Tip1/CLIP-170 Protein Is Required for Correct Chromosome Poleward Movement in Fission Yeast

Sherilyn Goldstone,Sylvie Tournier,Céline Reyes,Marion Dubarry,Thibault Courthéoux,Guillaume Gay,Yannick Gachet

doi:10.1371/journal.pone.0010634

Abstract

The plus-end microtubule binding proteins (+TIPs) play an important role in the regulation of microtubule stability and cell polarity during interphase. In S. pombe, the CLIP-170 like protein Tip1, together with the kinesin Tea2, moves along the microtubules towards their plus ends. Tip1 also requires the EB1 homolog Mal3 to localize to the microtubule tips. Given the requirement for Tip1 for microtubule stability, we have investigated its role during spindle morphogenesis and chromosome movement. Loss of Tip1 affects metaphase plate formation and leads to the activation of the spindle assembly checkpoint. In the absence of Tip1 we also observed the appearance of lagging chromosomes, which do not influence the normal rate of spindle elongation. Our results suggest that S. pombe Tip1/CLIP170 is directly or indirectly required for correct chromosome poleward movement independently of Mal3/EB1.

Highlights

Over the last decade the convergent efforts of molecular genetics and live microscopy have revealed a number of proteins that localize to the more dynamic ‘‘plus’’ ends of the microtubules
We created strains deleted for tip1, tea2 or mal3, with each strain carrying a GFP-tagged version of the non-essential a-tubulin gene (Atb2-GFP) in order to follow their progression through mitosis (Figure 1A, B, C, D)
We determined the length of time that each of these strains spent in mitosis by live video microscopy, from the time the spindle reached a length of approximately 2 mm up to full spindle elongation

Summary

Introduction

Over the last decade the convergent efforts of molecular genetics and live microscopy have revealed a number of proteins that localize to the more dynamic ‘‘plus’’ ends of the microtubules. Observation of these proteins as GFP fusions has revealed that they are often associated with the control of microtubule dynamics (growing, shrinking or pausing) This wellconserved family of proteins, called the plus-end microtubule tracking proteins (+TIPs), includes CLIP-170, an endosomemicrotubule linker [1] known as Tip in S.pombe [2] or Bik in S.cerevisiae [3] [4], as well as the EB1 protein (Mal in S.pombe, Bim in S.cerevisae) [5] [6]. The molecular actors of this checkpoint pathway include the Mad, Mad, Mad, and Bub, Bub, and Mps1/ Mph proteins [9,13,14,15,16] During mitosis, these proteins are recruited to unattached kinetochores, where they inhibit the activation of the anaphase promoting complex, blocking the activation of separase, the destruction of cohesion and cyclin B, and thereby the onset of anaphase, until correct attachment is achieved. The exact nature of the defect in the kinetochoremicrotubule interactions sensed by the SAC remains unclear but is known to involve problems in kinetochore/microtubule attachment, or lack of tension between attached sister kinetochores [11,17,18]

Methods

Results

Conclusion