Limits...
Stable morphology, but dynamic internal reorganisation, of interphase human chromosomes in living cells.

Müller I, Boyle S, Singer RH, Bickmore WA, Chubb JR - PLoS ONE (2010)

Bottom Line: This contrasted with the behaviour of specific loci on labelled chromosomes, which showed more progressive reorganisation, and revealed that "looping out" of chromatin from chromosome territories is a dynamic state.Chromosome structure showed tremendous resistance to inhibitors of transcription, histone deacetylation and chromatin remodelling.Together, these data indicate steric constraints determine structure, rather than innate chromosome architecture or function-driven anchoring, with interphase chromatin organisation governed primarily by opposition between needs for decondensation and the space available for this to happen.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee, UK.

ABSTRACT
Despite the distinctive structure of mitotic chromosomes, it has not been possible to visualise individual chromosomes in living interphase cells, where chromosomes spend over 90% of their time. Studies of interphase chromosome structure and dynamics use fluorescence in-situ hybridisation (FISH) on fixed cells, which potentially damages structure and loses dynamic information. We have developed a new methodology, involving photoactivation of labelled histone H3 at mitosis, to visualise individual and specific human chromosomes in living interphase cells. Our data revealed bulk chromosome volume and morphology are established rapidly after mitosis, changing only incrementally after the first hour of G1. This contrasted with the behaviour of specific loci on labelled chromosomes, which showed more progressive reorganisation, and revealed that "looping out" of chromatin from chromosome territories is a dynamic state. We measured considerable heterogeneity in chromosome decondensation, even between sister chromatids, which may reflect local structural impediments to decondensation and could potentially amplify transcriptional noise. Chromosome structure showed tremendous resistance to inhibitors of transcription, histone deacetylation and chromatin remodelling. Together, these data indicate steric constraints determine structure, rather than innate chromosome architecture or function-driven anchoring, with interphase chromatin organisation governed primarily by opposition between needs for decondensation and the space available for this to happen.

Show MeSH

Related in: MedlinePlus

Labelling individual human interphase chromosomes in live cells.A) Activation of H3 PA-GFP (green) on a single chromosome in mitosis. Using H2B mRFP, mitotic cell with separated chromosomes were identified and separated chromosomes were scanned using a 405 nm laser. A z-stack was used to confirm single chromosome labelling (right image, projection of 3D stack, 5 µm bar). B–E) Sample maximal projections of different pairs of daughter nuclei (red) showing single interphase chromosomes (green). Bar 15 µm. Images captured between 1 and 4 hours after mitosis. F) Quantification of decondensation of mitotic chromosomes into 4 h interphase chromosomes. Box plots show data range of chromosome volumes (µm3), the two mid-quartiles, and the median. Mitotic chromosomes n = 20, interphase n = 39.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2903487&req=5

pone-0011560-g001: Labelling individual human interphase chromosomes in live cells.A) Activation of H3 PA-GFP (green) on a single chromosome in mitosis. Using H2B mRFP, mitotic cell with separated chromosomes were identified and separated chromosomes were scanned using a 405 nm laser. A z-stack was used to confirm single chromosome labelling (right image, projection of 3D stack, 5 µm bar). B–E) Sample maximal projections of different pairs of daughter nuclei (red) showing single interphase chromosomes (green). Bar 15 µm. Images captured between 1 and 4 hours after mitosis. F) Quantification of decondensation of mitotic chromosomes into 4 h interphase chromosomes. Box plots show data range of chromosome volumes (µm3), the two mid-quartiles, and the median. Mitotic chromosomes n = 20, interphase n = 39.

Mentions: To visualise single interphase chromosomes, we photolabelled chromosomes during mitosis, when they are condensed and distinct (Figure 1A). The histone H3.1 variant (HIST1H3A) was chosen for labelling as it shows very slow turnover on chromatin, with approximately 80% of incorporated H3.1 showing no turnover after incorporation during S-phase [28]. In comparison, H2B has a major (40%) fraction with a half time for turnover of around 2 hours, and the replacement H3 variant, H3.3 was also expected to be more dynamic [29]. We initially tried photobleaching a H3-GFP fusion, but the extensive laser treatment meant chromosomes often moved before labelling was complete, and cells arrested in mitosis because of photodamage. Therefore, we generated human HT-1080 cell lines stably co-expressing Histone 2B mRFP (H2B-RFP) to identify cells in mitosis, and Histone H3 PA-GFP (H3-PA-GFP) fusions (Figure 1A). HT-1080 cells were selected as their chromosomes lack the translocations commonplace in other transformed human cell lines [30].


Stable morphology, but dynamic internal reorganisation, of interphase human chromosomes in living cells.

Müller I, Boyle S, Singer RH, Bickmore WA, Chubb JR - PLoS ONE (2010)

Labelling individual human interphase chromosomes in live cells.A) Activation of H3 PA-GFP (green) on a single chromosome in mitosis. Using H2B mRFP, mitotic cell with separated chromosomes were identified and separated chromosomes were scanned using a 405 nm laser. A z-stack was used to confirm single chromosome labelling (right image, projection of 3D stack, 5 µm bar). B–E) Sample maximal projections of different pairs of daughter nuclei (red) showing single interphase chromosomes (green). Bar 15 µm. Images captured between 1 and 4 hours after mitosis. F) Quantification of decondensation of mitotic chromosomes into 4 h interphase chromosomes. Box plots show data range of chromosome volumes (µm3), the two mid-quartiles, and the median. Mitotic chromosomes n = 20, interphase n = 39.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903487&req=5

pone-0011560-g001: Labelling individual human interphase chromosomes in live cells.A) Activation of H3 PA-GFP (green) on a single chromosome in mitosis. Using H2B mRFP, mitotic cell with separated chromosomes were identified and separated chromosomes were scanned using a 405 nm laser. A z-stack was used to confirm single chromosome labelling (right image, projection of 3D stack, 5 µm bar). B–E) Sample maximal projections of different pairs of daughter nuclei (red) showing single interphase chromosomes (green). Bar 15 µm. Images captured between 1 and 4 hours after mitosis. F) Quantification of decondensation of mitotic chromosomes into 4 h interphase chromosomes. Box plots show data range of chromosome volumes (µm3), the two mid-quartiles, and the median. Mitotic chromosomes n = 20, interphase n = 39.
Mentions: To visualise single interphase chromosomes, we photolabelled chromosomes during mitosis, when they are condensed and distinct (Figure 1A). The histone H3.1 variant (HIST1H3A) was chosen for labelling as it shows very slow turnover on chromatin, with approximately 80% of incorporated H3.1 showing no turnover after incorporation during S-phase [28]. In comparison, H2B has a major (40%) fraction with a half time for turnover of around 2 hours, and the replacement H3 variant, H3.3 was also expected to be more dynamic [29]. We initially tried photobleaching a H3-GFP fusion, but the extensive laser treatment meant chromosomes often moved before labelling was complete, and cells arrested in mitosis because of photodamage. Therefore, we generated human HT-1080 cell lines stably co-expressing Histone 2B mRFP (H2B-RFP) to identify cells in mitosis, and Histone H3 PA-GFP (H3-PA-GFP) fusions (Figure 1A). HT-1080 cells were selected as their chromosomes lack the translocations commonplace in other transformed human cell lines [30].

Bottom Line: This contrasted with the behaviour of specific loci on labelled chromosomes, which showed more progressive reorganisation, and revealed that "looping out" of chromatin from chromosome territories is a dynamic state.Chromosome structure showed tremendous resistance to inhibitors of transcription, histone deacetylation and chromatin remodelling.Together, these data indicate steric constraints determine structure, rather than innate chromosome architecture or function-driven anchoring, with interphase chromatin organisation governed primarily by opposition between needs for decondensation and the space available for this to happen.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee, UK.

ABSTRACT
Despite the distinctive structure of mitotic chromosomes, it has not been possible to visualise individual chromosomes in living interphase cells, where chromosomes spend over 90% of their time. Studies of interphase chromosome structure and dynamics use fluorescence in-situ hybridisation (FISH) on fixed cells, which potentially damages structure and loses dynamic information. We have developed a new methodology, involving photoactivation of labelled histone H3 at mitosis, to visualise individual and specific human chromosomes in living interphase cells. Our data revealed bulk chromosome volume and morphology are established rapidly after mitosis, changing only incrementally after the first hour of G1. This contrasted with the behaviour of specific loci on labelled chromosomes, which showed more progressive reorganisation, and revealed that "looping out" of chromatin from chromosome territories is a dynamic state. We measured considerable heterogeneity in chromosome decondensation, even between sister chromatids, which may reflect local structural impediments to decondensation and could potentially amplify transcriptional noise. Chromosome structure showed tremendous resistance to inhibitors of transcription, histone deacetylation and chromatin remodelling. Together, these data indicate steric constraints determine structure, rather than innate chromosome architecture or function-driven anchoring, with interphase chromatin organisation governed primarily by opposition between needs for decondensation and the space available for this to happen.

Show MeSH
Related in: MedlinePlus