Cacofurans A and B, new furanoditerpenes from a marine sponge.

Two new labdane-class diterpenes, cacofurans A (1) and B (2), have been isolated from a sponge Cacospongia sp. Their structures were determined by analyzing spectroscopic data, by chemical transformations, and by X-ray diffraction. Cacofurans 1 and 2 inhibited the development of fertilized sea urchin eggs at concentrations of 0.5 and 5 microg/mL and showed an actin-disrupting effect on the NBT-II cell line at 10 microg/mL.

Recruitment of cortexillin into the cleavage furrow is controlled by Rac1 and IQGAP-related proteins.

Cytokinesis in eukaryotic organisms is under the control of small GTP-binding proteins, although the underlying molecular mechanisms are not fully understood. Cortexillins are actin-binding proteins whose activity is crucial for cytokinesis in Dictyostelium. Here we show that the IQGAP-related and Rac1-binding protein DGAP1 specifically interacts with the C-terminal, actin-bundling domain of cortexillin I. Like cortexillin I, DGAP1 is enriched in the cortex of interphase cells and translocates to the cleavage furrow during cytokinesis. The activated form of the small GTPase Rac1A recruits DGAP1 into a quaternary complex with cortexillin I and II. In DGAP1(-) mutants, a complex can still be formed with a second IQGAP-related protein, GAPA. The simultaneous elimination of DGAP1 and GAPA, however, prevents complex formation and localization of the cortexillins to the cleavage furrow. This leads to a severe defect in cytokinesis, which is similar to that found in cortexillin I/II double-null mutants. Our observations define a novel and functionally significant signaling pathway that is required for cytokinesis.

Community thrombolysis in the Coromandel region. Audit of the "Cardiac Events in the Coromandel-Assessment Strategy and Triage" (CE-COAST) pilot program.

AIM: To audit the experience of a pilot program for community thrombolysis undertaken within the Coromandel region. METHODS: Community thrombolysis for patients suffering acute myocardial infarction (MI) was undertaken in areas within the Coromandel peninsula greater than half an hour by road from Thames Hospital. Thrombolytic therapy (Retelapse) was given following a discussion and review of a digitally transmitted ECG with the cardiology registrar. Treatment times and patients demographics were prospectively recorded. Subsequent clinical events were by chart review. Comparison of treatment times were made with an historical cohort for the same population which had received in-hospital thrombolysis between 1993 and 1998. RESULTS: Between July 1998 and December 1999, nineteen patients received thrombolysis in the community. There were no arrhythmic events during transportation and no deaths or reinfarctions during hospital stay. Median time from pain onset to thrombolysis was 135 (mean 175.5 +/- 144.9 SD) minutes which equated to a reduction in median time delay of 135 minutes compared to that experienced by the historical cohort (median 270, mean 316.7 +/- 145.8 SD minutes), p=0.0003. CONCLUSION: Community thrombolysis is logistically feasible within the New Zealand setting and results in major time reductions in the treatment of patients with acute MI.

Local photorelease of caged thymosin beta4 in locomoting keratocytes causes cell turning.

The broad aim of this work was to explore the feasibility of using light-directed perturbation techniques to study cell locomotion. Specifically, a caged form of thymosin beta4 (Tbeta4) was photoactivated in a defined local region of locomoting fish scale keratocytes and the resulting perturbation of locomotion was studied. Purified Tbeta4 was produced in an inactive form by "caging" with ([n-nitroveratryl]oxy)chlorocarbamate. In vitro spectrophotofluorometric assays indicated that caged Tbeta4 did not change the normal actin polymerization kinetics, whereas photoactivated Tbeta4 significantly inhibited actin polymerization. With an a priori knowledge of the cytoplasmic diffusion coefficient of Tbeta4 as measured by fluorescence recovery after photobleaching experiments, the rapid sequestration of actin monomers by uncaged Tbeta4 and the consequent reduction in the diffusional spread of the Tbeta4-actin complex were predicted using Virtual Cell software (developed at the Center for Biomedical Imaging Technology, University of Connecticut Health Center). These simulations demonstrated that locally photoactivating Tbeta4 in keratocytes could potentially elicit a regional locomotory response. Indeed, when caged Tbeta4 was locally photoactivated at the wings of locomoting keratocytes, specific turning about the irradiated region was observed, whereas various controls were negative. Additionally, loading of exogenous Tbeta4 into both keratocytes and fibroblasts caused very rapid disassembly of actin filaments and reduction of cellular contractility. Based on these results, a mechanical model is proposed for the turning behavior of keratocytes in response to photoreleased Tbeta4.

Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3.

BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. RESULTS: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. CONCLUSIONS: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.

Involvement of ezrin/moesin in de novo actin assembly on phagosomal membranes.

The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non-specific actin polymerization during the assay, fluorescent G-actin was mixed with thymosin beta4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to membrane fusion.

Protein phosphatase 2A inhibitors, phoslactomycins. Effects on the cytoskeleton in NIH/3T3 cells.

Protein phosphorylation is a key regulatory mechanism of the organization and dynamics of the actin cytoskeleton during cell motility, differentiation, and cytokinesis. The level of protein phosphorylation is dependent on the relative activities of both protein kinases and protein phosphatases. In this paper, we examined the effect of phoslactomycins (PLMs) on the regulation of the cytoskeleton of NIH/3T3 fibroblasts. Treatment of cells with PLM-F (10 microM) induced actin filament depolymerization after 4 h. This effect was reversible and actin filaments were reformed 1 h after removal of the inhibitors. As PLM-F had no effect at all on polymerization of purified actin in vitro, it is thought that PLMs induce actin depolymerization through an indirect mechanism. An in vitro assay showed PLMs inhibited protein phosphatase 2A at lower concentrations (IC50 4.7 microM) than protein phosphatase 1. An in situ phosphorylation assay also revealed that PLM-F treatment stimulated the phosphorylation of intracellular vimentin. These results suggest that phoslactomycins are protein phosphatase 2A-specific inhibitors and that protein phosphatase 2A is involved in regulation of the organization of the actin cytoskeleton.

Phototactic migration of Dictyostelium cells is linked to a new type of gelsolin-related protein.

The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100-insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.

The suitability and application of a GFP-actin fusion protein for long-term imaging of the organization and dynamics of the cytoskeleton in mammalian cells.

The product of a GFP-actin gene fusion, permanently or transiently transfected in diverse mammalian cell lines, was shown to be a suitable, intrinsic probe of both the organization and dynamics of the actin cytoskeleton. In live Swiss 3T3 and NIH 3T3 cells, the fusion protein was found to accumulate in lamellipodia, filopodia, focal contacts and stress fibers. Furthermore, comparisons of fluorescence images of GFP-actin and Cy3.5-phalloidin, an independent marker of F-actin, in permeabilized cells showed a complete overlap of the two fluorescence signals. In GFP-actin-transfected Hela cells that had been infected with Listeria monocytogenes, the fluorescence of the fusion protein was shown to dynamically associate in the F-actin rich comet tail that formed behind a motile bacterium. In stable transfectants of PC12 cells, GFP-actin constituted on the average 5% of the total actin - these cells exhibited normal growth behavior and responded to treatment with nerve growth factor by extending neurite-like extensions, the filopodia-like tips of which were densely packed with filamentous GFP-actin. Finally, the photobleaching decay time of GFP-actin in live cells of 63 seconds was much longer than that of fluorescein-labeled actin conjugates and little or no damage to the cytoskeleton was found during the photobleaching of GFP-actin. Having shown the suitability of GFP-actin as a probe of the cytoskeleton, its fluorescence was used in long-term imaging studies aimed at documenting changes in the cytoskeleton of rat bladder NBT-II carcinoma cells during the 24-hour growth factor-mediated epithelia to mesenchyme transformation. The intrinsic fluorescent probe was also used to investigate the organization of the actin cytoskeleton and behavior of individual mesenchyme NBT-II cells slowly migrating through a colony of epithelia cells.

Synthesis and applications of heterobifunctional photocleavable cross-linking reagents.

This study designed, synthesized, and characterized a number of new heterobifunctional photocleavable cross-linking reagents that may be used to photomodulate the activity of proteins or to prepare caged fluorescent dyes. Biomolecules or fluorophores caged via a thiol group with the BNBASE reagent can be covalently linked to a second protein, ligand, or derivatized surface through the activated carboxyl group. Members of the new class of photocleavable cross-linking reagent can be used to cage amino groups in the molecule of interest, which can then be covalently linked to a second molecule through the thiol-reactive oxirane group. These crosslinking reagents may be used for the following applications: (1) to cage the activity of a protein by masking its active site with a second macromolecule, e.g., aminodextran; (2) to prepare a protein conjugate exhibiting an enhanced or new activity that is lost on irradiation with near-ultraviolet light, e.g., cross-linked actin dimer; (3) to target the caged compound to a specific site by cross linking to a specific antibody; (4) to attach the caged compound to a thiol or amino derivatized surface; and (5) to render the caged compound fluorescent in order to image or to quantify the yield of the photoactivation reaction.

DdLIM is a cytoskeleton-associated protein involved in the protrusion of lamellipodia in Dictyostelium.

DdLim, a multi-domain member of the cysteine-rich family of LIM domain proteins, was isolated from Dictyostelium cells where it localizes in lamellipodia and at sites of membrane ruffling. The transcription and expression of DdLim are developmentally regulated, and the timing of its increased association with the actin cytoskeleton coincides with the acquisition in starved cells of a motile, chemotactic behavior. Vegetative cells that overexpress DdLim contain large lamella and exhibit ruffling at the cortex. The high frequency of large, multinucleated mutant cells found in suspension culture suggests that excess DdLim interferes with cytokinesis. DdLim was also identified as a protein in a Dictyostelium cell lysate that associated indirectly, but in a guanosine triphosphate-dependent manner, with a GST-rac1 fusion protein. The data presented suggest that DdLim acts as an adapter protein at the cytoskeleton-membrane interface where it is involved in a receptor-mediated rac1-signaling pathway that leads to actin polymerization in lamellipodia and ultimately cell motility.

Preparation and photoactivation of caged fluorophores and caged proteins using a new class of heterobifunctional, photocleavable cross-linking reagents.

The design, synthesis, and spectroscopic and chemical properties of four members of a new class of heterobifunctional photocleavable (caged) cross-linking reagents were described. One of the two reactive groups of the cross-linker reacted with amino groups to form the corresponding photolabile carbamates. Amino group containing compounds or proteins caged with these reagents can be coupled through the thiol reactive oxirane group of the cross-linker to a different biomolecule or to a thiol-derivatized surface. The 3,4-dimethoxy-6-nitrophenyl photoisomerization group of the reagent was physically and chemically isolated from the cross-linking functionality, and the high extinction coefficient and red-shifted action spectrum of this chromophore make it suitable for photoactivation applications of caged compounds on surfaces or in living cells. The bifunctional, photocleavable cross-linking reagents were used to prepare a thiol reactive caged rhodamine 110. The new reagents and conjugation procedures described may be used as part of a general procedure to cage the activity of proteins by physically masking binding sites.

Cooperative association of actin protomers and crosslinked actin oligomers in filaments at low ionic strength.

F-Actin treated with a half molar equivalent of the heterobifunctional cross-linker, 4-bromomethyl-3-nitrobenzoic acid succinimide ester (BNBA-SE), produced a population of actin oligomers containing 2-14 or more protomers and a significant amount of uncrosslinked actin protomers. The crosslinking reaction is presumed to occur between Cys 374 of one actin protomer with Lys 191 of an adjacent protomer on the genetic helix of F-actin, in a similar manner to N-maleimidobenzoic acid succinimidyl ester, which shares similar reactive groups and crosslinking dimensions. Crosslinked oligomers and uncrosslinked protomers were found to form filaments that sediment after high-speed centrifugation, even in a buffer of low ionic strength [G-buffer: 2 mM tris-hydroxymethylaminomethane (pH 8.0), 0.2 mM CaCl2, 0.2 mM ATP, 0.3 mM NaN3, 5 mM 2-mercaptoethanol] where affinity between actin protomers usually becomes weak, leading to the depolymerization of native F-actin. By performing the crosslinking reaction in the presence of an environment-sensitive fluorescent label, 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), which competes with BNBA-SE for Cys 374 of actin protomers, fluorescent, crosslinked F-actin filaments were obtained which were also stable in G-buffer. Fluorometric analysis of actin labeled with acrylodan (prodan-actin) in these depolymerization-resistant filaments suggested that the molecular environment around Cys 374 of actin protomers is similar to that of actin protomers in native F-actin in F-buffer (G-buffer plus 0.1 M KCl and 1 mM MgCl2). When G-actin labeled with acrylodan was co-polymerized with non-fluorescent crosslinked F-actin, some of the labeled actin was incorporated into filaments that were resistant to depolymerization in G-buffer. The emission spectrum of the prodan-actin in these filaments measured in G-buffer was almost identical to that of prodan-actin in native F-actin in F-buffer. Our interpretation of this result is that actin protomers are locked into an F-actin-like conformation in the depolymerization-resistant filament by the subunit interactions they make with crosslinked oligomers. We also present evidence which suggests that the depolymerization rate in G-buffer of filaments made with crosslinked oligomers is much slower than that of native actin because the ends of the depolymerization-resistant filaments are capped with crosslinked oligomers.

Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein.

BACKGROUND: The microfilament system in the cortex of highly motile cells, such as neutrophils and cells of the eukaryotic microorganism Dictyostelium discoideum, is subject to rapid re-organization, both spontaneously and in response to external signals. In particular, actin polymerization induced by a gradient of chemoattractant leads to local accumulation of filamentous actin and protrusion of a 'leading edge' of the cell in the direction of the gradient. In order to study the dynamics of actin in these processes, actin was tagged at its amino terminus with green fluorescent protein (GFP) and observed with fluorescence microscopy in living cells of D. discoideum. RESULTS: Purified GFP-actin was capable of copolymerizing with actin. In the transfected cells of D. discoideum studied, GFP-actin made up 10-20% of the total actin. Microfilaments containing GFP-actin were capable of generating force with myosin in an in vitro assay. Observations of single living cells using fluorescence microscopy showed that the fusion protein was enriched in cell projections, including filopodia and leading edges, and that the fusion protein reflected the dynamics of the microfilament system in cells that were freely moving, being chemotactically stimulated, or aggregated. When confocal sections of fixed cells containing GFP-actin were labeled with fluorescent phalloidin, which binds only to filamentous actin, there was a correlation between the areas of GFP-actin and phalloidin fluorescence, but there were distinct sites in which GFP-actin was more prominent. CONCLUSIONS: Double labeling with GFP-actin and other probes provides an indication of the various states of actin in motile cells. A major portion of the actin assemblies visualized using GFP-actin are networks or bundles of filamentous actin. Other clusters of GFP-actin might represent stores of monomeric actin in the form of complexes with actin-sequestering proteins.

Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes.

A protein purified from cytoskeletal fractions of Dictyostelium discoideum proved to be a member of the fimbrin/plastin family of actin-bundling proteins. Like other family members, this Ca(2+)-inhibited 67-kDa protein contains two EF hands followed by two actin-binding sites of the alpha-actinin/beta-spectrin type. Dd plastin interacted selectively with actin isoforms: it bound to D. discoideum actin and to beta/gamma-actin from bovine spleen but not to alpha-actin from rabbit skeletal muscle. Immunofluorescence labeling of growth phase cells showed accumulation of Dd plastin in cortical structures associated with cell surface extensions. In the elongated, streaming cells of the early aggregation stage, Dd plastin was enriched in the front regions. To examine how the bundled actin filaments behave in myosin II-driven motility, complexes of F-actin and Dd plastin were bound to immobilized heavy meromyosin, and motility was started by photoactivating caged ATP. Actin filaments were immediately propelled out of bundles or even larger aggregates and moved on the myosin as separate filaments. This result shows that myosin can disperse an actin network when it acts as a motor and sheds light on the dynamics of protein-protein interactions in the cortex of a motile cell where myosin II and Dd plastin are simultaneously present.

Australian Diabetes Screening Study: impaired glucose tolerance and non-insulin-dependent diabetes mellitus.

In preventing non-insulin-dependent diabetes mellitus (NIDDM) and its complications, screening high-risk individuals complements public health measures. Our screening instrument for patients of general practitioners was a questionnaire for self-determined high-risk groups plus a laboratory measurement of a random venous plasma glucose level. Collaborating practitioners evaluated 100 consecutive outpatients aged 40 years or older. The questionnaire identified patients with two or more diabetic symptoms or with two or more risk factors, and they were recommended to have their blood tested. For those with a random plasma glucose greater than 5.5 mmol/L, oral glucose tolerance tests (OGTTs) were advised. Of 50,859 subjects completing the study, there were 1,013 cases (2.0%) of new diabetes, 1,704 cases (3.4%) of impaired glucose tolerance (IGT), and 5,508 cases (10.8%) of previously diagnosed diabetes. Symptoms alone were a relatively poor discriminant. Almost all newly identified NIDDM and IGT patients had two or more risk factors for NIDDM. The risk ratios for abnormal glucose tolerance were as follows: high blood pressure, 2.4; overweight, 2.0; and positive family history, 1.7. Selection of cutoff points higher than 5.5 mmol/L would have substantially reduced the rate of newly discovered NIDDM and IGT. Screening for NIDDM and IGT in general practice is feasible and can be achieved with little disruption of office procedures. In preventive programs of this nature, the low screening threshold of 5.5 mmol/L for random venous plasma glucose maximizes the case-finding rate.

Dynamics and morphology of the in vitro polymeric form of elongation factor Tu from Escherichia coli.

Elongation factor Tu from Escherichia coli is known to polymerize at slightly acidic pH and low ionic strength. The structure and dynamics of these aggregates have been examined using imaging and spectroscopic methodologies. Electron microscopy provides evidence for two-dimensional sheets and bundled filaments of EF-Tu, whereas fluorescence microscopy of EF-Tu covalently labeled with tetramethylrhodamine isothiocyanate showed highly branched polymers of EF-Tu several microns in diameter. These polymers were studied using quasi-elastic light scattering to determine the evolution of the translational diffusion coefficient during the polymerization process. The rotational dynamics of the aggregate were investigated using phosphorescence anisotropy of EF-Tu covalently labeled with erythrosin isothiocyanate. A high infinite-time anisotropy was observed, suggesting a lack of motion or entanglement of EF-Tu polymers. A sub-microsecond motion which was slowed in the presence of glycerol may be due to local flexibility of the polymers. The possible relevance of polymeric EF-Tu to its function in vivo is discussed.

Light-directed generation of the actin-activated ATPase activity of caged heavy meromyosin.

An understanding of the molecular mechanism of muscle contraction will require a complete description of the kinetics of the myosin motor in vitro and in vivo. To this end chemical relaxation studies employing light-directed generation of ATP from caged ATP have provided detailed kinetic information in muscle fibers. A more direct approach would be to trigger the actin-activated ATPase activity from a caged myosin, i.e., myosin whose activity is blocked upon derivatization with a photolabile protection group. Herein we report that a new type of caged reagent can be used to prepare a caged heavy meromyosin by modification of critical thiol groups, i.e., a chemically modified motor without activity that can be reactivated at will using a pulse of near-ultraviolet light. Heavy meromyosin modified at Cys-707 with the thiol reactive reagent 1-(bromomethyl)-2-nitro-4,5-dimethoxybenzene does not exhibit an actin-activated ATPase activity and may be viewed as a caged protein. Absorption spectroscopy showed that the thioether bond linking the cage group to Cys-707 is cleaved following irradiation (340-400 nm) via a transient aci-nitro intermediate which has an absorption maximum at 440 nm and decays with a rate constant of 45.6 s(-1). The in vitro motility assay showed that caged heavy meromyosin cannot generate the force necessary to move actin filaments although following irradiation of the image field with a 30 ms pulse of 340-400 nm light the caged group was removed with the concomitant movement of most filaments at a velocity of 0.5-2 micron/s compared to 3-4 micron/s for unmodified HMM. The specificity and simplicity of labeling myosin with the caged reagent should prove useful in studies of muscle contraction in vivo.