Correlations of three-dimensional motion of chromosomal loci in yeast revealed by the double-helix point spread function microscope.
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.
Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305.Show MeSH
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Mentions: Because association with common transcriptional regulators has been implicated in distant genomic correlations and in the colocalization of genomic loci in discrete transcriptional factories (Sutherland and Bickmore, 2009), we sought to examine whether the two copies of the GAL locus in the nucleus of a diploid yeast cell showed correlated movements or colocalization in either activating or repressive conditions. To simultaneously track two gene loci in live yeast cells, we applied precise, two-color, wide-field 3D fluorescence localization microscopy using the double-helix point spread function (DH-PSF) microscope (Pavani et al., 2009; Thompson et al., 2010). Details of how the DH-PSF works are given in the references and in Materials and Methods. Briefly, the DH-PSF microscope enables 3D position estimation from a single image by converting the light emitted from a single point into two closely spaced lobes in the image. The lobes assume different angles between the line connecting their center points and the vertical as a function of z-position relative to the focal plane (Figure 1A). Thus, as a tracked locus moves in z, the two lobes in its image revolve around one another, effectively tracing out a double-helix shape, without the need for any scanning or image stack recording. From a single snapshot, it is possible to fit a double-Gaussian function (i.e., the sum of two two-dimensional [2D] Gaussians plus a constant offset) to the two lobes using least squares. The midpoint between the positions of the Gaussians gives the lateral (x, y) position, and comparison of the angle of revolution to a calibration curve gives the z-position, all to within subdiffraction precision. Previous studies of 3D chromatin tracking in yeast often used confocal scanning methods (Gartenberg et al., 2004; Bystricky et al., 2005; Cabal et al., 2006; Neumann et al., 2012), which are excellent for time-lapse tracking over longer periods of time but limit the attainable temporal resolution, spatial resolution, and throughput. As compared with confocal scanning methods, our wide-field/nonscanning dual-color DH-PSF approach allows for improved temporal resolution (10-Hz imaging rate) and throughput (∼100 cells) over a 2-μm z-range without scanning, enabling the examination of aspects of the 3D organization and dynamics of chromatin in living cells with subdiffraction precision.
Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305.