Spector, J. | Search bioRxiv

bioRxiv2024

Multi-camera Simultaneous Total Internal Reflection and Interference Reflection Microscopy

Interference Reflection Microscopy (IRM) is an optical technique that relies on the interference between the reflected light from an incident beam as it passes through materials of different refractive indices. This technique has been successfully used to image microtubules, biologically important biofilaments with a diameter of 25 nm. However, it is often desirable to image both the microtubule and microtubule interacting proteins simultaneously. Here we present a simple modification to a standard multi-color total internal reflection fluorescence (TIRF) microscope that enables simultaneous high-speed IRM and single molecule TIRF imaging. Our design utilizes a camera for each channel (IRM and TIRF) allowing independent optimization of camera parameters for the two different modalities. We illustrate its application by imaging unlabeled microtubules and GFP-labeled end-binding protein EB1 which forms comets on the tips of polymerizing microtubules. Our design is easily implemented, and with minimal cost, making it accessible to any laboratory with an existing fluorescence microscope.

biophysics↗

bioRxiv2024

The TTLL10 polyglycylase is stimulated by tubulin glutamylation and inhibited by polyglycylation

Microtubules in cells have complex and developmentally stereotyped posttranslational modifications that support diverse processes such as cell division, ciliary growth and axonal specification. Glycylation, the addition of glycines, singly (monoglycylation) or in chains (polyglycylation), is primarily found on axonemal microtubules where it functions in cilia maintenance and motility. It is catalyzed by three enzymes in the tubulin tyrosine ligase-like family, TTLL3, 8 and 10. We show that TTLL8 monoglycylates both - and {beta}-tubulin, unlike TTLL3 which prefers {beta}-tubulin. Microscopy and mass spectrometry show that TTLL10 requires monoglycylation for high affinity microtubule binding and elongates polyglycine chains only from pre-existing glycine branches. Surprisingly, tubulin polyglycylation inhibits TTLL10 recruitment to microtubules proportional with the number of posttranslationally added glycines, suggesting an autonomous mechanism for polyglycine chain length control. In contrast, tubulin glutamylation, which developmentally precedes polyglycylation in cilia, increases TTLL10 recruitment to microtubules, suggesting a mechanism for sequential deposition of tubulin modifications on axonemes. Our work sheds light on how the tubulin code is written by establishing the substrate preference and regulation of TTLL glycylases and provides a minimal system for generating differentially glycylated microtubules for in vitro analyses of the tubulin code.

biochemistry↗