Effects of Exogenous Proteins on Cytoplasmic Streaming

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 fluoresceinHMM (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 ofmuscle have been firmly established . Although the presence of actin and myosin has been demonstrated in a wide variety ofboth 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 THE JOURNAL OF CELL BIOLOGY " VOLUME 93 JUNE 1982 735-742 © The Rockefeller University Press " 0021-9525/82/06/0735/08 $1 .00 organized, rapid cytoplasmic streaming and, at the same time, can be subjected to a variety of mechanical and biochemical treatments without immediate stoppage of streaming . It is widely assumed that actin and myosin function together to produce streaming in these cells, although only limited direct evidence is available to support this assumption (3). The presence of actin-containing bundles attached to the chloroplast files at the interface between the stationary ectoplasm and moving endoplasm has been firmly established (4-7). The fact that these subcortical actin bundles are unidirectional (8) and are located at the site thought to be the seat of motive force (9) support the suggestion of an actin-myosin-based streaming mechanism . This hypothesis has been further supported by the observation of submicron-sized cytoplasmic particles which exhibit ATP-dependent (10) attachment to and motion along these bundles (11) . Motile filaments long observed in cellfragment preparations (12) have now been shown to contain actin and are probably fragments of the subcortical bundles (13) . Biochemical extraction has verified the presence of a 735 on A ril 0, 2017 D ow nladed fom Published June 1, 1982