Effects of Actin Cytoskeleton Disruption on Electroporation In Vitro.

The role of actin fibers in cellular responses to external electric pulses is not clear yet. In this study, we utilized the blocker of actin polymerization, cytochalasin D (cytoD), and investigated its effects on the electropore generation. Eight 100 µs electric pulses of sub-kilovolt per centimeter voltage with 100 ms intervals were applied to adhered cells in vitro, and the membrane permeability was quantified using membrane-impermeable propidium iodide (PI) dye. With cytoD application, the transfer of PI dye decreased significantly in all the applied voltages. At the same time, the roughness of cells increased, the membrane stiffness decreased, and the transmembrane resting potential decreased. Our result supports that actin fibers have clear effects on electroporation through modulating membrane properties including transmembrane resting potential.

Biosynthesis and mass spectral fragmentation pathways of (13)C and (15)N labeled cytochalasin D produced by Xylaria arbuscula.

The fungus Xylaria arbuscula was isolated as an endophyte from Cupressus lusitanica and has shown to be a prominent producer of cytochalasins, mainly cytochalasins C, D and Q. Cytochalasins comprise an important class of fungal secondary metabolites that have aroused attention due to their uncommon molecular structures and pronounced biological activities. Due to the few published studies on the ESI-MS/MS fragmentation of this important class of secondary metabolites, in the first part of our work, we studied the cytochalasin D fragmentation pathways by using an ESI-Q-ToF mass spectrometer coupled with liquid chromatography. We verified that the main fragmentation routes were generated by hydrogen and McLafferty rearrangements which provided more ions than just the ones related to the losses of H2 O and CO as reported in previous studies. We also confirmed the diagnostic ions at m/z 146 and 120 as direct precursor derived from phenylalanine. The present work also aimed the production of structurally diverse cytochalasins by varying the culture conditions used to grow the fungus X. arbuscula and further insights into the biosynthesis of cytochalasins. HPLC-MS analysis revealed no significant changes in the metabolic profile of the microorganism with the supplementation of different nitrogen sources but indicated the ability of X. arbuscula to have access to inorganic and organic nitrogen, such as nitrate, ammonium and amino acids as a primary source of nitrogen. The administration of 2-13 C-glycine showed the direct correlation of this amino acid catabolism and the biosynthesis of cytochalasin D by X. arbuscula, due to the incorporation of three labeled carbons in cytochalasin chemical structure. Copyright © 2017 John Wiley & Sons, Ltd.

Fluid flow facilitates inward rectifier K+ current by convectively restoring [K+] at the cell membrane surface.

The inward rectifier Kir2.1 current (IKir2.1) was reported to be facilitated by fluid flow. However, the mechanism underlying this facilitation remains uncertain. We hypothesized that during K+ influx or efflux, [K+] adjacent to the outer mouth of the Kir2.1 channel might decrease or increase, respectively, compared with the average [K+] of the bulk extracellular solution, and that fluid flow could restore the original [K+] and result in the apparent facilitation of IKir2.1. We recorded the IKir2.1 in RBL-2H3 cells and HEK293T cells that were ectopically over-expressed with Kir2.1 channels by using the whole-cell patch-clamp technique. Fluid-flow application immediately increased the IKir2.1, which was not prevented by either the pretreatment with inhibitors of various protein kinases or the modulation of the cytoskeleton and caveolae. The magnitudes of the increases of IKir2.1 by fluid flow were driving force-dependent. Simulations performed using the Nernst-Planck mass equation indicated that [K+] near the membrane surface fell markedly below the average [K+] of the bulk extracellular solution during K+ influx, and, notably, that fluid flow restored the decreased [K+] at the cell surface in a flow rate-dependent manner. These results support the "convection-regulation hypothesis" and define a novel interpretation of fluid flow-induced modulation of ion channels.

Morphological and Mechanical Properties of Osteosarcoma Microenvironment Cells Explored by Atomic Force Microscopy.

Cell mechanical properties that depend on cytoskeleton architecture are critical to the mechanotransduction process, and have great potential for cancer diagnosis and therapy. In this study, the morphological and mechanical properties of typical osteosarcoma microenvironment cells, including mesenchymal stem cells (MSC), normal human osteoblast cells (NHOst) and osteosarcoma cells (MG-63), were compared using atomic force microscopy (AFM). The MG-63 cells were smaller and thicker than the MSC and NHOst cells. The membrane roughness of MG-63 cells was higher than that of MSC and NHOst cells. The MG-63 cells had lower stiffness than their normal counterparts due to their reduced organization of the cytoskeleton structure. The cell stiffness influenced the mechanotransduction. The MG-63 cells had a lower percentage of nuclear YAP/TAZ compared with the MSC and NHOst cells. The F-actin assembly was disrupted by the cytochalasin D (cyto D) treatment used to investigate its influence on mechanotransduction. Disruption of the cytoskeleton leaded to a decrease of the cell stiffness, and reduced the nuclear YAP/TAZ percentage, indicating its inhibition in the cell mechanotransduction process. This study would shed light on the development of a novel cancer diagnosis strategy and would contribute to reveal the relationship between the cytoskeleton structure and the cell mechanical properties.

RhoA mediates the expression of acidic extracellular pH-induced matrix metalloproteinase-9 mRNA through phospholipase D1 in mouse metastatic B16-BL6 melanoma cells.

Solid tumors are characterized by acidic extracellular pH (pHe). The present study examined the contribution of small GTP-binding proteins to phospholipase D (PLD) activation of acidic pHe-induced matrix metalloproteinase-9 (MMP-9) production. Acidic pHe-induced MMP-9 production was reduced by C3 exoenzyme, which inhibits the Rho family of GTPases; cytochalasin D, which inhibits actin reorganization; and simvastatin, which inhibits geranylgeranylation of Rho. Small interfering RNA (siRNA) against RhoA, but not against Rac1 or Cdc42, significantly inhibited acidic pHe induction of MMP-9. Pull-down assays showed that acidic pHe increased the activated form of RhoA. Forced expression of constitutively active RhoA induced MMP-9 production, even at neutral pHe. RhoA siRNA also reduced acidic pHe induced PLD activity. Specific inhibition of PLD1 and Pld1 gene knockout significantly reduced acidic pHe-induced MMP-9 expression. In contrast, PLD2 inhibition or knockout had no effect on MMP-9 expression. These findings suggested that RhoA-PLD1 signaling is involved in acidic pHe induction of MMP-9.

Regulation of L-selectin-dependent hydrodynamic shear thresholding by leukocyte deformability and shear dependent bond number.

BACKGROUND: During inflammation leukocyte attachment to the blood vessel wall is augmented by capture of near-wall flowing leukocytes by previously adherent leukocytes. Adhesive interactions between flowing and adherent leukocytes are mediated by L-selectin and P-selectin Glycoprotein Ligand-1 (PSGL-1) co-expressed on the leukocyte surface and ultimately regulated by hydrodynamic shear thresholding. OBJECTIVE: We hypothesized that leukocyte deformability is a significant contributory factor in shear thresholding and secondary capture. METHODS: Cytochalasin D (CD) was used to increase neutrophil deformability and fixation was used to reduce deformability. Neutrophil rolling on PSGL-1 coated planar surfaces and collisions with PSGL-1 coated microbeads were analyzed using high-speed videomicroscopy (250 fps). RESULTS: Increased deformability led to an increase in neutrophil rolling flux on PSGL-1 surfaces while fixation led to a decrease in rolling flux. Abrupt drops in flow below the shear threshold resulted in extended release times from the substrate for CD-treated neutrophils, suggesting increased bond number. In a cell-microbead collision assay lower flow rates were correlated with briefer adhesion lifetimes and smaller adhesive contact patches. CONCLUSIONS: Leukocyte deformation may control selectin bond number at the flow rates associated with hydrodynamic shear thresholding. Model analysis supported a requirement for both L-selectin catch-slip bond properties and multiple bond formation for shear thresholding.

Kv1.3 channel blockade enhances the phagocytic function of RAW264.7 macrophages.

This study aimed to comprehend the largely unknown role of voltage-gated potassium channel 1.3 (Kv1.3) in the phagocytic function of macrophages. We found that blocking of the Kv1.3 channel with 100 pmol L(-1) Stichodactyla helianthus neurotoxin (ShK) enhanced the phagocytic capacities of both resting and lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages in the chicken erythrocyte system. In the fluorescein isothiocyanate (FITC)-labeled Escherichia coli k-12 system, ShK increased the phagocytic capacities of resting RAW264.7 cells, but not of the LPS-stimulated cells, as LPS alone stimulated almost saturated phagocytosis of the macrophages. ShK increased the nitric oxide (NO) production in LPS-activated cells, but not in resting RAW264.7 cells. There was no effect of ShK alone on the cytokine secretions in resting RAW264.7 cells, but it suppressed IL-1ß secretion in LPS-stimulated RAW264.7 cells. At a concentration of 100 pmol L(-1), ShK did not affect the viability of the tested cells. Kv1.3 was expressed in RAW264.7 cells; this expression was downregulated by LPS, but significantly upregulated by disrupting caveolin-dependent endocytosis with filipin III. In addition, cytochalasin D, an inhibitor of actin polymerization, did not affect the Kv1.3 expression. Thus, blocking of the Kv1.3 channel enhances the phagocytic capacity and NO production of this cell line. Our results suggest that Kv1.3 channel serves as a negative regulator of phagocytosis in macrophages and can therefore be a potential target in the treatment of macrophage dysfunction.

Imaging the elastic modulus of human platelets during thrombin-induced activation using scanning ion conductance microscopy.

Platelet activation plays a critical role in haemostasis and thrombosis. It is well-known that platelets generate contractile forces during activation. However, their mechanical material properties have rarely been investigated. Here, we use scanning ion conductance microscopy (SICM) to visualise morphological and mechanical properties of live human platelets at high spatial resolution. We found that their mean elastic modulus decreases during thrombin-induced activation by about a factor of two. We observed a similar softening of platelets during cytochalasin D-induced cytoskeleton depolymerisation. However, thrombin-induced temporal and spatial modulations of the elastic modulus were substantially different from cytochalasin D-mediated changes. We thereby provide new insights into the mechanics of haemostasis and establish SICM as a novel imaging platform for the ex vivo investigation of the mechanical properties of live platelets.

Dissecting the contribution of actin and vimentin intermediate filaments to mechanical phenotype of suspended cells using high-throughput deformability measurements and computational modeling.

Mechanical cell properties play an important role in many basic biological functions, including motility, adhesion, proliferation and differentiation. There is a growing body of evidence that the mechanical cell phenotype can be used for detection and, possibly, treatment of various diseases, including cancer. Understanding of pathological mechanisms requires investigation of the relationship between constitutive properties and major structural components of cells, i.e., the nucleus and cytoskeleton. While the contribution of actin und microtubules to cellular rheology has been extensively studied in the past, the role of intermediate filaments has been scarcely investigated up to now. Here, for the first time we compare the effects of drug-induced disruption of actin and vimentin intermediate filaments on mechanical properties of suspended NK cells using high-throughput deformability measurements and computational modeling. Although, molecular mechanisms of actin and vimentin disruption by the applied cytoskeletal drugs, Cytochalasin-D and Withaferin-A, are different, cell softening in both cases can be attributed to reduction of the effective density and stiffness of filament networks. Our experimental data suggest that actin and vimentin deficient cells exhibit, in average, 41% and 20% higher deformability in comparison to untreated control. 3D Finite Element simulation is performed to quantify the contribution of cortical actin and perinuclear vimentin to mechanical phenotype of the whole cell. Our simulation provides quantitative estimates for decreased filament stiffness in drug-treated cells and predicts more than two-fold increase of the strain magnitude in the perinuclear vimentin layer of actin deficient cells relatively to untreated control. Thus, the mechanical function of vimentin becomes particularly essential in motile and proliferating cells that have to dynamically remodel the cortical actin network. These insights add functional cues to frequently observed overexpression of vimentin in diverse types of cancer and underline the role of vimentin targeting drugs, such as Withaferin-A, as a potent cancerostatic supplement.

The effectiveness of different functional fibrinogen polymerization assays in eliminating platelet contribution to clot strength in thromboelastometry.

BACKGROUND: Viscoelastic tests such as functional fibrinogen polymerization assays (FFPAs) in thrombelastography (TEG®) or thromboelastometry (ROTEM®) measure clot elasticity under platelet inhibition. Incomplete platelet inhibition influences maximum clot firmness (MCF) of FFPAs. We compared the ability of existing and newly developed FFPAs to eliminate the platelet contribution to clot strength. METHODS: MCF of whole blood (WB), platelet-rich plasma (PRP), and platelet-poor plasma samples was recorded using a ROTEM device with different FFPAs, including the TEG functional fibrinogen test (FFTEG) and different ROTEM-based assays: the standard fib-tem reagent (FIBTEM), a lyophilized single-portion reagent fib-tem S (FIBTEM-S), a newly developed reagent FIBTEM PLUS, as well as FIBTEM or the standard extrinsic activation reagent ex-tem® (EXTEM) combined with 10-µg abciximab (FIBTEM-ABC/EXTEM-ABC). RESULTS: In WB (platelet count [mean ± SD], 183 ± 37 × 10/µL; plasma fibrinogen concentration, 2.49 ± 0.58 g/L), FFTEG and EXTEM-ABC showed higher MCF (15.7 ± 2.8 mm) than FIBTEM or FIBTEM-S (11.4 ± 3.3 mm, P < 0.001), whereas FIBTEM-ABC and FIBTEM PLUS resulted in lower MCF (9.3 ± 2.8 mm, P < 0.001). In 2 different PRP samples, with platelet counts of 407 ± 80 × 10/µL and 609 ± 127 × 10/µL, FIBTEM-ABC and FIBTEM PLUS reduced platelet contribution to clot strength within 95% confidence interval limits of -1.4 to 0.1 mm and -1.2 to 0.4 mm, respectively. Using all FFPAs it was observed that the Pearson correlation coefficient between plasma fibrinogen concentration and WB MCF was high (range, 0.75-0.93) and significant, regardless of the underlying platelet inhibiting component. Evaluating differences in the interception of regression lines by using analysis of covariance, we compared platelet-poor plasma and both PRP samples within the same assays and found that in contrast to the FIBTEM-ABC and FIBTEM PLUS assays, the FFTEG, EXTEM-ABC, FIBTEM, and FIBTEM-S methods still detected residual platelet activity and grossly overestimated fibrin clot strength in samples with high platelet counts. CONCLUSIONS: FFPAs based solely on glycoprotein-IIb/IIIa inhibition, such as FFTEG or EXTEM-ABC, are less effective than cytochalasin D-based assays, such as FIBTEM or FIBTEM-S, at inhibiting the platelet component of clot strength. The FIBTEM PLUS assay, and the combination of FIBTEM and abciximab, sufficiently inhibits platelet contribution to clot elasticity. The combination of a glycoprotein-IIb/IIIa receptor blocker and cytochalasin D allows evaluation of functional fibrinogen polymerization without platelet "noise." In a clinical setting, the significance of potent platelet inhibition ensures a more accurate assessment of MCF and therefore the need for fibrinogen supplementation therapy. Further studies are necessary to investigate the application and impact of these tests in a clinical situation.

Longer storage of red blood cells is associated with increased in vitro erythrophagocytosis.

BACKGROUND AND OBJECTIVES: Refrigerated storage of red blood cells (RBCs) induces numerous changes that may target the cells for erythrophagocytosis following transfusion. The influence of storage upon the phagocytosis of unseparated and fractionated young and old stored RBCs was investigated using two in vitro quantitative phagocytosis assays. MATERIALS AND METHODS: Leucocyte-depleted RBC units were sampled at day 1 or 42 of storage. Young and old RBCs were fractionated at day 1 by density centrifugation and stored in paediatric packs for up to 42 days. RBCs were labelled with the fluorescent dye PKH26 and incubated with the human monocytic cell line THP-1. Erythrophagocytosis was quantified by flow cytometry and plate fluorometric assays. RESULTS: A higher proportion of THP-1 cells phagocytosed RBCs stored for 42 days compared with 1 day (41% and 24% respectively; P<0·0001). This was associated with an increased mean number of RBCs phagocytosed per THP-1 cell (5·2±0·6 and 3·3±0·2 respectively; P<0·002). Erythrophagocytosis of fractionated young and old RBCs increased with longer storage duration up to 28 days (P<0·05). However, no significant differences were observed between erythrophagocytosis of young and old RBCs. CONCLUSION: The susceptibility of stored RBCs to erythrophagocytosis significantly increased with longer storage time of the RBC units. Storage duration of RBCs had a greater influence on in vitro erythrophagocytosis than the chronological age of the RBCs at donation.

A mechanism for actin filament severing by malaria parasite actin depolymerizing factor 1 via a low affinity binding interface.

Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.

Optical mapping of optically paced embryonic hearts.

Conduction maps of early embryonic hearts with optical mapping point to heterogeneous conduction patterns that quickly evolve over time. In adult hearts, electrical pacing is utilized to determine the anisotropy of the conduction patterns and the susceptibility of the tissue to arrhythmias. However, studying electrophysiology in developing hearts is limited due to their size. Electrical pacing creates an electrical artifact that obscures recordings from the entirety of early embryonic hearts. In this study, optical point stimulation using a 1440-nm near-infrared diode laser with a 12-µm diameter beam waist was used to pace embryonic quail hearts. Electrical activity was recorded across the surface of the embryonic hearts by high resolution optical mapping using di-4-ANEPPS and cytochalasin D. While there were no electrical artifacts produced by the optical point stimulation, an optical artifact due to thermal lensing did obscure the optical mapping near the point of stimulation. The optical artifact can be minimized by optimizing the stimulation parameters to minimize the energy deposited and can be further reduced by signal processing. Despite the presence of the optical artifact, the electrical activity over the majority of the heart can be obtained.

Spreading area and shape regulate apoptosis and differentiation of osteoblasts.

The in vivo observations have indicated that at the remodeling sites of bone, the spreading area or shape of preosteoblasts is confined by the mineralized matrix. But it remains unknown whether this spreading confinement regulates the differentiation or apoptosis of osteoblasts. In the present study, osteoblast-like cells (MC3T3-E1) were seeded on micropatterned islands with different area and shape. The expression of three osteogenic differentiation markers was measured by immunofluorescence staining and apoptotic cells were detected using a terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling assay kit. The membrane fluorescence staining results showed that the actual spreading area of micropatterned osteoblasts coincided with the designed value. When the area of a micropatterned cell was confined as 314 or 615 µm(2), which was lower than that of freely spreading osteoblasts, the circular shape promoted the expression of osteogenic differentiation markers and the percentage of apoptotic osteoblasts compared with the branched shape. This shape-regulated differentiation and apoptosis of osteoblasts with confined spreading area were abolished when actin polymerization was inhibited by cytochalasin D. The present study gives an insight into the roles of spreading morphology on osteoblastic differentiation and apoptosis.

Mechanics of spreading cells probed by atomic force microscopy.

Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.

Analyzing cellular internalization of nanoparticles and bacteria by multi-spectral imaging flow cytometry.

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed. Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStream(X) system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

Passive and active contributions to generated force and retraction in heart valve tissue engineering.

In tissue engineered heart valves, cell-mediated stress development during culture results in leaflet retraction at time of implantation. This tissue retraction is partly active due to traction forces exerted by the cells and partly passive due to release of residual stress in the extracellular matrix and the cells. Within this study, we unraveled the passive and active contributions of cells and matrix to generated force and retraction in engineered heart valve tissues. Tissue engineered rectangular strips, fabricated from PGA/P4HB scaffolds and seeded with human myofibroblasts, were cultured for 4 weeks, after which the cellular contribution was changed at different levels. Elimination of the active cellular traction forces was achieved with Cytochalasin D and inhibition of the Rho-associated kinase pathway. Both active and passive cellular contributions were eliminated by lysation and/or decellularization of the tissue. Maximum cell activity was reached by increasing the fetal bovine serum concentration to 50%. The generated force decreased ~20% after elimination of the active cellular component, ~25% when the passive cellular component was removed as well and remained unaffected by increased serum concentrations. Passive retraction accounted for ~60% of total retraction, of which ~15% was residual stress in the matrix and ~45% was passive cell retraction. Cell traction forces accounted for the remainder ~40% of the retraction. Full activation of the cells increased retraction by ~45%. These results illustrate the importance of the cells in the process of tissue retraction, not only actively retracting the tissue, but also in a passive manner to a large extent.

[Secondary metabolites of mangrove endophytic fungus BL321 in the South China Sea].

OBJECTIVE: To study the secondary metabolites of mangrove endophytic fungus BL321. METHODS: The compounds were isolated by chromatographic technique. The structures were identified by comprehensive physico-chemical properties and spectral methods. RESULTS: Five compounds were isolated and identified as 3,4a-dimethyl-2-oxo-2,4,4a,5,6,7-hexahydronaphtho[2,3-b]furan-5-carboxylic acid(1), cytochalasin C(2), cytochalasin D(3), 19,20-epoxycytochalasin C(4), ergosterol(5). CONCLUSION: Compound 1 is isolated from nature for the first time. Further more, several kinds of strong bioactive compounds were islolate from this fungus indicate that it may develop to be medical source microorganism.

Formation of N-type (Cav2.2) voltage-gated calcium channel membrane microdomains: Lipid raft association and clustering.

Voltage-gated calcium channels (Ca(v)s) comprise a pore-forming α1 with auxiliary α2δ and ß subunits which modulate Ca(v) function and surface expression. Ca(v)α1 and α2δ are present in signalling complexes termed lipid rafts but it is unclear whether α2δ is obligatory for targeting Ca(v)s to rafts or to what extent this influences cell surface organisation of Ca(v)s. Here, we have used imaging, biochemistry and electrophysiology to determine localisation and raft-partitioning of WT and functionally active HA-epitope tagged α2δ-1 and Ca(v)2.2 subunits expressed in COS-7 cells. We show that α2δ-1 not only partitions into lipid rafts itself but also mediates raft-partitioning of Ca(v)2.2/ß(1b) complexes. Ca(v)α2δ-1, Ca(v)2.2/ß(1b) and Ca(v)2.2/ß(1b)/α2δ-1 complexes are all organised into cell surface clusters although only in the presence of α2δ-1 do they co-localise with raft markers, caveolin and flotillin. Such clusters persist in the presence of 3-methyl-ß-cyclodextrin even though the raft markers disperse. However, clustering is profoundly sensitive to disruption of the actin-based cytoskeleton by cytochalasin-D. We conclude that α2δ-1, and likely other α2δ subunits, is necessary and sufficient for targeting Ca(v)s to lipid rafts. However, formation of clusters supporting "hotspots" of Ca(v) activity requires aggregation of macromolecular complexes containing raft components, stabilised by interactions with the cytoskeleton.