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Monoclonal antibodies that recognize individual polypeptides of the outer arm dyneins of Chlamydomonas flagella were obtained and used to study the structural relationships between the various polypeptides. Immunoblot analysis showed that the y heavy chain of 12S dynein and the a and /3 heavy chains and M, 69 The dynein heavy chains migrate very close to one another in NaDodSO4/polyacrylamide gels and are well resolved only at very low protein loadings (2). Consequently, characterization of our antibodies by immunoblot analysis required (i) a reliable procedure for efficiently transferring high molecular weight polypeptides from NaDodSO4/polyacrylamide gels to nitrocellulose sheets without loss of resolution and (i) a sensitive method for detecting the transferred proteins on the nitrocellulose. The techniques that we have developed to fulfill these requirements were essential for unambiguous identification of the chains recognized by our antibodies and should be useful to a wide range of investigators needing to electrotransfer high molecular weight proteins or to detect nanogram amounts of protein on nitrocellulose. MATERIALS AND METHODSPreparation of Axonemes and Purification of Dynein. The isolation of flagella from Chlamydomonas reinhardtii (wildtype strain 1132D-) by the dibucaine method, preparation of axonemes, extraction with 0.6 M KCl, and subsequent purification of 12S and 18S dyneins by sucrose gradient centrifugation and hydroxylapatite column chromatography were as described (2, 4).Ram sperm tails were prepared from ejaculated sperm by modifications of methods described by Tash and Means (6) and Harrison (7). A pellet containing sperm tails and midpieces was treated with 0.05% Nonidet P-40 (BDH) in 30 mM Hepes, pH 7.5/5 mM MgSO4/1 mM dithiothreitol/0.5 mM EDTA/25 mM KCl/1 mM phenylmethylsulfonyl fluoride to remove the membranes. The demembranated axonemes were then collected by centrifugation at 30,500 x g and resuspended in electrophoresis sample buffer.Axonemes and latent-activity dynein-1 (LAD-1) from T. gratilla were the gift of

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Two distinctly different ATPases have been reported to be endogenous to the mitotic apparatus: a Mg 2+-ATPase resembling axonemal dynein, and a Ca t+ -ATPase postulated to be bound in membranes. To examine the nature of the Mgt+ -ATPase, we isolated membranefree mitotic spindles from Strongylocentrotus droebachiensis embryos by rapidly lysing them in a calcium-chelating, low-ionic-strength buffer (5 mM EGTA, 0.5 MM M9Cl 2, 10 mM PIPES, pH 6.8) that contained 1% Nonidet P-40. The fibrous isolated mitotic spindles closely resembled spindles in living cells, both in general morphology and in birefringence. In electron micrographs, the spindles were composed primarily of microtubules, free from membranes and highly extracted of intermicrotubular cytoplasmic ground substance. As analyzed by SDSpolyacrylamide gel electrophoresis (SIDS-PAGE), the pelleted spindles contain 18% tubulin, variable amounts of actin (2-8%), and an unidentified protein of 55 kdaltons in a constant weight ratio to tubulin (1 :2 .5) . The isolated spindles also contained two polypeptides, larger than 300 kdaltons, that comigrated with egg dynein polypeptides, and ATPase activity (0 .02 pmol Pi/mg-min) that closely resembled both flagellar and egg dynein . The spindle Mgt+-ATPase showed a ratio of Cat+ -/Mg 2+ -ATPase = 0.85, had minimal activity in KCI and EDTA, and cleaved GTP at 35% of the rate of ATP. The Mg2+ -ATPase was insensitive to ouabain or oligomycin . The spindle Mg 2+-ATPase was inhibited by sodium vanadate but, like egg dynein, was less sensitive to vanadate than flagellar dynein . The spindle Mg 2+-ATPase does not resemble the mitotic Ca t+-ATPase described by others . We propose that the spindle Mg2 +-ATPase is egg dynein . Bound carbohydrate on the two high-molecular-weight polypeptides of both egg dynein and the spindle enzyme suggest that these proteins may normally associate with membranes in the living cell .

We have constructed a computational platform suitable for examining emergence of shape homeostasis in simple three-dimensional cellular systems. An embryo phenotype results from a developmental process starting with a single cell and its genome. When coupled to an evolutionary search, this platform can evolve embryos with particular stable shapes and high capacity for self-repair, even though repair is not genetically encoded or part of the fitness criteria. With respect to the genome, embryo shape and self-repair are emergent properties that arise from complex interactions among cells and cellular components via signaling and gene regulatory networks, during development or during repair. This report analyzes these networks and the underlying mechanisms that control embryo growth, organization, stability, and robustness to injury.

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Trifluoperazine (TFP), a drug that binds to Ca2+-calmodulin (CaM) complexes, altered swimming behavior not only in living paramecia, but also in reactivated, Triton-extracted "models" of the ciliate. By comparing the responses of living cells and models, we have ascertained that two sites of drug action exist in paramecium cilia. Swimming movements were recorded in darkfield stroboscopic flash photomicrographs; this permitted accurate quantitation of velocities and body-shape parameters. When living paramecia were incubated in a standard buffer containing 10 microM TFP, their speed of forward swimming fell over several minutes and their bodies shortened. Untreated paramecia backed up repeatedly and frequently upon transfer to a solution containing barium ions (the "barium dance"), but cells preincubated in TFP did not "dance." Instead they swam forward slowly for long periods of time without reversing and occasionally then exhibited abnormally prolonged reversals. W7 effects on swimming mimicked low doses of TFP, and the analog W5 did not visibly alter normal swimming patterns. These results suggest that TFP induces a decrease in the intracellular pCa of living paramecia, perhaps by reducing the efficiency of a calmodulin-activated calcium pump in the cell membrane. Paramecia extracted with Triton X-100 and reactivated to swim forward (7 greater than or equal to pCa greater than or equal to 6) were not affected by addition of up to 40 microM TFP to the reactivation medium. We conclude that the main drug effect in living cells is probably not at the axoneme. However, at low pCa, TFP directly affected the ciliary axoneme to shift its behavior to one characteristic of a higher pCa: TFP inhibited backward swimming in models reactivated at pCa less than 6; instead they swam forward or rocked in place. The mechanism of ciliary reversal in paramecium may therefore depend on an axonemal Ca2+-sensor, possibly bound CaM, which is affected by TFP only at low pCa, as has been postulated for other types of cilia.