Cell, Vol. 40, 729-730, April 1985. Copyright 0 1985 by MIT In Vitro Translocation of Organelles along Micmtubules Minireview Ttina A. Schroer and Regis B. Kelly Department of Biochemistry and BiOphySiC8 University of California San Francisco, California 94143 The rapidly growing list of biological processes that can be reconstituted in vitro now includes the fast transport of membranous organelles in axons. When membrane vesi- cles from squid axon are added to purified microtubules, the vesicles can be seen to move along the microtubules at speeds of about 2 rmlsec, if ATP and soluble factors from the squid axoplasm are also present (Vale et al., Cell 40, 559-569, 1965). This velocity of vesicle movement is comparable with that reported for in vivo fast axonal trans- port (Grafstein and Forman, Physiol. Rev. 60, 1167-1263, 1960). Since fast directional transport of vesicles along microtubules is not a unique property of axons, but a prop- erty of many cell8 (Schliwa, in Cell Muscle Motility, Vol. 5, ed. J. W. Shaw, Plenum Press, 1964, pp. l-62) what we learn from these studies may be applicable to a wide range of intracellular organelle movements. Until recently, fast axonal transport was conventionally studied by injecting radioactive precursors in the vicinity of nerve cell bodies in living animals. By sacrificing the an- imal after a suitable period of time, it was possible to mea- sure the rate of movement of the radioactive front down the axon, and in some cases identify the proteins that moved. The conclusion of such studies was that mem- brane organelles are transported down the axon at two to three different rates, ranging from 0.2 to 5 rmlsec (Willard et al., PNAS 77, 2163-2167, 1974). Early attempts to study membrane movement in the light microscope could only detect large organelles, which exhibited a slow, jumping (saltatory) movement. The new era in axonal transport began when the range of the light microscope was extended to include organelles the size of synaptic vesicle8 (50 nm). Using video-enhancement techniques to improve the contrast and image processing to reduce the “noise” and image defects, Allen et al. (Cell Mot. 7,291-302,196l) and lnoue (JCB 89,346-356,196l) succeeded in observing organelles that were less than 0.2 pm in diameter. In contrast to the slow, saltatory movement of the large organelles, the small vesicles moved without interruption and at a velocity of about 2 pmlsec-close to the speed of fast axonal transport measured by earlier techniques (Allen et al., Science 278, 1127-1129, 1962). These findings confirmed that there are several rates of membrane vesicle transport and suggested an inverse relationship between organelle size and velocity. The powerful video-enhancement techniques can be used to great advantage to observe vesicle movement in extruded squid axoplasm. Axoplasm, squeezed like tooth- paste from the axolemmal casing, continues to transport endogenous vesicles (Brady et al., Science 278, 1129- 1130, 1962). Visualization of vesicle transport is improved when the axoplasm is dissociated slightly by diluting the buffer twofold (Vale et al., Cell 40, 449-454, 1965). Under these conditions, individual filaments that “fray” out from the bulk of the axoplasm still transport vesicles when ATP is present. Surprisingly, particles with diameters ranging from less than 0.2 pm (the limit of resolution of the light microscope) to 3.6 pm all move with the same velocity of about 2 pm/set. The correlation between organelle speed and size observed in vivo is presumably due to sieving of large particles by the axoplasmic cytoskeleton. Invoking different transport mechanisms to explain the different transport rates found by Willard et al. no longer seems necessary. Another noteworthy conclusion from the work on dis- sociated aXOplasm iS that movement along a Single microtubule is bidirectional (Schnapp et al., Cell 40, 455-462, 1965). This was discovered when the transport- ing linear filaments were examined in the electron micro- scope, where single axonal microtubules can be resolved from bundles of microtubules. Organelles can move in both directions along a single microtubule and pass each other without apparent collision, implying that there may be directional tracks on individual filaments. Bidirectional- ity on a single linear filament is also seen in protozoa (Koonce and Schliwa, JCB 700,322-326,1965) and in fi- broblasts (Hayden and Allen, JCB 99, 1765-1793, 1964). Since all the tubulin monomers in a microtubule are be- lieved to have the same polarity, this is a rather astonish- ing result. Variations on these reconstitution experiments may allow identification of the molecular elements involved in intracellular transport. Gilbert and Sloboda (JCB 99, 445-452, 1964) have found that a membrane-enriched fraction of squid axoplasm show8 ATP-dependent move- ment when added back to extruded axoplasm. Movement is lost if the vesicles are first treated with proteases. Vale et al. (Cell 40,559-569,1965) have reconstituted transport using a better defined microtubule preparation. Mem- brane vesicle8 are isolated from squid axoplasm, after first removing tubulin and microtubule associated proteins by taxol-induced polymerization. When the membrane frac- tion from a sucrose density gradient is added to the microtubules polymerized from pure optic lobe tubulin, movement is not observed. On the other hand, addition of the soluble (tubulin-depleted) fraction from the top of the gradient restores ATP-dependent movement. Protease treatment of the vesicles or the soluble fraction destroys activity. The velocity of movement and its sensitivity to in- hibitors are indistinguishable from that of endogenous vesicles. The movement differs from that seen in axons or in dispersed axOplaSm Only in being undirectional. One observation in these reconstitution experiments is especially curious. Not only do vesicles move along microtubules in the presence of soluble factors and ATP, but free microtubules move along the glass slide. Vale et al. propose that a 8OlUble ATP-driven motor element ad- sorbs to the glass. Adsorbed motors which have the cor- rect polarity relative to the microtubule can generate