Dynactin p27^DNC-6 is required for dynein–dynactin recruitment to kinetochores. (A, left) Cartoon of the C. elegans dynactin pointed-end complex. (Right) Schematic of the endogenous p27^dnc-6 locus modified with a C-terminal 3×flag tag. Stop codons (red stars) and a frameshift mutation (green lettering) were subsequently introduced to create a null allele, dnc-6(−). (B) Genetics of the p27^dnc-6-null allele: F0 mothers heterozygous for dnc-6(−) contain a balancer chromosome with a wild-type copy, dnc-6(wt). F1 progeny homozygous for dnc-6(−) that survive to adulthood on maternally provided p27^DNC-6 produce F2 dnc-6(−/−) embryos that completely lack p27^DNC-6. (C) Immunoblots of F1 adults homozygous for dnc-6(−), demonstrating that levels of dynactin p150^DNC-1 and p50^DNC-2 are unaffected by the absence of p27^DNC-6. Molecular mass is indicated in kilodaltons. (D) Immunofluorescence images of F2 dnc-6(−/−) embryos at the two-cell stage stained with anti-p150^DNC-1 antibody, showing that dynactin without p27^DNC-6 is recruited to the nuclear envelope (arrowheads), the cell cortex (arrows), and centrosomes (asterisks). Blowups show evidence of chromosome missegregation and aneuploidy in F2 dnc-6(−/−) embryos. (E) Immunofluorescence images showing successful bipolar spindle assembly in F2 dnc-6(−/−) embryos. The arrow indicates chromosome missegregation. (F) Selected time-lapse sequence frames of the first mitotic division in embryos expressing mCherry::histone H2B. F2 dnc-6(−/−) embryos exhibit severe chromosome segregation defects that are similar to those observed for the Spindly^SPDL-1 F199A mutant. The defects are significantly ameliorated after depletion of ROD-1, as predicted by the inhibitory cross-talk between the kinetochore dynein module and NDC-80 (summarized in schematic on the left). The frequencies of embryos in which chromosomes congress to metaphase plates are indicated in the percentages and absolute numbers of embryos in parentheses. Time is relative to nuclear envelope breakdown. (G) Selected frames from a time-lapse sequence in F2 dnc-6(−/−) embryos expressing GFP::p50^DNC-2, demonstrating that dynactin without p27^DNC-6 localizes normally to centrosomes and the mitotic spindle (arrowheads) but is absent from kinetochores (arrow). (H, left) Experimental strategy to generate monopolar spindles in F2 dnc-6(−/−) embryos expressing GFP::p50^DNC-2 by depleting the centriole duplication kinase ZYG-1. (Right) Stills from a time-lapse sequence showing that dynactin failed to accumulate on unattached kinetochores of monopolar spindles in the absence of p27^DNC-6. Bars, 5 µm.