Ultra-Structural Imaging Provides 3D Organization of 46 Chromosomes of a Human Lymphocyte Prophase Nucleus

Abstract

Three dimensional (3D) ultra-structural imaging is an important tool for unraveling the organizational structure of individual chromosomes at various stages of the cell cycle. Performing hitherto uninvestigated ultra-structural analysis of the human genome at prophase, we used serial block-face scanning electron microscopy (SBFSEM) to understand chromosomal architectural organization within 3D nuclear space. Acquired images allowed us to segment, reconstruct, and extract quantitative 3D structural information about the prophase nucleus and the preserved, intact individual chromosomes within it. Our data demonstrate that each chromosome can be identified with its homolog and classified into respective cytogenetic groups. Thereby, we present the first 3D karyotype built from the compact axial structure seen on the core of all prophase chromosomes. The chromosomes display parallel-aligned sister chromatids with familiar chromosome morphologies with no crossovers. Furthermore, the spatial positions of all 46 chromosomes revealed a pattern showing a gene density-based correlation and a neighborhood map of individual chromosomes based on their relative spatial positioning. A comprehensive picture of 3D chromosomal organization at the nanometer level in a single human lymphocyte cell is presented.

Single-Molecule Micromanipulation and Super-Resolution Imaging Resolve Nanodomains Underlying Chromatin Folding in Mitotic Chromosomes

“…To enable comparison with current understandings of the mitotic chromosome, it is critical to determine the DNA content of the MNDs. To this end, we utilized a topographic technique frequently used to determine the volumes of biomolecular structures, AFM, − to quantify the physical volume of the MNDs, and used the DNA density determined in previous SBFSEM studies , to quantify the DNA content within the MNDs. We confirmed that the unperturbed (not extended) mitotic chromosome imaged by AFM consists of granular structures (CGs) that are roughly 100 to 200 nm in diameter (median 153 ± 19 nm), consistent with the 3D-SIM and BALM results described above, even with any potential contribution of tip broadening in these measurements (see Materials and Methods; Figures S7A,B and S8).…”

“…From these topographic AFM images, we determined the volume of each MND and, with the DNA density measured by SBFSEM (5.8 nm 3 /bp), , found that most domains encompass between 20 kb and 200 kb DNA, with a median content of 76 kb (Figure D). This amount of DNA is quite similar to the predicted size of the inner chromatin loops (80 kb) of the recent nested loop model .…”

“…Figure 5C shows that, like the axial and lateral dimensions, there is a narrow range in the heights of these MNDs: roughly 91 ± 24 nm, quite similar to the axial and lateral sizes (Figure S9). From these topographic AFM images, we determined the volume of each MND and, with the DNA density measured by SBFSEM (5.8 nm 3 /bp), 16,17 found that most domains encompass between 20 kb and 200 kb DNA, with a median content of 76 kb (Figure 5D). This amount of DNA is quite similar to the predicted size of the inner chromatin loops (80 kb) of the recent nested loop model.…”

“…Under such conditions, we found that there are exclusively ∼90 nm domains (called mitotic nanodomains (MNDs)), as revealed by both BALM and atomic force microscopy (AFM). On the basis of AFM volume measurements and previous serial-block face scanning electron microscopy (SBFSEM) data of the DNA density within mitotic chromosomes, − we estimate that the MNDs contain ∼80 kb DNA, which is surprisingly close to the size of the inner loops in the nested loop model. Further, if we assume that the entire human genome is packed in these MNDs, the total volume is only 12% different from the volume measured by SBFSEM .…”

“…The volume of each MND was calculated assuming an ellipsoidal shape. The estimation of the DNA content in each MND was based on the previously measured DNA density (5.8 nm 3 /bp) of mitotic chromosomes. , The estimate of the number of MNDs per cluster (each with a diameter of 153 nm according to the AFM results) was obtained assuming close-packing (0.74) of the MNDs (identified by AFM).…”