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Biomedical subjects

F Buss

Publications and source records attributed to F Buss.

16 recordsLinked to original sources

Myosin VI: a multifunctional motor.

Myosin VI moves towards the minus end of actin filaments unlike all the other myosins so far studied, suggesting that it has unique properties and functions. Myosin VI is present in clathrin-coated pits and vesicles, in membrane ruffles and in the Golgi complex, indicating that it has a wide variety of functions in the cell. To investigate the cellular roles of myosin VI, we have identified a variety of myosin VI-binding partners and characterized their interactions. As an alternative approach, we have studied the in vitro properties of intact myosin VI. Previous studies assumed that myosin VI existed as a dimer but our biochemical characterization and electron microscopy studies reveal that myosin VI is a monomer. Using an optical tweezers force transducer, we showed that monomeric myosin VI is a non-processive motor with a large working stroke of 18 nm. Potential roles for myosin VI in cells are discussed.

Actins↗

Axial flow catheter pump for circulatory support.

AIM: The development of a percutaneously insertable catheter pump for temporary left ventricular and circulatory support in patients with acute ischemic cardiomyopathy. MATERIAL AND METHODS: The expandable axial flow pump (introduction diameter 9-F, expanded diameter 6.5 mm) is mounted at the tip of a catheter, designed to be placed in aortic transvalvular position. Hydrodynamic tests were performed in a static flow model and in a pulsatile mock circuit (Medos VAD, Stolberg, Germany). Cardiogenic shock was simulating in vitro and in vivo. RESULTS: At a rotation speed of 32,000 rpm the catheter pump delivered a maximum flow of more than 4.0 L/min against a systemic mean pressure of 60 mmHg. Under shock simulation the pump significantly improved output and systemic pressure. CONCLUSION: A percutaneously insertable catheter pump is technically feasible. Hydrodynamic efficacy allows sufficient cardiac support under simulated shock condition.

Animals↗

Myosin VI, a new force in clathrin mediated endocytosis.

The integrity of the actin cytoskeleton and associated motor proteins are essential for the efficient functioning of clathrin mediated endocytosis at least in polarised cells. Myosin VI, the only motor protein so far identified that moves towards the minus end of actin filaments, is the first motor protein to be shown to associate with clathrin coated pits/vesicles at the plasma membrane and to modulate clathrin mediated endocytosis. Recent kinetic studies suggest that myosin VI may move processively along actin filaments providing clues about its functions in the cell. The possible role(s) of myosin VI in the sequential steps involved in receptor mediated endocytosis are discussed.

Actin Cytoskeleton↗

Myosin VI isoform localized to clathrin-coated vesicles with a role in clathrin-mediated endocytosis.

Myosin VI is involved in membrane traffic and dynamics and is the only myosin known to move towards the minus end of actin filaments. Splice variants of myosin VI with a large insert in the tail domain were specifically expressed in polarized cells containing microvilli. In these polarized cells, endogenous myosin VI containing the large insert was concentrated at the apical domain co-localizing with clathrin- coated pits/vesicles. Using full-length myosin VI and deletion mutants tagged with green fluorescent protein (GFP) we have shown that myosin VI associates and co-localizes with clathrin-coated pits/vesicles by its C-terminal tail. Myosin VI, precipitated from whole cytosol, was present in a protein complex containing adaptor protein (AP)-2 and clathrin, and enriched in purified clathrin-coated vesicles. Over-expression of the tail domain of myosin VI containing the large insert in fibroblasts reduced transferrin uptake in transiently and stably transfected cells by >50%. Myosin VI is the first motor protein to be identified associated with clathrin-coated pits/vesicles and shown to modulate clathrin-mediated endocytosis.

Amino Acid Sequence↗

Localization of myosin Va is dependent on the cytoskeletal organization in the cell.

Myosin V plays an important role in membrane trafficking events. Its implication in the transport of pigment granules in melanocytes and synaptic vesicles in neurons is now well established. However, less is known about its function(s) in other cell types. Finding a common function is complicated by the diversity of myosin V expression in different tissues and organisms and by its association with different subcellular compartments. Here we show that myosin V is present in a variety of cells. Within the same cell type under different physiological conditions, we observed two main cellular locations for myosin V that were dependent on the dynamics of the plasma membrane: in cells with highly dynamic membranes, myosin V was specifically concentrated at the leading edge in membrane ruffles, whereas in cells with less dynamic membranes, myosin V was enriched around the microtubule-organizing center. The presence of myosin V in the leading ruffling edge of the cell was induced by growth factor stimulation and was dependent on the presence of a functional motor domain. Moreover, myosin V localization at the microtubule-organizing center was dependent on the integrity of the microtubules. In polarized epithelial cells (WIF-B), where the microtubule-organizing region is close to the actin-rich apical surface, one single pool of myosin V, sensitive to the integrity of both microtubules and actin filaments, was observed.

Actins↗

Activation of neuroendocrine L-type channels (alpha1D subunits) in retinal pigment epithelial cells and brain neurons by pp60(c-src).

The aim of this study is to characterize the subtype of tyrosine kinase-regulated L-type Ca(2+) channels in retinal pigment epithelial (RPE) cells. Ca(2+) channel alpha1D-subunits were enriched by immunoprecipitation from membrane proteins isolated from rat RPE cells. Western blot analysis of the precipitates revealed coprecipitation of pp60(c-src). In addition, in precipitates obtained with antibodies against pp60(c-src), alpha1D-subunits were identified. The same was observed in immunoprecipitations from rat brain neurons. Tyrosine phosphorylation of alpha1D-subunits was confirmed using anti-phosphotyrosine antibodies. Ba(2+) currents through L-type channels in cultured rat RPE cells were increased by intracellular application of active pp60(c-src) (30 U/ml) (heat-inactivated pp60(c-src) had no effect). Thus, L-type channels of the neuroendocrine subtype can be expressed in epithelial cells and are activated by tyrosine kinase of the src subtype. This kind of regulation is also suggested for brain-derived neurons.

Animals↗

The localization of myosin VI at the golgi complex and leading edge of fibroblasts and its phosphorylation and recruitment into membrane ruffles of A431 cells after growth factor stimulation.

Myosin VI is an unconventional myosin that may play a role in vesicular membrane traffic through actin rich regions of the cytoplasm in eukaryotic cells. In this study we have cloned and sequenced a cDNA encoding a chicken intestinal brush border myosin VI. Polyclonal antisera were raised to bacterially expressed fragments of this myosin VI. The affinity purified antibodies were highly specific for myosin VI by immunoblotting and immunoprecipitation and were used to study the localization of the protein by immunofluorescence and immunoelectron microscopy. It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool. In A431 cells in which cell surface ruffling was stimulated by EGF, myosin VI was phosphorylated and recruited into the newly formed ruffles along with ezrin and myosin V. In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain. These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.

Actins↗

Macromolecular interactions in the nucleoporin p62 complex of rat nuclear pores: binding of nucleoporin p54 to the rod domain of p62.

Nuclear pore complexes are constructed from a large number of different proteins, called collectively nucleoporins. One of these nucleoporins, p62, has an alpha-helical coiled-coil COOH-terminal rod domain linked to an NH2-terminal domain that contains a series of degenerate pentapeptide repeats. In nuclear pores p62 forms a tight complex with at least two other proteins, p58 and p54, which can be extracted from isolated rat liver nuclei (Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. J. Cell Biol. 114:169-183). We have used a range of methods to demonstrate a strong binding between p62 and p54 in this complex and show that the rod domain of p62 appears to constitute the principal binding site for p54. Whole p62 and its rod domain expressed in Escherichia coli both bind strongly to p54 in blot-overlay assays. Most of the epitopes on the p62 rod recognized by polyclonal antisera are masked in the complex, whereas epitopes on the NH2-terminal domain of p62 are still exposed, both in the isolated complex and also in nuclear pores stained in situ by immunofluorescence in isolated rat nuclei. Moreover, it has been possible to exchange recombinant p62 rod for some of the native p62 in complexes partially dissociated by 4 M urea. Overall these results suggest a key role for the p62 rod domain in maintaining the structural integrity of the complex and also suggest a molecular model for the complex. This model is consistent with data that indicate that the analogous coiled-coil region of yeast nucleoporin NSP1 may function in a similar way.

Animals↗

Role of different domains in the self-association of rat nucleoporin p62.

We have expressed rat nucleoporin p62 cDNA in Escherichia coli to obtain material for structural and self-association studies. Electron microscopy and circular dichroism spectroscopy are consistent with a rod-shaped molecule with an alpha-helical coiled-coil domain at its C terminus and a cross-beta structure at its N terminus, separated by a threonine-rich linker, which has a less-defined secondary structure. Electron microscopy and the solubility properties of fragments produced using thrombin and CNBr digestion indicate that p62 molecules associate to form linear chains and that a small region near the C terminus is an important determinant of assembly. This association may have important consequences for pore structure and function; for example, one way p62 could associate would be to form rings in nuclear pores that could function like barrel hoops.

Amino Acid Sequence↗

Distribution of profilin in fibroblasts correlates with the presence of highly dynamic actin filaments.

We have used polyclonal and monoclonal antibodies raised against calf thymus profilin to localize the corresponding protein in translocating, spreading, and stationary rat fibroblasts. Immunofluorescence of whole cells and immunogold labeling on ventral membranes of lysis-squirted cells showed that profilin was markedly enriched in the highly dynamic lamellipodia or pseudopodial lobes. Within these regions, a significant fraction was colocalized with dynamic actin filaments organized in actin ribs, cortical filaments, or stress fiber-like bundles, and little profilin was found in membrane areas appearing free of actin. In contrast, stress fibers of stationary cells as well as actin arcs and ring-like bundles of spreading and migrating cells showed very little label. These results are discussed in context with the proposed role of profilin in regional membrane dynamics typical for fibroblasts and are compared to previous data (Hartwig et al.: J. Cell Biol. 109:1571-1579, 1989) on profilin distribution in platelets and granulocytes.

Actins↗

Long-term follow-up after percutaneous transluminal coronary angioplasty in patients with single-vessel disease.

Seven hundred ninety-eight patients with symptomatic single-vessel disease who underwent percutaneous transluminal coronary angioplasty (PTCA) between 1977 and 1985 were reevaluated by questionnaire 78 +/- 23 months after dilatation. Indication for PTCA was stenosis of > or = 70%, anginal symptoms, and objective signs of myocardial ischemia. The immediate success rate was 81.2%, and severe complications occurred in 7.1%, which included two fatal complications (0.3%). Repeat angiograms were performed in 582 of 648 patients who underwent successful dilatation and showed restenosis in 143 cases (24.6%). Within 1 year after the first dilatation, 586 patients had been successfully revascularized by PTCA (i.e., there was no evidence of restenosis or redilatation was successful), and 113 patients had undergone bypass surgery. The remaining 99 patients were treated medically if PTCA was unsuccessful or if restenosis (> or = 70%) that was not amenable to redilatation was present. The 8-year overall survival probability was 91.7%, and cardiac survival was 95.5%. The 8-year event-free survival probability was 52.7% for all patients: 62.5% in patients who had successful PTCA and 14.5% in patients who had unsuccessful PTCA (p = 0.0000). The cardiac survival probabilities of patients with lasting PTCA success at 1 year and of surgically treated patients were significantly better than those of patients who did not have successful revascularization (at 8 years 97.2% and 98.1% vs 88.9%; p < 0.04). Late events (> or = 1 year) occurred more often in patients who did not have successful revascularization compared with patients who had successful PTCA (at 8 years 57.9% were event-free vs 74.4%; p < 0.0001); even fewer late events were observed in surgically treated patients (at 8 years 88.2% were event-free; p < 0.004). Cox's proportional hazards regression analysis revealed left ventricular ejection fraction and revascularization status at 1 year as determinants of overall, cardiac, infarct-free, and event-free survival probabilities. At the time of reevaluation significantly more patients in the successful PTCA subgroup were still free of symptoms or had experienced improvement than patients in the bypass or medical subgroups (86.8% vs 68.9% and 59.5%, respectively; p < 0.0001), and more patients in the successful PTCA subgroup were still working (75.4% vs 53.3% and 56.9%, respectively; p < 0.001). We concluded that patients with single-vessel disease who have undergone successful dilatation have an excellent long-term prognosis with regard to survival, cardiac symptoms, and vocational status.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

Caldesmon-induced polymerization of actin from profilactin.

We have investigated the effect of caldesmon, a Ca2+/calmodulin-regulated actin-binding protein, on the complex between profilin and G-actin (profilactin). We found that smooth muscle caldesmon dissociates this complex rapidly and induces the polymerization of the released actin. Native profilactin (e.g. the complex isolated from calf thymus) proved more resistant to the attack of caldesmon than a heterologous complex reconstituted from calf thymus profilin and skeletal muscle actin. The mode of caldesmon-induced profilactin dissociation was similar to that described for Mg2+, and 2 mM MgCl2 potentiated the caldesmon effect. Since both caldesmon and profilin have been found enriched in ruffling membranes of animal cells, our in vitro findings may be relevant to the regulation of actin filaments in living cells.

Actins↗

Cortical microfilament proteins and the dynamics of the plasma membrane.

The microfilament system is thought to provide motor elements needed for plasma membrane dynamics. This article focuses on two protein components that may play key roles in this process: (1) Profilin, a G-actin binding protein which is considered as the source of actin subunits necessary for rapid changes in the amount of actin filaments. Our data demonstrate that profilin is synthesized even in terminally differentiated blood cells of a high dynamic potential. In addition, we show that plasma membrane-associated profilin in fibroblasts is unevenly distributed and is concentrated in areas that are highly motile. (2) The filament-forming myosin which is the classical motor protein in the microfilament system. We show that interfering with myosin filaments by microinjecting antibodies causes brush border-type microvilli on epithelial cells to loose their upright position. This result, together with our previous observations on the effects of anti-myosin injection into fibroblastic and epithelial cells (loss of stress fibers and cellular contact sites, increase in locomotory activity, delay of cytokinesis), suggests that bipolar myosin filaments are needed to maintain a specific cortical tension which is lost upon antibody binding.

Actins↗

Tissue-specific expression of profilin.

Expression of profilin and profilin:actin ratios in vertebrates were determined with polyclonal antibodies against profilin and actin. Profilin was detected in a wide variety of bovine tissues and was enriched in smooth muscle of bovine, porcine and avian origin. The protein was purified from pig stomach muscle tissue. Smooth muscle profilin was found to be more effective in inhibiting the polymerization of skeletal muscle actin than thymus profilin purified by the same method.

Actins↗

Immunological and biochemical studies on the relationship between two actin-binding proteins, phosphofructokinase and gelsolin.

Phosphofructokinase and gelsolin-like proteins coexist in many muscle and non-muscle tissues. They are both actin-binding proteins, and some of their biochemical parameters are remarkably similar. In a previous report [Füchtbauer, A., Jockusch, B. M., Leberer, E. & Pette, D. (1986) Proc. Natl Acad. Sci. USA 83, 9502-9506] it was shown that phosphofructokinase preparations contained actin-filament-severin activities characteristic for gelsolin. Therefore, we investigated a possible relationship between these proteins with respect to their actin-binding properties. Immunoblotting experiments with specific and non-cross-reacting antibodies to both proteins revealed two distinct polypeptides with slightly different molecular mass in SDS-PAGE of crude extracts from rabbit skeletal muscle, indicating that phosphofructokinase and gelsolin are not identical. An actin-filament-severing activity as well as the component detected by anti-gelsolin were found to copurify with phosphofructokinase during its preparation. However, the presumptive gelsolin was completely eliminated after a heat-denaturation step leaving the phosphofructokinase activity unaffected. Purified phosphofructokinase had no effects on the polymer state of preformed actin filaments. Unlike gelsolin, phosphofructokinase did not promote nucleation of actin polymerization but delayed the nucleation step. We therefore conclude that phosphofructokinase and gelsolin are functionally and structurally distinct proteins.

Actins↗