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C R Hopkins

Publications and source records attributed to C R Hopkins.

At least 19 recordsLinked to original sources
PubMed1998

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells.

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.

Animals↗
PubMed1998

In polarized MDCK cells basolateral vesicles arise from clathrin-gamma-adaptin-coated domains on endosomal tubules.

Human transferrin receptors (TR) and receptors for polymeric immunoglobulins (pIgR) expressed in polarized MDCK cells maintain steady-state, asymmetric distributions on the separate basolateral and apical surfaces even though they are trafficking continuously into and across these cells. The intracellular mechanisms required to maintain these asymmetric distributions have not been located. Here we show that TR and pIgR internalize from both surfaces to a common interconnected endosome compartment that includes tubules with buds coated with clathrin lattices. These buds generate vesicles that carry TR to the basolateral border. The lattices contain gamma-adaptin and are dispersed by treatment with brefeldin A (BFA). Since BFA treatment abrogates the vectorial trafficking of TR in polarized MDCK cells, we propose that the clathrin-coated domains of the endosome tubules contain the polarized sorting mechanism responsible for their preferential basolateral distribution.

Adaptor Protein Complex gamma Subunits↗
PubMed1998

Signal-dependent trafficking of beta-amyloid precursor protein-transferrin receptor chimeras in madin-darby canine kidney cells.

We have investigated the intracellular trafficking of a chimeric molecule consisting of the cytoplasmic domain of the beta-amyloid precursor protein (APP) and the transmembrane region and external domain of the human transferrin receptor (TR) in Madin-Darby canine kidney cells. Newly synthesized APP-TR chimeras are selectively targeted to the basolateral surface by a tyrosine-dependent sorting signal in the APP cytoplasmic tail. APP-TR chimeras are then rapidly internalized from the basolateral surface and a significant fraction ( approximately 20-30%) are degraded. Morphological studies show that APP-TR chimeras internalized from the basolateral surface are found in tubulo-vesicular endosomal elements, internal membranes of multivesicular bodies, and lysosomes. APP-TR chimeras are also found in 60-nm diameter vesicles previously shown to selectively deliver wild-type TR to the basolateral surface; this result is consistent with the fact that 90% of internalized chimeras that are not degraded are selectively recycled back to the basolateral surface. APP-TR chimeras internalized from the apical surface are selectively transcytosed to the basolateral surface underscoring the importance of basolateral sorting in the endocytic pathway for maintaining the polarized phenotype. Tyr-653, an important element of the YTSI internalization signal in the APP cytoplasmic domain, is required for basolateral sorting in the biosynthetic and endocytic pathways. However, the structural features for basolateral sorting differ from those required for internalization.

Amino Acid Sequence↗
PubMed1998

The kinetics of mannose 6-phosphate receptor trafficking in the endocytic pathway in HEp-2 cells: the receptor enters and rapidly leaves multivesicular endosomes without accumulating in a prelysosomal compartment.

We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor-epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome-lysosome pathway in these cells.

Biological Transport↗
PubMed1996

Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes.

We have followed the transfer of EGF-EGF receptor (EGFR) complexes from endosomal vacuoles that contain transferrin receptors (TfR) to lysosome vacuoles identified by their content of HRP loaded as a 15-min pulse 4 h previously. We show that the HRP-loaded lysosomes are lysosomal-associated membrane protein-1 (LAMP-1) positive, mannose-6-phosphate receptor (M6PR) negative. and contain active acid hydrolase. EGF-EGFR complexes are delivered to these lysosomes intact and are then rapidly degraded. Preactivating the HRP contained within the preloaded lysosomes inhibits the delivery of EGFR and degradation of EGF, and results in the accumulation of EGFR-containing multivesicular bodies (MVB). With time these accumulating MVB undergo a series of maturation changes that include the loss of TfR, the continued recruitment of EGFR, and the accumulation of internal vesicles, but they remain LAMP-1 and M6PR negative. The mature MVB are often seen to make direct contact with lysosomes containing preactivated HRP, but their perimeter membranes remain intact. Together our observations suggest that the transfer of EGF-EGFR complexes from the TfR-containing endosome compartment to the lysosomes that degrade them employs a single vacuolar intermediate, the maturing MVB, and can be achieved by a single heterotypic fusion step.

Antigens, CD↗
PubMed1996

Apical and basolateral endosomes of MDCK cells are interconnected and contain a polarized sorting mechanism.

We have evaluated transcytotic routes in MDCK cells for their ability to generate a polarized surface distribution of trafficking proteins by following the intracellular sorting of transferrin receptors (TRs). We find that the selective basolateral expression of TRs is maintained in the face of extensive trafficking between the apical and basolateral surfaces. Biochemical studies of receptors loaded with tracer under conditions approaching steady state indicate that TRs internalized from the two surfaces are extensively colocalized within MDCK cells and that both populations of receptors are selectively delivered to the basolateral surface. Tailless TRs in which the cytoplasmic domain has been deleted display an unpolarized cell surface distribution and recycle in an unpolarized fashion. We show by EM that wild-type receptors internalized from each surface are colocalized within endosomal elements distributed throughout the cytoplasm. By preloading endosomal elements directly accessible from the basolateral surface with transferrin (Tf)-HRP, we show that apically internalized TRs rapidly enter the same compartment. We also show that both transcytosing (apically internalized) and recycling (basolaterally internalized) TRs are delivered to the basolateral border by a distinctive subset of exocytotic, 60-nm-diam vesicles. Together, the biochemical and morphological data show that apical and basolateral endosomes of MDCK cells are interconnected and contain a signal-dependent polarized sorting mechanism. We propose a dynamic model of polarized sorting in MDCK cells in which a single endosome-based, signal-dependent sorting step is sufficient to maintain the polarized phenotype.

Amino Acid Sequence↗
PubMed1995

Newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell surface.

It is well established that a proportion of newly synthesized lysosomal enzymes and class II major histocompatibility complex antigens are delivered directly to the endocytic pathway from the Golgi complex. Here we show that a significant proportion of newly synthesized transferrin receptors can be detected in endosomes before reaching the cell surface. These newly synthesized transferrin receptors are delivered to the endosome more efficiently than either constitutively secreted soluble proteins or glycophosphatidylinositol-anchored plasma membrane proteins suggesting that their transfer to the endosome is signal-dependent. Identification of a signal-dependent transfer step for proteins like the transferrin receptor operating on the exocytic pathway has important implications for membrane biogenesis, especially in the establishment of cell surface polarity.

Biological Transport↗
PubMed1995

Thyroid and parathyroid imaging.

This article reviews the current roles of imaging in the diagnosis of thyroid and parathyroid disorders, with an emphasis on ultrasound evaluation. Imaging of the thyroid and parathyroid can be performed with nuclear medicine, ultrasound, CT, and MRI. Indications for thyroid and parathyroid imaging studies have recently changed. The availability of experienced endocrine surgeons, as well as the development of accurate laboratory tests, fine-needle aspiration (FNA) biopsy, and high-resolution ultrasound, have dramatically influenced the evaluation of thyroid and parathyroid disease. In patients with thyroid nodular disease, a clinical examination by an experienced clinician with appropriate lab values and palpation-guided FNA is the current diagnostic protocol of choice. Ultrasound evaluation of high-risk patients and ultrasound-guided FNA both augment this protocol when necessary. In patients with diffuse thyroid glandular disease, radionuclide imaging and color Doppler sonography both can be used for evaluation. When preoperative imaging is clinically necessary, sonography or scintigraphy can be used for parathyroid adenoma localization in patients with primary hyperparathyroidism. The recent development of technetium-99m sestamibi as a parathyroid imaging agent has improved the sensitivity of scintigraphy for parathyroid adenoma localization. Ultrasound and radionuclide imaging have also become valuable imaging techniques for parathyroid localization in patients with recurrent or persistent hyperparathyroidism.

Adenoma↗
PubMed1995

Anterograde and retrograde traffic between the rough endoplasmic reticulum and the Golgi complex.

The transfer of newly synthesized membrane proteins moving from the rough endoplasmic reticulum (RER) to the Golgi complex has been studied by electron microscopy in HEp-2 cells transfected with cDNAs for chimeric proteins. These proteins consist of a reporter enzyme, horseradish peroxidase (HRP), anchored to the transmembrane domains of two integral membrane proteins, the transferrin receptor and sialyl-transferase. The chimeras are distributed throughout the nuclear envelope, RER, vesicular tubular clusters (VTCs) and a network of tubules in the cis-Golgi area. At 20 degrees C tubules containing chimera connect the RER to the VTCs and to the cis-Golgi network. On transfer to 37 degrees C in the presence of dithiothreitol (DTT), the chimeras are seen to move from the RER and through the Golgi stack. With this temperature shift the direct connections with the RER are lost and free vesicles form; some of these vesicles contain HRP reaction product which is much more concentrated than in the adjacent RER while others lack reaction product entirely. In cells expressing SSHRPKDEL, DAB reaction product remains distributed throughout the RER, the VTCs, and the cis-Golgi network for prolonged periods in the presence of DTT and almost all of the vesicles which form at 37 degrees C are DAB-positive. Together these observations demonstrate that all three chimeras are transported from the RER to the cis-Golgi in free, 40-60-nm vesicles at 37 degrees C. They also suggest that the retrograde traffic which carries SSHRPKDEL back to the RER is probably mediated by vesicles with a similar morphology but which, in cells expressing membrane-anchored chimeras, lack detectable reaction product.

Amino Acid Sequence↗
PubMed1995

Membrane protein trafficking through the common apical endosome compartment of polarized Caco-2 cells.

By raising monoclonal antibodies to the apical surface of Caco-2 cells we have identified a membrane protein (p100) that internalizes and recycles constitutively between the apical plasma membrane and endosomes in the apical cytoplasm. By applying tracers bound to the transferrin receptor, which internalizes and recycles back to the basolateral border, we demonstrate that the apical endosomes containing p100 include a subset of multivesticular bodies (MVB), which are also accessible to proteins arriving from the basolateral endosome. Tracers bound to EGF receptors and alpha-2-macroglobulin, which internalize from the basolateral border and are degraded, probably in lysosomes, also pass through the p100-containing MVB. These studies therefore suggest that the apical cytoplasm of Caco-2 cells contains a population of MVB capable of receiving membrane proteins trafficking in from both apical and basolateral borders and then routing them to a variety of cell surface and intracellular destinations. The differential distribution of apical and basolateral tracers within the 50-nm-diameter tubules connected to these p100-positive apical MVB suggests that the destination of proteins trafficking from the MVB back to apical and basolateral surfaces is determined by the tubules to which they gain access.

Cell Polarity↗
PubMed1994

In migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma membrane of the leading lamella.

By following the intracellular processing of recycling transferrin receptors and the selective sorting of a-2 macroglobulin in chick embryo fibroblasts, we have shown that the concentration of 60 nm diam tubules which surrounds the centrioles represents a distal compartment on the recycling pathway. In migrating cells transferrin receptor tracers can be loaded into this compartment and then chased to the cell surface. When they emerge the recycling transferrin receptors are distributed over the surface of the leading lamella.

Animals↗
PubMed1994

Sorting signals in the MHC class II invariant chain cytoplasmic tail and transmembrane region determine trafficking to an endocytic processing compartment.

Targeting of MHC class II molecules to the endocytic compartment where they encounter processed antigen is determined by the invariant chain (Ii). By analysis of Ii-transferrin receptor (TR) chimera trafficking, we have identified sorting signals in the Ii cytoplasmic tail and transmembrane region that mediate this process. Two non-tyrosine-based sorting signals in the Ii cytoplasmic tail were identified that mediate localization to plasma membrane clathrin-coated pits and promote rapid endocytosis. Leu7 and Ile8 were required for the activity of the signal most distal to the cell membrane whereas Pro15 Met16 Leu17 were important for the membrane-proximal signal. The same or overlapping non-tyrosine-based sorting signals are essential for delivery of Ii-TR chimeras, either by an intracellular route or via the plasma membrane, to an endocytic compartment where they are rapidly degraded. The Ii transmembrane region is also required for efficient delivery to this endocytic processing compartment and contains a signal distinct from the Ii cytoplasmic tail. More than 80% of the Ii-TR chimera containing the Ii cytoplasmic tail and transmembrane region is delivered directly to the endocytic pathway by an intracellular route, implying that the Ii sorting signals are efficiently recognized by sorting machinery located in the trans-Golgi.

Amino Acid Sequence↗
PubMed1994

Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase.

We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.

Animals↗
PubMed1993

ATP-dependent translocation of ricin across the membrane of purified endosomes.

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 125I-ricin translocation was found to be strictly ATP- (Km approximately 4 mM) and temperature-dependent, with up to 30% endosomal 125I-ricin appearing in the external medium after 2 h at 37 degrees C. 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 125I-ricin to translocate increases with its depth in the endocytic system (i.e. 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.

Animals↗
PubMed1993

Annexin I is phosphorylated in the multivesicular body during the processing of the epidermal growth factor receptor.

We have previously shown that an active epidermal growth factor receptor (EGF-R) kinase is necessary for efficient sorting of the EGF-R to the lysosome, and we have shown that this occurs in the multivesicular body (MVB), where EGF-R are sorted away from recycling receptors by being removed to the internal vesicles of the MVB. The aim of the present study was to identify substrates of the EGF-R kinase associated with MVBs which might play a role in this sorting process. We used a density shift technique to isolate MVBs and show that the major substrates phosphorylated in vitro within MVBs which contain an active EGF-R kinase are the EGF-R itself and annexin I. Annexin I is associated with both plasma membrane and MVBs in a calcium-independent manner but can be phosphorylated in vitro only in MVBs. Phosphorylation of calcium-independent annexin I in isolated MVBs converts it to a form that requires calcium for membrane association. In cells with an active EGF-R kinase the amount of calcium-independent annexin I in MVBs is reduced, suggesting that a phosphorylation-induced conversion of the calcium independent to the calcium-dependent form also occurs in vivo. Our observations, together with the known properties of annexin I in mediating membrane fusion, suggest that inward vesiculation in MVBs is induced by the EGF-R and is mediated by phosphorylated annexin I.

3T3 Cells↗
PubMed1992

Selective membrane protein trafficking: vectorial flow and filter.

Membrane proteins trafficking along cellular pathways encounter molecular filters. These filters can introduce them to new pathways and thus direct them towards new destinations. Many proteins carry molecular signals within their cytoplasmic domains that allow them to be selected by the filters. The detailed characterization of these signals is providing new insights into the pathways themselves and indicating the locations of other, as yet unidentified filters.

Amino Acid Sequence↗