Using elemental staining and mapping techniques for simultaneous visualization of biological structures in the nucleus by multichannel electron microscopy.

Transmission electron microscopy (TEM) has been essential in defining the structural organization of the cell due to its ability to image cell structures at molecular resolution. However, the absence of colour has made it very difficult to compare the distributions and relationships of two or more types of biomolecules simultaneously if they lack clear morphological distinctions. Furthermore, single-channel information limits functional analysis, particularly in the nucleoplasm, where fibrillar material could be chromatin, ribonucleic acid or protein. Where specific stains exist to discriminate among these molecules, they cannot be combined because conventional TEM is a single-channel technology. A potential path around this barrier is through electron spectroscopic imaging (ESI). ESI can map the distributions of chemical elements within an ultrathin section. Here, we present methods to stain specific molecules with elements that ESI can visualize to enable multichannel electron microscopy.

Heterogeneity of Organization of Subcompartments in DSB Repair Foci.

Cells assemble compartments around DNA double-strand breaks (DSBs). The assembly of this compartment is dependent on the phosphorylation of histone H2AX, the binding of MDC1 to phosphorylated H2AX, and the assembly of downstream signaling and repair components. The decision on whether to use homologous recombination or nonhomologous end-joining repair depends on competition between 53BP1 and BRCA1. A major point of control appears to be DNA replication and associated changes in the epigenetic state. This includes dilution of histone H4 dimethylation and an increase in acetylation of lysine residues on H2A and H4 that impair 53BP1 binding. In this article, we examined more closely the spatial relationship between 53BP1 and BRCA1 within the cell cycle. We find that 53BP1 can associate with early S-phase replicated chromatin and that the relative concentration of BRCA1 in DSB-associated compartments correlates with increased BRCA1 nuclear abundance as cells progress into and through S phase. In most cases during S phase, both BRCA1 and 53BP1 are recruited to these compartments. This occurs for both IR-induced DSBs and breaks targeted to an integrated LacO array through a LacI-Fok1-mCherry fusion protein. Having established that the array system replicates this heterogeneity, we further examined the spatial relationship between DNA repair components. This enabled us to precisely locate the DNA containing the break and map other proteins relative to that DNA. We find evidence for at least three subcompartments. The damaged DNA, single-stranded DNA generated from end resection of the array, and nuclease CtIP all localized to the center of the compartment. BRCA1 and 53BP1 largely occupied discrete regions of the focus. One of BRCA1 or 53BP1 overlaps with the array, while the other is more peripherally located. The array-overlapping protein occupied a larger volume than the array, CtIP, or single-stranded DNA (ssDNA). Rad51 often occupied a much larger volume than the array itself and was sometimes observed to be depleted in the array volume where the ssDNA exclusively localizes. These results highlight the complexity of molecular compartmentalization within DSB repair compartments.