Monitoring MLCK

In This Issue In This Issue Monitoring MLCK yosin light chain kinase (MLCK) is a busy protein— it phosphorylates myosin's regulatory light chain (and thus activates myosin) during nonmuscle cell contraction, cytokinesis, stress fiber formation, and motility. In general, MLCK is known to be present at the cellular sites implicated in these events, but the results of Chew et al. (page 543) are some of the first hints at the dynamics of that localization and the in situ activity of the kinase. M Visualizing vesicles arbero et al. have visualized the movement of individual vesicles from late endosomes to the trans Golgi by using GFP-marked Rab9 (page 511). Their results suggest that vesicles rather than tubules are used to deliver mannose 6-phosphate receptors (MPRs) in their return to the Golgi, after these proteins have transported enzymes from the Golgi to prelysosomes. The detailed localization of the GFP-Rab9 is also telling. Rab GTPases are thought to contribute delivery address information for many trafficking vesicles, but Barbero et al. note that Rab9 is present on the endosome-derived vesicles from budding through docking. This is consistent with the group's earlier experiments suggesting B that Rab9 has a function during budding. Perhaps as part of this budding process Rab9 clusters in particular regions, as the authors note that Rab9 and Rab7 (a GTPase involved in late endosome fusion) are present in largely nonoverlapping domains within a given late endosome. Once Barbero et al. have overcome some protein engineering problems, they hope to visualize MPRs and the Rab9-binding cargo collector TIP47. They predict that MPRs will be present in the transport vesicles, and that TIP47 will be released from the vesicles after budding, perhaps to be replaced by an unknown docking factor. ᭿ Rab9 (red) and Rab7 (green) localize to distinct domains on late endosomes. Chew et al. track MLCK by adding a module with a calcium–calmodulin-binding domain flanked by BFP and GFP. In the absence of calcium, the BFP and GFP are close enough to each other to allow fluorescence resonance energy transfer (FRET) between them. When calcium–calmodulin binds, however, this disrupts FRET. Chew et al. suspect that when there is sufficient calmodulin to bind to the added cassette, there will also be calmodulin binding to a domain in MLCK, an event that activates the kinase. Thus, the absence of FRET is interpreted as a sign of an activated kinase. The authors measure both …