Label Free Microtubule Microscopy

January5

The exciting work of Mohammed Mahamdeh and Joe Howard (J.Microsc. https://doi.org/10.1111/jmi.12744) prompted me in the December break from teaching in 2018 to try and reproduce it, using the Nikon TiE inverted epifluorescence microscopy in the lab. With the able presence of a talented Masters student Yash Jawale (now PhD student in the Netherlands), we attempted to uncover its secret levers of light attentuation. After frustrating months, we had a visitor from France, Dr. Kheya Sengupta from CINAM Marseille. As an expert on IRM and RICM (a related method) she helped us achieve it- label free microscopy of the 30 nm wide single MT filaments in (IRM)! And since then we have been toying with it, getting better images with passing days. Now if only the remaining experiments would work!

One, many and the nanometer collective: Dynein gliding assays show the way

January8

Our 5 year long study on dynein can be summarized in the “one, many and collective” phrase. We have been able to show that teams of motors on a surface in a gliding assay appear to transport different lengths of microtubules differenty. In challenging experiments that went beyond qualitative, Kunalika Jain from the lab optimized a quantitative gliding assay that allows her to infer the change in 2D directionality of transpoted MTs.

Setup of the dynein gliding assay in simulation and experiment

Dynein Collective Transport: Simulation & Experiment (Jain et al. 2019 Soft Matter)

Given the details available at a single molecule level, we asked whether counter-intuitive inferences could be made about the role of length-dependent transport. Turns out due to the peculiar detachment mechanics and “search and capture”, MTs are indeed transported in a more directional manner when 3 um long filaments encounter approximately 10 motors or more. Why 10, why 3 um,.. read more in our paper by Jain, Khetan and Athale (2019) Soft Matter.