Fig 1.
Live-cell imaging visualizes dynamics of meiosis and spermatid differentiation.
(A) A diagram showing the spermatid differentiation process in C. elegans. (B) Time-lapse analysis of meiosis and differentiation in 2 connected secondary spermatocytes dissected from him-5 males. White arrow indicates the pseudo-cleavage furrow between 2 undifferentiated spermatids. Double-headed arrow indicates RB expansion during spermatid differentiation. Yellow arrows point to cleavage furrows between spermatids and RB. Dashed line boxes indicate 1 haploid spermatid at each differentiation step. Numbers of released spermatids/total spermatids were quantified and are shown in the top right corner. (C–F) Time-lapse analysis of meiosis and differentiation in 2 connected secondary spermatocytes dissected from him-5 males. Spermatocytes in (C) and (E) are from animals expressing Histone::GFP and RPL-5::GFP, respectively. Spermatocytes in (D) and (F) are stained by MitoTracker Red and SiR-actin, respectively. White arrow in (C) indicates the pseudo-cleavage furrow. In (F), white lines indicate the distribution and enrichment of SiR-actin signal during RB formation. The inset and white arrow show an enlarged view of a moving SiR-actin punctum with a magnification of 4×, and yellow arrows indicate the closure of SiR-actin at the scission site. Scale bars: 5 μm. GFP, green fluorescent protein; RB, residual body; RPL-5, ribosomal protein 5, large subunit; SiR, silicon-rhodamine.
Fig 2.
Myosin II and myosin VI play distinct roles to regulate spermatid and RB formation.
(A) Time-lapse analysis of meiosis and differentiation in 2 connected secondary spermatocytes dissected from him-5 males expressing NMY-2::GFP. Arrow indicates accumulation of NMY-2 at the pseudo-cleavage furrow between 2 undifferentiated spermatids. Double-headed arrow indicates expansion of RB and NMY-2 during spermatid differentiation. (B) Time-lapse analysis of meiosis and differentiation in secondary spermatocytes dissected from him-5 or nmy-2;him-5 males at the nonpermissive temperature of 25°C. White arrows indicate pseudo-cleavage furrow ingression in the middle of the dividing him-5 secondary spermatocyte. Double-headed arrow indicates RB expansion in him-5. Yellow arrows point to the midpoint of the dividing secondary spermatocyte in nmy-2;him-5 animals. White lines mark the sperm–RB boundaries and indicate the sites of spermatid budding from the remaining cell body in nmy-2;him-5 animals. (C and D) Time-lapse images of secondary spermatocytes from him-5 males at different developmental stages in the presence of Latrunculin A. White arrows point to the midpoint of a secondary spermatocyte. Yellow arrows indicate the pseudo-cleavage furrow between 2 undifferentiated spermatids. (E) Time-lapse images of 2 connected secondary spermatocytes dissected from spe-15(hc75) nmy-2(ne3409);him-5(e1490) males stained by the DNA label DRAQ5 at the nonpermissive temperature of 25°C. The DRAQ5-labeled chromosomes were segregated, but differentiation of haploid spermatids was blocked. (F) Time-lapse images of meiosis and differentiation in 2 connected secondary spermatocytes dissected from him-5 males expressing GFP::SPE-15. Yellow arrows indicate accumulation of SPE-15 at the spermatid–RB boundary. (G) Time-lapse analysis of meiosis and differentiation in 2 connected secondary spermatocytes dissected from nmy-2(ne3409);him-5(e1490) males expressing GFP::SPE-15 at the nonpermissive temperature (25°C). (H) Time-lapse analysis of differentiation in connected spermatids dissected from spe-15(hc75);him-5(e1490) males expressing NMY-2::GFP at 20°C. White arrows point to NMY-2 puncta that appeared in spermatids when the RB was formed. The percentage of secondary spermatocytes with the representative pattern is shown at the top right corner. “n” indicates the number of secondary spermatocytes that were followed and quantified. Scale bars: 5 μm. DRAQ5, deep red anthraquinone 5; GFP, green fluorescent protein; NMY-2, non-muscle myosin 2; RB, residual body; SPE-15, defective spermatogenesis 15.
Fig 3.
Myosin II and myosin VI regulate cytoplasm segregation in different manners.
(A) Time-lapse analysis of meiosis and differentiation in 2 connected secondary spermatocytes dissected from him-5(e1490) and nmy-2(ne3409);him-5(e1490) males expressing Histone::GFP and stained by MitoTracker Red at the nonpermissive temperature (25°C). Numbers of mitochondria in RBs were quantified, and the results are shown in the table below the images. Underlying data can be found in S1 Data. (B) Time-lapse images of meiosis and differentiation in 2 connected secondary spermatocytes dissected from him-5(e1490) and spe-15(hc75);him-5(e1490) males expressing Histone::GFP and stained by MitoTracker Red at 20°C. Numbers of mitochondria in newly formed RBs and late RBs were quantified, and the results are shown in the table below the images. Underlying data can be found in S1 Data. (C and D) Time-lapse images of meiosis and differentiation in 2 connected secondary spermatocytes dissected from the indicated strains expressing RPL-5::GFP at 25°C (C) or 20°C (D). Arrows point to cortical ribosomes retained in spermatids of spe-15(hc75);him-5(e1490) animals. The percentage of secondary spermatocytes with the representative pattern is shown at the top right corner. n indicates the number of secondary spermatocytes that were followed. Scale bars: 5 μm. GFP, green fluorescent protein; RB, residual body; RPL-5, ribosomal protein 5, large subunit.
Fig 4.
Spermatid release is achieved through myosin VI–mediated cytokinesis.
(A) Time-lapse analysis of meiosis and differentiation in 2 connected secondary spermatocytes dissected from spe-15(hc75);him-5(e1490) and spe-15(ok153);him-5(e1490) males. White arrows indicate the pseudo-cleavage furrow between 2 undifferentiated spermatids. Double-headed arrows indicate RB expansion during spermatid differentiation. Yellow arrows point to the spermatid–RB boundary and indicate defective cytokinesis. Numbers of released spermatids/total spermatids were quantified and are shown in the top right corner. (B) Time-lapse analysis of spermatids and RBs that undergo cytokinesis in the indicated strains expressing Histone::GFP and stained by SiR-actin. White arrows indicate sites of cytokinesis. Numbers of sealed spermatids/total spermatids were quantified and are shown in the top right corner. DIC images of spermatids and RBs at the start time point (0 min, insets) are shown. (C and D) Light and fluorescence images of an RB with 4 attached spermatids stained by anti-SPE-15 antibody and phalloidin (C) or expressing NMY-2::GFP and stained by SiR-actin (D). Arrows point to the connection between the spermatid and RB. (E) Time-lapse images of meiosis and differentiation in 2 connected secondary spermatocytes dissected from him-5 males expressing GFP::SPE-15 and NMY-2::mCherry. White arrows indicate the pseudo-cleavage furrow with enriched NMY-2. Yellow arrows indicate accumulation of SPE-15 at the cleavage furrow between spermatids and RB. Scale bars: 5 μm. DIC, differential interference contrast; GFP, green fluorescent protein; NMY-2, non-muscle myosin 2; RB, residual body; SiR, silicon-rhodamine; SPE-15, defective spermatogenesis 15.
Fig 5.
Myosin VI and F-actin form an actomyosin VI contractile ring to control cytokinesis.
(A and B) Time-lapse images of the secondary spermatocytes (dissected from him-5 males) that undergo cytokinesis in the presence of Latrunculin A or Jasplakinolide. Arrows indicate the spermatid–RB connection and defective cytokinesis. The percentage of connections with the representative pattern is shown at the top right corner. n indicates the number of connections that were followed and quantified. (C) Time-lapse images showing differentiation of a spermatid dissected from a him-5 male expressing GFP::SPE-15 and further stained by SiR-actin. White arrows indicate movement of a SPE-15–positive SiR-actin punctum from the spermatid pole to the RB. (D) Reconstituted 3D time-lapse images showing differentiation of connected spermatids dissected from him-5 males expressing GFP::SPE-15 and further stained by SiR-actin. Arrows indicate contraction of the SPE-15 ring and sealing of the cortical actin. (E) Reconstituted 3D time-lapse images showing resistance of the actomyosin VI ring to detergent treatment; 2D images in the bottom row show disruption of spermatids and RBs by detergent. Scale bars: 5 μm. GFP, green fluorescent protein; RB, residual body; SiR, silicon-rhodamine; SPE-15, defective spermatogenesis 15.
Fig 6.
GIPC interacts with myosin VI to regulate actomyosin VI ring formation and spermatid release.
(A and B) GIPC-1 (A) or GIPC-2 (B) is coprecipitated by the CBD of wild-type SPE-15 but not the AAA SPE-15 mutant (R1067A, R1068A, and L1069A). (C and D) Time-lapse images of meiosis and differentiation in 2 connected secondary spermatocytes dissected from gipc-1;gipc-2;him-5 (C) or spe-15(qx529);him-5(e1490) (D) males. White arrows indicate the pseudo-cleavage furrow between 2 undifferentiated spermatids. Double-headed arrows indicate RB expansion during spermatid differentiation. Yellow arrows point to the spermatid–RB connections and indicate cytokinesis defects. Numbers of released spermatids/total spermatids were quantified and are shown in the top right corner. (E) Time-lapse analysis of the cytokinesis process in SiR-actin–stained spermatids/RBs from the indicated strains. White arrows indicate sites of cytokinesis. Numbers of sealed spermatids/total spermatids were quantified and are shown in the top right corner. DIC images of spermatids and RBs at the start time point (0 min, inset) are shown. (F) Fluorescence images of RBs with attached spermatids in the indicated strains stained by anti-GIPC and anti-SPE-15 antibody. White arrows point to spermatid–RB connections. (G) Fluorescence images of an RB with 4 attached spermatids dissected from him-5 or gipc-1;gipc-2;him-5 males expressing GFP::SPE-15. White arrows point to a spermatid–RB connection. Scale bars: 5 μm. CBD, cargo-binding domain; DIC, differential interference contrast; GFP, green fluorescent protein; GIPC, RGS-GAIP-interacting protein C terminus; IB, immunoblotting; IP, immunoprecipitation; RB, residual body; SiR, silicon-rhodamine; SPE-15, defective spermatogenesis 15.
Fig 7.
Proposed models showing the role of myosin II and myosin VI in spermatid differentiation.
(A) Proposed model of the role of myosin II and myosin VI in spermatid differentiation. Myosin II and myosin VI play distinct roles to regulate RB formation and cytoplasm segregation. Dashed boxes show the differentiation process of a single haploid spermatid in him-5 animals. Residual body formation is achieved by myosin II–mediated RB expansion and myosin VI–mediated spermatid budding. Spermatids are separated from RBs by myosin VI–mediated cytokinesis. Cellular contents are differentially segregated into spermatids and RBs during RB expansion, regulated by myosin II and myosin VI, respectively. Green arrows point to the pseudo-cleavage furrow that is enriched in actomyosin II. Blue arrows indicate accumulation of actomyosin VI at the cleavage furrow between the spermatid and the RB. Green dashed arrows and blue dashed arrows indicate the movements of NMY-2 and SPE-15, which are in opposite directions. In spe-15 nmy-2 double mutants, spermatid differentiation is completely blocked. (B) Proposed model of actomyosin VI ring formation and contraction in separating spermatids from the RB. GIPC may promote myosin VI dimerization or oligomerization, which is required for actomyosin VI ring formation. Head domains in SPE-15 dimers or oligomers may localize to anti-parallel actin bundles. Their movement towards the minus end of actin bundles may generate a pulling force and induce actin sliding, which drive contraction of the actomyosin VI ring. Other actin regulators, including formins, Arp2/3, and capping proteins, may regulate the architecture and dynamics of the actin network that are required for actomyosin VI ring formation and contraction. Arp2/3, actin-related proteins 2 and 3; GIPC, RGS-GAIP-interacting protein C terminus; NMY-2, non-muscle myosin 2; RB, residual body; SPE-15, defective spermatogenesis 15.