A comparative study of fixation techniques in the open Bankart operation using either a cannulated screw or suture-anchors

Neuroendocrine PC12 cells contain small microvesicles that closely resemble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous size and their unique protein composition. Since synaptic vesicles arise by endocytosis from the plasma membrane, nerve terminals and PC12 cells must contain the molecular machinery to sort synaptic vesicles from other membrane proteins and pinch off vesicles of the correct diameter from a precursor compartment. A cell-free reconstitution system was developed that generates vesicles from PC12 membrane precursors in the presence of ATP and brain cytosol and is temperature dependent. At 15 degrees C, surface-labeled synaptic vesicle proteins accumulate in a donor compartment, while labeled synaptic vesicles cannot be detected. The block of synaptic vesicle formation at 15 degrees C enables the use of the monoclonal antibody, KT3, a specific marker for the epitope-tagged synaptic vesicle protein, VAMP-TAg, to label precursors in the synaptic vesicle biogenesis pathway. From membranes labeled in vivo at 15 degrees C, vesicles generated in vitro at 37 degreesC had the sedimentation characteristics of neuroendocrine synaptic vesicles on glycerol velocity gradients, and excluded the transferrin receptor. Therefore, vesiculation and sorting can be studied in this cell-free system.     

Differential localization of vesicular acetylcholine and monoamine transporters in PC12 cells but not CHO cells

Previous studies have indicated that neuro-endocrine cells store monoamines and acetylcholine (ACh) in different secretory vesicles, suggesting that the transport proteins responsible for packaging these neurotransmitters sort to distinct vesicular compartments. Molecular cloning has recently demonstrated that the vesicular transporters for monoamines and ACh show strong sequence similarity, and studies of the vesicular monoamine transporters (VMATs) indicate preferential localization to large dense core vesicles (LDCVs) rather than synaptic-like microvesicles (SLMVs) in rat pheochromocytoma PC12 cells. We now report the localization of the closely related vesicular ACh transporter (VAChT). In PC12 cells, VAChT differs from the VMATs by immunofluorescence and fractionates almost exclusively to SLMVs and endosomes by equilibrium sedimentation. Immunoisolation further demonstrates colocalization with synaptophysin on SLMVs as well as other compartments. However, small amounts of VAChT also occur on LDCVs. Thus, VAChT differs in localization from the VMATs, which sort predominantly to LDCVs. In addition, we demonstrate ACh transport activity in stable PC12 transformants overexpressing VAChT. Since previous work has suggested that VAChT expression confers little if any transport activity in non-neural cells, we also determined its localization in transfected CHO fibroblasts. In CHO cells, VAChT localizes to the same endosomal compartment as the VMATs by immunofluorescence, density gradient fractionation, and immunoisolation with an antibody to the transferrin receptor. We have also detected ACh transport activity in the transfected CHO cells, indicating that localization to SLMVs is not required for function. In summary, VAChT differs in localization from the VMATs in PC12 cells but not CHO cells.     

GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15 degrees and 37 degrees C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.     

Vesiculation and sorting from PC12-derived endosomes in vitro

Formation of small vesicles resembling synaptic vesicles can be reconstituted in vitro by incubating labeled homogenates of PC12 cells with ATP and two cytoplasmic proteins, AP3 and ARF1 [Faúndez, V., Horng, J.-T. & Kelly, R. B. (1998) Cell 93, 423-432]. To determine whether AP3 was mediating budding from plasma membranes or endosomes the organelle that generated the synaptic vesicles was characterized. The budding activity was enriched in organelles that labeled at 15 degrees C, but not at 4 degrees C, that excluded a marker of plasma membranes and that contained internalized transferrin, indicating that the precursor was an endosome. Vesicles formed from the endosomal precursor in vitro excluded transferrin. We conclude that ARF-mediated vesiculation into synaptic vesicle-sized organelles uses an endosomal precursor and occurs simultaneously in vitro with sorting of synaptic vesicle proteins from other membrane protein constituents of the endosome.     

Differential ultrastructural distribution of synapsin and synaptophysin proximal to a ligation in bovine splenic nerve

Synaptophysin and synapsin, closely correlated on synaptic vesicles in terminals, may show a differential distribution at synapse formation and maturation. In order to disclose the fine structural details of these differences, synapsin and synaptophysin distribution was studied by immunocytochemistry on ligated bovine splenic axons in vitro and compared with terminals in the vas deferens. In the synaptic differentiations taking place proximally synapsin could only be detected on the accumulating elements of the axonal reticulum. Large dense granules and clusters of small synaptic vesicles were negative. Synaptophysin was restricted to these clusters. In the vas deferens, co-localization of synapsin and synaptophysin could be seen on small vesicles. From their formation small synaptic vesicles carry synaptophysin. Synapsin may be involved in the dynamic membrane changes taking place at the ligation. At a functional terminal, synapsin shifts to small synaptic vesicles.     

In vitro binding of synaptic vesicles to the synaptic plasma membrane: lack of effect of beta-bungarotoxin

To help characterize the mechanisms of neurotransmitter release, and the role of the specific neurotoxin beta-bungarotoxin in inhibiting release, the interaction of synaptic vesicles with the synaptic plasma membrane was investigated using two in vitro systems. Binding of radiolabeled synaptic vesicles to immobilized synaptic plasma membrane was specific, protein-dependent, and modulated by phosphorylation of membrane proteins. Stimulation of phosphorylation by phorbol ester increased binding, and reduction of phosphorylation by alkaline phosphatase or staurosporine reduced binding. beta-Bungarotoxin did not alter basal binding of synaptic vesicles to synaptic plasma membrane, nor did it affect the increase in binding induced by phorbol esters. Under conditions which stimulate acetylcholine release from synaptosomes, both phorbol ester and 4-aminopyridine caused an increase in attachment of the synaptic vesicle marker protein synaptophysin to the synaptic plasma membrane. beta-Bungarotoxin did not alter the change in localization of synaptophysin induced by either drug, under conditions in which it inhibits ACh release induced by 4-aminopyridine. It is concluded that beta-bungarotoxin inhibition probably does not occur at the level of the interaction of the synaptic vesicle and the synaptic plasma membrane, but occurs at an earlier stage in the neurotransmission process.     

Targeting of the synaptic vesicle protein synaptobrevin in the axon of cultured hippocampal neurons: evidence for two distinct sorting steps

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10- amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61-70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.     

Morphology of the yeast endocytic pathway

Positively charged Nanogold (Nanoprobes, Stony Brook, NY) has been developed as a new marker to follow the endocytic pathway in yeast. Positively charged Nanogold binds extensively to the surface of yeast spheroplasts and is internalized in an energy-dependent manner. Internalization of gold is blocked in the end3 mutant. During a time course of incubation of yeast spheroplasts with positively charged Nanogold at 15 degrees C, the gold was detected sequentially in small vesicles, a peripheral, vesicular/tubular compartment that we designate as an early endosome, a multivesicular body corresponding to the late endosome near the vacuole, and in the vacuole. Experiments examining endocytosis in the sec18 mutant showed an accumulation of positively charged Nanogold in approximately 30-50 nm diameter vesicles. These vesicles most likely represent the primary endocytic vesicles as no other intermediates were detected in the mutant cells, and they correspond in size to the first vesicles detected in wild-type spheroplasts at 15 degrees C. These data lend strong support to the idea that the internalization step of endocytosis in yeast involves formation of small vesicles of uniform size from the plasma membrane.     

Pantophysin is a ubiquitously expressed synaptophysin homologue and defines constitutive transport vesicles

Certain properties of the highly specialized synaptic transmitter vesicles are shared by constitutively occurring vesicles. We and others have thus identified a cDNA in various nonneuroendocrine cell types of rat and human that is related to synaptophysin, one of the major synaptic vesicle membrane proteins, which we termed pantophysin. Here we characterize the gene structure, mRNA and protein expression, and intracellular distribution of pantophysin. Its mRNA is detected in murine cell types of nonneuroendocrine as well as of neuroendocrine origin. The intron/exon structure of the murine pantophysin gene is identical to that of synaptophysin except for the last intron that is absent in pantophysin. The encoded polypeptide of calculated mol wt 28,926 shares many sequence features with synaptophysin, most notably the four hydrophobic putative transmembrane domains, although the cytoplasmic end domains are completely different. Using antibodies against the unique carboxy terminus pantophysin can be detected by immunofluorescence microscopy in both exocrine and endocrine cells of human pancreas, and in cultured cells, colocalizing with constitutive secretory and endocytotic vesicle markers in nonneuroendocrine cells and with synaptophysin in cDNA-transfected epithelial cells. By immunoelectron microscopy, the majority of pantophysin reactivity is detected at vesicles with a diameter of < 100 nm that have a smooth surface and an electron-translucent interior. Using cell fractionation in combination with immunoisolation, these vesicles are enriched in a light fraction and shown to contain the cellular vSNARE cellubrevin and the ubiquitous SCAMPs in epithelial cells and synaptophysin in neuroendocrine or cDNA-transfected nonneuroendocrine cells and neuroendocrine tissues. Pantophysin is therefore a broadly distributed marker of small cytoplasmic transport vesicles independent of their content.     

Transmembrane movement of phosphatidylcholine in mitochondrial outer membrane vesicles

One of the steps in the import of phosphatidylcholine (PC) in mitochondria is transmembrane movement across the outer membrane. This process was investigated in vitro using isolated mitochondrial outer membrane vesicles (OMV) from rat liver. 14C-Labeled PC was introduced into the OMV from small unilamellar vesicles by a PC-specific transfer protein (PCTP). The membrane topology of the newly introduced PC was determined from its accessibility to phospholipase A2. Under conditions where the OMV stay intact, externally added phospholipase A2 is able to hydrolyze up to 50% of both the introduced [14C]PC and the endogenous PC. Pool size calculations showed that close to 100% of the PC in the OMV can be exchanged by PCTP. A back-exchange experiment revealed that the introduction of the labeled PC is reversible. The results demonstrate that newly introduced PC molecules readily equilibrate over both leaflets of the OMV membrane. The kinetics of the PCTP-mediated exchange process indicate that the t1/2 of the transmembrane movement at 30 degrees C is 2 min or less.     

Surgical removal of pulmonary metastasis: prospective study in 182 patients]

Dystrophic neurites are major components of neuritic (both immature and mature) senile plaques in Alzheimer disease. Previous studies have shown strong immunoreactivity for different neuropeptides, and chromogranin A, a protein associated with dense-core vesicles, in dystrophic neurites. In the present study, antibodies to synaptophysin, synapsin, Rab3a and synaptotagmin (synaptic vesicle proteins), and SNAP-25 (synaptosomal-associated protein of 25 kD) and syntaxin (presynaptic plasma membrane proteins) have been used to learn about the dystrophic neurite equipment of proteins that are necessary for the docking and fusion of synaptic vesicles, and then for exocytosis and neurotransmission. The present results have shown that, although most neuritic senile plaques have chromogranin A- and SNAP-25-immunoreactive dystrophic neurites, only a percentage of them contain synaptophysin, and a minority contain synaptotagmin and Rab3a. Dystrophic neurites do not contain synapsin and syntaxin. These results show that dystrophic neurites of senile plaques are defective in proteins that control exocytosis and neurotransmission.     

Posttraumatic stress and maladjustment among adult burn survivors 1 to 2 years postburn. Part II: the interview data

Using native plasma membrane vesicle suspensions from the rat cerebral cortex under conditions designed to alter intravesicular [Ca2+], we found that Ca2+ induced 47 +/- 5% more influx of [3H]GABA, [3H]D-aspartate and [3H]glycine at 37 degrees C with half-times 1.7 +/- 0.5, 1.3 +/- 0.4 and 1.3 +/- 0.4 min, respectively. We labelled GABA transporter sites with the uptake inhibitor, [3H]-(R,S)-N-[4,4-bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid and found that Ca2+ induced a partial dissociation of the bound inhibitor from GABA transporter sites with a similar half-time. By means of rapid kinetic techniques applied to native plasma membrane vesicle suspensions, containing synaptic vesicles stained with the amphipathic fluorescent styryl membrane probe N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyrid inium dibromide, we have measured the progress of the release and reuptake of synaptic vesicles in response to Ca2+ and high-[K+] depolarization in the 0.0004-100 s range of time. Synaptic vesicle exocytosis, strongly influenced by external [Ca2+], appeared with the kinetics accelerated by depolarization. These results are consistent with the potential involvement of Ca2+ in taking low-affinity transporters to the plasma membrane surface via exocytosis.     

Structural destabilization of synaptosomal particles by lysis and sequential chemical treatments

Synaptosomes from rat brains were subjected to a sequence of treatments: osmotic lysis, buffered saline wash, nonionic detergent, EGTA and EDTA. After each treatment, particulate samples were fixed in 2% glutaraldehyde-1% formaldehyde and centrifuged to form pellets which were then processed for and examined by electron microscopy. Five morphological classes of synaptic particle were defined in terms of character and presence of synaptic vesicles, flocculent and stranded material, designated as intervesicular scaffolding (IVS), and presynaptic membrane. During osmotic lysis, the presynaptic compartment was altered by loss of most, but not all, small synaptic vesicles, by increase in proportion of large vesicles, and by disappearance of the presynaptic densities. The retention of vesicles was interpreted in terms of IVS struts interconnecting anchorage sites on synaptic vesicles and the presynaptic junctional membrane. Treatment of lysed synaptosomes with nonionic detergent or EGTA resulted in loss of vesicles and IVS from the junctional region in most particles. The apposition of pre-and postsynaptic junctional membranes along the synaptic cleft was disrupted more by EGTA than by detergent. The final result of the sequential treatments was a sediment containing a high proportion of synaptic particles, about half of which had lost their presynaptic junctional membranes.     

Immunohistochemistry of synapsin I and synaptophysin in human nervous system and neuroendocrine tumors. Applications in diagnostic neuro-oncology

Synapsin I is a phosphoprotein localized to the cytoplasmic surface of synaptic vesicles and is one of the best characterized neuron-specific proteins. Synaptophysin is an integral membrane glycoprotein, also located on presynaptic vesicles, which has been shown to be a useful immunohistochemical marker for neuroendocrine/neuronal differentiation in tumor diagnosis. The sensitivity and specificity of immunohistochemical staining for these two proteins in formalin-fixed, paraffin-embedded tissues was studied in a series of 67 neuroectodermal, neuroendocrine, and non-neural tumors. Intense immunoreactivity for both synapsin I and synaptophysin was observed in tumors containing well-differentiated neurons (gangliocytoma, ganglioglioma, neurocytoma). In these tumors, immunostaining was primarily concentrated along the outer surface of the cell membrane of the neuronal cells. Primitive neuroectodermal tumors (PNETs) (cerebral PNET, medulloblastoma, neuroblastoma) and most neuroendocrine tumors generally showed less intense and more variable immunoreactivity for these proteins. In most cases, immunostaining for synapsin I was sharper and often more intense than for synaptophysin. Some PNETs and neuroendocrine tumors that were immunoreactive for synapsin I did not stain for synaptophysin. We conclude that synapsin I is a reliable, sensitive immunohistochemical marker for neuronal/neuroendocrine differentiation in human neoplasms and may offer some advantages over synaptophysin when applied to formalin-fixed, paraffin-embedded tissues, particularly in the evaluation of primitive neuroectodermal tumors and neuroendocrine tumors.     

Regulation of the major detoxication functions by phenobarbital and 3-methylcholanthrene in co-cultures of rat hepatocytes and liver epithelial cells

We have examined the rotational diffusion of the luteinizing hormone (LH) receptors binding human chorionic gonadotropin (hCG) or ovine luteinizing hormone (oLH) in MA-10 Leydig tumor cells using time-resolved phosphorescence anisotropy techniques. LH receptors binding erythrosin isothiocyanate (ErITC)-derivatized oLH were rotationally mobile with rotational correlation times of 62 micros, 48 micros, 38 micros, and 29 micros at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C, respectively. ErITC-hCG bound to the LH receptor was rotationally immobile, showing no anisotropy decay at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C. To determine whether cytoskeletal components influenced the rotational diffusion of LH receptors, we measured rotational diffusion of LH receptors on MA-10 cells treated with 20 microg/ml cytochalasin D and on plasma membrane preparations. Following 1 h exposure to cytochalasin D, the rotational correlation times for hCG-occupied LH receptors were typically 11 micros at 37 degrees C compared to > 1000 micros on untreated cells. Treatment of MA-10 cells with cytochalasin B or colchicine had no affect on LH receptor rotational diffusion. Rotational correlation times for LH-occupied receptors decreased from 29 micros to 12 micros at 37 degrees C following cytochalasin D treatment. The rotational diffusion of LH receptors on plasma membrane preparations was similar to that observed for LH- and hCG-occupied receptors on intact cells treated with cytochalasin D. These various results indicate that there are differential effects of LH and hCG binding on the interactions of LH receptors with plasma membrane proteins and that microfilaments anchor the hCG- and LH-occupied receptors.     

Acyl chain-length asymmetry alters the interfacial elastic interactions of phosphatidylcholines

Phosphatidylcholines (PCs) with stearoyl (18:0) sn-1 chains and variable-length, saturated sn-2 acyl chains were synthesized and investigated using a Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at various constant temperatures between 10 degrees C and 30 degrees C. Over this temperature range, 18:0-10:0 PC displayed only liquid-expanded behavior. In contrast, di-14:0 PC displayed liquid-expanded behavior at 24 degrees C and 30 degrees C, but two-dimensional phase transitions were evident at 20 degrees C, 15 degrees C, and 10 degrees C. The average molecular area of 18:0-10:0 PC was larger than that of liquid-expanded di-14:0 PC at equivalent surface pressures, and the shapes of their liquid expanded isotherms were somewhat dissimilar. Analysis of the elastic moduli of area compressibility (Cs(-1)) as a function of molecular area revealed shallower slopes in the semilog plots of 18:0-10:0 PC compared to di-14:0 PC. At membrane-like surface pressures (e.g., 30 mN/m), 18:0-10:0 PC was 20-25% more elastic (in an in-plane sense) than di-14:0 PC. Other PCs with varying degrees of chain-length asymmetry (18:0-8:0 PC, 18:0-12:0 PC, 18:0-14:0 PC, 18:0-16:0 PC) were also investigated to determine whether the higher in-plane elasticity of fluid-phase 18:0-10:0 PC is a common feature of PCs with asymmetrical chain lengths. Two-dimensional phase transitions in 18:0-14:0 PC and 18:0-16:0 PC prevented meaningful comparison with other fluid-phase PCs at 30 mN/m. However, the Cs(-1) values for fluid-phase 18:0-8:0 PC and 18:0-12:0 PC were similar to that of 18:0-10:0 PC (85-90 mN/m). These values showed chain-length asymmetrical PCs to have 20-25% greater in-plane elasticity than fluid-phase PCs with mono- or diunsaturated acyl chains.     

Separation of early steps in endocytic membrane transport

We describe a simple subcellular fractionation scheme aimed at separating early endosomes from the plasma membrane in view of studying the possible arrival of plasma membrane-bound toxins, proteins or other extracellular ligands in endosomes. Plasma membrane proteins were labeled with the impermeable reagent sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC) biotin at 4 degrees C. In a separate set of cells, early endosomes were labeled by internalization of horseradish peroxidase from the medium for 5 min. The first step of the purification, which consists of a step sucrose gradient, led to three fractions, respectively: enriched in biosynthetic membranes (interface 3), in plasma membrane and early endosomes (interface 2), and in late endosomes (interface 1). The second step, in which interface 2 was loaded at the bottom of a 17% Percoll gradient, led to the separation of the plasma membrane, including caveolae and cholesterol-glycolipid rafts, from early endosomes. Western blot analysis of the fractions from the Percoll gradient showed that the transferrin receptor, the small GTPases rab5 and Arf6, as well as annexin II were present both at the plasma membrane and in early endosomes, whereas the caveolar marker caveolin, 1co, migrated only with the biotinylated plasma membrane proteins. We used this fractionation procedure to show that the pore-forming toxin aerolysin does not reach the endocytic compartments of baby hamster kidney (BHK) cells. The procedure should be generally useful in rapidly determining whether extracellular proteins or ligands reach endosomes.     

Moving GLUT4: the biogenesis and trafficking of GLUT4 storage vesicles

The GLUT4 system in muscle and fat cells plays an important role in whole-body glucose homeostasis. Insulin stimulates the translocation of GLUT4 from an intracellular storage compartment to the cell surface. The nature of this compartment remains largely unknown. We review recent studies describing the biogenesis and molecular constituents of the GLUT4 storage compartment and conclude that it is segregated from the endosomal and biosynthetic pathways. Further, we present evidence to suggest that the GLUT4 storage compartment moves directly to the plasma membrane in response to insulin and, hence, is analogous to small synaptic vesicles in neurons. We propose that the GLUT4 storage compartment be referred to as GLUT4 storage vesicles or GSVs.     

A constitutively internalizing and recycling mutant of the mu-opioid receptor

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation, whereas certain other receptor types recycle constitutively, e.g., the transferrin receptor. To investigate structural domains involved in mu-opioid receptor internalization, we constructed two truncation mutants bracketing a Ser/Thr-rich domain (354ThrSerSerThrIleGluGlnGlnAsn362) unique to the C-terminus of the mu-opioid receptor (mutants Trunc354 and Trunc363). Ligand binding did not differ substantially, and G protein coupling was slightly lower for these mu-receptor constructs, in particular for Trunc363. To permit localization of the receptor by immunocytochemistry, an epitope tag was added to the N-terminus of the wild-type and mutant receptors. Both the wild-type mu-opioid receptor and Trunc363 resided largely at the plasma membrane and internalized into vesicles upon stimulation with the agonist [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin. Internalization occurred into vesicles that contain transferrin receptors, as shown previously, as well as clathrin, but not caveolin. In contrast, even without any agonist present, Trunc354 colocalized in intracellular vesicles with clathrin and transferrin receptors, but not caveolin. On blocking internalization by hyperosmolar sucrose or acid treatment, Trunc354 translocated to the plasma membrane, indicating that the mutant internalized into clathrin-coated vesicles and recycled constitutively. Despite agonist-independent internalization of Trunc354, basal G protein coupling was not elevated, suggesting distinct mechanisms for coupling and internalization. Furthermore, a portion of the C-terminus, particularly the Ser/Thr domain, appears to suppress mu-receptor internalization, which can be overcome by agonist stimulation. These results demonstrate that a mutant GPCR can be constructed such that internalization, normally an agonist-dependent process, can occur spontaneously without concomitant G protein activation.     

Subverted transferrin trafficking in Leishmania-infected macrophages

The intracellular fate of human transferrin (HTf) in macrophages infected by Leishmania was investigated. Binding of HTf-gold complexes at 4 degrees C was competitively inhibited by native holoHTf but not by apoHTf. Infected and uninfected macrophages displayed rather distinct HTf trafficking. Pulse-chase experiments using uninfected macrophages loaded with 15-nm gold-conjugated bovine serum albumin (BSA) and then incubated with 5-nm gold-conjugated HTf revealed a remarkable segregation of these tracers in distinct compartments. Nevertheless, Leishmania-infected macrophages presented extensive particle colocalization at both 60 min and 18 h. Light and electron microscopy immunolabeling indicated that HTf was delivered to the parasitophorous vacuole, formed patches on the amastigote surface, and was endocytosed via the flagellar pocket. Double-staining assays showed the colocalization of biotinylated HTf and its receptor in association with the parasitophorous vacuole. To approach the Tf-binding sites of amastigotes we performed HTf-fluorescein isothiocyanate (FITC) assays. Staining was diffuse at 4 degrees C and punctate at 35 degrees C, and only the former was sensitive to ethidium bromide, indicating an eventual temperature-dependent endocytic process. Within parasites, HTf was found in cysteine-proteinase-rich structures, suggesting that the protein can be endocytosed by intracellular amastigotes and sorted to the parasite endosomal-lysosomal compartments rather than being recycled. The treatment of infected macrophages with holoHTf, but not apoHTf, promoted the parasite's intracellular survival. These results suggest that Leishmania amastigotes can exploit and subvert the host-cell endocytic system and indicate the role of Tf-carried iron in the outcome of leishmanial infection.