AP-1 Membrane–Cytoplasm Recycling Regulated by μ1A-Adaptin

The adaptor protein complex AP-1 mediates vesicular protein sorting between the trans Golgi network and endosomes. AP-1 recycles between membranes and the cytoplasm together with clathrin during transport vesicle formation and vesicle uncoating. AP-1 recycles independent of clathrin, indicating binding to unproductive membrane domains and premature termination of vesicle budding. Membrane recruitment requires ADP ribosylation factor-1-GTP, a transmembrane protein containing an AP-1-binding motif and phosphatidyl-inositol phosphate (PI-4-P). Little is known about the regulation of AP-1 membrane–cytoplasm recycling. We identified the N-terminal domain of μ1A-adaptin as being involved in the regulation of AP-1 membrane–cytoplasm recycling by constructing chimeras of μ1A and its homologue μ2. The AP-1* complex containing this μ2–μ1A chimera had slowed down recycling kinetics, resulting in missorting of mannose 6-phosphate receptors. The N-terminal domain is only accessible from the cytoplasmic AP-1 surface. None of the proteins known to influence AP-1 membrane recycling bound to this μ1A domain, indicating the regulation of AP-1 membrane–cytoplasm recycling by an yet unidentified cytoplasmic protein.     

Internalization of a major group human rhinovirus does not require cytoplasmic or transmembrane domains of ICAM-1.

Intercellular adhesion molecule-1 (CD54), a cell adhesion molecule and the receptor for the major group of rhinoviruses, is a class 1 membrane protein with five Ig-like domains in its extracellular region, a transmembrane domain, and a short cytoplasmic domain. The amino-terminal domains (D1 and D2) are sufficient for virus binding and the first is most important (1). We have investigated whether other extracellular domains, transmembrane or cytoplasmic domains are required for virus entry as determined by postinfection virion protein biosynthesis. We demonstrate that cytoplasmic, transmembrane, and Ig-like domains 3, 4, and 5 are not essential for rhinovirus entry into transfected COS cells. The efficiency of rhinovirus infection directly correlates with the efficiency of rhinovirus binding and a form of intercellular adhesion molecule-1 that is glycophosphatidyl-inositol anchored, and thus does not extend into the inner leaflet of the membrane bilayer or the cytoplasm efficiently supports virus entry.     

Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations

This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Furthermore, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C delta 1 labeled with green fluorescent protein on ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area-to-volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible.     

OBSERVATIONS ON THE STRUCTURE OF RHODOSPIRILLUM MOLISCHIANUM

The lamellae of the bacterium Rhodospirillum molischianum originate as extensions of the cytoplasmic membrane into the cytoplasm of the cell. Initially, these extensions are narrow folds and occur independently of one another. The first lamellae to appear average about 80 A in width, representing one side of the infolded cytoplasmic membrane, or 160 A when the two sides of the fold are closely appressed. The 160-A lamellae increase in number and may associate to form larger lamellae, which represent varying degrees of association between adjacent folds. Later, the space within each fold increases; the two appressed regions of the cytoplasmic membrane in each fold separate to form distinct invaginations, and the lamellae observed at this stage are formed by an association of the sides of adjacent invaginations.     

Membrane events involved in myoblast fusion

Myoblast fusion has been studied in cultures of chick embryonic muscle utilizing ultrastructural techniques. The multinucleated muscle cells (myotubes) are generated by the fusion of two plasma membranes from adjacent cells, apparently by forming a single bilayer that is particle-free in freeze-fracture replicas. This single bilayer subsequently collapses, and cytoplasmic continuity is established between the cells. The fusion between the two plasma membranes appears to take place primarily within particle-free domains (probably phospholipid enriched), and cytoplasmic unilamellar, particle-free vesicles are occasionally associated with these regions. These vesicles structurally resemble phospholipid vesicles (liposomes). They are present in normal myoblasts, but they are absent in certain fusion-arrested myoblast popluations, such as those treated with either 5-bromo-deoxyuridine (BUdR), cycloheximide (CHX), or pospholipase C (PLC). The unilamellar, particle-free vesicles are present in close proximity to the plasma membranes, and physical contact is observed frequently between the vesicle membrane and the plasma membrane. The regions of vesicle membrane-plasma membrane interaction are characteristically free of intramembrane particles. A model for myoblast fusion is presented that is based onan interpretation of these observations. This model suggests that the cytoplasmic vesicles initiate the generation of particle-depleted membrane domains, both being essential components in the fusion process.     

AP-1 membrane-cytoplasm recycling regulated by mu1A-adaptin.

The adaptor protein complex AP-1 mediates vesicular protein sorting between the trans Golgi network and endosomes. AP-1 recycles between membranes and the cytoplasm together with clathrin during transport vesicle formation and vesicle uncoating. AP-1 recycles independent of clathrin, indicating binding to unproductive membrane domains and premature termination of vesicle budding. Membrane recruitment requires ADP ribosylation factor-1-GTP, a transmembrane protein containing an AP-1-binding motif and phosphatidyl-inositol phosphate (PI-4-P). Little is known about the regulation of AP-1 membrane-cytoplasm recycling. We identified the N-terminal domain of micro1A-adaptin as being involved in the regulation of AP-1 membrane-cytoplasm recycling by constructing chimeras of micro1A and its homologue micro2. The AP-1* complex containing this mu2-micro1A chimera had slowed down recycling kinetics, resulting in missorting of mannose 6-phosphate receptors. The N-terminal domain is only accessible from the cytoplasmic AP-1 surface. None of the proteins known to influence AP-1 membrane recycling bound to this micro1A domain, indicating the regulation of AP-1 membrane-cytoplasm recycling by an yet unidentified cytoplasmic protein.     

Recycling of MUC1 is dependent on its palmitoylation

MUC1 is a mucin-like transmembrane protein expressed on the apical surface of epithelia, where it protects the cell surface. The cytoplasmic domain has numerous sites for phosphorylation and docking of proteins involved in signal transduction. In a previous study, we showed that the cytoplasmic YXXphi motif Y20HPM and the tyrosine-phosphorylated Y60TNP motif are required for MUC1 clathrin-mediated endocytosis through binding AP-2 and Grb2, respectively (Kinlough, C. L., Poland, P. A., Bruns, J. B., Harkleroad, K. L., and Hughey, R. P. (2004) J. Biol. Chem. 279, 53071-53077). Palmitoylation of transmembrane proteins can affect their membrane trafficking, and the MUC1 sequence CQC3RRK at the boundary of the transmembrane and cytoplasmic domains mimics reported site(s) of S-palmitoylation. [3H]Palmitate labeling of Chinese hamster ovary cells expressing MUC1 with mutations in CQC3RRK revealed that MUC1 is dually palmitoylated at the CQC motif independent of RRK. Lack of palmitoylation did not affect the cold detergent solubility profile of a chimera (Tac ectodomain and MUC1 transmembrane and cytoplasmic domains), the rate of chimera delivery to the cell surface, or its half-life. Calculation of rate constants for membrane trafficking of wild-type and mutant Tac-MUC1 indicated that the lack of palmitoylation blocked recycling, but not endocytosis, and caused the chimera to accumulate in a EGFP-Rab11-positive endosomal compartment. Mutations CQC/AQA and Y20N inhibited Tac-MUC1 co-immunoprecipitation with AP-1, although mutant Y20N had reduced rates of both endocytosis and recycling, but a normal subcellular distribution. The double mutant chimera AQA+Y20N had reduced endocytosis and recycling rates and accumulated in EGFP-Rab11-positive endosomes, indicating that palmitoylation is the dominant feature modulating MUC1 recycling from endosomes back to the plasma membrane.     

Protein zero of peripheral nerve myelin: biosynthesis, membrane insertion, and evidence for homotypic interaction.

Protein zero (P0), an integral membrane glycoprotein synthesized by Schwann cells, is the major glycoprotein of peripheral nerve myelin. The predicted disposition of P0 with respect to the membrane bilayer postulates the existence of extracellular and intracellular domains, that mediate compaction of the myelin lamellae. We used in vitro translations programmed with sciatic nerve mRNA and cells transfected with a P0 cDNA construct to study the biosynthesis and topology of P0 in the bilayer. The behavior of P0 at the cell surface, when expressed under physiological conditions, was also examined. We have verified the topological predictions of an earlier model, derived from analysis of a P0 cDNA, and provide evidence that the extracellular domain of P0 mediates homotypically cell-cell interactions in the transfectants.     

The origin and fate of annulate lamellae in maturing sand dollar eggs

Electron micrograph evidence is presented that the nuclear envelope of the mature ovum of Dendraster excentricus is implicated in a proliferation of what appear as nuclear envelope replicas in the cytoplasm. The proliferation is associated with intranuclear vesicles which apparently coalesce to form comparatively simple replicas of the nuclear envelope closely applied to the inside of the nuclear envelope. The envelope itself may become disorganized at the time when fully formed annulate lamellae appear on the cytoplasmic side and parallel with it. The concept of interconvertibility of general cytoplasmic vesicles with most of the membrane systems of the cytoplasm is presented. The structure of the annuli in the annulate lamellae is shown to include small spheres or vesicles of variable size embedded in a dense matrix. Dense particles which are about 150 A in diameter are often found closely associated with annulate lamellae in the cytoplasm. Similar structures in other echinoderm eggs are basophilic. In this species, unlike other published examples, the association apparently takes place in the cytoplasm only after the lamellae have separated from the nucleus. If 150 A particles are synthesized by annulate lamellae, as their close physical relationship suggests, then in this species at least the necessary synthetic mechanisms and specificity must reside in the structure of annulate lamellae.     

The structure of the gills of the elasmobranch,Scyliorhinus canicula (L.)

Summary The anatomy of the blood supply to the gills of the dogfish, Scyliorhinus canicula, is described. The anatomical basis for a counter-current exchange system at the respiratory surfaces is reported. Within the interbranchial septum there is a capillary network joining all the afferent branchial arterioles of the gill. The structure of the walls of the corpus cavernosum is found to be of smooth muscle cells supported by a basal lamina and connective tissue and lined by endothelial cells containing phagocytic vesicles. Both the capillary network and corpus cavernosum are suggested to function in smoothing the pressure pulses of the blood flow. Pre- and post-lamellar vessels and pre- and post-lamellar sphincters are described. The sphincters are thought to control the number of secondary lamellae physiologically in the respiratory circuit, and by retaining blood within nonperfused lamellae to act in conjunction with pillar cells (contracting in antagonism to the hydrostatic skeleton of the blood) to maintain the rigidity of secondary lamellae in the water current.Whorls of cells of unknown function are found within the interbranchial septum. In the epithelium lining the water channel large cells having a complexly branching plasma membrane and a very large central vacuole occurs. The cytoplasm lining the lumen contains numerous vacuoles each surrounded by a double membrane.This work formed part of a thesis submitted for the degree of Master of Science at the University of Bristol. I should like to thank Professor G.M. Hughes for the use of facilities in the Department of Zoology, University of Bristol.     

The Origin and Fate of Annulate Lamellae in Maturing Sand Dollar Eggs

Electron micrograph evidence is presented that the nuclear envelope of the mature ovum of Dendraster excentricus is implicated in a proliferation of what appear as nuclear envelope replicas in the cytoplasm. The proliferation is associated with intranuclear vesicles which apparently coalesce to form comparatively simple replicas of the nuclear envelope closely applied to the inside of the nuclear envelope. The envelope itself may become disorganized at the time when fully formed annulate lamellae appear on the cytoplasmic side and parallel with it. The concept of interconvertibility of general cytoplasmic vesicles with most of the membrane systems of the cytoplasm is presented. The structure of the annuli in the annulate lamellae is shown to include small spheres or vesicles of variable size embedded in a dense matrix. Dense particles which are about 150 A in diameter are often found closely associated with annulate lamellae in the cytoplasm. Similar structures in other echinoderm eggs are basophilic. In this species, unlike other published examples, the association apparently takes place in the cytoplasm only after the lamellae have separated from the nucleus. If 150 A particles are synthesized by annulate lamellae, as their close physical relationship suggests, then in this species at least the necessary synthetic mechanisms and specificity must reside in the structure of annulate lamellae.     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary The first stages of the oogenesis of Triops cancriformis have been studied. At the outset the oocyte is smaller than the nurse cells. Meiosis begins with typical synaptonemal complexes. The significance of these complexes and of some other peculiar structures of germ cells, i.e., pore complexes and annuli within the nucleus, and annulate lamellae within the cytoplasm are discussed. The morphofunctional uniformity of some cytoplasmic structures (annulate lamellae, concentrically arranged ER, and yolk globules) in the oocyte as well as its nurse cells is also discussed.     

The Cdc42 Guanine Nucleotide Exchange Factor FGD6 Coordinates Cell Polarity and Endosomal Membrane Recycling in Osteoclasts

The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation.     

Desmosome assembly in MDCK epithelial cells does not require the presence of functional microtubules.

Desmosomes, complex multisubunit structures that assemble at sites of cell-cell contact, are important components of the epithelial junctional complex. Desmosome assembly requires the coordinated interaction at the plasma membrane of at least 8 cytoplasmic and integral membrane proteins organized into two structurally and functionally distinct domains, the cytoplasmic plaque and membrane core. Previous studies (Pasdar et al., J. Cell Biol., 113:645-655) provided evidence that cytokeratin filaments and microtubules may regulate transfer and assembly of cytoplasmic plaque and membrane core proteins, respectively. To determine directly the role of microtubules in these processes, Madin-Darby canine kidney (MDCK) cells were treated with nocodazole or colchicine to disrupt the microtubular network. Biochemical analysis of the different components of the cytoplasmic plaque and membrane core domains revealed little or no effect of nocodazole or colchicine on the kinetics of synthesis, post-translational modifications, transfer of proteins to the plasma membrane or their metabolic stability in the presence or absence of cell-cell contact. Likewise, immunofluorescence analysis of desmosome formation demonstrated an apparently normal desmosome assembly in the presence of nocodazole or colchicine upon induction of cell-cell contact. These results indicate that an intact microtubular network is not necessary for the processing or transport of the desmosomal membrane core glycoproteins to the plasma membrane in the absence or presence of cell-cell contact. Furthermore, the integration of the cytoplasmic plaque and membrane core domains induced by cell-cell contact at the plasma membranes of adjacent cells does not require the presence of functional microtubules.     

Is the nuclear envelope a ‘generator’ of membrane?

Summary Early diplotene oocytes from Necturus maculosus ranging from 0.2 to 0.5 mm in diameter were examined by electron microscopy. In the smallest oocytes of this range, the cytoplasm is largely devoid of membranes, but contains primarily ribosomes and mitochondria. In slightly larger oocytes, smooth-surfaced cytomembranes first appear in the perinuclear cytoplasm. At this time, the outer layer of the germinal vesicle nuclear envelope (GVNE) shows frequent connections with long membranous lamellae that extend for considerable, but variable distances into the juxtanuclear ooplasm. The number of smooth membranous lamellae increases tremendously as the oocytes increase in diameter. In such oocytes as well, frequent continuities are observed between the outer membrane of the GVNE and many of the cytoplasmic membranes. Eventually, as the ooplasm becomes populated with extensive numbers of membranous lamellae, instances of continuity between the membranous lamellae and nuclear envelope now become sparse and eventually non-existent. The frequent connections observed between membranous lamellae and the outer membrane of the GVNE during a circumscribed interval of diplotene strongly implicate the GVNE in the generation of extensive amounts of cytoplasmic membrane. The ooplasm of larger oocytes in the size range indicated contain numerous Golgi complexes and large quantities of annulate lamellae most of which are positioned in the peripheral or subcortical ooplasm, as well as extensive quantities of smooth membranes of the endoplasmic reticulum and lipid droplets.     

Relationship between Cell Wall, Cytoplasmic Membrane, and Bacterial Motility

High-resolution electron microscopy of polarly flagellated bacteria revealed that their flagella originate at a circular, differentiated portion of the cytoplasmic membrane approximately 25 nm in diameter. The flagella also have discs attaching them to the cell wall. These attachment discs are extremely resistant to lytic damage and are firmly bound to the flagella. The cytoplasm beneath the flagellum contains a granulated basal body about 60 nm in diameter, and a specialized polar membrane. The existence of membrane-bound basal bodies is shown to be an artifact arising from adherence of cell wall and cytoplasmic membrane fragments to flagella in lysed preparations. Based on structures observed, a mechanism to explain bacterial flagellar movement is proposed. Flagella are considered to be anchored to the cell wall and activated by displacement of underlying cytoplasmic membrane to which they are also firmly attached. An explanation for the membrane displacement is given.     

Mutational analysis of the human KDEL receptor: distinct structural requirements for Golgi retention, ligand binding and retrograde transport.

The KDEL receptor is a seven-transmembrane-domain protein that is responsible for the retrieval of endoplasmic reticulum (ER) proteins from the Golgi complex. It is a temporary resident of the Golgi apparatus: upon binding a KDEL-containing ligand, it moves to the ER, where the ligand is released. We have expressed mutant forms of the human receptor in COS cells and examined their intracellular locations and ligand-binding capacities. We show that ligand binding is dependent on charged residues within the transmembrane domains. Surprisingly, retrograde transport of occupied receptor is unaffected by most mutations in the cytoplasmic loops, but is critically dependent upon an aspartic acid residue in the seventh transmembrane domain. Retention in the Golgi apparatus requires neither ligand binding nor this aspartate residue, and thus is independent of receptor recycling. We suggest that movement of the receptor is controlled by conformational changes and intermolecular interactions within the membrane bilayer.     

Culture of human endometrial cells under polarizing conditions

Glandular epithelial and stromal cells were isolated from human endometrial biopsies and cultured in a dual-chambered system (Millicell; Millipore, Bedford, Ma., USA) that provides access of the medium to both sides of a membrane coated with reconstituted basement membrane material (Matrigel; Collaborative Research Inc., Bedford, Ma., USA). Examination by electron microscopy revealed that the epithelial cells formed a polarized cuboidal-columnar monolayer on the Matrigel surface. The cells exhibited apical microvilli, basal nuclei, and numerous cytoplasmic structures consistent with a well-differentiated cytoplasm; they were joined basally by interdigitating processes and apically by tight junctions and desmosomes. In contrast, epithelial cells cultured in parallel on plastic dishes were flattened, had fewer microvilli and cytoplasmic structures, and no junctional complexes.