QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 2 December 2003
doi: 10.1242/jcs.00844

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pitt, C. W. Articles by Doonan, J. H. Search for Related Content PubMed PubMed Citation Articles by Pitt, C. W. Articles by Doonan, J. H. Social Bookmarking                         What's this?

The pot1⁺ homologue in Aspergillus nidulans is required for ordering mitotic events

Christopher W. Pitt^*, Eric Moreau, Patricia A. Lunness and John H. Doonan

John Innes Centre, Colney, Norwich, NR4 7UH, UK

View larger version (11K): [in a new window]   Fig. 1. nimU24 strains fail to complete mitosis. Chromatin is shown in blue (DAPI staining) and spindle pole bodies in pink (indirect immunofluorescence with anti- tubulin antibody). (A) A nimU24 cell (EM4) with multiple separated spindle pole bodies (SPBs) but foci of chromatin connected by strands of chromatin. In this image the chromatin appears condensed. (B) A uninucleate nimU24 cell (EM4) with two separated SPBs. (C) A wild-type cell (GR5) containing two normal interphase nuclei, each with a single SPB. (D) SPB separation was scored in a nimU24 strain (EM4) and a wild-type strain (GR5) through the time course indicated, at the restrictive temperature (42°C). Scale bar: 5 µm.

View larger version (26K): [in a new window]   Fig. 2. Mitotic failure in the absence of nimU function. (A) Chromosome mitotic index was scored in wild-type (GR5), nimU24 (EM4), and nimU (GR5/15) cells at nimU24 permissive and restrictive temperatures. (B) After 10 hours at the restrictive temperature a typical wild-type germling has undergone three mitoses to produce eight nuclei (top panel). Under the same conditions a nimU24 germling has an enlarged, misshapen nucleus (lower panel).

View larger version (111K): [in a new window]   Fig. 3. Sequence comparison of NIMU and its higher eukaryotic homologues with the telomere end-binding protein (TEBP) subunits and ß of ciliated protozoa. Multiple sequences alignments were obtained using the 3D-PSSM program [(Kelley et al., 2000) online at http://www.sbg.bio.ic.ac.uk/servers/3dpssm/] where amino acids are placed into the following conserved groups based on secondary structure predictions: LIVM, PSTAG, QNEHDRK, CW, FY, followed by minor manual adjustment. The mammalian and plant sequences were manually aligned to the TEBPß. TEBPa, telomere end binding protein subunit; TEBPb, telomere end binding protein ß subunit. The organisms are abbreviated as follows: An, Aspergillus nidulans; At, Arabidopsis thaliana (accession no. NM_120714.1); Hs, Homo sapiens (Baumann and Cech, 2001); Mc, Moneuplotes crassus (Wang et al., 1992); Mf, Macaca fascicularis (accession no. AB066545.1); Mm, Mus musculus (accession no. NM_133931.1); Nc, Neurospora crassa (accession no. CAC28643.1); On, Oxytricha nova (Gray et al., 1991; Hicke et al., 1990); Ot, Oxytricha trifallax (Prescott et al., 1998; DuBois and Prescott, 1997); Sl, Stylonychia lemnae (accession no. AAF87600); Sm, Stylonychia mytilis (Fang and Cech, 1991); Sp, Schizosaccharomyces pombe (Baumann and Cech, 2001). (A) Domain structure of the three most highly conserved regions of homology between the NIMU/Pot1 proteins and the TEBP and ß subunits of protozoa. The positions of oligonucleotide/oligosaccharide-binding (OB) folds of O. nova TEBP and ß are indicated by black lines below the domain structures. Two strong regions of homology (black and cross hatched boxes) were identified between the NIMU/Pot1proteins and the TEBP subunits corresponding to regions overlapping the first two OB folds. A third region of homology (white box) was found between the NIMU/Pot1 proteins and the TEBPß subunits which overlap with the OB fold of TEBPß. (B,C) Multiple sequence alignments of the two subunit domains (black and cross hatched boxes in A). Starting and ending amino acid numbers are shown. Residues conserved in at least four sequences (including both ciliate and non-ciliate sequences) are shown as white letters shaded in black. Residues conserved in at least four of the non-ciliate sequences and representing at least two kingdoms are shown as black letters shaded in grey. (D) Multiple sequence alignment of the ß subunit domain (white box in A). Shading as in C. An asterisk indicates the L536Q mutation in nimU24. Sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession numbers AJ567920 (nimU genomic DNA) and AJ567922 (nimU cDNA).

View larger version (45K): [in a new window]   Fig. 4. nimU is an essential gene. (A) Wild type, nimU24 and nimU spores were grown for 14 hours at permissive and restrictive temperatures for nimU24, then fixed and stained with DAPI. The wild-type strain grew normally at both temperatures, producing multinucleate hyphae. nimU24 spores produced multinucleate hyphae at the permissive temperature, but only grossly enlarged nuclei at the restrictive temperature. The nimU spores produced grossly enlarged nuclei at both temperatures.

View larger version (15K): [in a new window]   Fig. 5. Early mitotic exit when nimU function is lost. Mitotic progression in the presence and absence of nimU function was monitored by following G2/M synchronised cells (strains CP1/1 and CP1/6) through mitosis. Synchrony was achieved by utilising the temperature sensitivity of the nimA5 allele. Samples were taken through the time course indicated and the chromosome mitotic index (A), the spindle mitotic index (B), and the rate of entry into mitosis (C), were determined.

View larger version (30K): [in a new window]   Fig. 6. The spindle assembly checkpoint cannot delay mitotic exit in the absence of nimU. (A) The G2/M block/release experiment was repeated in the presence of benomyl and the chromosome mitotic index determined. (B) Conidiospores were spot-inoculated onto rich medium with or without benomyl and germinated at semi-permissive (32°C) or restrictive (34.5°C) temperatures for the nimU24 mutant for several days. sldA744 and sldB937 are loss of function mutations in the spindle checkpoint components BUB1 and BUB3, respectively.

View larger version (18K): [in a new window]   Fig. 7. Mitotic arrest but premature spindle elongation in nimU24 bimE7 cells. (A) bimE7 and nimU24 bimE7 cells were germinated overnight at the permissive temperature and then shifted to the restrictive temperature and fixed at 0.5 hourly intervals. The chromosome mitotic index was determined after staining with DAPI. (B) The same samples were also stained for mitotic spindles (anti- tubulin immunofluorescence) and 100 mitotic spindles were measured for each time point using IP Lab software. Values plotted are arbitrary units and represent the mean and standard error for the 100 measurements.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter