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Chromosome movements promoted by the mitochondrial protein SPD-3 are required for homology search during Caenorhabditis elegans meiosis.

Labrador L, Barroso C, Lightfoot J, Müller-Reichert T, Flibotte S, Taylor J, Moerman DG, Villeneuve AM, Martinez-Perez E - PLoS Genet. (2013)

Bottom Line: Preventing SC assembly in spd-3 mutants does not improve homolog pairing, demonstrating that SPD-3 is required for homology search at the start of meiosis.However, quantitative analysis of SUN-1 aggregate movement in spd-3 mutants demonstrates a clear reduction in mobility, although this defect is not as severe as that seen in sun-1(jf18) mutants, which also show a stronger pairing defect, suggesting a correlation between chromosome-end mobility and the efficiency of pairing.Our work reveals how chromosome mobility impacts the different early meiotic events that promote homolog pairing and suggests that efficient homology search at the onset of meiosis is largely dependent on motor-driven chromosome movement.

View Article: PubMed Central - PubMed

Affiliation: MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, London, United Kingdom.

ABSTRACT
Pairing of homologous chromosomes during early meiosis is essential to prevent the formation of aneuploid gametes. Chromosome pairing includes a step of homology search followed by the stabilization of homolog interactions by the synaptonemal complex (SC). These events coincide with dramatic changes in nuclear organization and rapid chromosome movements that depend on cytoskeletal motors and are mediated by SUN-domain proteins on the nuclear envelope, but how chromosome mobility contributes to the pairing process remains poorly understood. We show that defects in the mitochondria-localizing protein SPD-3 cause a defect in homolog pairing without impairing nuclear reorganization or SC assembly, which results in promiscuous installation of the SC between non-homologous chromosomes. Preventing SC assembly in spd-3 mutants does not improve homolog pairing, demonstrating that SPD-3 is required for homology search at the start of meiosis. Pairing center regions localize to SUN-1 aggregates at meiosis onset in spd-3 mutants; and pairing-promoting proteins, including cytoskeletal motors and polo-like kinase 2, are normally recruited to the nuclear envelope. However, quantitative analysis of SUN-1 aggregate movement in spd-3 mutants demonstrates a clear reduction in mobility, although this defect is not as severe as that seen in sun-1(jf18) mutants, which also show a stronger pairing defect, suggesting a correlation between chromosome-end mobility and the efficiency of pairing. SUN-1 aggregate movement is also impaired following inhibition of mitochondrial respiration or dynein knockdown, suggesting that mitochondrial function is required for motor-driven SUN-1 movement. The reduced chromosome-end mobility of spd-3 mutants impairs coupling of SC assembly to homology recognition and causes a delay in meiotic progression mediated by HORMA-domain protein HTP-1. Our work reveals how chromosome mobility impacts the different early meiotic events that promote homolog pairing and suggests that efficient homology search at the onset of meiosis is largely dependent on motor-driven chromosome movement.

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SPD-3 is required for chiasma formation and localizes to mitochondria.(A) Projection of diakinesis oocytes stained with DAPI. Note that while WT oocytes display 6 DAPI-stained bodies, corresponding to 6 bivalents, the spd-3(me85) mutant oocyte displays 9 DAPI-stained bodies, demonstrating a partial failure in chiasma formation. (B) Diagram of the spd-3 gene indicating the position of the me85 mutation and two deletion alleles. (C) Western blot probed with anti-SPD-3 antibodies, arrowheads point to the endogenous SPD-3 protein, arrow points the SDP-3::GFP fusion protein, and the asterisks labels a band that may correspond to a truncated SPD-3 protein made by spd-3(me85) mutants. The western blot shown at the bottom was probed with anti-tubulin antibodies and was used as a loading control. (D) Pachytene region of an ex-vivo germ line from a worm expressing SPD-3::GFP, which shows a localization pattern similar to that observed in (E) for immunolocalization of the mitochondrial protein cytochrome C in the pachytene region of fixed germ lines. (F) Western blots from cytosolic and mitochondrial extracts stained with the indicated antibodies. Note that SPD-3 is only found in the mitochondrial extract. (G) Electron micrographs of mitochondria purified from worms expressing SDP-3::GFP, and labeled with anti-GFP (left, 10 nm gold) and anti-NDUFS3 antibodies (right, 18 nm gold). (H) Whole germ lines from 16–18 hours post L4 worms stained with DAPI.
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pgen-1003497-g001: SPD-3 is required for chiasma formation and localizes to mitochondria.(A) Projection of diakinesis oocytes stained with DAPI. Note that while WT oocytes display 6 DAPI-stained bodies, corresponding to 6 bivalents, the spd-3(me85) mutant oocyte displays 9 DAPI-stained bodies, demonstrating a partial failure in chiasma formation. (B) Diagram of the spd-3 gene indicating the position of the me85 mutation and two deletion alleles. (C) Western blot probed with anti-SPD-3 antibodies, arrowheads point to the endogenous SPD-3 protein, arrow points the SDP-3::GFP fusion protein, and the asterisks labels a band that may correspond to a truncated SPD-3 protein made by spd-3(me85) mutants. The western blot shown at the bottom was probed with anti-tubulin antibodies and was used as a loading control. (D) Pachytene region of an ex-vivo germ line from a worm expressing SPD-3::GFP, which shows a localization pattern similar to that observed in (E) for immunolocalization of the mitochondrial protein cytochrome C in the pachytene region of fixed germ lines. (F) Western blots from cytosolic and mitochondrial extracts stained with the indicated antibodies. Note that SPD-3 is only found in the mitochondrial extract. (G) Electron micrographs of mitochondria purified from worms expressing SDP-3::GFP, and labeled with anti-GFP (left, 10 nm gold) and anti-NDUFS3 antibodies (right, 18 nm gold). (H) Whole germ lines from 16–18 hours post L4 worms stained with DAPI.

Mentions: In order to identify genes required for crossover formation, we performed a forward genetic screen to isolate mutants that displayed diakinesis oocytes with reduced numbers of chiasmata (Text S1). In wild-type oocytes, the six pairs of homologs are linked by chiasmata and therefore six DAPI-stained bodies are present (Figure 1A). In contrast, me85 mutants displayed diakinesis oocytes with up to twelve DAPI-stained bodies, demonstrating deficient chiasma formation (Figure 1A). Mapping of the me85 mutation using a comparative genome hybridization array [40, Text S1] suggested the presence of a mutation in the spd-3 gene, and sequencing of the spd-3 gene in me85 mutants confirmed the presence of an early STOP codon predicted to remove the last 61 amino acids of SPD-3 (Figure 1B). We next performed complementation tests between me85 mutants and two strains carrying spd-3 deletion alleles (tm2969 and ok1817, which are expected to be spd-3 alleles), as well as a transgenic strain expressing a functional GFP-tagged version of SPD-3 [41]. Both spd-3 deletion alleles failed to complement the me85 mutation, and the spd-3::GFP transgene fully rescued the defects of me85 homozygous worms. Furthermore, western blot analysis demonstrated that protein extracts from me85 mutants lacked a band at the expected molecular weight for SPD-3, while SPD-3-positive bands were clearly visible in extracts from wild-type worms and from worms carrying the spd-3::GFP transgene (Figure 1C). These results confirm that the phenotypes seen in me85 mutants are due to the early STOP codon in spd-3.


Chromosome movements promoted by the mitochondrial protein SPD-3 are required for homology search during Caenorhabditis elegans meiosis.

Labrador L, Barroso C, Lightfoot J, Müller-Reichert T, Flibotte S, Taylor J, Moerman DG, Villeneuve AM, Martinez-Perez E - PLoS Genet. (2013)

SPD-3 is required for chiasma formation and localizes to mitochondria.(A) Projection of diakinesis oocytes stained with DAPI. Note that while WT oocytes display 6 DAPI-stained bodies, corresponding to 6 bivalents, the spd-3(me85) mutant oocyte displays 9 DAPI-stained bodies, demonstrating a partial failure in chiasma formation. (B) Diagram of the spd-3 gene indicating the position of the me85 mutation and two deletion alleles. (C) Western blot probed with anti-SPD-3 antibodies, arrowheads point to the endogenous SPD-3 protein, arrow points the SDP-3::GFP fusion protein, and the asterisks labels a band that may correspond to a truncated SPD-3 protein made by spd-3(me85) mutants. The western blot shown at the bottom was probed with anti-tubulin antibodies and was used as a loading control. (D) Pachytene region of an ex-vivo germ line from a worm expressing SPD-3::GFP, which shows a localization pattern similar to that observed in (E) for immunolocalization of the mitochondrial protein cytochrome C in the pachytene region of fixed germ lines. (F) Western blots from cytosolic and mitochondrial extracts stained with the indicated antibodies. Note that SPD-3 is only found in the mitochondrial extract. (G) Electron micrographs of mitochondria purified from worms expressing SDP-3::GFP, and labeled with anti-GFP (left, 10 nm gold) and anti-NDUFS3 antibodies (right, 18 nm gold). (H) Whole germ lines from 16–18 hours post L4 worms stained with DAPI.
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Related In: Results  -  Collection

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pgen-1003497-g001: SPD-3 is required for chiasma formation and localizes to mitochondria.(A) Projection of diakinesis oocytes stained with DAPI. Note that while WT oocytes display 6 DAPI-stained bodies, corresponding to 6 bivalents, the spd-3(me85) mutant oocyte displays 9 DAPI-stained bodies, demonstrating a partial failure in chiasma formation. (B) Diagram of the spd-3 gene indicating the position of the me85 mutation and two deletion alleles. (C) Western blot probed with anti-SPD-3 antibodies, arrowheads point to the endogenous SPD-3 protein, arrow points the SDP-3::GFP fusion protein, and the asterisks labels a band that may correspond to a truncated SPD-3 protein made by spd-3(me85) mutants. The western blot shown at the bottom was probed with anti-tubulin antibodies and was used as a loading control. (D) Pachytene region of an ex-vivo germ line from a worm expressing SPD-3::GFP, which shows a localization pattern similar to that observed in (E) for immunolocalization of the mitochondrial protein cytochrome C in the pachytene region of fixed germ lines. (F) Western blots from cytosolic and mitochondrial extracts stained with the indicated antibodies. Note that SPD-3 is only found in the mitochondrial extract. (G) Electron micrographs of mitochondria purified from worms expressing SDP-3::GFP, and labeled with anti-GFP (left, 10 nm gold) and anti-NDUFS3 antibodies (right, 18 nm gold). (H) Whole germ lines from 16–18 hours post L4 worms stained with DAPI.
Mentions: In order to identify genes required for crossover formation, we performed a forward genetic screen to isolate mutants that displayed diakinesis oocytes with reduced numbers of chiasmata (Text S1). In wild-type oocytes, the six pairs of homologs are linked by chiasmata and therefore six DAPI-stained bodies are present (Figure 1A). In contrast, me85 mutants displayed diakinesis oocytes with up to twelve DAPI-stained bodies, demonstrating deficient chiasma formation (Figure 1A). Mapping of the me85 mutation using a comparative genome hybridization array [40, Text S1] suggested the presence of a mutation in the spd-3 gene, and sequencing of the spd-3 gene in me85 mutants confirmed the presence of an early STOP codon predicted to remove the last 61 amino acids of SPD-3 (Figure 1B). We next performed complementation tests between me85 mutants and two strains carrying spd-3 deletion alleles (tm2969 and ok1817, which are expected to be spd-3 alleles), as well as a transgenic strain expressing a functional GFP-tagged version of SPD-3 [41]. Both spd-3 deletion alleles failed to complement the me85 mutation, and the spd-3::GFP transgene fully rescued the defects of me85 homozygous worms. Furthermore, western blot analysis demonstrated that protein extracts from me85 mutants lacked a band at the expected molecular weight for SPD-3, while SPD-3-positive bands were clearly visible in extracts from wild-type worms and from worms carrying the spd-3::GFP transgene (Figure 1C). These results confirm that the phenotypes seen in me85 mutants are due to the early STOP codon in spd-3.

Bottom Line: Preventing SC assembly in spd-3 mutants does not improve homolog pairing, demonstrating that SPD-3 is required for homology search at the start of meiosis.However, quantitative analysis of SUN-1 aggregate movement in spd-3 mutants demonstrates a clear reduction in mobility, although this defect is not as severe as that seen in sun-1(jf18) mutants, which also show a stronger pairing defect, suggesting a correlation between chromosome-end mobility and the efficiency of pairing.Our work reveals how chromosome mobility impacts the different early meiotic events that promote homolog pairing and suggests that efficient homology search at the onset of meiosis is largely dependent on motor-driven chromosome movement.

View Article: PubMed Central - PubMed

Affiliation: MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, London, United Kingdom.

ABSTRACT
Pairing of homologous chromosomes during early meiosis is essential to prevent the formation of aneuploid gametes. Chromosome pairing includes a step of homology search followed by the stabilization of homolog interactions by the synaptonemal complex (SC). These events coincide with dramatic changes in nuclear organization and rapid chromosome movements that depend on cytoskeletal motors and are mediated by SUN-domain proteins on the nuclear envelope, but how chromosome mobility contributes to the pairing process remains poorly understood. We show that defects in the mitochondria-localizing protein SPD-3 cause a defect in homolog pairing without impairing nuclear reorganization or SC assembly, which results in promiscuous installation of the SC between non-homologous chromosomes. Preventing SC assembly in spd-3 mutants does not improve homolog pairing, demonstrating that SPD-3 is required for homology search at the start of meiosis. Pairing center regions localize to SUN-1 aggregates at meiosis onset in spd-3 mutants; and pairing-promoting proteins, including cytoskeletal motors and polo-like kinase 2, are normally recruited to the nuclear envelope. However, quantitative analysis of SUN-1 aggregate movement in spd-3 mutants demonstrates a clear reduction in mobility, although this defect is not as severe as that seen in sun-1(jf18) mutants, which also show a stronger pairing defect, suggesting a correlation between chromosome-end mobility and the efficiency of pairing. SUN-1 aggregate movement is also impaired following inhibition of mitochondrial respiration or dynein knockdown, suggesting that mitochondrial function is required for motor-driven SUN-1 movement. The reduced chromosome-end mobility of spd-3 mutants impairs coupling of SC assembly to homology recognition and causes a delay in meiotic progression mediated by HORMA-domain protein HTP-1. Our work reveals how chromosome mobility impacts the different early meiotic events that promote homolog pairing and suggests that efficient homology search at the onset of meiosis is largely dependent on motor-driven chromosome movement.

Show MeSH
Related in: MedlinePlus