1. Cell Biology

CYK-4 functions independently of its centralspindlin partner ZEN-4 to cellularize oocytes in germline syncytia

  1. Kian-Yong Lee
  2. Rebecca A Green
  3. Edgar Gutierrez
  4. J Sebastian Gomez-Cavazos
  5. Irina Kolotuev
  6. Shaohe Wang
  7. Arshad Desai
  8. Alex Groisman
  9. Karen Oegema  Is a corresponding author
  1. University of California, San Diego, United States
  2. University of Rennes 1, France
https://doi.org/10.7554/eLife.36919
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Cite this article
  1. Kian-Yong Lee
  2. Rebecca A Green
  3. Edgar Gutierrez
  4. J Sebastian Gomez-Cavazos
  5. Irina Kolotuev
  6. Shaohe Wang
  7. Arshad Desai
  8. Alex Groisman
  9. Karen Oegema
(2018)
CYK-4 functions independently of its centralspindlin partner ZEN-4 to cellularize oocytes in germline syncytia
eLife 7:e36919.
https://doi.org/10.7554/eLife.36919
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8 figures, 1 video, 2 tables and 1 additional file

Figures

Figure 1 with 2 supplements
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Both centralspindlin subunits localize to intercellular bridges throughout C. elegans germline development.

(A) (Left) Schematics highlight the domain structure of the two molecular components, CYK-4 and ZEN-4, of the heterotetrameric centralspindlin complex, and the location of the temperature sensitive …

https://doi.org/10.7554/eLife.36919.002
Figure 1—source data 1

Rachis bridges increase in diameter during oocyte loading prior to their closure during oocyte cellularization.

https://doi.org/10.7554/eLife.36919.005
Figure 1—figure supplement 1
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Generation of a functional single-copy transgene encoding CYK-4::mNeonGreen and in situ-tagged GFP::ZEN-4.

(A) (Left) Schematic of the RNAi-resistant cyk-4::mNeonGreen transgene integrated into a specific site (Mos transposon insertion) on Chromosome II. (Right) Graph plotting embryonic viability (mean Â± â€¦

https://doi.org/10.7554/eLife.36919.003
Figure 1—figure supplement 1—source data 1

The RNAi-resistant transgene encoding CYK-4::mNeonGreen rescues depletion of endogenous CYK-4.

https://doi.org/10.7554/eLife.36919.006
Figure 1—figure supplement 2
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Structure of the nascent syncytial germline and rachis in the L1 larva.

(A) Timelapse images collected using the worm trap described in Figure 4A over a 3-hr period of the germline in an L1 stage worm. At this stage, the intercellular bridge connecting the two nuclear …

https://doi.org/10.7554/eLife.36919.004
Figure 2
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CYK-4 is required for oocyte production by the adult germline.

(A) The CYK-4 C-terminus is not required for compartment proliferation during germline development. (Upper left) Larvae were upshifted to the non-permissive temperature (25°C) at the L1 stage and …

https://doi.org/10.7554/eLife.36919.008
Figure 2—source data 1

The CYK-4 C-terminal region is not required for germline compartment proliferation.

https://doi.org/10.7554/eLife.36919.009
Figure 2—source data 2

The CYK-4 C-terminal region is required for oocyte production.

https://doi.org/10.7554/eLife.36919.010
Figure 3 with 1 supplement
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ZEN-4 is not essential for embryo production by the adult syncytial germline.

(A) (Top) Schematic outline of the imaging experiment following dsRNA injection. (Bottom) Single plane confocal images of adult germlines expressing a GFP-tagged plasma membrane probe (shown in red) …

https://doi.org/10.7554/eLife.36919.011
Figure 3—source data 1

ZEN-4 is required for embryo viability but not production.

https://doi.org/10.7554/eLife.36919.013
Figure 3—source data 2

The CYK-4—ZEN-4 interaction is not required for embryo production.

https://doi.org/10.7554/eLife.36919.014
Figure 3—figure supplement 1
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The interaction between the CYK-4 and ZEN-4 dimers is globally required for worm growth and development between the L1 and L4 stages.

(A) Low-resolution images of worms grown as indicated in the schematic. Briefly, L1 worms were picked from asynchronous populations grown at 16°C, and pictures were snapped immediately before …

https://doi.org/10.7554/eLife.36919.012
Figure 3—figure supplement 1—source data 1

The CYK-4—ZEN-4 interaction is required for larval growth and germline pathfinding.

https://doi.org/10.7554/eLife.36919.015
Figure 3—figure supplement 1—source data 2

Aberrant germlines in L1-L4 upshifted centralspindlin assembly mutants fail to produce embryos following downshift.

https://doi.org/10.7554/eLife.36919.016
Figure 4 with 2 supplements
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CYK-4 is required for oocyte cellularization.

Individual CYK-4 mutant worms were longitudinally monitored using a custom vacuum-actuated microfluidic device (the ‘worm trap’). (A) Schematics summarize the experimental procedure for mounting …

https://doi.org/10.7554/eLife.36919.017
Figure 4—figure supplement 1
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Design of the worm trap microfluidic chip.

Schematic of microfluidic device (left) and 4X zoom of left-most imaging chamber (right). The worm trap microfluidic device is assembled out of a PDMS chip with microchannels engraved on its surface …

https://doi.org/10.7554/eLife.36919.018
Figure 4—figure supplement 2
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Measurement of the rachis bridges in the CYK-4 C-terminal mutant cyk-4(or749ts).

(Top left) Schematic shows the temperature shift procedure for the experiment. (Top right and middle panels) Schematic and single plane confocal images of adult germlines in control and cyk-4(or749ts…

https://doi.org/10.7554/eLife.36919.019
Figure 4—figure supplement 2—source data 1

Rachis bridges in the pachytene region are wider in upshifted cyk-4(or749ts) mutant worms than in controls.

https://doi.org/10.7554/eLife.36919.020
Figure 5 with 1 supplement
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CYK-4 requires both its C1 domain and the Rho GTPase interaction interface of its GAP to target to the rachis surface and bridges and promote oocyte cellularization.

(A) (Left) Schematic illustrates the set of single copy untagged RNAi-resistant cyk-4 transgenes inserted into a specific location on Chr. II generated to analyze the role of the C1 and GAP domains. …

https://doi.org/10.7554/eLife.36919.021
Figure 5—source data 1

Both the C1 domain and the GTPase binding interface of the GAP domain are required for the germline function of CYK-4.

https://doi.org/10.7554/eLife.36919.023
Figure 5—figure supplement 1
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The WT, GTPase binding interface mutant, and ΔC1 mutant CYK-4 proteins encoded by the single copy untagged transgenes are expressed at levels comparable to endogenous CYK-4.

(A) Schematic of the RNAi-resistant untagged cyk-4 transgene integrated into a specific site (Mos transposon insertion) on Chromosome II. (B) Full versions of the blots in Figure 5B. L374A and GG …

https://doi.org/10.7554/eLife.36919.022
Figure 5—figure supplement 1—source data 1

Both the C1 and the GTPase binding interface of the GAP domain are required for embryonic viability.

https://doi.org/10.7554/eLife.36919.024
Figure 5—figure supplement 1—source data 2

The RNAi-resistant transgene encoding CYK-4::mCherry rescues depletion of endogenous CYK-4.

https://doi.org/10.7554/eLife.36919.025
Figure 6 with 2 supplements
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The Rho GTPase binding interface of the CYK-4 GAP domain may recruit CYK-4 to the rachis surface/bridges by binding RhoARHO-1.

(A) Central plane confocal images acquired in adult worms expressing CYK-4::mNeonGreen (green) and an mCherry-tagged plasma membrane probe (red) before and after photobleaching of the …

https://doi.org/10.7554/eLife.36919.026
Figure 6—source data 1

Depletion of RhoA or CYK-4 leads to a comparable reduction in embryo production.

https://doi.org/10.7554/eLife.36919.030
Figure 6—figure supplement 1
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The anillin homolog, ANI-2, is not required to recruit CYK-4 recruitment to the rachis surface/bridges.

Maximum intensity projections of germlines in adult control and ani-2(RNAi) (48 hr after introduction of dsRNA by soaking) animals expressing CYK-4::mNeonGreen and an mCherry-tagged plasma membrane …

https://doi.org/10.7554/eLife.36919.027
Figure 6—figure supplement 2
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Depletion of RacCED-10 or CDC-42 cannot rescue the effects of mutants in the Rho GTPase binding interface on the germline.

Graph shows the number of embryos laid by the worms (mean Â± SD) 24–48 hr post-injection for the indicated strains and RNAi conditions. N = number of worms.

https://doi.org/10.7554/eLife.36919.028
Figure 6—figure supplement 2—source data 2

Depletion of Racor CDC-42 does not rescue the germline defects of the CYK-4 Rho GTPase binding interface mutants.

https://doi.org/10.7554/eLife.36919.029
Figure 7
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Active RhoARHO-1 localization to the rachis surface/bridges depends on ECT-2 and CYK-4.

(A) Maximum intensity projections (top) and single central plane images (middle) of the germline in adult worms expressing GFP::RGA-3 (green) and an mCherry-tagged plasma membrane probe (red) …

https://doi.org/10.7554/eLife.36919.031
Figure 7—source data 1

RGA-3/4 depletion cannot rescue the effect of the R459A mutation on embryo production.

https://doi.org/10.7554/eLife.36919.032
Figure 8
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A C-terminal C1 domain-GAP module targets CYK-4 to the rachis surface/bridges to enable oocyte celluarization.

(A) Schematics compare the closure of compartment bridges during oocyte cellularization in the germline (left) to cytokinesis (shown here in a two-cell stage embryo, right). (Left) During bridge …

https://doi.org/10.7554/eLife.36919.033

Videos

Video 1
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Structure of the syncytial germline at the two-compartment stage in an L1 larva.

Video shows images of a stack of serial 100 nm serial sections of an L1 germline that were collected and imaged by transmission electron microscopy. Images are shown without and then with …

https://doi.org/10.7554/eLife.36919.007

Tables

Table 1
C. elegans strains used in this study.
https://doi.org/10.7554/eLife.36919.034
Strain #GenotypeFigure
N2wild type (ancestral)1S1, 1S2B, 2B, 3B, 5B, 5C, 5E, 5S1, 6B, 6S2
OD95unc-119(ed3) III; ltIs37 [pAA64; Ppie-1::mCherry::his-58; unc-119 (+)] IV;
ltIs38 [pAA1; Ppie-1::GFP::PH(PLC1delta1); unc-119(+)] III		1S2, 2A, 2B, 3A, 3E, 3F, 3S1, 4S2, 6B
OD239cyk-4(or749ts) III; ltIs38 [pAA1; Ppie-1::GFP::PH(PLC1delta1) unc-119 (+)] III;
ltIs37 [pAA64; Ppie-1::mCherry::H2B his-58; unc-119 (+)] IV		2A, 2B, 3S1, 4B, 4S2
OD241cyk-4(t1689ts) III; ltIs38 [pAA1; Ppie-1::GFP::PH(PLC1delta1) unc-119 (+)] III;
ltIs37 [pAA64; Ppie-1::mCherry::H2B his-58; unc-119 (+)] IV		3E, 3F, 3S1, 4B
OD1176unc-119(ed3) III; ItSi346 [pKL3; Pcyk-4::CYK-4reencoded::mCherry;
cb-unc-119(+)] IV		5D, 5E, 5S1D, 6C, 7C
OD1178unc-119(ed3) III; ItSi348 [pKL4; Pcyk-4::CYK-4reencoded(R459A)::mCherry;
cb-unc-119(+)] IV		5D, 5E, 7C
OD1211ItSi346 [pKL3; Pcyk-4::CYK-4reencoded::mCherry; cb-unc-119(+)] IV; ltIs38 [pAA1;
Ppie-1::GFP::PH(PLC1delta1); unc-119 (+)] III		6C
OD1364unc-119(ed3) III; ItSi432[pKL33; Pcyk-4::CYK-4reencoded(∆C1)::mCherry;
cb-unc-119(+)] IV		5D, 5E
OD1970ltSi835 [pKL62; Pcyk-4::CYK-4reencoded; cb-unc-119(+)]II; unc-119(ed3) III5B, 5S1B, 5S1C, 6S2
OD1972ltSi837 [pKL64; Pcyk-4::CYK-4reencoded(R459A);
cb-unc-119(+)] II; unc-119(ed3) III		5B, 5S1B, 5S1C, 6S2
OD1974ltSi839 [pKL65; Pcyk-4::CYK-4reencoded(R459A/K495A/R499E);
cb-unc-119(+)] II; unc-119(ed3) III		5B, 5S1B, 5S1C, 6S2
OD1978ltSi843 [pKL67; Pcyk-4::CYK-4reencoded(∆C1); cb-unc-119(+)] II; unc-119(ed3) III5B, 5S1B, 5S1C
OD2064ltSi849[pKL120; Pmex-5::mCherry-PH::tbb-2 3'UTR; cb-unc-119(+)]I;
ItSi641[pKL89; Pcyk-4::CYK-4reencoded::GFP::MEI-1 (1–224); cb-unc-119(+)]I;
unc-119(ed3)III; ltIs37[pAA64; pie-1/mCherry::H2B his-58; unc-119(+)] IV		5C
OD2083ltSi849 [pKL120; Pmex-5::mCherry::PH(PLC1delta1)::tbb-2 3'UTR;
cb-unc-119(+)] I; ItSi641 [pKL89; Pcyk-4::CYK-4reencoded::GFP::MEI-1 (1–224);
cb-unc-119(+)] I; ltSi835 [pKL62; Pcyk-4::CYK-4reencoded; cb-unc-119(+)]II;
unc-119(ed3) III; ltIs37 [pAA64; Ppie-1::mCherry::H2B his-58; unc-119(+)] IV		5C
OD2084ltSi849 [pKL120; Pmex-5::mCherry:: PH(PLC1delta1)::tbb-2 3'UTR;
cb-unc-119(+)] I; ItSi641 [pKL89; Pcyk-4::CYK-4reencoded::GFP::MEI-1 (1–224);
cb-unc-119(+)] I; ltSi837 [pKL64; Pcyk-4::CYK-4reencoded(R459A);
cb-unc-119(+)] II; unc-119(ed3) III; ltIs37 [pAA64; Ppie-1::mCherry::H2B his-58;
unc-119(+)] IV		5C
OD2085ltSi849 [pKL120; Pmex-5::mCherry::PH(PLC1delta1)::tbb-2 3'UTR;
cb-unc-119(+)] I; ItSi641 [pKL89; Pcyk-4::CYK-4reencoded::GFP::MEI-1 (1–224);
cb-unc-119(+)] I; ltSi839 [pKL65; Pcyk-4::CYK-4reencoded(R459A/K495A/R499E);
cb-unc-119(+)] II; unc-119(ed3) III; ltIs37 [pAA64; Ppie-1::mCherry::H2B his-58;
unc-119(+)] IV		5C
OD2087ltSi849 [pKL120; Pmex-5::mCherry::PH(PLC1delta1)::tbb-2 3'UTR; cb-unc-119(+)]I;
ItSi641 [pKL89; Pcyk-4::CYK-4reencoded::GFP::MEI-1 (1–224); cb-unc-119(+)] I;
ltSi843 [pKL67; Pcyk-4::CYK-4reencoded(∆C1); cb-unc-119(+)] II; unc-119(ed3) III;
ltIs37 [pAA64; pie-1::mCherry::H2B his-58; unc-119(+)] IV		5C
OD2127ltSi220 [pOD1249/pSW077; Pmex-5::GFP-tbb-2-operon-linker-mCherry-his-11;
cb-unc-119(+)] I; ltSi849 [pKL120; Pmex-5::mCh-PH::tbb-2 3'UTR; cb-unc-119(+)] I		3G
OD2286unc-119(ed3) III; ItSi867 [pKL142; Pcyk-4::CYK-4reencoded(R459A/K495A/R499E)::mCherry;
cb-unc-119(+)] IV		5D, 5E
OD3639ltSi849 [pKL120; Pmex-5::mCherry::PH(PLC1delta1)::tbb-2 3'UTR; cb-unc-119(+)] I;
ltSi17 [pOD928/EZ-36; Prga-3::GFP::RGA-3; cb-unc-119(+)] II; unc-119(ed3) III		7A, 7B
OD3640ltSi849 [pKL120; Pmex-5::mCherry::PH(PLC1delta1)::tbb-2 3'UTR; cb-unc-119(+)] I;
unc-119(ed3) III(?); zen-4(lt30[GFP::loxP::zen-4]) IV		1B, 3C
OD3686ltSi849 [pKL120; Pmex-5::mCherry::PH(PLC1delta1)::tbb-2 3'UTR; cb-unc-119(+)] I;
ltSi1124[pKL177/pSG092; Pcyk-4::CYK-4reencoded::mNeonGreen::cyk-4 3'-UTR;
cb- unc-119(+)] II; unc-119(ed3) III		1B, 1C, 3D, 6A, 6D, 6S1
JCC754unc-119(ed3) III?; ltIs38 [pAA1; Ppie-1::GFP::PH(PLC1delta1); unc-119 (+)] III;
zen-4(or153ts)IV; ltIs37 [pAA64; Ppie-1::mCherry::his-58; unc-119 (+)] IV		3E, 3F, 3S1
Table 2
Oligos used for dsRNA production.
https://doi.org/10.7554/eLife.36919.035
GeneOligonucleotide 1 (5’→3’)Oligonucleotide 2 (5→3’)TemplateConcentration (mg/ml)
cyk-4 (K08E3.6)CGTAATACGACTCACTATAGGTGTCA
AAGACACTCAGAAAC		CGTAATACGACTCACTATAGGCCTC
TTCGAATTGGCAGCAGC		N2 cDNA2.0
zen-4 (M03D4.1)AATTAACCCTCACTAAAGGAATTGGT
TATGGCTCCGAGA		TAATACGACTCACTATAGGATTGGA
GCTGTTGGATGAGC		N2 cDNA1.3
ect-2
(T19E10.1)		TAATACGACTCACTATAGGTCTCCGA
TAAATCTGTGGGG		AATTAACCCTCACTAAAGGCAGCAG
TTTGCGAAAATGAA		N2 genomic DNA2.0
spd-1
(Y34D9A.4)		TAATACGACTCACTATAGGTCGTTGA
CGCGTACTCAACT		AATTAACCCTCACTAAAGGGAATTC
GAAATCCGACTCCA		N2 cDNA1.8
rga-3/4
(K09H11.3/Y75B7AL.4)		TAATACGACTCACTATAGGCCTTCCT
GAGCACGACTTTC		AATTAACCCTCACTAAAGGAGCTTT
CGCGACCTTAAACA		N2 genomic DNA2.6
rho-1 (Y51H4A.3)AATTAACCCTCACTAAAGGATCGTC
TGCGTCCACTCTCT		TAATACGACTCACTATAGGCTCGGC
TGAAATTTCCAAAA		N2 genomic DNA1.9
ced-10 (C09G12.8)AATTAACCCTCACTAAAGGATCGCC
TCATCGA AAACTTG		TAATACGACTCACTAT AGGTCAAAT
GTGTCGT CGTTGGT		N2 cDNA2.0
cdc-42 (R07G3.1)AATTAACCCTCACTAAAGGGTTTGG
CATTTTTCAGGGAA		TAATACGACTCACTATAGGACGTGT
GCGTGCACATTTAT		N2 genomic DNA2.0
hyls-1 (C05C8.9)AATTAACCCTCACTAAAGGTGGCA
AATTTTACCACTGAAA		TAATACGACTCACTATAGGTGATATC
TTGTGACCGGATCA		N2 cDNA2.0
gfpAATTAACCCTCACTAAAGGCCAA
CACTTGTCACTACTTTCTGTTATGG		TAATACGACTCACTATAGGGTATAGT
TCATCCATGCCATGTGTAATCCC		Plasmid2.0

Additional files

Transparent reporting form
https://doi.org/10.7554/eLife.36919.036

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