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The CSN/COP9 signalosome regulates synaptonemal complex assembly during meiotic prophase I of Caenorhabditis elegans.

Brockway H, Balukoff N, Dean M, Alleva B, Smolikove S - PLoS Genet. (2014)

Bottom Line: In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes.The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers.The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Program in Genetics, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
The synaptonemal complex (SC) is a conserved protein structure that holds homologous chromosome pairs together throughout much of meiotic prophase I. It is essential for the formation of crossovers, which are required for the proper segregation of chromosomes into gametes. The assembly of the SC is likely to be regulated by post-translational modifications. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via the ubiquitin degradation/proteasome pathway. Here we examine the role of the CSN/COP9 signalosome in SC assembly in the model organism C. elegans. Our work shows that mutants in three subunits of the CSN/COP9 signalosome fail to properly assemble the SC. In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes. The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers. The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes. We also find a marked increase in apoptosis in csn mutants that specifically eliminates nuclei with aggregated SC proteins. csn mutants exhibit defects in germline proliferation, and an almost complete pachytene arrest due to an inability to activate the MAPK pathway. The work described here supports a previously unknown role for the CSN/COP9 signalosome in chromosome behavior during meiotic prophase I.

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Apoptosis and MPK-1 expression are altered in csn mutants.A) Quantification of the number of nuclei with CED-1::GFP present in late pachytene of the C. elegans gonad. Red bars represent the total number of apoptotic nuclei in the late pachytene region. The blue bars represent the total number of nuclei in the late pachytene region. Apoptosis is increased in csn-2 mutants, but not in csn-5 mutants, *pMW<0.0005. There is also a reduction of overall nuclei in the late pachytene region of the gonad in both csn mutants, **pMW<0.0005. wild-type n = 25 gonads; csn-2 n = 19; and csn-5, n = 18, B) Analysis of SYP-1 aggregate phenotype in csn mutants and apoptosis checkpoint double mutants. Bypassing the apoptotic checkpoints reduces the number of nuclei with aggregates. Total number of nuclei counted and p-values can be found in Sup. Table 5. C) Quantification of dpMPK-1 expression via IF analyses. csn mutants lack MPK-1 staining in late pachytene and in diakinesis, *pFET<0.0005. wild-type n = 75, csn-2 n = 32, csn-5, n = 27, syp-1(me17) n = 32, D) Western Blot confirming the lack of expression of MPK-1B in csn mutants. MPK-1A is mostly somatic and MPK-1B is germline specific. Normalization values (α-MPK-1/α-TUB) shown are the average of 3 different experiments. Normalized intensities: wild-type 2.27±1.03, csn-2 0.96±0.42 and csn-5 0.99±0.09.
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pgen-1004757-g006: Apoptosis and MPK-1 expression are altered in csn mutants.A) Quantification of the number of nuclei with CED-1::GFP present in late pachytene of the C. elegans gonad. Red bars represent the total number of apoptotic nuclei in the late pachytene region. The blue bars represent the total number of nuclei in the late pachytene region. Apoptosis is increased in csn-2 mutants, but not in csn-5 mutants, *pMW<0.0005. There is also a reduction of overall nuclei in the late pachytene region of the gonad in both csn mutants, **pMW<0.0005. wild-type n = 25 gonads; csn-2 n = 19; and csn-5, n = 18, B) Analysis of SYP-1 aggregate phenotype in csn mutants and apoptosis checkpoint double mutants. Bypassing the apoptotic checkpoints reduces the number of nuclei with aggregates. Total number of nuclei counted and p-values can be found in Sup. Table 5. C) Quantification of dpMPK-1 expression via IF analyses. csn mutants lack MPK-1 staining in late pachytene and in diakinesis, *pFET<0.0005. wild-type n = 75, csn-2 n = 32, csn-5, n = 27, syp-1(me17) n = 32, D) Western Blot confirming the lack of expression of MPK-1B in csn mutants. MPK-1A is mostly somatic and MPK-1B is germline specific. Normalization values (α-MPK-1/α-TUB) shown are the average of 3 different experiments. Normalized intensities: wild-type 2.27±1.03, csn-2 0.96±0.42 and csn-5 0.99±0.09.

Mentions: Both csn-2 and csn-5 mutants had lower average numbers of nuclei in late pachytene (Figure 6A wild-type average = 52.2; csn-2 = 26.7; and csn-5 = 30.9). However, only csn-2 had a significant increase in apoptosis (4-fold) while csn-5 had apoptotic levels similar to wild-type (Figure 6A wild-type average = 2.96; csn-2 = 8.2; and csn-5 = 3.3 apoptosis levels were not examined in csn-6 mutants). When normalized for the number of nuclei in late pachytene, both mutants showed increased apoptosis, and, as expected, csn-2 mutants showed a larger increase. This is the more appropriate analysis since csn mutants have less germline nuclei.


The CSN/COP9 signalosome regulates synaptonemal complex assembly during meiotic prophase I of Caenorhabditis elegans.

Brockway H, Balukoff N, Dean M, Alleva B, Smolikove S - PLoS Genet. (2014)

Apoptosis and MPK-1 expression are altered in csn mutants.A) Quantification of the number of nuclei with CED-1::GFP present in late pachytene of the C. elegans gonad. Red bars represent the total number of apoptotic nuclei in the late pachytene region. The blue bars represent the total number of nuclei in the late pachytene region. Apoptosis is increased in csn-2 mutants, but not in csn-5 mutants, *pMW<0.0005. There is also a reduction of overall nuclei in the late pachytene region of the gonad in both csn mutants, **pMW<0.0005. wild-type n = 25 gonads; csn-2 n = 19; and csn-5, n = 18, B) Analysis of SYP-1 aggregate phenotype in csn mutants and apoptosis checkpoint double mutants. Bypassing the apoptotic checkpoints reduces the number of nuclei with aggregates. Total number of nuclei counted and p-values can be found in Sup. Table 5. C) Quantification of dpMPK-1 expression via IF analyses. csn mutants lack MPK-1 staining in late pachytene and in diakinesis, *pFET<0.0005. wild-type n = 75, csn-2 n = 32, csn-5, n = 27, syp-1(me17) n = 32, D) Western Blot confirming the lack of expression of MPK-1B in csn mutants. MPK-1A is mostly somatic and MPK-1B is germline specific. Normalization values (α-MPK-1/α-TUB) shown are the average of 3 different experiments. Normalized intensities: wild-type 2.27±1.03, csn-2 0.96±0.42 and csn-5 0.99±0.09.
© Copyright Policy
Related In: Results  -  Collection

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pgen-1004757-g006: Apoptosis and MPK-1 expression are altered in csn mutants.A) Quantification of the number of nuclei with CED-1::GFP present in late pachytene of the C. elegans gonad. Red bars represent the total number of apoptotic nuclei in the late pachytene region. The blue bars represent the total number of nuclei in the late pachytene region. Apoptosis is increased in csn-2 mutants, but not in csn-5 mutants, *pMW<0.0005. There is also a reduction of overall nuclei in the late pachytene region of the gonad in both csn mutants, **pMW<0.0005. wild-type n = 25 gonads; csn-2 n = 19; and csn-5, n = 18, B) Analysis of SYP-1 aggregate phenotype in csn mutants and apoptosis checkpoint double mutants. Bypassing the apoptotic checkpoints reduces the number of nuclei with aggregates. Total number of nuclei counted and p-values can be found in Sup. Table 5. C) Quantification of dpMPK-1 expression via IF analyses. csn mutants lack MPK-1 staining in late pachytene and in diakinesis, *pFET<0.0005. wild-type n = 75, csn-2 n = 32, csn-5, n = 27, syp-1(me17) n = 32, D) Western Blot confirming the lack of expression of MPK-1B in csn mutants. MPK-1A is mostly somatic and MPK-1B is germline specific. Normalization values (α-MPK-1/α-TUB) shown are the average of 3 different experiments. Normalized intensities: wild-type 2.27±1.03, csn-2 0.96±0.42 and csn-5 0.99±0.09.
Mentions: Both csn-2 and csn-5 mutants had lower average numbers of nuclei in late pachytene (Figure 6A wild-type average = 52.2; csn-2 = 26.7; and csn-5 = 30.9). However, only csn-2 had a significant increase in apoptosis (4-fold) while csn-5 had apoptotic levels similar to wild-type (Figure 6A wild-type average = 2.96; csn-2 = 8.2; and csn-5 = 3.3 apoptosis levels were not examined in csn-6 mutants). When normalized for the number of nuclei in late pachytene, both mutants showed increased apoptosis, and, as expected, csn-2 mutants showed a larger increase. This is the more appropriate analysis since csn mutants have less germline nuclei.

Bottom Line: In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes.The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers.The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Program in Genetics, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
The synaptonemal complex (SC) is a conserved protein structure that holds homologous chromosome pairs together throughout much of meiotic prophase I. It is essential for the formation of crossovers, which are required for the proper segregation of chromosomes into gametes. The assembly of the SC is likely to be regulated by post-translational modifications. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via the ubiquitin degradation/proteasome pathway. Here we examine the role of the CSN/COP9 signalosome in SC assembly in the model organism C. elegans. Our work shows that mutants in three subunits of the CSN/COP9 signalosome fail to properly assemble the SC. In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes. The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers. The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes. We also find a marked increase in apoptosis in csn mutants that specifically eliminates nuclei with aggregated SC proteins. csn mutants exhibit defects in germline proliferation, and an almost complete pachytene arrest due to an inability to activate the MAPK pathway. The work described here supports a previously unknown role for the CSN/COP9 signalosome in chromosome behavior during meiotic prophase I.

Show MeSH
Related in: MedlinePlus