Fig 2: Missing MZT1 modules bind on the outer surface of the recombinant human ?-TuRC.a Two views on the cryo-EM reconstruction of the recombinant human ?-TuRC. Spokes are numbered and coloring is indicated. The positioning of all MZT1 modules is highlighted. The position of the MZT1 module on GCP3(14) was not visible in this structure due to substoichiometry and structural heterogeneity, but is resolved in other reconstructions at the indicated position (black circle). Substoichiometry of the ?-TuSC(1,2) and ?-TuSC(13,14) units in this reconstruction is indicated by grey coloring in the schematic representation. b Structure and composition of the lumenal bridge. Atomic model (PDB 6X0U) superposed to the segmented density of the lumenal bridge. Coloring as indicated. c Cryo-EM density of the MZT1-GCP3 module associated with GCP3(6). An atomic model of the MZT1-GCP3 module from the lumenal bridge (PDB 6X0U) was docked as a rigid body. MZT1-GCP3 modules directly interact with the adjacent ?-TuSC units via the GCP2 C-terminus.

Fig 3: The tubgcp3 mutant embryos exhibit decreased expression of proliferation and differentiation markers in CMZ cells. (A–D) ISH analyses show that the expression of vsx2 is normal in the central retina but significantly reduced in the CMZ in tubgcp3 mutant retinae at 3 dpf and 5 dpf. (E–H) ISH analyses exhibit that ccnd1 is highly expressed in wild-type sibling CMZ (M,O) but clearly reduced in tubgcp3 mutant CMZ (N,P) at 3 dpf and 5 dpf. (I–P) atoh7 and cdkn1c (associated with retinal cell differentiation) are expressed in the central CMZ of wild-type sibling retina but significantly reduced in tubgcp3 mutant CMZ at 3 dpf and almost disappeared at 5 dpf. (Q–T) Immunostaining analyses displaying normal ZPR-1 staining (green/red double cone photoreceptors marker) in the central area of the tubgcp3 mutant retina. Arrows indicate the CMZ of the retina. Scale bars: 100 µm (A–T).

Fig 4: The tubgcp3 mutant CMZ cells arrest in M-phase showing monopolar spindles and abnormal distributed centrioles and ?-tubulin. (A–F) Immunostaining analysis of cell proliferation in zebrafish retina at 3 dpf using DNA replication marker (BrdU, red) and mitotic marker (PH3, green). Embryos are incubated with BrdU for 6 h before being collected at 72 hpf for the analysis. Almost all cells in wild-type sibling CMZ are BrdU+ with several PH3+ cells among them (A,C,E). In the (tubgcp3 mutant retina, PH3+ cells are significantly increased (B,F), but BrdU+ cells are markedly decreased (D,F). Note that PH3+ BrdU- cells are detected in the tubgcp3 mutant retina (F) but absent in the wild-type sibling (E). (G) Bar chart analyses depicting quantification of BrdU- and PH3-labeled cells in wild-type sibling and tubgcp3 mutant retinae. Data are mean + SEM from 50 retinal sections for each group. Student’s t-test: **P < 0.01. (H–M) Immunostaining of 3 dpf retinal cryosections with anti-a-tubulin (red) and anti-PH3 (green) displaying bipolar spindles formed in mitotic cells in wild-type siblings (H,J,L). In the tubgcp3 mutant retina, many mitotic RPCs exhibit monopolar spindles (I,K,M). Insets indicate high-magnification images of mitotic RPCs in rectangles in (H–M). (N) Bar charts depicting quantification of mitotic cells with monopolar spindles in wild-type sibling (0.12 per section, n = 43 sections) and the tubgcp3 mutant retinae (8.14 per section, n = 36 sections). (O–Q) Immunostaining analyses displaying a pair of centrioles at each pole of the bipolar spindle in mitotic cells in wild-type sibling CMZ (O). In the tubgcp3 mutant retinae, centrioles are distributed at the center of the M-phase arrested cells (57.6%, n = 59 M-phase arrested cells) (P) or randomly scatter in these cells (42.4%, n = 59 M-phase arrested cells) (Q). (R–T) Immunostaining analyses exhibiting ?-tubulin at the spindle poles in mitotic cells in wild-type sibling (R). In tubgcp3 mutant retinae, ?-tubulin localizes at the center of the M-phase arrested cells, showing a single focus (62.5%, n = 80 M-phase arrested cells) (S) or scattered foci (37.5%, n = 80 M-phase arrested cells) (T). (U) Schematic representation of the structure of ?-TuSC and ?-TuRC. (V) Co-immunoprecipitation (IP) assays showing Tubgcp3 interacts with ?-tubulin through its C terminal domain. HEK293T cells were transfected with plasmids to express GFP-tagged zebrafish ?-tubulin and Myc-tagged zebrafish Tubgcp3 fragments, including full length (1–906 aa) Tubgcp3, N terminal (1–551 aa) Tubgcp3 and C terminal (552–906 aa) Tubgcp3. Then the cell samples were performed by immunoprecipitation with anti-Myc antibody and analyzed by immunoblotting (IB) with anti-Myc and anti-GFP antibodies. ß-Actin was used as the loading control. Arrowheads indicate the IgG heavy chain (~50 kDa) and IgG light chain (~25 kDa). Scale bars: 20 µm (A–F); 20 µm (H–M); 2 µm (O–T).

Fig 5: The tubgcp3 mutants exhibit developmental defects in the CMZ of the retina. (A–D) Hematoxylin and Eosin (HE) staining displaying significant defects in the CMZ of the tubgcp3 mutant retinae at 3 dpf and 5 dpf. (E–H) Higher-magnification images of the CMZ in blue dotted rectangles in (A–D). (I–L) Higher-magnification image of the orange dotted rectangles in (A–D). The tubgcp3 mutants exhibit normal retinal laminar structures in the central retina. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Scale bars: 100 µm (A–D); 20 µm (E–L).