Radial Microtubule Organization by Histone H1 on Nuclei of Cultured Tobacco BY-2 Cells

Takashi Hotta,Takateru Nakayama,Koichi Mizuno,Takeharu Ishii

doi:10.1074/jbc.m705764200

Abstract

In acentriolar higher plant cells, the surface of the nucleus acts as a microtubule-organizing center, substituting for the centrosome. However, the protein factors responsible for this microtubule organization are unknown. The nuclear surfaces of cultured tobacco BY-2 cells possess particles that generate microtubules. We attempted to isolate the proteins in these particles to determine their role in microtubule organization. When incubated with plant or mammalian tubulin, some, but not all, of the isolated nuclei generated abundant microtubules radially from their surfaces. The substance to induce the formation of radial microtubules was confirmed by SDS-PAGE to be a protein with apparent molecular mass of 38 kDa. Partial analysis of the amino acid sequences of the peptide fragments suggested it was a histone H1-related protein. Cloning and cDNA sequence analysis confirmed this and revealed that when the recombinant protein was incubated with tubulin, it could organize microtubules as well as the 38-kDa protein. Histone H1 and tubulin formed complexes immediately, even on ice, and then clusters of these structures were formed. These clusters generated radial microtubules. This microtubule-organizing property was confined to histone H1; all other core histones failed to act as organizers. On immunoblot analysis, rabbit antibodies raised against the 38-kDa protein cross-reacted with histone H1 proteins from tobacco BY-2 cells. These antibodies virtually abolished the ability of the nucleus to organize radial microtubules. Indirect immunofluorescence showed that the antigen was distributed at the nuclear plasm and particularly at nuclear periphery independently from DNA.

Highlights

Some biochemical approaches have provided limited evidence of the potential role of protein factor(s) as candidates for MT-organizing particles (MTOPs) on nuclei
Microtubules Protruding from the Surfaces of Isolated Nuclei or Nuclear Fragments—More than 50% of the tobacco BY-2 cells cultured for 5 days contained nuclei with surface-associated radial MTs extending to the cell periphery (Fig. 1A)
Days were incubated with MT-associated proteins (MAPs)-free tubulin in polymerization buffer at 27 °C for 30 min, radial MT assembly occurred on the surfaces of the nuclei (Fig. 1, B and C) at a tubulin concentration of 0.4 mg/ml, which is below the critical tubulin concentration (ϳ1 mg/ml) for spontaneous assembly

Summary

EXPERIMENTAL PROCEDURES

Preparation of Nuclei—Tobacco BY-2 cells were cultured at 27 °C in the dark with shaking in Linsmaier and Skoog medium and subcultured at intervals of 7 days. A mixture of monoclonal antibody raised against ␣-tubulin from chick brain (at a dilution of 1:500; Amersham Biosciences) and rabbit antibodies raised against the 38-kDa protein from the nuclei of tobacco BY-2 cells (at a dilution of 1:500) was applied for 1 h at 36 °C for indirect immunofluorescence double-staining of cells. For the determination of 38-kDa protein in aster-like structures, the fixed preparation was applied on polylysinecoated coverslips and was treated with a mixture of monoclonal antibody raised against ␣-tubulin from chick brain (at a dilution of 1:500; Amersham Biosciences) and rabbit antibodies raised against the 38-kDa protein from the nuclei of tobacco BY-2 cells (at a dilution of 1:500) as above. The dialyzed preparation of recombinant 38-kDa protein was centrifuged at 100,000 ϫ g for 30 min at 4 °C to remove aggregations, and its ability to form aster-like structures was determined as noted under “Assay for MTOC.”

RESULTS

Regions of Peptide Fragments of

DISCUSSION