Microtubule Sliding in Swimming Sperm Flagella: Direct and Indirect Measurements on Sea Urchin and Tunicate Spermatozoa

Direct measurements of microtubule sliding in the flagella of actively swimming, demembranated, spermatozoa have been made using submicron diameter gold beads as markers on the exposed outer doublet microtubules . With spermatozoa of the tunicate, Ciona, these measurements confirm values of sliding calculated indirectly by measuring angles relative to the axis of the sperm head. Both methods of measurement show a nonuniform amplitude of oscillatory sliding along the length of the flagellum, providing direct evidence that "oscillatory synchronous sliding" can be occurring in the flagellum, in addition to the metachronous sliding that is necessary to propagate a bending wave. Propagation of constant amplitude bends is not accomplished by propagation of a wave of oscillaEA urchin and tunicate spermatozoa propagate nearly planar bending waves along their flagella . The propagation ofthe bending waves is associated with propaga tion of regions of sliding between axonemal microtubules, with the direction of sliding reversing as the direction ofcurvature of the flagellum reverses (Brokaw, 1971) . This distribution of microtubule sliding in flagella has not been deduced by direct observations of sliding, but by observations of bending ofthe flagellum . As illustrated in Fig . 1, the sliding between outer doublet microtubules can be calculated from the bent configuration of the flagellum, assuming that there is no twisting of the axoneme or longitudinal compliance of the microtubules. With these assumptions, the amount of sliding between two outer doublet microtubules at a particular locus on the flagellum is proportional to the difference in angular orientation between that locus and the basal end of the flagellum, where there is assumed to be no sliding (Satir, 1968 ; Warner and Satir, 1974 ; Gibbons, 1981) . The constant of proportionality is the "doublet separation" : the distance, in the plane ofbending, between the two sliding outer doublet microtubules . Since this will vary depending upon the choice of microtubules, it is convenient to describe the pattern of sliding in terms of "shear angle," measured in angular units (rads), without introducing a proportionality constant . Sets ofshear angle curves, showing the shear angle © The Rockefeller University Press, 0021-9525/91/09/1201/15 $2.00 The Journal of Cell Biology, Volume 114, Number 6, September 1991 1201-1215 1201 tory sliding of constant amplitude, and therefore appears to require a mechanism for monitoring and controlling the bend angle as bends propagate. With sea urchin spermatozoa, the direct measurements of sliding do not agree with the values calculated by measuring angles relative to the head axis . The oscillation in angular orientation of the sea urchin sperm head as it swims appears to be accommodated by flexure at the head-flagellum junction and does not correspond to oscillation in orientation of the basal end of the flagellum . Consequently, indirect calculations of sliding based on angles measured relative to the longitudinal axis of the sperm head can be seriously inaccurate in this species . as a function of position along the flagellum, for various times in the beat cycle, thus provide a useful description of the sliding occurring in a flagellum . Fig . 2 A shows a set of shear angle curves for a hypothetical flagellar bending pattern composed of circular arcs . It shows the pattern ofpropagated sliding that has been referred to as "metachronous" sliding (Brokaw and Gibbons, 1975 ; Goldstein, 1976), with a uniform amplitude of oscillatory sliding at each position in the distal portion of the flagellum . Sliding associated with the growth (i .e ., increase in angle) of a bend at the base of a flagellum has been referred to as "synchronous" sliding, because it corresponds to a constant velocity of sliding throughout the portion of the flagellum distal to the growing bend. The basal region of these sperm flagella normally contains two developing bends. In many cases, these two developing bends have approximately equal rates ofincrease ofbend angle in opposite directions, so that the summed synchronous sliding required by these two developing bends is relatively small, and does not greatly alter the pattern ofprimarily metachronous sliding in bends propagating along the distal regions of the flagellum (Goldstein, 1976) . With light microscopy, determining the orientation of the basal end ofa flagellum is difficult . In dark-field photomicrographs, the sperm head image interferes with observation of on Jne 6, 2017 D ow nladed fom Published September 15, 1991