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Kinesin-1 is a molecular transporter that trafficks along microtubules. There is some evidence that kinesin-1 targets specific cellular sites, but it is unclear how this spatial regulation is achieved. To investigate this process, we used a combination of in vivo imaging of kinesin heavy-chain Kif5c (an isoform of kinesin-1) fused to GFP, in vitro analyses and mathematical modelling. GFP-Kif5c fluorescent puncta localised to a subset of microtubules in live cells. These puncta moved at speeds of up to 1 μm second–1 and exchanged into cortically labelled clusters at microtubule ends. This behaviour depended on the presence of a functional motor domain, because a rigor-mutant GFP-Kif5c bound to microtubules but did not move along them. Further analysis indicated that the microtubule subset decorated by GFP-Kif5c was highly stable and primarily composed of detyrosinated tubulin. In vitro motility assays showed that the motor domain of Kif5c moved detyrosinated microtubules at significantly lower velocities than tyrosinated (unmodified) microtubules. Mathematical modelling predicted that a small increase in detyrosination would bias kinesin-1 occupancy towards detyrosinated microtubules. These data suggest that kinesin-1 preferentially binds to and trafficks on detyrosinated microtubules in vivo, providing a potential basis for the spatial targeting of kinesin-1-based cargo transport.

Cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) is a calcium-sensitive enzyme involved in receptor-mediated eicosanoid production. In resting cells, cPLA(2)-alpha is present in the cytosol and nucleus and translocates to membranes via its calcium-dependent lipid-binding (CaLB) domain following stimulation. cPLA(2)-alpha is also regulated by phosphorylation on several residues, which results in enhanced arachidonic acid release. Little is known about the factors controlling the nuclear localisation of cPLA(2)-alpha. Here the nuclear localisation of cPLA(2)-alpha in the EA.hy.926 human endothelial cell line was investigated. Nuclear localisation was dependent on proliferation, with subconfluent cells containing higher levels of nuclear cPLA(2)-alpha than contact-inhibited confluent or serum-starved cells. The broad-range protein kinase inhibitor staurosporine caused a decrease in the nuclear level of cPLA(2)-alpha, whereas the protein phosphatase inhibitor okadaic acid increased the level of nuclear cPLA(2)-alpha. Using inhibitors for specific mitogen-activated protein (MAP) kinases, both p42/44(MAPK) and p38(MAPK) were shown to be important in modulating nuclear localisation. Finally, inhibition of nuclear import and export using Agaricus bisporus lectin and leptomycin B, respectively, demonstrated that cPLA(2)-alpha contains functional nuclear localisation and export signals. Thus we have identified a novel mode of regulation of cPLA(2)-alpha. This, together with the increasing body of evidence supporting the role of nuclear lipid second messengers in gene expression and proliferation, may have important implications for controlling the growth of endothelial cells in angiogenesis and tumour progression.