The organization of membranous structures in the cell, such as the Golgi body or endoplasmic reticulum, depends on motor proteins and the microtubules along which they move. Of the two major protein families involved, the dyneins move towards the ‘minus’ end of the microtubules, generally clustered close to the nucleus, whereas the kinesins chiefly move towards the ‘plus’ ends of the microtubules, distributed about the periphery of the cell.
Although organelle transport is a central function of microtubule-based motors, these motor proteins were originally identified for other reasons, such as their ability to make microtubules glide over glass coverslips. Now, however, Ron Vale and colleagues present a more direct approach (J. Cell Biol. 147, 493–505; 1999). By specifically assaying organelle transport they have identified two new kinesin-like motors from the slime mould Dictyostelium discoideum.
To do this, the authors mixed Dictyostelium cell extracts with organelles and assembled microtubules. Then, using video-enhanced differential interference microscopy, Vale and colleagues assessed the extracts' ability to reconstitute organelle movement, and also determined the direction of that movement. An example of this assay is shown in the picture. Video stills separated by half a second are overlaid and colour-coded to show the direction of motion (purple–cyan).
Using a number of chromatography steps, two plus-end-directed activities were purified associated with proteins of relative molecular masses (Mr) 245,000 and 170,000. Peptide analysis allowed the identification, cloning and sequencing of the Mr 245,000 protein. It proved to be homologous to known kinesins from mice and nematode worms, and was named DdUnc104 after the worm homologue. When Vale and colleagues knocked out the DdUnc104 gene, the mutant mould had disrupted organelle transport, confirming the in vivo role of DdUnc104 in this process.
The importance of this work is not so much in the identification of two previously unknown kinesins, but in the establishment of a system for dissecting the mechanics of organelle transport. Because Dictyostelium is so amenable to genetic manipulation, it should be possible to investigate not only the motors involved, but also the accessory proteins and receptors that allow them to identify and transport specific organelles.
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physica status solidi (b) (2005)
The American Biology Teacher (2000)