Structure of the dynein-1 outer arm in sea urchin sperm flagella. I. Analysis by separation of subunits.

Our current thinking about the driving force for the movements of intracellular particles or chromosomes has been dominated by our knowledge ofthe two most common contractile systems, those based upon actin-myosin interactions or upon microtubule-dynein interactions. Abundant quantities of material in well-ordered arrays have allowed considerable progress to be made in the investigation of the mechanisms responsible for movement in these systems . There are many examples of the involvement of cytoplasmic actin and myosin in nonmuscle cell motility (1, 2). Evidence in favor ofa cytoplasmic dynein in microtubule-dependent motility is beginning to accumulate, and recent work from several laboratories has provided preliminary evidence that a dyneinlike ATPase is present in the mitotic spindle of sea urchin eggs (3-5). In addressing the question of a cytoplasmic dyneinlike ATPase, it is primarily important to establish a general definition ofdynein and to characterize those features that distinguish it from myosin and other ATPases. Dynein has been loosely defined as an ATPase that associates with microtubules and has high molecular weight polypeptides in excess of 300 kdaltons (6) . A more meaningful definition includes its direct role in generating a force for movement coupled to the hydrolysis of ATP, which must be approached in terms of the biology as well as the biochemistry of the system . In this review we will summarize the important biochemical properties of ciliary dynein in terms of a comparison with myosin and relate the results to the potentially distinguishing features of a cytoplasmic dynein .

Section: Structure Of Dynein: a Unified Viewmentioning

confidence: 86%

Section: Dynein Polypeptide Compositionmentioning

confidence: 99%

Mechanism of force production for microtubule-dependent movements.

Johnson¹,

Porter²,

Shimizu³

1984

“…For gel electrophoresis, specimens were prepared in the sample buffer described in Tang et al, (47). High molecular weight polypeptides were separated by SDS-PAGE in a 3-6% linear acrylamide gradient and with a linear O-8-M urea gradient as described in Piperno and Luck (33).…”

Section: Miscellaneous Proceduresmentioning

confidence: 99%

Direction of force generated by the inner row of dynein arms on flagellar microtubules.

Fox

Sale

1987

Abstract. Our goal was to determine the direction of force generation of the inner dynein arms in flageUar axonemes. We developed an efficient means of extracting the outer row of dynein arms in demembranated sperm tail axonemes, leaving the inner row of dynein arms structurally and functionally intact. Sperm tail axonemes depleted of outer arms beat at half the beat frequency of sperm tails with intact arms over a wide range of ATP concentrations. The isolated, outer armdepleted axonemes were induced to undergo microtubule sliding in the presence of ATP and trypsin. Electron microscopic analysis of the relative direction of microtubule sliding (see Sale, W. S. and P. Satir, 1977, Proc. Natl. A_cad. Sci. USA, 74:2045USA, 74: -2049 revealed that the doublet microtubule with the row of inner dynein arms, doublet N, always moved by sliding toward the proximal end of the axoneme relative to doublet N+I. Therefore, the inner arms generate force such that doublet N pushes doublet N+I tipward. This is the same direction of microtubule sliding induced by ATP and trypsin in axonemes having both inner and outer dynein arms. The implications of this result for the mechanism of ciliary bending and utility in functional definition of cytoplasmic dyneins are discussed.

“…Pfister et al (7,8) demonstrated that all three HCs in Chlamydomonas 18S and 12S dynein have ATP-binding sites. Sea urchin 21S dynein has been shown to have two ATPases (10,13). ,…”

Section: Multiple Atpase Sites On Tetrahymena 22s Dyneinmentioning

confidence: 99%

Chymotryptic digestion of Tetrahymena ciliary dynein. II. Pathway of the degradation of 22S dynein heavy chains.

Toyoshima¹

1987

Abstract. As shown in the preceding paper (Toyoshima, Y. Y., 1987, J. Cell Biol., 105:887-895) threeheaded Tetrahymena 22S dynein consists of three heavy chains (HCs) and is decomposed into twoheaded (H) and one-headed (L) fragments by chymotryptic digestion. To accurately determine the presence of multiple ATPases and ultimately the location of various domains, it is necessary to determine the identity of each HC fragment relative to the original HCs in 22S dynein. The degradation pathway of each HC was determined by peptide mapping and immunoblotting.The three HCs (As, Aa, and A0 were inununologically different; although SDS-urea gel electrophoresis showed that Ay HC was apparently resistant to the digestion, actually three distinct HCs contributed to the same band alternately. H fragment was derived from Aa and Av HCs, whereas L fragment originated from A, HC. Since both fragments were associated with ATPase activity, these results directly demonstrate the presence of multiple ATPase sites in Tetrahymena 22S dynein. FROM the recent electron microscopic studies, it has been established that Tetrahymena ciliary 22S dynein consists of three globular heads and thin stalks (2, 3, 12). SDS-urea gel electrophoresis showed that three heavy chains (HCs) 1 are present in the 22S dynein preparation (12). To study the functions of these HCs and their relationships to the heads, we must decompose the molecule into functional units. As shown in the preceding paper, chymotryptic digestion splits the molecule into two portions, both associated with ATPase activity: one consisting of two globular heads linked by stalks (H fragment), and the other consisting of a single head (L fragment). This does not necessarily mean that 22S dynein has multiple ATPase sites, since the L fragment might come from the H fragment. To examine this possibility, we must determine the degradation pathway of the HCs.In this work, I determined the pathway by peptide mapping and immunoblotting. The HC constituting L fragment originated from A,-HC and was not a digestion product of H fragment. Thus the presence of multiple ATPase sites is demonstrated directly. Further, these results indicate that the three HCs are different species and strongly suggest that one 22S dynein molecule consists of three distinct HCs, each of which would correspond to one head.