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H P Moore

Publications and source records attributed to H P Moore.

At least 19 recordsLinked to original sources
PubMed1999

Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H+-K+-ATPase.

Stimulation of the gastric parietal cell results in a massive redistribution of H+-K+-ATPase from cytoplasmic tubulovesicles to the apical plasma membrane. Previous studies have implicated the small GTPase rab11 in this process. Using matrix-assisted laser desorption mass spectrometry, we confirmed that rab11 is associated with H+-K+-ATPase-enriched gastric microsomes. A stoichiometry of one rab11 per six copies of H+-K+-ATPase was estimated. Furthermore, rab11 exists in at least three forms on rabbit gastric microsomes: the two most prominent resemble rab11a, whereas the third resembles rab11b. Using an adenoviral expression system, we expressed the dominant negative mutant rab11a N124I in primary cultures of rabbit parietal cells under the control of the tetracycline transactivator protein (tTA). The mutant was well expressed with a distribution similar to that of the H+-K+-ATPase. Stimulation of these cultures with histamine and IBMX was assessed by measuring the aminopyrine (AP) uptake relative to resting cells (AP index). In experiments on six culture preparations, stimulated uninfected cells gave an AP index of 10.0 +/- 2.9, whereas parallel cultures expressing rab11a N124I were poorly responsive to stimulation, with a mean AP index of 3.2 +/- 0. 9. Control cultures expressing tTA alone or tTA plus actin responded equally well to stimulation, giving AP index values of 9.0 +/- 3.1 and 9.6 +/- 0.9, respectively. Thus inhibition by rab11a N124I is not simply due to adenoviral infection. The AP uptake data were confirmed by immunocytochemistry. In uninfected cells, H+-K+-ATPase demonstrated a broad cytoplasmic distribution, but it was cleared from the cytoplasm and associated with apically derived membranes on stimulation. In cells expressing rab11a N124I, H+-K+-ATPase maintained its resting localization on stimulation. Furthermore, this effect could be alleviated by culturing infected cells in the presence of tetracycline, which prevents expression of the mutant rab11. We therefore conclude that rab11a is the prominent GTPase associated with gastric microsomes and that it plays a role in parietal cell activation.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Cellubrevin-targeted fluorescence uncovers heterogeneity in the recycling endosomes.

The pH and trafficking of recycling endosomes have previously been studied using transferrin. We have used another approach, one in which the vesicle transport protein cellubrevin was appended with a luminal IgG epitope to allow targeting of fluorescein-5'-isothiocyanate (FITC)-labeled anti-IgG F(ab) antibodies to the recycling endosomes in living cells. FITC-F(ab) was specifically internalized by COS cells transfected with cellubrevin-Ig, which at steady state accumulated in a pericentriolar region similar to rhodamine-transferrin. Confocal microscopic analysis showed that endosome labeling by these two markers was heterogeneous. This differential distribution was not induced by the IgG tag, since endogenous Cb and Tf were also partitioned into separate endosomal populations. We used fluorescence ratio imaging of internalized FITC-F(ab) to measure the pH of cellubrevin-enriched recycling endosomes (pHCb) and FITC-transferrin to measure the pH of transferrin-enriched recycling endosomes (pHTf). In COS cells, cellubrevin endosomes (mean pHCb 6.1 +/- 0.05; range, 5.2-6.6) were more acidic than transferrin endosomes (mean pHTf 6.5 +/- 0.05; range, 5.6-7.2). Similar results were obtained in Chinese hamster ovary cells. Treatment with the vacuolar H+-ATPase inhibitor bafilomycin A1 caused pHTf to increase (DeltapHTf = 1.2 pH units) to a greater extent than pHCb (DeltapHCb = 0.5 pH units). Furthermore, inhibition of the Na+/K+-ATPase by ouabain or acetylstrophanthidin caused pHTf to decrease by 0.6 pH units but had no effect on pHCb. Based on the combination of these morphological and functional data, we suggest that the recycling endosomes are heterogeneous in their biochemical compositions, ion transport properties, and pH values.

Animals↗
PubMed1998

The epithelial sodium-hydrogen antiporter Na+/H+ exchanger 3 accumulates and is functional in recycling endosomes.

Na+/H+ exchangers (NHEs) mediate electroneutral exchange of Na+ for H+ and thereby play a central role in pH regulation and Na+ homeostasis. NHE3, the predominant epithelial isoform, is found in apical membranes of renal and intestinal epithelial cells, where it contributes to NaCl (re)absorption. NHE activity has been detected in endomembrane vesicles of epithelial cells, but the precise compartment involved and its functional role have not been defined. Many aspects of the targeting machinery that defines the compartmentation and polarity of epithelia are also functional in nonepithelial cells. We therefore compared the targeting of NHE1, the basolateral isoform, with that of NHE3 in Chinese hamster ovary cells. To circumvent the confounding effects of endogenous exchangers, epitope-tagged constructs of NHE1 and NHE3 were stably expressed in antiport-deficient (AP-1) cells. While NHE1 was found almost exclusively in the surface membrane, NHE3 was also found intracellularly, accumulating in a juxtanuclear compartment. Confocal microscopy showed this compartment to be distinct from the Golgi, trans-Golgi network, and lysosomes. Instead, NHE3 colocalized with transferrin receptors and with cellubrevin, markers of recycling endosomes. The activity of NHE3 in endomembranes was assessed by targeting pH-sensitive probes to the recycling endosomes using a chimeric cellubrevin construct with an accessible extracellular epitope. Fluorescence ratio imaging indicated that cellubrevin resides intracellularly in an acidic compartment. In AP-1 cells, endosomal acidification was unaffected by omission of Na+ but was dissipated entirely by concanamycin, a blocker of H(+)-ATPases. In contrast, the cellubrevin compartment was more acidic in NHE3 transfectants, and the acidification was only partially reduced by concanamycin. Moreover, removal of extracellular Na+ resulted in a significant alkalization of the endocytic compartment. These results indicate that NHE3 is present and active in recycling endosomes. By recruiting NHE3 to the plasma membrane, modulation of vesicular traffic could contribute to the regulation of Na+ reabsorption across epithelia.

Animals↗
PubMed1998

Analysis of constitutive and constitutive-like secretion in semi-intact pituitary cells.

To study biosynthetic transport through the constitutive and regulated secretory pathways, we have designed a semi-intact mammalian cell system that restores the transport of secretory proteins from the trans-Golgi/trans-Golgi network (TGN) to the cell surface. The mouse pituitary AtT-20 cell line is a suitable model to biochemically analyze molecular sorting in the secretory pathway. The prohormone proopiomelanocortin is sulfated on N-linked carbohydrate chains in the trans-Golgi prior to proteolytic processing in the secretory granule. Radiolabeling with [35S]sulfate therefore provides a convenient tool to selectively follow molecular events in the regulated secretory pathway without interference from earlier steps. Likewise, transport through the constitutive secretory pathway may be monitored using sulfate-labeled glycosaminoglycan chains. We show that export from the TGN is efficiently reconstituted in cells made semi-intact with streptolysin O, and is dependent on temperature, ATP and GTP hydrolysis, and cytosol. Packaging of proopiomelanocortin into immature secretory granules also activates the proteolytic processing machinery which eventually converts the prohormone to its bioactive mature product, adrenocorticotropic hormone. In addition, a large fraction of incompletely processed proopiomelanocortin is secreted as the processing intermediates from immature secretory granules. This process of constitutive-like secretion can be clearly distinguished from direct constitutive secretion from the trans-Golgi network by kinetic and compositional criteria. Furthermore, we have found that specific inhibitors of different protein phosphatases and kinases are potent blockers of constitutive and constitutive-like secretion. This experimental model should provide a valuable system to elucidate the molecular mechanism regulating post-Golgi traffic during secretory granule biogenesis.

Adenosine Triphosphate↗
PubMed1997

Targeting of leptin to the regulated secretory pathway in pituitary AtT-20 cells.

Leptin, a key regulator of fat homeostasis, is the product of the obese gene [1-3], and is secreted from adipocytes and binds to receptor sites in the choroid plexus [4-5]. Several studies have implicated serum insulin levels in the upregulation of leptin gene expression [6-8]. It is currently not known whether leptin levels are also subject to regulation at the level of secretion. Leptin is normally produced in adipocytes, the secretory pathways of which are not well characterized. Here, we used pituitary AtT-20 cells, which serve as a model system for both regulated and constitutive secretory pathways, to examine the intracellular targeting and secretion of leptin. Confocal immunofluorescence analysis of AtT-20 cells expressing an epitope-tagged human leptin (FLAG-leptin) demonstrated that FLAG-leptin colocalized with endogenous adrenocorticotrophic hormone (ACTH) at the tips of processes extended from these cells, where regulated secretory granules accumulate. FLAG-leptin secretion was increased in the presence of 8-Br-cAMP, which stimulates the secretion of ACTH. For FLAG-leptin, the calculated sorting index, a quantitative measure of the efficiency of protein sorting to the regulated pathway, was similar to those of other regulated secretory proteins. These results demonstrate that FLAG-leptin behaves like a regulated protein in cells with a biosynthetic regulated secretory pathway.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Distinct molecular events during secretory granule biogenesis revealed by sensitivities to brefeldin A.

The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their temporal order and interrelationships, we have developed a pulse-chase protocol that follows the synthesis and packaging of a sulfated hormone, pro-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated into POMC predominantly on N-linked endoglycosidase H-resistant oligosaccharides. Subcellular fractionation and pharmacological studies confirm that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sulfation, POMC undergoes a number of molecular events before final storage in dense-core granules. The first step involves the transfer of POMC from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20 degrees C block prevents its proteolytic conversion to mature adrenocorticotropic hormone. Proteolytic cleavage products were found in vesicular fractions corresponding to ISGs, suggesting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little unregulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage event within the maturing granule. The second step of granule biogenesis thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregulated release to one that favors intracellular storage. By using BFA, we show that the two processes occurring in ISGs may be uncoupled: although the unregulated secretion from ISGs is impaired by BFA, proteolytic processing of POMC within this organelle proceeds unaffected. The finding that BFA impairs constitutive secretion from both the TGN and ISGs also suggests that these secretory processes may be related in mechanism. Finally, our data indicate that the unusually high levels of unregulated secretion often associated with endocrine tumors may result, at least in part, from inefficient storage of secretory products at the level of ISGs.

Animals↗
PubMed1996

A biosynthetic regulated secretory pathway in constitutive secretory cells.

It has frequently been proposed that while the constitutive secretory pathway is present in all cells, the regulated secretory pathway is found only in specialized cells such as neuronal, endocrine, or exocrine types. In this study we provide evidence that suggests that this distinction is not as restrictive as proposed. We have identified a population of post-Golgi storage vesicles in several constitutive secretory cells using [35S]SO4-labeled glycosaminoglycan (GAG) chains as a marker. A fraction of this pool of vesicles can undergo exocytosis in response to stimuli such as cytoplasmic Ca2+ and phorbol esters. The effect of Ca2+ was demonstrated both in intact cells in the presence of the ionophore A23187 and in streptolysin-O-permeabilized semi-intact cells. N-ethylmaleiimide, under conditions known to block regulated and constitutive secretion, inhibited the stimulated secretion from these cells, suggesting that the observed release of labeled GAG chains was not due to a leakage artefact. Subcellular fractionation revealed that the stored GAG chains were in low-density membrane granules (d approximately 1.12 g/ml), whose size was greater than that of synaptic-like vesicles found in PC12 cells. In addition, in CHO cells that express epitope-tagged rab 3D, the labeled GAG chains were found to cofractionate with the exogenous rab protein. When expressed in the regulated cell line AtT-20, this tagged rab protein was found to colocalize with ACTH-containing dense-core granules by indirect immunofluorescence. Taken together, these results provide evidence for the presence of a cryptic regulated secretory pathway in "constitutive" cells and suggest that the regulated secretory pathway is more widespread amongst different cell types than previously believed.

Animals↗
PubMed1995

Ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in AtT-20 cells.

Newly synthesized prohormones and their processing enzymes transit through the same compartments before being packaged into regulated secretory granules. Despite this coordinated intracellular transport, prohormone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 cell line, conversion of pro-opiomelanocortin (POMC) to mature adrenocorticotropic hormone involves the prohormone convertase PC1. The mechanism by which this proteolytic processing is restricted to late secretory compartments is unknown; PC1 activity could be regulated by compartment-specific activators/inhibitors, or through changes in the ionic milieu that influence its activity. By arresting transport in a semi-intact cell system, we have addressed whether metabolically labeled POMC trapped in early secretory compartments can be induced to undergo conversion if the ionic milieu in these compartments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi network did not result in processing, thereby supporting the theory that processing is normally a post-Golgi/trans-Golgi network event. However, acidification of these compartments allowed effective processing of POMC to the intermediate and mature forms. The observed processing increased sharply at a pH below 6.0 and required millimolar calcium, regardless of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is predominantly restricted to acidifying secretory granules, and that a change in pH within these granules is both necessary and sufficient to activate POMC processing.

Adrenocorticotropic Hormone↗
PubMed1994

Synthesis and targeting of insulin-like growth factor-I to the hormone storage granules in an endocrine cell line.

Export of growth factors is generally believed to be restricted to the constitutive secretory pathway, whereas peptide hormones are typically secreted in a regulated manner. Here we show that insulin-like growth factor (IGF)-I, a growth factor released constitutively from the liver, is synthesized and secreted from the mouse pituitary AtT-20 cell line via the regulated pathway. IGF-I production is 1500-fold less than the peptide hormone ACTH. Secretagogue induces IGF-I secretion in a manner similar to ACTH. Like ACTH, IGF-I is sorted into the regulated pathway >35-fold more efficiently than a constitutively secreted protein. Dense core granules isolated from cells transfected with a human IGF-I cDNA contain both ACTH and human IGF-I. AtT-20 cells also synthesize IGF-binding proteins, and at least one of these is secreted by the regulated pathway. Human IGF-I does not exhibit milieu-induced, concentration-dependent aggregation, in contrast to secretogranin II which sorts by a proposed aggregation mechanism. These data suggest that 1) growth factors are not solely released from tissues via the constitutive pathway, 2) IGF-I may contain information for correct granular targeting, and 3) IGF-I may be sorted by a mechanism distinct from that proposed for the secretogranins.

Adrenocorticotropic Hormone↗
PubMed1994

Transport via the regulated secretory pathway in semi-intact PC12 cells: role of intra-cisternal calcium and pH in the transport and sorting of secretogranin II.

To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi-intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 microM [Ca2+]free to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 microM GTP gamma S, 50 microM AlF3-5 or 5 micrograms/ml BFA. When added after immature granules had formed, GTP gamma S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SgII via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SgII into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.

Adenosine Triphosphate↗
PubMed1994

Regulated Cl transport, K and Cl permeability, and exocytosis in T84 cells.

We measured stimulant-induced changes of exocytosis that are associated with increases in Cl secretion (i.e., short circuit current, ISC, in microA/cm2) and apical (ap) Cl permeability (PCl) and basolateral (bl) K permeability (PK) (both in cm/s) in T84 monolayers. PCl and PK were measured by permeabilizing the bl or ap membrane with nystatin. PCl was also measured with a fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). A noninvasive and sensitive method (release of 35SO4-labeled glycosaminoglycan [GAG], a fluid-phase marker of Golgi-derived vesicles) was used to measure exocytosis at both ap and bl membranes. At rest, ISC = 3.6, PK = 0.8 x 10(-6), PCl = 2.1 x 10(-6) with SPQ and 2.4 x 10(-6) electrically, and there was constitutive GAG secretion (i.e., exocytosis) to both ap and bl sides (bl > 2 x ap). Carbachol (C) increased: ISC (delta = 18.6), PK (6.5x), PCl (1.8-2.9x), and exocytosis to both ap (2.2-3.5x) and bl (2.0-3.0x) membranes. Forskolin (F) increased ISC (delta = 29), PCl (5.5-11x) and ap exocytosis (1.5-2x), but had no effect on PK or bl exocytosis. Synergistic effects on ISC occurred when C was added to F-treated cells but not vice versa, even though the characteristic effects of F+C on PCl, PK, and/or GAG secretion were identical to those exhibited when stimulants were added individually. Cl secretion results from coordinated activation of channels at ap and bl membranes, and exocytosis may play a role in these events.

Carbachol↗
PubMed1993

Molecular analysis of SAR1-related cDNAs from a mouse pituitary cell line.

Vesicular transport between the endoplasmic reticulum (ER) and the Golgi in the yeast Saccharomyces cerevisiae requires a Ras-like, small GTP-binding protein, Sar1p [1-3]. Whether a functional homologue operates in export from the ER in mammalian cells is unknown, nor is it clear if transport in other branches of the secretory pathway requires member(s) of a gene family. In this study, we used a PCR approach to examine the complexity of SAR1-related sequences expressed in mammalian cells that possess multiple secretory pathways. Amplification of cDNA sequences from rodent pituitary cells with primers corresponding to two conserved GTP binding domains of Sar1p yielded several clones with sequences homologous to Sar1 and/or the closely related ADP-ribosylation factor (ARF) family. Of these, only two showed closer homologies to S. cerevisiae Sar1 than members of the ARF family and are designated as mSARa and mSARb. Northern blot analysis shows that mSARa is expressed in most tissues including liver, heart, brain, skeletal muscle and kidney. In contrast, mSARb is preferentially expressed in skeletal muscle and liver. The full-length cDNA of mSARa isolated from a mouse pituitary AtT-20 cDNA library encodes a protein of 198 amino acids, and is 61.6% identical to Sar1p from S. cerevisiae. Thus in contrast to the large rab family of GTP-binding proteins, vesicular transport in mammalian cells appears to be mediated by a relatively small number of Sar1-related proteins.

Amino Acid Sequence↗
PubMed1993

Gene cloning and characterization of a GTP-binding Rab protein from mouse pituitary AtT-20 cells.

The Rab proteins constitute a family of Ras-like, low-molecular-weight GTP-binding proteins that are involved in vesicular transport in mammalian cells. Several members of this family have been localized to specific cellular compartments, indicating that they may control vesicular transfer between discrete portions of the endomembrane systems. To examine the complexity of the Rab proteins produced in cells with both regulated and constitutive secretory pathways, we used a PCR-based strategy to clone members of this gene family from rodent pituitary. Here, we report the identification and studies of one of the clones, RAB18. The full-length cDNA clone from a mouse pituitary AtT-20 cDNA library encodes a 23.5-kDa protein with four consensus GTP-binding domains. The deduced amino acid sequence is 42% and 40% identical to Rab2 and Sec4p, respectively, and contains sequence motifs characteristic of the Rab family. Northern blot analysis shows that the level of expression is high in the brain, moderate in the pituitary, and low in the liver. Rab18 may be involved in membrane traffic events during biogenesis and/or recycling steps of secretory vesicle components.

Amino Acid Sequence↗
PubMed1993

Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.

Small GTP-binding proteins of the YPT/SEC4/Rab family have been shown to play an essential role in intracellular membrane trafficking. In mammals, Rab8 and Rab10 are the two small GTP-binding proteins identified so far that are closest to SEC4, an essential gene product involved in post-Golgi constitutive secretion in the yeast Saccharomyces cerevisiae. To study the localization of Rab proteins, we have expressed the cDNAs with an influenza virus hemagglutinin (HA) epitope tag at the N terminus. The feasibility of this method was tested by using yeast SEC4. HA-tagged SEC4 functionally complemented a temperature-sensitive sec4 mutant similarly to wild-type SEC4, indicating that the modified protein retained functional integrity. Monoclonal antibody 12CA5, raised against the HA tag, was used to determine the expression and localization of HA-tagged proteins after transfection. In stably transfected CHO and Swiss 3T3 cells, HA-tagged Rab8 was localized to the cell periphery, with the highest concentration in the ruffling areas. In contrast, epitope-tagged Rab10 expressed in CHO and BHK cells was concentrated on membranes in the perinuclear region. By light microscopy, the staining partially overlapped with that of a Golgi marker, beta-COP. Thus, despite the high degree homology of Rab8 and Rab10 (66% identity), the two proteins are localized to distinct cellular compartments. This approach should provide a general tool for the analyses of other members of the YPT/SEC4/rab gene family.

Animals↗
PubMed1992

Regulated and constitutive secretion. Differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways.

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

The establishment of polarized membrane traffic in Xenopus laevis embryos.

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin was polarized. Oogenic prolactin was secreted only into the blastocoel (from the cleavage membrane), none could be detected in the external medium (from the original oocyte membrane). These results provide the first direct evidence that the oocyte synthesizes a cache of vesicles for specific recruitment to the embryonic cleavage membranes which are polarized beginning with the first cleavage division.

Animals↗