Figure 8. Introducing the Arl2 GTP-locked Q73L mutation induced pausing of dynamic MTs in vivo.
(A) Expression of cin4-Q73L elicits dominant benomyl sensitivity and MT polymerization defects. Wild type or cin4∆ mutant cells transformed with plasmids expressing the indicated genes under galactose-inducible promoters were plated on inducing media without benomyl or with 10 µg/ml benomyl, and grown at 30°C for 4 days. (B) Expression of cin4-Q73L interferes with MT dynamics and activates pausing. Right panel, wild type cells expressing MT labeled with Tub1-GFP. Strain: yJM0562. Second panel, wild type yeast cells expressing Tub1-GFP transformed with a cin4-Q73L expression plasmid and treated with galactose to induce expression for 90 min before imaging. Third panel, a separate population of wild type cells transformed with a cin4-Q73L expression plasmid and induced for 180 min before imaging. Right panel, cells constitutively expressing cin4-Q73L from the chromosomal locus. Images are 2D projections of 13 Z planes separated by 400 nm. (C) Representative raw fields of yeast cells in each condition with MTs labeled with GFP-tub. Videos 4–7 accompany these panels. (D) Representative lifeplots of astral MT dynamics in wild type cells (top panel), wild type cells expressing cin4-Q73L for 90 min (middle panel), and cin4-Q73L mutants (bottom panel). Astral MT length was measured over time by plotting the distance between the spindle and the distal end of the MT. Strains: wild type, yJM0562; cin4-Q73L, yJM1375. (E) MT rescue frequencies from time-lapse imaging of wild type (n = 5), wild type expressing cin4-Q73L for 90 min (n = 7), and constitutive cin4-Q73L mutant (n = 10) cells. Asterisks indicate statistical significance (p<0.01) determined by t-test, compared to wild type. Strains: wild type, yJM0562; cin4-Q73L, yJM1375. (F) Durations of pause events from time-lapse imaging of wild type (black), wild type expressing cin4-Q73L for 90 min, and constitutive cin4-Q73L mutant cells (shown in pink). Asterisks indicate statistical significance (p<0.01) determined by t-test, compared to wild type. Strains: wild type, yJM0562; cin4-Q73L, yJM1375. (G) Average MT disassembly rates from time-lapse imaging of wild type (black), wild type expressing cin4-Q73L for 90 min, and constitutive cin4-Q73L mutant cells (shown in pink). Asterisks indicate statistical significance (p<0.01) determined by t-test, compared to wild type. Strains: wild type, yJM0562; cin4-Q73L, yJM1375. (H) Average length of astral MTs (aMT) per yeast cell measured, for wild type (black), wild type expressing cin4-Q73L for 90 min, and constitutive cin4-Q73L mutant cells (shown in pink). Asterisks indicate statistical significance (p<0.01) determined by t-test, compared to wild type. Strains: wild type, yJM0562; cin4-Q73L, yJM1375.