They pair in prophase I, and then separate in the first division. Each cell now has only one sex chromosome, like a haploid cell. One way of thinking about ploidy is the number of possible alleles for each gene a cell can have.
Right after meiosis I, the homologous chromosomes have separated into different cells. Each homolog carries one copy of the gene, and each gene could be a different allele, but these two homologs are now in two different cells.
Though it looks like there are two of each chromosome in each cell, these are duplicated chromosomes; ie, it is one chromosome which has been copied, so there is only one possible allele in the cell just two copies of it.
The second meiotic division is where sister duplicated chromatids separate. It resembles mitosis of a haploid cell. At the start of the second division, each cell contains 1N chromosomes, each consisting of a pair of sister chromatids joined at the centromere. Here is a simplified diagram illustrating the overall process and products of meiosis:. Meiosis Overview from Wikipedia by Rdbickel. And here is a video that walks through the steps of meiosis: It is very important that you recognize how and why cells become haploid after meiosis I.
To confirm for yourself that you understand meiosis, work through one or more of these interactive tutorials:. Chromosomes by definition contain the DNA that makes up the fundamental genome of the cell.
In a prokaryote, the genome is usually packaged into one circular chromosome consisting of a circular DNA molecule of a few million base pairs Mbp. In eukaryotes, the genome is packaged into multiple linear chromosomes, each consisting of a linear DNA molecule of tens or hundreds of Mbp. Chromosomes exist at all different phases of the cell cycle. The chromosome number, N, in eukaryotes, refers to the number of chromosomes in a haploid cell, or gamete sperm or egg cell.
Diploid cells all the cells in our body except our gametes have 2N chromosomes, because a diploid organism is created by union of 2 gametes each containing 1N chromosomes. A pair of sister chromatids is one chromosome because it has genetic information alleles inherited from only one parent. A pair of homologous chromosomes, each consisting of a single chromatid in a daughter cell at the end of mitosis, has alleles from the father and from the mother, and counts as 2 chromosomes.
This chromosome number stays the same after chromosome replication during S phase: each chromosome entering cell division now consists of a pair of sister chromatids joined together at the centromere. Then in mitosis, the sister chromatids of each chromosome separate, so each daughter cell receives one chromatid from each chromosome. The result of mitosis is two identical daughter cells, genetically identical to the original cell, all having 2N chromosomes.
So during a mitotic cell cycle, the DNA content per chromosome doubles during S phase each chromosome starts as one chromatid, then becomes a pair of identical sister chromatids during S phase , but the chromosome number stays the same.
A chromatid, then, is a single chromosomal DNA molecule. The number of chromatids changes from 2X in G1 to 4X in G2 and back to 2X, but the number of chromosomes stays the same.
Kinetochore microtubules attach the chromosomes to the spindle pole; interpolar microtubules extend from the spindle pole across the equator, almost to the opposite spindle pole; and astral microtubules extend from the spindle pole to the cell membrane.
Metaphase leads to anaphase , during which each chromosome's sister chromatids separate and move to opposite poles of the cell. Enzymatic breakdown of cohesin — which linked the sister chromatids together during prophase — causes this separation to occur. Upon separation, every chromatid becomes an independent chromosome. Meanwhile, changes in microtubule length provide the mechanism for chromosome movement.
More specifically, in the first part of anaphase — sometimes called anaphase A — the kinetochore microtubules shorten and draw the chromosomes toward the spindle poles. Then, in the second part of anaphase — sometimes called anaphase B — the astral microtubules that are anchored to the cell membrane pull the poles further apart and the interpolar microtubules slide past each other, exerting additional pull on the chromosomes Figure 2.
Figure 2: Types of microtubules involved in mitosis During mitosis, several types of microtubules are active. The motor proteins associated with the interpolar microtubules drive the assembly of the spindle. Note the other types of microtubules involved in anchoring the spindle pole and pulling apart the sister chromatids. Figure Detail. Cytokinesis is the physical process that finally splits the parent cell into two identical daughter cells. During cytokinesis, the cell membrane pinches in at the cell equator, forming a cleft called the cleavage furrow.
The position of the furrow depends on the position of the astral and interpolar microtubules during anaphase. The cleavage furrow forms because of the action of a contractile ring of overlapping actin and myosin filaments. As the actin and myosin filaments move past each other, the contractile ring becomes smaller, akin to pulling a drawstring at the top of a purse. When the ring reaches its smallest point, the cleavage furrow completely bisects the cell at its center, resulting in two separate daughter cells of equal size Figure 3.
Figure 3: Mitosis: Overview of major phases The major stages of mitosis are prophase top row , metaphase and anaphase middle row , and telophase bottom row. This page appears in the following eBook. Aa Aa Aa. What Are the Phases of Mitosis? Figure 1: Drawing of chromosomes during mitosis by Walther Flemming, circa What Happens during Prophase? What Happens during Prometaphase?
Each microtubule is highly dynamic, growing outward from the centrosome and collapsing backward as it tries to locate a chromosome. Eventually, the microtubules find their targets and connect to each chromosome at its kinetochore , a complex of proteins positioned at the centromere. The actual number of microtubules that attach to a kinetochore varies between species, but at least one microtubule from each pole attaches to the kinetochore of each chromosome. A tug-of-war then ensues as the chromosomes move back and forth toward the two poles.
What Happens during Metaphase and Anaphase? Figure 2: Types of microtubules involved in mitosis. During mitosis, several types of microtubules are active. What Happens during Telophase? During telophase , the chromosomes arrive at the cell poles, the mitotic spindle disassembles, and the vesicles that contain fragments of the original nuclear membrane assemble around the two sets of chromosomes.
These studies also enabled us to address whether our inability to observe an effect of cohesin knockdown in interphase cells resulted from inadvertent disruption of the cell cycle; for example, arrest in G1, prior to S phase, would necessarily preclude sister chromatid separation.
Evidence against this explanation was the fact that, while Rad21 knockdown caused an increased mitotic index, cells continued cycling, albeit with a delay as compared to control cells S6A Fig , consistent with published results [ 28 , 84 ].
These observations argue that the apparent maintenance of cohesion following cohesin knockdown cannot be explained by a paucity of G2 nuclei. Thus, in conjunction with the findings described above, our studies indicate that the well FISH format cannot explain why cohesin was not identified as a candidate gene in our screen [ 64 ], and further, that neither inefficient knockdown nor a paucity of G2 nuclei can explain why cohesin RNAi treatment does not disrupt sister chromatid cohesion or homolog pairing in interphase cells.
As the three loci we initially examined by FISH were all located within pericentric heterochromatin, it was possible that the reduced requirement for cohesin we observed in interphase cells was specific to repetitive or heterochromatic sequences. Therefore, we used FISH to target eleven euchromatic regions in a variety of genomic locations following Rad21 knockdown Fig 3.
Applying Oligopaint [ 91 ] FISH probes to control and Rad21 RNAi-treated cells, we targeted eight euchromatic loci ranging in size from tens to hundreds of kilobases and representing all major Drosophila chromosomes: 5A X chromosome, target size These data suggest that the reduced requirement of cohesin protein to maintain interphase cohesion and homolog pairing is a property of both single-copy euchromatic as well as pericentric repetitive regions.
Considering the possibility that cohesin might be required to maintain sister chromatid cohesion and homolog pairing on a more global scale in ways not obvious from the analysis of short chromosomal regions, we also tested cohesin knockdowns with Oligopaints targeting three large regions on the right arm of chromosome 2 3. Examining a large region minimized the chances of visualizing only late-replicating regions where, in early G2, sister chromatids may not yet have formed. Additionally, large FISH targets allowed a greater dynamic range in the size of the FISH signals, permitting us to more easily measure the area of the FISH signals in maximum-Z projections, in addition to counting the number of signals.
Following knockdown of Rad21, neither the number of FISH signals nor the area of the image covered by these signals showed a significant increase, contrary to what might have been expected if sister chromatids had simply separated Fig 3C ; the combined area of the FISH signals was The decrease in signal areas we observed following Rad21 knockdown was unexpected, and could indicate an interesting role for cohesin in antagonizing compaction of chromatin, though further experiments are necessary to confirm this trend.
Regardless, our data suggest that sister chromatids can maintain cohesion and homologs can remain paired across all chromosome arms with very little or no Rad We next addressed whether homolog pairing can contribute to the cohesion of sister chromatids in interphase, reducing the requirement for cohesin proteins.
For example, the mechanisms that pair homologs might also act directly between sister chromatids, holding them together even in the absence of cohesin. Alternatively, it is possible that, because homologs are paired in G1, the replication products of these chromosomes can remain closely associated in G2 without mechanisms acting directly to hold sisters together Fig 4A.
To test this second possibility, we studied a chromosome that does not have a homolog, that is, the single X chromosome in a diploid XY male cell. A Cartoon showing theoretical interactions between chromosomes. Homologs and sister chromatids can be held together by a combination of homolog-homolog and sister-sister interactions left or just homolog-homolog interactions that indirectly hold sister chromatids together middle, right. B Karyotype of Clone 8 cells, which have two copies of the autosomes long arrows and a single X chromosome arrowhead.
The cell marked by the arrow is in G2 and depleted for cohesin, but FISH indicates that sister chromatid cohesion is unperturbed, both for an autosome dodeca, 3rd Chr. For these studies we selected Drosophila Clone 8 Cl. Consistent with our results in other cell lines, the percentage of nuclei with a single FISH signal at dodeca was not significantly different between control G2 cells and those treated with Rad21 RNAi Remarkably, we found that sister chromatid cohesion at 16E on the X chromosome was also unaffected by cohesin knockdown, with These observations argue that, barring intrinsic features that may be specific to the X chromosome, cohesion between sister chromatids can be maintained with little to no cohesin protein even when these chromosomes have never experienced homolog pairing.
Thus, while the rDNA of the X and Y chromosomes can support local pairing [ 92 ], we consider it unlikely that pairing of the rDNA loci accounts for cohesion with little to no cohesin at 16E. That being said, it remains possible that inter-chromosomal associations occurring near the centromere might influence the organization of a chromosome arm. Although cohesion of sister chromatids in cohesin-depleted G2 cells may not require the presence of a homolog, it could still depend on mechanisms that also participate in homolog pairing.
To test this idea, we combined cohesin knockdown with knockdown of Slmb, a gene which is required for homolog pairing; Slmb is a negative regulator of condensin II, and Slmb knockdown leads to an increased number of FISH signals and thus a decrease in the percentage of nuclei with a single FISH signal [ 64 , 70 ]. If Slmb is also required for cohesin-independent cohesion, we might expect simultaneous knockdown of both Slmb and cohesin to disrupt cohesion as well as homolog pairing, leading to even more FISH signals than when Slmb alone is knocked down.
Therefore, pairing levels were similarly reduced whether we knocked down only Slmb or both Rad21 and Slmb; however, when unpairing did occur, we often observed more FISH signals when both Rad21 and Slmb were knocked down Fig 5A and S9 Fig.
We reasoned that the "extra" FISH signals likely represented the separation of sister chromatids and applied this approach in subsequent analyses. As this assay requires homolog pairing as well as the separation of sister chromatids, our measure of sister chromatid separation is likely an underestimate.
B Quantification of results for experiments illustrated in A showing percentages of nuclei with a single FISH signal, indicating nuclei where homolog pairing and sister chromatid cohesion are intact. C Quantification of results for experiments illustrated in A showing percentages of nuclei with more than 3 AACAC signals or more than 4 dodeca signals, indicating nuclei with possible sister chromatid separation in addition to homolog unpairing.
Using this metric for identifying instances of sister chromatid separation, we observed that there is little sister chromatid separation following knockdown of Slmb alone; the percentages of nuclei with more than three FISH signals at AACAC or more than four FISH signals at dodeca were 4.
In contrast, these percentages were These findings suggest that, unlike knockdown of either Slmb or Rad21 alone, the double knockdown of Slmb and Rad21 results in sister chromatid separation as well as homolog unpairing. As the extra FISH signals could be explained by aneuploidy, we analyzed metaphase spreads following knockdowns, but did not find evidence for increased aneuploidy after double knockdown of Rad21 and Slmb as compared to knockdown of Slmb alone S10 Fig. The extra FISH signals were also unlikely to reflect decompaction or fragmentation of heterochromatin, as double knockdowns of Rad21 and Slmb increased the number of signals at three out of five euchromatic loci studied S11 Fig.
The relatively modest effects observed at euchromatic as versus heterochromatic loci may stem from the overall higher levels of homolog pairing at euchromatin [ 64 , 93 , 94 ]. We also considered the possibility that the increase of nuclei with extra FISH signals represented the arrest of cells in mitosis, when sister chromatid cohesion is lost following cohesin knockdown.
Furthermore, immunofluorescence for cyclin B confirmed that the increase in the number of FISH signals in the double knockdown was not caused by an enrichment of G2 cells S12 Fig ; knockdowns decreased the proportion of G2 cells from Therefore, while we cannot rule out any contribution of aneuploidy, disorganization of heterochromatin, or cell cycle arrest, we favor the hypothesis in which the extra FISH signals in the double knockdowns of Rad21 and Slmb are caused by sister chromatid separation in interphase.
This interpretation suggests that Slmb contributes to cohesion independently of cohesin. However, we also note that, even if Slmb does not regulate cohesion, any of the alternative explanations mentioned would still indicate an interesting relationship between Rad21 and Slmb and, therefore, between cohesion and homolog pairing. Finally, we considered the role that Slmb plays in the negative regulation of condensin II [ 70 ] and asked whether condensin II might contribute to sister chromatid separation in our assays.
Importantly, this effect was not due to a reduction in the percentage of G2 cells in the triple knockdown S12 Fig. This suppression of the extra FISH signals by the triple knockdown also argues that the extra FISH signals observed in the double knockdown of Rad21 and Slmb were not an artifact of the double knockdown.
Taken together, these results suggest that condensin II contributes to sister chromatid separation when Rad21 is compromised, raising the possibility that condensin II may contribute to sister chromatid separation under normal conditions, perhaps by removing cohesin-independent connections between sister chromatids. In light of the contributions of both condensin II and Slmb to homolog pairing [ 64 , 69 — 71 ], these data further suggest that the mechanisms mediating cohesin-independent cohesion may be similar to, or the same as, those that mediate homolog pairing.
Our results show that Drosophila interphase cells with little to no cohesin display levels of sister chromatid and homolog alignment comparable to that of control cells, as assayed by FISH at resolutions allowed by light-microscopy. If indeed the phenotypes we observed are the result of residual cohesin protein, it suggests that, at least in interphase, cohesin is not required at a high density to maintain alignment of sister chromatids along their length, while in metaphase, wild-type levels of cohesin are required for proper cohesion.
This in turn raises the question of why Drosophila and other vertebrates have more cohesin loaded onto the chromosomes in G2 than in metaphase, since the majority of cohesin in these organisms is removed during prophase [ 96 , 97 ]. Perhaps the majority of cohesin protein present on sister chromatids in G2 participates in functions other than cohesion.
As mentioned previously, the idea that there may be cohesin-independent mechanisms that contribute to segregation of sister chromatids is not new. However, in most instances where a reduced dependence on cohesin has been observed, it has been documented in mitosis and only at specific regions [ 44 ].
As for studies of interphase in organisms other than Drosophila, those that have used FISH to assay the impact of cohesin loss have detected an increase in the number of FISH signals, increased distance between signals, or abnormally shaped signals [ 27 , 98 — ].
These data indicate that in most organisms, cohesin loss is sufficient to cause chromatid separation in G2, and that cohesin-independent mechanisms, if they do contribute to cohesion, do so in a locus-specific manner. Here we suggest that cohesin-independent mechanisms may be widespread in Drosophila, contributing to the pairing of homologs as well as to the cohesion of sister chromatids in G2 at 3 heterochromatic and 11 euchromatic loci, and therefore may act genome-wide.
Based on our results, we cannot rule out that cohesin-independent mechanisms contributing to chromatid alignment are induced in response to cohesin knockdown. Nevertheless, these results demonstrate the potential for sister chromatids to remain aligned in interphase with little to no cohesin. It may well be no coincidence that Drosophila also supports extensive pairing of homologous chromosomes in somatic cells.
We have also observed a genetic interaction between Rad21 and Slmb, a gene required for homolog pairing [ 64 , 70 ]. This finding suggests that homolog pairing and sister chromatid cohesion might be regulated by common mechanisms, consistent with a model that has been proposed by Ono et al.
In particular, we favor a model in which the higher levels of condensin II activity caused by Slmb knockdown [ 70 ] separate sister chromatids as well as homologs in the absence of cohesin. This could happen if condensin II negatively regulates residual cohesin, or if cohesin-independent connections exist between sister chromatids as well as between homologs and condensin II antagonizes those connections Fig 6.
The latter model is supported by evidence from organisms other than Drosophila implicating condensin in the resolution of cohesin-independent connections between sister chromatids, including at the budding yeast rDNA locus [ 47 — 51 ].
In Drosophila, in addition to condensin II and several of its regulators being involved in homolog pairing [ 69 — 74 ], the condensin I subunits Barren [ — ], Cap-G [ , ], and Cap-D2 [ ], as well as Smc4 present in both complexes [ ], are required for the complete resolution of sister chromatids in mitosis.
In human cells, condensin II is necessary for sister chromatid resolution beginning in late S-phase [ 77 ], and a significant amount of chromatid resolution by condensin II also takes place during prophase [ ]. Consistent with these findings, our data suggest that condensin II-regulated mechanisms contribute to sister chromatid cohesion in interphase, and that this mechanism of cohesion is related to homolog pairing in Drosophila.
Hypothetical cohesin-independent connections that form between chromosomes and the timing of their removal. In this model, the cohesin-independent connections that form between sister chromatids in Drosophila potentially, DNA catenations also form between homologs.
As shown, cohesin-independent connections may be resolved later in the cell cycle in Drosophila than in other organisms; alternatively, it is possible that cohesin-independent connections are formed more efficiently in Drosophila, so that they are more widespread. Condensin II may antagonize inter-homolog or inter-sister interactions in a number of ways: they may remove residual cohesin, recruit topoisomerases to remove catenations between chromosomes, or, as pictured here, compact chromosomes in cis , limiting their ability to participate in trans interactions [ 13 , 64 , 69 , 71 ].
Further, it is possible that condensin II cooperates with condensin I during mitosis in the process of resolving sister chromatids see S13 Fig.
Note that, in the top panel representing Drosophila, all cohesin-independent linkages are resolved by metaphase, but in theory, some connections between homologs might remain in metaphase and in anaphase. Similarly, in the lower panel representing other organisms, all cohesin-independent connections between sisters have been resolved by G2, but it is possible that they remain in certain regions.
As for whether cohesin-independent mechanisms contribute to cohesion in vivo , this possibility is supported by studies showing that cohesin cleavage induced via a TEV protease in Drosophila larvae does not noticeably disrupt polytene chromosome alignment [ 83 ]. In contrast, overexpression of the condensin II component Cap-H2 disrupted polytene alignment in the same cell type [ 69 ].
It will be of great interest to determine whether similar phenotypes are observed in actively dividing fly tissues. Our observations suggest that the mechanisms that act between sister chromatids may also act between homologs.
This model is consistent with the idea that recognition of a pairing partner is based on DNA sequence or chromatin structure, as exemplified by the fact that chromosomal translocations can pair e. Our work also pertains to the question of whether or not cells distinguish sisters from homologs e. If, as our work suggests, there are some aspects of chromosomal organization that do not distinguish sister chromatids from homologs, sister chromatids may influence gene expression beyond their contribution to chromosome copy number.
For example, as homolog pairing can influence the communication between regulatory elements and promoters in cis as well as in trans [ 56 — 58 ], sister chromatids may be able to join and influence this dialogue [ 93 ].
Indeed, just as transvection can occur between paired homologs, so might it occur between sister chromatids, making it possible for these two forms of transvection to be synergistic or mutually inhibitory during G2 S14 Fig. Importantly, inter-homolog communication and the contribution of sister chromatids to that process could vary by cell type, depending on the levels of cohesin-independent connections between sisters and homologs.
Our data also address the long-standing question of when in the cell cycle homolog pairing can be established. While some studies have shown that levels of homolog pairing are higher in G1 than in G2, suggesting that S-phase is a stage when pairing is more dynamic and possibly disrupted [ 64 , ], other work shows that pairing levels are similar in G1 and G2 [ 93 ], possibly reflecting variability between cell types. Our experiments in the male diploid Clone 8 cell line suggest that cohesin-independent G2 cohesion of sisters, if it occurs, is not dependent on the presence of a homolog, which would indicate that cohesin-independent cohesion could be established de novo in G2.
As such, perhaps the pairing of homologs can also be established at this stage. A major question concerns the potential nature of a cohesin-independent connection between chromosomes. There are several possibilities, including the contribution of factors, such as proteins or RNA, that function similarly to cohesin in keeping chromosomes together, but act specifically in interphase.
For example, cohesion in somatic cells may involve multiple novel cohesin complexes, as is known to be the case in Drosophila meiosis [ — ]. Alternatively, cohesin-independent cohesion might involve direct connections between chromosomes themselves without any need for bridging factors, perhaps involving nontraditional base pairing or DNA catenations resulting from replication.
In fact, one of the earliest models for cohesion, proposed before cohesin proteins were known, posited that sister chromatids could be held together by DNA catenations [ — ]. Consistent with this model, topoisomerase II, an enzyme that removes catenations formed during replication and other processes, is known to regulate the segregation of sister chromatids reviewed by [ ].
Furthermore, recent work has shown not only that catenations contribute to cohesion but also that cohesin may play a role in maintaining catenations [ 34 , ]. These observations raise the questions of whether catenations might be sufficient to maintain cohesion in interphase Drosophila cells in the absence of cohesin, and how condensin II might act to resolve catenations. The role of condensin II in regulating these sorts of topological connections may be to recruit or activate topoisomerase II; alternatively, condensin II may play a role in separating chromosomes independently of topoisomerase II reviewed by [ ], see also [ 76 , , ].
It is also possible that the activity of condensin II in compacting chromosomes, by forming more intra-chromosomal interactions, suppresses inter-chromosomal interactions Fig 6 [ 13 , 64 , 69 , 71 ].
Given the mechanistic relatedness we have observed between homolog pairing and cohesion, it is possible that homolog pairing is also mediated at least in part by DNA catenations or entanglements [ 59 , 64 , 93 , , ]. If so, the widespread nature of homolog pairing in Drosophila cells might imply that these cells are more permissive for the formation of catenations.
For example, homologs may become catenated when they are replicated in close proximity, perhaps via replication fork collapse and repair, or when they recombine, especially at the repetitive sequences of pericentric heterochromatin [ ]. Interestingly, inhibition of topoisomerase II reduces levels of homolog pairing in Drosophila cells, which may reflect different roles for topoisomerase in sister chromatid cohesion as versus homolog pairing [ 93 ], see discussion within.
Drosophila cells may also differ from other organisms in the timing of the resolution of catenations; for example, retention of catenations until mitosis, when perhaps they are resolved in response to spindle formation [ ] or other mitosis-specific factors, might explain why cohesin knockdown did not perturb the cohesion of sister chromatids in G2. Indeed, the recent identification of ultrafine bridges in human cells demonstrates that catenations can remain until anaphase at certain regions reviewed by [ ].
If cohesin-independent connections exist between sister chromatids, why maintain another mechanism of cohesion in the form of the highly conserved and essential cohesin proteins? One explanation may be the requirement for unique connections between sister chromatids in order to ensure their segregation. Such connections may be provided by cohesin, whose establishment is coupled to DNA replication [ 18 — 20 ], while cohesin-independent mechanisms may contribute to genome organization in other ways.
Secondly, cohesin-independent connections may allow cohesion to be maintained at chromosomal regions where cohesin protein is not always bound at a high density. This would enable cohesin binding to be spatially and temporally dynamic [ 42 , ] and permit additional roles of cohesin in interphase, such as in the regulation of transcription and DNA repair [ 14 , ].
Thus, cohesin-independent mechanisms contributing to cohesion, perhaps including the maintenance of catenations, may be especially important in cell types having a long G2 stage, such as the cells used in this study. Finally, having a diversity of cohesion mechanisms may allow for a more layered regulation of cohesion removal as cells enter mitosis [ 75 ]. In fact, in higher eukaryotes, cohesin proteins are removed from different parts of the chromosome at different times; while a small population of cohesin is retained at the centromeres and cleaved at anaphase [ 96 , 97 , ], the bulk of cohesin on the chromosome arms is removed during prophase by Wapl and Pds5 [ 17 , 97 , — ].
Telomeric cohesion involves yet additional regulation [ — ]. Thus, cohesin-independent cohesion may constitute a further layer to be removed during the segregation of sister chromatids, the regulation of which may be useful in determining the order of segregation [ 51 ] or the length of the cell cycle. Since all these processes must be coordinated with the condensation of chromosomes prior to mitosis, perhaps it is not surprising that condensin proteins are involved in antagonizing cohesion reviewed by [ ] or that cohesin and cohesin regulators play a role in condensation [ 11 , 46 , — ].
Overall, these observations and the work presented here indicate interesting interactions between different SMC complexes in the maintenance of interphase nuclear organization as well as the ways in which homologous DNA sequences interact with each other, whether between sister chromatids or between maternal and paternal homologs.
RNAi treatments were started in each case one day after the cells had been split as part of their regular passaging. RNAi treatments lasted for four days unless otherwise specified. When using Effectene, the amount of dsRNA was reduced to 1.
Cells were collected after four days of RNAi and their protein levels were analyzed by Western blot according to standard protocols. Blots were probed using a rabbit anti-Rad21 antibody generous gift from Dr.
Band intensities were estimated using the gel analysis tools in ImageJ [ ]. Secondary antibodies used Jackson ImmunoResearch Laboratories : Cy3-conjugated anti-rabbit , conjugated anti-mouse , Cy5-conjugated anti-mouse Our protocol for fluorescence in situ hybridization has been previously published [ 64 , 91 ] and was adapted from standard protocols [ 85 , , ].
In cases where both IF and FISH were carried out, generally the two protocols were carried out in succession and the slides imaged afterwards. For some more sensitive antibodies, the cells were imaged following IF, then washed, used for FISH, and re-imaged, using software-assisted stage navigation to relocate the same fields. Metaphase cells were prepared using protocols adapted from published methods [ , ]. Cells were obtained from actively growing cultures without the use of drugs to increase mitotic index unless otherwise specified.
The cells were incubated at room temperature for 30 minutes. We then added 1 mL of cold fixative methanol: glacial acetic acid solution , spun down the cells, and washed three more times in 10 mL of the same fixative. FISH probes were added and the cells were allowed to hybridize without any additional denaturing, followed by our standard FISH washes.
When scoring sister chromatid separation in metaphase spreads without FISH , each spread was examined for the presence of single chromatids not attached to a sister along the entire chromosome arm. If unattached chromatids were visible, that metaphase was scored as having premature loss of cohesion, while if no unpaired chromatids were visible, it would be scored as having intact cohesion.
All images were obtained using an Olympus IX83 epifluorescence microscope with a 60x oil objective and the CellSens acquisition software. All uniquely identifiable foci of fluorescent signal above background were counted as FISH signals, regardless of the distance between them. The number of FISH signals and the area of FISH signals following cohesin knockdown was used as a measure of cohesion, defined as the close alignment of sister chromatids in interphase.
When assaying the number of FISH signals in a nucleus, a whole population of cells was scored and each nucleus classified as either having one signal homolog pairing as well as sister chromatid cohesion intact or more than one signal homologs have become unpaired or sisters have lost cohesion.
This type of analysis was also used when examining nuclei with higher numbers of FISH signals i. Finally, when examining the number of FISH signals in metaphase spreads, a Mann-Whitney U test was used to compare the different conditions. Relative mRNA levels were normalized to levels of rp49, a ribosomal gene, in each sample, and each sample was then normalized to levels in LacZ dsRNA-treated cells. A Western blot showing that knockdown is efficient after four days of RNAi.
B Immunofluorescence for Rad21 confirms knockdown in individual cells. Western blots prepared from cells treated for four days with either lacZ dsRNA or Rad21 dsRNA, using A various concentrations of anti-Rad21 antibody for probing, and B various exposure times when imaging the blots.
At high antibody concentrations and exposure times, residual cohesin can be observed. While a more accurate estimate of knockdown efficiency would be obtained following an antibody titration, our results, especially when combined with the immunofluorescence and qPCR data, indicate that the knockdown of Rad21 in these cells is fairly efficient.
Spreads were prepared following four days of RNAi without use of any drugs to increase mitotic index. Rad21 knockdown caused a more severe loss-of-cohesion phenotype as compared to knockdowns of Smc1 and Smc3.
Rad21 knockdown caused a slight cell cycle delay compared to untreated cells. C Timecourse showing gradual onset of the premature loss of cohesion phenotype in response to Rad21 RNAi in Kc cells.
Immunofluorescence after Rad21 RNAi demonstrates that some cells are depleted for Rad21 and only show background levels of fluorescence, while other cells show fluorescence intensities that are comparable to that of control cells. Data shown is the same as in Fig 5B and 5C , but nuclei are sorted by the actual number of FISH signals per nucleus rather than the proportion having more than three signals, etc. Frequency was calculated by dividing the number of nuclei having a specific number of FISH signals by the total amount of nuclei scored.
A Representative control cells stained with antibodies against cyclin B. A Rad21 knockdown leads to an increase in the number of FISH signals in mitotic nuclei, which are identified by staining for phosphorylated histone H3 pH3. B Rad21 knockdown also leads to abnormal morphologies of mitotic nuclei, including multi-lobed structures with breaks in the mass of chromosomes formed at mitosis dotted line; DAPI perimeter.
C Depletion of condensin protein Smc2, present in both condensin I and II, partially rescues defects observed after Rad21 knockdown; nuclear morphology resembles that of control cells, consistent with published results [ 29 , ]. D Quantification of results for experiments illustrated in B and C.
Mitotic nuclei were classified as either having Discontinuous or not having Continuous discontinuities in structure, as revealed by pH3 staining. Significant rescues were observed when condensin I proteins were knocked down in addition to Rad21, but not with condensin II. This finding suggests that whatever function is played by condensin II in antagonizing sister chromatid cohesion in interphase is at least partially redundant with that played by condensin I in mitosis.
Metaphase spreads were scored as either having intact cohesion all visible chromatids were attached to a sister chromatid or not unattached chromatids were visible, indicative of premature loss of sister chromatid cohesion.
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