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

First published online 12 September 2007
doi: 10.1242/jcs.010215

This Article Summary Full Text Full Text (PDF) Supplementary Material A correction has been published 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 Maple, J. Articles by Møller, S. G. Search for Related Content PubMed PubMed Citation Articles by Maple, J. Articles by Møller, S. G. Social Bookmarking                         What's this?

Interdependency of formation and localisation of the Min complex controls symmetric plastid division

Centre for Organelle Research, Department of Mathematics and Natural Sciences, University of Stavanger, 4036 Stavanger, Norway

View larger version (41K): [in this window] [in a new window]   Fig. 1. Division site placement in E. coli. (A) At the polar zone of the cell, MinD-ATP (circle) associates with the inner membrane and recruits MinC (oval), to create a zone of inhibition of FtsZ polymerisation (broken arrow). MinE (diamond) associates with MinD at the leading edge of the polar zone and stimulates MinD ATPase activity, leading to the release of phosphate (Pi). MinD-ADP is released from the membrane and the MinCD inhibition complex subsequently relocates to the opposite pole of the cell (B). The process is repeated and oscillation of the Min proteins from pole to pole of the cell results in formation of the Z-ring at the cell midpoint (C) and subsequent symmetric cell division (D).

View larger version (30K): [in this window] [in a new window]   Fig. 2. The formation of the Arabidopsis Min complex. (A) Yeast two-hybrid analysis of AtMinE1 and AtMinD1 interactions. Domains are represented as follows: TP, transit peptide; AMD, anti-MinCD domain; hatched box, AtMinD1-interacting domain; TSD, topological specificity domain; red arrow, -sheet; red cylinder, -helix; CTE, C-terminal extension. Full-length AtMinE1 or AtMinD1 fused to the GAL4 DNA-binding domain (BD) and truncations of AtMinE1 fused to the GAL4 activation domain (AD) were expressed in HF7c yeast cells and monitored for growth on synthetic drop-out media lacking tryptophan and leucine (–TL) and synthetic drop-out media lacking tryptophan, leucine and histidine (–HTL). Relative interaction strengths are represented as three classes based on the ratio of growth on –TL to –HTL: ++, ratio of 0.6-1.0; +, ratio of 0.3-0.6; –, ratio equal to the relevant control (<0.3 in all cases). (B) BiFC assays. AtMinE1 and AtMinD1 were fused to the non-fluorescent YFP155-238 (CY) and were coexpressed in tobacco chloroplasts with truncations of AtMinE1 fused to YFP1-154 (NY). Interactions of the full-length AtMinE1 and AtMinD1 fusions were used as positive controls. Extended-focus images of reconstituted YFP fluorophore (YFP) and chlorophyll autofluorescence (chlorophyll) were captured by epifluorescence microscopy using Volocity II software. Bar, 5 µm; +, positive reconstitution of YFP; –, no reconstitution of YFP.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Analysis of the effects of amino acid substitutions in AtMinE135-141 on the interaction with AtMinD1. (A) Alignment of amino acids 120-140 of AtMinE1 with amino acids 11-33 of E. coli MinE reveals that this region of the anti-MinCD domain (AMD) is highly conserved. Asterisks indicate the amino acids substituted in this study. (B) A helical-wheel projection of amino acids 120-140 of AtMinE1. The colours indicate the effects of mutations on the BD-AtMinE135-141 and AD-AtMinD1 interaction in yeast two-hybrid assays. BD-AtMinE135-141 and AD-AtMinD1 were expressed in HF7c yeast cells and monitored for growth on synthetic drop-out media lacking tryptophan and leucine (–TL) and synthetic drop-out media lacking tryptophan, leucine and histidine (–HTL). Relative interaction strengths are represented as two classes based on the ratio of growth on –TL to growth on –HTL media: red, ratio of >0.8; blue, ratio equal to the relevant control (<0.3 in all cases); black, not tested. The letters a-g represent the positions of the amino acids in the helical wheel.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Domains mediating the localisation of AtMinE1. Domains represented as follows: TP, transit peptide; AMD, anti-MinCD domain; hatched box, AtMinD1-interacting domain; TSD, topological specificity domain; red arrow, -sheet; red cylinder, -helix; CTE, C-terminal extension. For the analysis of N-terminal truncations in living chloroplasts, the transit peptide region from AtABC1 (TP) was fused to AtMinE1117-229 and AtMinE1142-229. The localisation of AtMinE1-YFP, AtMinE11-197-YFP, AtMinE11-169-YFP, AtMinE11-141-CFP, TP.AtMinE1117-229-YFP and TP.AtMinE1142-229-YFP was analysed in tobacco and Arabidopsis by biolistic transformation. Extended-focus images of YFP were captured by epifluorescence microscopy using Volocity II software. Bars, 2 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 5. Overexpression of AtMinE1 affects chloroplast division. (A) Chloroplast phenotypes in mesophyll and petiole cells of wild-type (WT), 35S-AtMinE1, 35S-AtMinE11-197, 35S-AtMinE11-169, 35S-AtMinE11-141, 35S-TP.AtMinE1117-229 and 35S-TP.AtMinE1142-229 Arabidopsis primary transformants. Extended-focus images of chlorophyll autofluorescence in mesophyll and petiole cells were captured by epifluorescence microscopy using Volocity II software. White arrowheads indicate septation events. Bar, 25 µm. (B) 21-day-old WT and Arabidopsis primary transformants analysed for AtMinE1 and actin expression by RT-PCR. Transcript levels were quantified using imageJ software and the relative amounts of AtMinE1 transcript to actin transcript shown as the means±s.e.m. (with WT=1).

View larger version (57K): [in this window] [in a new window]   Fig. 6. Functional conservation of AtMinE1. (A) AtMinE1 partially complements a minE– E. coli mutant. Cells were examined by microscopy after growth in 0.05 mM IPTG or 4 mM IPTG. minB::KanRPlacMinCD/PaddA-vector control cells show a filamentous phenotype 3 hours after MinCD induction. minB::KanRPlacMinCD/PaddA-AtMinE1 are, on average, shorter and symmetric (black arrowheads) and asymmetric septation events (black arrows) and minicell formation (white arrows) are observed. Cell length was measured using ImageJ software and WT cell length estimated from the average length of 100 minE+ cells. The lengths of >100 cells are represented graphically. (B) The localisation of the AtMinE1 domains in E. coli. Expression of the fusion proteins was induced using 0.05 mM IPTG and single-plane images of YFP captured from cells containing the empty pRSET-A vector (PT7), expressing YFP alone (PT7-YFP) or AtMinE1, AtMinE11-197, AtMinE11-169, AtMinE11-141, TP.AtMinE1117-229 and TP.AtMinE1142-229 fused to YFP or CFP. (C) Affect of expression of AtMinE1 truncations on E. coli cell-division phenotypes. Cells containing the empty pRSET-A vector (PT7), or expressing YFP alone (PT7-YFP) or AtMinE1, AtMinE11-197, AtMinE11-169, AtMinE11-141, TP.AtMinE1117-229 and TP.AtMinE1142-229 fused to YFP or CFP were induced with 2 mM IPTG. Symmetric (black arrowheads) and asymmetric septation events (black arrows) and minicell formation (white arrowheads) are observed. Bars, 2 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 7. A model of AtMinE1 interaction and localisation. (A) AtMinE1 interacts with AtMinD1 through one face of an -helix (amino acids 118-140; blue) of the AMD. AtMinE1 dimerisation is mediated through the TSD (amino acids 142-229; red). The minimal region required for dimerisation is amino acids 142-169, although determinants within the rest of the TSD might affect stability. (B) AtMinE1 dimerises through the TSD (red) and interacts with AtMinD1 (yellow) through the AMD (blue). The Min complex localises to one or two spots at the pole of the chloroplast. (C) The AMD alone localises in association with the entire chloroplast inner envelope. (D) The TSD localises to diffuse patches in the chloroplast stroma. AMD, anti-MinCD domain; TSD, topological specificity domain.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter