Quantitative analysis of microtubule lattice damage in NF1-deficient HER2-positive breast cancer under mitotic and pharmacological stress

Our understanding of microtubule (MT) dynamics has recently expanded to include remodeling events within the lattice, where mechanical stress generates discontinuities repaired through intra-lattice tubulin incorporation and EB1 redistribution. Only two factors, CLASP1 and SSNA1, are known to recognize and modulate this form of MT damage. Methods to reliably estimate MT damage and repair are not established, leaving their contribution to tumorigenesis and anticancer drug activity unclear. In our previous work, we demonstrated that loss of the tumor suppressor NF1, a new MT-associated protein (MAP), leads to increased MT damage in HER2-positive breast cancer cells. This in turn causes mitotic defects (potentially contributing to tumorigenesis) but also sensitization to maytansinoids like DM1, a drug class frequently used in Antibody-Drug Conjugates (ADC, T-DM1 and Mirvetuximab Soravtansine). To investigate MT damage and repair upon pharmacological and mechanical stress, we analyzed the distribution of EB1, normally confined to growing MT tips but known to redistribute along the MT body upon structural disruption or when repair factors like CLASP are ablated. We developed a novel method to systematically quantify EB1 and tubulin signal along single MTs. To achieve the spatial precision required to resolve EB1 we combined DeepSIM and expansion microscopy (ExM) with two complementary analytical strategies: manual 3D MT tracing using SNT ImageJ plugin (designed for neurite tracing) and an automated pipeline for MT segmentation and EB1 signal extraction. Upon DM1 treatment, EB1 showed progressive re-localization from the tip to the MT body, consistent with partial intra-lattice disruption in addition to its established de-polymerizing activity at the tip. In NF1 deficient cells, an initial increase in intra-lattice EB1 was followed by a sharp reduction in overall EB1 signal and pronounced MT fragmentation, indicating faster DM1-induced MT fracturing and depolymerization; this was abolished by NF1 re-expression. These results directly show how failed MT repair, more prominent in NF1 deficient cells, creates further intra-lattice DM1 binding sites explaining increased sensitivity. In mitosis, NF1 deficient cells showed strongly increased EB1 signal on the MT shaft, consistent with failure of repair under strong mechanical stress. EB1 shaft localization and mitotic defects (chromosome missegregation, aneuploidy) were rescued by NF1 re-expression.By establishing a quantitative framework to measure EB1 redistribution at single MT resolution, our study provides a previously unavailable strategy to monitor intra-lattice damage in cancer cells. These findings establish for the first time a direct causative link between unrepaired MT damage and chromosomal instability, providing a new model to understand maytansinoid mechanism of action.