Effects of exogenous proteins on cytoplasmic streaming in perfused Chara cells.

Abstract

Cytoplasmic streaming in characean algae is thought to be generated by interaction between subcortical actin bundles and endoplasmic myosin . Most of the existing evidence supporting this hypothesis is of a structural rather than functional nature . To obtain evidence bearing on the possible function of actin and myosin in streaming, we used perfusion techniques to introduce a number of contractile and related proteins into the cytoplasm of streaming Chara cells. Exogenous actin added at concentrations as low as 0.1 mg/ml is a potent inhibitor of streaming. Deoxyribonuclease I (DNase I), an inhibitor of amoeboid movement and fast axonal transport, does not inhibit streaming in Chara . Fluorescein-DNase I stains stress cables and microfilaments in mammalian cells but does not bind to Chara actin bundles, thus suggesting that the lack of effect on streaming is due to a surprising lack of DNase I affinity for Chara actin bundles. Heavy meromyosin (HMM) does not inhibit streaming, but fluorescein-HMM (FL-HMM), having a partially disabled EDTA ATPase, does . Quantitative fluorescence micrography provides evidence that inhibition of streaming by FL-HMM may be due to a tendency for FL-HMM to remain bound to Chara actin bundles even in the presence of MgATP. Perfusion with various control proteins, including tubulin, ovalbumin, bovine serum albumin, and irrelevant antibodies, does not inhibit streaming . These results support the hypothesis that actin and myosin function to generate cytoplasmic streaming in Chara .The central roles played by actin and myosin in the contraction of muscle have been firmly established . Although the presence of actin and myosin has been demonstrated in a wide variety of both plant and animal cells, the active involvement of these proteins in the generation of nonmuscle cell motility has not been demonstrated directly (1) . Recent experiments have shown clearly that actin-myosin-containing stress fibers of fibroblastic cells are capable of contraction (2), yet even these observations do not establish that such contractions are essential for cell motility. It has been suggested that conclusive evidence for the involvement of actin and myosin in nonmuscle cell motility might come from experiments with cells having genetically defective actin or myosin (1) . Until such mutants have been identified and characterized for a variety of organisms, less direct experiments can continue to provide valuable evidence on the question of actin-myosin involvement in nonmuscle cell motility. Use of cells that are both highly motile and readily manipulable facilitates such experiments .The giant intemodal cells of characean algae exhibit highly

ATP-dependent movement of myosin in vitro: characterization of a quantitative assay.

“…Another weakness of the assay is that Nitella actin could differ in important ways from other actins. For example, Nothnagel et al (20) suggest that Nitella actin does not bind DNase I. However, others (Vale, R., A. G. Szent-Gy6rgyi, and M. P. Sheetz, manuscript submitted for publication) have been able to confer calcium dependence on 1870 THE JOURNAL OF CELL BIOLOGY • VOLUME 99, 1984 skeletal myosin movement by adding the tropomyosin-troponin complex from rabbit skeletal muscle to the Nitella actin cables.…”

Hydrodynamic models of viscous coupling between motile myosin and endoplasm in characean algae.

“…The hydrodynamics presented will be modeled for the geometry of cytoplasmic streaming in characean algae. Various microscopic, ultrastructural, and biochemical observations (2)(3)(4)(5) have provided evi-dence that this streaming is driven by actin-myosin. Although other mechanisms have been proposed (6), most available evidence (3) suggests that characean streaming is driven by interaction between motile endoplasmic myosin and stationary subcortical actin bundles (Fig.…”