Abstract
The sliding tubule model of ciliary motion requires that active sliding of microtubules occur by cyclic cross-bridging of the dynein arms. When isolated, demembranated Tetrahymena cilia are allowed to spontaneously disintegrate in the presence of ATP, the structural conformation of the dynein arms can be clearly resolved by negative contrast electron microscopy. The arms consist of three structural subunits that occur in two basic conformations with respect to the adjacent B subfiber. The inactive conformation occurs in the absence of ATP and is characterized by a uniform, 32 degrees base-directed polarity of the arms. Inactive arms are not attached to the B subfiber of adjacent doublets. The bridged conformation occurs strictly in the presence of ATP and is characterized by arms having the same polarity as inactive arms, but the terminal subunit of the arms has become attached to the B subfiber. In most instances the bridged conformation is accompanied by substantial tip-directed sliding displacement of the bridged doublets. Because the base-directed polarity of the bridged arms is opposite to the direction required for force generation in these cilia and because the bridges occur in the presence of ATP, it is suggested that the bridged conformation may represent the initial attachment phase of the dynein cross-bridge cycle. The force-generating phase of the cycle would then require a tip-directed deflection of the arm subunit attached to the B subfiber.
Highlights
The sliding tubule model of ciliary motion requires that active sliding of microtubules occur by cyclic cross-bridging of the dynein arms
Several independent studies confirm the sliding tubule mechanism of ciliary and flagellar motion [20, 21, 25]; we as yet do not understand how the adenosine triphosphatase or dynein arms participate in the mechanochemical activity that must be responsible for linear displacement of microtubules
An important contribution to the continuing development of the sliding tubule model comes from Sale and Satir [19] who observed that active sliding in Tetrahymena cilia was a distally or tipdirected phenomenon, the net force being directed away from the proximally directed angle of the dynein arms
Summary
Cilia from the protozoan Tetrahymena pyriformis strain B-III were isolated and purified according to the detailed procedures reported previously [26]. In order to visualize the microtubule sliding phenomenon and associated dynein arm conformations, it was first necessary to allow the cilia to actively disintegrate in the presence of ATP [21]. Isolated, demembranated cilia were resuspended in a solution consisting of 40 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES), 0.15 M KC1, 2 mM MgSO4, 0.5 mM EDTA, and 1 mM dithiothreitol (DTT) at pH 7.8 [9]. For Tetrahymenacilia, active sliding disintegration does not require external proteolysis. Purified cilia disintegrate spontaneously in the presence of 0.1 mM ATP [19, 26], the amount of disintegration can vary between 20 and 100% in different preparations. Disintegration was monitored by both dark-field and phase-contrast light microscopy and spectrophotometry (350 nm)
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