Leptotene/zygotene chromosome movement via the SUN/KASH protein bridge in Caenorhabditis elegans

Abstract

The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2-dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates.

Figure 1. Dynamics of SUN-1 aggregates.

(A) Frames from a movie showing the movement of SUN-1::GFP (green); chromatin stained with Hoechst 33342 (blue). White arrowhead highlights protrusion of chromatin. (B) Three-dimensional reconstruction of SUN-1::GFP displacement track dynamics. View from top (i), 30° z-axis rotation and 60° x-axis rotation (ii), and view from right side (iii) and left side (iv). (C) (i) Each line represents the distribution of the projected speed of all SUN-1 aggregates inside a nucleus from individual movies (yellow and orange: tracks from first movie, seven nuclei shown; light and dark blue: tracks from second movie, seven nuclei shown). (ii) Arcs show distance traveled for each SUN-1 track inside a nucleus. Yellow and orange: tracks from first movie, seven nuclei shown; light and dark blue: tracks from second movie, seven nuclei shown). Nuclei in the distal (D), central (E), and proximal (F) TZ, with projection of the cumulative movement of SUN-1::GFP (i), displacement tracks with different colors for each track (ii), distribution of speed for each track, using the same color code as for displacement tracks (iii), and number of SUN-1 aggregates as a function of time (iv). See Table 1 for number of nuclei analyzed. Scale bar: 2 µm.

Figure 2. Dynamics of fusion/splitting events of SUN-1 aggregates for all genotypes studied (15 min recording).

(A) Number of SUN-1 fusion/splitting events grouped into classes. (B) Quantification of the coalescence time (t) grouped into classes (t<1 min, 1 min≤t<3 min, and t≥3 min).

Figure 3. Disruption of the SUN/KASH bridge abrogates SUN-1 aggregate movement.

Restrained movement in SUN-1(G311V)::GFP (A), displacement tracks (B), distribution of the projected speed of all SUN-1 aggregates inside a nucleus (C), and arcs representing the traveled distance for each track inside a nucleus (D). Blue lines represent values from the first movie, orange lines from the second. Eight out of the eight nuclei analyzed are shown. See Table 1 for number of nuclei analyzed. Scale bar: 2 µm.

Figure 4. Impact of SC components on the dynamics of SUN-1 aggregates.

him-3(gk149) (A), htp-1(gk174) (B), syp-2(ok307) (C), and syp-3(me42) (D), with projection of cumulative movement (i), displacement tracks (ii), and distribution of the projected speed (iii). Arcs represent traveled distance (iv). Blue lines represent values from the first movie, orange lines from the second. (C) syp-2(ok307) (i), (ii), (iii), and (iv) from the distal part of the extended TZ and i', ii', iii', and iv' from the proximal part. See Table 1 for number of nuclei analyzed. Scale bar: 2 µm.

Figure 5. Restrained movement of SUN-1 aggregates in htp-1(gk174) is due to precocious synapsis.

Projection of the cumulative movement of SUN-1::GFP in htp-1(gk174); syp-1(RNAi) (A, A'), displacement tracks (B, B'), distribution of the projected speed (C, C'), and arcs (D, D'). Blue lines represent values from the first movie; orange lines values from the second. (A, B, C, D) from distal TZ, (A', B', C', D') from proximal zone where SUN-1 aggregates move. See Table 1 for number of nuclei analyzed. Scale bar: 2 µm.

Figure 6. Influence of meiotic regulators on dynamics of SUN-1 aggregates.

prom-1(ok1140) (A), him-19(jf6) (B), irradiated wild type (C), and cra-1(tm2144) (D), showing projection of the cumulative movement of SUN-1::GFP aggregates (i), displacement tracks (ii), distribution of the projected speed (iii), and arcs representing travelled distances (iv). Blue lines represent values from the first movie, orange lines from the second. See Table 1 for number of nuclei analyzed. Scale bar: 2 µm.

Figure 7. Effect of DSB formation on SUN-1 aggregate dynamics.

spo-11(me44) (A), 2-d-old spo-11(me44) (B) 2 hours after irradiation with projection of cumulative movement (i), displacement tracks (ii), and distribution of the projected speed (iii). Arcs represent traveled distance (iv). Blue lines represent values from the first movie, orange lines from the second. See Table 1 for number of nuclei analyzed. (C) Distribution of SUN-1 foci and patches formed in wild type, spo-11(me44), 2-d-old wild type 2 hours after irradiation and 2-d-old spo-11(me44) 2 hours after irradiation. >400 SUN-1 aggregates counted per genotype. Scale bar: 2 µm.

Figure 8. Establishment of synapsis and formation of high speed via shuffling of chromosome ends through SUN-1 patches.

Chromosome axes (green lines) support binding of PC proteins (red or violet shapes) that connect chromosome ends (red, blue, and violet loops) to SUN-1 (brown ellipses), directly or indirectly, (unknown factor, blue circle). ZYG-12 (pink ellipses) and SUN-1 bridge chromosomes to cytoplasmic forces (orange arrow) to move chromosome ends. (A) SUN-1 patch containing two nonhomologous chromosome ends (red and blue loops) fuses with SUN-1 focus carrying a single chromosome end, a homolog (red loops). After fusion, chromosome ends will be shuffled inside the newly formed SUN-1 patch. When the homologous chromosome is found, synapsis overcomes the cytoplasmic forces and synapsis can be established. (B) The same scenario is depicted, except that ends of nonhomologous chromosomes are in the SUN-1 aggregate (violet loops). After fusion, chromosome ends will be shuffled. However, as the cytoplasmic forces overcome the attempt to synapse, one SUN-1 focus will be driven out of the patch. This tension-generated splitting event is one of the factors leading to the formation of high speed in the distribution of SUN-1 aggregates. Our data support other factors as sources for the high speed aggregates: recombination intermediates (C) and chromosomes entanglements (D).