Figure 2 Functional comparisons of wt and mutated human espin 3A using the LLC‐PK1‐CL4 epithelial cell transfection model. Differentiated LLC‐PK1‐CL4 epithelial cells were transfected with GFP‐wt human espin 3A or the designated mutated construct, labeled for F‐actin with Texas Red‐phalloidlin and examined by confocal microscopy. Multiple examples are shown. A‐C, wt. D‐F, S719R. G‐I, D744N. J‐L, R774Q. M‐O, delK848. All images are of the apical (microvillar) surface of the monolayer, except O, which is a z‐section through the middle of the cells in N highlighting the nuclear accumulation of the delK848 construct. The F‐actin‐rich junctional complexes adjointing the apical‐lateral boundaries of neighboring cells are often evident as red lines. Note that the wt construct (A‐C) is colocalized (yellow) in microvilli that are much longer than the brush border microvilli (red) of surrounding control (untransfected) cells. The R774Q (J‐L) is indistinguishable from wt. The S719R (D‐F) and D744N (G‐I) constructs are targeted to microvilli and cause microvillar elongation, but the ong microvilli frequently appear in irregular patches that occupy only a small percentage of the apical surface. The delK848 construct (M‐O) is severely impaired in microvillar elongation and shows abnormally high accumulation in the nucleus (O). The objects within the nucleus that exclude the GFP‐delK848 construct are nucleoli. Bar, 5 µm.