Motor-Induced Microtubule Lattice Renewal and Fresh-Kinesin Readout: An Experimental Protocol and Computational Validation Framework

This publication presents an integrated experimental protocol and computational validation framework for investigating whether motor-induced microtubule lattice renewal produces persistent functional alterations detectable by subsequent kinesin transport. Building upon published experimental evidence of motor-induced microtubule lattice damage, tubulin incorporation, and kinesin sensitivity to lattice defects, the study proposes a controlled writer–reader experimental system. In this approach, kinesin motors interact with microtubules in the presence of soluble tubulin, followed by verified removal of the original motors and introduction of freshly labeled kinesin to evaluate subsequent transport behavior. The proposed experimental measurements include motor landing rates, velocity, pausing, trajectory survival, and spatial associations with incorporation-marked lattice regions. Randomized controls, residual-motor detection, imaging calibration, and statistical analysis are incorporated to distinguish genuine functional alterations from experimental artifacts. The publication also includes mathematical models and reproducible computational benchmarks examining photobleaching, motor crowding, tracking errors, trajectory crossings, and informative censoring. These simulations evaluate potential sources of bias in single-molecule fluorescence measurements and establish methodological considerations for interpreting future experimental results. The accompanying supplementary archive contains Python source code, synthetic datasets, numerical outputs, and reproducibility documentation. Scientific scope: This work presents an experimentally testable research hypothesis and computational methodology. It does not claim that persistent microtubule memory has been experimentally demonstrated, nor does it establish a new biological mechanism. Its objective is to provide a transparent, falsifiable framework that experimental laboratories can independently evaluate, reproduce, and extend. Keywords: Microtubules; Kinesin-1; Microtubule Lattice Repair; Tubulin Incorporation; Molecular Motors; Single-Molecule Imaging; Motor Transport; Computational Modeling; Photobleaching; Survival Analysis; Experimental Protocol; Reproducibility.