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Annotated Classic Kinesin Motors on Microtubules Annotated Classic Kinesin Motors on Microtubules Trina A. Schroer1,* 1Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA *Correspondence: [email protected] We are pleased to present a series of Annotated Classics celebrating 40 years of exciting biology in the pages of Cell. This installment revisits “Identification of a Novel Force-Generating Protein, Kinesin, Involved in Microtubule-Based Motility” by Ronald D. Vale, Thomas S. Reese, and Michael P. Sheetz. Here, Trina Schroer comments on the work that led to the purification of a force-generating protein from the giant axons of squid. The motor, named kinesin in this work, depended on nucleotide hydrolysis and was suggested to be involved in subcellular organellar transport. Vale and Sheetz collaborated on this and four other papers that appeared in Cell in 1985 (see below), which, in aggregate, provided a foundation for the microtubule- associated motor field. Each Annotated Classic offers a personal perspective on a groundbreaking Cell paper from a leader in the field with notes on what stood out at the time of first reading and retrospective comments regarding the longer term influence of the work. To see Trina A. Schroer’s thoughts on different parts of the manuscript, just download the PDF and then hover over or double- click the highlighted text and comment boxes. You can also view Schroer’s annotation by opening the Comments navigation panel in Acrobat. Schnapp, B.J., Vale, R.D., Sheetz, M.P., Reese, T.S. (1985). Single Microtubules from Squid Axoplasm Support Bidirectional Movement of Organelles. Cell 40, 455–462. http://dx.doi.org/10.1016/0092-8674(85)90160-6. Vale, R.D., Schnapp, B.J., Reese, T.S., Sheetz, M.P. (1985). Movement of Organelles along Filaments Dissociated from the Axoplasm of the Squid Giant Axon. Cell 40, 449–454. http://dx.doi.org/10.1016/0092-8674(85)90159-X. Vale, R.D., Schnapp, B.J., Reese, T.S., Sheetz, M.P. (1985). Organelle, Bead, and Microtubule Translocations Promoted by Soluble Factors from the Squid Giant Axon. Cell 40, 559–569. http://dx.doi.org/10.1016/0092-8674(85)90204-1. Vale, R.D., Schnapp, B.J., Mitchison, T., Steuer, E., Reese, T.S., Sheetz, M.P. (1985). Different axoplasmic proteins generate movement in opposite directions along microtubules in vitro. Cell 43, 623–632. http://dx.doi.org/10.1016/0092-8674(85)90234-X. Cell 157, April 24, 2014, 2014 ©2014 Elsevier Inc. Cell, Vol. 42, 39-50, August 1985, Copyright © 1985 by MIT 0092-8674/85/080039-12 $02.00/0 Identification of a Novel Force-Generating Protein, Kinesin, Involved in Microtubule-Based Motility Ronald D. Vale,*tt Thomas S. Reese,* and fluorescence using antitubulin antibodies (Schnapp et al., Michael P. Sheetz,*l§ 1985). Microtubules also support organelle movements in * Laboratory of Neurobiology, a variety of nonneuronal cells (Schliwa, 1984; Hayden et National Institute of Neurological and al., 1983). Communicative Disorders and Stroke Directed organelle movement within cells depends on at the Marine Biological Laboratory ATP (Forman et al., 1984; Schliwa, 1984), and it has been Woods Hole, Massachusetts 02543 generally assumed that movement is powered by an ATP- tDepartment of Physiology ase. Since all organelles move at the same velocity along University of Connecticut Hea,,h Center isolated microtubules, the same type of molecular motor, Farmington, Connecticut 06032 or "translocator" may interact with a variety of organelles Department of Neurobiology (Vale et al., 1985a). However, it is not known which pro- Stanford University School of Medicine teins comprise the translocator. Dynein is the only mole- Stanford, California 94305 cule known to interact with microtubules to generate mo- tive force (Gibbons, 1981), but attempts to identify dynein in neural tissue, where organelle movements are com- Summary mon, have been unsuccessful (Murphy et al., 1983). Biochemical identification of the proteins involved in or- Axoplasm from the squid giant axon contains a solu- ganelle movement requires an assay system for studying ble protein translocator that induces movement of this phenomenon in vitro. Recently, we showed that iso- microtubules on glass, latex beads on microtubules, lated axoplasmic organelles move on purified squid optic and axoplasmic organelles on microtubules. We now lobe microtubules and that movement is enhanced by a report the partial purification of a protein from squid soluble protein or proteins in axoplasm (Vale et al., 1985b). giant axons and optic lobes that induces these This experiment suggests that the translocator is a protein microtubule-based movements and show that there is that binds reversibly to organelles and to microtubules. a homologous protein in bovine brain. The purifica- Organelle movement in the reconstituted system is similar tion of the translocator protein depends primarily on to that in dissociated axoplasm except that movement its unusual property of forming a high affinity complex along single microtubules in the reconstituted system is with microtubules in the presence of a nonhydrolyz- primarily unidirectional (Vale et al., 1985a; Schnapp et al., able ATP analog, adenylyl imidodiphosphate. The pro- 1985). tein, once released from microtubules with ATP, mi- The soluble fraction from axoplasm also induces move- grates on gel filtration columns with an apparent ment of purified microtubules along a glass coverslip and molecular weight of 600 kilodaltons and contains movement of carboxylated latex beads along microtu- 110-120 and 60-70 kilodalton polypeptides. This pro- bules (Vale et al., 1985b). Native microtubules in extruded tein is distinct in molecular weight and enzymatic be- squid axoplasm also move along a glass coverslip (Allen havior from myosin or dynein, which suggests that it et al., 1985). The soluble translocator from axoplasm that belongs to a novel class of force-generating mole- induces bead movement might also be responsible for cules, for which we propose the name kL;esin. moving inert beads after they are microinjected into axons or cultured cells (Adams and Bray, 1983; Beckerle, 1984). Introduction The velocities of microtubule and bead movements are the same (0.4/~m/sec), but both movements are 3 to 4 fold Most animal cells transport organelles within their cyto- slower than organelle movements (Vale et al., 1985b). plasm (Schliwa, 1984). In neurons, bidirectional organelle However, their similar sensitivities to vanadate suggest transport within axons is thought to be the basis of fast that organelle, bead, and microtubule movements may be axonal transport (Smith, 1980; Tsukita and Ishikawa, driven by the same ATPase. 1980). The squid giant axon, because of its large size, has We describe the purification, from squid axoplasm and been particularly useful for studying organelle movement optic lobes, of a translocator protein that induces move- (Allen et al., 1982; Brady et al., 1982). Axoplasm from the ment of microtubules on glass and movement of beads giant axon also can be dissociated, allowing bidirectional along microtubules. We also have identified a homologous organelle movements to be visualized along individual cy- protein in bovine brain. The characteristics of these pro- toplasmic filaments with video-enhanced differential inter- teins are distinct from myosin and dynein and appear to ference contrast microscopy (Vale et al., 1985a; Allen et define a novel class of force-generating molecules. al., 1985). These cytoplasmic filaments were identified as single microtubules by electron microscopy and immuno- Results In Vitro Movement Assays § To whom all correspondenceshould be addressed at his present ad- dress: Departmentof Cell Biologyand Physiology,Washington Univer- A high speed supernatant from axoplasm ($2 superna- sity School of Medicine, St. Louis, Missouri 63110. tant) promoted linear, ATP-dependent movement of purl- Cell 40 Table 1. Movements Supported by Squid and Bovine Translocators Microtubule Microtubule Movement Movement Sample along Glass in Solution Bead Movement Organelle Movementb Buffer - - + $2 axoplasmic supernatant a 0.44 _+ 0.07/~m/sec + 0.52 ± 0.06/~m/sec + + + to + + + + Squid gel filtration 0.35 _+ 0.06 #m/sec + 0.34 _+ 0.05 Hm/sec + + to + + + + Squid hydroxyapatite 0.37 -4- 0.03 ~m/sec + 0.37 ± 0.06/~m/sec + + to + + + + Bovine gel filtration 0.41 ± 0.05/~m/sec + 0.59 ± 0.01 ~m/sec + to + + Microtubule, bead, and organelle movements promoted by $2 axoplasmic supernatant or purified squid or bovine translocator; movement occurred consistently in preparations for which rates are shown. Squid gel filtration peak fractions (n = 7; equivalent to lane 30 in Figure 4) and hydroxyapa- tite peak fractions (n = 3; equivalent to lane 40, 41 in Figure 5) were tested in motility buffer or 100 mM KCI, 50 mM Tris (pH 7.6), 5 mM MgCI2, 0.5 mM EDTA, 2 mM ATP at final protein concentrations between 10 and 150/~g/ml. Bovine gel filtration peak fractions (n = 3; equivalent to lane 30 in Figure 7) were tested in KCI/Tris buffer at protein concentrations between 20 and 50/~g/ml. Organelle movement was assessed by viewing a 20 ~m x 20 p.m field of microtubules for 2.5-15 min and counting the number of different organeiles that made directed movements along microtubules. The following rating system was employed: -, no movement in all preparations; +, 0-3 movements/min; + +, 3-7 move- ments/min; + + +, 7-15 movements/min; + + + +, 15-34 movements/min. The range of organelle movement in different preparations is reported. Microtubule, bead, or organelle movement assays are described in Experimental Procedures. a Values from Vale et al. (1985b). b The velocity of organelle movement in all samples was approximately 1.64 _+ 0.24/~m/sec. fled microtubules (freed of microtubule-associated pro- microtubules, it might be possible to bind the translocator teins by 1 M NaCI extraction) at 0.4/~m/sec along a glass to microtubules with AMP-PNP to provide a means of af- coverslip (Table 1).
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