Shin Lin

Dynamics and segregation of cell–matrix adhesions in cultured fibroblasts

Here we use time-lapse microscopy to analyse cell–matrix adhesions in cells expressing one of two different cytoskeletal proteins, paxillin or tensin, tagged with green fluorescent protein (GFP). Use of GFP–paxillin to analyse focal contacts and GFP–tensin to study fibrillar adhesions reveals that both types of major adhesion are highly dynamic. Small focal contacts often translocate, by extending centripetally and contracting peripherally, at a mean rate of 19 micrometres per hour. Fibrillar adhesions arise from the medial ends of stationary focal contacts, contain α5β1 integrin and tensin but not other focal-contact components, and associate with fibronectin fibrils. Fibrillar adhesions translocate centripetally at a mean rate of 18 micrometres per hour in an actomyosin-dependent manner. We propose a dynamic model for the regulation of cell–matrix adhesions and for transitions between focal contacts and fibrillar adhesions, with the ability of the matrix to deform functioning as a mechanical switch.

Specific interaction of vinculin with α-actinin

Abstract Vinculin and α-actinin are cytoskeletal proteins present at focal contacts of the ventral surface of cultured fibroblasts. We labelled α-actinin with an acceptor fluorophore and vinculin with a donor. A mixture of vinculin and α-actinin showed a 28% quench, due to energy transfer, suggesting an interaction. Quench of vinculin was dependent on the concentration of α-actinin; Scatchard analysis gives a dissociation constant in the μM range. Quench was inhibited by excess unlabelled α-actinin, and by reaction of the acceptor protein with p-chloromercuribenzoate. We found that vinculin had a slightly greater elution volume in a gel filtration column equilibrated with α-actinin, indicating a higher effective Stokes radius due to the interaction of the two proteins.

High-affinity interaction of vinculin with actin filaments in vitro

Immunofluorescence and microinjection experiments have shown that vinculin (molecular weight 130,000) is localized at adhesion plaques of fibroblasts spread on a solid substrate. We found that this protein affects actin filament assembly and interactions in vitro at substoichiometric levels. Vinculin inhibits the rate of actin polymerization under conditions that limit nuclei formation, indicating an effect on the filament elongation step of the reaction. Vinculin also reduces actin filament--filament interactions measured with a low-shear viscometer. Scatchard plot analysis of the binding of 3H-labeled vinculin to actin filaments showed that there is one high-affinity binding site (dissociation constant=20 nM) for every 1,500-2,000 actin monomers. These results suggested that vinculin interacts with a specific site located at the growing ends of actin filaments in a cytochalasin-like manner, a property consistent with its proposed function as a linkage protein between filaments and the plasma membranes.

Detection of high molecular weight vinculin binding proteins in muscle and nonmuscle tissues with an electroblot-overlay technique

In this study, we examined binding of radiolabelled vinculin to proteins separated on sodium dodecyl sulfate-polyacrylamide gels, and then electrophoretically transferred onto nitrocellulose sheets. We detected saturable binding of vinculin to polypeptides with apparent Mrs of 215,000, 205,000 and 185,000 in a low ionic strength extract from chicken gizzard membranes. Binding of vinculin to proteins with apparent Mrs of 205,000, 185,000, and 165,000 in human platelets was also detected. In addition, we found that [125I]vinculin binds to unlabelled vinculin and to alpha-actinin, although these interactions appear to be of lower affinity than those with the higher molecular weight proteins.

A platelet inhibitor protein with cytochalasin-like activity against actin polymerization in vitro

We have obtained an inhibitor fraction containing cytochalasin-like activity from human platelets. Using a procedure involving DEAE-cellulose, hydroxyapatite and gel filtration column chromatography, we obtained a fraction from human platelets which apparently can compete with 3H--cytochalasin B for binding to spectrin-actin complexes from human erythrocytes. The inhibitor activity is nondialyzable, sensitive to heat and to trypsin and has a Stokes radius of 40 A. This fraction stops nuclei-induced actin polymerization in 0.4 mM MgCl2 and reduces the viscosity of F actin to that of G actin, which suggests depolymerization of the filaments. These results suggest that the inhibitor fraction contains a protein which interacts with actin filaments and nuclei in a manner similar to that of cytochalasin B. It is possible that such a protein is involved in the control of cell motility by affecting assembly and disassembly of actin-containing microfilaments in vivo.

Reversal of profilin inhibition of actin polymerization invitro by erythrocyte cytochalasin-binding complexes and cross-linked actin nuclei

Abstract Actin polymerization in 2 mM MgCl 2 is known to be inhibited by profilin. We found that small amounts of cytochalasin-binding complexes from human red cell membranes or actin nuclei cross-linked by p - NN′ -phenylenebismaleimide can reverse the inhibitory action of profilin, leading to the rapid polymerization of the actin. This type of polymerization is inhibited by low concentrations of cytochalasin B. These results indicate that (a) the complexes and nuclei promote actin polymerization in the presence of profilin by providing sites onto which actin monomers can be added, and (b) profilin and cytochalasin B affect two distinct steps (i.e. nucleus formation and filament elongation, respectively) in the polymerization reaction.

Opiate receptors in highly purified neuronal cell populations isolated in bulk from embryonic chick brain

A bulk isolation procedure was used to obtain neuronal and non-neuronal cell populations from embryonic chick brain. The procedure utilizes the differences in cell-substrate adhesiveness of the two populations and the ability of the former to form homotypic aggregates in the presence of intermittent mechanical agitation. [3H]-Thymidine incorporation, semi-quantification of S-100 protein, and recycling of radioactively labelled putative non-neuronal cells through the isolation procedure indicate that the neuronal fraction contained less than 1% of non-neuronal cells. The binding of [3H]-naloxone to the isolated neuronal cells was characterized. These cells were found to have opiate receptors which are very similar to those found in adult rat brain with respect to stereospecificity of [3H]-naloxone binding, the ability of several opiate agonists and antagonists of varying analgesic activity to displace bound [3H]-naloxone, and the differential response of these drugs to Na+. Isolated non-neuronal cells, in contrast, had no specific opiate binding activity. Total binding activity of both populations of cells was greatly increased after 24 hr in culture. However, while a major portion of the activity of the neuronal cells was of the specific type, all of the activity in the non-neuronal cells remained nonspecific.

A Forty-Five Year Follow-Up EEG Study of Qigong Practice

A follow-up EEG study was conducted on a subject with 50 years of experiences in Qigong. Resting EEG at present showed frontally dominant alpha-1 as compared to occipitally dominant alpha-2 described in 1962. During the Qigong practice alph-1 enhanced quickly and became far more prominent than 50 years ago. Compared with baseline, these activities remained to be higher at rest after the Qigong practice. These results suggest that extended practice in meditation may change the EEG pattern and its underlying neurophysiology. It remains to be explored as to what biological significance and clinical relevance do these physiological changes might mean.

A rapid assay for actin-associated high-affinity cytochalasin binding sites based on isoelectric precipitation of soluble protein.

Abstract When protein from a red cell ghost extract precipitates in the presence of [3H]dihydrocytochalasin B (a drug which inhibits cell motility in many eucaryotic cells), it carries along with it a saturable amount of the labeled drug. This phenomenon, which probably reflects the isoelectric precipitation of the cytochalasin: receptor complex, occurs at around pH 5, is independent of the type of acid used to adjust the pH, and is inhibited by high salt concentrations and by pretreatment of extract protein with chymotrypsin. A simple, rapid, and quantitative assay based on the isoelectric precipitation reaction has been devised. This assay was used to demonstrate the presence of actin-associated high-affinity binding sites for dihydrocytochalasin B in extracts of a variety of eucaryotic cells.

Purification and characterization of an inhibitor protein with cytochalasin-like acitvity from bovine adrenal medulla

Abstract A protein preparation with cytochalasin-like activity has been obtained from bovine adrenal medulla. Analysis by electrophoresis in SDS-polyacrylamide gels and chromatography in a Sephacryl S-200 column indicated that the inhibitor activity coincided with a 90 000 dalton polypeptide. The inhibitor decreased high-affinity binding of [3H]cytochalasin B to actin nuclei, apparently by competing with the drug for thesame binding site. At substoichometric levels, the inhibitor had a potent effect on actin filament elongation and on actin-dependent gelation of cell extracts in vitro. These results suggest that the inhibitor may be involved in the control of actin filament assembly and interaction in the adrenal medulla.