We report a motiliy in the flagella of the green alga Chlamydomonas that is unrelated to dynein-based flgelar beating. This motility, referred to as intraflageUar transport, was observed as the rapid bidirectional movement of granule-like particles along the length of the ftgella. IntraflageUar transport could be experimentally separated from other, previously reported, nonbeat fgellar motilities. EM of flageUla showed groups of nonvesicular, lollipop-shaped structures positioned between the outer doublet microtubules and the flagellar membrane. Movement of these complexes along the length of the flagella may be responsible for intraflagellar transport. While reexamining these three motilities with videoenhanced differential interference-contrast (DIC) microscopy, we were surprised to find a fourth, previously unobserved, nonbeat motility within the flagella of Chlamydomonas. In prior light microscopic studies, the flagella of Chlamydomonas have always been observed as relatively featureless high-contrast rods. Therefore, the visualization of granule-like particles moving bidirectionally along the length ofthe flagella, apparently between the microtubular axoneme and flagellar membrane, was striking. In addition, improved fixation methods for EM have allowed for the routine observation, in thin section, of complexes between the flagellar membrane and the axonemal microtubules. The movement of these complexes may account for this motility, referred to as intraflagellar transport (IFT). for video-enhanced DIC microscopy because their flagella are =50%o longer than those of C. reinhardtii. The paralyzed flagellar dynein triple-mutant ida2 ida4 oda6 of C. reinhardtii was from R. Kamiya (Nagoya University). Logarithmicphase, synchronously dividing cultures were grown in minimal medium (MI) (8) on a 12 hr:12 hr light/dark cycle with continuous aeration. MATERIALS AND METHODSFlagellar Regeneration and Resorption. Flagellar regeneration was induced by pH-shock (9). After deflagellation, the cells were washed with fresh MI medium. Flagellar resorption was induced by placing the cells in a low-Ca2+, high-Na+ medium (10) or also, with C. reinhardtii, by adding 3-isobutyl-l-methylxanthine to MI medium at a final concentration of 0.5 mM (11).Video Microscopy. For all experiments, cultures were diluted 1:5 with double-distilled water and placed between two acid-washed no. 1 coverslips (Corning) supported with 1-mm plastic shims affixed with Vaseline. When required, polystyrene beads with a 0.3-,&m diameter (Polysciences), washed four times with double-distilled water, were mixed with the cells to a final dilution of 1:500. Dilution of the culture medium enhanced the attachment of beads to the flagellar membrane. All experiments were done at room temperature using a Zeiss Axiovert microscope. The optics and method ofdigital data acquisition have been detailed (12), except that the light source used in this study was passed through a fiber optic scrambler to obtain full and even illumination of the condenser (1.4 N.A.) ...

Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions--regulatory and structural. These interactions comprise at least three conserved 'mechanochemical activity modules' that perform similar roles in these diverse cell functions.

Abstract. Actions of cytochalasin B (CB) on cytoskeletons and motility of growth cones from cultured Aplysia neurons were studied using a rapid flow perfusion chamber and digital video light microscopy. Living growth cones were observed using differential interference contrast optics and were also fixed at various time points to assay actin filament (F-actin) and microtubule distributions. Treatment with CB reversibly blocked motility and eliminated most of the phalloidin-stainable F-actin from the leading lamella.

Retrograde actin flow works in concert with cell adhesion to generate traction forces that are involved in axon guidance in neuronal growth cones. Myosins have been implicated in retrograde flow, but identification of the specific myosin subtype(s) involved has been controversial. Using fluorescent speckle microscopy (FSM) to assess actin dynamics, we report that inhibition of myosin II alone decreases retrograde flow by 51% and the remaining flow can be almost fully accounted for by the 'push' of plus-end actin assembly at the leading edge of the growth cone. Interestingly, actin bundles that are associated with filopodium roots elongated by approximately 83% after inhibition of myosin II. This unexpected result was due to decreased rates of actin-bundle severing near their proximal (minus or pointed) ends which are located in the transition zone of the growth cone. Our study reveals a mechanism for the regulation of actin-bundle length by myosin II that is dependent on actin-bundle severing, and demonstrate that retrograde flow is a steady state that depends on both myosin II contractility and actin-network treadmilling.