Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest D.; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony

doi:10.1093/nar/gku726

Cited by 34 publications

(33 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Briefly, this method exploits motion blurring to selectively eliminate signals arising from rapidly diffusing molecules, allowing visualisation of low mobility signals derived from DNA-associated molecules (Fig 2F). Previously we reported that low mobility PCNA (mEos3.1-Pcn1) is notably enriched during S phase [27]. Deletion of rad18 significantly reduced the fraction of these molecules (Fig 2F, right), thus confirming that PCNA-K164 ubiquitylation results in increased amounts of loaded PCNA during unperturbed S phase.…”

Section: Resultssupporting

confidence: 74%

PCNA ubiquitylation ensures timely completion of unperturbed DNA replication in fission yeast

et al. 2017

Self Cite

View full text Add to dashboard Cite

PCNA ubiquitylation on lysine 164 is required for DNA damage tolerance. In many organisms PCNA is also ubiquitylated in unchallenged S phase but the significance of this has not been established. Using Schizosaccharomyces pombe, we demonstrate that lysine 164 ubiquitylation of PCNA contributes to efficient DNA replication in the absence of DNA damage. Loss of PCNA ubiquitylation manifests most strongly at late replicating regions and increases the frequency of replication gaps. We show that PCNA ubiquitylation increases the proportion of chromatin associated PCNA and the co-immunoprecipitation of Polymerase δ with PCNA during unperturbed replication and propose that ubiquitylation acts to prolong the chromatin association of these replication proteins to allow the efficient completion of Okazaki fragment synthesis by mediating gap filling.

show abstract

Section: Resultssupporting

confidence: 74%

PCNA ubiquitylation ensures timely completion of unperturbed DNA replication in fission yeast

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…For the FtsZ imaging we used slower acquisition mode to exclude cytosolic FtsZ molecules—as they diffuse much faster than FtsZ built into the ring structures, their point-spread functions are blurred, thus most of the cytosolic FtsZ molecules are not included in further analysis steps. This approach was first used by Etheridge et al in their work [ 32 ]. mEos3.2-A69T molecules were converted by primed conversion, while PAmCherry was photoactivated using UV.…”

Section: Methodsmentioning

confidence: 99%

Combining Primed Photoconversion and UV-Photoactivation for Aberration-Free, Live-Cell Compliant Multi-Color Single-Molecule Localization Microscopy Imaging

Virant

Turkowyd

Balinovic

et al. 2017

IJMS

View full text Add to dashboard Cite

Super-resolution fluorescence microscopy plays a major role in revealing the organization and dynamics of living cells. Nevertheless, single-molecule localization microscopy imaging of multiple targets is still limited by the availability of suitable fluorophore combinations. Here, we introduce a novel imaging strategy which combines primed photoconversion (PC) and UV-photoactivation for imaging different molecular species tagged by suitable fluorescent protein combinations. In this approach, the fluorescent proteins can be specifically photoactivated/-converted by different light wavelengths using PC and UV-activation modes but emit fluorescence in the same spectral emission channel. We demonstrate that this aberration-free, live-cell compatible imaging method can be applied to various targets in bacteria, yeast and mammalian cells and can be advantageously combined with correlative imaging schemes.

show abstract

“…Although no changes in dissociation time were observed (Extended Data Figures 9b,c), trajectories were likely truncated by photobleaching (our mean trajectory lengths were 8-11 frames). We therefore took advantage of 'motion blurring' when recording 500 ms trajectories (see below) to detect only chromatin bound proteins 1,46 , and combined this with time-lapse imaging for different lengths of time, in an attempt to determine the dissociation time of the NuRD complex. To our surprise this showed that the dissociation times were much longer than we expected (greater than 100 seconds for MBD3, Extended Data Figure 9d), such that it proved impossible to track individual molecules for long enough in order to determine reliable rates.…”

Section: Chromatin Association Of the Deacetylase Sub-complex Requirementioning

confidence: 99%

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

Basu

Shukron²,

Ponjavic³

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Enhancer-promoter dynamics are critical for the spatiotemporal control of gene expression, but it remains unclear how these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. Here, we use Hi-C experiments to show that the intact NuRD complex increases CTCF/Cohesin binding and the probability of the interaction of intermediate-range (~1Mb) genomic sequences.To understand how NuRD alters 3D genome structure in this way, we developed an approach to segment and extract key biophysical parameters from trajectories of the NuRD complex determined using live-cell 3D singlemolecule imaging. Unexpectedly, this revealed that the intact NuRD complex decompacts chromatin structure and makes NuRD-bound sequences move faster, thus increasing the overall volume of the nucleus that these sequences explore. Interestingly, we also uncovered a rare fast-diffusing state of chromatin that exhibits directed motion. The intact NuRD complex reduces the amount of time that enhancers/promoters remain in this fastdiffusing state, which we propose would otherwise re-organise enhancer-promoter proximity. Thus, we uncover an intimate connection between a chromatin remodeller and the spatial dynamics of the local region of the genome to which it binds.3D genome organisation is thought to be critical for the spatiotemporal control of gene expression. However, little is known about the multi-scale chromatin dynamics of cis-regulatory elements or how they relate to genome organisation. To probe these dynamics at the single-cell level, one of two complementary methods are typically used: either live-cell imaging 1-4 or single nucleus versions of chromosome conformation capture experiments (such as Hi-C), which reveal the proximity of DNA sequences in different individual fixed cells [5][6][7][8][9][10][11][12][13][14][15][16] . For example, live-cell tracking of enhancers and promoters has revealed fast diffusion (or 'stirring') during transcription 4 , but the mechanisms underlying these changes in enhancer dynamics or how they relate to Hi-C measurements of enhancer-promoter proximity remain unclear.The nucleosome remodelling and deacetylase (NuRD) complex is a highly conserved 1 MDa multisubunit protein complex that we have previously shown clusters in 3D space in the nucleus with active enhancers and promoters 16 . It combines two key enzymatic activities -nucleosome remodelling via its helicasecontaining ATPase (predominantly CHD4 in ES cells) and lysine deacetylation via its HDAC1/2 subunits [16][17][18][19][20][21][22] .These activities are thought to be present in two sub-complexes (Figure 1a). HDAC1/2 associates, along with the histone chaperones RBBP4/7, with the core scaffold proteins MTA1/2/3 (metastasis tumour antigens) to form a stable sub-complex with deacetylase activity 23 . The nucleosome remodeller CHD4 interacts with chromatin by itself and may also form a second sub-complex with GATAD2A/B and DOC1 (CDK2AP1) 23-25 .The methyl-CpG DNA binding domain proteins MBD2/3 ...

show abstract

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

Cited by 34 publications

References 32 publications

PCNA ubiquitylation ensures timely completion of unperturbed DNA replication in fission yeast

PCNA ubiquitylation ensures timely completion of unperturbed DNA replication in fission yeast

Combining Primed Photoconversion and UV-Photoactivation for Aberration-Free, Live-Cell Compliant Multi-Color Single-Molecule Localization Microscopy Imaging

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

Contact Info

Product

Resources

About