Figure 5. Spindly binds dynein LIC and... | Rockefeller University Press

Figure 5.

$Figure 5. Spindly binds dynein LIC and the dynactin pointed-end complex. (A) Cartoon of dynein, composed of two heavy chains (HCs), two ICs, two LICs, and six light chains (LCs). The C-terminal region of LIC binds dynein adaptors involved in membrane transport. The two point mutants of human Spindly used throughout this figure are indicated. (B) Sequence alignment showing a conserved region in the first coiled-coil segment of Spindly and other dynein adaptors (CC1 box). The two alanines mutated to valine in Spindly (A23/A24) and BICD2 (A43/A44) are marked with asterisks. (C) Coomassie-stained gels of purified Spindly fragments fused to Strep-tag II (StTgII) as well as GST-tagged full-length (FL) and C-terminal LIC1. (D) Immunoblots of GST pull-downs using the proteins in C. Protein fractions bound to beads were detected on blots with Strep-Tactin. The same membrane was then reprobed with anti-GST antibody. (E) Cartoon of dynactin with the pointed-end complex highlighted. (F) Sequence alignment showing that several dynein adaptors possess a motif that in Spindly is implicated in dynein–dynactin recruitment to kinetochores. The phenylalanine mutated to alanine in Spindly (F258) is marked with an asterisk. (G) Coomassie-stained gels of the purified recombinant dynactin pointed-end complex. The p25 subunit is tagged with 6×His. Arp11 appears as two distinct bands. We verified that both bands correspond to Arp11 by expressing Arp11::6×His (unpublished data). (H) Coomassie-stained gel and immunoblot of Strep-tag II pull-downs using the purified proteins in C and G. (I) Immunoblots of Strep-tag II pull-downs from porcine brain lysate using the purified proteins in C. The same membrane was probed for dynactin p150, dynein IC, and Strep-tagged Spindly. Molecular mass is indicated in kilodaltons.$

Spindly binds dynein LIC and the dynactin pointed-end complex. (A) Cartoon of dynein, composed of two heavy chains (HCs), two ICs, two LICs, and six light chains (LCs). The C-terminal region of LIC binds dynein adaptors involved in membrane transport. The two point mutants of human Spindly used throughout this figure are indicated. (B) Sequence alignment showing a conserved region in the first coiled-coil segment of Spindly and other dynein adaptors (CC1 box). The two alanines mutated to valine in Spindly (A23/A24) and BICD2 (A43/A44) are marked with asterisks. (C) Coomassie-stained gels of purified Spindly fragments fused to Strep-tag II (StTgII) as well as GST-tagged full-length (FL) and C-terminal LIC1. (D) Immunoblots of GST pull-downs using the proteins in C. Protein fractions bound to beads were detected on blots with Strep-Tactin. The same membrane was then reprobed with anti-GST antibody. (E) Cartoon of dynactin with the pointed-end complex highlighted. (F) Sequence alignment showing that several dynein adaptors possess a motif that in Spindly is implicated in dynein–dynactin recruitment to kinetochores. The phenylalanine mutated to alanine in Spindly (F258) is marked with an asterisk. (G) Coomassie-stained gels of the purified recombinant dynactin pointed-end complex. The p25 subunit is tagged with 6×His. Arp11 appears as two distinct bands. We verified that both bands correspond to Arp11 by expressing Arp11::6×His (unpublished data). (H) Coomassie-stained gel and immunoblot of Strep-tag II pull-downs using the purified proteins in C and G. (I) Immunoblots of Strep-tag II pull-downs from porcine brain lysate using the purified proteins in C. The same membrane was probed for dynactin p150, dynein IC, and Strep-tagged Spindly. Molecular mass is indicated in kilodaltons.