Round Tubulin Droplet Formation in Presence of Cross-linkers

Phase separation phenomena in proteins have emerged as an important activity in biological systems in recent years. In cell, it has been found that cytoplasmic and nuclear bodies, such as nucleoli, cajal bodies, stress granules, germ granules, centrosome are membraneless organelles formed by phase separation. These organelles can spontaneously form when certain external factor is triggered. Prior work points to unstructured and highly charged domains being responsible for liquid phase separation, but proteins with a well-formed structure should not do this. Surprisingly, we have observed that tubulin, a well-characterized enzyme that polymerizes into microtubules, can display droplet-like phase separation in the presence of a small molecule crosslinker, Sulfo-SMCC, and a microtubule-associated protein, MAP65. These droplets are driven by the presence of MAP65, which co-localizes with the droplets. We expect that the sulfo-SMCC introduces multi-valency via cystine residues in the tubulin which might help in the phase transition. Prior work showed that Sulfo-SMCC can stop microtubules from annealing by crosslinking within the tubulin dimer. Although the droplets are initially liquid, they age quickly and become viscoelastic blobs that show little to no liquidity at later time.