Extended Data Fig. 6: Mapping of CEP104 and TOGARAM1 interaction.
a, b, Co-immunoprecipitation of either wildtype CEP104 with indicated TOGARAM1 constructs (a) or wildtype TOGARAM1 with indicated CEP104 constructs (b), the two proteins interact between the linker of TOGARAM1 and the zinc finger of CEP104. Assays were repeated independently at least two times. c, Additional kymographs (scale bars 2 µm and 60 s) illustrating MT dynamics from GMPCPP-stabilized seed with GFP-EB3, mCherry-TOGARAM1, and GFP-CEP104. Assay was repeated independently three times. d, Kymographs illustrating mobility of DmKHC(1-421) on slow growing MT with all CTM proteins (top) and DmKHC (bottom) proving that the slow growing end of the MT is the plus end. Scale bars 2 µm and 60 s for both kymographs. Assay was repeated independently three times e, Additional kymographs (scale bars 2 µm and 60 s) illustrating MT dynamics from GMPCPP-stabilized seeds of the entire CTM with 20 nM GFP-EB3. Assay was repeated independently three times. f, FRAP analysis of CEP104 at slow growing MT plus ends with the entire CTM. Arrowhead marks point of photobleaching in representative kymograph (top), scale bars 2 µm and 60 s. Plot (bottom) show average curve with exponential fit. Number of FRAP measurements, n=19 Error bars represent s.e.m. Assay was repeated independently three times. g-k, Fields of view (left, scale bar 2 µm) and kymographs (right, scale bars 2 µm and 60 s) illustrating MT dynamics from GMPCPP-stabilized seeds with 20 nM GFP-EB3 and indicated concentrations and colors of CTM proteins. Assays were repeated independently three times.
