Super-Resolution Microscopy Reveals Diversity of Plant Centromere Architecture

Abstract

Centromeres are essential for proper chromosome segregation to the daughter cells during mitosis and meiosis. Chromosomes of most eukaryotes studied so far have regional centromeres that form primary constrictions on metaphase chromosomes. These monocentric chromosomes vary from point centromeres to so-called "meta-polycentromeres", with multiple centromere domains in an extended primary constriction, as identified in Pisum and Lathyrus species. However, in various animal and plant lineages centromeres are distributed along almost the entire chromosome length. Therefore, they are called holocentromeres. In holocentric plants, centromere-specific proteins, at which spindle fibers usually attach, are arranged contiguously (line-like), in clusters along the chromosomes or in bands. Here, we summarize findings of ultrastructural investigations using immunolabeling with centromere-specific antibodies and super-resolution microscopy to demonstrate the structural diversity of plant centromeres. A classification of the different centromere types has been suggested based on the distribution of spindle attachment sites. Based on these findings we discuss the possible evolution and advantages of holocentricity, and potential strategies to segregate holocentric chromosomes correctly.

Keywords: CENH3; CENP-A; Cuscuta; Lathyrus; Luzula; Pisum; Rhynchospora; clustered centromere; holocentromere; microtubule; monocentromere; structured illumination microscopy.

Figure 1

Different centromere types of somatic plant metaphase chromosomes. Images were obtained via global chromatin labelling by DAPI. After surface rendering of structured illumination microscopy (SIM) image stacks [40] using the Imaris 8.0 software, the centromere structure variability of different plant species becomes visible. Regional monocentromeres are characterized by a district primary constriction (white arrows). Meta-polycentromeres represent an elongated primary constriction (region indicated by dashes). Line-like holocentromeres are characterized by the arrangement of centromere-specific proteins in a distinct line within a groove (red arrows), as found in Luzula and Rhynchospora (Figure 3). Holocentromeres in Cuscuta europaea are structures where spindle fibres attach along the whole chromosome at centromere-specific histone H3 (CENH3)/centromere protein A (CENP-A)-chromatin as well as at CENH3/CENP-A-free regions (see also Figure 2 and Figure 4), but the surface is relatively smooth without a specific constriction.

Figure 2

Different centromere types labeled by centromere-specific histone markers and tubulin. These markers, such as different CENH3/CENP-A variants and H2A phosphorylated at threonine 120 H2AT120ph, intermingle in regional monocentromeres. Spindle fibers attach to H2AT120ph-containing regions of line-like Luzula holocentromeres and CENH3/CENP-A-containing and CENH3/CENP-A-free regions of C. europaea holocentromeres, respectively, along the entire chromosomes. The arrow marks chromosome 1 of C. europaea with a chromosome-wide distribution of tubulin and restricted amount of CENH3/CENP-A. Chromosomes are counterstained with DAPI (in blue).

Figure 3

Centromere formation differs between mitosis and meiosis of Rhynchospora pubera. Whereas line-like holocentromeres appear in mitosis, cluster-like holocentromeres become established in meiosis. The process of global chromatin condensation and the dynamics of CENH3/CENP-A arrangement is visualized by DAPI staining and immunolabeling with CENH3/CENP-A-specific antibodies. Surface rendering of SIM image stacks clearly indicates the presence of grooves (arrowheads) at somatic metaphase chromosomes, but their absence at metaphase I bivalents. The merged side-view of the metaphase I cell reveals CENH3/CENP-A at the surface, but not inside the bivalents.

Figure 4

Models of the different mono- and holocentromere types appearing in different plant species indicate the possible centromere plasticity during mitosis and meiosis. The classification is based on the distribution of the spindle fibre attachment sites. In mono- and meta-polycentromeres, the microtubules (tubulin) form branching bundles and attach mainly at the flanks of the CENH3/CENP-A clusters, but not at H2AT120ph. The bundle formation is less pronounced at holocentromeres. In line-like holocentromeres, spindle fibres attach mainly as single microtubules at the rim along the entire groove containing CENH3/CENP-A and H2AT120ph, as is clearly visible in the cross-section [41]. The CENH3/CENP-A-containing domains in meta-polycentromeres are usually well discernible (the upper model), but may also fuse into one line-like domain (bottom model). In C. europaea holocentromeres, the spindle fibres also attach to CENH3/CENP-A-free chromatin.

Figure 5

Models to expand centromeres. (A) “Telomere to centromere” model based on Villasante et al. [76,77]. (1) Different retroelements are mobilized to heal the DNA ends of a broken chromosome. (2) The most effective telomeric retrotransposons become selected. (3) Retrotransposons with a more effective capping capability are selected. (4) Subtelomeric repeats appear. (5) Subtelomeric repeats are amplified and become centromeric repeats. Ribonucleoprotein complexes are formed after transcription of theses repeats. (6) Centromeric repeats become a protocentromere after being recognized by microtubules. (7) Chromosome wide extension of the centromere to form a holocentromere. (B) “Centromere drive model” of Henikoff et al. [2]. The expansion of a satellite that binds CENH3/CENP-A provides more microtubule attachment sites. This stronger centromere drives in female meiosis into the egg cell.

Figure 6

A model illustrating possible karyotype alterations after fragmentation of holocentric chromosomes based on the interplay between holocentricity and telomere healing. (A) Irradiation of holocentric chromosomes (arrowhead) induces chromosome fragments. Centromere activity is present along all chromosome fragments. (B) Broken ends lack telomere repeats and become gradually healed (C) by de novo telomere syntheses. Based on Jankowska et al. [86].