Cytoplasmic microtubules are critical for establishing and maintaining cell shape and polarity. Our investigations of kinesin-like proteins (klps) and morphological mutants in the fission yeast Schizosaccharomyces pombe have identified a kinesin-like gene, tea2 +, that is required for cells to generate proper polarized growth. Cells deleted for this gene are often bent during exponential growth and initiate growth from improper sites as they exit stationary phase. They have a reduced cytoplasmic microtubule network and display severe morphological defects in genetic backgrounds that produce long cells. The tip-specific marker, Tea1p, is mislocalized in both tea2-1 and tea2Δ cells, indicating that Tea2p function is necessary for proper localization of Tea1p. Tea2p is localized to the tips of the cell and in a punctate pattern within the cell, often coincident with the ends of cytoplasmic microtubules. These results suggest that this kinesin promotes microtubule growth, possibly through interactions with the microtubule end, and that it is important for establishing and maintaining polarized growth along the long axis of the cell.

Kinesins are microtubule-based motor proteins that transport cargo to specific locations within the cell. However, the mechanisms by which cargoes are directed to specific cellular locations have remained elusive. Here, we investigated the in vivo movement of the Schizosaccharomyces pombe kinesin Tea2 to establish how it is targeted to microtubule tips and cell ends. Tea2 is loaded onto microtubules in the middle of the cell, in close proximity to the nucleus, and then travels using its intrinsic motor activity primarily at the tips of polymerizing microtubules. The microtubule-associated protein Mal3, an EB1 homologue, is required for loading and/or processivity of Tea2 and this function can be substituted by human EB1. In addition, the cell-end marker Tea1 is required to anchor Tea2 to cell ends. Movement of Tea1 and the CLIP170 homologue Tip1 to cell ends is abolished in Tea2 rigor (ATPase) mutants. We propose that microtubule-based transport from the vicinity of the nucleus to cell ends can be precisely regulated, with Mal3 required for loading/processivity, Tea2 for movement and Tea1 for cell-end anchoring.

Cytoplasmic microinjection of murine Mx mRNA synthesized in vitro or nuclear microinjection of Mx cDNA under the control of a constitutive promoter into murine Mx- cells led to the accumulation of Mx protein in the nucleus and inhibited the replication of influenza virus but not of vesicular stomatitis virus (VSV). Similar results were also found with dog, rat, chicken, and monkey cells. A human lung fibroblast cell line (A549) was exceptional in that Mx protein was located predominantly in the cytoplasm and showed antiviral activity. Truncation of the 19 last residues of murine Mx protein almost completely abolished accumulation of Mx protein in the nucleus; however the activity against influenza virus was at least partially retained. The truncated region contains a segment rich in basic amino acids, similar to that reported for several nuclear location signals.

Tea2 is a kinesin family member from Schizosaccharomyces pombe that is targeted to microtubule tips and cell ends in a process that depends on Mal3. Constructs of Tea2 containing the motor domain only or the motor domain plus the N-terminal extension are monomeric, whereas a construct including the first predicted coiled coil region is dimeric. These constructs have a low basal rate of ATP hydrolysis of <0.1 s ؊1 , but microtubules stimulate the rate of ATP hydrolysis to a maximum of ϳ15