Extracts from syncytial wild type embryos were immunoprecipitated with anti-KLP61F serum or mock-precipitated (no Ab IP) and western blotted for KLP61F (A) or dWee1 (B). Samples equivalent to 0.6% and 1.2% of starting extracts were loaded for comparison.

(A–B) Pupal lethality of klp61f³ mutants (A) and level of aneuploidy in klp61f³ mutant neuroblasts (B) are rescued by a Myc-KLP61F^WT transgene ([WT]; klp61f³) but not a Myc-KLP61F^3YF transgene ([3YF]; klp61f³). Data is represented as percentage of dead pupae (mean +/− SD from 3 embryo collections) in (A) and as percentage of aneuploid nuclei (mean +/− SD of nuclei from 3 larval brains) in (B). * indicates p < 0.01.

(A) Schematic representation of KLP61F with head domain (red), coiled-coil (blue) and tail domain that contains the BimC box (yellow). His-tagged polypeptides used in kinase assays are depicted. (B) His-KLP61F was incubated with varying amounts of GST-dWee1 in kinase reactions in vitro and analyzed for ³²P incorporation. (C) Reaction with equimolar GST-dWee1 and His-KLP61F was western blotted with anti-phosphotyrosine antibody after in vitro kinase assays. (D–E) GST-dWee1 and His-KLP61F polypeptides were incubated in in vitro kinase reactions and analyzed for ³²P incorporation (left panels) and subsequently by western blots for phosphotyrosines (right panels). (F) KLP61F head domain with Y23, Y152 and Y207 mutated to phenylalanines, His-Head^3YF, was incubated with GST-dWee1 in in vitro kinase reactions and analyzed for ³²P incorporation. none = no kinesin; GST-KHC = conventional Kinesin Heavy Chain head domain; His-Head^WT = wild type head domain. (G–H) Western blots for GST (G) or His tags (H) show equivalent amounts of substrates in kinase reactions in (F).

Embryos from mothers that are wild type, klp61f³ heterozygotes (klp61f), klp61f³ heterozygotes with Myc-KLP61F^WT ([WT]) and klp61f³ heterozygotes with Myc-KLP61F^3YF ([3YF]) were fixed and stained for DNA (blue), α-tubulin (green), and centrosomin (red). Representative images show a normal spindle (A) and defects significantly different among the genotypes: a monopolar spindle (B), an anastral spindle (C), and spindles with multiple MTOCs (F–G) from interior divisions, and a promiscuous microtubule interaction during anaphase in cortical divisions (H). Scale bar = 5 µm. Defects in (A–C) and (F–H) are quantified in (D) and (E) respectively. See Table 1 for numbers. * p < 0.01, ** p < 0.001, and ^ p < 0.05. (I) A model for regulation of KLP61F to maintain mitotic spindle integrity and prevent promiscuous MT interactions. dWee1 may regulate KLP61F activity to bundle parallel (not shown) and/or anti-parallel microtubules to create a more robust spindle (left). Without dWee1 regulation, KLP61F activity is reduced on the spindle, leading to an unstable spindle with microtubule spurs. These microtubule spurs can then interact with neighboring spindles in a syncytium (spur-spindle interaction is not depicted). Reduced KLP61F activity on the spindle is depicted as reduced protein levels on the spindle, but it remains possible that similar level of protein associate with the spindle but display reduced activity.