SPIRAL2 stabilises endoplasmic microtubule minus ends in the moss Physcomitrella patens

Abstract

Stabilisation of minus ends of microtubules (MTs) is critical for organising MT networks in land plant cells, in which all MTs are nucleated independent of centrosomes. Recently, Arabidopsis SPIRAL2 (SPR2) protein was shown to localise to plus and minus ends of cortical MTs, and increase stability of both ends. Here, we report molecular and functional characterisation of SPR2 of the basal land plant, the moss Physcomitrella patens. In protonemal cells of P. patens, where non-cortical, endoplasmic MT network is organised, we observed SPR2 at minus ends, but not plus ends, of endoplasmic MTs and likely also of phragmoplast MTs. Minus end decoration was reconstituted in vitro using purified SPR2, suggesting that moss SPR2 is a minus end-specific binding protein (-TIP). We generated a loss-of-function mutant of SPR2, in which frameshift-causing deletions/insertions were introduced into all four paralogous SPR2 genes by means of CRISPR/Cas9. Protonemal cells of the mutant showed instability of endoplasmic MT minus ends. These results indicate that moss SPR2 is a MT minus end stabilising factor.