate glycolytic flux, controlling which one of the two pyruvate pools is metabolized in the tricarboxylic acid cycle when substrates more oxidized or reduced than glucose are used.

Animals↗
PubMed1997

Compartmentation of the type I regulatory subunit of cAMP-dependent protein kinase in cardiac ventricular muscle.

The species-dependent compartmentation of type I cAMP-dependent protein kinase (PKA I) and its dissociated regulatory subunit (RI) was examined in the heart by biochemical and immunohistochemical means. PKA I and RI were resolved from type II cAMP-dependent protein kinase and its regulatory subunit by DEAE-Sephacel chromatography of the supernatant and Triton X-100 soluble particulate fractions of heart homogenates. The relative amounts of holoenzymes and subunits were determined by cAMP-binding, protein kinase, 8-N3-[32P]cAMP photoaffinity labeling, and Western blot assays. Rat, rabbit, and guinea pig hearts all contained PKA I to varying degrees, but only in the supernatant fractions. Significant amounts of dissociated RI were found in the supernatant fractions, and to a lesser extent the particulate fractions, of these species. In contrast, though no PKA I was detected in the supernatant or particulate fractions of pig and beef heart, half of the cAMP-binding activity in the particulate fraction was attributed to RI. The results suggest that RI may associate with membrane fractions when it is not associated with the PKA catalytic subunit. Immunohistochemical studies of tissue sections from pig, beef, and rat cardiac ventricle using antibodies directed against RI also revealed species-dependent localization of RI. Cardiac myocyte intercalated discs were stained in pig and beef sections with additional sarcolemmal staining in beef sections. Rat ventricle, which contained large amounts of supernatant PKA I, showed nuclear staining. The localization of RI to cardiac myocyte intercalated discs and sarcolemma in certain species suggests a role(s) for this subunit in mediating cAMP-regulated events in these regions.

Animals↗
PubMed2001

Mag-indo1 affinity for Ca(2+), compartmentalization and binding to proteins: the challenge of measuring Mg(2+) concentrations in living cells.

A physicochemical study of the Mag-indo1 binding to Ca(2+) in solution showed that: (i) the characteristic fluorescence spectra of Ca(2+)-bound and Mg(2+)-bound Mag-indo1 are identical; (ii) two successive equilibria occur for increasing Ca(2+) concentrations; and (iii) the value of the dissociation constant of the first one, as determined by using a probe dilution protocol, amounts to 780 nM. In order to investigate the fluorescence level of Mag-indo1 trapped in cell organelles, fluorescence spectra of Mag-indo1-loaded fibroblasts were recorded before and after a digitonin permeabilization. Their resolution into cation-bound, protein-bound, and free Mag-indo1 characteristic spectra allowed measurement of the fluorescence intensities of these species. The intensities emitted from whole cells were compared to those emitted from organelles (assumed to be endoplasmic reticulum according to a DiOC(6) loading). The cation-bound Mag-indo1 fluorescence resulted partially (20 to 50%) from the cytosol for 30% of the cells, and totally from compartments for 70% of the cells. We found a concentration value of 500 nM for compartmentalized Ca(2+) and concluded that the Mag-indo1 binding to Ca(2+) is likely to affect drastically the Mg(2+) concentration measurements in cells. Moreover, we showed that the amount variation of protein-bound Mag-indo1 also affects Mg(2+) measurements when using the two-wavelength ratio method.

3T3 Cells↗
PubMed1997

Metabolic compartmentation in living cells: structural association of aldolase.

The glycolytic enzyme aldolase is concentrated in a domain around stress fibers in living Swiss 3T3 cells, but the mechanism by which aldolase is localized has not been revealed. We have recently identified a molecular binding site for F-actin on aldolase, and we hypothesized that this specific binding interaction, rather than a nonspecific mechanism, is responsible for localizing aldolase in vivo. In this report, we have used fluorescent analog cytochemistry of a site-directed mutant of aldolase to demonstrate that actin-binding activity localizes this molecule along stress fibers in quiescent cells and behind active ruffles in the leading edge of motile cells. The specific cytoskeletal association of aldolase could play a structural role in cytoplasm, and it may contribute to metabolic regulation, metabolic compartmentation, and/or cell motility. Functional duality may be a widespread feature among cytosolic enzymes.

3T3 Cells↗
PubMed2001

Subcellular compartmentalization and trafficking of the insulin-responsive glucose transporter, GLUT4.

Insulin increases glucose transport into cells of target tissues, primarily striated muscle and adipose. This is accomplished via the insulin-dependent translocation of the facilitative glucose transporter 4 (GLUT4) from intracellular storage sites to the plasma membrane. Insulin binds to the cell-surface insulin receptor and activates its intrinsic tyrosine kinase activity. The subsequent activation of phosphatidylinositol 3-kinase (PI 3-K) is well known to be necessary for the recruitment of GLUT4 to the cell surface. Both protein kinase B (PKB) and the atypical protein kinase C(lambda/zeta) (PKClambda/zeta) appear to function downstream of PI 3-K, but how these effectors influence GLUT4 translocation remains unknown. In addition, emerging evidence suggests that a second signaling cascade that functions independently of the PI 3-K pathway is also required for the insulin-dependent translocation of GLUT4. This second pathway involves the Rho-family GTP binding protein TC10, which functions within the specialized environment of lipid raft microdomains at the plasma membrane. Future work is necessary to identify the downstream effectors that link TC10, PKB, and PKClambda/zeta to GLUT4 translocation. Progress in this area will come from a better understanding of the compartmentalization of GLUT4 within the cell and of the mechanisms responsible for targeting the transporter to specialized insulin-responsive storage compartments. Furthermore, an understanding of how GLUT4 is retained within and released from these compartments will facilitate the identification of downstream signaling molecules that function proximal to the GLUT4 storage sites.

Adipocytes↗
PubMed1994

Control of nucleo-cytoplasmic HLA-DRA mRNA partitioning by interaction of a retention signal with compartmentalized proteins.

Transport of mRNA from the nucleus to the cytoplasm is still a poorly understood process in which RNA signal sequences and cognate RNA-binding proteins may be involved. We have analysed the transport of the mRNA encoded by HLA-DRA, a member of the immunologically important MHC class II multigene family. We report that, in transient transfection experiments, HLA-DRA mRNA molecules encompassing a signal situated in the 3' untranslated region predominantly accumulate in the nucleus. We also show that the RNA sequence involved interacts with compartmentalized proteins of either nuclear or cytoplasmic origin. Deletion of the mRNA region encompassing this retention site results both in the abrogation of protein binding and in the release of HLA-DRA mRNA into the cytoplasm. In addition, we have found that the distribution of these HLA-DRA mRNA binding proteins is different in different cell types; in particular, their pattern of expression in Ntera-2, a human teratocarcinoma cell line, is distinct from that observed in Raji, a human B-lymphoma cell line, and is modulated by growth in retinoic acid. We conclude that recognition of a mRNA retention signal by proteins located in different compartments on either side of the nuclear membrane may regulate the nucleo-cytoplasmic partitioning of HLA-DRA transcripts and, perhaps, of MHC class II mRNA in general.

Base Sequence↗
PubMed1999

Compartmentalization of the mitogen-activated protein kinase (MAPK) in hepatic endosomes: association with the internalized epidermal growth factor (EGF) receptor.

A pool of MAPK was found in hepatic plasma membrane (PM) and endosomes (ENs). After injection of a single dose of EGF (10 microg/100 g body weight), MAPK was detected in EGF receptor (EGFR) immunoprecipitates prepared from ENs. MAPK was detected in a time-dependent manner in EGFR immunoprecipitates that was coincident with the progressive concentration of the EGFR. The EGFR-associated MAPK was also detected by using an anti-phospho-MAPK suggesting that it was active. MAPK was present in wheat-germ agglutinin (WGA) eluates prepared from ENs and was maximally tyrosine-phosphorylated at the time peak of EGFR internalization. MAPK therefore is compartmentalized in PM and ENs of rat liver. A fraction of the endosomal MAPK was found to be associated with the internalized EGFR complexes, suggesting that it plays a role in the control of the EGFR activity at this locus.

Animals↗
PubMed1986

Compartmentation and functional mechanisms in myocardial failure and myocardial infarction.

Changes in compartmentation and specific mechanism in acute myocardial failure due to global ischemia and in regional myocardial ischemia in dog hearts are described. Ischemic failure was produced by periodic arrest of flow to supported heart preparations perfused with a fluorocarbon (FC-43). Sarcolemmal vesicles (SL) prepared from ischemic failing heart preparations exhibited diminished Ca++ binding and phosphorylation. TA-064, a beta-1-agonist partially abolished the reduction in Ca++ binding and phosphorylation of SL vesicles. The addition of cyclic-AMP (cAMP) and of protein kinase (PK) increased phosphorylation of SL vesicles obtained from non failing heart preparations. Combination of cAMP and of PK had the greatest effect. In contrast to myocardial failure, myocardial infarction is known to produce a large variety of specific disturbances in intermediary cardiac metabolism. Apparently in ischemic failing heart preparations, Ca++ binding and phosphorylation by SL are deficient. The results with TA-064 and isoproterenol suggest that phosphorylation of SL may play a role in the positive inotropic effect of beta-1-agonists.

Adenosine Triphosphate↗
PubMed1988

Effects of bone marrow ablation on compartmental prostaglandin synthesis by mononuclear phagocytes.

Mononuclear phagocyte functions were studied in mice selectively deprived of bone marrow and rendered profoundly monocytopenic by the administration of the bone seeking isotope, 89Sr. Characteristics of such mice include severe impairment of monocyte-M phi elicitation, ablation of C. parvum induction of PGSM but the persistence of resident peritoneal and pulmonary alveolar M phi populations; splenic M phi increase in number concomitantly with splenic hemopoiesis. Studies on compartmental regulation in this model suggest that the capacity of splenic M phi to synthesize and release PGE2 is dependent upon a function of the bone marrow and is not wholly determined by the local environment. The relationship of blood monocytes to PGSM is uncertain. In contrast to splenic M phi, the capacity of resident peritoneal M phi for eicosanoid synthesis appears to be independent of bone marrow function. Monocyte influx, moreover, does not appear necessary for the maintenance of the resident peritoneal and alveolar M phi populations. We do not yet know whether bone marrow ablation destroys a migratory precursor of PGSM or the source of a crucial regulatory agent. In conclusion, the observations discussed show that prostaglandin metabolism within the spleen is subject to extracompartmental influence. It is clearly important to determine the regulatory characteristics of individual M phi compartments and generalizations about functional properties of mononuclear phagocytes should be made with circumspection.

Animals↗
PubMed1995

Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypanoplasma borelli: an evolutionary scenario of subcellular compartmentation in kinetoplastida.

In Trypanoplasma borelli, a representative of the Bodonina within the Kinetoplastida, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in both the cytosol and glycosomes. This situation is similar to that previously found in Trypanosomatidae, belonging to a different Kinetoplastida suborder. In Trypanosomatidae different isoenzymes, only distantly related, are responsible for the activity in the two cell compartments. In contrast, immunoblot analysis indicated that the GAPDH activity in cytosol and glycosomes of T. borelli should be attributed to identical or at least very similar proteins related to the glycosomal GAPDH of Trypanosomatidae. Moreover, only genes related to the glycosomal GAPDH genes of Trypanosomatidae could be detected. All attempts to identify a gene related to the one coding for the trypanosomatid cytosolic GAPDH remained unsuccessful. Two tandemly arranged genes were found which are 95% identical. The two encoded polypeptides differ in 17 residues. Their sequences are 72-77% identical to the glycosomal GAPDH of the other Kinetoplastida and share with them some characteristic features: an excess of positively charged residues, specific insertions, and a small carboxy-terminal extension containing the sequence -AKL. This tripeptide conforms to the consensus signal for targeting of proteins to glycosomes. One of the two gene copies has undergone some mutations at positions coding for highly conserved residues of the active site and the NAD(+)-binding domain of GAPDH. Modeling of the protein's three-dimensional structure suggested that several of the substitutions compensate each other, retaining the functional coenzyme-binding capacity, although this binding may be less tight. The presented analysis of GAPDH in T. borelli gives further support to the assertion that one isoenzyme, the cytosolic one, was acquired by horizontal gene transfer during the evolution of the Kinetoplastida, in the lineage leading to the suborder Trypanosomatina (Trypanosoma, Leishmania), after the divergence from the Bodonina (Trypanoplasma). Furthermore, the data clearly suggest that the original GAPDH of the Kinetoplastida has been compartmentalized during evolution.

Amino Acid Sequence↗
PubMed1986

Compartmentation of the rough endoplasmic reticulum.

It has become evident during recent years that a wide variety of proteins are synthesized on membrane-bound polysomes, very many of which are not ultimately secreted from the cell. The majority of proteins appear to go through some form of post-translational modification before the final appearance of an 'active' product, and in some cases the polypeptide chain may be modified before the completed protein molecule is released from the ribosome. This then raises the question concerning the possibility of the organization of the rough endoplasmic reticulum into individual domains, or compartments, each of which may have the responsibility of performing definite and well defined functions. During recent years the behaviour of two subfractions of the rough endoplasmic reticulum in a variety of cell types and under a variety of conditions has been studied in order to gain insight into a possible compartmentation of this organelle. Throughout the studies disruption of cells has been performed by nitrogen cavitation. This technique was chosen in order to provide conditions of homogenization which were extremely reproducible since shearing forces, mechanical damage and the effects of local heating were eliminated. Endoplasmic reticulum (ER) membranes isolated from the post-mitochondrial supernatant have been separated into subfractions by centrifugation on discontinuous sucrose gradients. By virtue of their high density imparted by the association of ribosomes, rough ER (RER) membranes penetrate 1.4 M sucrose accumulating above either 2.0 M sucrose (light rough -LR membranes) or a cushion of 2.3 M sucrose (heavy rough -HR membranes). Smooth (S) membranes, which are virtually devoid of ribosomes, collect above 1.4 M sucrose. The HR, LR and S subfractions in MPC-11 cells differ in a number of respects: RNA/protein and RNA/phospholipid ratios, polysome profiles and marker enzymes. When cells were homogenized in buffer containing 25 mM KCl then all three ER subfractions were observed, however, when the buffer contained 100 mM KCl then only the LR and S subfractions were observed in gradients, radioactivity equivalent to that in the HR fraction was not recovered in the other two subfractions. Four times as many light chain immunoglobulin polypeptides were found associated with polysomes of HR membranes compared to LR membranes. The nuclear associated ER (NER), though very active in protein synthesis, was only 20% as active in the synthesis of light chain as the combined LR/HR fraction. Studies with MPC-11 cells showed that the relative amounts of the three ER subfractions were related to the phase of the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Phenotypic variations dictate the intracellular compartmentalization of doxorubicin in normal human bone marrow cells.

In vitro accumulation of doxorubicin in intracellular compartments of normal bone marrow cells was studied with the use of fluorescent microscopy. Both the cytoplasmic and nuclear compartments had distinguishable drug accessibility in the diverse hemopoietic series and in different stages of maturation of each lineage. Nuclei appeared to be more sheltered in the myelogranulocytic series than in the nucleated erythroid cells. Nuclei of activated phagocytic cells of the myelogranulocytic series and macrophages appeared to be the least accessible to doxorubicin uptake. These observations establish that phenotypic variations dictate the patterns of anthracyclines' subcellular compartmentalization. They also suggest that the molecular mechanism contributing to the intracellular trafficking of doxorubicin deserves more substantial investigation that may contribute to our understanding of drug resistance and sensitivity.

Bone Marrow↗
PubMed1990

Lactate compartmentation in hippocampal slices: evidence for a transporter.

Lactic acid accumulation has been implicated in the evolution of brain damage after ischemia. Since compartmentation of lactate may play a role in acid-base balance, lactate release from gerbil hippocampal slices was examined during a number of metabolic stresses including elevated [K+]e, ischemia, anoxia, and aglycemia. Slices were preincubated for 1 hr in artificial cerebrospinal fluid (ACSF) equilibrated with 95% O2/5% CO2 (pH 7.4 at 37 degrees C) and then transferred to tubes containing 300 microliters of test medium. The rate of lactate release in control slices was 9.64 nmol/min/mg protein and increased 2.6- and 3.2-fold in the presence of 60 mM potassium and anoxia, whereas the rate of lactate release was decreased by 50 and 25% during ischemia and aglycemia. Lactate release was temperature dependent and was only minimally influenced by removing Ca2+ or by adding 5 mM d-lactate to the ACSF. In contrast, pyruvate inhibited lactate release with an apparent Ki of 2.4 mM. The results suggest that lactate can be released from cells via a saturable and stereospecific lactate transporter with an apparent Km of 10.7 mM and Vmax of 43.7 nmol/mg protein/min. Such a relatively high-capacity transporter system can rapidly equilibrate brain lactate but is probably not involved in regulating intracellular acid-base balance.

Adenosine Triphosphate↗
PubMed1980

Endocytosis and compartmentation of peroxidases and cationized ferritin in neuroblastoma cells.

To investigate further the selectivity of retrograde axonal transport of horseradish peroxidase (HRP) isoenzymes previously observed in the rat central visual pathways, cultured mouse neuroblastoma cells (clone N18) were examined for selectivity of endocytosis of peroxidases and cationized ferritin in vitro. Differentiating N18 cells were incubated with proteins in various timing and pulse-chase experiments, and examined for the ultrastructural localization of endocytosed protein using cytochemical techniques. Semi-quantitative morphometry was performed on some of the samples. Major findings were as follows. (1) The protein was internalized into vesicles (coated and uncoated), short tubules and occasional small cup-shaped bodies within the first few minutes, and transferred via tubules and uncoated vesicles to secondary lysosome-like organelles (vacuoles, multivesicular bodies and dense bodies) from 5 to 15 min, with dense bodies representing the final site of protein accumulation. All the proteins tested were endocytosed through the same pathways in the soma and neurite of the cell. (2) There was no indication of a net orthograde or retrograde neuritic transport proteins. (3) There were induced increases in both vesicle and tubule formation as a result of protein endocytosis, with HRP isoenzyme C showing the greatest effect. (4) The apparent rate of internalization of HRP isoenzyme C into vesicles during the initial 5 min was significantly greater than for other peroxidases amd cationized ferritim. (5) Relatively more tubules than vesicles were involved in the uptake of protein as endocytosis was prolonged from 5 min to 8 h, with HRP isoenzyme C showing the largest effect. (6) There were indications of preferential compartmentation of endocytosed protein into certain organelles.

Animals↗