Fig. 5

NEK7 and Eg5 limit retrograde microtubule growth in distal dendrites. a Hippocampal cultures were infected with control or Nek7 shRNA virus at 3 DIV, transfected with EB3-tomato at 8 DIV, and analyzed by time-lapse microscopy at 9 DIV. Kymographs show EB3-tomato comet dynamics in distal dendrites. White arrowheads indicate retrograde EB3 comets. b Quantification of EB3 comet density in proximal and distal segments of dendrites in control or Nek7-depleted neurons; n = 3 independent experiments. Total of 29 (Control shRNA) and 26 (shNek7) dendrites. *P < 0.05 by two-tailed t-test. c Quantification of the polarity of comets analyzed in (b). Statistics as in (b). d Neurons were treated with DMSO or STLC at 5 DIV and transfected with EB3-tomato reporter and analyzed as in (a). Kymographs show EB3-tomato comet dynamics in distal dendrites. White arrowheads indicate retrograde EB3 comets. e Quantification of EB3 comet density in proximal and distal segments of dendrites in DMSO or STLC-treated neurons; n = 3 independent experiments. Total of 29 (DMSO) and 27 (STLC) dendrites. *P < 0.05 by two-tailed t-test. f Quantification of the polarity of EB3 comets in (e). Statistics as in (e). g Hippocampal cultures were co-transfected with GFP or GFP-CDK5RAP2 51–100, and the reporter EB3-tomato at 6 DIV, and analyzed by time-lapse microscopy at 9 DIV. Kymographs show EB3-tomato comet dynamics in distal dendrites. White arrowheads indicate retrograde EB3 comets. h Quantification of EB3 comet density in proximal and distal segments of dendrites in neurons expressing GFP or GFP-CDK5RAP2 51–100; n = 3 independent experiments. Total of 24 dendrites (GFP) and 25 dendrites (GFP-C5R2 51–100). *P < 0.05 by two-tailed t-test. i Quantification of EB3 comet polarity in the segments analyzed in (h). Statistics as in (h). j Nine DIV neurons expressing GFP or GFP-CDK5RAP2 51–100 were stained with GFP antibody. Scale bar, 25 μm. k Quantification of total dendrite length of neurons as in (j); n = 3 independent experiments, total number of neurons: 71 (GFP) and 36 (GFP-CDK5RAP2 51–100). *P < 0.05 by two-tailed t-test. l Mean number of dendrite intersections in neurons as in (j). Statistics as in (k). Error bars: s.e.m. Columns in all graphs show means and dot overlays individual data points