Figure 1. Generation of KNL-1 microtubule-binding mutants and development of an in vivo system to analyze KNL-1 functions. (A) Primary sequence features of KNL-1. A conserved basic patch is highlighted. Mutations engineered to neutralize the basic patch (4A) or delete it (Δ9) are indicated. (B) Purification of WT and mutant KNL-11–505 proteins from bacteria using the indicated steps. The final purified proteins were analyzed by SDS-PAGE and Coomassie staining. (C) Microtubule-bundling analysis of recombinant KNL-11–505 proteins. 1 µM taxol-stabilized rhodamine microtubules (MTs) was imaged either alone or in the presence of the indicated 2-µM KNL-11–505 variants. Bar, 10 µm. (D) Analysis of recombinant KNL-11–505 proteins by gel filtration chromatography. Fractions were analyzed by SDS-PAGE followed by immunoblotting with an anti–KNL-1 antibody. Ipt, input. (E) Bead assay to analyze microtubule-binding activity of KNL-1. 20 fields were photographed, and the number of bound beads was quantified. (F) A schematic of the KNL-1 transgene (KNL-1RR::mCherry) targeted to a single Mos transposon insertion on chromosome II (Chr II). The transgene has the endogenous knl-1 promoter and 3′ untranslated region (UTR), mCherry fused to the C terminus, and exon 4 modified to preserve coding information but alter nucleotide sequence, thereby enabling RNAi-mediated depletion of endogenous KNL-1. (G) dsRNA targeted to the recoded region selectively depletes >95% of endogenous KNL-1. α-Tubulin serves as a loading control. (H–K) Single-copy transgene insertion–encoded KNL-1RR::mCherry is fully functional. Kinetochore localization (H), embryonic viability (I; n = number of embryos scored), chromosome segregation phenotype (J), and kinetic analysis of spindle pole separation (K) are shown. (H and J) GFP::H2b and GFP::γ-tubulin were crossed into the KNL-1RR::mCherry transgenic strain to visualize chromosomes (arrow) and spindle poles (arrowheads), respectively. Bars, 5 µm. (J) Frames from time-lapse sequences aligned relative to NEBD (t = 0); the mCherry signal is not shown. (K) Pole–pole distance measured at 10-s intervals, aligned relative to NEBD, averaged for the indicated number (n) of embryos, and plotted versus time. Inset shows the time of anaphase onset in control (nontransgenic) and transgenic endogenous KNL-1–depleted one-cell embryos. Error bars represent the SEM with a 95% confidence interval.
Figure 1.

Generation of KNL-1 microtubule-binding mutants and development of an in vivo system to analyze KNL-1 functions. (A) Primary sequence features of KNL-1. A conserved basic patch is highlighted. Mutations engineered to neutralize the basic patch (4A) or delete it (Δ9) are indicated. (B) Purification of WT and mutant KNL-11–505 proteins from bacteria using the indicated steps. The final purified proteins were analyzed by SDS-PAGE and Coomassie staining. (C) Microtubule-bundling analysis of recombinant KNL-11–505 proteins. 1 µM taxol-stabilized rhodamine microtubules (MTs) was imaged either alone or in the presence of the indicated 2-µM KNL-11–505 variants. Bar, 10 µm. (D) Analysis of recombinant KNL-11–505 proteins by gel filtration chromatography. Fractions were analyzed by SDS-PAGE followed by immunoblotting with an anti–KNL-1 antibody. Ipt, input. (E) Bead assay to analyze microtubule-binding activity of KNL-1. 20 fields were photographed, and the number of bound beads was quantified. (F) A schematic of the KNL-1 transgene (KNL-1RR::mCherry) targeted to a single Mos transposon insertion on chromosome II (Chr II). The transgene has the endogenous knl-1 promoter and 3′ untranslated region (UTR), mCherry fused to the C terminus, and exon 4 modified to preserve coding information but alter nucleotide sequence, thereby enabling RNAi-mediated depletion of endogenous KNL-1. (G) dsRNA targeted to the recoded region selectively depletes >95% of endogenous KNL-1. α-Tubulin serves as a loading control. (H–K) Single-copy transgene insertion–encoded KNL-1RR::mCherry is fully functional. Kinetochore localization (H), embryonic viability (I; n = number of embryos scored), chromosome segregation phenotype (J), and kinetic analysis of spindle pole separation (K) are shown. (H and J) GFP::H2b and GFP::γ-tubulin were crossed into the KNL-1RR::mCherry transgenic strain to visualize chromosomes (arrow) and spindle poles (arrowheads), respectively. Bars, 5 µm. (J) Frames from time-lapse sequences aligned relative to NEBD (t = 0); the mCherry signal is not shown. (K) Pole–pole distance measured at 10-s intervals, aligned relative to NEBD, averaged for the indicated number (n) of embryos, and plotted versus time. Inset shows the time of anaphase onset in control (nontransgenic) and transgenic endogenous KNL-1–depleted one-cell embryos. Error bars represent the SEM with a 95% confidence interval.

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