Placentation in the rock cavy, Kerodon rupestris (Wied)

Rock cavies are rodents found in the semi-arid caatinga of Brazil. We studied the structure of the rock cavy placenta by light and transmission electron microscopy. The exchange area of the labyrinth was organized in lobes separated by interlobular areas. The interhaemal barrier was syncytial haemomonochorial. The syncytiotrophoblast had recesses in the basal membrane and some invaginations of the apical membrane, but transtrophoblastic channels could not be found. The interlobular regions comprised syncytiotrophoblast, enclosing maternal venous blood channels, and cytotrophoblast. There was a prominent subplacenta composed of cytotrophoblast and syncytiotrophoblast. Microvilli projected into spaces between the cytotrophoblasts and into lacunae within the syncytiotrophoblast. The yolk sac epithelium exhibited coated pits, endocytotic vesicles and larger vacuoles, consistent with a role in protein uptake from the uterine lumen. Tight junctions between these cells provided a barrier to diffusion by the intercellular route. The reproductive biology of the rock cavy differs from other members of the family, including the guinea pig, but the architecture of the placenta remains remarkably constant. (c) 2004 Elsevier Ltd. All rights reserved.

Estudo de transcriptomas por RNAseq em tecidos de cabeça e glândula salivar de Rhodnius montenegrensis e Rhodnius robustus (Hemiptera, Reduviidade, Triatominae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expressions- und Funktionsanalysen von "Tetraspan Vesicle Membrane Proteins" in Caenorhabditis elegans

Tetraspan vesicle membrane proteins (TVPs) sind ubiquitäre Komponenten von Transportvesikeln. Bei den Säugetieren unterscheidet man drei Familien, die Physine, Gyrine und SCAMPs (secretory carrier-associated membrane proteins). Ihre Funktion ist weitgehend unbekannt, es wird jedoch vermutet, dass sie eine Rolle bei der Vesikelbildung und der Vesikelrezirkulierung spielen. In Caenorhabditis elegans existiert von jeder Familie jeweils nur ein einziges Polypeptid: für die Physine Synaptophysin (SPH-1), für die Gyrine Synaptogyrin (SNG-1) und für die SCAMPs SCAMP (SCM-1). Ziel der Arbeit war es die Verteilung der C. elegans TVPs zu untersuchen und ihre Funktion unter besonderer Berücksichtigung der vesikelvermittelten synaptischen Kopplung zu bestimmen. Wenn die C. elegans TVPs in humanen Epithelzellen synthetisiert werden, lokalisieren sie in zytoplasmatischen Vesikeln. In Kotransfektionsexperimenten wurde gezeigt, dass sie größtenteils in den gleichen Strukturen enthalten sind. In C. elegans synthetisierte TVP-Reporterkonstrukte können in unterschiedlichen Geweben nachgewiesen werden. Dabei ist SNG-1 fast ausschließlich in Neuronen zu finden. SPH-1 und SCM-1 hingegen weisen komplexe und teilweise überlappende Verteilungsmuster auf. Während für SPH-1 eine starke Fluoreszenz im Pharynx, auf der apikalen Seite der Darmzellen oberhalb des sog. terminal webs und in adluminalen Regionen von exkretorischen Geweben gefunden wurde, war SCM-1 stark in der Muskulatur und den Coelomozyten vertreten. Die Expression von SCM-1 in Pharynx und Darm war deutlich schwächer. Die C. elegans TVPs werden früh in der Entwicklung ab der Gastrulation (SPH-1 und SCM-1) bzw. ab der Neurulation im sog. Komma-Stadium (SNG-1) produziert. Um die Funktion der TVPs in C. elegans zu untersuchen, wurden TVP-Mutanten analysiert. Durch Kombination aller drei TVP-Gen-Mutanten wurden TVP-Dreifachmutanten generiert. Diese wiesen keinen offensichtlichen Defekt im Bewegungsmuster auf, entwickelten sich normal und bildeten ein normales Nervensystem aus. Auch auf unterschiedliche chemische und physikalische Reize in sensorischen Tests reagierten die TVP-Dreifachmutanten in gleicher Weise wie Wildtyptiere. Ebenso zeigen die TVP-Dreifachmutanten elektrophysiologisch unter normalen Bedingungen keine anormalen Reaktionsmuster. In ultrastrukturellen Untersuchungen wurde lediglich eine signifikant erhöhte Anzahl Clathrin-ummantelter Vesikel in cholinergen Synapsen gefunden. Erst unter Stressbedingungen, hervorgerufen durch den GABA-Antagonisten Pentylentetrazol (PTZ), wiesen sowohl die TVP-Dreifach- als auch die TVP-Einzelmutanten eine deutlich erhöhte Krampfbereitschaft auf. Zusammengenommen zeigen die Analysen, dass TVPs zwar für grundlegende neuronale Prozesse nicht notwendig sind, dass sie aber auf der anderen Seite vermutlich an alternativen redundanten Wegen der Neurotransmitterfreisetzung beteiligt sind.

Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages.

Precise knowledge regarding cellular uptake of nanoparticles is of great importance for future biomedical applications. Four different endocytotic uptake mechanisms, that is, phagocytosis, macropinocytosis, clathrin- and caveolin-mediated endocytosis, were investigated using a mouse macrophage (J774A.1) and a human alveolar epithelial type II cell line (A549). In order to deduce the involved pathway in nanoparticle uptake, selected inhibitors specific for one of the endocytotic pathways were optimized regarding concentration and incubation time in combination with fluorescently tagged marker proteins. Qualitative immunolocalization showed that J774A.1 cells highly expressed the lipid raft-related protein flotillin-1 and clathrin heavy chain, however, no caveolin-1. A549 cells expressed clathrin heavy chain and caveolin-1, but no flotillin-1 uptake-related proteins. Our data revealed an impeded uptake of 40 nm polystyrene nanoparticles by J774A.1 macrophages when actin polymerization and clathrin-coated pit formation was blocked. From this result, it is suggested that macropinocytosis and phagocytosis, as well as clathrin-mediated endocytosis, play a crucial role. The uptake of 40 nm nanoparticles in alveolar epithelial A549 cells was inhibited after depletion of cholesterol in the plasma membrane (preventing caveolin-mediated endocytosis) and inhibition of clathrin-coated vesicles (preventing clathrin-mediated endocytosis). Our data showed that a combination of several distinguishable endocytotic uptake mechanisms are involved in the uptake of 40 nm polystyrene nanoparticles in both the macrophage and epithelial cell line.

GIPC, a PDZ domain containing protein, interacts specifically with the C terminus of RGS-GAIP

We have identified a mammalian protein called GIPC (for GAIP interacting protein, C terminus), which has a central PDZ domain and a C-terminal acyl carrier protein (ACP) domain. The PDZ domain of GIPC specifically interacts with RGS-GAIP, a GTPase-activating protein (GAP) for Gαi subunits recently localized on clathrin-coated vesicles. Analysis of deletion mutants indicated that the PDZ domain of GIPC specifically interacts with the C terminus of GAIP (11 amino acids) in the yeast two-hybrid system and glutathione S-transferase (GST)-GIPC pull-down assays, but GIPC does not interact with other members of the RGS (regulators of G protein signaling) family tested. This finding is in keeping with the fact that the C terminus of GAIP is unique and possesses a modified C-terminal PDZ-binding motif (SEA). By immunoblotting of membrane fractions prepared from HeLa cells, we found that there are two pools of GIPC–a soluble or cytosolic pool (70%) and a membrane-associated pool (30%). By immunofluorescence, endogenous and GFP-tagged GIPC show both a diffuse and punctate cytoplasmic distribution in HeLa cells reflecting, respectively, the existence of soluble and membrane-associated pools. By immunoelectron microscopy the membrane pool of GIPC is associated with clusters of vesicles located near the plasma membrane. These data provide direct evidence that the C terminus of a RGS protein is involved in interactions specific for a given RGS protein and implicates GAIP in regulation of additional functions besides its GAP activity. The location of GIPC together with its binding to GAIP suggest that GAIP and GIPC may be components of a G protein-coupled signaling complex involved in the regulation of vesicular trafficking. The presence of an ACP domain suggests a putative function for GIPC in the acylation of vesicle-bound proteins.

A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA

The topology of signal transduction is particularly important for neurons. Neurotrophic factors such as nerve growth factor (NGF) interact with receptors at distal axons and a signal is transduced by retrograde transport to the cell body to ensure survival of the neuron. We have discovered an organelle that may account for the retrograde transport of the neurotrophin signal. This organelle is derived from endocytosis of the receptor tyrosine kinase for NGF, TrkA. In vitro reactions containing semi-intact PC12 cells and ATP were used to enhance recovery of a novel organelle: small vesicles containing internalized NGF bound to activated TrkA. These vesicles were distinct from clathrin coated vesicles, uncoated primary endocytic vesicles, and synaptic vesicles, and resembled transport vesicles in their sedimentation velocity. They contained 10% of the total bound NGF and almost one-third of the total tyrosine phosphorylated TrkA. These small vesicles are compelling candidates for the organelles through which the neurotrophin signal is conveyed down the axon.

High-Affinity Binding Of The AP-1 Adaptor Complex to Trans-Golgi Network Membranes Devoid Of Mannose 6-Phosphate Receptors

The GTP-binding protein ADP-ribosylation factor (ARF) initiates clathrin-coat assembly at the trans-Goli network (TGN) by generating high-affinity membrane-binding sites for the AP-1 adaptor complex. Both transmembrane proteins, which are sorted into the assembling coated bud, and novel docking proteins have been suggested to be partners with GTP-bound ARF in generating the AP-1-docking sites. The best characterized, and probably the major transmembrane molecules sorted into the clathrin-coated vesicles that form on the TGN, are the mannose 6-phosphate receptors (MPRs). Here, we have examined the role of the MPRs in the AP-1 recruitment process by comparing fibroblasts derived from embryos of either normal or MPR-negative animals. Despite major alterations to the lysosome compartment in the MPR-deficient cells, the steady-state distribution of AP-1 at the TGN is comparable to that of normal cells. Golgi-enriched membranes prepared from the receptor-negative cells also display an apparently normal capacity to recruit AP-1 in vitro in the presence of ARF and either GTP or GTPγS. The AP-1 adaptor is recruited specifically onto the TGN and not onto the numerous abnormal membrane elements that accumulate within the MPR-negative fibroblasts. AP-1 bound to TGN membranes from either normal or MPR-negative fibroblasts is fully resistant to chemical extraction with 1 M Tris-HCl, pH 7, indicating that the adaptor binds to both membrane types with high affinity. The only difference we do note between the Golgi prepared from the MPR-deficient cells and the normal cells is that AP-1 recruited onto the receptor-lacking membranes in the presence of ARF1·GTP is consistently more resistant to extraction with Tris. Because sensitivity to Tris extraction correlates well with nucleotide hydrolysis, this finding might suggest a possible link between MPR sorting and ARF GAP regulation. We conclude that the MPRs are not essential determinants in the initial steps of AP-1 binding to the TGN but, instead, they may play a regulatory role in clathrin-coated vesicle formation by affecting ARF·GTP hydrolysis.

Inhibition of Endosome Fusion by Wortmannin Persists in the Presence of Activated rab5

Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5Q79L or of a xanthosine triphosphate-binding mutant, rab5D136N, in the presence of the nonhydrolysable analogue XTPγS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPγS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5Q79L, beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPγS.

ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.

Vesicle-associated Membrane Protein 4 is Implicated in Trans-Golgi Network Vesicle Trafficking

The trans-Golgi network (TGN) plays a pivotal role in directing proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the vesicle-associated membrane protein (VAMP) family of trafficking proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport.

Testosterone Signaling through Internalizable Surface Receptors in Androgen Receptor-free Macrophages

Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca2+], which is due to release of Ca2+ from intracellular Ca2+ stores. This Ca2+ mobilization is also inducible by plasma membrane-impermeable testosterone-BSA. It is not affected by the AR blockers cyproterone and flutamide, whereas it is completely inhibited by the phospholipase C inhibitor U-73122 and pertussis toxin. Binding sites for testosterone are detectable on the surface of intact IC-21 cells, which become selectively internalized independent on caveolae and clathrin-coated vesicles upon agonist stimulation. Internalization is dependent on temperature, ATP, cytoskeletal elements, phospholipase C, and G-proteins. Collectively, our data provide evidence for the existence of G-protein-coupled, agonist-sequestrable receptors for testosterone in plasma membranes, which initiate a transcription-independent signaling pathway of testosterone.

Uptake and Intracellular Transport of Acidic Fibroblast Growth Factor: Evidence for Free and Cytoskeleton-anchored Fibroblast Growth Factor Receptors

Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment.

Ubiquitously Expressed Dynamin-II Has a Higher Intrinsic GTPase Activity and a Greater Propensity for Self-assembly Than Neuronal Dynamin-I

To begin to understand mechanistic differences in endocytosis in neurons and nonneuronal cells, we have compared the biochemical properties of the ubiquitously expressed dynamin-II isoform with those of neuron-specific dynamin-I. Like dynamin-I, dynamin-II is specifically localized to and highly concentrated in coated pits on the plasma membrane and can assemble in vitro into rings and helical arrays. As expected, the two closely related isoforms share a similar mechanism for GTP hydrolysis: both are stimulated in vitro by self-assembly and by interaction with microtubules or the SH3 domain-containing protein, grb2. Deletion of the C-terminal proline/arginine-rich domain from either isoform abrogates self-assembly and assembly-dependent increases in GTP hydrolysis. However, dynamin-II exhibits a ∼threefold higher rate of intrinsic GTP hydrolysis and higher affinity for GTP than dynamin-I. Strikingly, the stimulated GTPase activity of dynamin-II can be >40-fold higher than dynamin-I, due principally to its greater propensity for self-assembly and the increased resistance of assembled dynamin-II to GTP-triggered disassembly. These results are consistent with the hypothesis that self-assembly is a major regulator of dynamin GTPase activity and that the intrinsic rate of GTP hydrolysis reflects a dynamic, GTP-dependent equilibrium of assembly and disassembly.

Circulation of the Plasma Membrane in Dictyostelium

We have developed a fluorimetric assay with the use of the dye FM1-43 to determine the rate at which Dictyostelium amoebae endocytose their surface membrane. Our results show that they do so about once each 4–10 min. A clathrin null mutant takes its surface up only ∼30% more slowly, showing that this membrane uptake cannot be caused by clathrin-coated vesicles. Surprisingly, Ax2 and its parent, NC4, which differ in their rates of fluid-phase internalization by ∼60-fold, take up their surfaces at the same rates. These results show that, in axenic cells, the uptake of fluid and of surface area are separate processes. The large activity of this new endocytic cycle in both Ax2 and NC4 amoebae appears capable of delivering sufficient new surface area to advance the cells’ fronts during migration.

Subcellular Distribution and Tissue Expression of Phospholipase Dα, Dβ, and Dγ in Arabidopsis1

Three phospholipase Ds (PLDs; EC 3.1.4.4) have been cloned from Arabidopsis, and they exhibit two distinct types of activities: polyphosphoinositide-requiring PLDβ and PLDγ, and polyphosphoinositide-independent PLDα. In subcellular fractions of Arabidopsis leaves, PLDα and PLDγ were both present in the plasma membrane, intracellular membranes, mitochondria, and clathrin-coated vesicles, but their relative levels differed in these fractions. In addition, PLDγ was detected in the nuclear fraction. In contrast, PLDβ was not detectable in any of the subcellular fractions. PLDα activity was higher in the metabolically more active organs such as flowers, siliques, and roots than in dry seeds and mature leaves, whereas the polyphosphoinositide-dependent PLD activity was greater in older, senescing leaves than in other organs. PLDβ mRNA accumulated at a lower level than the PLDα and PLDγ transcripts in most organs, and the expression pattern of the PLDβ mRNA also differed from that of PLDα and PLDγ in different organs. Collectively, these data demonstrated that PLDα, PLDβ, and PLDγ have different patterns of subcellular distribution and tissue expression in Arabidopsis. The present study also provides evidence for the presence of an additional PLD that is structurally more closely related to PLDγ than to the other two PLDs.