Fig. 5: Multimers of CENP-V bind with high affinity to, diffuse along and bundle microtubules. | Nature Communications

Fig. 5: Multimers of CENP-V bind with high affinity to, diffuse along and bundle microtubules.

From: CENP-V is required for proper chromosome segregation through interaction with spindle microtubules in mouse oocytes

Fig. 5

a Binding of recombinant CENP-V-eGFP (top panel row) to taxol-stabilised ATTO 647N-labelled microtubules (bottom panel row) at the indicated CENP-V concentrations. Please refer to Sup. Figure 7A for pictures of the full concentration range and supplementary. Mov. S5. Bar equals 10 µm. b Saturation binding curve of CENP-V-eGFP showing the concentration-dependent fluorescence intensity of microtubule-associated CENP-V-eGFP in arbitrary units (arb.units) per micrometer. The dissociation constant (Kd) and the Hill-coefficient (h) are given. Error bars indicate the standard error of the mean of three independent experiments. Blue line is a Hill-, dashed grey line a Michaelis-Menten-model fit. (c) Representative kymographs (I-V) showing the behaviour of 1 nM CENP-V-eGFP on the lattice of taxol-stabilised ATTO 647N-labelled microtubules in BRB80 at 25 °C. Blue arrowheads indicate transitions between static and motile periods. White arrowheads indicate fast shift events during slow diffusion episodes. Scheme to the right indicates the extend of the respective microtubule lattice (red) and the microtubule ends (asterix). (d) Mean square displacement (MSD) analysis of motile CENP-V-eGFP particles. Data is fitted with a linear regression (black line); blue error bars are the standard error of the mean. (e) Histogram showing the initial fluorescence intensity of motile CENP-V-eGFP particles – i.e., the mean intensity during the first (in average75) movie frames. Blue lines indicate Gaussian fits to the multiple peaks of the histogram adding up to the cumulative Gaussian fit shown as a green line. Dashed lines indicate the local maxima of the respective fits. The respective peak intensity values in arbitrary units (a.u.) ± standard error of the mean are given above. (f) Plot showing the intensity loss during single bleaching events (n = 50) of motile CENP-V-eGFP particles (n = 25). Solid and dashed lines indicate median and mean (98.9 a.u. and 118.3 a.u. ± 8.2 s.e.m. respectively). (g) Surface immobilised axol-stabilised ATTO 647N-labelled microtubules after pre-incubation with either buffer, 50 nM CENP-V or CENP-V-eGFP (top panel row). The corresponding eGFP-channels are shown in the bottom panel row. Scale bar = 20 µm. Representative images of three independent experiments are shown.

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