The formation of the central element of the synaptonemal complex may occur by multiple mechanisms: the roles of the N- and C-terminal domains of the Drosophila C(3)G protein in mediating synapsis and recombination

Abstract

In Drosophila melanogaster oocytes, the C(3)G protein comprises the transverse filaments (TFs) of the synaptonemal complex (SC). Like other TF proteins, such as Zip1p in yeast and SCP1 in mammals, C(3)G is composed of a central coiled-coil-rich domain flanked by N- and C-terminal globular domains. Here, we analyze in-frame deletions within the N- and C-terminal regions of C(3)G in Drosophila oocytes. As is the case for Zip1p, a C-terminal deletion of C(3)G fails to attach to the lateral elements of the SC. Instead, this C-terminal deletion protein forms a large cylindrical polycomplex structure. EM analysis of this structure reveals a polycomplex of concentric rings alternating dark and light bands. However, unlike both yeast and mammals, all three proteins deleted for N-terminal regions completely abolished both SC and polycomplex formation. Both the N- and C-terminal deletions significantly reduce or abolish meiotic recombination similarly to c(3)G null homozygotes. To explain these data, we propose that in Drosophila the N terminus, but not the C-terminal globular domain, of C(3)G is critical for the formation of antiparallel pairs of C(3)G homodimers that span the central region and thus for assembly of complete TFs, while the C terminus is required to affix these homodimers to the lateral elements.

Figure 1.—

(A) Model of the SC showing the orientation of the C(3)G protein. LEs, which line the length of homologous chromosomes, are interconnected across the central region of the SC by C(3)G homodimers, which make up the TFs. The C(3)G homodimers are oriented with the C-terminal domain (blue) aligned along the LEs and the N-terminal domain (red) interconnecting the C(3)G homodimers along the central element. (B) Full-length and deletion constructs of C(3)G. On the basis of protein secondary structure predictions for coiled-coil (tan cylinders) and non-coiled-coil (red lines) structure, C(3)G consists of a non-coiled-coil N-terminal domain, a central coiled-coil-rich region (CC) that includes a prominent short segment of coiled-coil (CC1), a non-coiled-coil gap near the N-terminal end, and a non-coiled-coil C-terminal domain. In-frame deletions of C(3)G are represented by open regions. FLAG epitopes are not drawn to scale.

Figure 2.—

IF images of Drosophila pro-oocyte nuclei expressing the indicated full-length or in-frame deletions of C(3)G stained with anti-C(3)G antibody. (A, A′, and A″) y w (c(3)G⁺ control). (B, B′, and B″) P{UASP-c(3)G^FL}3. (C, C′, and C″) P{UASP-c(3)G^CFLAG}85. (D, D′, and D″) P{UASP-c(3)G^Ndel}3. (E, E′, and E″) P{UASP-c(3)G^NCdel}4. (F, F′, and F″) P{UASP-c(3)G^CC1del}5. (G, G′, and G″) P{UASP-c(3)G^Cdel}4. The C(3)G proteins were expressed in a c(3)G⁶⁸ mutant background, so that localization of the antibody detects only the expression of the transgene-encoded protein in each case. (A–G) Anti-C(3)G immunofluorescence. (A′–G′) Nuclear DNA stained with DAPI. (A″–G″) A merge of the anti-C(3)G IF (green) and DAPI (blue) images. Bar, 1 μm.

Figure 3.—

IF images of Drosophila pro-oocytes of the genotype expressing the C(3)G^Cdel protein. (A) Single optical section of a w ; P{w[+], gc(2)M-myc}II.5 ; c(3)G⁺ pro-oocyte nucleus in which C(3)G^Cdel is not expressed. (B–D) Single optical sections of three different pro-oocyte nuclei of the genotype y w ; P{UASP-c(3)G^Cdel}4/P{w[+], gc(2)M-myc}II.5 ; c(3)G⁶⁸ e/nos-GAL4∷VP16 cu c(3)G⁶⁸. A–D show the localization of C(2)M-myc, detected using anti-c-myc (green), and C(3)G, detected using anti-C(3)G (red) and DAPI (blue). (E) Single optical section of a y w nanos-GAL4∷VP16/y w ; P{UASP-c(3)G^Cdel}4/+ ; c(3)G⁺ pro-oocyte nucleus, in which both C(3)G^Cdel and wild-type C(3)G are present. The images in E show the localization of C(3)G^Cdel, detected using anti-FLAG (green), and of C(3)G [both wild-type C(3)G and C(3)G^Cdel], detected using anti-C(3)G (red) and DAPI (blue). Bar, 1 μm.

Figure 4.—

Transmission electron micrographs and interpretive models of PCs from Drosophila females expressing the C(3)G^Cdel deletion protein (A–E) compared to a more typical PC from Drosophila (F). (A) Series of five consecutive sections through a symmetrical cylindrical PC from a germarium expressing C(3)G^Cdel. The PC is sectioned parallel to the cylindrical axis, showing the pattern of alternating light and dark bands and the central space running from end to end of the PC. (B) Oblique section through a second PC demonstrating the hole within the PC, the alternating light and dark bands, and the general circular shape of the entire PC. (C and D) Diagrams of the proposed model of PC structure as a thick-walled, hollow cylinder consisting of alternating dark and light layers. In C, two different planes of section are illustrated. Plane 1 corresponds to the plane of section of the micrographs in A, with the middle micrograph most closely corresponding to the diagrammed plane. Plane 2 is an oblique cut through a PC that is similar to the section shown in B. The diagram in D is labeled to indicate the dimensions of the PCs that were measured. The thickness of the walls (a) is ∼200–300 nm and the diameter of the central hole (b) is ∼150–160 nm. Both the length (c) and the diameter (d) are ∼750 nm. A small section of the PC has been enlarged to show one possible orientation of the C(3)G^Cdel proteins in spatial relationship to the PC layers. “N” represents the N-terminal globular domain and the vertical lines represent the coiled-coil-rich domain of the C(3)G^Cdel proteins. The appearance of these unusual PCs can be explained if a large flat sheet of banded SC-like material interacts with itself (indicated here by a vertical white line through the model) to form a hollow cylinder. (E) Magnification of the far left section of the series in A, showing the alternate dark and light bands of the PC and the lack of narrow LE-like bands, which are found in typical PCs in Drosophila. (F) Example of a PC with typical structure in Drosophila (Rasmussen 1975; Anderson et al. 2005). The PC appears as stacked, closely apposed SCs with a narrow LE-like band between two central regions. Although E and F are both at the same magnification, the ovaries were not prepared in the same way, so direct physical measurements comparing the two cannot be made. However, comparison of E and F allows the differences in structure between typical PCs and the C(3)G^Cdel PCs to be distinguished. Bar, 500 nm in A and B and 100 nm in E and F.