A discontinuous-sucrose-gradient procedure for isolating endosomes from mouse lymphoma cells has ... more A discontinuous-sucrose-gradient procedure for isolating endosomes from mouse lymphoma cells has been developed. After centrifugation, most organelles (especially mitochondria and lysosomes) are recovered in the denser fractions of the gradient, whereas a mixture of plasma membrane and endosomes is present at lighter densities. The endosome recovery in this fraction can be increased (by 100%) by (a) a mild trypsin treatment of the postnuclear supernatant and (b) loading the cell endosomes with a saturating concentration of low-density lipoproteins. Removal of the plasma-membrane contamination was achieved by preincubating the cells with a gold-ricin complex at 4 degrees C. On centrifugation, the gold-loaded membranes sediment to the bottom of the gradient. The endosome preparation isolated by these procedures is less than 6% contaminated by other organelles and contains 42% of internalized 125I-transferrin. We show that these isolated endosomes are functional, as displayed by their ...
Complexes of cell-surface receptors and their ligands are commonly internalized by endocytosis an... more Complexes of cell-surface receptors and their ligands are commonly internalized by endocytosis and enter a prelysosomal endosomal pathway for further processing. Fluorescence microscopy and video recording of living cells to trace the passage of ligand-receptor complexes has identified the endosomal compartment as an extensive network of tubular cisternae. Endocytosed material entering this reticulum enters discrete swellings, identified as multivesicular bodies by electron microscopy, which move along the reticulum towards the pericentriolar area.
... Permissions & Reprints. Chimeric molecules employing horseradish peroxidase as reporter e... more ... Permissions & Reprints. Chimeric molecules employing horseradish peroxidase as reporter enzyme for protein localization in the electron microscope. Colin Hopkins, Ade`le Gibson, Jane Stinchcombe and Clare Futter. Available online 16 December 2003. Excerpt. ...
The p75 neurotrophin receptor (p75NTR) plays multiple roles in neuronal physiology through intera... more The p75 neurotrophin receptor (p75NTR) plays multiple roles in neuronal physiology through interactions with many ligands and coreceptors. However, its intracellular neuronal trafficking prior to and after neurotrophin activation is still poorly characterized. We have previously shown that in response to nerve growth factor (NGF), p75NTR is retrogradely transported along the axons of motor neurons (MNs) in carriers shared with NGF, brain‐derived neurotrophic factor and the tyrosine kinase receptor TrkB. Here, we report that NGF does not enhance the internalization or degradation of p75NTR, which undergoes a rapid dynamin‐dependent and clathrin‐independent recycling process in MNs. Instead, incubation of cells with NGF leads to the redirection of a pool of plasma membrane p75NTR into clathrin‐coated pits. The subsequent internalization of p75NTR via clathrin‐mediated endocytosis, as well as the activity of Rab5, are essential for the sorting of the p75NTR‐containing endosomes to the ...
A new procedure has been developed for dissociating anterior pituitary tissue and producing a via... more A new procedure has been developed for dissociating anterior pituitary tissue and producing a viable suspension of single cells. The procedure involves incubation of small tissue blocks in 1 mg/ml trypsin (15 min), followed by incubation in 8 µg/ml neuraminidase and 1 mM EDTA (15 min), followed by mechanical dispersion. Cell yields are ∼55%, based on recovered DNA. By electron microscopy five types of secretory cells (somatotrophs, mammotrophs, thyrotrophs, gonadotrophs, and corticotrophs) plus endothelial and follicular cells can be identified and are morphologically well preserved up to 20 h after dissociation. Throughout this period, the cells incorporate linearly [3H]leucine into protein for up to 4 h at a rate 90% greater than hemipituitaries, and they synthesize, transport intracellularly, and release the two major pituitary secretory products, growth hormone and prolactin. Immediately after dissociation the cells' ability to respond to secretogogues (high K+ and dibutyryl...
With the use of poly-L-lysine, a method has been developed which induces acutely dissociated rat ... more With the use of poly-L-lysine, a method has been developed which induces acutely dissociated rat anterior pituitary cells to attach to glass and polyacrylamide surfaces. In these attached cells the recovery of the secretory response, which is impaired in acutely dissociated cells, has been followed, and it has been established that, in terms of their ability to secrete luteinizing hormone (LH) in response to the specific secretogogue luteinizing-hormone-releasing hormone (LHRH), the cells become maximally responsive after 48 h. The attached cells also allow the short-term kinetics of LH secretion to be followed with great facility; and, when cells allowed to recover for 48 h are used, it is shown that in response to LHRH the pattern of LH release is biphasic.
Rab GTPases are regulators of intracellular membrane traffic. We report a possible function of Ra... more Rab GTPases are regulators of intracellular membrane traffic. We report a possible function of Rab27a, a protein implicated in several diseases, including Griscelli syndrome, choroideremia, and the Hermansky-Pudlak syndrome mouse model, gunmetal. We studied endogenous Rab27a and overexpressed enhanced GFP-Rab27a fusion protein in several cultured melanocyte and melanoma-derived cell lines. In pigmented cells, we observed that Rab27a decorates melanosomes, whereas in nonpigmented cells Rab27a colocalizes with melanosome-resident proteins. When dominant interfering Rab27a mutants were expressed in pigmented cells, we observed a redistribution of pigment granules with perinuclear clustering. This phenotype is similar to that observed by others in melanocytes derived from the ashen and dilute mutant mice, which bear mutations in the Rab27a and MyoVa loci, respectively. We also found that myosinVa coimmunoprecipitates with Rab27a in extracts from melanocytes and that both Rab27a and myos...
Heptahelical, or G-protein-coupled, receptors control many cellular functions and normally consis... more Heptahelical, or G-protein-coupled, receptors control many cellular functions and normally consist of one polypeptide chain. In contrast, heptahelical receptors that belong to the long N-terminus, group B (LNB) family are cleaved constitutively into two fragments. The N-terminal fragments (NTFs) resemble cell-adhesion proteins and the C-terminal fragments (CTFs) are typical G-protein-coupled receptors (GPCRs) with seven transmembrane regions. However, the functional roles of this cleavage and of any subsequent NTF-CTF interactions remain to be identified. Using latrophilin, a well-studied member of the LNB family, we now demonstrate that cleavage is critical for delivery of this receptor to the cell surface. On the plasma membrane, NTF and CTF behave as separate membrane proteins involved, respectively, in cell-surface reception and signalling. The two fragments can also internalise independently. However, separated NTF and CTF can reassociate on solubilisation. Agonist binding to NTF on the cell surface also induces re-association of fragments and provokes signal transduction via CTF. These findings define a novel principle of structural and functional organisation of the cleaved, two-subunit GPCRs.
Synaptogenesis requires formation of trans‐synaptic complexes between neuronal cell‐adhesion rece... more Synaptogenesis requires formation of trans‐synaptic complexes between neuronal cell‐adhesion receptors. Heterophilic receptor pairs, such as neurexin Iβ and neuroligin, can mediate distinct intracellular signals and form different cytoplasmic scaffolds in the pre‐ and post‐synaptic neuron, and may be particularly important for synaptogenesis. However, the functions of neurexin and neuroligin depend on their distribution in the synapse. Neuroligin has been experimentally assigned to the post‐synaptic membrane, while the localization of neurexin remains unclear. To study the subcellular distribution of neurexin Iβ and neuroligin in mature cerebrocortical synapses, we have developed a novel method for the physical separation of junctional membranes and their direct analysis by western blotting. Using urea and dithiothreitol, we disrupted trans‐synaptic protein links, without dissolving the lipid phase, and fractionated the pre‐ and post‐synaptic membranes. The purity of these fractions...
The unusual adhesion G-protein-coupled receptors (aGPCRs) contain large extracellular N-terminal ... more The unusual adhesion G-protein-coupled receptors (aGPCRs) contain large extracellular N-terminal domains, which resemble cell-adhesion receptors, and C-terminal heptahelical domains, which may couple to G-proteins. These receptors are cleaved posttranslationally between these domains into two fragments (NTF and CTF). Using the aGPCR latrophilin 1, we previously demonstrated that the fragments behave as independent cell-surface proteins. Upon binding the agonist, ␣-latrotoxin (LTX), latrophilin fragments reassemble and induce intracellular signaling. Our observations raised important questions: is the aGPCR signaling mediated by reassembled fragments or by any non-cleaved receptors? Also, can the fragments originating from distinct aGPCRs form hybrid complexes? To answer these questions, we created two types of chimerical constructs. One contained the CTF of latrophilin joined to the NTF of another aGPCR, EMR2; the resulting protein did not bind LTX but, similar to latrophilin, could couple to G-proteins. In another construct, the NTF of latrophilin was fused with the C terminus of neurexin; this chimera bound LTX but could not signal via G-proteins. Both constructs were efficiently cleaved in cells. When the two constructs were co-expressed, their fragments could cross-interact, as shown by immunoprecipitation. Furthermore, LTX N4C induced intracellular Ca 2؉ signaling only in cells expressing both constructs but not each individual construct. Finally, we demonstrated that fragments of unrelated aGPCRs can be cross-immunoprecipitated from live tissues. Thus, (i) aGPCR fragments behave as independent proteins, (ii) the complementary fragments from distinct aGPCRs can cross-interact, and (iii) these cross-complexes are functionally active. This unusual cross-assembly of aGPCR fragments could couple cell-surface interactions to multiple signaling pathways.
Using gold complexes stabilized with a monoclonal antibody specific for the human transferrin rec... more Using gold complexes stabilized with a monoclonal antibody specific for the human transferrin receptor, the distribution of transferrin receptors on the surfaces of human epidermoid carcinoma A431 cells has been mapped at high resolution. On prefixed cells and cells incubated at 5°C, the receptors are predominantly within and around clathrin-coated microdomains near the free cell margin. By preincubating the cells with saturating concentrations of free antibody at 5°C and warming them to 37°C in the presence of the gold complexes, the appearance of new receptors in the membrane has been followed. The majority first appear near the free cell margin and then move centripetally. At first, they are monodisperse, but as they move toward the site of internalization they form loose aggregates. Within the immediate vicinity of the clathrin-coated microdomains the migrating receptors form closely packed, ordered aggregates. These observations indicate recycling transferrin receptors move to their site of internalization without cross-linking .
Biochemical and Biophysical Research Communications, 1981
Summary Exposure of quiescent cultures of bovine granulosa cells to epidermal growth factor (EGF)... more Summary Exposure of quiescent cultures of bovine granulosa cells to epidermal growth factor (EGF) stimulates the cells to enter S-phase. Addition of the calmodulin inhibitor, trifluoperazine (TFP), reduces the rate at which these cells enter S-phase. Half-maximal inhibition occurs with a concentration of 3 × 10−6M TFP. The inhibitory effect of TFP only occurs if the drug is added to the cell cultures during the first 5–6 hours of G1, but not if it is added during the second half of G1. Similarly, addition of TFP during S-phase does not affect the number of cells entering S-phase, however, it does reduce the rate of incorporation of 3H-thymidine into acid-precipitable material. TFP does not inhibit the binding of EGF to these cells.
In the prolactin cell of the eel adenohypophysis acid phosphatase activity occurs within the majo... more In the prolactin cell of the eel adenohypophysis acid phosphatase activity occurs within the majority of the Golgi cisternae and developing secretory granules. Acid phosphatase is also present within larger membrane-bound bodies, most of which are similar to the lyric dense bodies described in other cell types. In discussing the functional significance of this enzyme distribution particular attention is paid to its association with the secretory mechanisms of the prolactin cell. 23 Cell Sci. 3
Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endo-cytosi... more Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endo-cytosis in human epidermoid carcinoma (A431) cells. The isolation protocol involves density modification of endosome elements in A431 cells, caused by the receptor-dependent binding and internalization at 20 °C of colloidal gold-transferrin receptor antibody (B3/25) particles. The use of 125I-labelled gold–B3/25 provides a radioactive marker for the endosome compartment, the major peak being recovered at the bottom of a continuous sucrose gradient at a density of 1·23 g ml−1. Enzyme markers characteristic of other cytoplasmic compartments are present only in negligible amounts in this fraction and L-[35S]methionine-labelling of the cells indicates approximately a 200-fold enrichment of bilabelled gold–B3/25 versus protein. Electron microscopy of the endosome-rich fraction reveals that we have isolated a highly purified population of small gold-containing vesicles and tubules from which the tra...
The aim of the present study was to isofate different parts of the endocytic pathway in order to ... more The aim of the present study was to isofate different parts of the endocytic pathway in order to examine the rofe of epidermal growth factor (EGF)-receptor internalisation in mediating the biofogical effects of EGF. We have used an antibody to the transferrin receptor complexed with cofloidal gold to modify the density of the endocytic compartments so that they can be purified by sucrose density centrifugation. Using this technique, we have been able to isofate a highly purified preparation of endocytic vesicles from H.Ep.2 cells that contain internalised EGF. By employing pulse-chase protocofs, it is possible to isofate the different parts of the endocytic pathway and show that they are temporally distinct with regard to the processing of EGF. It should now be possible to examine interactions between the EGF receptor and intracellular substrates in different parts of the endocytic pathway.
Ricin translocation was demonstrated (using both fluorescence-and radiolabel-based assays) across... more Ricin translocation was demonstrated (using both fluorescence-and radiolabel-based assays) across the membrane of endosomes purified from mouse lymphocytes. Selectivity of the process was shown by the absence of translocation activity of transferrin and horseradish peroxidase used as membrane-bound and fluid-phase endosome labels, respectively. Endocytosed "'I-ricin translocation was found to be strictly ATP-(K , = 4 mM) and temperature-dependent, with up to 30% endosomal"'1-ricin appearing in the external medium after 2 h at 37 OC. No treatments neutralizing the acidic endosome pH (ammonium chloride, nigericin, chloroquine) significantly impaired ricin translocation, and the pH gradient across the endosome membrane is not required for this process. Chase experiments showed that the ability of "'I-ricin to translocate increases with its depth in the endocytic system (Le. plasma membrane << early endosomes < late endosomes). Both A and B ricin chains displayed translocation ability as demonstrated by the results of our assay on ricin, ricin B, transferrin-ricin A, and transferrin-ricin B conjugates. Biological activity of both ricin chains is preserved after translocation as shown by the inhibitory effect of the A chain on cell-free protein synthesis and the binding of the B chain to lactose-agarose. Ricin and diphtheria toxin (DT)' are powerful cellular poisons since a single molecule of these proteins is believed to be capable of killing a cell (1, 2). These toxins are made of polypeptide chains A and B. The B chain enables binding at the plasma membrane via recently identified receptors for DT (3) and galactose residues for ricin (1, 4, 5). The A chain is an enzyme which, once in the cytosol, inactivates protein * This work was supported by the Leukemia Research Fund, the Commission of The European Communities, and the Ministere des Affaires Etrangeres. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A discontinuous-sucrose-gradient procedure for isolating endosomes from mouse lymphoma cells has ... more A discontinuous-sucrose-gradient procedure for isolating endosomes from mouse lymphoma cells has been developed. After centrifugation, most organelles (especially mitochondria and lysosomes) are recovered in the denser fractions of the gradient, whereas a mixture of plasma membrane and endosomes is present at lighter densities. The endosome recovery in this fraction can be increased (by 100%) by (a) a mild trypsin treatment of the postnuclear supernatant and (b) loading the cell endosomes with a saturating concentration of low-density lipoproteins. Removal of the plasma-membrane contamination was achieved by preincubating the cells with a gold-ricin complex at 4 degrees C. On centrifugation, the gold-loaded membranes sediment to the bottom of the gradient. The endosome preparation isolated by these procedures is less than 6% contaminated by other organelles and contains 42% of internalized 125I-transferrin. We show that these isolated endosomes are functional, as displayed by their ...
Complexes of cell-surface receptors and their ligands are commonly internalized by endocytosis an... more Complexes of cell-surface receptors and their ligands are commonly internalized by endocytosis and enter a prelysosomal endosomal pathway for further processing. Fluorescence microscopy and video recording of living cells to trace the passage of ligand-receptor complexes has identified the endosomal compartment as an extensive network of tubular cisternae. Endocytosed material entering this reticulum enters discrete swellings, identified as multivesicular bodies by electron microscopy, which move along the reticulum towards the pericentriolar area.
... Permissions & Reprints. Chimeric molecules employing horseradish peroxidase as reporter e... more ... Permissions & Reprints. Chimeric molecules employing horseradish peroxidase as reporter enzyme for protein localization in the electron microscope. Colin Hopkins, Ade`le Gibson, Jane Stinchcombe and Clare Futter. Available online 16 December 2003. Excerpt. ...
The p75 neurotrophin receptor (p75NTR) plays multiple roles in neuronal physiology through intera... more The p75 neurotrophin receptor (p75NTR) plays multiple roles in neuronal physiology through interactions with many ligands and coreceptors. However, its intracellular neuronal trafficking prior to and after neurotrophin activation is still poorly characterized. We have previously shown that in response to nerve growth factor (NGF), p75NTR is retrogradely transported along the axons of motor neurons (MNs) in carriers shared with NGF, brain‐derived neurotrophic factor and the tyrosine kinase receptor TrkB. Here, we report that NGF does not enhance the internalization or degradation of p75NTR, which undergoes a rapid dynamin‐dependent and clathrin‐independent recycling process in MNs. Instead, incubation of cells with NGF leads to the redirection of a pool of plasma membrane p75NTR into clathrin‐coated pits. The subsequent internalization of p75NTR via clathrin‐mediated endocytosis, as well as the activity of Rab5, are essential for the sorting of the p75NTR‐containing endosomes to the ...
A new procedure has been developed for dissociating anterior pituitary tissue and producing a via... more A new procedure has been developed for dissociating anterior pituitary tissue and producing a viable suspension of single cells. The procedure involves incubation of small tissue blocks in 1 mg/ml trypsin (15 min), followed by incubation in 8 µg/ml neuraminidase and 1 mM EDTA (15 min), followed by mechanical dispersion. Cell yields are ∼55%, based on recovered DNA. By electron microscopy five types of secretory cells (somatotrophs, mammotrophs, thyrotrophs, gonadotrophs, and corticotrophs) plus endothelial and follicular cells can be identified and are morphologically well preserved up to 20 h after dissociation. Throughout this period, the cells incorporate linearly [3H]leucine into protein for up to 4 h at a rate 90% greater than hemipituitaries, and they synthesize, transport intracellularly, and release the two major pituitary secretory products, growth hormone and prolactin. Immediately after dissociation the cells' ability to respond to secretogogues (high K+ and dibutyryl...
With the use of poly-L-lysine, a method has been developed which induces acutely dissociated rat ... more With the use of poly-L-lysine, a method has been developed which induces acutely dissociated rat anterior pituitary cells to attach to glass and polyacrylamide surfaces. In these attached cells the recovery of the secretory response, which is impaired in acutely dissociated cells, has been followed, and it has been established that, in terms of their ability to secrete luteinizing hormone (LH) in response to the specific secretogogue luteinizing-hormone-releasing hormone (LHRH), the cells become maximally responsive after 48 h. The attached cells also allow the short-term kinetics of LH secretion to be followed with great facility; and, when cells allowed to recover for 48 h are used, it is shown that in response to LHRH the pattern of LH release is biphasic.
Rab GTPases are regulators of intracellular membrane traffic. We report a possible function of Ra... more Rab GTPases are regulators of intracellular membrane traffic. We report a possible function of Rab27a, a protein implicated in several diseases, including Griscelli syndrome, choroideremia, and the Hermansky-Pudlak syndrome mouse model, gunmetal. We studied endogenous Rab27a and overexpressed enhanced GFP-Rab27a fusion protein in several cultured melanocyte and melanoma-derived cell lines. In pigmented cells, we observed that Rab27a decorates melanosomes, whereas in nonpigmented cells Rab27a colocalizes with melanosome-resident proteins. When dominant interfering Rab27a mutants were expressed in pigmented cells, we observed a redistribution of pigment granules with perinuclear clustering. This phenotype is similar to that observed by others in melanocytes derived from the ashen and dilute mutant mice, which bear mutations in the Rab27a and MyoVa loci, respectively. We also found that myosinVa coimmunoprecipitates with Rab27a in extracts from melanocytes and that both Rab27a and myos...
Heptahelical, or G-protein-coupled, receptors control many cellular functions and normally consis... more Heptahelical, or G-protein-coupled, receptors control many cellular functions and normally consist of one polypeptide chain. In contrast, heptahelical receptors that belong to the long N-terminus, group B (LNB) family are cleaved constitutively into two fragments. The N-terminal fragments (NTFs) resemble cell-adhesion proteins and the C-terminal fragments (CTFs) are typical G-protein-coupled receptors (GPCRs) with seven transmembrane regions. However, the functional roles of this cleavage and of any subsequent NTF-CTF interactions remain to be identified. Using latrophilin, a well-studied member of the LNB family, we now demonstrate that cleavage is critical for delivery of this receptor to the cell surface. On the plasma membrane, NTF and CTF behave as separate membrane proteins involved, respectively, in cell-surface reception and signalling. The two fragments can also internalise independently. However, separated NTF and CTF can reassociate on solubilisation. Agonist binding to NTF on the cell surface also induces re-association of fragments and provokes signal transduction via CTF. These findings define a novel principle of structural and functional organisation of the cleaved, two-subunit GPCRs.
Synaptogenesis requires formation of trans‐synaptic complexes between neuronal cell‐adhesion rece... more Synaptogenesis requires formation of trans‐synaptic complexes between neuronal cell‐adhesion receptors. Heterophilic receptor pairs, such as neurexin Iβ and neuroligin, can mediate distinct intracellular signals and form different cytoplasmic scaffolds in the pre‐ and post‐synaptic neuron, and may be particularly important for synaptogenesis. However, the functions of neurexin and neuroligin depend on their distribution in the synapse. Neuroligin has been experimentally assigned to the post‐synaptic membrane, while the localization of neurexin remains unclear. To study the subcellular distribution of neurexin Iβ and neuroligin in mature cerebrocortical synapses, we have developed a novel method for the physical separation of junctional membranes and their direct analysis by western blotting. Using urea and dithiothreitol, we disrupted trans‐synaptic protein links, without dissolving the lipid phase, and fractionated the pre‐ and post‐synaptic membranes. The purity of these fractions...
The unusual adhesion G-protein-coupled receptors (aGPCRs) contain large extracellular N-terminal ... more The unusual adhesion G-protein-coupled receptors (aGPCRs) contain large extracellular N-terminal domains, which resemble cell-adhesion receptors, and C-terminal heptahelical domains, which may couple to G-proteins. These receptors are cleaved posttranslationally between these domains into two fragments (NTF and CTF). Using the aGPCR latrophilin 1, we previously demonstrated that the fragments behave as independent cell-surface proteins. Upon binding the agonist, ␣-latrotoxin (LTX), latrophilin fragments reassemble and induce intracellular signaling. Our observations raised important questions: is the aGPCR signaling mediated by reassembled fragments or by any non-cleaved receptors? Also, can the fragments originating from distinct aGPCRs form hybrid complexes? To answer these questions, we created two types of chimerical constructs. One contained the CTF of latrophilin joined to the NTF of another aGPCR, EMR2; the resulting protein did not bind LTX but, similar to latrophilin, could couple to G-proteins. In another construct, the NTF of latrophilin was fused with the C terminus of neurexin; this chimera bound LTX but could not signal via G-proteins. Both constructs were efficiently cleaved in cells. When the two constructs were co-expressed, their fragments could cross-interact, as shown by immunoprecipitation. Furthermore, LTX N4C induced intracellular Ca 2؉ signaling only in cells expressing both constructs but not each individual construct. Finally, we demonstrated that fragments of unrelated aGPCRs can be cross-immunoprecipitated from live tissues. Thus, (i) aGPCR fragments behave as independent proteins, (ii) the complementary fragments from distinct aGPCRs can cross-interact, and (iii) these cross-complexes are functionally active. This unusual cross-assembly of aGPCR fragments could couple cell-surface interactions to multiple signaling pathways.
Using gold complexes stabilized with a monoclonal antibody specific for the human transferrin rec... more Using gold complexes stabilized with a monoclonal antibody specific for the human transferrin receptor, the distribution of transferrin receptors on the surfaces of human epidermoid carcinoma A431 cells has been mapped at high resolution. On prefixed cells and cells incubated at 5°C, the receptors are predominantly within and around clathrin-coated microdomains near the free cell margin. By preincubating the cells with saturating concentrations of free antibody at 5°C and warming them to 37°C in the presence of the gold complexes, the appearance of new receptors in the membrane has been followed. The majority first appear near the free cell margin and then move centripetally. At first, they are monodisperse, but as they move toward the site of internalization they form loose aggregates. Within the immediate vicinity of the clathrin-coated microdomains the migrating receptors form closely packed, ordered aggregates. These observations indicate recycling transferrin receptors move to their site of internalization without cross-linking .
Biochemical and Biophysical Research Communications, 1981
Summary Exposure of quiescent cultures of bovine granulosa cells to epidermal growth factor (EGF)... more Summary Exposure of quiescent cultures of bovine granulosa cells to epidermal growth factor (EGF) stimulates the cells to enter S-phase. Addition of the calmodulin inhibitor, trifluoperazine (TFP), reduces the rate at which these cells enter S-phase. Half-maximal inhibition occurs with a concentration of 3 × 10−6M TFP. The inhibitory effect of TFP only occurs if the drug is added to the cell cultures during the first 5–6 hours of G1, but not if it is added during the second half of G1. Similarly, addition of TFP during S-phase does not affect the number of cells entering S-phase, however, it does reduce the rate of incorporation of 3H-thymidine into acid-precipitable material. TFP does not inhibit the binding of EGF to these cells.
In the prolactin cell of the eel adenohypophysis acid phosphatase activity occurs within the majo... more In the prolactin cell of the eel adenohypophysis acid phosphatase activity occurs within the majority of the Golgi cisternae and developing secretory granules. Acid phosphatase is also present within larger membrane-bound bodies, most of which are similar to the lyric dense bodies described in other cell types. In discussing the functional significance of this enzyme distribution particular attention is paid to its association with the secretory mechanisms of the prolactin cell. 23 Cell Sci. 3
Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endo-cytosi... more Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endo-cytosis in human epidermoid carcinoma (A431) cells. The isolation protocol involves density modification of endosome elements in A431 cells, caused by the receptor-dependent binding and internalization at 20 °C of colloidal gold-transferrin receptor antibody (B3/25) particles. The use of 125I-labelled gold–B3/25 provides a radioactive marker for the endosome compartment, the major peak being recovered at the bottom of a continuous sucrose gradient at a density of 1·23 g ml−1. Enzyme markers characteristic of other cytoplasmic compartments are present only in negligible amounts in this fraction and L-[35S]methionine-labelling of the cells indicates approximately a 200-fold enrichment of bilabelled gold–B3/25 versus protein. Electron microscopy of the endosome-rich fraction reveals that we have isolated a highly purified population of small gold-containing vesicles and tubules from which the tra...
The aim of the present study was to isofate different parts of the endocytic pathway in order to ... more The aim of the present study was to isofate different parts of the endocytic pathway in order to examine the rofe of epidermal growth factor (EGF)-receptor internalisation in mediating the biofogical effects of EGF. We have used an antibody to the transferrin receptor complexed with cofloidal gold to modify the density of the endocytic compartments so that they can be purified by sucrose density centrifugation. Using this technique, we have been able to isofate a highly purified preparation of endocytic vesicles from H.Ep.2 cells that contain internalised EGF. By employing pulse-chase protocofs, it is possible to isofate the different parts of the endocytic pathway and show that they are temporally distinct with regard to the processing of EGF. It should now be possible to examine interactions between the EGF receptor and intracellular substrates in different parts of the endocytic pathway.
Ricin translocation was demonstrated (using both fluorescence-and radiolabel-based assays) across... more Ricin translocation was demonstrated (using both fluorescence-and radiolabel-based assays) across the membrane of endosomes purified from mouse lymphocytes. Selectivity of the process was shown by the absence of translocation activity of transferrin and horseradish peroxidase used as membrane-bound and fluid-phase endosome labels, respectively. Endocytosed "'I-ricin translocation was found to be strictly ATP-(K , = 4 mM) and temperature-dependent, with up to 30% endosomal"'1-ricin appearing in the external medium after 2 h at 37 OC. No treatments neutralizing the acidic endosome pH (ammonium chloride, nigericin, chloroquine) significantly impaired ricin translocation, and the pH gradient across the endosome membrane is not required for this process. Chase experiments showed that the ability of "'I-ricin to translocate increases with its depth in the endocytic system (Le. plasma membrane << early endosomes < late endosomes). Both A and B ricin chains displayed translocation ability as demonstrated by the results of our assay on ricin, ricin B, transferrin-ricin A, and transferrin-ricin B conjugates. Biological activity of both ricin chains is preserved after translocation as shown by the inhibitory effect of the A chain on cell-free protein synthesis and the binding of the B chain to lactose-agarose. Ricin and diphtheria toxin (DT)' are powerful cellular poisons since a single molecule of these proteins is believed to be capable of killing a cell (1, 2). These toxins are made of polypeptide chains A and B. The B chain enables binding at the plasma membrane via recently identified receptors for DT (3) and galactose residues for ricin (1, 4, 5). The A chain is an enzyme which, once in the cytosol, inactivates protein * This work was supported by the Leukemia Research Fund, the Commission of The European Communities, and the Ministere des Affaires Etrangeres. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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