Direct is correct

A challenge to conventional thinking has been met and answered by two new articles in this issue. Hua et al. (page 1035< / A r t R e f >) and Paladino et al. (page < A r t R e f v o l = " 1 7 2 " I s s = " 7 " > 1023< / A r t R e f >) have imaged live three-dimensional cells to reaffi rm that a class of proteins takes a direct route to the apical membrane. The suggestion that GPI-linked apical proteins traffi c directly from the Golgi to the apical plasma membrane was made—and widely accepted—years ago based on classical biochemical experiments. But a recent article proposed instead that, in MDCK cells, this class of proteins were delivered fi rst to the basolateral membrane, and then endocytosed and sent across the cell to the apical membrane. Striking images were presented to support this view, but the work also involved a somewhat controversial new assay. The new papers suggest that the new assay may have been problematic. Paladino found that it caused a partial depolarization of the cells. Both new papers also used live cell imaging to track the transport of newly synthesized GPI-anchored proteins. This required some substantial technical breakthroughs. Most live cell imaging is performed on fl at, nearly two-dimensional cell preparations. But fully polarized MDCK cells are at least 10-μm tall, meaning multiple focal planes have to be imaged all at once. To minimize the main problem of photobleaching, Paladino et al. used the quicker spinning disc confocal technology, but this approach did not permit quantitation. Hua and colleagues stuck with laser scanning microscopy, optimizing every parameter, and carefully quantified the dynamics of transport. Although resolution was limited in both strategies, fl uores-cent signals can be followed for the longer periods of time required for traffi cking through the taller cells. Both groups saw GPI-GFP move directly from the Golgi, through the trans-Golgi network (TGN), and to the apical membrane. Very little protein ever reached the basolateral membrane. These new imaging techniques, although powerful in resolving this controversy, may really shine when used to test the requirement for putative traffi cking components. T he Stu2p microtubule-associated protein (MAP) clamps around tubulin heterodimers, as revealed by electron micrograph images from Al-Bassam et al. (page < A r t R e f v o l = …