Figure S3.
Expression pattern, localization and severing activity of kat-60L1. (A)Kat-60L1-short-gal4 (kat-60L1-short-gal4; uas-cd4-tdgfp) drove strong expression in the central nervous system (red arrow, right panel) and weak expression in peripheral sensory neurons (left; confocal images). The expression can only be detected in a few haltere campaniform receptors, and no signal was observed in larval da neurons. Scale bars, 10 µm (left) and 100 µm (right). (B)Kat-60L1-long-gal4 (kat-60L1-long-gal4; uas-cd4-tdgfp) drove strong expression in both central nervous system (right panel) and peripheral sensory neurons (left panel and red arrows, right panel; confocal images). Scale bars, 10 µm (left) and 100 µm (right). Note that in A and B, the representative images for haltere and larva are juxtaposed for display. (C and D) RFP-Kat-60L1-long showed an enriched signal at the basal body and diffusive signals in the outer segment in wing (C) and leg (D) campaniform mechanoreceptors (confocal images). Genotype, uas-rfp-kat-60L1-long; kat-60L1-long-gal4/uas-cd4-tdgfp. Scale bars, 5 µm. (E) A protein gel image (Coomassie blue staining) of purified MBP-GFP-kat-60L1 (long isoform, indicated with a black arrowhead). Lane 3 was overloaded to check the protein purity. (F) The protein gel image of the microtubule spin-down assay. Kat-60L1 was able to disassemble taxol-stabilized microtubules in vitro in the presence of ATP. In the presence of ADP, kat-60L1 bound to microtubules but could not disassemble them. s, supernatant; p, pellet. (G) Representative TIRF images showing that kat-60L1 (300 nM) was able to disassemble taxol-stabilized microtubules in the presence of ATP (1 mM) but not ADP (1 mM). The microtubules indicated with yellow arrowheads are enlarged in H. Scale bar, 10 µm. (H) TIRF images showing that kat-60L1 was able to sever microtubules in the presence of ATP (1 mM; lower) but not ADP (1 mM; upper). Scale bar, 5 µm. (I) Statistical quantification of the severing activity of kat-60L1 in the presence of ATP (1 mM; n = 4 assays) or ADP (1 mM). The total length of microtubules at each time point was measured to reflect the mass of microtubules. Data are presented as mean ± SEM (n = 4 assays). BB, basal body. OS, outer segment.

Expression pattern, localization and severing activity of kat-60L1. (A) Kat-60L1-short-gal4 (kat-60L1-short-gal4; uas-cd4-tdgfp) drove strong expression in the central nervous system (red arrow, right panel) and weak expression in peripheral sensory neurons (left; confocal images). The expression can only be detected in a few haltere campaniform receptors, and no signal was observed in larval da neurons. Scale bars, 10 µm (left) and 100 µm (right). (B)Kat-60L1-long-gal4 (kat-60L1-long-gal4; uas-cd4-tdgfp) drove strong expression in both central nervous system (right panel) and peripheral sensory neurons (left panel and red arrows, right panel; confocal images). Scale bars, 10 µm (left) and 100 µm (right). Note that in A and B, the representative images for haltere and larva are juxtaposed for display. (C and D) RFP-Kat-60L1-long showed an enriched signal at the basal body and diffusive signals in the outer segment in wing (C) and leg (D) campaniform mechanoreceptors (confocal images). Genotype, uas-rfp-kat-60L1-long; kat-60L1-long-gal4/uas-cd4-tdgfp. Scale bars, 5 µm. (E) A protein gel image (Coomassie blue staining) of purified MBP-GFP-kat-60L1 (long isoform, indicated with a black arrowhead). Lane 3 was overloaded to check the protein purity. (F) The protein gel image of the microtubule spin-down assay. Kat-60L1 was able to disassemble taxol-stabilized microtubules in vitro in the presence of ATP. In the presence of ADP, kat-60L1 bound to microtubules but could not disassemble them. s, supernatant; p, pellet. (G) Representative TIRF images showing that kat-60L1 (300 nM) was able to disassemble taxol-stabilized microtubules in the presence of ATP (1 mM) but not ADP (1 mM). The microtubules indicated with yellow arrowheads are enlarged in H. Scale bar, 10 µm. (H) TIRF images showing that kat-60L1 was able to sever microtubules in the presence of ATP (1 mM; lower) but not ADP (1 mM; upper). Scale bar, 5 µm. (I) Statistical quantification of the severing activity of kat-60L1 in the presence of ATP (1 mM; n = 4 assays) or ADP (1 mM). The total length of microtubules at each time point was measured to reflect the mass of microtubules. Data are presented as mean ± SEM (n = 4 assays). BB, basal body. OS, outer segment.

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