When can you see chromatin in mitosis

Z occupancy Kumar and Wigge, ; Pecinka et al. Severe heat stress modulates chromatin structure, by increasing histone acetylation and decreasing H3K9me2, and eventually induces programmed cell death Z. Wang et al. Surprisingly, cold stress also leads to general chromatin de-condensation, as suggested by Hi-C analysis in rice, but specific regions may be subject to chromatin condensation and gene silencing Liu et al.

Taken together, the data suggest that at a range of optimal temperatures, which are species specific, chromatin is normally condensed, and de-condenses under suboptimal conditions. However, this hypothesis needs to be tested for a broader range of species and temperatures.

Vernalization—acquisition of competence to flower only in response to a period of cold—is a well-known example of cold-induced chromatin change. However, vernalization evolved multiple times in plants and its mechanism differs between species Reeves et al.

Temperature changes also lead to selective and transient activation of repetitive sequences Steward et al. Recent studies suggested that this is due to the presence of the canonical cis -regulatory elements in the long terminal repeats LTRs of specific stress-responsive transposon families Cavrak et al. This could represent an evolutionary mechanism of dispersal for cis -regulatory elements in the genome and foundation of novel gene expression patterns Ito et al. Reduced water availability negatively influences yield and resistance to other stresses.

The effect of water stress on plant chromatin is not well understood, but data suggest that the responses are species specific. In contrast, no consistent water stress-induced DNA methylation changes were observed in Arabidopsis and maize Eichten and Springer, ; Ganguly et al. Attacks of crops by pathogens may have severe consequences on plant vitality and yield, and can even cause lethality.

Biotic stress defense mechanisms are fast evolving to match the evolutionary innovations on the pathogen side, which leads to a constant race between the host and the pathogen. Following infection by biotrophic or necrotrophic pathogens, plants typically reprogram gene expression from growth to defense Moore et al.

Some pathogens developed strategies to directly affect chromatin modifiers. For example, the necrotrophic fungus Alternaria brassicola produces a toxin that inhibits the enzyme histone deacetylase HDA activity during infection Matsumoto et al.

In contrast, some HDAs regulate innate immunity positively Latrasse et al. Although it is clear that histone acetylation and de-acetylation plays an important role in the regulation of defense-related genes, it is still not clear how HAT and HDAs are targeted to the target loci to allow genome-wide changes in gene expression Ramirez-Prado et al. The effects of viruses on plant chromatin remain only poorly understood. The replicated virus dsDNAs are packed with nucleosomes and form tiny chromosome-like structures.

In summary, this section shows that responses of chromatin to various stresses are diverse and in some cases highly adaptive. In many cases, we have only a basic description of the stress-induced chromatin changes, and we are still lacking information on the persistence of these changes after recovery from the stress and about their heritability through mitosis and meiosis. Therefore, we expect that many future studies will focus on the identification of the underlying mechanisms.

In addition, it is expected that more groups of chromatin modifiers such as histone de methyltransferases and de ubiquitinylases will be firmly connected with stress-induced chromatin responses Dhawan et al. Understanding the involvement of chromatin in adjusting plant adaptation to diverse environmental challenges is of interest to a broad audience of plant scientists, considering that stresses are generally predicted to become exacerbated due to climate change and that they can strongly affect crop yields.

Chromatin undergoes drastic changes affecting its degree of compaction during the cell cycle. At the onset of cell divisions, the NE disassembles, allowing the access of cytoplasmic proteins to the nucleoplasm, including proteins which contribute to further chromatin condensation and spindle formation. Chromatin condensation is critical for the individualization of chromosome in order to guarantee the proper distribution of genetic information between daughter cells.

After segregation, chromatin is decondensed to restore its interphase state. In maize, histone H3S28p and H3S50p delineate the pericentromeric and centromeric regions during chromosome segregation, respectively Zhang et al. In the same species, changes in the level of histone H3S10p regulate sister chromatid cohesion Kaszas and Cande, , and an increase of H3 phosphorylation is linked to reduced acetylation levels at Lys9 residues in histone H3 Edmondson et al.

A survey of 17 plants species revealed that the distribution of histone H4K5ac differs between small and large genome species Feitoza et al. The condensin complex is another main player in chromosome organization Hirano et al. Condensin II accesses the cell nucleus before mitosis and its reduction partially reduces early H3 phosphorylation Ono et al. Subsequently, condensin I contributes to prophase chromatin compaction. Cohesion is established at the onset of S phase and persists until the metaphase—anaphase transition, and it is essential to resist the force of the spindle microtubules while chromosomes are aligned at the equatorial plate, allowing their accurate segregation to opposite poles Fig.

At the beginning of anaphase, cohesin is released from chromosomes in two steps Nasmyth, During prophase and prometaphase, cohesin is removed from chromosome arms. In the second step, before the onset of anaphase, the remaining cohesin is released from centromeres, allowing separation of sister chromatids. Several studies have demonstrated that cohesin plays additional roles in DNA double-strand break repair DSBR and regulation of gene expression Yuan et al.

Overview of chromosome organization during mitosis and meiosis. At the onset of mitosis, chromatin condensation is necessary to disassemble the interphase chromatin in a process driven by specific post-translational modifications PTMs in H3 and condensin complexes.

In addition, the cohesin complex is essential for defining chromosome structure by providing a physical linkage between sister chromatids until their segregation at anaphase. Throughout meiosis, condensin complexes I and II are required to maintain the structural integrity of chromosomes. During leptonema, the histone variant H2A. X at double-strand break DSB sites.

The synaptonemal complex SC forms between paired chromosomes at zygonema, and full synapsis is reached at pachynema. During anaphase I, loss of cohesion between the arms of sister chromatids allows the segregation of homologous chromosomes to the opposite poles. Centromeric cohesion is released at the onset of anaphase II, and sister chromatids segregate to form a tetrad. There are remarkable differences in chromatin condensation and organization between mitosis and meiosis Fig.

Meiotic chromosome condensation proceeds simultaneously with alignment of homologous chromosomes, programmed DSB formation, repair through homologous recombination HR , and establishment and dissolution of the synaptonemal complex SC. These processes are associated with striking morphological changes including dynamic variations in histone PTMs Nasuda et al. X Shroff et al. X is completely lost from fully synapsed chromosomes.

This is also likely to be the case for barley as DSBs and H3K4me3 are strongly localized towards the telomeres, whereas they are quite low in pericentromeric regions Baker et al. The influence of these PTMs in meiotic HR has been highlighted in a recent work in which the disruption of H3K9me2 and DNA methylation pathways produces the epigenetic activation of meiotic recombination near centromeres Choi et al.

These are regions normally suppressed for COs in order to avoid aneuploidies in the offspring Rockmill et al. Entangling of meiotic prophase I chromosomes results in interlocks Gelei, , which could compromise chromatin integrity and result in chromosome mis-segregation.

At the onset of meiosis, telomeres attach to the NE and cluster, forming a characteristic bouquet arrangement Bass et al. The mechanism of bouquet formation is not well understood and, although it is widely conserved among eukaryotes, a characteristic bouquet arrangement is apparently not formed in Arabidopsis Armstrong et al.

In Arabidopsis, telomeres present a complex behavior and are associated with the nucleolus throughout meiotic interphase and early prophase I. Clustering of telomeres around the nucleolus allows pairing at the same time as when axial elements of the SC are assembled Roberts et al.

However, in other species, the subtelomeric regions undergo differential behavior during pre-meiotic G 2 and prophase I Colas et al. In the large genome of cereals, the telomere bouquet precedes chromosomes synapsis Phillips et al.

SMC complexes are essential during meiosis. Both condensin I and II complexes are important for maintaining the structure of meiotic chromosomes. In addition, the cohesin complex is indispensable for proper pairing and HR Golubovskaya et al. In rice, if centromere cohesion is compromised, chromatids separate prematurely at anaphase I and chromosomes are intertwined, leading to chromosome bridges and fragmentation Shao et al.

This function is most probably conserved in both mitosis and meiosis, as shown in rice Wang et al. Furthermore, mutations in the two Arabidopsis WAPL genes, with a significant role in the removal of cohesin, lead to alterations in the organization of heterochromatin and delayed cohesin removal during prophase I De et al. Most of the information on the behavior of chromatin in meiosis derives from studies with fixed cells.

However, innovative methodologies are being developed to enable the dynamic analysis of meiotic processes in live meiocytes. In a pioneer study, prophase I has been analyzed within PMCs of intact anthers in maize Sheehan and Pawlowski, and recently live microscopy of male meiosis was performed at high resolution in Arabidopsis Prusicki et al. Such advancements in technology will allow an in-depth analysis of the dynamics of meiotic processes.

Finally, the link between chromatin conformation and gene regulation during meiosis is still very obscure despite the number of genomic and transcriptomic studies in various plant species Zhou and Pawlowski, However, most of these analyses have mainly been done with tissue covering the overall meiosis rather than specific meiotic stages, which is necessary to understand the gene expression pattern. In Angiosperms, sexual reproduction starts with the development of flowers, when the SAM is transformed into the inflorescence meristem IM continuously producing the floral meristems FMs.

Remarkably, the FM switches from an indeterminate fate to a determinate fate to give rise to all the organs of the flower, the gametes, and the fruit. All reproductive development transitions are controlled by endogenous, hormonal, or external environmental signaling pathways, which require complex gene regulatory networks involving transcription factors and epigenetic mechanisms. In Arabidopsis, FT expression is subjected to photoperiod and ambient temperature, and is under a complex balance of active and repressive chromatin modifications involving both Polycomb Repressive Complex PRC 1 and 2 He, Thereafter, AG represses WUS activity to ensure termination of the FM, and to promote all the finely tuned developmental transitions required for the proper development of floral organs.

The repression of WUS is a perfect example to illustrate the importance of epigenetic regulatory mechanisms during FM termination. After meiosis see the previous section , the male haploid gametophyte microspore undergoes an asymmetric division to produce a generative cell GC and a vegetative cell VC , and the GC divides once more to produce two sperm cells SCs representing the male gametes reviewed, for example, by Berger and Twell, SCs and VCs have very different chromatin characteristics, which also determine their fate, genome integrity, and capacity to divide Slotkin et al.

The functional significance of such extensive epigenetic reprogramming is still debated, but the activation of TEs in VCs may represent a non-autonomous silencing mechanism, which switches off any potentially active transposons in the germline and thus preserves the genome integrity of the next generation.

However, to what extent this is typical for plants other than Arabidopsis remains unknown. In addition, there seem to be a specific variant of the largest subunit of Pol V in grasses Trujillo et al. The high and medium numbers of copies of putative orthologs of H3K27 and H3K4 demethylases, respectively, indicates that rice SCs may require more extensive reprogramming of repressive marks Anderson et al.

The replacement of canonical histones by specific variants is also characteristic of epigenetic control at male gametogenesis. This variant has been correlated with the loss of H3K27me3 methylation, due to the composition of the adjacent amino acid residues Borg and Berger, Histones H3. Z are the most remarkable. Histone H3. Replacement of histones also occurs in the Arabidopsis VC, since CenH3 is progressively lost in centromeric heterochromatin when it begins to de-condense, while there is a loss of H3K9me2 marks, indicating a state of terminal differentiation Schoft et al.

In Brassica rapa , H3K4me3 and H3K27me3 deposition is necessary for the regulation of the pollen wall construction Shen et al. In megasporogenesis, the diploid megaspore mother cell undergoes meiosis, resulting in four haploid megaspores. One megaspore develops into the female gametophyte, while the others die. The formation and differentiation of the different cell types in the reproductive lineage are characterized by global changes in chromatin organization.

Histone modifications were observed via cytogenetic and chromatin reporter studies in Arabidopsis megaspores and also in the surrounding nucellar cells in maize Garcia-Aguilar et al. Genetic analyses have identified DNA methylation acting upon establishment of the megaspore fate, and also the action of small RNAs silencing TEs in the female gametes in Arabidopsis and maize Garcia-Aguilar et al. The multicellular embryo sac consists of the egg cell, the central cell, two synergid cells, and three antipodal cells.

The female gametes exhibit chromatin dimorphism as they express different histone H3 proteins, with the egg cell expressing only the H3. Due to the technically limiting accessibility to the female gametophyte, gene-level resolution of the chromatin perturbations has not been reported to date.

The histone modifications observed suggest a global epigenetic reprogramming phase during development of the female gametophyte. The epigenetic dimorphism of the two female gametes at the DNA methylation level, with the global demethylation of the central cell versus the non-CG DNA methylation of the egg cell, highlights the different roles which these two cell types are going to play in seed development Pillot et al.

For an extensive review on the dynamics of the chromatin landscape on the female gametophyte development follow Baroux and Autran In the zygote, the parentally derived histone H3 variants are replaced before the first division of the embryo to reflect the content found in sporophytic cells Ingouff and Berger, Two maternal epigenetic pathways are acting in the early embryo to regulate the paternal transcripts, the RdDM pathway and the histone chaperone complex chromatin assembly factor 1 CAF1.

These pathways do not regulate genomic imprinting Autran et al. The central cell will give rise upon fusion with one sperm cell nucleus to the endosperm. It should be noted that endosperm development is sensitive to parental genome dosage, and the majority of imprinted genes reported are expressed from the maternal genome in the endosperm reviewed, for example, in Gehring and Satyaki, Endosperm chromatin is characterized by a looser structure, DNA hypomethylation, and decreased levels of H3K9me2, when compared with somatic tissues and embryo Baroux et al.

In contrast to embryo development, extensive demethylation occurs during endosperm development and this dynamic process allows for imprinting variation observed in maize and Arabidopsis Gehring et al. In maize, HDA and members of different chromatin-remodeling complexes affect endosperm transfer cells leading to an alteration in the kernel size Yang et al.

Kernels of hda hda plants showed a strong defective phenotype with fully or partially empty pericarp. Starchy endosperm tissue failed to accumulate starch or developed only partially in defective kernels, while the embryo showed abnormalities that varied from the presence of an undifferentiated aborted embryo to a defective embryo blocked at the coleoptilar stage Forestan et al. Seeds are embedded in fruits, many of which are an important source of food for humans.

The best understood development of fleshy fruits is that of tomato, which displays remarkable characteristics related to chromosome structure, chromatin organization, and chromatin dynamics Bourdon et al. A major developmental feature is an increase in nuclear DNA content due to endoreduplication leading to cell hypertrophy, thereby influencing fruit growth and size Chevalier et al. Whether chromatin modifications are associated with endoreduplication still remains largely unknown.

However, it was shown in Arabidopsis that endoreduplicated nuclei have less condensed heterochromatin Schubert et al.

In tomato, DNA methylation decreases in the highly endoreduplicated pericarp tissue and is significantly reduced at the onset of fruit maturation and during ripening Teyssier et al. Currently, there is increasing evidence for epigenetic control during fruit organogenesis, and epigenome dynamics play an important role during fruit maturation and ripening in tomato reviewed in Giovannoni et al. Decades of breeding and selection have narrowed down the pool of genetic variability in many crops Palmgren et al.

Crop breeding programs have classically relied on sequence-based genetic variability of either natural or induced origin. These efforts have allowed the generation of varieties with an increased and more stable yield, and relatively well adapted to biotic and abiotic stresses. However, the exploitation of genetic variability existing within gene pools has been limited.

Furthermore, not all the heritable phenotypic diversity can be explained by sequence variation, and has been termed the missing heritability Maher, ; Gallusci et al. Such variation could have an epigenetic basis. The applicability of chromatin modifications for the purpose of crop improvement Fig. Epigenetic modifications may be of interest for breeders only if their regulatory effects are maintained through mitosis and ideally through meiosis.

Here, DNA methylation and specific histone PTMs are the prime candidates for crop improvement, as they were mitotically transmittable for at least a limited time in several species Hyun et al. This raises the possibility of employing them as tools for breeding in clonally propagated crops, such as many fruit trees.

However, for seed-propagated crops, specific chromatin modifications need to pass the epigenetic resetting barriers during gametogenesis and seed development in order to pass to the next generation Pecinka and Mittelsten Scheid, ; Grossniklaus et al. Here, DNA methylation seems to be the best candidate due to its stability and because PTMs are lost due to gametogenesis specific-removal and replacement of the parental nucleosomes Ingouff et al. Applications of epigenetic variation for the purposes of plant breeding.

Natural epigenetic variation is relatively little explored and known cases were often selected by the phenotype and only later described to have an epigenetic basis.

Presumably, genome-wide screening for natural epigenetic variation will allow less biased use of the naturally occurring germplasms in the future. In contrast, induced epigenetic variation is provoked by humans either in a targeted manner towards a specific genomic locus or in an untargeted manner with subsequent identification and selection of the modified loci. Choice of the method s is guided by the purpose, the species, and its available resources.

Some of the artificially produced epialleles fall under the GMO regulations. Plant developmental processes determine a great number of traits of agronomic interest that have been targeted for selection in crops. Some of them are epigenetically regulated, either by DNA methylation or histone PTMs such as leaf shape, flowering time and flower development, male fertility, oil yield, fruit ripening, grain size, plant stature, inflorescence structure, branching plant architecture, boll setting rate, abscission rate, photoperiod responses, etc.

Zhang, ; Ong-Abdullah et al. Expanding evidence also shows that epigenetic control has an important role in the fine-tuning of the responses to biotic and abiotic stress Gourcilleau et al. This raises the possibility of generating or selecting variability of epigenetic changes to assist plant breeding.

Stably inherited epialleles have been characterized for genes controlling some developmental processes. Examples of such epialleles in crops include: the tomato CNR locus controlling fruit ripening Manning et al.

One possible approach is to select epigenetic variants among the natural diversity by exploiting DNA methylation states in different germplasms Takuno et al.

This type of analysis has revealed large amounts of epigenetic variability in ecotypes, cultivars, landraces, and wild relatives Eichten et al. However, it requires good reference genomes and can be more time-consuming and tedious than mining genetic polymorphisms. The easiest way to link DNA methylation polymorphisms with phenotypes is to simultaneously monitor gene expression Eichten et al. However, this may be challenging for genes with tissue-specific transcription.

Epialleles can also be generated artificially. Untargeted approaches employ cell culture Mittelsten Scheid et al. In addition, this can be achieved by the generation of epigenetic recombinant inbred lines epiRILs from crosses between the wild type and maintenance DNA methylation mutants.

However, the current trends are directed towards controlled induction of the chromatin states. In addition, there are studies demonstrating that the modified CRISPR clustered regularly interspaced short palindromic repeat system using Cas9 or related nucleases such as Cfp1 offers wide possibilities to change chromatin at specific loci Liu and Moschou, ; Xie et al.

We predict that the number of dCas9-induced modifications will grow rapidly in the model plants as well as in crops. This approach has a great potential to shed more light on how the chromatin states are established, maintained, and erased in plants.

In addition, this could improve agriculturally relevant developmental or stress resistance-related traits in crops; however, the legal restrictions will most probably remain the main hurdle towards practical use of such inventions world-wide.

Chromatin modifications have emerged as a complementary source of variability contributing to plant phenotypic plasticity Fig. It could also address new challenges in crop improvement, including adaptive responses to environmental stresses.

Since the emergence and inheritance of epigenetic variation differs from the genetic variants, current methods of trait mapping miss substantial phenotype-determining variation and thus may have reduced efficacy. Therefore, the relative contribution of genetic versus epigenetic variation remains unknown Pecinka et al. However, plant breeding using chromatin traits can be assisted by newly developed tools including process-based models Hu et al.

Classical plant breeding harnesses the genetic variation that is generated by homologous recombination during meiosis. In this context, a better understanding about the influence of the epigenetic make up on meiotic recombination would contribute to development of novel strategies to modify the recombination pattern and to generate new elite crop varieties Fig.

The ever-increasing knowledge drawn from epigenetics studies in model and crop plants paves the way to applied perspectives and foreseen plant breeding strategies. The exploitation of epigenetic diversity is the forthcoming challenge for the next plant breeding strategies, since chromatin modifications are tightly intertwined with plant phenotypic plasticity reviewed in Pecinka et al.

During interphase, the cell grows and the nuclear DNA is duplicated. Interphase is followed by the mitotic phase.

During the mitotic phase, the duplicated chromosomes are segregated and distributed into daughter nuclei. The cytoplasm is usually divided as well, resulting in two daughter cells. The first stage of interphase is called the G 1 phase first gap because, from a microscopic aspect, little change is visible. However, during the G 1 stage, the cell is quite active at the biochemical level.

The cell grows and accumulates the building blocks of chromosomal DNA and the associated proteins as well as sufficient energy reserves to complete the task of replicating each chromosome in the nucleus. The synthesis phase of interphase takes the longest because of the complexity of the genetic material being duplicated. Throughout interphase, nuclear DNA remains in a semi-condensed chromatin configuration. In the S phase, DNA replication results in the formation of identical pairs of DNA molecules, sister chromatids, that are firmly attached to the centromeric region.

The centrosome is duplicated during the S phase. The two centrosomes will give rise to the mitotic spindle, the apparatus that orchestrates the movement of chromosomes during mitosis. At the center of each animal cell, the centrosomes of animal cells are associated with a pair of rod-like objects, the centrioles, which are at right angles to each other. Centrioles help organize cell division.

Centrioles are not present in the centrosomes of other eukaryotic species, such as plants and most fungi. In the G 2 phase, the cell replenishes its energy stores and synthesizes proteins necessary for chromosome manipulation. Some cell organelles are duplicated, and the cytoskeleton is dismantled to provide resources for the mitotic phase. There may be additional cell growth during G 2.

The final preparations for the mitotic phase must be completed before the cell is able to enter the first stage of mitosis. During the multistep mitotic phase, the cell nucleus divides, and the cell components split into two identical daughter cells.

The mitotic phase is a multistep process during which the duplicated chromosomes are aligned, separated, and move into two new, identical daughter cells. The first portion of the mitotic phase is called karyokinesis or nuclear division. The second portion of the mitotic phase, called cytokinesis, is the physical separation of the cytoplasmic components into the two daughter cells.

Histones carry positive charges and bind negatively charged DNA in a specific conformation. In particular, a segment of the DNA double helix wraps around each histone core particle a little less than twice. The exact length of the DNA segment associated with each histone core varies from species to species, but most such segments are approximately base pairs in length. Furthermore, each histone molecule within the core particle has one end that sticks out from the particle.

These ends are called N-terminal tails , and they play an important role in higher-order chromatin structure and gene expression. Figure 4: The nucleosome structure within chromatin.

Each nucleosome contains eight histone proteins blue , and DNA wraps around these histone structures to achieve a more condensed coiled form. Figure 5: To better fit within the cell, long pieces of double-stranded DNA are tightly packed into structures called chromosomes.

Although nucleosomes may look like extended "beads on a string" under an electron microscope, they appear differently in living cells. In such cells, nucleosomes stack up against one another in organized arrays with multiple levels of packing. The first level of packing is thought to produce a fiber about 30 nanometers nm wide.

These 30 nm fibers then form a series of loops, which fold back on themselves for additional compacting Figure 5. The multiple levels of packing that exist within eukaryotic chromosomes not only permit a large amount of DNA to occupy a very small space, but they also serve several functional roles. For example, the looping of nucleosome-containing fibers brings specific regions of chromatin together, thereby influencing gene expression.

In fact, the organized packing of DNA is malleable and appears to be highly regulated in cells. Chromatin packing also offers an additional mechanism for controlling gene expression. Specifically, cells can control access to their DNA by modifying the structure of their chromatin.

Highly compacted chromatin simply isn't accessible to the enzymes involved in DNA transcription , replication , or repair. Thus, regions of chromatin where active transcription is taking place called euchromatin are less condensed than regions where transcription is inactive or is being actively inhibited or repressed called heterochromatin Figure 6.

Figure 6: The structure of chromatin in interphase Heterochromatin is more condensed than euchromatin. Typically, the more condensed chromatin is, the less accessible it is by transcription factors and polymerases. The dynamic nature of chromatin is regulated by enzymes. For example, chromatin can be loosened by changing the position of the DNA strands within a nucleosome.

This loosening occurs because of chromatin remodeling enzymes, which function to slide nucleosomes along the DNA strand so that other enzymes can access the strand. This process is closely regulated and allows specific genes to be accessed in response to metabolic signals within the cell. Another way cells control gene expression is by modifying their histones with small chemical groups, such as methyl and acetyl groups in the N-terminal tails that extend from the core particle.

Different enzymes catalyze each kind of N-terminal modification. Scientists occasionally refer to the complex pattern of histone modification in cells as a "histone code. In electron micrographs, eukaryotic interphase chromatin appears much like a plate of spaghetti — in other words, there is no obvious pattern of organization. In recent years, however, investigators have begun using fluorescent probes for each of the different interphase chromosomes.

It is now well-established that spindles are bipolar arrays of microtubules composed of tubulin Figure 5 and that the centrosomes nucleate the growth of the spindle microtubules. During mitosis, many of the spindle fibers attach to chromosomes at their kinetochores Figure 6 , which are specialized structures in the most constricted regions of the chromosomes.

The length of these kinetochore-attached microtubules then decreases during mitosis, pulling sister chromatids to opposite poles of the spindle. Other spindle fibers do not attach to chromosomes, but instead form a scaffold that provides mechanical force to separate the daughter nuclei at the end of mitosis. From his many detailed drawings of mitosen, Walther Flemming correctly deduced, but could not prove, the sequence of chromosome movements during mitosis Figure 7.

Flemming divided mitosis into two broad parts: a progressive phase, during which the chromosomes condensed and aligned at the center of the spindle, and a regressive phase, during which the sister chromatids separated. Our modern understanding of mitosis has benefited from advances in light microscopy that have allowed investigators to follow the process of mitosis in living cells. Such live cell imaging not only confirms Flemming's observations, but it also reveals an extremely dynamic process that can only be partially appreciated in still images.

Mitosis begins with prophase, during which chromosomes recruit condensin and begin to undergo a condensation process that will continue until metaphase. In most species , cohesin is largely removed from the arms of the sister chromatids during prophase, allowing the individual sister chromatids to be resolved. Cohesin is retained, however, at the most constricted part of the chromosome, the centromere Figure 9. During prophase, the spindle also begins to form as the two pairs of centrioles move to opposite poles and microtubules begin to polymerize from the duplicated centrosomes.

Prometaphase begins with the abrupt fragmentation of the nuclear envelope into many small vesicles that will eventually be divided between the future daughter cells. The breakdown of the nuclear membrane is an essential step for spindle assembly. Because the centrosomes are located outside the nucleus in animal cells, the microtubules of the developing spindle do not have access to the chromosomes until the nuclear membrane breaks apart.

Prometaphase is an extremely dynamic part of the cell cycle. Microtubules rapidly assemble and disassemble as they grow out of the centrosomes, seeking out attachment sites at chromosome kinetochores, which are complex platelike structures that assemble during prometaphase on one face of each sister chromatid at its centromere. As prometaphase ensues, chromosomes are pulled and tugged in opposite directions by microtubules growing out from both poles of the spindle, until the pole-directed forces are finally balanced.

Sister chromatids do not break apart during this tug-of-war because they are firmly attached to each other by the cohesin remaining at their centromeres. At the end of prometaphase, chromosomes have a bi-orientation, meaning that the kinetochores on sister chromatids are connected by microtubules to opposite poles of the spindle.

Next, chromosomes assume their most compacted state during metaphase, when the centromeres of all the cell's chromosomes line up at the equator of the spindle. Metaphase is particularly useful in cytogenetics , because chromosomes can be most easily visualized at this stage. Furthermore, cells can be experimentally arrested at metaphase with mitotic poisons such as colchicine. Video microscopy shows that chromosomes temporarily stop moving during metaphase.

A complex checkpoint mechanism determines whether the spindle is properly assembled, and for the most part, only cells with correctly assembled spindles enter anaphase. Figure 10 Figure Detail. Figure 9. The progression of cells from metaphase into anaphase is marked by the abrupt separation of sister chromatids. A major reason for chromatid separation is the precipitous degradation of the cohesin molecules joining the sister chromatids by the protease separase Figure Two separate classes of movements occur during anaphase.

During the first part of anaphase, the kinetochore microtubules shorten, and the chromosomes move toward the spindle poles. During the second part of anaphase, the spindle poles separate as the non-kinetochore microtubules move past each other. These latter movements are currently thought to be catalyzed by motor proteins that connect microtubules with opposite polarity and then "walk" toward the end of the microtubules.

Mitosis ends with telophase, or the stage at which the chromosomes reach the poles. The nuclear membrane then reforms, and the chromosomes begin to decondense into their interphase conformations.

Telophase is followed by cytokinesis, or the division of the cytoplasm into two daughter cells. The daughter cells that result from this process have identical genetic compositions.

Cheeseman, I. Molecular architecture of the kinetochore-microtubule interface. Nature Reviews Molecular Cell Biology 9 , 33—46 doi Cremer, T. Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nature Reviews Genetics 2 , — doi