Fig. 3. The C-terminal domain of the long Shot isoforms (C-Shot L) binds to microtubules. (A) C-Shot L contains two EF-hand motifs (hatched) (Ikura, 1996) and homology to GAS2 (black) (Brancolini et al., 1992). EGY48 yeast cells (Gyuris et al., 1993) grow on leucine-depleted selective medium when they express the C-Shot L-lexA fusion and an 
-tubulin-activation domain fusion, but do not grow when they express a bicoid-lexA fusion and an -tubulin-activation domain fusion. The cDNA inserts of the two clones encode the C-terminal regions of tubulin 1 (amino acids 344 to 450, Accession Number, P06603) and tubulin 3 (amino acids 343 to 450, Accession Number P06605) (Theurkauf et al., 1986). EGY48 yeast also do not grow on selective medium when they contain the C-Shot L-lexA fusion and the activation domain alone (data not shown). (B) Summary of the qualitative ability of C-Shot L fragments to associate with microtubules in transfected cells. ++, as in E,K; +/–, as in H. (C-E) An NIH3T3 cell transfected with a C-Shot L-GFP expression vector. (F-H) A cell transfected with a GFP fusion to sequences C-terminal to the GAS2 sequence. (I-K) Cells treated with 1 µM nocodazole for 20 minutes. All cells transfected with the GAS2 motif-GFP expression vector (arrows) still contain microtubules, whereas no control cells (arrowhead) contain detectable microtubules. (L-N) Cells transfected with a GFP fusion to an N-terminal fragment from isoform Shot L(C) that contains the second calponin homology repeat and the plakin-like domain. (C,F,I,L) Microtubule distribution. (D,G,J,M) GFP expression and distribution. (E,H,K,N) Overlap (yellow) between microtubule (red) and GFP (green) distributions. Only GFP fusions with C-Shot L fragments that contain the GAS2 motif associate strongly with microtubules (E,K) and stabilize microtubules in transfected cells against nocodazole treatment (I-K). GFP fusions with a C-terminal fragment lacking the GAS2 motif associate weakly (H, arrow), and GFP fusions with the Shot plakin-like domain do not associate with microtubules (N). Images in C-N are 1 µm confocal sections. Scale bars: 10 µm.
