Fig. 6: Tubulin and filamin A form a complex with CLIP170, which elicits increased binding of paclitaxel to microtubules.

A Spatial colocalization of alpha-tubulin and filamin A in MDA-MB-231 cells (Pearson’s colocalization coefficient = 0.5, n = 76 cells). Scale bars: 10 µm. B Filamin A pull-down in MDA-MB-231 WT or CDK4 cell lines. For each isolated protein, the X axis represents the Log₂ of the average ratio of the protein isolated in the anti-filamin A pull-down and the protein isolated in the IgG control antibody. The Y axis, conversely, represents the Log₁₀ of the sum of the average intensities found bound to the anti-filamin-A antibody and the IgG isotype control. C Same as in (B) for tubulin pull-downs. D Coimmunoprecipitation of CLIP-170 and filamin A. Filamin A was immunoprecipitated in whole-cell lysates from the three cell lines: MDA-MB-231 WT, CDK4 and FLNA. Three samples are shown for each cell line: total lysate, immunoprecipitated with anti-filamin A, and immunoprecipitated with isotype IgG control. Samples derive from the same experiment and blots were processed in parallel. Experiment was repeated 3 times with similar results. E Schematic representing the proposed mechanism. On the left-hand side, low CDK4 levels lead to average filamin A expression, which does not enhance the binding of CLIP-170 to microtubules. On the right-hand side, tumour cells with increased CDK4 levels would lead to the overexpression of filamin A. Filamin A would recruit an excess of CLIP-170 to tubulin, which ultimately leads to increased binding of paclitaxel and microtubule acetylation and hyperstabilization. F Paclitaxel-binding experiment. Fluorescently labelled paclitaxel was added to live cultures of MDA-MB-231 WT, CDK4 or FLNA cells. MDA-MB-231 CDK4 cells with filamin A knockdown were added to the experiment as well. The greater the green signal is, the higher the amount of paclitaxel bound to microtubules. It can be appreciated how both CDK4- and filamin A-overexpressing cell lines display both earlier and higher paclitaxel binding. Scale bar: 75 µm. The chart on the right-hand side depicts the signal (in fluorescent surface units) tracing paclitaxel accumulation over the 48-h time course, displaying a clear increase in the two overexpressing transfectants (CDK4 and FLNA) compared to the parental cell line and a reversion of the phenotype by filamin A knockdown in MDA-MB-231 CDK4 cells. Each dot represents mean signal intensity of six independent fields. Source data are provided as a Source data file.