4D map of the tubulin code in the human neuron

National Institute Of Neurological Disorders And Stroke

Investigators

Abstract

Life depends on the efficient and organized transport of cellular cargo on microtubules, polymers composed of alpha/beta-tubulins. No cell type is more dependent on this process than the neuron which needs to support fast, efficient communication between compartments that can be more than 1 m apart. As a result, neuronal processes are one of the densest cellular environments, with microtubules spaced as close as 20 nm that support the large continuous volume of bidirectional cellular traffic necessary for survival and optimal function. Neurons have evolved means to organize and differentiate these microtubule tracks by functionalizing tubulin with chemically diverse posttranslational modifications. An emerging paradigm is that these modifications act as a tubulin code to regulate microtubule dynamics, mechanics and recruitment of microtubule-associated proteins and microtubule-based motors. This project leverages the expertise of our groups on the tubulin code (Roll-Mecak, NINDS) and high-resolution cellular imaging (Hari Shroff, NIBIB and AIM) to generate a complete single-molecule resolution 4D map of the tubulin code and its readers in the human neuron by developing methods for expansion microscopy coupled with single molecule imaging. Since the inception of this award we succeeded in refining the expansion protocols, developing new protocols for expansion of i3Neurons on a glial support, we validated antibodies that are compatible for expansion microscopy, generated knock-in and knock-out lines of tubulin code components, and developed protocols necessary for imaging these expanded samples, which included changes to our microscopy setup to improve sample stability and increase camera sensitivity and resolution, as well as quantitative image analysis pipelines to analyze tubulin modification patterns at the mm scale. The methods we have developed in the past year are broadly applicable to other questions that require imaging at nanoscale resolution and molecular contrast.