Single-Molecule Tracking of Chromatin-Associated Proteins in the C. elegans Gonad

Abstract

Biomolecules are distributed within cells by molecular-scale diffusion and binding events that are invisible in standard fluorescence microscopy. These molecular search kinetics are key to understanding nuclear signaling and chromosome organization and can be directly observed by single-molecule tracking microscopy. Here, we report a method to track individual proteins within intact C. elegans gonads and apply it to study the molecular dynamics of the axis, a proteinaceous backbone that organizes meiotic chromosomes. Using either fluorescent proteins or enzymatically ligated dyes, we obtain multisecond trajectories with a localization precision of 15-25 nm in nuclei actively undergoing meiosis. Correlation with a reference channel allows for accurate measurement of protein dynamics, compensating for movements of the nuclei and chromosomes within the gonad. We find that axis proteins exhibit either static binding to chromatin or free diffusion in the nucleoplasm, and we separately quantify the motion parameters of these distinct populations. Freely diffusing axis proteins selectively explore chromatin-rich regions, suggesting they are circumventing the central phase-separated region of the nucleus. This work demonstrates that single-molecule microscopy can infer nanoscale-resolution dynamics within living tissue, expanding the possible applications of this approach.

Figure 1.

Imaging in the ex vivo gonad. A. Schematic of C. elegans with both gonad arms marked. Meiosis progresses linearly along the gonad (gray arrows), with the pachytene stage occurring in approximately the middle third (blue line). B. Schematic of gonad extrusion. Top, manual dissection of C. elegans extrudes the gonad into solution while remaining supported by attachment to the main body of the worm. Bottom, many extruded gonads are immobilized between an agarose pad and a coverslip mounted on a microscope slide. C. Approximate geometry of the six meiotic chromosomes of C. elegans during pachytene. Blue, chromatin; green, axis; gray, nucleolus. D. The nanoscale geometry of looped chromosomal DNA (blue) and the meiotic axis (green). In worms, two major axis protein components are HTP-3 and HIM-3. E. Meiotic nuclei maintain function for tens of minutes following dissection. Images are partial maximum intensity projections of nuclei expressing GFP-labeled HIM-3, which marks the meiotic axis. Arrows highlight the chromosome motion that characterizes this stage of meiosis. F. The gonad is permeable for the synthetic dye JF549 functionalized with a HaloTag ligand (HTL), allowing labeling of HTP-3 fused to the HaloTag enzyme. Partial maximum intensity projections of nuclei are shown for clarity. The image contrast for the GFP and JF549 channels is kept the same between conditions where the HTP-3-HaloTag fusion is present (“HaloTag”) and the dye is or is not added (“JF549-HTL”). Note the colocalization of GFP-HIM-3 and HTP-3-HaloTag, indicating unperturbed localization of HTP-3-HaloTag.

Figure 2.

Single-molecule imaging of axis proteins. A. Left, diffraction-limited image of mMaple3-HIM-3 before activation. Right, trajectory of a single photoactivated mMaple-3-HIM-3 molecule associated with the axis. Diffraction limited data are from a single epifluorescence image. B. Selected 100 ms frames of raw data from the molecular trajectory shown in A. Frame borders are colored according to the time colormap in A. One plane of biplane data is shown. C. Left, diffraction-limited image of GFP-HIM-3 reference channel. Right, trajectory of a single photoactivated PAJF549 molecule linked to HTP-3-HaloTag associated with the axis. D. Histogram of total photon counts and corresponding exponential fits for trajectories of mMaple3-HIM-3 and HTP-3-HaloTag (PAJF549), means indicated at top. E. Histogram of total lengths in seconds and corresponding exponential fits for trajectories of mMaple3-HIM-3 and HTP-3-HaloTag (PAJF549), means indicated at top. All imaging performed with 100 ms integration time.

Figure 3.

Single-molecule tracking of HTP-3 subpopulations with different dynamics. A. Single-molecule trajectories of HTP-3-HaloTag molecules overlaid onto diffraction-limited reference images of GFP-HIM-3, marking the axis in pachytene nuclei. Top, a trajectory displaying diffusion through the nucleoplasm. Bottom, a static trajectory within a single axis. B. the cumulative distribution function (CDF) of mean jump displacements (JDs) for HTP-3-HaloTag molecules. Dots on the CDF curve mark the mean jump displacements of the two tracks shown in A. C., mean squared displacements as a function of lag time for the mobile and bound trajectory populations marked in B. Top, mobile fraction (N = 39 trajectories). The bound fraction is also plotted for reference, virtually overlapping with the x axis at this scale. Bottom, the bound fraction (N = 163 trajectories), with MSD scaled by a factor of 1000. Error bars of each MSD value represent standard error estimated from bootstrapping tracks. Precision of D estimate is the standard deviation when bootstrapping tracks.

Figure 4.

The nucleolus excludes HTP-3 molecules. A. Confocal imaging of free axis proteins (HTP-3) relative to DNA (DAPI, mChy-H2B) and the nucleolus (DAO-5) in premeiotic nuclei. Top, immunolabeling of fixed gonads. Bottom, imaging in live extruded gonads. White circles: nuclei used to generate profiles of radial density, shown on the right. Each line, mean of density profiles in indicated nuclei; shaded areas, standard deviation. B. Distribution of single-molecule tracks of HTP-3 in meiotic (pachytene) nuclei. Left, example ellipse drawn onto diffraction-limited GFP-HIM-3 reference image to define nuclear coordinates. Middle, five tracks registered within the shared polar coordinate system. Right, distribution of mobile HTP-3 molecule localizations from 39 tracks. C. Schematic of axis protein dynamics within pachytene chromosomes. Left, three-dimensional nuclear geometry, with cross-section shown at right. Mobile axis proteins are excluded from the nucleolus and must diffuse in the nuclear periphery (black arrows) to find binding sites on chromosomes.