FYCO1 and autophagy control the integrity of the haploid male germ cell-specific RNP granules - PubMed (original) (raw)

FYCO1 and autophagy control the integrity of the haploid male germ cell-specific RNP granules

Matteo Da Ros et al. Autophagy. 2017 Feb.

Abstract

Ribonucleoprotein (RNP) granules play a major role in compartmentalizing cytoplasmic RNA regulation. Haploid round spermatids that have exceptionally diverse transcriptomes are characterized by a unique germ cell-specific RNP granule, the chromatoid body (CB). The CB shares many characteristics with somatic RNP granules but also has germline-specific features. The CB appears to be a central structure in PIWI-interacting RNA (piRNA)-targeted RNA regulation. Here, we identified a novel CB component, FYCO1, which is involved in the intracellular transport of autophagic vesicles in somatic cells. We demonstrated that the CB is associated with autophagic activity. Induction of autophagy leads to the recruitment of lysosomal vesicles onto the CB in a FYCO1-dependent manner as demonstrated by the analysis of a germ cell-specific Fyco1 conditional knockout mouse model. Furthermore, in the absence of FYCO1, the integrity of the CB was affected and the CB was fragmented. Our results suggest that RNP granule homeostasis is regulated by FYCO1-mediated autophagy.

Keywords: FYCO1; RNP granule; autophagy; chromatoid body; germ cell; knockout; lysosome; posttranscriptional; spermatogenesis; vesicle.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Expression of a novel CB component FYCO1 during spermatogenesis. (A) FYCO1 coprecipitates with CBs. CB isolation was performed from cross-linked testicular lysate using anti-DDX4 antibody. FYCO1 was detected in the CB fraction (CB IP) but not in the negative control immunoprecipitated with rabbit IgG (CTRL IP). LYS, testicular cell lysate; SUP, CB-free supernatant fraction after low-speed centrifugation; PEL, CB-containing pellet fraction after low-speed centrifugation; FP, filtered pellet fraction. Immunoblotting with anti-DDX4 confirmed the successful CB isolation. (B) Immunoblotting of tissue lysates using anti-FYCO1 antibody. T, testis; E, epididymis; O, ovary; B, brain; K, kidney; S, spleen; H, heart;; L, liver. GAPDH antibody was used as a loading control. (C) Expression of FYCO1 during the first wave of spermatogenesis. Testis samples were collected from juvenile mice at different time points (1 wk, 2 wk, 3 wk, 4 wk, 10 wk/adult). In 1-wk-old testes, spermatogonia exist together with somatic cells. At 2 wk of age, pachytene spermatocytes have appeared; at 3 wk, round spermatids are present; and at 4 wk, spermatid elongation has begun. Anti-GAPDH was used for normalization. Error bars represent standard deviations. (D) Expression of Fyco1 mRNA during first wave of spermatogenesis as detected by RT-qPCR. Error bars represent s.e.m. of 3 biological replicates. (E) Immunofluorescence analysis of the PFA-fixed paraffin-embedded testis samples (10 wk old) using anti-FYCO1 antibody (red). FYCO1 has granular cytoplasmic staining in round spermatids (white arrows, stages II-V and VI-VII), late pachytene spermatocytes (blue arrows, stage IX-X) and step 9 to 14 elongating spermatids (yellow arrows, stages IX-X, XII and II-V). No FYCO1 signal was detected in step 15 to 16 elongating spermatids at stage VI-VIII. Nuclei were stained with DAPI (blue). PSpc, pachytene spermatocyte; RS, round spermatid; ES, elongating spermatid; M, meiotic metaphase; 2nd Spc, secondary spermatocyte. Scale bar: 10 μm.

Figure 2.

Figure 2.

FYCO1 localizes to the CBs. (A) Coimmunostaining of FYCO1 (red) with different CB markers DDX4, DDX25 and PIWIL1 (green) in round spermatids. FYCO1 signal is strongly associated with CBs (arrows), which are detected in close contact with round spermatid nuclei (asterisks). Costaining of FYCO1 with TSKS (green) showed the localization of FYCO1 in TSKS-positive late CBs in elongating spermatids (arrowheads) in addition to the localization of the CBs in round spermatids that are negative for TSKS (arrows). Nuclei were stained with DAPI (blue). (B) FYCO1 colocalizes with CB markers DDX25 and DDX4 already in cytoplasmic granules of late pachytene spermatocytes (arrowheads). (C) The FYCO1 signal is not concentrated on the core CB matrix but in the peripheral areas of the CB, dashed circles are drawn as a reference. Some non-CB-associated small FYCO1 granules can be detected in the cytoplasm or round spermatids (arrowheads). Upper and lower images in (C) represent different slices of a stack obtained by laser-scanning confocal microscopy of the same sample. Scale bars: 5 μm. (D) 3D modeling of the FYCO1 (red) localization in relation to the PIWIL2-positive CB (green). In the confocal image, red arrows point to the FYCO1-positive areas at the periphery of the CB that are not overlapping with the PIWIL2 signal. Scale bar: 5 µm. Middle and right panels show 2 different views of the 3D modeling of the confocal z stack immunostained with anti-PIWIL2 (green) and anti-FYCO1 (red) antibodies.

Figure 3.

Figure 3.

Inhibition of phosphoinositide 3-kinases affects the CB localization of FYCO1. (A) Stage-specific pieces of seminiferous tubules (II-V) from wildtype mice were incubated with DMSO or wortmannin for 6 h. After cultures, squash preparations were made and immunostained with anti-DDX25 (green) and anti-FYCO1 (red) antibodies. Nuclei were stained with DAPI. (B) A closer look at the DMSO- and wortmannin-treated tubules immunostained with anti-DDX25 and anti-FYCO1 antibodies. Arrows indicate the CB domain with accumulated FYCO1 signal after wortmannin treatment, and DDX25-positive CB domains devoid of FYCO1 signal are circled with dashed green line. Scale bars: 10 µm. Intensity line graphs show fluorescence intensity of FYCO1 (red) and DDX25 (green) along the white lines drawn on the images. (C) Quantification of the colocalization of FYCO1 and DDX25 after wortmannin treatment using Manders coefficients. M1 represents the relative amount of red pixels (FYCO1) colocalizing with green pixels (DDX25), while M2 represents the relative amount of green pixels colocalizing with red pixels. Error bars represent standard deviations. Significant reduction of colocalization was detected in both groups (*P value ≤ 0.05).

Figure 4.

Figure 4.

FYCO1 interacts with CB components and proteins involved in vesicle trafficking and microtubule-mediated transport. (A) Coimmunoprecipitation using anti-FYCO1 antibody followed by western blotting with anti-FYCO1, anti-RUVBL1, anti-RUVBL2, anti-EIF4A3, anti-PIWIL2 and anti-PIWIL1 antibodies. Immunoprecipitation using rabbit IgG (CTRL IP) was used as a negative control. LYS, protein lysate before immunoprecipitation. (B) Twenty most significant GO terms for FYCO1-interacting proteins. The graph shows the total number of proteins listed under each GO term (light gray), the number of FYCO1-interacting proteins found in each GO term (dark gray), and the number of proteins expected to be found by chance (black). For better comparability, the total numbers are log transformed. (C) FYCO1-interacting proteins contained 20 CB proteins, 46 proteins under the GO term “vesicle” and 38 proteins under the GO term “microtubule-based process.” 12 proteins that are listed under both GO terms are shown in red.

Figure 5.

Figure 5.

FYCO1 is required for the integrity of the CB. (A) Immunoblotting of adult testicular lysate using anti-FYCO1 antibody validated the dramatically lowered amount of FYCO1 in the germ cell-specific Fyco1 cKO testes. DDX4 expression levels were unaltered in cKO testes. Anti-GAPDH immunoblotting demonstrated the equal loading of proteins in each lane. CTRL: Fyco1(fx/fx);Neurog3 Cre−, HEZ: Fyco1(fx/wt);Neurog3 Cre+, cKO: Fyco1(fx/fx);Neurog3 Cre+. (B) Immunofluorescence staining using anti-FYCO1 antibody (red) confirmed the absence of FYCO1 expression in the Fyco1 cKO round spermatids. Nuclei were stained with DAPI. Scale bar: 10 μm. (C) The integrity of the CB was examined by immunofluorescence analysis using CB markers DDX4 (green) and DDX25 (red). Nuclei were stained with DAPI. CBs in wild-type samples are indicated with white arrows. Fragmented CBs in cKO tissues are indicated with arrowheads; fragments representing the CB of one cell are marked with arrowheads of the same color . Scale bar: 5 μm. (D) The number of CB fragments (DDX4-positive granules) per nucleus at different phases of differentiation (stages I-II, II-V, VII-VIII) was increased in Fyco1 cKO compare with control round spermatids. The change was significant at stage I-II (**P value ≤ 0.01, 2-tailed t -test) and stage II-V (*P value ≤ 0.05, 2-tailed t test). For each stage, 50 nuclei were counted in each of 3 replicates. For a second graph, DDX25-positive granules were segmented using BioimageXD to calculate the average volume (in voxels) of DDX25-positive CBs (**P value ≤ 0.01, 2-tailed t test). For a third graph, DDX4-positive granules were segmented and the total volume of DDX4-positive CBs (in voxels) was normalized by the number of nuclei marked by DAPI (***P value ≤ 0.001, 2-tailed t test). Error bars represent standard deviations. (E) Pieces of stage IV-VI seminiferous tubules from control and Fyco1 cKO mice were cultured for 6 h in the absence (DMSO) or presence of the microtubule disrupting chemical nocodazole. Immunofluorescence staining of the squashed preparations from the cultured seminiferous tubules was performed with anti-DDX4 antibody (red). Scale bar: 10 µm. Quantification demonstrates the number of DDX4-postitive fragments per nucleus after nocodazole treatment in control and Fyco1 cKO round spermatids. For each condition, 50 nuclei were counted in each of 3 replicates. (F) PFA-fixed paraffin-embedded testis sections were immunostained with anti-TUBULIN (green) and anti-DDX25 (red). Nuclei were stained with DAPI. Scale bar: 10 µm.

Figure 6.

Figure 6.

The CB associates with vesicles in a FYCO1-dependent manner. (A) In electron microscopy, some of the CB-associated vesicles resemble phagophores (red circles in left and middle panel), although smaller in size than non-CB-associated phagophores (red circles in right panel). Arrows point to the nuclear envelope. Scale bar: 500 nm. (B) Electron tomography visualized the cup-like shape of CB-associated vesicles (arrowheads). Left panel: electron tomography, scale bar: 500 nm, middle panel: segmentation of the CB (red) and small cytoplasmic vesicles (green), right panels: 3D surface reconstruction of 2 representative vesicles. (C) Immunofluorescence analysis of paraffin-embedded testis sections with anti-DDX25 (green) and anti-LC3B (red) antibodies. LC3B-positive structures (arrows) can be found in the CB (upper and middle images) and accumulated in a close proximity of the CB (bottom images). Nuclei were stained with DAPI. (D) Accumulations of the LC3B signal close to the CB in control round spermatids (CTRL) are indicated with red dashed circles. These LC3B-positive clouds were absent in Fyco1 cKO spermatids, and only small LC3B vesicles were found distributed in the cytoplasm, sometimes making contact with the CB (white arrows). Scale bar: 10 µm. A graph shows the significant reduction (***P value ≤ 0.001, 2-tailed t -test) in CBs that overlap with LC3B signal in cKO round spermatids. (E) Anti-FYCO1 immunoprecipitation was performed from the round spermatid vesicle-containing fraction. Immunoblotting with anti-LC3B showed a coprecipitation of both LC3-I and LC3-II with FYCO1 (IP FYCO1), but not with rabbit IgG-bound beads (IP CTRL). Testis LYS, input lysate that was used as a starting material for immunoprecipitation. HeLa LYS, lysate from starved HeLa cells. (F) Electron microscopic analysis of control and Fyco1 cKO CBs. Two different stages of the seminiferous epithelial cycle containing either early- (stage I-II) or late-round spermatids (stage VII) are shown. CB-associated vesicles are painted in red. G, Golgi complex; Nu, nucleus. Scale bar: 500 nm. Quantification of CB-associated vesicle at 3 different developmental stages (stages I, II-V and VI-VIII) revealed a dramatic reduction of vesicles surrounding Fyco1 cKO CBs. Error bars represent standard deviations. P values range from *P value ≤ 0.05 to ***P value ≤ 0.001 (2-tailed t test) analyzed by GraphPad Prism 7.0.

Figure 7.

Figure 7.

Lysosomal vesicles are not recruited to the CB in the absence of FYCO1. (A) Stage-specific (II-V) pieces of the control (CTRL) or Fyco1 cKO (cKO) seminiferous tubules were cultured in the presence of vehicle (DMSO), rapamycin or Baf. After cultures, squash preparations were made and immunostained with anti-DDX25 (red) and anti-LAMP1 (green) antibodies. Nuclei were stained with DAPI (blue). Arrows point to the LAMP1-signal recruited in the CB area after rapamycin treatment. Accumulations of LAMP1-positive vesicles next to the CB after Baf treatment are indicated by arrowheads. Scale bar: 10 μm. Intensity of LAMP1 and DDX25 signal was measured along the yellow lines to reveal the spatial localization of LAMP1-positive vesicles and the CB. (B) Colocalization of LAMP1 with DDX25 in control and cKO testis treated with rapamycin, Baf or the vector (DMSO) was measured using Manders coefficients. Error bars represent standard deviations.

Figure 8.

Figure 8.

PIWIL2 is downregulated in Fyco1 cKO testes. (A) Western blot images for expression of FYCO1, PIWIL2, PIWIL1, DDX4 and GAPDH in control and Fyco1 cKO testes (3 biological replicates per genotype). Quantification revealed significantly lowered amount of PIWIL2 in Fyco1 cKO testes (**P value ≤ 0.01; 2-tailed t test). The expression levels of PIWIL1 and DDX4 were unaltered. Anti-GAPDH signal was used for the normalization. Error bars represent s.e.m. (B) Immunofluorescence analysis of paraffin-embedded testis sections at stage II-V (PIWIL1: red, PIWIL2: green) and stage I (PIWIL2: green) in control (CTRL) and Fyco1 cKO (cKO) mice. Nuclei were stained with DAPI. PSpc, pachytene spermatocyte; RS, round spermatid; ES, elongating spermatid. The roman numbers in parentheses after RS or ES represent the step of spermatid differentiation. Scale bar: 10 µm. (C) Segments of seminiferous tubules of control and Fyco1 cKO mice were incubated with DMSO, rapamycin (Rap) or Baf (30 segments/treatment) and PIWIL2 was detected with a specific antibody. The western blotting image represents one of the 3 biological replicates that were used for quantification. Anti-GAPDH signal was used for normalization. The average of 3 biological replicates are shown. Error bars represent s.e.m. *P value ≤ 0.05; 2-tailed t test.

References

    1. Soumillon M, Necsulea A, Weier M, Brawand D, Zhang X, Gu H, Barthès P, Kokkinaki M, Nef S, Gnirke A, et al.. Cellular source and mechanisms of high transcriptome complexity in the mammalian testis. Cell Rep 2013; 3:2179-90; PMID:23791531; http://dx.doi.org/10.1016/j.celrep.2013.05.031 - DOI - PubMed
    1. Chalmel F, Lardenois A, Evrard B, Rolland AD, Sallou O, Dumargne MC, Coiffec I, Collin O, Primig M, Jégou B. High-resolution profiling of novel transcribed regions during rat spermatogenesis. Biol Reprod [Internet] 2014. [cited 2015February25]; 91:5 Available from: http://www.ncbi.nlm.nih.gov/pubmed/24740603 - PubMed
    1. Laiho A, Kotaja N, Gyenesei A, Sironen A. Transcriptome profiling of the murine testis during the first wave of spermatogenesis. PLoS One [Internet] 2013; 8:e61558 Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3629203&tool=p...; http://dx.doi.org/10.1371/journal.pone.0061558 - DOI - PMC - PubMed
    1. Idler RK, Yan W. Control of messenger RNA fate by RNA-binding proteins: an emphasis on mammalian spermatogenesis. J Androl 2012; 33:309-37; PMID:21757510; http://dx.doi.org/10.2164/jandrol.111.014167 - DOI - PMC - PubMed
    1. Paronetto MP, Sette C. Role of RNA-binding proteins in mammalian spermatogenesis. Int J Androl 2010; 33:2-12; PMID:19281489; http://dx.doi.org/10.1111/j.1365-2605.2009.00959.x - DOI - PubMed

MeSH terms

Substances

LinkOut - more resources