NCAM and PSA-NCAM dependent membrane spreading and F-actin reorganization in suspended adhering neural cells

Mediation of synchronous cell-cell interactions by NCAM and PSA-NCAM is examined here in aggregates (monolayers) of C6 polysialylated embryonic neural cells, formed rapidly (within 30 s) in suspension in an ultrasound trap. These cells express all three main isoforms of neural cell adhesion molecule (NCAM). The rate of extension of perimeter contact (i.e., membrane spreading) between closely adjacent cells and the temporal reinforcement of the Filamentous (F)-actin cytoskeleton at those regions were measured. Enzymatic removal of the cell-cell repelling polysialic acid (PSA) increases the rate of NCAM-induced membrane spreading, while removal of NCAM-120 had no detectable effect. Competitive peptide inhibition of the third immunoglobulin domain of NCAM significantly reduced the rate of membrane spreading, while NCAM siRNA transfected cells lost their ability to spread. It is argued that NCAM induced contact is the initial requirement for membrane spreading and facilitates conditions for subsequent cytoskeletal reorganization in these neural cells.     

Distinctive PSA-NCAM and NCAM Hallmarks in Glutamate-Induced Dendritic Atrophy and Synaptic Disassembly

Dendritic and synapse remodeling are forms of structural plasticity that play a critical role in normal hippocampal function. Neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) participate in neurite outgrowth and synapse formation and plasticity. However, it remains unclear whether they contribute to dendritic retraction and synaptic disassembly. Cultured hippocampal neurons exposed to glutamate (5 µM) showed a reduced MAP-2 (+) area in the absence of neuronal death 24 h after the insult. Concomitantly, synapse loss, revealed by decreased synaptophysin and post-synaptic density-95 cluster number and area, together with changes in NCAM and PSA-NCAM levels were found. Dendritic atrophy and PSA-NCAM reduction proved NMDA-receptor dependent. Live-imaging experiments evidenced dendritic atrophy 4 h after the insult; this effect was preceded by smaller NCAM clusters (1 h) and decreased surface and total PSA-NCAM levels (3 h). Simultaneously, total NCAM cluster number and area remained unchanged. The subsequent synapse disassembly (6 h) was accompanied by reductions in total NCAM cluster number and area. A PSA mimetic peptide prevented both the dendritic atrophy and the subsequent synaptic changes (6 h) but had no effect on the earliest synaptic remodeling (3 h). Thus, NCAM-synaptic reorganization and PSA-NCAM level decrease precede glutamate-induced dendritic atrophy, whereas the NCAM level reduction is a delayed event related to synapse loss. Consequently, distinctive stages in PSA-NCAM/NCAM balance seem to accompany glutamate-induced dendritic atrophy and synapse loss.     

Expression of the neural cell adhesion molecule NCAM in endocrine cells

We examined the expression of the neural cell adhesion molecule NCAM in a number of endocrine tissues of adult rat and in an endocrine tumor cell line. NCAM was found by immunoelectron microscopy to be present on the surface of all endocrine cells in the three lobes of the hypophysis, although staining was relatively less intense in the intermediate lobe, and in pancreatic islets. Pituicytes, hypophyseal glial cells, were also labeled for NCAM. A rat insulinoma cell line (RIN A2) also expressed NCAM as judged by immunocytochemistry. Analysis of NCAM antigenic determinants (Mr 180, 140, and 120 KD) revealed large variations in the relative proportions of NCAM polypeptides present in the different tissues. Although all tissues and cell lines expressed NCAM-140, NCAM-180 was not detected in the adenohypophysis, pancreas, or adrenal medulla, and NCAM-120 was found in none of the endocrine tissues or cell lines except at low levels in the neurohypophysis. The tumor cell line expressed significant levels of NCAM-180, which was most abundant in the neurohypophysis. These results show that NCAM expression appears to be a general property of endocrine cells, although the antigenic composition differs markedly from that in brain tissue. These data are discussed with regard to the embryological origins of the different endocrine tissues, and possible functional implications are suggested.     

Association of the growth-arrest-specific protein Gas7 with F-actin induces reorganization of microfilaments and promotes membrane outgrowth.

The growth-arrest-specific gene, Gas7, is required for neurite outgrowth in cerebellar neurons. Here we report that Gas7 can induce the formation of extended cellular processes in NIH3T3 cells by interacting with actin and mediating reorganization of microfilaments. The Gas 7 protein, which increased markedly during growth arrest of NIH3T3 cells and persisted transiently at high levels upon reentry of cells into the cell cycle, localized near the plasma membrane and selectively colocalized with microfilaments in membrane ruffles. Process extensions induced by ectopic overexpression of Gas7 were blocked by the actin-depolymerizing agent cytochalasin D, suggesting that membrane extensions produced by Gas7 require actin polymerization. Association of endogenous Gas7 protein with microfilaments was verified by F-actin affinity chromatography; direct binding of purified His-Gas7 to actin also was demonstrated and shown to be mediated by the Gas7 C-terminal domain. Similarly, localization of Gas7 in membrane ruffles was mediated by the C-terminal domain, although neither this region nor the N-terminal domain was individually sufficient to induce process formation. Biochemical studies and electron microscopy showed that both full-length Gas7 protein and its C-terminal region can promote actin assembly as well as the crosslinking of actin filaments. We propose that Gas7 localized near the plasma membrane induces the assembly of actin and the membrane outgrowth.     

Alterations in dysadherin expression and F-actin reorganization: a possible mechanism of hypericin-mediated photodynamic therapy in colon adenocarcinoma cells

Dysadherin is a recently found anti-adhesion molecule, therefore detection and down regulation of its expression is promising in cancer treatment. The up-regulation of dysadherin contributes to colon cancer recurrence and metastasis. Dysadherin also has connections with cytoskeletal proteins and it can cause alterations in the organisation of filamentous actin (F-actin) in metastatic cancers. In this study, hypericin (HYP)-mediated photodynamic therapy (PDT) was performed in two different grade colon adenocarcinoma cell lines HT-29 (Grade I) and Caco-2 (Grade II). Cells were treated with 0.04, 0.08 or 0.15 μM HYP concentrations and irradiated with (4 J/cm²) fluorescent lamps. The effects of HYP was examined 16 and 24 h after the activation. We investigated for the first time the effect of HYP-mediated PDT on the expression of dysadherin and F-actin organisation. According to the results, HYP mediated PDT caused a decrease in gene expression and immunofluorescence staining of dysadherin and an increase in actin stress fibers and actin aggregates in HT-29 and Caco-2 cell lines. Besides, cytotoxicity, number of floating cells and apoptotic index changed depending on the cell type, HYP concentration and incubation time. We have demonstrated for the first time that dysadherin and F-actin could be target molecules for HYP-mediated PDT in HT-29 and Caco-2 colon cancer cell lines.     

Selenium nanoparticles induced membrane bio-mechanical property changes in MCF-7 cells by disturbing membrane molecules and F-actin

Selenium nanoparticles (Se NPs) have been served as promising materials for biomedical applications, especially for cancer treatment. The anti-cancer effects of Se NPs against cancer cells have been widely studied in recent years, but whether Se NPs can induce the changes of cell membrane bio-mechanical properties in cancer cells still remain unexplored. In this Letter, we prepared Se NPs for investigating the intracellular localization of Se NPs in MCF-7 cells and determined the effects of Se NPs on apoptosis and necrosis in MCF-7 cells. Especially, we reported for the first time about the effects of Se NPs on the bio-mechanical properties of cancer cells and found that Se NPs could remarkably decrease the adhesion force and Young’s modulus of MCF-7 cells. To further understand the potential mechanisms about how Se NPs affect the bio-mechanical properties of MCF-7 cells, we also investigated the expression of CD44 molecules, the structure and the amounts of F-actin. The results indicated that the decreased adhesion force between AFM tip and cell membrane was partially due to the changes of membrane molecules induced by Se NPs, such as the down-regulation of trans-membrane CD44 molecules. Additionally, the decrease of Young’s modulus of MCF-7 cells was due to the dis-organization and down-regulation of F-actin induced by Se NPs. These results collectively suggested that cell membrane was of vital importance in Se NPs induced toxicity in cancer cells, which could be served as a potential target for cancer treatment by Se NPs.     

Hamster pulmonary endocrine cells with neural cell adhesion molecule (NCAM) immunostaining

Pulmonary endocrine cells of Syrian golden hamster were stained for neural cell adhesion molecule (NCAM) with indirect fluorescent immunostaining and observed with a confocal laser scanning microscope equipped with an argon laser. Sections 100 m thick of hamster lung fixed with phosphate-buffered 4% paraformaldehyde were prepared. The sections were incubated with rat monoclonal antibody against NCAM, followed by fluorescence-labeled antibody against rat immunoglobulin. Some were doubly immunostained for NCAM and one of the following endocrine markers: neuron-specific enolase, calcitonin gene-related peptide and serotonin. Expression of NCAM in the hamster airway epithelium was seen in cell nests resembling neuroepithelial bodies (NEBs). NCAM immunostaining was positive at the lateral cell borders between the cells composing the nest, but negative at the border with the adjacent, presumably non-endocrine cells. Double immunostaining confirmed that the grouped cells with NCAM immunoreactivity were of an endocrine nature, but that single endocrine cells did not show NCAM immunoreactivity. An electron microscopic study with NCAM immunostaining confirmed the light microscopic study. These suggest that NCAM expression could be important for the morphogenesis of NEBs. A confocal laser microscope was used to make theee-dimensional images of NEBs after NCAM immunostaining and the spatial interaction between NEBs and the surrounding microenvironment was studied.     

Compensatory neural reorganization in Tourette syndrome

Children with neurological disorders may follow unique developmental trajectories whereby they undergo compensatory neuroplastic changes in brain structure and function that help them gain control over their symptoms. We used behavioral and brain imaging techniques to investigate this conjecture in children with Tourette syndrome (TS). Using a behavioral task that induces high levels of intermanual conflict, we show that individuals with TS exhibit enhanced control of motor output. Then, using structural (diffusion-weighted imaging) brain imaging techniques, we demonstrate widespread differences in the white matter (WM) microstructure of the TS brain that include alterations in the corpus callosum and forceps minor (FM) WM that significantly predict tic severity in TS. Most importantly, we show that task performance for the TS group (but not for controls) is strongly predicted by the WM microstructure of the FM pathways that lead to the prefrontal cortex and by the functional magnetic resonance imaging blood oxygen level-dependent response in prefrontal areas connected by these tracts. These results provide evidence for compensatory brain reorganization that may underlie the increased self-regulation mechanisms that have been hypothesized to bring about the control of tics during adolescence.     

p59Hck isoform induces F-actin reorganization to form protrusions of the plasma membrane in a Cdc42- and Rac-dependent manner

Hck is a protein kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, localized at the plasma membrane and lysosomes, respectively. Their individual involvement in functions ascribed to Hck, phagocytosis, cell migration, and lysosome mobilization, is still unclarified. To investigate the specific role of p59Hck, a constitutively active variant in fusion with green fluorescent protein (p59Hck(ca)) was expressed in HeLa cells. p59Hck(ca) was found at focal adhesion sites and triggered reorganization of the actin cytoskeleton, leading to plasma membrane protrusions where it co-localized with F-actin. Similarly, microinjection of p59Hck(ca) cDNA in J774.A1 macrophages induced membrane protrusions. Whereas kinase activity and membrane association of p59Hck were dispensable for location at focal adhesions, p59Hck-induced membrane protrusions were dependent on kinase activity, plasma membrane association, and Src homology 2 but not Src homology 3 domain and were inhibited by dominant-negative forms of Cdc42 or Rac but not by blocking Rho activity. A dominant negative form of p59Hck inhibited the Cdc42- and Rac-dependent FcgammaRIIa-mediated phagocytosis. Expression of the Cdc42/Rac-interacting domain of p21-activated kinase in macrophages abolished the p59Hck(ca)-induced morphological changes. Therefore, p59Hck-triggered remodeling of the actin cytoskeleton depends upon the activity of Cdc42 and Rac to promote formation of membrane protrusions necessary for phagocytosis and cell migration.     

Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow

Nonmuscle myosin IIA (NMM-IIA) is involved in the formation of focal adhesions and neurite retraction. However, the role of NMM-IIA in these functions remains largely unknown. Using RNA interference as a tool to decrease NMM-IIA expression, we have found that NMM-IIA is the major myosin involved in traction force generation and retrograde F-actin flow in mouse embryonic fibroblast cells. Quantitative analyses revealed that approximately 60% of traction force on fibronectin-coated surfaces is contributed by NMM-IIA and approximately 30% by NMM-IIB. The retrograde F-actin flow decreased dramatically in NMM-IIA-depleted cells, but seemed unaffected by NMM-IIB deletion. In addition, we found that depletion of NMM-IIA caused cells to spread at a higher rate and to a greater area on fibronectin substrates during the early spreading period, whereas deletion of NMM-IIB appeared to have no effect on spreading. The distribution of NMM-IIA was concentrated on the dorsal surface and approached the ventral surface in the periphery, whereas NMM-IIB was primarily concentrated around the nucleus and to a lesser extent at the ventral surface in cell periphery. Our results suggest that NMM-IIA is involved in generating a coherent cytoplasmic contractile force from one side of the cell to the other through the cross-linking and the contraction of dorsal actin filaments.     

F-actin aggregates in transformed cells

Polymerized actin has been found aggregated into distinctive patches inside transformed cells in culture. The F-actin-specific fluorescent probe, nitrobenzoxadiazole-phallacidin, labels these F-actin aggregates near the ventral cell surface of cells transformed by RNA or DNA tumor viruses, or by chemical mutagens, or spontaneously. Their appearance in all eight transformed cell types studied suggests their ubiquity and involvement in transformation morphology. Actin patches developed in normal rat kidney (NRK) cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (LA23-NRK) within 30 min after a shift from the nonpermissive (39 degrees C) to the permissive temperature (32 degrees C). Patch appearance paralleling viral src gene expression tends to implicate pp60src kinase activity in destabilizing the cytoskeleton. However, appearance of the actin aggregates in cells not transformed by retrovirus calls for alternative mechanisms, perhaps involving an endogenous kinase, for this apparently common trait.     

Phagocytosis of bacteria by polymorphonuclear leukocytes suspended in liquid or adhering to a surface

Phagocytosis of Staphylococcus aureus by polymorphonuclear leukocytes (PNL) was studied in healthy men. PNL suspended in nutrient medium did not practically ingest bacteria. The intake of bacteria got considerably intensified if leukocytes and bacteria ran into each other by turning over the test tubes during incubation. A still greater rise of phagocytic activity was discovered under the conditions favouring the attachment of PNL to the surface and the possibility of chemotaxis. These conditions were created by introducing a gelatin-coated film into the test tube.     

Voltage dependent membrane conductances in cultured renal distal cells.

Cultured Na(+)-transporting epithelia from amphibian renal distal tubule (A6) were impaled with microelectrodes and analyzed at short-circuit and after transepithelial voltage perturbation to evaluate the influence of voltage on apical and basolateral membrane conductances. For equivalent circuit analysis, amiloride was applied at each setting of transepithelial potential. At short-circuit, apical and basolateral membrane conductances averaged 88 and 497 microS/cm2, respectively (n = 10). Apical membrane conductance, essentially due to Na(+)-specific pathways, decreased after depolarization of the apical membrane. The drop was considerably larger than predicted by the Goldman-Hodgkin-Katz (GHK) constant-field equation. This suggests decrease in permeability of the apical Na+ channels upon depolarization. Basolateral membrane conductance, preferentially determined by K+ channels, increased after hyperpolarization of the basolateral membrane. This behavior is contrary to the prediction of the GHK constant field equation and reflects inward rectification of the K+ channels. The observed rectification patterns can be valuable for maintenance of cellular homeostasis.     

Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol.     

Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering.

The GTPase Rho is known to mediate the assembly of integrin-containing focal adhesions and actin stress fibers. Here, we investigate the role of Rho in regulating the distribution of the monocyte-binding receptors E-selectin, ICAM-1, and VCAM-1 in human endothelial cells. Inhibition of Rho activity with C3 transferase or N19RhoA, a dominant negative RhoA mutant, reduced the adhesion of monocytes to activated endothelial cells and inhibited their spreading. Similar effects were observed after pretreatment of endothelial cells with cytochalasin D. In contrast, dominant negative Rac and Cdc42 proteins did not affect monocyte adhesion or spreading. C3 transferase and cytochalasin D did not alter the expression levels of monocyte-binding receptors on endothelial cells, but did inhibit clustering of E-selectin, ICAM-1, and VCAM-1 on the cell surface induced by monocyte adhesion or cross-linking antibodies. Similarly, N19RhoA inhibited receptor clustering. Monocyte adhesion and receptor cross-linking induced stress fiber assembly, and inhibitors of myosin light chain kinase prevented this response but did not affect receptor clustering. Finally, receptor clusters colocalized with ezrin/moesin/ radixin proteins. These results suggest that Rho is required in endothelial cells for the assembly of stable adhesions with monocytes via the clustering of monocyte-binding receptors and their association with the actin cytoskeleton, independent of stress fiber formation.     

Modulation of the F-actin cytoskeleton by c-Abl tyrosine kinase in cell spreading and neurite extension

The nonreceptor tyrosine kinase encoded by the c-Abl gene has the unique feature of an F-actin binding domain (FABD). Purified c-Abl tyrosine kinase is inhibited by F-actin, and this inhibition can be relieved through mutation of its FABD. The c-Abl kinase is activated by physiological signals that also regulate the actin cytoskeleton. We show here that c-Abl stimulated the formation of actin microspikes in fibroblasts spreading on fibronectin. This function of c-Abl is dependent on kinase activity and is not shared by c-Src tyrosine kinase. The Abl-dependent F-actin microspikes occurred under conditions where the Rho-family GTPases were inhibited. The FABD-mutated c-Abl, which is active in detached fibroblasts, stimulated F-actin microspikes independent of cell attachment. Moreover, FABD-mutated c-Abl stimulated the formation of F-actin branches in neurites of rat embryonic cortical neurons. The reciprocal regulation between F-actin and the c-Abl tyrosine kinase may provide a self-limiting mechanism in the control of actin cytoskeleton dynamics.