TPX2 performs multiple roles in microtubule organization. Previously, it was shown that plant AtTPX2 binds AtAurora1 kinase and colocalizes with microtubules in a cell cycle-specific manner. To elucidate the function of TPX2 further, this work analysed Arabidopsis cells overexpressing AtTPX2-GFP. Distinct arrays of bundled microtubules, decorated with AtTPX2-GFP, were formed in the vicinity of the nuclear envelope and in the nuclei of overexpressing cells. The microtubular arrays showed reduced sensitivity to anti-microtubular drugs. TPX2-mediated formation of nuclear/perinuclear microtubular arrays was not specific for the transition to mitosis and occurred independently of Aurora kinase. The fibres were not observed in cells with detectable programmed cell death and, in this respect, they differed from TPX2-dependent microtubular assemblies functioning in mammalian apoptosis. Colocalization and co-purification data confirmed the interaction of importin with AtTPX2-GFP. In cells with nuclear foci of overexpressed AtTPX2-GFP, strong nuclear signals for Ran and importin diminished when microtubular arrays were assembled. This observation suggests that TPX2-mediated microtubule formation might be triggered by a Ran cycle. Collectively, the data suggest that in the acentrosomal plant cell, in conjunction with importin, overexpressed AtTPX2 reinforces microtubule formation in the vicinity of chromatin and the nuclear envelope.
- Klíčová slova
- Arabidopsis thaliana, AtTPX2, Aurora kinase, Ran., fibres, importin, microtubules, nuclei, γ-tubulin,
- MeSH
- apoptóza MeSH
- Arabidopsis cytologie enzymologie metabolismus MeSH
- buněčné jádro metabolismus MeSH
- centrozom metabolismus MeSH
- chromatin metabolismus MeSH
- jaderný obal metabolismus MeSH
- karyoferiny metabolismus MeSH
- kinasy aurora metabolismus MeSH
- mikrotubuly metabolismus MeSH
- mitóza MeSH
- počítačová simulace MeSH
- proteiny asociované s mikrotubuly metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- rostlinné buňky metabolismus MeSH
- subcelulární frakce metabolismus MeSH
- transport proteinů MeSH
- tubulin metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zobrazování trojrozměrné MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chromatin MeSH
- karyoferiny MeSH
- kinasy aurora MeSH
- proteiny asociované s mikrotubuly MeSH
- proteiny huseníčku MeSH
- TPX2 protein, Arabidopsis MeSH Prohlížeč
- tubulin MeSH
- zelené fluorescenční proteiny MeSH
BACKGROUND: RanBPM (Ran-binding protein in the microtubule-organizing centre) was originally reported as a centrosome-associated protein in human cells. However, RanBPM protein containing highly conserved SPRY, LisH, CTLH and CRA domains is currently considered as a scaffolding protein with multiple cellular functions. A plant homologue of RanBPM has not yet been characterized. RESULTS: Based on sequence similarity, we identified a homologue of the human RanBPM in Arabidopsis thaliana. AtRanBPM protein has highly conserved SPRY, LisH, CTLH and CRA domains. Cell fractionation showed that endogenous AtRanBPM or expressed GFP-AtRanBPM are mainly cytoplasmic proteins with only a minor portion detectable in microsomal fractions. AtRanBPM was identified predominantly in the form of soluble cytoplasmic complexes ~230-500 kDa in size. Immunopurification of AtRanBPM followed by mass spectrometric analysis identified proteins containing LisH and CRA domains; LisH, CRA, RING-U-box domains and a transducin/WD40 repeats in a complex with AtRanBPM. Homologues of identified proteins are known to be components of the C-terminal to the LisH motif (CTLH) complexes in humans and budding yeast. Microscopic analysis of GFP-AtRanBPM in vivo and immunofluorescence localization of endogenous AtRanBPM protein in cultured cells and seedlings of Arabidopsis showed mainly cytoplasmic and nuclear localization. Absence of colocalization with γ-tubulin was consistent with the biochemical data and suggests another than a centrosomal role of the AtRanBPM protein. CONCLUSION: We showed that as yet uncharacterized Arabidopsis RanBPM protein physically interacts with LisH-CTLH domain-containing proteins. The newly identified high molecular weight cytoplasmic protein complexes of AtRanBPM showed homology with CTLH types of complexes described in mammals and budding yeast. Although the exact functions of the CTLH complexes in scaffolding of protein degradation, in protein interactions and in signalling from the periphery to the cell centre are not yet fully understood, structural conservation of the complexes across eukaryotes suggests their important biological role.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- Arabidopsis chemie genetika metabolismus MeSH
- cytoskeletální proteiny genetika metabolismus MeSH
- Eukaryota chemie klasifikace genetika MeSH
- jaderné proteiny genetika metabolismus MeSH
- konzervovaná sekvence MeSH
- lidé MeSH
- molekulární evoluce * MeSH
- molekulární sekvence - údaje MeSH
- proteiny huseníčku chemie genetika metabolismus MeSH
- rostliny chemie klasifikace genetika MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adaptorové proteiny signální transdukční MeSH
- cytoskeletální proteiny MeSH
- jaderné proteiny MeSH
- proteiny huseníčku MeSH
- Ran binding protein 9 MeSH Prohlížeč
