Revised and Improved Procedure for Immunolocalization of Male Meiotic Chromosomal Proteins and Spindle in Plants without the Use of Enzymes

Immunolocalization studies to visualize the distribution of proteins on meiotic chromosomes have become an integral part of studies on meiosis in the model organism Arabidopsis thaliana. These techniques have been used to visualize a wide range of meiotic proteins involved in different aspects of meiosis, including sister chromatid cohesion, recombination, synapsis, and chromosome segregation. However, the analysis of meiotic spindle structure by immunofluorescence is of outstanding importance in plant reproductive biology and is very challenging. In the following report, we describe the complete and easy protocol for the localization of proteins to the male meiotic spindle and male meiotic chromosomes. The protocol is fast, improved, and robust without the use of any harsh enzymes.

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Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains

10.1101/2021.09.24.461725 ◽

2021 ◽

Author(s):

Rachael E Barton ◽

Lucia F Massari ◽

Daniel Robertson ◽

Adele L Marston

Keyword(s):

Chromosome Segregation ◽

Sister Chromatid ◽

Meiotic Chromosome ◽

Chromatin Loops ◽

Meiotic Chromosomes ◽

Sister Chromatids ◽

Gamete Formation ◽

Chromosome Domains ◽

Chromatid Cohesion ◽

Cohesin Subunit

Cohesin organizes the genome by forming intra-chromosomal loops and inter-sister chromatid linkages. During gamete formation by meiosis, chromosomes are reshaped to support crossover recombination and two consecutive rounds of chromosome segregation. Here we show that Eco1 acetyltransferase positions both chromatin loops and sister chromatid cohesion to organize meiotic chromosomes into functional domains in budding yeast. Eco1 acetylates the Smc3 cohesin subunit in meiotic S phase to establish chromatin boundaries, independently of DNA replication. Boundary formation by Eco1 is critical for prophase exit and for the maintenance of cohesion until meiosis II, but is independent of the ability of Eco1 to antagonize the cohesin-release factor, Wpl1. Conversely, prevention of cohesin release by Wpl1 is essential for centromeric cohesion, kinetochore monoorientation and co-segregation of sister chromatids in meiosis I. Our findings establish Eco1 as a key determinant of chromatin boundaries and cohesion positioning, revealing how local chromosome structuring directs genome transmission into gametes.

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Multiple Subunits of the Caenorhabditis elegans Anaphase-Promoting Complex Are Required for Chromosome Segregation During Meiosis I

Genetics ◽

10.1093/genetics/160.2.805 ◽

2002 ◽

Vol 160 (2) ◽

pp. 805-813 ◽

Cited By ~ 3

Author(s):

Edward S Davis ◽

Lucia Wille ◽

Barry A Chestnut ◽

Penny L Sadler ◽

Diane C Shakes ◽

...

Keyword(s):

Rna Interference ◽

Caenorhabditis Elegans ◽

Chromosome Segregation ◽

Sister Chromatid ◽

Sister Chromatid Cohesion ◽

Anaphase Promoting Complex ◽

Genetic Screens ◽

Chromatid Cohesion ◽

Interference Studies ◽

Meiosis I

Abstract Two genes, originally identified in genetic screens for Caenorhabditis elegans mutants that arrest in metaphase of meiosis I, prove to encode subunits of the anaphase-promoting complex or cyclosome (APC/C). RNA interference studies reveal that these and other APC/C subunits are essential for the segregation of chromosomal homologs during meiosis I. Further, chromosome segregation during meiosis I requires APC/C functions in addition to the release of sister chromatid cohesion.

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Double or nothing: a Drosophila mutation affecting meiotic chromosome segregation in both females and males.

Genetics ◽

10.1093/genetics/136.3.953 ◽

1994 ◽

Vol 136 (3) ◽

pp. 953-964 ◽

Cited By ~ 2

Author(s):

D P Moore ◽

W Y Miyazaki ◽

J E Tomkiel ◽

T L Orr-Weaver

Keyword(s):

Cell Death ◽

Chromosome Segregation ◽

Sister Chromatid ◽

Meiotic Chromosome ◽

Sister Chromatid Cohesion ◽

Aberrant Chromosome ◽

Chromatid Cohesion ◽

Meiotic Nondisjunction ◽

Lethal Allele ◽

Meiosis I

Abstract We describe a Drosophila mutation, Double or nothing (Dub), that causes meiotic nondisjunction in a conditional, dominant manner. Previously isolated mutations in Drosophila specifically affect meiosis either in females or males, with the exception of the mei-S332 and ord genes which are required for proper sister-chromatid cohesion. Dub is unusual in that it causes aberrant chromosome segregation almost exclusively in meiosis I in both sexes. In Dub mutant females both nonexchange and exchange chromosomes undergo nondisjunction, but the effect of Dub on nonexchange chromosomes is more pronounced. Dub reduces recombination levels slightly. Multiple nondisjoined chromosomes frequently cosegregate to the same pole. Dub results in nondisjunction of all chromosomes in meiosis I of males, although the levels are lower than in females. When homozygous, Dub is a conditional lethal allele and exhibits phenotypes consistent with cell death.

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A quantitative analysis of cohesin decay in mitotic fidelity

The Journal of Cell Biology ◽

10.1083/jcb.201801111 ◽

2018 ◽

Vol 217 (10) ◽

pp. 3343-3353 ◽

Cited By ~ 6

Author(s):

Sara Carvalhal ◽

Alexandra Tavares ◽

Mariana B. Santos ◽

Mihailo Mirkovic ◽

Raquel A. Oliveira

Keyword(s):

Quantitative Analysis ◽

Chromosome Segregation ◽

Sister Chromatid ◽

Mitotic Spindle ◽

Sister Chromatid Cohesion ◽

Force Balance ◽

Chromosome Organization ◽

Centromeric Chromatin ◽

Chromatid Cohesion ◽

Chromosome Alignment

Sister chromatid cohesion mediated by cohesin is essential for mitotic fidelity. It counteracts spindle forces to prevent premature chromatid individualization and random genome segregation. However, it is unclear what effects a partial decline of cohesin may have on chromosome organization. In this study, we provide a quantitative analysis of cohesin decay by inducing acute removal of defined amounts of cohesin from metaphase-arrested chromosomes. We demonstrate that sister chromatid cohesion is very resistant to cohesin loss as chromatid disjunction is only observed when chromosomes lose >80% of bound cohesin. Removal close to this threshold leads to chromosomes that are still cohered but display compromised chromosome alignment and unstable spindle attachments. Partial cohesin decay leads to increased duration of mitosis and susceptibility to errors in chromosome segregation. We propose that high cohesin density ensures centromeric chromatin rigidity necessary to maintain a force balance with the mitotic spindle. Partial cohesin loss may lead to chromosome segregation errors even when sister chromatid cohesion is fulfilled.

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ConnectingGCN5’s centromeric SAGA to the mitotic tension-sensing checkpoint

Molecular Biology of the Cell ◽

10.1091/mbc.e17-12-0701 ◽

2018 ◽

Vol 29 (18) ◽

pp. 2201-2212 ◽

Cited By ~ 1

Author(s):

Emily L. Petty ◽

Masha Evpak ◽

Lorraine Pillus

Keyword(s):

Chromosome Segregation ◽

Sister Chromatid ◽

Spindle Assembly Checkpoint ◽

Binding Function ◽

Chromatid Cohesion ◽

Histone H3 Variant ◽

Spindle Microtubules ◽

Low Tension ◽

Centromere Histone H3 ◽

Segregation Of Chromosomes

Multiple interdependent mechanisms ensure faithful segregation of chromosomes during cell division. Among these, the spindle assembly checkpoint monitors attachment of spindle microtubules to the centromere of each chromosome, whereas the tension-sensing checkpoint monitors the opposing forces between sister chromatid centromeres for proper biorientation. We report here a new function for the deeply conserved Gcn5 acetyltransferase in the centromeric localization of Rts1, a key player in the tension-sensing checkpoint. Rts1 is a regulatory component of protein phopshatase 2A, a near universal phosphatase complex, which is recruited to centromeres by the Shugoshin (Sgo) checkpoint component under low-tension conditions to maintain sister chromatid cohesion. We report that loss of Gcn5 disrupts centromeric localization of Rts1. Increased RTS1 dosage robustly suppresses gcn5∆ cell cycle and chromosome segregation defects, including restoration of Rts1 to centromeres. Sgo1’s Rts1-binding function also plays a key role in RTS1 dosage suppression of gcn5∆ phenotypes. Notably, we have identified residues of the centromere histone H3 variant Cse4 that function in these chromosome segregation-related roles of RTS1. Together, these findings expand the understanding of the mechanistic roles of Gcn5 and Cse4 in chromosome segregation.

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Organization of Chromosomal DNA by SMC Complexes

Annual Review of Genetics ◽

10.1146/annurev-genet-112618-043633 ◽

2019 ◽

Vol 53 (1) ◽

pp. 445-482 ◽

Cited By ~ 44

Author(s):

Stanislau Yatskevich ◽

James Rhodes ◽

Kim Nasmyth

Keyword(s):

Chromosome Segregation ◽

Sister Chromatid ◽

Mitotic Chromosome ◽

Sister Chromatid Cohesion ◽

Chromosomal Dna ◽

Chromosome Architecture ◽

Chromatid Cohesion ◽

Dna Translocases ◽

Structural Maintenance Of Chromosomes

Structural maintenance of chromosomes (SMC) complexes are key organizers of chromosome architecture in all kingdoms of life. Despite seemingly divergent functions, such as chromosome segregation, chromosome maintenance, sister chromatid cohesion, and mitotic chromosome compaction, it appears that these complexes function via highly conserved mechanisms and that they represent a novel class of DNA translocases.

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Cohesins Determine the Attachment Manner of Kinetochores to Spindle Microtubules at Meiosis I in Fission Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.23.11.3965-3973.2003 ◽

2003 ◽

Vol 23 (11) ◽

pp. 3965-3973 ◽

Cited By ~ 76

Author(s):

Shihori Yokobayashi ◽

Masayuki Yamamoto ◽

Yoshinori Watanabe

Keyword(s):

Fission Yeast ◽

Chromosome Segregation ◽

Sister Chromatid ◽

Specific Requirement ◽

Spindle Poles ◽

Chromatid Cohesion ◽

Spindle Microtubules ◽

Yeast Meiosis ◽

Meiosis I ◽

Random Division

ABSTRACT During mitosis, sister kinetochores attach to microtubules that extend to opposite spindle poles (bipolar attachment) and pull the chromatids apart at anaphase (equational segregation). A multisubunit complex called cohesin, including Rad21/Scc1, plays a crucial role in sister chromatid cohesion and equational segregation at mitosis. Meiosis I differs from mitosis in having a reductional pattern of chromosome segregation, in which sister kinetochores are attached to the same spindle (monopolar attachment). During meiosis, Rad21/Scc1 is largely replaced by its meiotic counterpart, Rec8. If Rec8 is inactivated in fission yeast, meiosis I is shifted from reductional to equational division. However, the reason rec8Δ cells undergo equational rather than random division has not been clarified; therefore, it has been unclear whether equational segregation is due to a loss of cohesin in general or to a loss of a specific requirement for Rec8. We report here that the equational segregation at meiosis I depends on substitutive Rad21, which relocates to the centromeres if Rec8 is absent. Moreover, we demonstrate that even if sufficient amounts of Rad21 are transferred to the centromeres at meiosis I, thereby establishing cohesion at the centromeres, rec8Δ cells never recover monopolar attachment but instead secure bipolar attachment. Thus, Rec8 and Rad21 define monopolar and bipolar attachment, respectively, at meiosis I. We conclude that cohesin is a crucial determinant of the attachment manner of kinetochores to the spindle microtubules at meiosis I in fission yeast.

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Meiotic Cohesion Requires Accumulation of ORD on Chromosomes before Condensation

Molecular Biology of the Cell ◽

10.1091/mbc.e02-06-0332 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3890-3900 ◽

Cited By ~ 17

Author(s):

Eric M. Balicky ◽

Matthew W. Endres ◽

Cary Lai ◽

Sharon E. Bickel

Keyword(s):

Chromosome Segregation ◽

Wild Type ◽

Chromatin Compaction ◽

Meiotic Chromosomes ◽

Sister Chromatids ◽

Chromatid Cohesion ◽

Meiotic Nondisjunction ◽

Males And Females ◽

Mitosis And Meiosis

Cohesion between sister chromatids is a prerequisite for accurate chromosome segregation during mitosis and meiosis. To allow chromosome condensation during prophase, the connections that hold sister chromatids together must be maintained but still permit extensive chromatin compaction. In Drosophila, null mutations in the orientation disruptor (ord) gene lead to meiotic nondisjunction in males and females because cohesion is absent by the time that sister kinetochores make stable microtubule attachments. We provide evidence that ORD is concentrated within the extrachromosomal domains of the nuclei ofDrosophila primary spermatocytes during early G2, but accumulates on the meiotic chromosomes by mid to late G2. Moreover, using fluorescence in situ hybridization to monitor cohesion directly, we show that cohesion defects first become detectable inord null spermatocytes shortly after the time when wild-type ORD associates with the chromosomes. After condensation, ORD remains bound at the centromeres of wild-type spermatocytes and persists there until centromeric cohesion is released during anaphase II. Our results suggest that association of ORD with meiotic chromosomes during mid to late G2 is required to maintain sister-chromatid cohesion during prophase condensation and that retention of ORD at the centromeres after condensation ensures the maintenance of centromeric cohesion until anaphase II.

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Human kinetochores are swivel joints that mediate microtubule attachments

eLife ◽

10.7554/elife.16159 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 21

Author(s):

Chris A Smith ◽

Andrew D McAinsh ◽

Nigel J Burroughs

Keyword(s):

Chromosome Segregation ◽

Sister Chromatid ◽

Mitotic Spindle ◽

Three Dimensional ◽

Organisational Change ◽

Mechanical Process ◽

Spindle Poles ◽

Chromatid Cohesion ◽

Sister Kinetochore ◽

Dynamic Microtubule

Chromosome segregation is a mechanical process that requires assembly of the mitotic spindle – a dynamic microtubule-based force-generating machine. Connections to this spindle are mediated by sister kinetochore pairs, that form dynamic end-on attachments to microtubules emanating from opposite spindle poles. This bi-orientation generates forces that have been reported to stretch the kinetochore itself, which has been suggested to stabilise attachment and silence the spindle checkpoint. We reveal using three dimensional tracking that the outer kinetochore domain can swivel around the inner kinetochore/centromere, which results in large reductions in intra-kinetochore distance (delta) when viewed in lower dimensions. We show that swivel provides a mechanical flexibility that enables kinetochores at the periphery of the spindle to engage microtubules. Swivel reduces as cells approach anaphase, suggesting an organisational change linked to checkpoint satisfaction and/or obligatory changes in kinetochore mechanochemistry may occur before dissolution of sister chromatid cohesion.

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NudCL2 is an Hsp90 cochaperone to regulate sister chromatid cohesion by stabilizing cohesin subunits

10.1101/358911 ◽

2018 ◽

Author(s):

Yuehong Yang ◽

Wei Wang ◽

Min Li ◽

Wen Zhang ◽

Yuliang Huang ◽

...

Keyword(s):

Atpase Activity ◽

Chromosome Segregation ◽

Sister Chromatid ◽

Mammalian Cells ◽

Ectopic Expression ◽

Sister Chromatid Cohesion ◽

Chaperone Function ◽

Chromatid Cohesion ◽

Protein Instability ◽

The Stability

AbstractSister chromatid cohesion plays a key role in ensuring precise chromosome segregation during mitosis, which is mediated by the multisubunit complex cohesin. However, the molecular regulation of cohesin subunits stability remains unclear. Here, we show that NudCL2 (NudC-like protein 2) is essential for the stability of cohesin subunits by regulating Hsp90 ATPase activity in mammalian cells. Depletion of NudCL2 induces mitotic defects and premature sister chromatid separation and destabilizes cohesin subunits that interact with NudCL2. Similar defects are also observed upon inhibition of Hsp90 ATPase activity. Interestingly, ectopic expression of Hsp90 efficiently rescues the protein instability and functional deficiency of cohesin induced by NudCL2 depletion, but not vice versa. Moreover, NudCL2 not only binds to Hsp90, but also significantly modulates Hsp90 ATPase activity and promotes the chaperone function of Hsp90. Taken together, these data suggest that NudCL2 is a previously undescribed Hsp90 cochaperone to modulate sister chromatid cohesion by stabilizing cohesin subunits, providing a hitherto unrecognized mechanism that is crucial for faithful chromosome segregation during mitosis.

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