(A) A schematic illustration of exogenous gene expression of Mem-BFP-iLID and NuMA-Nano fusions from the AAVS1 and Rosa 26 loci, respectively. Whereas Mem-BFP-iLID is stably expressed, NuMA-Nano fusions are conditionally expressed following the treatment with Doxycycline (Dox). The SNAP-tag was also inserted at the DHC1 gene loci. (B) Schematic of experimental procedures. LGN siRNA, Dox, and SiR-647 were used to deplete endogenous LGN, to induce expression of NuMA-Nano fusions, and to label endogenous DHC-SNAP, respectively. Cells were treated with RO-3306 and MG132 to synchronize at G2 and to arrest cells at metaphase, respectively. Cells were observed by microscopy 1 hr after the release from G2 arrest, and analyzed for 3 hr. (C) Left: the tested NuMA truncation fragments. Right: A summary of the cortical dynein recruitment. (D–G) Live fluorescent images of indicated NuMA constructs (upper) and DHC-SNAP (lower). NuMA (1-505) was sufficient to recruit dynein to the cell cortex, and both its N-terminal globular domain (aa: 1–213) and short coiled-coil region (aa: 414–505) were required for cortical dynein recruitment. Similar to NuMA (1-705) WT, NuMA (1-505), but not other truncated fragments, accumulated around spindle pole adjacent to light-illuminated region following cortical recruitment. (H) Merged images of NuMA(1-705)-RFP-Nano (magenta) and DHC-SNAP (green) from Figure 4F. NuMA(1-705)-RFP-Nano WT, but not 4A mutant, asymmetrically accumulates around the spindle pole, indicating that NuMA N-terminal fragments containing the Spindly motif are sufficient to recruit dynein-dynactin to the mitotic cell cortex, and likely to activate dynein’s motility, which in turn transports these NuMA fragments toward the spindle pole. (I) The definition of spindle displacement. For cases where Dmax (maximal spindle displacement distance) is longer than Dt=0 (starting pole-to-cortex distance)×1/2, the spindle is assessed as displaced. (J) Scatterplots of Dmax as a percentage of Dt=0 for each condition. Cells with displaced spindles are plotted to the right side of the red line, which indicates 50% of the spindle displacement distance. For NuMA fragments, all samples (except #2 and #11) show statistical difference (p<0.05) when compared to control (#1) using the Mann-Whitney test. Purple lines indicate mean ±SD. See Figure 4C and Figure 5B for the number of cells. (K) Genomic PCR showing the genotype of clones after Blasticidin (BSD) selection. Both clones display a single 4.6 kb band, indicating that the SNAP (BSD) cassette was inserted in both DHC1 gene loci. The clone No.11 was used as a parent in the 3rd selection. (L) Genomic PCR showing the genotype of clones after Hygromycin (Hygro) selection. A 1.6 kb band confirms the insertion of NuMA-RFP-Nano (Hygro) cassettes with different NuMA fragments at the Rosa 26 locus. The following clones were used; ΔNLS (#1: No.7), 1–1700 (#2: No.28), 1–705 (#3: No.5), 1–705 4A (#4: No.18). 214–705 (#5: No.2), 1–505 (#6: No.21), 1–413 (#7: No.23), and 1–213 (#8: No.8), Scale bars = 10 μm.