S. pombe CLASP needs dynein, not EB1 or CLIP170, to induce microtubule instability and slows polymerization rates at cell tips in a dynein-dependent manner

Abstract

The Schizosaccharomyces pombe CLIP170-associated protein (CLASP) Peg1 was identified in a screen for mutants with spindle formation defects and a screen for molecules that antagonized EB1 function. The conditional peg1.1 mutant enabled us to identify key features of Peg1 function. First, Peg1 was required to form a spindle and astral microtubules, yet destabilized interphase microtubules. Second, Peg1 was required to slow the polymerization rate of interphase microtubules that establish end-on contact with the cortex at cell tips. Third, Peg1 antagonized the action of S. pombe CLIP170 (Tip1) and EB1 (Mal3). Fourth, although Peg1 resembled higher eukaryotic CLASPs by physically associating with both Mal3 and Tip1, neither Tip1 nor Mal3 was required for Peg1 to destabilize interphase microtubules or for it to associate with microtubules. Conversely, neither Mal3 nor Tip1 required Peg1 to associate with microtubules or cell tips. Consistently, while mal3.⌬ and tip1.⌬ disrupted linear growth, corrupting peg1 + did not. Fifth, peg1.1 phenotypes resembled those arising from deletion of the single heavy or both light chains of fission yeast dynein. Furthermore, all interphase phenotypes arising from peg1 + manipulation relied on dynein function. Thus, the impact of S. pombe CLASP on interphase microtubule behavior is more closely aligned to dynein than EB1 or CLIP170.[Keywords: CLASP; dynein; S. pombe; fission yeast; CLIP170; EB1] Supplemental material is available at http://www.genesdev.org.

Interphase microtubule bundles use global cell shape to guide spindle alignment in fission yeast

“…We therefore analyzed spindle orientation in a mutant with altered interphase MT organization. Deletion of the S. pombe CLIP170 homologue tip1 results in cells with short MTs that often do not extend the entire length of the cell, and therefore are only partially aligned with the long axis of the cells (Brunner and Nurse, 2000;Carazo-Salas and Nurse, 2006;Daga et al, 2006b;Grallert et al, 2006). We observed in tip1Δ cells, which have short, poorly aligned MTs, that the frequency of spindle misalignment increased significantly, consistent with our hypothesis that interphase MTs aligned along the axis of the cell influence spindle orientation (Fig.…”

Stress-regulated kinase pathways in the recovery of tip growth and microtubule dynamics following osmotic stress inS. pombe

“…These bundles are generated and maintained by MAPs such as Ase1 and microtubule motor proteins such as Klp2 (Loiodice et al, 2005;Yamashita et al, 2005;Carazo-Salas et al, 2005). Exploitation of one tubulin-GFP imaging system found no impact of the fission yeast CLASP homologue Peg1 upon interphase microtubule function (Bratman and Chang, 2007), whereas the exploitation of a different GFP-tubulin expression system found, in an internally controlled study, that the normal behaviour of microtubule bundles relies upon dynein and Peg1 (Grallert et al, 2006). Individual microtubules polymerize from the end of these bundles, invariably reaching the cell tips before depolymerizing (Drummond and Cross, 2000;Grallert et al, 2006;Hoog et al, 2007;Tran et al, 2001).…”

“…Exploitation of one tubulin-GFP imaging system found no impact of the fission yeast CLASP homologue Peg1 upon interphase microtubule function (Bratman and Chang, 2007), whereas the exploitation of a different GFP-tubulin expression system found, in an internally controlled study, that the normal behaviour of microtubule bundles relies upon dynein and Peg1 (Grallert et al, 2006). Individual microtubules polymerize from the end of these bundles, invariably reaching the cell tips before depolymerizing (Drummond and Cross, 2000;Grallert et al, 2006;Hoog et al, 2007;Tran et al, 2001). It is this contact with the cell tip that promotes the deposition of polarity factors at cell tips (Feierbach and Chang, 2001;Martin et al, 2005;Martin et al, 2007;Snaith and Sawin, 2003).…”

In vivo movement of the type V myosin Myo52 requires dimerisation but is independent of the neck domain

“…Live cells were grown, visualised and images were processed as described previously (Grallert et al, 2006). Cells were visualised on a Deltavision Spectris system (Applied Precision Instruments), based around an Olympus IX71 microscope.…”