Lattice Instability Drives Formation of Protofilament Clusters at the Microtubule Plus End Tips

Microtubules (MTs) are dynamic cytoskeletal filaments composed of α- and β-tubulin protein dimers. They are crucial for maintaining cell structure, facilitating intracellular transport, and ensuring proper chromosome segregation among other things. These biological functions are influenced by the dynamic instability of the MT plus-end tip. Recent work has challenged traditional models in which GTP (guanosine-5'-triphosphate) hydrolysis directly regulates microtubule lattice stability. To explore these notions further, in this work, we have constructed, from bottom up, a relatively high resolution coarse-grained (CG) molecular dynamics (MD) model for tubulins with 20 CG sites per tubulin monomer and then performed extensive CG MD simulations on MT lattices with 8 and 40 layers of heterodimers. Our findings demonstrate that, independent of nucleotide state, MT plus-end tips can form distinct, similarly curved protofilament (PF) clusters. These clusters are stable up to tens of microseconds of CG MD simulation time during spontaneous outward bending relaxation, and their formation is driven by an intrinsic thermodynamic instability of the MT lattice. Such lattice instability accumulates in longer microtubules and further facilitates protofilament cluster formation with outward bending relaxation; however, in longer MTs the lattice instability can also be released via lattice curvature and in-lattice twisting. Our systematic parametrization of the CG forcefield from atomic scale interactions also confirms weakened lateral interactions in GDP-MTs, which lead to faster bending relaxation and the formation of more PF clusters in both 8-layer and 40-layer GDP-bound MT lattices. A comprehensive all-atom MD analysis reveals that the formation of PF clusters is initiated by relaxation in the longitudinal interactions and later stabilized by relaxation in the lateral interactions. Our findings highlight the critical role of lattice instability in microtubule dynamics and offer new insight on the behavior of MT plus-end tips.