Abstract
Mal3p and Tip1p are the fission yeast (Schizosaccharomyces pombe) homologues of EB1 and CLIP-170, two conserved microtubule plus end tracking proteins (+TIPs). These proteins are crucial regulators of microtubule dynamics. Using electron tomography, we carried out a high-resolution analysis of the phenotypes caused by mal3 and tip1 deletions. We describe the 3-dimensional microtubule organization, quantify microtubule end structures and uncover novel defects of the microtubule lattices. We also reveal unexpected structural modifications of the spindle pole bodies (SPBs), the yeast microtubule organizing centers. In both mutants we observe an increased SPB volume and a reduced number of MT/SPB attachments. The discovered defects alter previous interpretations of the mutant phenotypes and provide new insights into the molecular functions of the two protein families.
Highlights
Microtubule (MT) dynamic instability is regulated by a wealth of MT associated proteins (MAPs)
We have shown that the absence of either one of the two +TIP proteins, Tip1p or Mal3p, alters the morphology of the spindle pole bodies (SPBs), and the anchoring of MTs to the SPB
We hypothesize that this could result from three different causes; 1) both +TIP mutants have short MTs which could affect the SPB morphology by decreased pushing forces
Summary
Microtubule (MT) dynamic instability is regulated by a wealth of MT associated proteins (MAPs). A subgroup of MAPs has been found to localize to MT plus ends and are commonly called +TIPs [1]. EB1 has been described as the master controller of the +TIPs, recruiting other proteins such as CLIP-170 to the MT plus end [2,3,4,5]. EB-class proteins preferentially bind to MT plus ends directly, by interacting with GTP-tubulin [6], and bind along the MT seam [1,7]. The MT seam is the position along the tube were neighboring protofilaments align in a different orientation, A lattice, than along the rest of the tube, which consists of B lattice [8]. It has been suggested that EB1 binding at the A lattice may stabilize this potential weak spot in MTs [7]
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