Ventral Membrane Research Articles

Cortactin is a component of clathrin-coated pits and participates in receptor-mediated endocytosis.

The actin cytoskeleton is believed to contribute to the formation of clathrin-coated pits, although the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an F-actin-associated protein, localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction with a component of the endocytic machinery suggests that cortactin may participate in one or several endocytic processes. Therefore, the goal of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis. Morphological experiments with either anti-cortactin antibodies or expressed red fluorescence protein-tagged cortactin revealed a striking colocalization of cortactin and clathrin puncta at the ventral plasma membrane. Consistent with these observations, cells microinjected with these antibodies exhibited a marked decrease in the uptake of labeled transferrin and low-density lipoprotein while internalization of the fluid marker dextran was unchanged. Cells expressing the cortactin Src homology three domain also exhibited markedly reduced endocytosis. These findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. Thus, cortactin provides a direct link between the dynamic actin cytoskeleton and the membrane pinchase dynamin that supports vesicle formation during receptor-mediated endocytosis.

Change of the Topography of Ventral Cell Surface during Spreading of Fibroblasts as Revealed by Evanescent Wave-Excited Fluorescence Microscopy: Effect of Contractility and Microtubule Integrity

Evanescent wave-excited fluorescence microscopy, which selectively probes the ventral membranes of cells adhered to glass substrate, was utilized to observe the change in the topography of the ventral plasma membranes of Swiss 3T3 fibroblasts during spreading. In the initial stage of the spreading (up to 2 hours after seeding), the ventral membrane was close (<100nm) to the substrate in the peripheral and the central regions. About 4 hrs after seeding, the ventral surface assumed a flat topography for a short period and then gradually became uneven, displaying streak pattern of cell-to-substrate contact (6-8 hours after seeding). By 24 hours after seeding, cells gained polygonal shape and most regions except for the focal adhesions were separated from the substrate. Within these well-spread cells actin stress fibers were found to emanate obliquely from the focal adhesions, as previously reported. When cells were grown in the presence of 2, 3-butanedione monoxime (BDM), an inhibitor of actomyosin-based contraction of stress fibers and the cell, the ventral membranes in majority of the cells displayed flat topography, and the tilt of the stress fibers decreased. Cells grown in the presence of colchicine, a microtubule-depolymerizing agent also possessed flat ventral membrane and less tilted stress fibers. These results suggest that the contraction of stress fibers and integrity of microtubules are important in the formation of the uneven topography of ventral membrane and the tilt of stress fibers.

Dynamin Participates in Focal Extracellular Matrix Degradation by Invasive Cells

The degradation of extracellular matrix (ECM) by matrix metalloproteases is crucial in physiological and pathological cell invasion alike. Degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Herein, we show that the dynamin 2 (Dyn2), a GTPase implicated in the control of actin-driven cytoskeletal remodeling events and membrane transport, is necessary for focalized matrix degradation at invadopodia. Dynamin was inhibited by using two approaches: 1) expression of dominant negative GTPase-impaired or proline-rich domain-deleted Dyn2 mutants; and 2) inhibition of the dynamin regulator calcineurin by cyclosporin A. In both cases, the number and extension of ECM degradation foci were drastically reduced. To understand the site and mechanism of dynamin action, the cellular structures devoted to ECM degradation were analyzed by correlative confocal light-electron microscopy. Invadopodia were found to be organized into a previously undescribed ECM-degradation structure consisting of a large invagination of the ventral plasma membrane surface in close spatial relationship with the Golgi complex. Dyn2 seemed to be concentrated at invadopodia.

Dystrophin isoform Dp71 is present in lamellipodia and focal complexes in human astrocytoma cells U-373 MG

Dp71 is the most abundant product of the dmd gene in the brain. There are at least 2 isoforms derived from alternative splicing of exon 78 (Dp71d, which contains exon 78 and Dp71f, the spliced isoform) but the precise localization and function of each isoform is still unknown. In the present study, we demonstrate by RT-PCR that the Dp71f isoform is present in an astrocytoma cell line U-373 MG, and its subcellular localization was determined in the cytoplasm, particularly in perinuclear areas, with lower amounts towards the periphery but increasing in the leader borders of lamellipodia and focal complexes. Double labeling indirect immunofluorescence showed that Dp71f colocalized with actin-like beta-dystroglycan and beta-1 integrin. We also demonstrated by triple labeling that Dp71f was colocalized with actin and two members of integrin complexes, alpha-actinin and vinculin, in focal complexes. Ventral plasma membranes were enriched and in those containing focal complex proteins, we found colocalization of Dp71f, actin and vinculin. It is concluded that U-373 MG cells express Dp71f as part of lamellipodia and focal complex proteins, and possibly connected via distroglycan complexes to integrin complexes.

3N1015 単離基底側細胞膜を用いた細胞接着タンパク質インテグリンの分布調節機構の研究(12.細胞生物学的課題,一般講演,日本生物物理学会第40回年会)

3N1015 単離基底側細胞膜を用いた細胞接着タンパク質インテグリンの分布調節機構の研究(12.細胞生物学的課題,一般講演,日本生物物理学会第40回年会)

Components of cell-matrix adhesions.

Focal adhesions (FAs), also known as ‘focal contacts’, are specialized, extracellular matrix (ECM) attachment and signaling organelles, usually measuring a few square microns, located along the ventral plasma membrane of adherent cultured cells. Interference reflection microscopy, electron

A new genus of viviparous gyrodactylid (Monogenea) from the Great Barrier Reef, Australia with descriptions of seven new species

Acanthoplacatus gen. nov., a new genus of viviparous gyrodactylid, is described from the fins and skin of siganid fishes from the Great Barrier Reef, Australia. The genus is characterized by a muscular, tube-like haptor with 16 marginal hooks on the posterior margin. The ventral lobe of the haptor is located anteriorly relative to the dorsal lobe and contains a pair of hamuli and a ventral bar with posteriorly-projecting ventral bar membrane. A dorsal bar is absent. Five pairs of posterior gland cells surround the posterior terminations of the gut. The male copulatory organ is a muscular, non-eversible bulb with several spines around the distal opening. Species of Acanthoplacatus have a bilateral excretory system consisting of six pairs of flame cells and a pair of excretory bladders. Seven new species are described: Acanthoplacatus adlardi sp. nov. and A. amplihamus sp. nov. from Siganus punctatus (Forster, 1801), A. brauni sp. nov. from S. corallinus (Valenciennes, 1835), A. parvihamus sp. nov. from S. vulpinus (Schlegel and Mueller, 1845), A. puelli sp. nov. from S. puellus Schlegel, 1852, A. shieldsi sp. nov. from S. lineatus (Valenciennes, 1835) and A. sigani sp. nov. from S. fuscescens (Houttuyn, 1782). Species can be discriminated by shape and size of the hamuli, marginal hooks and ventral bar and by male copulatory organ sclerite morphology. Three species (A. brauni sp. nov., A. shieldsi sp. nov. and A. sigani sp. nov.) were assessed for seasonal variation of sclerite size. Ten of thirteen morphological characters showed seasonal variation in size for at least one of the species. The characters were longer in winter except dorsal root tissue cap width. Only one character, marginal hook length, showed significant seasonal variation for all three species. Species of Acanthoplacatus were observed to attach using only the marginal hooks and the role of hamuli in attachment is unclear. The dorsal fin of the host is the preferred site for most species but the anal fin, caudal fin and body surfaces are preferred by some species. Prevalences for species range from 57 to 100%.

3P200細胞接着タンパク質インテグリンの基底膜内における分布変化観察 : 開放細胞系を用いた研究

3P200細胞接着タンパク質インテグリンの基底膜内における分布変化観察 : 開放細胞系を用いた研究

Discovery of Mycangia and Mucus in Adult Female Xiphydriid Woodwasps (Hymenoptera: Xiphydriidae) in Japan

Abstract Dissection of emerging adult xiphydriid woodwasps, Xiphydria ogasawarai Matsumura, occurring in eastern, central, and western Japan during a 2- to 4-yr period, revealed that adult females possessed mycangia filled with fungal spores, whereas the mycangia were absent in males. Two masses of the spore were located on the slit-like 1st and 2nd valvulae of the ovipositor sting directly below the subgenital plate, and were covered with the ventral membranes. This is a new type of mycangia in woodwasps. The spores were globular or ovoid in shape and ≈5–30 μm in diameter, and had no clamp connections. All isolates from the mycangia of the xiphydriid populations, originating from different sites and years, developed the same colonies on potato-dextrose agar. This suggests that female adults of X. ogasawarai carry a single fungal species in their mycangia. It was also found that X. ogasawarai produced a slime-like secretion from glandular organs in the body cavity (mucus reservoir), which is homologous to...

Titanium dioxide ceramics control the differentiated phenotype of cardiac muscle cells in culture

A new approach, the cultivation of heart muscle cells on biocompatible scaffolds made from titanium dioxide ceramics was established to provide a mechanism for in vitro engineering of a vital heart tissue. Terminally differentiated ventricular myocytes isolated from hearts of adult rats were kept in primary culture for long periods of time and used as an experimental model. The microenvironmental properties of titanium dioxide ceramics helped to maintain the tissue-like structural organisation of the cardiac cells in vitro. Coating of the cell substrata with fine-grained titanium dioxide ceramics imitating cell surface topography favoured the formation of focal adhesion complexes in the ventral plasma membrane of cardiomyocytes. It also promoted the cellular expression of vinculin, a protein that connects the ECM integrin receptors to the network of cytoplasmic filaments, which define cell shape. This topographical reinforcement of cell–material interactions led to stabilisation of the molecular linkage between the extracellular contacts and the intracellular cytoskeleton and thus assisted the preservation and maintenance of the heart muscle cell differentiated phenotype in long-term primary culture. The results of this work demonstrate a promising pathway for the regulation of cellular organisation in vitro by local geometric control.

Syndesmos, a protein that interacts with the cytoplasmic domain of syndecan-4, mediates cell spreading and actin cytoskeletal organization.

Syndecan-4 is a cell surface heparan sulfate proteoglycan which, in cooperation with integrins, transduces signals for the assembly of focal adhesions and actin stress fibers in cells plated on fibronectin. The regulation of these cellular events is proposed to occur, in part, through the interaction of the cytoplasmic domains of these transmembrane receptors with intracellular proteins. To identify potential intracellular proteins that interact with the cytoplasmic domain of syndecan-4, we carried out a yeast two-hybrid screen in which the cytoplasmic domain of syndecan-4 was used as bait. As a result of this screen, we have identified a novel cellular protein that interacts with the cytoplasmic domain of syndecan-4 but not with those of the other three syndecan family members. The interaction involves both the membrane proximal and variable central regions of the cytoplasmic domain. We have named this cDNA and encoded protein syndesmos. Syndesmos is ubiquitously expressed and can be myristylated. Consistent with its myristylation and syndecan-4 association, syndesmos colocalizes with syndecan-4 in the ventral plasma membranes of cells plated on fibronectin. When overexpressed in NIH 3T3 cells, syndesmos enhances cell spreading, actin stress fiber and focal contact formation in a serum-independent manner.

1J0930 細胞膜底膜構造の形成機構 : エバネセント波励起蛍光顕微鏡による解析

1J0930 細胞膜底膜構造の形成機構 : エバネセント波励起蛍光顕微鏡による解析

Regulation of GTPase and adenylate cyclase activity by amyloid β-peptide and its fragments in rat brain tissue

Modulation of GTPase and adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) activity by Alzheimer's disease related amyloid β-peptide, Aβ(1–42), and its shorter fragments, Aβ(12–28), Aβ(25–35), were studied in isolated membranes from rat ventral hippocampus and frontal cortex. In both tissues, the activity of GTPase and adenylate cyclase was upregulated by Aβ(25–35), whereas Aβ(12–28) did not have any significant effect on the GTPase activity and only weakly influenced adenylate cyclase. Aβ(1–42), similar to Aβ(25–35), stimulated the GTPase activity in both tissues and adenylate cyclase activity in ventral hippocampal membranes. Surprisingly, Aβ(1–42) did not have a significant effect on adenylate cyclase activity in the cortical membranes. At high concentrations of Aβ(25–35) and Aβ(1–42), decreased or no activation of adenylate cyclase was observed. The activation of GTPase at high concentrations of Aβ(25–35) was pertussis toxin sensitive, suggesting that this effect is mediated by G i/G o proteins. Addition of glutathione and N-acetyl- l-cysteine, two well-known antioxidants, at 1.5 and 0.5 mM, respectively, decreased Aβ(25–35) stimulated adenylate cyclase activity in both tissues. Lys-Aβ(16–20), a hexapeptide shown previously to bind to the same sequence in Aβ-peptide, and prevent fibril formation, decreased stimulation of adenylate cyclase activity by Aβ(25–35), however, NMR diffusion measurements with the two peptides showed that this effect was not due to interactions between the two and that Aβ(25–35) was active in a monomeric form. Our data strongly suggest that Aβ and its fragments may affect G-protein coupled signal transduction systems, although the mechanism of this interaction is not fully understood.

Attachment of growth cones on substrate observed by multi-mode light microscopy.

Evanescent light illumination was introduced into a multi-mode microscope to construct a new type of total internal reflection fluorescence microscope (TIRFM). This microscope, capable of TIRFM, high resolution video-enhanced differential interference contrast (DIC), epifluorescence, interference reflection (IR) imaging, was combined with an image acquisition system for time-lapse microscopy. Neuronal growth cones of a rat hippocampal neuron were stained with membrane labeling fluorescence dyes (DiI or octadecyl rhodamine B). Dynamic changes of the cell substrate contact of the neuronal growth cone were observed using the multi-imaging capacities of this system. When growth cone regions were stimulated by pressure ejection of a high potassium solution, TIRFM intensity at the basal membrane of the growth cone increased, suggesting that basal membrane of growth cone approaches the glass substrate when excited. The approach of the ventral membrane to the substrate during excitatory stimulation was also observed with IR microscope. The functional importance of cell/substrate contact in growth cones is discussed.

Rac GTPases localize at sites of actin reorganization during dynamic remodeling of the cytoskeleton of normal embryonic fibroblasts.

Rac GTP-binding proteins are implicated in the dynamic organization of the actin cytoskeleton, and the mechanisms utilized for this purpose are not understood yet. In this paper we have analysed the effects of the expression of Rac proteins on the organization of the cytoskeleton, and their subcellular distribution in chicken embryo fibroblasts. In these cells, overexpression of wild-type Rac GTPases induces disassembly of stress fibers, and production of long, highly branched actin-rich protrusions, with consequent dramatic changes in cell morphology. The formation of these protrusions is mediated by adhesion to the substrate, and is prevented by incubation with anti-(beta)1 function-blocking antibodies. Rac-mediated cell shape changes require a wild-type GTPase, since expression of constitutively active V12-Rac proteins affects actin organization differently in these cells, without causing alterations in their morphology. Localization studies performed on ventral plasma membranes from fibroblasts transfected with wild-type or mutant GTPases show codistribution of Rac along stress fibers, before their disassembly and the formation of the actin-rich protrusions. These data show a link between Rac protein distribution, and their effects on the actin cytoskeleton. Altogether, our results are indicative of an active role of Rac proteins in stress fiber disassembly, and show that Rac, which can cycle its bound nucleotide, produces unique dynamic effects on actin organization.

Behavioral aspects of sperm competition in cuttlefish, Sepia officinalis (Sepioidea: Cephalopoda)

Sexual selection studies in cephalopods indicate that sperm competition is a central feature of their mating systems, yet this has not been studied experimentally in any detail. In 1998 we staged 20 matings of the cuttlefish, Sepia officinalis L., in the laboratory. Males rapidly initiated mating in the “head-to-head” position, with no apparent courtship. Mating lasted an average of 10 min (range 7 to 14 min). For the first 6 min (on average 63% of the mating duration), the male flushed strong jets of water directly at the female's buccal membrane, which sometimes resulted in the expulsion of parts of spermatangia placed there in recent matings. Then, in a single discrete movement that lasted an average of only 14 s, the male's modified fourth left arm – the hectocotylus – wrapped around a single large bundle of spermatophores (ca. 150 to 300) and transferred them to the female's buccal membrane. For the remainder of the mating (average 3 min, range 1.5 to 5.0 min), the hectocotylus repeatedly broke the spermatophores open, and manipulated them, so that sperm were released and many spermatangia were attached along the ventral buccal membrane, near the paired seminal receptacles. Approximately 140 spermatangia were attached in rows 3 to 5 deep around the ventral buccal membrane in a single mating; the rest were usually discarded during mating. Histology revealed that each of the seminal receptacles consists of a series of sperm storage bulbs connected by a central duct, which leads to a single pore at the surface of the buccal membrane. Baseline data on sperm motility were obtained, but the mechanism by which sperm enter the seminal receptacle remains unknown. Females seemed to initiate termination of mating, then males guarded their mates temporarily. These results, combined with other recent laboratory experiments, provide evidence that sperm competition may be a major feature of the mating system of S. officinalis.

Ectoparasites of Brazilian Free-Tailed Bats with Emphasis on Anatomical Site Preferences forChiroptonyssus robustipes(Acari: Macronyssidae)

Seven species of arthropods were recovered from 45 Brazilian free-tailed bats, Tadarida brasiliensis (I. Geoffrey), captured from May 1994 to May 1995 from a roost in Jenkins County, GA. Six species of mites were found infesting the bats, representing 5 families: Macronyssidae, Cheyletidae, Sarcoptidae, Rosensteiniidae, and Uropodidae. The macronyssid mite chiroptonyssus robustipes (Ewing) and the hemipteran bat bug, Cimex adjunctus (Barber), were collected from the host and the roost. C. robustipes was the most abundant mite (2,508 specimens) and the macronyssid Steatonyssus ceratognathus (Ewing) (189 specimens) was the 2nd most abundant mite found infesting the bats. The predatory cheyletid mite Cheyletonella vespertilionis Womersley (51 specimens) was also collected from the bats, and this is thought to be the 1st report of this mite from Georgia. In addition, Teinocoptes sp. (Sarcoptidae, 8 specimens), Nycteriglyphus sp. (Rosensteiniidae, 8 specimens), and uropodid mites (5 specimens) were removed from the bats. Of the 45 bats examined, all 30 females and 14 of 15 males were infested with C. robustipes. Twenty-three bat anatomical areas were designated and all C. robustipes mites were counted to ascertain anatomical preference. This mite was significantly more abundant on the ventral wing membrane areas posterior to the radiusulna and on the lower torso. Protonymphs of C. robustipes predominated on the wing membranes of the bats, whereas adult mites were more concentrated on the torso and head.

A cell-free system to study regulation of focal adhesions and of the connected actin cytoskeleton.

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca2+ concentrations induce quasi-reversible diffusion of beta1 integrins out of focal adhesions, whereas low Ca2+ concentrations induce irreversible recruitment of beta1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated beta1 receptors show that the cytoplasmic portion of beta1 is required for low Ca2+-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified alpha-actinin colocalizes and redistributes with beta1 receptors on ventral plasma membranes depleted of actin, implicating binding of alpha-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.

Gyrodactylus longidactylus n. sp., a monogenean from Pomatoschistus lozanoi (de Buen) (Gobiidae) from the North Sea

Gyrodactylus longidactylus n. sp., a gyrodactylid monogenean parasitising the gills of Pomatoschistus lozanoi (de Buen, 1923) in the North Sea is described. It is a species with rather small anchors which are only connected to a ventral bar which lacks a ventral bar membrane. The marginal hooks have long handles, which are always longer than the total length of the anchor. The pharynx has long pharyngeal processes. Measurements, drawings of the opisthaptoral hard parts, penis and pharynx, and SEM micrographs of this monogenean are presented. G. longidactylus n. sp. is the first monogenean species described from P. lozanoi.

Binding of [3H]PD 128907, a Putatively Selective Ligand for the D3 Dopamine Receptor, in Rat Brain: A Receptor Binding and Quantitative Autoradiographic Study

[3H]PD 128907 has been proposed as a selective ligand for the D3 dopamine receptor. This study characterizes the binding of this radioligand in rat brain using in vitro radioligand binding and autoradiographic methods. In radioligand binding studies, [3H]PD 128907 exhibited 0.3 nmol/L affinity for a single, low density site in ventral striatal membranes. The pharmacological profile for [3H]PD 128907 was similar to that of [3H](+)-7-OH-DPAT with the rank order of potency for dopamine agonists being PD 128907 ≈ 7-OH-DPAT ≈ quinpirole ⩾ dopamine; for antagonists, spiperone > (+)-butaclamol ≈ domperidone ⩾ haloperidol > SCH 23390. Guanyl nucleotides had no effect on the binding of either ligand. These observations indicate labeling of a dopaminergic site with characteristics consistent with the D3 receptor. In autoradiographic studies, highest densities of [3H]PD 128907–labeled sites were observed in islands of Calleja followed by the nucleus accumbens, nucleus of the horizontal limb of the diagonal band, the molecular layer of cerebellar lobule X, and the ventral caudate/putamen.