Affinity chromatography of immobilized actin and myosin

Bottomley, Robin C.; Trayer, Ian P.

doi:10.1042/bj1490365

Cited by 39 publications

(24 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The force-generating interaction between the presumptive endoplasmic myosin and the subcortical actin bundles may be competitively inhibited by this same released actin. Interaction between the presumptive endoplasmic myosin and free actin monomers (9) and/or oligomers could not itself generate the organized, directed force required to drive streaming. This mechanism inhibits streaming without gross disruption of the subcortical actin bundles .…”

Section: Discussionmentioning

confidence: 99%

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara.

Nothnagel¹,

Barak²,

Sanger³

et al. 1981

The Journal of Cell Biology

View full text Add to dashboard Cite

Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin . To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB . Use of two fluorescent actin probes, fluorescein isothiocyanateheavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phalIacid in (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB . Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment . NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions .The highly organized cytoplasmic streaming exhibited by the giant internodal cells of characean algae has long made these cells an attractive system for the study of cell motility (4) . Accumulated evidence has suggested that motility in a variety of eukaryotic cells is actomyosin-dependent (21) . Measurements of streaming-velocity profiles in characean cells have suggested that the motive force is generated primarily at the interface between the stationary ectoplasm and motile endoplasm (17) . Early ultrastructural studies have revealed the presence of microfilament bundles attached to the chloroplasts at this interface (26) . Subsequent electron and, more recently, fluorescence microscope studies using labeling by heavy meromyosin (20, 27, 29) or antiactin antibodies (45) have demonstrated the presence of actin in these subcortical microfilament bundles and established the unidirectional orientation of the actin filaments (19) . Other light microscope studies have suggested the presence of endoplasmic filaments that appeared to branch from the subcortical actin bundles and participate in the generation of the motive force (1, 2) .The demonstration of actin in characean cells has encouraged workers to search for evidence of myosin in these cells as well . Using a perfusion technique that permitted manipulation of internal pH and ion and ATP concentrations, Williamson observed the ATP-dependent moti...

show abstract

Section: Discussionmentioning

confidence: 99%

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara.

Nothnagel¹,

Barak²,

Sanger³

et al. 1981

The Journal of Cell Biology

View full text Add to dashboard Cite

show abstract

“…Affinity Chromatography-Synthetic peptides were coupled to Sepharose 4B by the cyanogen bromide procedure as described previously (39,40). The affinity columns were equilibrated with 50 mM Tris buffer, pH 7.5.…”

Section: Methodsmentioning

confidence: 99%

The Identification of a Second Cofilin Binding Site on Actin Suggests a Novel, Intercalated Arrangement of F-actin Binding

Renoult

Ternent²,

Maciver³

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

The cofilins are members of a protein family that binds monomeric and filamentous actin, severs actin filaments, and increases monomer off-rate from the pointed end. Here, we characterize the cofilin-actin interface. We confirm earlier work suggesting the importance of the lower region of subdomain 1 encompassing the N and C termini (site 1) in cofilin binding. In addition, we report the discovery of a new cofilin binding site (site 2) from residues 112-125 that form a helix toward the upper, rear surface of subdomain 1 in the standard actin orientation (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347, 37-44). We propose that cofilin binds "behind" one monomer and "in front" of the other longitudinally associated monomer, accounting for the fact that cofilin alters the twist in the actin (McGough, A., Pope, B., Chiu, W., and Weeds, A. (1997) J. Cell Biol. 138, 771-781). The characterization of the cofilin-actin interface will facilitate an understanding of how cofilin severs and depolymerizes filaments and may shed light on the mechanism of the gelsolin family because they share a similar fold with the cofilins (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagiki, F. (1996) Cell 85, 1047-1055).Many motile processes in cells require cyclic polymerization and depolymerization of actin filaments. In cell locomotion for example, actin is polymerized at the leading edge of the cell and is recycled by depolymerizing toward the cell center. The rate constants of pure actin have been established (1), and it is clear that a discrepancy exists between these known rates and those calculated from filament turnover in cells (2). A host of actinbinding proteins are known that dramatically alter the behavior of actin in vitro, and of these, the cofilins have been suggested to have the correct properties to increase filament turnover in cells (3). This view has been confirmed by studies with living Saccharomyces (4) and Dictyostelium (5) and by Listeria motility assays (6, 7).The cofilins are a group of low molecular mass (15-21 kDa), actin-binding proteins that depolymerize actin filaments (8).This group includes vertebrate cofilin (9) and ADF 1 (10), twinstar from Drosophila (11), depactin from echinoderms (12), ADFs from plants (13), Unc-60 from nematode (14), cofilins from Saccharomyces (15, 16) and Dictyostelium (17), and actophorin from Acanthamoeba castellanii (18).The mechanism by which cofilin depolymerizes actin filament has been contentious. Soon after the discovery of the first member of the family (10), several authors suggested that depolymerization occurred through severing (9, 18). Evidence for a severing mechanism later came from videomicroscopy (19,20), but this was later challenged (6) as it was shown (6, 21) that cofilins increased the off-rate from the pointed end of the filament. However, the two opinions are not necessarily exclusive (22-24), and a similar mechanism has been proposed for both events (23). We report the identification of a cof...

show abstract

“…Each immunoglobulin fraction was further purified by affinity chromatography with homologous antigen (22). Immunofluorescent staining was performed as described (20).…”

Section: Methodsmentioning

confidence: 99%

Appearance of cytoskeletal components on the surface of leukemia cells and of lymphocytes transformed by mitogens and Epstein-Barr virus.

Bachvaroff¹,

Miller²,

Rapaport³

1980

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Lactoperoxidase iodination and two-dimensional electrophoresis of the labeled proteins have demonstrated well-characterized cytoskeletal proteins (actin and tubulins) on the surface of human lymphocytes undergoing blastogenic transformation and of certain malignant human cells. Such proteins could not be detected on the surface of normal resting human lymphocytes. The most prominent cytoskeletal rotein identified on the surface membrane of mitogen-transformed T and B lymphocytes was actin. In Epstein-Barr virus genome-positive Burkitt's lymphoma and lymphoblastoid cell lines and in two leukemia cells, the major iodinated membrane protein components were actin and a,-, a-, and P-tubulins.These proteins were firmly connected to the cytoplasmic skeleton and could not be removed by Triton X-100. Concurrent immunofluorescence studies with specific antibodies and FRab')2 fragments confirmed the appearance of cytoskeletal components on the surface of live and fixed lymphocytes, in parallel with the biochemical data, and indicated that such cytoskeletal proteins formed distinctive patterns on the cell surface, ranging from small patches to large projections. Five-hour labeling with [asS]methionine indicates that all such cells released large quantities of labeled actin and tubulins into the culture medium. These materials were not readsorbed to the membrane surfaces of the cells.The cytoskeletal framework, composed principally of microfilaments and microtubules, is normally confined to the interior of the cell and to the inner surface of the cell membrane (1-4). Earlier studies have documented marked increases in relative rates of synthesis of major skeletal proteins (actin and tubulin) in polyclonally activated normal human T and B lymphocytes, as well as in Epstein-Barr virus (EBV) genome-positive lines and in three leukemia cell lines (5). Such cells also release large quantities of labeled actin and tubulin into the culture medium, with no evidence of a loss of cell viability (5). In view of recent reports of extensive shedding of membrane materials and submembrane anchorage components by mouse mastocytoma cells (6), an attempt has been made to ascertain whether a similar mechanism was operative in the transformed human lymphocytes studied in this laboratory. The results indicate that blastogenic stimulation of normal lymphocytes or malignant transformation of such cells (or both) is associated with the appearance of large concentrations of cytoskeletal proteins on the cell membrane surface. This observation is in marked contrast to the absence of such components on the surface of normal human lymphocytes. MATERIALS AND METHODSEnriched populations of T and B lymphocytes from healthy volunteers were prepared from defibrinated blood (7) and were stimulated in vitro with concanavalin A (Con A) (T cells) or pokeweed mitogen (PWM) (B cells) as described (8). EBV genome-positive cells used in the present study were: (i) Burkitt's lymphoma lines Daudi, Maku, and Solubo; (iH) AW-Ramos and EHRA-Ramos, lines that had...

show abstract

Affinity chromatography of immobilized actin and myosin

Cited by 39 publications

References 46 publications

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara.

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara.

The Identification of a Second Cofilin Binding Site on Actin Suggests a Novel, Intercalated Arrangement of F-actin Binding

Appearance of cytoskeletal components on the surface of leukemia cells and of lymphocytes transformed by mitogens and Epstein-Barr virus.

Contact Info

Product

Resources

About