IMAGE

FIGURE 5

ID
ZDB-IMAGE-190723-806
Antibodies
Source
Figures for Li et al., 2019
Image
Figure Caption

FIGURE 5

The tubgcp3 mutant CMZ cells arrest in M-phase showing monopolar spindles and abnormal distributed centrioles and γ-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-α-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 γ-tubulin at the spindle poles in mitotic cells in wild-type sibling (R). In tubgcp3 mutant retinae, γ-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 γ-TuSC and γ-TuRC. (V) Co-immunoprecipitation (IP) assays showing Tubgcp3 interacts with γ-tubulin through its C terminal domain. HEK293T cells were transfected with plasmids to express GFP-tagged zebrafish γ-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. β-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).

Figure Data
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Front. Mol. Neurosci.