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Correlations of three-dimensional motion of chromosomal loci in yeast revealed by the double-helix point spread function microscope.

Backlund MP, Joyner R, Weis K, Moerner WE - Mol. Biol. Cell (2014)

Bottom Line: As controls, we tracked pairs of loci along the same chromosome at various separations, as well as transcriptionally orthogonal genes on different chromosomes.This relative increase has potentially important biological implications, as it might suggest coupling via shared silencing factors or association with decoupled machinery upon activation.We also found that on the time scale studied (∼0.1-30 s), the loci moved with significantly higher subdiffusive mean square displacement exponents than previously reported, which has implications for the application of polymer theory to chromatin motion in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305.

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MSD and velocity autocorrelations for DLDC data. (A) Time-ensemble-averaged MSD for DLDC-dextrose (blue dots) and DLDC-galactose (red dots) plotted on linear scale. Upper bounds of contribution of nuclear translation to total MSD calculated as described in the Supplemental Material for dextrose (dashed blue line) and galactose (dashed red line). (B) Time-ensemble-averaged MSD for DLDC-dextrose (blue dots) and DLDC-galactose (red dots) plotted on log-log scale over the time scale used for subdiffusive parameter estimation. Fitted lines are overlaid in black, giving estimated parameters of D* = 0.0020 and α = 0.75 for dextrose and D* = 0.0018 and α = 0.64 for galactose. (C) Scaled velocity autocorrelation for DLDC-dextrose for δ on the interval [1, 10 s] at 0.1-s intervals. Curve fitting to Eq. 6 gives solid black line. (D) Same as in C but for DLDC-galactose.
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Figure 6: MSD and velocity autocorrelations for DLDC data. (A) Time-ensemble-averaged MSD for DLDC-dextrose (blue dots) and DLDC-galactose (red dots) plotted on linear scale. Upper bounds of contribution of nuclear translation to total MSD calculated as described in the Supplemental Material for dextrose (dashed blue line) and galactose (dashed red line). (B) Time-ensemble-averaged MSD for DLDC-dextrose (blue dots) and DLDC-galactose (red dots) plotted on log-log scale over the time scale used for subdiffusive parameter estimation. Fitted lines are overlaid in black, giving estimated parameters of D* = 0.0020 and α = 0.75 for dextrose and D* = 0.0018 and α = 0.64 for galactose. (C) Scaled velocity autocorrelation for DLDC-dextrose for δ on the interval [1, 10 s] at 0.1-s intervals. Curve fitting to Eq. 6 gives solid black line. (D) Same as in C but for DLDC-galactose.

Mentions: where is the 3D position, and again we have a choice in how we compute the average denoted by the angle brackets; we chose to pool displacements from all tracks and compute a total time-ensemble average akin to the T-EAVCC described earlier in order to obtain sufficient averaging. Unsurprisingly, previous studies found these MSDs to increase initially before plateauing at longer times due to confinement of the loci within subregions of the nucleus (Marshall et al., 1997; Heun et al., 2001; Drubin et al., 2006; Neumann et al., 2012). At times before this confinement becomes apparent, loci appear to move subdiffusively, that is, their MSDs are proportional to δα for some . Bacterial chromosomal loci tend to exhibit α ∼ 0.4 rather ubiquitously (Weber et al., 2010a; Javer et al., 2013). Previous studies in yeast determined α values around 0.5 for various loci (Hajjoul et al., 2013), and one early tracking study of the GAL locus reported α within the range 0.4–0.5 (Cabal et al., 2006). Thus we were surprised to find values of α = 0.75 for dextrose and α = 0.64 in galactose from analysis of the MSD (Figure 6, A and B) of the GAL locus in our study, indicating a higher degree of diffusivity than previously believed, at least on this time scale. Data shown here are taken only from the green channel since the signal-to-noise ratio (SNR) was markedly better. The values do not change significantly if we analyze the MSD averaged only with nonoverlapping frame intervals as well (Supplemental Figure S10 and Table S4). This finding also did not seem to be related to ploidy, as tracking in haploids gave α = 0.77 in dextrose (Table S3). Furthermore, all loci in the various strains tracked in this study showed α values between 0.6 and 0.75 (Supplemental Figure S9 and Supplemental Table S3), indicating that this behavior is more general than just applying to the GAL locus or pericentromeric loci alone. In light of this surprising result, we performed a control experiment in which we tracked freely diffusing fluorescent beads in 90% glycerol/water solution (Supplemental Figure S12) using the same experimental apparatus. With the same analysis software, we estimated α = 0.98, consistent with the expected result of α = 1. The larger α values also cannot be explained by the translation of the nucleus, since our dual-locus tracking scheme allowed us to bound the nuclear contribution to the MSD. Namely, we can set a bound for the contribution of nuclear translation to the apparent MSD by calculating the “cross MSD” (CMSD),(5)


Correlations of three-dimensional motion of chromosomal loci in yeast revealed by the double-helix point spread function microscope.

Backlund MP, Joyner R, Weis K, Moerner WE - Mol. Biol. Cell (2014)

MSD and velocity autocorrelations for DLDC data. (A) Time-ensemble-averaged MSD for DLDC-dextrose (blue dots) and DLDC-galactose (red dots) plotted on linear scale. Upper bounds of contribution of nuclear translation to total MSD calculated as described in the Supplemental Material for dextrose (dashed blue line) and galactose (dashed red line). (B) Time-ensemble-averaged MSD for DLDC-dextrose (blue dots) and DLDC-galactose (red dots) plotted on log-log scale over the time scale used for subdiffusive parameter estimation. Fitted lines are overlaid in black, giving estimated parameters of D* = 0.0020 and α = 0.75 for dextrose and D* = 0.0018 and α = 0.64 for galactose. (C) Scaled velocity autocorrelation for DLDC-dextrose for δ on the interval [1, 10 s] at 0.1-s intervals. Curve fitting to Eq. 6 gives solid black line. (D) Same as in C but for DLDC-galactose.
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Figure 6: MSD and velocity autocorrelations for DLDC data. (A) Time-ensemble-averaged MSD for DLDC-dextrose (blue dots) and DLDC-galactose (red dots) plotted on linear scale. Upper bounds of contribution of nuclear translation to total MSD calculated as described in the Supplemental Material for dextrose (dashed blue line) and galactose (dashed red line). (B) Time-ensemble-averaged MSD for DLDC-dextrose (blue dots) and DLDC-galactose (red dots) plotted on log-log scale over the time scale used for subdiffusive parameter estimation. Fitted lines are overlaid in black, giving estimated parameters of D* = 0.0020 and α = 0.75 for dextrose and D* = 0.0018 and α = 0.64 for galactose. (C) Scaled velocity autocorrelation for DLDC-dextrose for δ on the interval [1, 10 s] at 0.1-s intervals. Curve fitting to Eq. 6 gives solid black line. (D) Same as in C but for DLDC-galactose.
Mentions: where is the 3D position, and again we have a choice in how we compute the average denoted by the angle brackets; we chose to pool displacements from all tracks and compute a total time-ensemble average akin to the T-EAVCC described earlier in order to obtain sufficient averaging. Unsurprisingly, previous studies found these MSDs to increase initially before plateauing at longer times due to confinement of the loci within subregions of the nucleus (Marshall et al., 1997; Heun et al., 2001; Drubin et al., 2006; Neumann et al., 2012). At times before this confinement becomes apparent, loci appear to move subdiffusively, that is, their MSDs are proportional to δα for some . Bacterial chromosomal loci tend to exhibit α ∼ 0.4 rather ubiquitously (Weber et al., 2010a; Javer et al., 2013). Previous studies in yeast determined α values around 0.5 for various loci (Hajjoul et al., 2013), and one early tracking study of the GAL locus reported α within the range 0.4–0.5 (Cabal et al., 2006). Thus we were surprised to find values of α = 0.75 for dextrose and α = 0.64 in galactose from analysis of the MSD (Figure 6, A and B) of the GAL locus in our study, indicating a higher degree of diffusivity than previously believed, at least on this time scale. Data shown here are taken only from the green channel since the signal-to-noise ratio (SNR) was markedly better. The values do not change significantly if we analyze the MSD averaged only with nonoverlapping frame intervals as well (Supplemental Figure S10 and Table S4). This finding also did not seem to be related to ploidy, as tracking in haploids gave α = 0.77 in dextrose (Table S3). Furthermore, all loci in the various strains tracked in this study showed α values between 0.6 and 0.75 (Supplemental Figure S9 and Supplemental Table S3), indicating that this behavior is more general than just applying to the GAL locus or pericentromeric loci alone. In light of this surprising result, we performed a control experiment in which we tracked freely diffusing fluorescent beads in 90% glycerol/water solution (Supplemental Figure S12) using the same experimental apparatus. With the same analysis software, we estimated α = 0.98, consistent with the expected result of α = 1. The larger α values also cannot be explained by the translation of the nucleus, since our dual-locus tracking scheme allowed us to bound the nuclear contribution to the MSD. Namely, we can set a bound for the contribution of nuclear translation to the apparent MSD by calculating the “cross MSD” (CMSD),(5)

Bottom Line: As controls, we tracked pairs of loci along the same chromosome at various separations, as well as transcriptionally orthogonal genes on different chromosomes.This relative increase has potentially important biological implications, as it might suggest coupling via shared silencing factors or association with decoupled machinery upon activation.We also found that on the time scale studied (∼0.1-30 s), the loci moved with significantly higher subdiffusive mean square displacement exponents than previously reported, which has implications for the application of polymer theory to chromatin motion in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305.

Show MeSH
Related in: MedlinePlus