Tws is primarily cytoplasmic but shuttles through the nucleus. (A) D-Mel cells expressing Tws-GFP or Tws^NLSm-GFP (NLS mutant: K455I, K457M) were treated with 50 nM LMB (b and d) or 0.1% ethanol (EtOH; mock control; a and c) for 2 h before fixation and DNA staining with DAPI. Scale bar: 10 µm. (B) Cells expressing Tws-GFP or Tws^NLSm-GFP were treated with LMB or EtOH and imaged. Scale bar: 5 µm. (C) The nuclear/cytoplasmic ratio of GFP fluorescence intensity from video images in B was quantified through time (10 cells for each condition, mean ± SD). (D) Tws contains an NLS that is conserved in vertebrate B55 orthologues. Top: sequence alignment showing the NLS in magenta. Mutations introduced to inactivate the NLS in Tws are shown in red. Bottom: location of the identified NLS in the structure of the human PP2A-B55α complex bound to microcystin-LR (Protein Data Bank accession no. 3DW8; Xu et al., 2008). Dm, Drosophila melanogaster; Hs, Homo sapiens; Xl, Xenopus laevis.