Dynamic localization of SAF-A promotes normal chromosome segregation in anaphase. (a) SAF-A–AID–mCherry degron cells were transduced with lentivirus encoding either SAF-A^wt–GFP or the phosphomutant SAF-A^{S14A S26A}–GFP. (b) In this scheme, untreated cells express only SAF-A–AID–mCherry. Addition of doxycycline and auxin (IAA) induces SAF-A–GFP alleles while also causing degradation of SAF-A–AID–mCherry protein through induction of TIR1. (c) The SAF-A degron cell line was reconstituted with either SAF-A^wt–GFP or SAF-A^{S14 S26}–GFP. Cells were labeled with EU to detect RNA localization and immunostained for GFP to detect SAF-A–GFP alleles. Examples of cells in prometaphase and late anaphase are shown. (d) Quantitation of EU-RNA localization in SAF-A–GFP-reconstituted cell lines, expressed as a ratio of chromatin-associated RNA versus cytoplasmic RNA. The mean and SD are represented by the horizontal and diamond-shaped dashed lines, respectively. SAF-A^wt–GFP, n = 25; SAF-A^{S14A S26A}, n = 25. The P value was calculated using a Student’s t test. (e) Chromosome missegregation during anaphase was measured in cells with and without SAF-A–AID–mCherry depletion and in cells reconstituted with SAF-A^wt–GFP or SAF-A^{S14 S26}–GFP. Three biological replicates were performed with 200 anaphases scored in each experiment for the incidence of lagging chromosomes and chromosome bridge formation. Error bars indicate SD. An example of each type of aberrant anaphase is depicted beside the graph with CREST immunostaining to mark kinetochores. Scale bar = 10 µm.