▲ EXPERIMENTAL FIGURE 20-18 DIC microscopy demonstrates microtubule-based vesicle transport in vitro. (a) The cytoplasm was squeezed from a squid giant axon with a roller onto a glass coverslip. After buffer containing ATP was added to the preparation, it was viewed in a differential interference contrast microscope, and the images were recorded on videotape. In the sequential images shown, the two organelles indicated by open and solid triangles move in opposite directions (indicated by colored arrows) along the same filament, pass each other, and continue in their original directions. Elapsed time in seconds appears at the upper-right corner of each video frame. (b) A region of cytoplasm similar to that shown in part (a) was freeze dried, rotary shadowed with platinum, and viewed in the electron microscope. Two large structures attached to one microtubule are visible; these structures presumably are small vesicles that were moving along the microtubule when the preparation was frozen. [See B. J. Schnapp et al., 1985, Cell 40:455; courtesy of B. J. Schnapp, R. D. Vale, M. P Sheetz, and T S. Reese.]

the synaptic terminals and is associated with axonal growth and the delivery of synaptic vesicles. In the opposite, retrograde, direction, "old" membranes from the synaptic terminals move along the axon rapidly toward the cell body where they will be degraded in lysosomes. Findings from such experiments also revealed that different materials move at different speeds (Figure 20-17). The fastest-moving material, consisting of membrane-limited vesicles, has a velocity of about 250 mm/day, or about 3 ^m/s. The slowest-moving material, comprising tubulin subunits and neurofilaments, moves only a fraction of a millimeter per day. Organelles such as mitochondria move down the axon at an intermediate rate.

Axonal transport can be directly observed by video microscopy of cytoplasm extruded from a squid giant axon. The movement of vesicles along microtubules in this cell-free system requires ATP, its rate is similar to that of fast axonal transport in intact cells, and it can proceed in both the an-terograde and the retrograde directions (Figure 20-18a). Electron microscopy of the same region of the axon cytoplasm reveals vesicles attached to individual microtubules (Figure 20-18b). These pioneering in vitro experiments established definitely that organelles move along individual microtubules and that their movement requires ATP. As discussed shortly, these two observations led to the identification of micro-tubule motor proteins, which generate the movements.

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▲ EXPERIMENTAL FIGURE 20-19 Transport of GFP-tagged neurofilaments down axons exhibits periodic pauses. (a) A

segment of an axon is imaged after GFP-labeled neurofilament protein, NF-M, is expressed in a cultured neuronal cell. Bundles of labeled neurofilaments are separated by gaps within an axon. A GFP-labeled neurofilament (arrowhead) is seen to traverse a

15-pm gap between two labeled bundles. In this time series, each frame is taken at 5-second intervals. (b) A plot of the time-dependent distance traveled shows pauses (arrow) in neurofilament transport. Although the peak velocity (arrowheads) is similar to fast axonal transport, the average velocity is much lower. [From L. Wang et al., 2000, Nature Cell Biol. 2:137; courtesy of A. Brown.]

Findings from recent experiments in which neurofilaments tagged with green fluorescent protein (GFP) were injected into cultured cells suggest that neurofilaments pause frequently as they move down an axon (Figure 20-19). Although the peak velocity of neurofilaments is similar to that of fast-moving vesicles, their numerous pauses lower the average rate of transport. These findings suggest that there is no fundamental difference between fast and slow axonal transport, although why neurofilament transport stops periodically is unknown.

Kinesin I Powers Anterograde Transport of Vesicles in Axons

The first microtubule motor protein was identified by using a simple system consisting of microtubules assembled in vitro from purified tubulin subunits and stabilized by the drug taxol. When synaptic vesicles and ATP were added to these microtubules, the vesicles neither bound to the microtubules nor moved along them. However, the addition of a cytosolic extract of squid giant axon (free of tubulin) caused the vesicles to bind to the microtubules and to move along them, indicating that a soluble protein in the axonal cytosol is required for translocation.

When researchers incubated vesicles, axonal cytosol, and microtubules in the presence of AMPPNP, a nonhydrolyzable analog of ATP, the vesicles bound tightly to the microtubules but did not move. However, the vesicles did move when ATP was added. These results suggested that a motor protein in the cytosol binds to microtubules in the presence of ATP or AMPPNP, but movement requires ATP hydrolysis. To purify the soluble motor protein, scientists incubated a mixture of microtubules, cell or tissue extract, and AMPPNP, with the rationale that AMPPNP would promote tight binding between the microtubules and motor proteins in the extract. After incubation, the microtubules with any bound proteins were collected by centrifugation. Treatment of the microtubule-rich material in the pellet with ATP released one predominant protein back into solution; this protein is now known as kinesin I.

Kinesin I isolated from squid giant axons is a dimer of two heavy chains, each complexed to a light chain, with a total molecular weight of 380,000. The molecule comprises a pair of large globular head domains connected by a long central stalk to a pair of small globular tail domains, which contain the light chains (Figure 20-20). Each domain carries out a particular function: the head domain, which binds mi-crotubules and ATP, is responsible for the motor activity of kinesin, and the tail domain is responsible for binding to the membrane of vesicles, most likely through the kinesin light chain.

Kinesin-dependent movement of vesicles can be tracked by in vitro motility assays similar to those used to study myosin-dependent movements. In one type of assay, a vesicle

► FIGURE 20-20 Structure of kinesin. (a) Schematic model of kinesin showing the arrangement of the two heavy chains (each with a molecular weight of 110,000-135,000) and the two light chains (60,000-70,000 MW). (b) Three-dimensional structure of the kinesin dimer based on x-ray crystallography. Each head is attached to an a-helical neck region, which forms a coiled-coil dimer. Microtubules bind to the helix indicated; this interaction is regulated by the nucleotide (orange) bound at the opposite side of the domain. The distance between microtubule binding sites is 5.5 nm. [Part (b) courtesy of E. Mandelkow and E. M. Mandelkow, adapted from M. Thormahlen et al., 1998, J. Struc. Biol. 122:30.]

or a plastic bead coated with kinesin is added to a glass slide along with a preparation of microtubules. In the presence of ATP, the beads can be observed microscopically to move along a microtubule in one direction. By determining the polarity of the microtubules, researchers found that the beads coated with kinesin I always moved from the (—) to the (+) end of a microtubule (Figure 20-21). Thus kinesin I is a (+) end-directed microtubule motor protein. Because this direction corresponds to anterograde transport, kinesin I is implicated as a motor protein that mediates anterograde axonal transport.

Most Kinesins Are Processive (+) End-Directed Motor Proteins

To date, approximately 10 different kinesin subfamilies have been identified. All contain a globular head (motor) domain, but they differ in their tail domains and several other properties. In most kinesins, the motor domain is at the N-terminus (N-type) of the heavy chain but, in others, the motor domain is centrally located (M-type) or at the C-terminus (C-type). Both N- and M-type kinesins are (+) end-directed motors, whereas C-type kinesins are (—) end-directed motors. Although most kinesins have two heavy chains (e.g., kinesin I), others have a single heavy chain (e.g.,

KIF1) or four heavy chains (e.g., BimC). Tetrameric BimC has an unusual bipolar arrangement in which pairs of motor domains lie at opposite ends of a central rod segment.

Kinesins can be divided into two broad functional groups— cytosolic and mitotic kinesins—on the basis of the nature of the cargo that they transport (Table 20-2). The functional differences between kinesins are related to their unique tail domains, which determine their cargoes. Cytoso-lic kinesins take part in vesicle and organelle transport; they include the classic axonal kinesin I, which has been shown to transport lysosomes and other organelles. Some cytosolic ki-nesins, however, transport one specific cargo. For example, KIF1B and its close relative KIF1A transport mitochondria and synaptic vesicles, respectively, to nerve terminals. Other cytosolic kinesins mediate the transport of secretory vesicles to the plasma membrane and the radial movement of ER

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