Domain characterization of mitotic spindle protein CKAP2 and Dissecting how it regulates Microtubule dynamics In Vitro.

Abstract

This abstract contains text that is close to or identical to text found in my Junior Paper, Michael Ruttlen Jr, A proposal to analyze the mechanisms responsible for mitotic spindle protein, CKAP2’s, ability to regulate MT dynamics in vitro , Junior Independent Work Paper, Spring 2022.

Microtubule associated proteins (MAPs) are important proteins that bind to microtubules (MT) and regulate their dynamics, modulating MT growth, shrinkage, nucleation, and catastrophe rates. One such protein is mitotic spindle protein “cytoskeleton associated protein 2” (CKAP2), which is upregulated in many tumor cell lines and causes spindle defects as well as genomic instability disruption in its absence. Recently, CKAP2 was identified as a protein that strongly affects MT dynamics, increases nucleation frequency both spontaneously and from stabilized microtubule templates, and increases growth rates while suppressing catastrophe in vitro. Phase separation by MAPs has recently emerged as an important activity regulating spindle dynamics and suggests one potential mechanism for CKAP2’s effects on MT assembly and stability, given its intrinsically disordered N-terminus and microtubule binding activities. Although CKAP2 has properties known to be important for proper mitotic spindle assembly, the protein’s mechanism is poorly understood. In this study, I investigate which functional regions of CKAP2 are responsible for affecting MT dynamics in vitro. I show that full length CKAP2 concentrates tubulin on a GTP- or GDP-MT lattice in a concentration-dependent manner. The coating of the MT lattice likely occurs via condensation of CKAP2 with tubulin, which may suggest a mechanism to induce MT nucleation. Interestingly, the N-terminal half of CKAP2 binds the newly forming MT lattice, increases the rate of polymerization. Similar to full-length CKAP2, it may reduce catastrophe and prolong MT elongation, thereby increasing MT stability. It is also evident that the C-terminal half of this protein may regulate the activity of the N-terminal half given that it is a target for phosphorylation. Elucidating the function of both halves of this protein sheds light on how full-length CKAP2 functions within the cell, highlighting its mechanistic properties.