Neuroscience. Brain under surveillance: the microglia patrol.
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Adenosine Triphosphate↗
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Results for “Cell Surface Extensions”
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Glial membranes at the node of Ranvier prevent neurite outgrowth.
Nodes of Ranvier are regularly placed, nonmyelinated axon segments along myelinated nerves. Here we show that nodal membranes isolated from the central nervous system (CNS) of mammals restricted neurite outgrowth of cultured neurons. Proteomic analysis of these membranes revealed several inhibitors of neurite outgrowth, including the oligodendrocyte myelin glycoprotein (OMgp). In rat spinal cord, OMgp was not localized to compact myelin, as previously thought, but to oligodendroglia-like cells, whose processes converge to form a ring that completely encircles the nodes. In OMgp-null mice, CNS nodes were abnormally wide and collateral sprouting was observed. Nodal ensheathment in the CNS may stabilize the node and prevent axonal sprouting.
Animals↗
Characterization of Pseudomonas aeruginosa adherence to mouse corneas in organ culture.
The present study was designed to obtain further information on the nature of the corneal macromolecule(s) to which Pseudomonas aeruginosa adheres and how adherence might be prevented. Scarified adult mouse corneas in organ culture were treated with trypsin or lipase to determine whether the receptor molecule(s) was protein or lipid in nature. Trypsin (20 micrograms/ml) treatment of the cornea for 5 min had no significant effect on bacterial adherence, and longer periods of enzyme exposure resulted in extensive surface cell lysis. In contrast, lipase treatment (50,000 U/ml) for 1 h caused little visible cell lysis and significantly reduced bacterial adherence. To test further the lipid nature of the receptor, a highly purified monosialoganglioside (GM1) preparation (500 micrograms/ml) was used to preincubate (1 h) the cornea prior to bacterial application, and this also inhibited bacterial adherence. Similar corneal treatment with gangliotetraosylceramide (asialo GM1) (500 micrograms/ml) had little effect on ocular bacterial binding. Premixing of the bacterial inoculum with GM1 prior to corneal application had no significant effect on inhibiting bacterial binding, but similarly premixing the bacterial inoculum with asialo GM1 transiently decreased adherence. Lastly, premixing of the bacterial inoculum or preincubation of corneas with fibronectin (500 micrograms/ml for 1 h) both decreased bacterial adherence. These findings provide evidence that the receptor-adhesin interactions of P. aeruginosa at the ocular surface in organ culture are complex, involve a glycolipid moiety, and may be blocked by a ganglioside containing at least one sialosyl residue or by fibronectin, which may bind to membrane-associated gangliosides.
Animals↗
Caspase-3 regulates catalytic activity and scaffolding functions of the protein tyrosine phosphatase PEST, a novel modulator of the apoptotic response.
The protein tyrosine phosphatase PEST (PTP-PEST) is involved in the regulation of the actin cytoskeleton. Despite the emerging functions attributed to both PTPs and the actin cytoskeleton in apoptosis, the involvement of PTP-PEST in apoptotic cell death remains to be established. Using several cell-based assays, we showed that PTP-PEST participates in the regulation of apoptosis. As apoptosis progressed, a pool of PTP-PEST localized to the edge of retracting lamellipodia. Expression of PTP-PEST also sensitized cells to receptor-mediated apoptosis. Concertedly, specific degradation of PTP-PEST was observed during apoptosis. Pharmacological inhibitors, immunodepletion experiments, and in vitro cleavage assays identified caspase-3 as the primary regulator of PTP-PEST processing during apoptosis. Caspase-3 specifically cleaved PTP-PEST at the (549)DSPD motif and generated fragments, some of which displayed increased catalytic activity. Moreover, caspase-3 regulated PTP-PEST interactions with paxillin, leupaxin, Shc, and PSTPIP. PTP-PEST acted as a scaffolding molecule connecting PSTPIP to additional partners: paxillin, Shc, Csk, and activation of caspase-3 correlated with the modulation of the PTP-PEST adaptor function. In addition, cleavage of PTP-PEST facilitated cellular detachment during apoptosis. Together, our data demonstrate that PTP-PEST actively contributes to the cellular apoptotic response and reveal the importance of caspases as regulators of PTPs in apoptosis.
Amino Acid Sequence↗
The neurofibromatosis type 2 gene product, merlin, reverses the F-actin cytoskeletal defects in primary human Schwannoma cells.
Schwannoma tumors, which occur sporadically and in patients with neurofibromatosis, account for 8% of intracranial tumors and can only be treated by surgical removal. Most schwannomas have biallelic mutations in the NF2 tumor suppressor gene. We previously showed that schwannoma-derived Schwann cells exhibit membrane ruffling and aberrant cell spreading when plated onto laminin, indicative of fundamental F-actin cytoskeletal defects. Here we expand these observations to a large group of sporadic and NF2-related tumors and extend them to schwannomatosis-derived tumors. Mutation at NF2 correlated with F-actin abnormalities, but the extent of morphological change did not correlate with the type of NF2 mutation. We used a recently described molecular strategy, TAT-mediated protein transfer, to acutely introduce the NF2 protein, merlin, into primary human schwannoma cells in an attempt to reverse the cytoskeletal phenotype. Abnormal ruffling and cell spreading by cells with identified NF2 mutations were rapidly reversed by introduction of TAT-merlin. The effect is specific to TAT-merlin isoform 1, the growth-suppressive isoform of merlin. TAT-merlin isoform 2, a TAT-merlin mutant (L64P), and merlin lacking TAT were ineffective in reversing the cytoskeletal phenotype. Results show that merlin isoform 1 is sufficient to restore normal actin organization in NF2-deficient human tumor cells, demonstrating a key role for merlin in the NF2 phenotype. These results lay the foundation for epigenetic complementation studies in NF2 mouse models and possibly for experiments to evaluate the utility of merlin transduction into patients as protein therapy.
Actins↗
Antibodies in the small intestine: mucosal synthesis and deposition of anti-glycosyl IgA, IgM, and IgG in the enterocyte brush border.
Synthesis and deposition of immunoglobulins in the brush border was studied in organ-cultured pig small intestinal mucosal explants. Surprisingly, comparable amounts of IgM and IgA were synthesized during a 6-h pulse, and also newly made IgG was detected in media and explants, including the microvillar fraction. For IgA and IgM, this subcellular distribution is consistent with basolateral-to-apical transcytosis, mediated by the polymeric immunoglobulin receptor. IgG is a ligand for the Fc receptor FcRn, and beta2-microglobulin, the light chain of FcRn, coclustered in immunogold double labeling with IgG in subapical endosomes and in the basolateral membrane of enterocytes. In addition, beta2-microglobulin was copurified with IgG on protein G-Sepharose. Apical endocytosis of IgG, as judged by internalization of fluorescent protein G, was not detectable except in a few isolated cells. This suggests that IgG in the adult small intestine is transported across the enterocyte mainly in the basolateral to apical direction. Significant fractions of all immunoglobulins bound to lactoseagarose, indicating that "anti-glycosyl" antibodies, raised against commensal gut bacteria, are synthesized locally in the small intestine. By partial deposition in the brush border, these antibodies therefore may have a protective function by preventing lectin-like pathogens from gaining access to the brush border surface.
Animals↗
Oligodendrocyte progenitor proliferation and maturation is differentially regulated by male and female sex steroid hormones.
Using primary cultures of oligodendrocyte progenitors isolated from male and female neonatal rodent brains, we observed more oligodendrocytes in female-derived compared to male-derived cultures. To determine whether the observed differences were due to a differential effect of sex hormones on proliferation, we treated cultures with increasing doses of 17beta-estradiol, testosterone or progesterone and labeled cells with bromodeoxyuridine to identify cells in S phase. Treatment with 17beta-estradiol, but not progesterone or testosterone, delayed the exit of oligodendrocyte progenitor cells from the cell cycle. In addition, 17beta-estradiol treatment enhanced membrane sheet formation, while progesterone increased cellular branching. Interestingly, the estrogen modulator tamoxifen mimicked the effect of 17beta-estradiol on cell cycle exit, but not on membrane formation. Immunocytochemical localization of estrogen receptors (ERs) showed ERbeta mainly localized to the cytoplasm of oligodendrocytes, suggesting that the effect of 17beta-estradiol on membrane formation could be mediated by interaction with this receptor. We conclude that sex steroids differentially regulate oligodendrocyte progenitor number and myelin formation, possibly contributing to gender-specific differences in repair.
Animals↗
Aspirin and gastroduodenal injury.
Aspirin almost invariably causes acute mucosal injury to the stomach or duodenum as evidenced by erosions seen endoscopically, extensive surface cell disruption on histology, reduced mucosal potential difference and increased gastric bleeding. This injury is usually minor and transient, and not associated with symptoms. However, in the elderly recent aspirin intake may be associated with bleeding peptic ulcer, and along with other NSAIDs, may be causal in a third of cases. Regular chronic aspirin intake may also be associated with gastric ulceration. Such ulceration, however, although seemingly capable of healing despite continued aspirin or other NSAID use, may take longer to do so. Although aspirin and other NSAIDs have a strong connection with gastroduodenal problems the risk in an individual needs to be balanced by the likelihood of such events. Given their widespread use, the serious side effects of these drugs occur in only a small proportion of users. This, however, does not mitigate against careful prescription.
Aspirin↗
WASp deficiency in mice results in failure to form osteoclast sealing zones and defects in bone resorption.
No defects related to deficiency of the Wiskott-Aldrich Syndrome protein (WASp) have been described in osteoclasts. Here we show that there are significant morphologic and functional abnormalities. WASp-null cells spread over a much larger surface area and are highly polykaryotic. In their migratory phase, normal cells assemble clusters of podosomes behind their leading edges, whereas during the bone resorptive phase multiple podosomes are densely aggregated in well-defined actin rings forming the sealing zone. In comparison, WASp-null osteoclasts in either phase are markedly depleted of podosomes. On bone surfaces, this results in a failure to form actin rings at sealing zones. Complementation of WASp-null osteoclasts with an enhanced green fluorescent protein (eGFP)-WASp fusion protein restores normal cytoarchitecture. These structural disturbances translate into abnormal patterns of bone resorption both in vitro on bone slices and in vivo. Although physiologic steady-state levels of bone resorption are maintained, a major impairment is observed when WASp-null animals are exposed to a resorptive challenge. Our results provide clear evidence that WASp is a critical component of podosomes in osteoclasts and indicate a nonredundant role for WASp in the dynamic organization of these actin structures during bone resorption.
Actin Cytoskeleton↗
Cell surface tissue transglutaminase is involved in adhesion and migration of monocytic cells on fibronectin.
Expression of tissue transglutaminase (transglutaminase II, tTG) was shown to increase drastically during monocyte differentiation into macrophages; however, its role in monocytic cells remains largely unknown. This study describes a novel function of cell surface tTG as an adhesion and migration receptor for fibronectin (Fn). Two structurally related transglutaminases, tTG and the A subunit of factor XIII (FXIIIA), are expressed on the surface of monocytic cells, whereas only surface tTG is associated with multiple integrins of the beta1 and beta3 subfamilies. Both surface levels of tTG and the amounts of integrin-bound tTG are sharply up-regulated during the conversion of monocytes into macrophages. In contrast, a reduction in biosynthesis and surface expression of FXIIIA accompanies monocyte differentiation. Cell surface tTG is colocalized with beta1- and beta3-integrins in podosomelike adhesive structures of macrophages adherent on Fn. Down-regulation of surface tTG by expression of antisense tTG construct or its inhibition by function-blocking antibodies significantly decreases adhesion and spreading of monocytic cells on Fn and, in particular, on the gelatin-binding fragment of Fn consisting of modules I6II1,2I7-9. Likewise, interfering with the adhesive function of surface tTG markedly reduces migration of myeloid cells on Fn and its gelatin-binding fragment. These data demonstrate that cell surface tTG serves as an integrin-associated adhesion receptor that might be involved in extravasation and migration of monocytic cells into tissues containing Fn matrices during inflammation.
Antigens, CD↗
Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation.
Cellular senescence suppresses cancer by arresting cells at risk of malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation and branching morphogenesis. Furthermore, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts - the ability to alter epithelial differentiation - that might also explain the loss of tissue function and organization that is a hallmark of aging.
Animals↗
Cdc42 controls the polarity of the actin and microtubule cytoskeletons through two distinct signal transduction pathways.
Cdc42, a Rho family GTPase, is a key regulator of cell polarity. In Saccharomyces cerevisiae, it is required for polarized bud formation and pheromone gradient sensing, while in higher eukaryotes, it participates in asymmetric cell division, directional sensing during migration, and morphogenesis. Using a scratch-induced fibroblast migration assay, we previously showed that Cdc42 controls the polarization of both membrane protrusions and the Golgi/centrosome. We now find that Golgi/centrosome polarity is mediated through activation of the Par6/aPKC complex, as previously described in astrocytes. However, this complex is not involved in Cdc42-dependent polarization of protrusions, which instead is mediated by Pak acting through the Rac guanine nucleotide exchange factor, betaPIX. Pak kinase activity is essential for spatially restricting Rac-dependent actin polymerization to the leading edge of the migrating cells, though it is not required for actin polymerization per se. We conclude that in migrating cells, Cdc42 co-ordinately regulates the polarity of the microtubule and actin cytoskeletons through two distinct pathways.
Actins↗
Invasion of endothelial cells by Neisseria meningitidis requires cortactin recruitment by a phosphoinositide-3-kinase/Rac1 signalling pathway triggered by the lipo-oligosaccharide.
Type-IV-pilus-mediated adhesion of Neisseria meningitidis (also known as meningococcus) to human endothelial cells induces the formation of membrane protrusions leading to bacterial uptake. We have previously shown that these protrusions result from a Rho- and Cdc42-dependent cortical actin polymerization, and from the activation of the ErbB2 tyrosine-kinase receptor and the Src kinase, leading to tyrosine phosphorylation of cortactin. We report here that N. meningitidis mutants expressing a deglycosylated lipo-oligosaccharide are poorly invasive. These mutants show structurally altered actin polymerization. Moreover, although they efficiently recruit and activate ErbB2 and Src, these mutants are defective in the recruitment and phosphorylation of cortactin. We demonstrate that phosphorylated cortactin controls the cortical actin polymerization, which leads to membrane protrusion formation. In addition, we show that cortactin recruitment is dependent on the activation of a phosphoinositide-3-kinase/Rac1-GTPase signalling pathway, which is required for actin polymerization and internalization of N. meningitidis, and is not activated by the mutant strains. Altogether, these results define a new role for the lipo-oligosaccharide in triggering a phosphoinositide-3-kinase/Rac1 signalling required to elicit an efficient uptake of N. meningitidis in non-phagocytic cells.
Actin Cytoskeleton↗
Phosphorylation of ezrin on threonine 567 produces a change in secretory phenotype and repolarizes the gastric parietal cell.
Phosphorylation of the membrane-cytoskeleton linker protein ezrin has been functionally linked to acid secretion and vesicle recruitment to the apical secretory membrane in gastric parietal cells. Phosphorylation of the conserved T567 residue of ezrin has been shown to alter the N/C oligomerization of ezrin and promote the formation of actin-rich surface projections in other cells. To test the importance of T567 as a regulatory site for ezrin in parietal cell activation, we incorporated wild-type (WT) and mutant forms of ezrin, including the nonphosphorylatable T567A mutation and a mutant mimicking permanent phosphorylation, T567D. All ezrin constructs included C-terminal cyan-fluorescent protein (CFP) and were incorporated into adenoviral constructs for efficient introduction into cultured parietal cells from rabbit stomach. Fluorescence microscopy was used to localize CFP-ezrin and monitor morphological responses. Accumulation of a weak base (aminopyrine) was used to monitor receptor-mediated acid secretory response of the cultured cells. Similar to endogenous ezrin, WT and T567A CFP-ezrin localized heavily to apical membrane vacuoles with considerably lower levels associated with the surrounding basolateral membrane. Interestingly, H,K-ATPase within cytoplasmic tubulovesicles was incorporated into the apical vacuoles along with WT and T567A mutant ezrin. In these parietal cells secretagogue stimulation produced a striking vacuolar expansion associated with HCl secretion and the secretory phenotype. Expression of T567D CFP-ezrin was quite different, being rarely associated with apical vacuoles. T567D was more typically localized to the basolateral membrane, often associated with long spikes and fingerlike projections. Moreover, the cells did not display secretagogue-dependent morphological changes and, to our surprise, H,K-ATPase was recruited to the T567D CFP-ezrin-enriched basolateral projections. We conclude that T567 phosphorylation, which is probably regulated through Rho signaling pathway, may direct ezrin to membrane-cytoskeletal activity at the basolateral membrane and away from apical secretory activity. The large basolateral expansion is predicted to recruit membranes from sources not normally targeted to that surface.
Adenoviridae↗
Fluid-phase uptake by macropinocytosis in Dictyostelium.
To study fluid-phase endocytosis in living cells and its relationship to changes in the cell cortex, we have used a green fluorescent protein (GFP)-tagged version of coronin, an actin-associated protein that localises to dynamic regions of the Dictyostelium cell cortex. In the confocal microscope, internalisation of fluorescently labelled dextran as a fluid-phase marker can be recorded simultaneously with the recruitment of the coronin-GFP fusion-protein from the cytoplasm of the phagocyte. At crown-shaped surface protrusions, extracellular medium is taken up into vesicles with an average diameter of 1.6 microns, which is significantly larger than the 0.1 microns diameter of clathrin-coated pinosomes. The observed frequency of macropinosome formation can account for a large portion, if not all, of the fluid-phase uptake. The redistribution of coronin-GFP strongly resembles cytoskeletal rearrangements during phagocytosis. Scanning-electron micrographs indicate that crown-shaped cell-surface extensions can undergo shape changes, without a particle bound, that are similar to shape changes that occur during phagocytosis. In quantitative assays, the uptake of particles and fluid are about equally dependent on F-actin and coronin.
Actins↗
Adhesion between cells and extracellular matrix with special reference to hepatic stellate cell adhesion to three-dimensional collagen fibers.
Hepatic stellate cells are located in the perisinusoidal space (space of Disse), and extend their dendritic, thin membranous processes and fine fibrillar processes into this space. The stellate cells coexist with a three-dimensional extracellular matrix (ECM) in the perisinusoidal space. In turn the three-dimensional structure of the ECM regulates the proliferation, morphology, and functions of the stellate cell. In this review, the morphology of sites of adhesion between hepatic stellate cells and extracellular matrix is described. Hepatic stellate cells cultured in polystyrene dishes spread well, whereas the cells cultured on or in type I collagen gel become slender and elongate their long cellular processes which adhere directly to the collagen fibers. Cells in type I collagen gel form a large number of adhesive structures, each adhesive area forming a face but not a point. Adhesion molecules, integrins, for the ECM are localized on the cell surface. Elongation of the cellular processes occurs via integrin-binding to type I collagen fibers. The signal transduction mechanism, including protein and phosphatidylinositol phosphorylation, is critical to induce and sustain the cellular processes. Information on the three-dimensional structures of ECM is transmitted via three-dimensional adhesive structures containing the integrins.
Animals↗
Maintenance of midgut epithelial cells from Dendroctonus valens (Coleoptera: Scolytidae) in vitro.
This article describes the culture of epithelial cells from anterior and posterior midgut regions of adult Dendroctonus valens. Culture conditions were established, and cell adherence was improved by means of a new technique that allowed the cells to grow between two glass coverslips. Cytoplasmic projections occur as anterior midgut cells grow to confluence; these projections were not observed in cells of the posterior midgut. The optimal culture medium for the maintenance of these epithelial cells was Roswell Park Memorial Institute 1640 medium at 25 degrees C. Cells in Grace's medium died in 24 h. Cultures did not require CO(2) atmosphere, but culture development was favored by the microaerophilic environment and the dark conditions in which the cells were grown, between the coverslips.
Animals↗
Transforming growth factor-beta induces osteoclast ruffling and chemotaxis: potential role in osteoclast recruitment.
Transforming growth factor-beta (TGF-beta) is released from the matrix during bone resorption and has been implicated in the pathogenesis of giant cell tumors of bone and the expansion of breast cancer metastases in bone. Because osteoclasts mediate tumor-induced osteolysis, we investigated whether TGF-beta stimulates osteoclast recruitment. Osteoclasts were isolated from rat long bones and time-lapse video microscopy was used to monitor their morphology and motility. Within 5 minutes, TGF-beta (0.1 nM) induced dynamic ruffling, with 65% of osteoclasts displaying membrane ruffles compared with 35% in untreated controls. Over a 2-h period, osteoclasts exhibited significant directed migration toward a source of TGF-beta, indicating chemotaxis. echistatin, an alphavbeta3 integrin blocker that inhibits macrophage colony-stimulating factor (M-CSF)-induced osteoclast migration, did not prevent the migration of osteoclasts toward TGF-beta. In contrast, a beta1 integrin blocking antibody inhibited osteoclast chemotaxis toward TGF-beta but not M-CSF. These data indicate the selective use of integrins by osteoclasts migrating in response to different chemotaxins. In addition, wortmannin and U0126 inhibited TGF-beta-induced chemotaxis, suggesting involvement of the phosphatidylinositol 3 (PI 3) kinase and mitogen-activated protein (MAP) kinase signaling pathways. Physiologically, TGF-beta, may coordinate osteoclast activity by recruiting osteoclasts to existing sites of resorption. Pathologically, TGF-beta-induced osteoclast recruitment may be critical for expansion of primary and metastatic tumors in bone.
Androstadienes↗