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W H Goldmann

Publications and source records attributed to W H Goldmann.

At least 37 records · Page 2Linked to original sources
PubMed1996

Viscoelasticity in wild-type and vinculin-deficient (5.51) mouse F9 embryonic carcinoma cells examined by atomic force microscopy and rheology.

We have been studying mouse F9 embryonic carcinoma cells which contain no detectable vinculin protein (5.51 cells), and compared them with F9 wild-type cells. Employing atomic force microscopy, we probed the elastic properties of individual F9 wild-type and 5.51 cells by measuring the dynamic response of controlled loads of the cantilever tip. An elastic modulus (Young) of approximately 3.8 and approximately 2.5 kPa was calculated for wild-type and 5.51 cells, respectively. Using disc rheometry, we detected a marked change in shear of a 1000g pellet of approximately 55 x 10(6) cells between wild-type and 5.51 mutants. These differences are attributed to the loss of vinculin and altered cytoskeletal organization in these cells.

Animals↗
PubMed1995

Determination of the affinity of talin and vinculin to charged lipid vesicles: a light scatter study.

Recent experimental findings have demonstrated that both talin and vinculin bind to phospholipids and insert into the hydrophobic region of lipid membranes. Here, we show that the light scatter method can be used for measuring the affinity of proteins to phospholipid membranes. Large unilamellar DMPC/DMPG vesicles were produced by the extrusion technique (LUVETs). We have used repeated heating/cooling scans between 15 degrees C and 35 degrees C to ensure protein-lipid interaction/insertion. A molar affinity of talin, K = 2.9 x 10(6) M-1 and of vinculin, K = 3.3 x 10(5) M-1 to lipid vesicles, respectively, was determined from the plot; light scatter signal at 380 nm against protein concentrations by fitting the term, ln (Io/I-1) = A-K x c to the data.

Dimyristoylphosphatidylcholine↗
PubMed1995

Motility of vinculin-deficient F9 embryonic carcinoma cells analyzed by video, laser confocal, and reflection interference contrast microscopy.

We have studied the motility of wild-type F9 and vinculin-deficient (5.51) mouse embryonal carcinoma cells. F9 cells extended filopodia at a rate of 61 ( +/- 18) nm/s over a distance of 3.18 (+/- 0.29) microns. In contrast, 5.51 cells exhibited filopodia which extended at a similar speed of 57 (+/- 17) nm/s but over a longer distance of 5.10 (+/- 2.14) microns. Cell-substratum contact areas of both cell types were examined by reflection interference contrast microscopy. Wild-type F9 cells had distinct close contacts (dark gray areas) at the cell periphery, whereas 5.51 cells had only a few light gray pinpoint contacts with the substrate. Confocal microscopy showed alpha-actinin to be localized along actin stress fibers in wild-type cells, and in 5.51 cells stress fibers were absent and alpha-actinin was associated with F-actin in the filopodia. beta 1-integrin, talin, and paxillin were concentrated in focal contacts in wild-type cells, but in 5.51 cells beta 1-integrin and talin were in patches under the plasma membrane and paxillin was diffusely distributed in the cytoplasm. We conclude that changes in cell shape and motility of 5.51 compared to wild-type F9 cells are due to the absence of vinculin even though there may be functions of other focal adhesion complex proteins, e.g., talin, linking the actin cytoskeleton to the plasma membrane.

Actinin↗
PubMed1995

Interaction of the 47-kDa talin fragment and the 32-kDa vinculin fragment with acidic phospholipids: a computer analysis.

In recent in vitro experiments, it has been demonstrated that the 47-kDa fragment of the talin molecule and the 32-kDa fragment of the vinculin molecule interact with acidic phospholipids. By using a computer analysis method, we determined the hydrophobic and amphipathic stretches of these fragments and, by applying a purpose-written matrix method, we ascertained the molecular amphipathic structure of alpha-helices. Calculations for the 47-kDa mouse talin fragment (residues 1-433; NH2-terminal region) suggest specific interactions of residues 21-39, 287-342, and 385-406 with acidic phospholipids and a general lipid-binding domain for mouse talin (primary amino acid sequence 385-401) and for Dictyostelium talin (primary amino acid sequence 348-364). Calculations for the 32-kDa chicken embryo vinculin fragment (residues 858-1066; COOH-terminal region) and from nematode vinculin alignment indicate for chicken embryo vinculin residues 935-978 and 1020-1040 interactions with acidic phospholipids. Experimental confirmation has been given for vinculin (residues 916-970), and future detailed experimental analyses are now needed to support the remaining computational data.

Amino Acid Sequence↗
PubMed1995

The influence of cations and ionic strength on actin polymerization in the presence/absence of alpha-actinin.

The presence of alpha-actinin has little influence on polymerizing actin with regards to the lag phase, rate and amplitude. Rising KCl or MgCl2 concentrations parallel an increase in polymerization rate and a decrease in lag phase for both protein species. The observed overall smaller actin fluorescence amplitudes in the presence of alpha-actinin is due to a higher ratio of free G-actin to F-actin at steady state.

Actinin↗
PubMed1994

Insertion of filamin into lipid membranes examined by calorimetry, the film balance technique, and lipid photolabeling.

The interaction of the actin-binding protein filamin with mixtures of zwitterionic and anionic phospholipids (DMPC, DMPG, PC, PS) was studied in reconstituted lipid monolayers and bilayers. Protein-lipid interactions were investigated by differential scanning calorimetry, the film balance technique, and hydrophobic photoradiolabeling. For calorimetric assays, multilamellar vesicles (MLVs) and large unilamellar vesicles produced by the extrusion technique (LUVETs) were used. With MLVs, filamin induced a pronounced drop in phase transition cooperativity. Mixed DMPC/DMPG LUVETs showed a linear decrease of the main phase transition enthalpy and a significant shift in temperature for the solidus and liquidus lines with increasing mole fractions of reconstituted filamin. The insertion of native filamin into uncharged and negatively charged lipid monolayers was measured in time/area diagrams with the film balance technique. Finally, we have newly synthesized a highly sensitive lipid analogue, [125]TID-PC/16, which selectively labels membrane-embedded hydrophobic domains of proteins, and which proved to label filamin, supporting evidence that this protein partially inserts into the hydrophobic domain of liposomes.

Calorimetry, Differential Scanning↗
PubMed1994

Identification of functional domains in the cytoskeletal protein talin.

The cytoskeletal protein talin potentially plays a key role in actin-membrane linkage. It is able to nucleate actin filament growth in vitro while binding simultaneously to lipid bilayers. Thrombin digestion of human platelet talin yields tow polypeptide domains of 200 kDa and 47 kDa. We have purified these fragments and analyzed their functional properties: the 200-kDa fragment was active in nucleating actin filament formation and reduced the viscosity of filamentous actin, comparable to the effects of the intact protein. The 47-kDa fragment was inactive in this respect. However, the 47-kDa polypeptide, but not the 200-kDa fragment, interacted specifically with large liposomes containing acidic phospholipids. This is demonstrated by selective, hydrophobic photolabeling of the 47-kDa fragment using phosphatidylserine liposomes containing trace amounts of a photoactivable phospholipid analogue and by selective co-sedimentation of this domain with the liposomes. The 200-kDa fragment, whether alone or in conjunction with the small fragment, neither incorporated significant amounts of label nor co-sedimented with the liposomes. We thus are able to attribute specialized functions to distinct domains on the talin molecule. These enable the protein to interact simultaneously with actin filaments and lipid membranes.

Actins↗
PubMed1994

Analysis of filamin-actin binding and cross-linking/bundling by kinetic method.

The reaction of smooth muscle filamin and skeletal muscle actin was kinetically examined by double exponential analysis. The overall rate of binding, k+1, is concentration and temperature dependent whilst the overall rate of cross-linking/bundling, k+2, is concentration independent. The activation energy, Ea = 99.5 kJ/mol, was calculated from the Arrhenius Plot.

Actins↗
PubMed1994

Computer analyses suggest interactions of non-muscle filamin with lipid membranes.

It is concluded from structure predictions of the primary amino acid sequence by computer analyses that two segments of non-muscle filamin could facilitate lipid membrane attachment or anchoring. Residues 49-71 of the amino-terminal may attach to phospholipid membranes, and residues 131-155 may anchor in the hydrophobic region of lipid membranes.

Contractile Proteins↗
PubMed1993

Analysis of filamin and alpha-actinin binding to actin by the stopped flow method.

We ascertained by the stopped flow method the overall association rate constant, k+1, of filamin and alpha-actinin to fluorescently labelled filamentous actin of approximately 1.3 x 10(6) M-1.s-1 and approximately 1.0 x 10(6) M-1.s-1 as well as the overall dissociation rate constant, k-1, of approximately 0.6 s-1 and approximately 0.4 s-1, respectively. The overall equilibrium constant, K, for filamin and alpha-actinin to actin deduced from the relation K = k+1/k-1 agree well with published data.

Actinin↗
PubMed1993

Interaction of NBD-talin with lipid monolayers. A film balance study.

Fluorescently labelled smooth muscle talin like native talin interacts with negatively or partly negatively charged lipid monolayers. This was measured in time/area diagrams using the film balance technique combined with fluorescence imaging after double photolabelling of talin and phospholipids.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

The viscoelasticity of entangled actin networks: the influence of defects and modulation by talin and vinculin.

Rheological measurements of the frequency-dependent complex elastic module G*(omega) of entangled F-actin solutions in the frequency range 10(-5)-1 Hz were carried out in three dynamic regimens: 1.) A terminal relaxation from gel-like to liquid-like behaviour measured at frequencies omega < or = tau d-1, 2.) a rubber-type plateau and 3.) a regime determined by chain conformational transitions at frequencies omega > tau i-1. A major point of interest was to clarify whether rheological, high precision measurements can yield quantitative information about the influence of talin and vinculin on the structure, chain dynamics, elasticity and viscoelasticity of actin filaments with time. We show that in the regime reflecting internal chain dynamics (10(-2) to 1 s time domain), F-actin behaves as a random coil of the Rouse type. This contrasts with dynamic light scattering and correlation spectroscopic studies of actin filament flickering, which indicate that filaments behave as semiflexible rods. The internal chain dynamics, which are determined by thermically excited bending undulations, exhibit a persistence length of 0.3-1 microns. Evidence is provided that this discrepancy is due to a cross-over of semiflexible rod behaviour at excitation wavelengths (lambda) below approximately 1 micron to random-coil behaviour at lambda >> 1 micron (expected at a frequency omega approximately 1 Hz). The random coil behaviour is largely determined by defects in actin filaments leading to sharp bends of the chain which act as semiflexible hinges. Talin produces drastic effects on the time course of viscoelasticity during actin polymerization. It promotes the rapid formation of short filament fragments (approximately 1 micron, within time scale of min) which anneal slowly into long filaments (within several hours), most probably by fusion. The viscoelasticity depends on the coexistence of short and very long filaments indicated by the elongation of the rubber plateau. The most dramatic effect is a reduction of the ratio of the terminal (tau d) to the Rouse relaxation time of tau i by more than one order of magnitude (tau d/tau i = 100 compared to ratio tau d/tau i = 2000 for pure actin). From this it is concluded that talin causes a remarkable decrease in the effective segment length of the macromolecule and, thus induces an increase in chain stiffness. Vinculin on the other hand shows no such effect.

Actins↗