Figure 2.
Figure 2. The entire N-terminal coiled-coil of Ndel1, but not the L IS1-binding domain alone, is sufficient to rescue microtubule aster formation in Xenopus egg extracts. (a) Schematic of truncated mouse Ndel1 (mNdel1) proteins used in rescue experiments. (b) Ndel1 proteins were immunodepleted greater than 90%. CSF-arrested extracts were diluted to the specified amount or control ΔIgG and ΔxNdel1 extracts were immunoblotted with affinity-purified polyclonal α-Ndel1 antibodies. (b′) Representative images of the predominant microtubule structures observed after rescuing xNdel1-depleted extracts (ΔxNdel1) with the indicated recombinant proteins. Structures such as those in ΔxNdel1+1–174 (mNdel11–174), ΔxNdel1+8–192 (mNdel18–192), and ΔNdel1+8–310 (mNdel18–310) would be classified as asters, whereas those seen in ΔxNdel1+BSA and ΔxNdel1+8–99 (mNdel18–99) are unfocused structures. (b′′) Quantification of three independent experiments after Ndel1 depletion and rescue with truncated mouse Ndel1 proteins; for each depletion and rescue condition over 100 microtubule structures were quantified; asterisks indicate statistically significant differences (Student’s t test with P < 0.05; *** indicates P < 0.0005). (c) Ndel1 coimmunoprecipitates LIS1, but not dynein and dynactin in Xenopus egg extracts, and α-Ndel1 antibodies do not deplete LIS1. α-Ndel1 antibodies were used to immunoprecipitate proteins from Xenopus CSF-arrested extracts. The resulting precipitates (IPs) and extracts from which the IP was performed (SUPS) were immunoblotted for indicated proteins. (d) Level of Ndel1 depletion from CSF-arrested egg extract as measured by immunoblot. (d′) LIS1-binding domain mNdel188–192 (88–192) is not sufficient to rescue aster formation in Ndel1-depleted extracts. (d′′) Quantification of microtubule structures from three independent experiments where Ndel1-depleted extracts were rescued with the indicated mNdel1 variants. Over 100 microtubule structures were quantified, asterisks indicate statistically significant differences (Student’s t test with P < 0.05; *** indicates P < 0.0005). Bar, 20 µm.

The entire N-terminal coiled-coil of Ndel1, but not the L IS1-binding domain alone, is sufficient to rescue microtubule aster formation in Xenopus egg extracts. (a) Schematic of truncated mouse Ndel1 (mNdel1) proteins used in rescue experiments. (b) Ndel1 proteins were immunodepleted greater than 90%. CSF-arrested extracts were diluted to the specified amount or control ΔIgG and ΔxNdel1 extracts were immunoblotted with affinity-purified polyclonal α-Ndel1 antibodies. (b′) Representative images of the predominant microtubule structures observed after rescuing xNdel1-depleted extracts (ΔxNdel1) with the indicated recombinant proteins. Structures such as those in ΔxNdel1+1–174 (mNdel11–174), ΔxNdel1+8–192 (mNdel18–192), and ΔNdel1+8–310 (mNdel18–310) would be classified as asters, whereas those seen in ΔxNdel1+BSA and ΔxNdel1+8–99 (mNdel18–99) are unfocused structures. (b′′) Quantification of three independent experiments after Ndel1 depletion and rescue with truncated mouse Ndel1 proteins; for each depletion and rescue condition over 100 microtubule structures were quantified; asterisks indicate statistically significant differences (Student’s t test with P < 0.05; *** indicates P < 0.0005). (c) Ndel1 coimmunoprecipitates LIS1, but not dynein and dynactin in Xenopus egg extracts, and α-Ndel1 antibodies do not deplete LIS1. α-Ndel1 antibodies were used to immunoprecipitate proteins from Xenopus CSF-arrested extracts. The resulting precipitates (IPs) and extracts from which the IP was performed (SUPS) were immunoblotted for indicated proteins. (d) Level of Ndel1 depletion from CSF-arrested egg extract as measured by immunoblot. (d′) LIS1-binding domain mNdel188–192 (88–192) is not sufficient to rescue aster formation in Ndel1-depleted extracts. (d′′) Quantification of microtubule structures from three independent experiments where Ndel1-depleted extracts were rescued with the indicated mNdel1 variants. Over 100 microtubule structures were quantified, asterisks indicate statistically significant differences (Student’s t test with P < 0.05; *** indicates P < 0.0005). Bar, 20 µm.

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