J. Mol. Biol. (1995) 251, 329–333 COMMUNICATION Re-examination of the Polarity of Microtubules and Sheets Decorated with Kinesin Motor Domain Keiko Hirose, Juan Fan and Linda A. Amos* Electron microscope images of microtubules and tubulin sheets decorated MRC Laboratory of Molecular with kinesin head domains have shown the main mass of the kinesin head Biology, Hills Road domain to be superimposed on one subunit of each tubulin dimer. We have Cambridge, CB2 2QH England polymerized brain tubulin extensions on to the ends of flagellar axonemes under varied conditions, in order to check the polarity of the tubulin–kinesin head complex. Since the polarity of axonemes incubated with normal brain tubulin may be ambiguous, we also tried 50% N-ethylmaleimide-treated tubulin which specifically blocks minus ends. Our conclusion, which conflicts with recently published results, is that the main mass of the kinesin head is associated with the tubulin subunit closer to the plus end of a microtubule.  1995 Academic Press Limited *Corresponding author Keywords: kinesin; microtubule; tubulin; axonemes; polarity Kinesin is a molecular motor molecule that moves along microtubules towards their plus ends, defined by their faster rate of assembly (Brady, 1985; Scholey et al. , 1985; Vale et al. , 1985). The N-terminal 30 to 40 kDa part of the heavy chain, the motor domain, binds to microtubules in a nucleotide-dependent manner. In electron microscope images, micro- tubules decorated with motor domain show a longitudinal periodicity of 8 nm, equivalent to the spacing of the tubulin dimer (Harrison et al. , 1993; Song & Mandelkow, 1993; Huang & Hackney, 1994; Kikkawa et al. , 1994). The kinesin–tubulin complex has a polar appear- ance that is clearly recognized in electron microscopy (EM) images when open-out microtubules (sheets) are decorated with kinesin heads. It is important to know how this polar appearance of kinesin decoration is related to the polarity of the underlying microtubules, in order to relate the structure of kinesin to its direction of movement. One way of determining the polarity of microtubules is to grow them from the ends of flagellar microtubule seeds, since the faster-growing plus ends are distinguish- able by longer extensions. Using this method, Song & Mandelkow (1995) reported that the main mass of the motor domain is superimposed on the monomer occupying the minus end of the tubulin heterodimer. We have repeated the experiments, preparing the decorated specimens under a range of conditions, and have come to a different conclusion. Three different procedures were used for preparing decorated specimens. In method 1, axonemes prepared from sea urchin sperm (Gibbons & Fronk, 1979) were mixed with tubulin (0.8 mg/PC tubulin/ml, prepared from porcine brain tissue (Mandelkow et al. , 1985)) in assembly buffer (80 mM K-Pipes, 1 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, 0.5 mM GTP (pH 6.8)) and incubated at 37°C. The axonemes with polymerized microtubules were diluted with Hepes buffer (20 mM Hepes, 1 mM EGTA, 2mM magnesium acetate, 40 mM potassium acetate, 1 mM DTT (pH 7.2)) containing bacterially- expressed rat kinesin motor domain K340 (Lockhart et al. , 1995) and 0.5 to 1.0 mM AMP-PNP, were incubated at room temperature for 5 to 10 minutes, and then were put on to carbon-coated grids to be negatively stained. Some axonemes showed assembly at both ends (see Figure 1C), while others had additional microtubules at only one end (Figure 1D). Some of the ends without additional microtubules seemed to be blocked by debris. In method 2, a similar mixture of the proteins and the same buffers were used, but the whole procedure was performed on an EM grid in order to avoid breakage of the microtubule–axoneme complex by pipetting. Also, taxol was added after assembly but before decoration, to prevent depolymerization of the extended microtubules. Tubulin and axonemes in the assembly buffer were mixed on a carbon-coated EM grid, which was incubated in a humid chamber at 37°C for 5 to 10 minutes to polymerize tubulin. The Abbreviations used: K340, kinesin heavy chain domain, residues 1 to 340; AMP-PNP, 5'-adenylylimidodiphosphate; NEM, N-ethylmaleimide; EM, electron microscopy. 0022–2836/95/330329–05 $12.00/0  1995 Academic Press Limited