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Why do I see so many kinetochores?

Why do I see so many kinetochores?


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I am analysing RPE-1 cells from humans and I do not understand why I see so many kinetochores by immunofluorescence (more than 100 in many cells). They are in prometaphase.


Chapter 12 - The Cell Cycle

  • The ability of organisms to reproduce their kind is the one characteristic that best distinguishes living things from nonliving matter.
  • The continuity of life is based on the reproduction of cells, or cell division.

Cell division functions in reproduction, growth, and repair.

  • The division of a unicellular organism reproduces an entire organism, increasing the population.
  • Cell division on a larger scale can produce progeny for some multicellular organisms.
  • This includes organisms that can grow by cuttings.
  • Cell division enables a multicellular organism to develop from a single fertilized egg or zygote.
  • In a multicellular organism, cell division functions to repair and renew cells that die from normal wear and tear or accidents.
  • Cell division is part of the cell cycle, the life of a cell from its origin in the division of a parent cell until its own division into two.

Concept 12.1 Cell division results in genetically identical daughter cells

  • Cell division requires the distribution of identical genetic material—DNA—to two daughter cells.
  • What is remarkable is the fidelity with which DNA is passed along, without dilution, from one generation to the next.
  • A dividing cell duplicates its DNA, allocates the two copies to opposite ends of the cell, and then splits into two daughter cells.
  • A cell’s genetic information, packaged as DNA, is called its genome.
    • In prokaryotes, the genome is often a single long DNA molecule.
    • In eukaryotes, the genome consists of several DNA molecules.
    • Every eukaryotic species has a characteristic number of chromosomes in each cell nucleus.
      • Human somatic cells (body cells) have 46 chromosomes, made up of two sets of 23 (one from each parent).
      • Human gametes (sperm or eggs) have one set of 23 chromosomes, half the number in a somatic cell.
      • Each single chromosome contains one long, linear DNA molecule carrying hundreds or thousands of genes, the units that specify an organism’s inherited traits.
      • The chromatids are initially attached by adhesive proteins along their lengths.
      • As the chromosomes condense, the region where the chromatids connect shrinks to a narrow area, the centromere.
      • Once the sister chromatids separate, they are considered individual chromosomes.
      • Each of us inherited 23 chromosomes from each parent: one set in an egg and one set in sperm.
      • The fertilized egg, or zygote, underwent cycles of mitosis and cytokinesis to produce a fully developed multicellular human made up of 200 trillion somatic cells.
      • These processes continue every day to replace dead and damaged cells.
      • Essentially, these processes produce clones—cells with identical genetic information.
      • Meiosis yields four nonidentical daughter cells, each with half the chromosomes of the parent.
      • In humans, meiosis reduces the number of chromosomes from 46 to 23.
      • Fertilization fuses two gametes together and doubles the number of chromosomes to 46 again.

      Concept 12.2 The mitotic phase alternates with interphase in the cell cycle

      • The mitotic (M) phase of the cell cycle alternates with the much longer interphase.
        • The M phase includes mitosis and cytokinesis.
        • Interphase accounts for 90% of the cell cycle.
        • During all three subphases, the cell grows by producing proteins and cytoplasmic organelles such as mitochondria and endoplasmic reticulum.
        • However, chromosomes are duplicated only during the S phase.
        • Of this time, the M phase would last less than an hour, while the S phase might take 10–12 hours, or half the cycle.
        • The rest of the time would be divided between the G1 and G2 phases.
        • The G1 phase varies most in length from cell to cell.
        • A nuclear membrane bounds the nucleus, which contains one or more nucleoli.
        • The centrosome has replicated to form two centrosomes.
        • In animal cells, each centrosome features two centrioles.
        • The nucleoli disappear.
        • The mitotic spindle begins to form.
          • It is composed of centrosomes and the microtubules that extend from them.
          • Each of the two chromatids of a chromosome has a kinetochore, a specialized protein structure located at the centromere.
          • Kinetochore microtubules from each pole attach to one of two kinetochores.
          • Nonkinetochore microtubules interact with those from opposite ends of the spindle.
          • Each is now pulled toward the pole to which it is attached by spindle fibers.
          • By the end, the two poles have equivalent collections of chromosomes.
          • Nuclear envelopes arise from the fragments of the parent cell’s nuclear envelope and other portions of the endomembrane system.
          • The chromosomes become less tightly coiled.

          The mitotic spindle distributes chromosomes to daughter cells: a closer look.

          • The mitotic spindle, fibers composed of microtubules and associated proteins, is a major driving force in mitosis.
          • As the spindle assembles during prophase, the elements come from partial disassembly of the cytoskeleton.
          • The spindle fibers elongate by incorporating more subunits of the protein tubulin.
          • Assembly of the spindle microtubules starts in the centrosome.
            • The centrosome (microtubule-organizing center) is a nonmembranous organelle that organizes the cell’s microtubules.
            • In animal cells, the centrosome has a pair of centrioles at the center, but the centrioles are not essential for cell division.
            • As the spindle microtubules grow from them, the centrioles are pushed apart.
            • By the end of prometaphase, they are at opposite ends of the cell.
            • The kinetochores of the joined sister chromatids face in opposite directions.
            • Once the chromosomes are separate, full-fledged chromosomes, they move toward opposite poles of the cell.
            • Meanwhile, the excess microtubule sections depolymerize at their kinetochore ends.
            • These microtubules interdigitate and overlap across the metaphase plate.
            • During anaphase, the area of overlap is reduced as motor proteins attached to the microtubules walk them away from one another, using energy from ATP.
            • As microtubules push apart, the microtubules lengthen by the addition of new tubulin monomers to their overlapping ends, allowing continued overlap.

            Cytokinesis divides the cytoplasm: a closer look.

            • Cytokinesis, division of the cytoplasm, typically follows mitosis.
            • In animal cells, cytokinesis occurs by a process called cleavage.
            • The first sign of cleavage is the appearance of a cleavage furrow in the cell surface near the old metaphase plate.
            • On the cytoplasmic side of the cleavage furrow is a contractile ring of actin microfilaments associated with molecules of the motor protein myosin.
              • Contraction of the ring pinches the cell in two.
              • The plate enlarges until its membranes fuse with the plasma membrane at the perimeter.
              • The contents of the vesicles form new cell wall material between the daughter cells.

              Mitosis in eukaryotes may have evolved from binary fission in bacteria.

              • Prokaryotes reproduce by binary fission, not mitosis.
              • Most bacterial genes are located on a single bacterial chromosome that consists of a circular DNA molecule and associated proteins.
              • While bacteria are smaller and simpler than eukaryotic cells, they still have large amounts of DNA that must be copied and distributed equally to two daughter cells.
              • The circular bacterial chromosome is highly folded and coiled in the cell.
              • In binary fission, chromosome replication begins at one point in the circular chromosome, the origin of replication site, producing two origins.
                • As the chromosome continues to replicate, one origin moves toward each end of the cell.
                • While the chromosome is replicating, the cell elongates.
                • When replication is complete, its plasma membrane grows inward to divide the parent cell into two daughter cells, each with a complete genome.
                • The movement is similar to the poleward movements of the centromere regions of eukaryotic chromosomes.
                • However, bacterial chromosomes lack visible mitotic spindles or even microtubules.
                • Several proteins have been identified and play important roles.
                • There is evidence that mitosis had its origins in bacterial binary fission.
                • Some of the proteins involved in binary fission are related to eukaryotic proteins.
                • Two of these are related to eukaryotic tubulin and actin proteins.
                • In dinoflagellates, replicated chromosomes are attached to the nuclear envelope.
                • In diatoms, the spindle develops within the nucleus.

                Concept 12.3 The cell cycle is regulated by a molecular control system

                • The timing and rates of cell division in different parts of an animal or plant are crucial for normal growth, development, and maintenance.
                • The frequency of cell division varies with cell type.
                  • Some human cells divide frequently throughout life (skin cells).
                  • Others have the ability to divide, but keep it in reserve (liver cells).
                  • Mature nerve and muscle cells do not appear to divide at all after maturity.

                  Cytoplasmic signals drive the cell cycle.

                  • The cell cycle appears to be driven by specific chemical signals present in the cytoplasm.
                  • Some of the initial evidence for this hypothesis came from experiments in which cultured mammalian cells at different phases of the cell cycle were fused to form a single cell with two nuclei.
                    • Fusion of an S phase cell and a G1 phase cell induces the G1 nucleus to start S phase.
                      • This suggests that chemicals present in the S phase nucleus stimulated the fused cell.
                      • Cyclically operating molecules trigger and coordinate key events in the cell cycle.
                      • The control cycle has a built-in clock, but it is also regulated by external adjustments and internal controls.
                      • The signals are transmitted within the cell by signal transduction pathways.
                      • Animal cells generally have built-in stop signals that halt the cell cycle at checkpoints until overridden by go-ahead signals.
                      • Many signals registered at checkpoints come from cellular surveillance mechanisms.
                      • These indicate whether key cellular processes have been completed correctly.
                      • Checkpoints also register signals from outside the cell.
                      • If the cell receives a go-ahead signal at the G1 checkpoint, it usually completes the cell cycle and divides.
                      • If it does not receive a go-ahead signal, the cell exits the cycle and switches to a nondividing state, the G0 phase.
                        • Most cells in the human body are in this phase.
                        • Liver cells can be “called back” to the cell cycle by external cues, such as growth factors released during injury.
                        • Highly specialized nerve and muscle cells never divide.
                        • These regulatory molecules include protein kinases that activate or deactivate other proteins by phosphorylating them.
                        • Levels of cyclin proteins fluctuate cyclically.
                        • Because of the requirement for binding of a cyclin, the kinases are called cyclin-dependent kinases, or Cdks.
                        • MPF promotes mitosis by phosphorylating a variety of other protein kinases.
                        • MPF stimulates fragmentation of the nuclear envelope by phosphorylation of various proteins of the nuclear lamina.
                        • It also triggers the breakdown of cyclin, dropping cyclin and MPF levels during mitosis and inactivating MPF.
                          • The noncyclin part of MPF, the Cdk, persists in the cell in inactive form until it associates with new cyclin molecules synthesized during the S and G2 phases of the next round of the cycle.

                          Internal and external cues help regulate the cell cycle.

                          • While research scientists know that active Cdks function by phosphorylating proteins, the identity of all these proteins is still under investigation.
                          • Scientists do not yet know what Cdks actually do in most cases.
                          • Some steps in the signaling pathways that regulate the cell cycle are clear.
                            • Some signals originate inside the cell, others outside.
                            • This ensures that daughter cells do not end up with missing or extra chromosomes.
                            • This keeps the anaphase-promoting complex (APC) in an inactive state.
                            • When all kinetochores are attached, the APC activates, triggering breakdown of cyclin and inactivation of proteins holding sister chromatids together.
                            • For example, cells fail to divide if an essential nutrient is left out of the culture medium.
                            • For example, platelet-derived growth factors (PDGF), produced by platelet blood cells, bind to tyrosine-kinase receptors of fibroblasts, a type of connective tissue cell.
                            • This triggers a signal-transduction pathway that allows cells to pass the G1 checkpoint and divide.
                            • Fibroblasts in culture will only divide in the presence of a medium that also contains PDGF.
                            • The resulting proliferation of fibroblasts helps heal the wound.
                            • Cultured cells normally divide until they form a single layer on the inner surface of the culture container.
                            • If a gap is created, the cells will grow to fill the gap.
                            • At high densities, the amount of growth factors and nutrients is insufficient to allow continued cell growth.
                            • To divide, they must be anchored to a substratum, typically the extracellular matrix of a tissue.
                            • Control appears to be mediated by pathways involving plasma membrane proteins and elements of the cytoskeleton linked to them.

                            Cancer cells have escaped from cell cycle controls.

                            • Cancer cells divide excessively and invade other tissues because they are free of the body’s control mechanisms.
                              • Cancer cells do not stop dividing when growth factors are depleted.
                              • This is either because a cancer cell manufactures its own growth factors, has an abnormality in the signaling pathway, or has an abnormal cell cycle control system.
                              • In contrast, nearly all mammalian cells divide 20 to 50 times under culture conditions before they stop, age, and die.
                              • HeLa cells from a tumor removed from a woman (Henrietta Lacks) in 1951 are still reproducing in culture.
                              • Normally, the immune system recognizes and destroys transformed cells.
                              • However, cells that evade destruction proliferate to form a tumor, a mass of abnormal cells.
                              • Most do not cause serious problems and can be fully removed by surgery.
                              • Cancer cells are abnormal in many ways.
                              • They may have an unusual number of chromosomes, their metabolism may be disabled, and they may cease to function in any constructive way.
                              • Cancer cells may secrete signal molecules that cause blood vessels to grow toward the tumor.
                              • These treatments target actively dividing cells.
                              • Chemotherapeutic drugs interfere with specific steps in the cell cycle.
                              • For example, Taxol prevents mitotic depolymerization, preventing cells from proceeding past metaphase.
                              • The side effects of chemotherapy are due to the drug’s effects on normal cells.
                              • The causes are diverse, but cellular transformation always involves the alteration of genes that influence the cell cycle control system.

                              Lecture Outline for Campbell/Reece Biology, 7th Edition, © Pearson Education, Inc. 12-1


                              The Truth About Drowning Worms

                              Conventional wisdom holds that earthworms head to the surface after rain because they can’t breathe. This is still taught to schoolkids, and you can find a lot of detailed explanation online. Most claim that worm trails and air pockets underground become submerged, and the earthworms can’t breathe. It makes sense.

                              Most researchers, though, dispute this explanation. As Chris Lowe, a researcher at the University of Central Lancashire, points out in Scientific American, earthworms breathe through their skin and require moisture to do so.

                              Humans drown when their lungs fill with water. This is not possible for earthworms as they lack lungs. Multiple studies have also shown that most earthworm species can survive being submerged in water for two weeks or more.


                              A Scrambled Mess

                              Karen Schindler
                              May 1, 2016

                              A light micrograph of a section of fetal ovary shows primordial follicles (light pink ovals) with oocytes (dark pink spots) that have already begun to mature into fertilizable eggs. But the process won&rsquot be complete for decades, during which time mistakes in chromosome division can occur. © TISSUEPIX/SCIENCE SOURCE

                              U p to a quarter of pregnancies are not carried to term oftentimes an embryo is aborted by the body before a woman even knows she&rsquos pregnant. The most common cause of miscarriage is egg aneuploidy&mdashthe oocyte contains too many or too few chromosomes. Aneuploidy is thus the leading genetic cause of infertility, and those embryos that are not miscarried can result in children with developmental disorders, such as Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), and Turner syndrome (monosomy X).

                              The life of an oocyte begins during female fetal development but does not finish for decades, providing multiple windows.

                              For more than 80 years, the scientific community has known that the incidence of Down syndrome births increases with maternal age and that female fertility rapidly declines after the age of 35. 1 These concerns can be bypassed by the use of donor eggs from younger women, however, suggesting that the eggs of older women are the source of the reproductive decline, not the mother’s reproductive system itself. Sure enough, up to 20 percent of eggs in healthy females may be aneuploid, and this number increases with age. But despite the ubiquity of egg aneuploidy, the cellular and genetic reasons for the phenomenon are poorly understood.

                              We now know that the multistage process of meiosis that forms a woman’s eggs is highly error prone. 2 While germ-line meiosis in males initiates at puberty and provides a fresh supply of haploid sperm cells until death, the life of an oocyte begins during female fetal development but does not finish for decades, providing multiple windows of opportunity for problems that compromise egg quality. And in the past two years, clinicians and basic scientists have started conducting analyses of human oocytes to get at the molecular details of this pervasive problem. Thanks to technical advances, such as genome-wide recombination mapping and high-resolution, live-cell imaging, we now have a clearer picture of how chromosomes behave during meiosis.

                              Once scientists understand the basic machinery that controls meiosis, they can develop appropriate diagnostics and interventions to help women achieve pregnancies with egg cells that have properly apportioned chromosomes. Currently, one in six couples is infertile, and about half of those cases are due to abnormalities on the female side. And as the average age at which a woman experiences her first pregnancy increases in the U.S. and other developed countries—in some nations, that age has reached 30—the challenges of aneuploidy will only become more common.

                              Divvying up the genome

                              ASYMMETRIC DIVISION: Just before ovulation, the first cell division of meiosis yields a large oocyte (green) and much smaller polar body (yellow). © PROF. P.M. MOTTA/UNIV. “La Sapienza”, ROME/SCIENCE SOURCE During female fetal development, the primordial germ cells that give rise to oocytes replicate their full diploid complement of DNA, with each chromosome forming two sister chromatids joined along the arms and centromeres by a protein complex known as cohesin. Homologous chromosomes then pair with each other and exchange bits of DNA through homologous recombination. The process involves breaking the chromosomes and swapping bits of DNA between nonsister chromatids of a homologous pair (homologs). During the swap, termed crossing over, linkages called chiasmata form between homologs and are maintained until the onset of anaphase I several decades later, when the chromosomes are pulled apart before division into two daughter cells. This marks the completion of the first stage of meiosis (Meiosis I). If chiasmata fail to form, the chromosomes may separate improperly, a phenomenon known as nondisjunction. (See illustration below and “Picturing Inheritance, 1916.”) Most cases of trisomy 21 are due to maternal nondisjunction.

                              Last year, Christian Ottolini in Eva Hoffman’s laboratory at the University of Kent in the U.K. and colleagues generated genome-wide recombination maps, dubbed “MeioMaps,” and found evidence that properly functioning recombination is indeed protective against chromosome segregation errors in human oocytes. Using single nucleotide polymorphism (SNP) arrays with some 300,000 genetic markers, the researchers pinpointed the sites of recombination in 13 human oocytes and their associated polar bodies—the nonfunctioning cells produced during meiosis that do not become the mature egg—as well as 10 embryo–polar body sets from patients undergoing in vitro fertilization (IVF). Notably, this is the first time researchers have assessed all the products from a complete meiosis. In addition, the researchers performed preimplantation genetic diagnosis of 29 embryos to diagnose aneuploidy. While the number of recombination events were highly variable between samples, they tended to decrease with age. And oocytes that underwent less recombination were more likely to be aneuploid. 3

                              Nondisjunction is not the only way to get eggs with an incorrect number of chromosomes. In fact, some data indicate that a more-frequent cause of aneuploidy is the premature separation of sister chromatids (PSSC). 4,5 Under normal conditions, cohesin is deposited along the length of chromosomes during premeiotic DNA replication to hold sister chromatids together. At the onset of anaphase during meiosis I, cohesin is cleaved along the chromosome arms, but it is protected at sister centromeres by a protein called shugoshin to ensure that sister chromatids remain together as homologs segregate. During anaphase of meiosis II, the remaining cohesin is cleaved, allowing sister chromatid separation and the formation of four fully haploid daughter cells. Therefore, to ensure proper sister chromatid associations throughout oocyte maturation, cohesin proteins laid down during fetal development must still be functional decades later. 6 If cohesin is lost or rendered dysfunctional at any point along the way, the sister chromatids can be pulled into different daughter cells prematurely.

                              Sure enough, as my colleagues and I as well as other groups have found, cohesin levels are reduced and sister chromatid centromeres begin to separate prematurely in oocytes from aged mice. 7,8,9 Similarly, the distance between sister chromatids in human oocytes increases with maternal age and aneuploidy rates go up. 10,11 These observations support the hypothesis that exhaustion of cohesin can lead to increased PSSC in human eggs.

                              Additionally, while in mice and other model organisms sister chromatid kinetochores—the two centromeric protein complexes that attach to the spindle microtubules extending from the cell’s poles during meiosis—are fused together, recent research suggests that the same may not be true of chromosomes in human eggs. Last year, two independent groups used high-resolution imaging to examine the geometry of the sister-chromatid kinetochores in human oocytes harvested for IVF and found that they were not fused, and thus did not act as a single unit as they do in mice and other organisms, where they serve as further insurance that both chromatids end up in the same daughter cell following the first meiotic division. 12,13 The distance between sister chromatid kinetochores in human oocytes increases with maternal age, but kinetochore separation is also frequently observed in younger women, possibly contributing to the fact that even young women can have high rates of meiotic aneuploidy. 14,15 (See “In the Genes” below.)

                              But high rates of PSSC do not rule out a role for recombination defects in aneuploidy. In 2006, Beth Rockmill, then in Shirleen Roeder’s lab at Yale University, and colleagues observed wild-type yeast strains engineered to harbor an extra copy of chromosome 3 containing selectable markers so that they could easily detect PSSC. After dissecting 1,300 tetrad spores—the equivalent of a mammalian egg and its three polar bodies—the researchers found a correlation between PSSC and crossovers that occurred close to the centromere, suggesting that where along their length homologous chromosomes recombine is important. If the crossover is too close to the centromere, it may interfere with sister chromatid cohesion, causing the sister chromatids to dissociate. 16 Ottolini and collaborators also found that some chromosomes in human eggs failed to suppress crossovers at or close to centromeres—consistent with the team’s observations of elevated PSSC.

                              MEIOTIC MYSTERIES: Meiosis in human females takes place over decades. At any point in this process, an incorrect number of chromosomes can be transferred to daughter cells, resulting in aneuploid gametes, the most common cause of miscarriage and the root of certain developmental disorders, such as Down syndrome.
                              See full infographic: WEB | PDF © 2016 MICA DURAN

                              All of these missegregation scenarios are chromosome-centric. What is missing from these pictures, however, is the behavior of the microtubules that connect the chromosomes to the spindle poles on opposite sides of the cell. Even if sister chromatids do separate prematurely, they may not segregate improperly if the microtubules hook up as they would if the chromatids were still attached. But if these connections are not correct, chromosomes are at risk of ending up in the wrong daughter cell. The attachment of sister kinetochores to microtubule fibers from opposite poles during meiosis I, for example, could cause sister chromatids to split up. As the distance between sister chromatids increases with maternal age, the risk of such aberrant microtubule attachment also likely increases.

                              By visualizing 100 human oocytes as they underwent spindle formation during meiosis I, Zuzana Holubcová in Melina Schuh’s laboratory at the Medical Research Council in Cambridge, U.K., and colleagues observed several abnormalities in building the spindle. 17 In some cases, the spindle structure was unstable and would either lack any poles or become multipolar. The researchers also noted chromosome segregation problems such as lagging chromosomes that would remain in the center of the spindle during anaphase I. They hypothesized that these lagging chromosomes resulted from errors in how the microtubules attached. Taking a snapshot of the microtubule connections, they found that 20 percent of sister chromatid kinetochores attached to both poles instead of a single pole. In mice, such attachment is a trial-and-error process in which aberrant connections are normally fixed. If human oocytes are inefficient at correcting such attachment errors, it could explain the high rate of chromosome missegregation during the formation of human eggs.

                              Additionally, all of the human oocytes Holubcová tracked lacked microtubule-organizing centers that help coordinate spindle assembly in mouse oocytes. Instead, chromosomes initiated microtubule growth. Moreover, the researchers discovered that human oocytes took an unusually long time to build the spindle—a whopping 16 hours, compared to just 5 hours in mouse oocytes and the 30 minutes it takes cells to build spindles for mitotic division. Such inefficient spindle formation could favor incorrect attachments that can lead to aneuploidy. Given the importance of microtubule attachments for proper chromosome segregation in human oocyte development, studying oocyte spindle biology will be critical to understanding why meiosis I is so error prone.

                              A closer look

                              Surprisingly, improper chromosome segregation doesn’t always lead to aneuploid oocytes. Ottolini’s team observed, for example, that some oocytes that had experienced PSSC still contained the proper number of chromosomes at the end of meiosis II. Specifically, these oocytes appeared to have completed meiosis backwards, separating sister chromatids in meiosis I and homologous chromosomes in meiosis II, as evidenced by the fact that their first polar bodies (formed during meiosis I) contained a pair of homologous chromosomes, each with just one sister chromatid. During the second meiotic division, then, the oocytes segregated those homologous chromatid pairs, resulting in a euploid cell, or one with a normal chromosome number. This phenomenon, which the authors termed “reverse segregation,” brings into question how ordered chromosome segregation actually is in human oocytes.

                              Only once scientists understand the basic machinery that controls meiosis can they develop appropriate diagnostics and inter­ventions to help women achieve pregnancies with egg cells that have properly apportioned chromosomes.

                              A similar phenomenon could also result when paired homologous chromosomes, or bivalents, separate prematurely. In the 1990s, Roslyn Angell at the University of Edinburgh examined 200 discarded oocytes from patients undergoing IVF and observed 61 cases of lone homologs (univalents) that had apparently separated precociously during metaphase of meiosis I, prior to the first meiotic cell division. 18,19 Last year, using live, high-resolution confocal microscopy to track individual kinetochores, Yogo Sakakibara in Tomoya Kitajima’s laboratory and colleagues at the RIKEN Center for Developmental Biology in Kobe, Japan, documented the same phenomenon in oocytes from young and old mice: homolog kinetochores were sometimes farther apart than normal, and this often led to univalent formation. 20

                              The resulting univalents had one of three fates during meiosis I, two of which involve unbalanced segregation: both chromatids of one univalent could segregate into one daughter cell, while the chromatids of the other homolog were separated, or all four chromatids of the two univalents could end up in the same daughter cell. Most of the time, however, the segregation was balanced, where the two sister chromatids of each homolog segregated into separate daughter cells. The resulting egg was euploid but with one sister chromatid from each homolog instead of both chromatids from a single homolog—just like the reverse segregation patterns observed by Ottolini’s team. (See illustration above.)

                              Sakakibara and colleagues also examined three human oocytes from donors over the age of 35 and again observed univalents prior to meiosis I segregation, suggesting that this separation of homologs may contribute to high rates of egg aneuploidy. But because a balanced division of the resulting univalents would result in a euploid egg, a chromosome analysis without watching the chromosome behavior would fail to detect any issue. Only through the power of live imaging can researchers detect improper, yet balanced, chromosome segregation.

                              Because these embryos are euploid, it is not known if they are developmentally equivalent to those derived from classical meiotic segregation. 21 Perhaps selection of these euploid embryos for transfer could help explain the low success rates of IVF procedures, in which fertilized eggs are screened for aneuploidy and other chromosomal abnormalities before being transplanted into the host uterus. If such reverse segregation is detrimental to the fetus, IVF screens must sample both embryos and polar bodies after fertilization to identify all cases where meiosis may have gone awry.

                              A grain of salt

                              While the study of oocytes retrieved from IVF clinics has greatly improved our understanding of mistakes that can occur during meiosis, the results must be interpreted with caution. Most of the patients have undergone hormonal stimulation to increase the number of oocytes retrieved, possibly recruiting oocytes of poorer quality. Moreover, eggs that successfully complete meiosis I are fertilized and developed into embryos, leaving those oocytes that have not yet completed meiosis I to be used for these types of studies. Therefore, it is possible that these discarded oocytes are not representative of how a healthy oocyte would behave.

                              Currently, most US states and other countries do not allow financial compensation to women to donate their oocytes for research. It is therefore rare that one would volunteer to undergo an invasive process for the sake of scientific advancement, thereby limiting the oocytes used in experiments to those from women undergoing IVF.

                              In addition to the remaining questions about how chromosomes in normal human oocytes (mis)behave, we are also left with trying to understand why. What molecular players are deficient in human oocytes compared to other organisms such as mice that have lower rates of aneuploidy? Can methods of gamete selection that aim to fertilize only the eggs that did everything right during meiosis I be improved? And is it possible to develop interventions to correct this error-prone process when patients are undergoing IVF?

                              Answering these questions will be essential for improving IVF outcomes. Hopefully, by coupling these observational experiments using human oocytes with genetic and cellular biological experiments that can be conducted in model systems, researchers in the field of human reproductive biology will soon solve these mysteries.

                              Karen Schindler is an assistant professor who studies reproductive biology in the Department of Genetics at Rutgers, The State University of New Jersey.

                              IN THE GENES
                              By Jacob Ohring

                              Although maternal age is clearly associated with the incidence of aneuploidy, it does not explain why some reproductively young women (<35 years of age) have higher than average levels of aneuploidy. Some population-based studies point to genetics as the missing link. For example, marriages between close relatives are associated with increased aneuploidy among children in specific populations. In 1970, an estimated 50 percent of all marriages among native Kuwaitis occurred between close family members, and 40 percent of non-native Kuwaitis living in the country were in familial marriages (Clin Genet, 27:483-86, 1985). Data from the 11,614 births that occurred that year in the Kuwait Obstetric Hospital supported the effects of increased maternal age, but also pointed to close kinship between the parents as causing an increase in the incidence of children born with Down syndrome. Bedouin Kuwaitis, who have higher rates of consanguineous marriages than urban Kuwaitis, had nearly double the risk of having a child with the disorder (3/1,000 births, compared with 1.6/1,000 births).

                              Analyses of Down syndrome in the U.S. between 1983 and 1990 have also linked genetics to rates of the disorder. Data from 17 state surveillance programs revealed higher rates of Down syndrome for Hispanic populations (1.8/1,000 births) than for white (0.92/1,000 births) and black populations (0.72/1,000 births), even when controlling for maternal age. The US Centers for Disease Control and Prevention blamed these discrepancies on the differential use of prenatal diagnostics, but this trend for Hispanic mothers was also identified in South American countries, where access to these services is more equal: in a remote hospital in Chile between 1997 and 2003, the prevalence of Down syndrome was 2.96/1,000 live births. These studies, and many others, support the hypothesis that some women are genetically predisposed to producing aneuploid gametes, even at a young age.

                              With the advent of embryo screening in IVF clinics, together with the decreasing costs of next-generation sequencing, it is easy to imagine that an evaluation of the genomes of patients who produce more or fewer aneuploid embryos could identify causal gene variants. In a genome-wide analysis of single nucleotide polymorphisms (SNPs) in 2,362 unrelated mothers, for example, researchers identified a region of chromosome 4 that is associated with a mistake in the first mitotic division after fertilization (Science, 348:235-38, 2015). Of the many genes contained in this region of chromosome 4, polo-like kinase 4 (PLK4) stands out as possibly important for maintaining the correct chromosome number in the developing embryo, as it is known to regulate spindle formation in other cell types. This functional connection has yet to be tested, however, and until more studies are conducted, the scientific community remains largely in the dark about the genes that underlie gamete quality.

                              Jacob Ohring is an undergraduate genetics major at Rutgers University.


                              Acknowledgments

                              We thank Alastair Simpson, Gordon Lax and Julius Lukeš for providing access to Euglenida, Diplonemida and/or Kinetoplastida transcriptomes and genomes before publication Svenja Hester in the Advanced Proteomics Facility for processing mass spectrometry samples Shabaz Mohammed for advice on crosslinking mass spectrometry and Keith Gull for providing an original electron micrograph of T. brucei kinetochores. We also thank Kim Nasmyth and David Sherratt for discussion. We thank members of the Akiyoshi and Waller labs for feedback.


                              Repurposing of Synaptonemal Complex Proteins for Kinetochores in Kinetoplastida

                              Kinetochores are macromolecular protein complexes that bring about the segregation of chromosomes during cell division. Kinetochores vary across eukaryotes in protein composition and sequence1. A striking example of divergence is seen in Kinetoplastida. Kinetoplastida are a group of unicellular flagellates including parasites like Trypanosoma and Leishmania spp. within the phylum Euglenozoa. Trypanosoma has a kinetochore made up of KKT/KKIP proteins functionally analogous to the canonical kinetochore found in model organisms, but lacks any direct homologs of its components. Why and how do proteins with such apparently critical cellular functions evolve so rapidly? More specifically, how did this novel/unique kinetoplastid kinetochore emerge? Were they acquired from endosymbionts or did they evolve by repurposing other cellular machinery?

                              Key findings and future directions

                              The canonical kinetochore has a mosaic origin, with many components sharing their ancestry with proteins performing a range of functions that include vesicular transport, DNA replication and repair and transcription 2 . Could this also be true for the kinetoplastids? While it was not possible to identify kinetochore protein homologs, many of the KKT proteins had generic domains shared with proteins involved in homologous recombination, DNA repair and cell cycle regulators like Polo kinases. This provided a clue about what might be going on in Kinetoplastids. Interestingly, KKT17/18, two components of the three member KKT16 subcomplex, that likely emerged through gene duplication, displayed a unique domain topology of Armadillo repeats (ARM) with a Pleckstrin homology (PH) domain followed by coiled coils. This domain arrangement is only found in SYCP2, a protein of the synaptonemal complex that links chromosomes during meiotic homologous recombination suggesting a shared evolutionary history and repurposing of meiotic components for different cellular functions in mitosis.

                              Figure 1: Evolutionary scenario of SYCP2-3 proteins (Figure 5 from preprint, provided under CC BY 4.0 International License)

                              Using an iterative alignment method that merged clade-specific alignments into a super alignment, the authors identified SYCP 2-3 family proteins that were normally difficult to detect or establish relationships with using only metazoan references across eukaryotes, including Giardia, microsporidia and diverse fungi, suggesting an ancient origin of these kinetoplastid kinetochores. Further, most lineages that lacked any SYCP 2-3 also have not revealed any synaptonemal complex structures. Therefore, the presence of SYCP 2-3 in a proteome could be a potential indicator of the presence of the synaptonemal complex in the organism.

                              While this gives us a glimpse into the evolutionary history of this unique kinetochore, its relationship with the other present day eukaryotic and ancient kinetochores is still unclear. Did the early kinetoplastids replace their canonical kinetochores with this new one? Or did early eukaryotes have a kinetochore different from all systems present today, that diverged along these many different paths? Well, only more such studies within the euglenozoa on close relatives like Diplonemida, and across eukaryotes more broadly, will provide a better picture of the ancestry of this unique and essential kinetochore complex.


                              Contents

                              Although the process of meiosis is related to the more general cell division process of mitosis, it differs in two important respects:

                              usually occurs between identical sister chromatids and does not result in genetic changes

                              Meiosis begins with a diploid cell, which contains two copies of each chromosome, termed homologs. First, the cell undergoes DNA replication, so each homolog now consists of two identical sister chromatids. Then each set of homologs pair with each other and exchange genetic information by homologous recombination often leading to physical connections (crossovers) between the homologs. In the first meiotic division, the homologs are segregated to separate daughter cells by the spindle apparatus. The cells then proceed to a second division without an intervening round of DNA replication. The sister chromatids are segregated to separate daughter cells to produce a total of four haploid cells. Female animals employ a slight variation on this pattern and produce one large ovum and two small polar bodies. Because of recombination, an individual chromatid can consist of a new combination of maternal and paternal genetic information, resulting in offspring that are genetically distinct from either parent. Furthermore, an individual gamete can include an assortment of maternal, paternal, and recombinant chromatids. This genetic diversity resulting from sexual reproduction contributes to the variation in traits upon which natural selection can act.

                              Meiosis uses many of the same mechanisms as mitosis, the type of cell division used by eukaryotes to divide one cell into two identical daughter cells. In some plants, fungi, and protists meiosis results in the formation of spores: haploid cells that can divide vegetatively without undergoing fertilization. Some eukaryotes, like bdelloid rotifers, do not have the ability to carry out meiosis and have acquired the ability to reproduce by parthenogenesis.

                              Meiosis does not occur in archaea or bacteria, which generally reproduce asexually via binary fission. However, a "sexual" process known as horizontal gene transfer involves the transfer of DNA from one bacterium or archaeon to another and recombination of these DNA molecules of different parental origin.

                              Meiosis was discovered and described for the first time in sea urchin eggs in 1876 by the German biologist Oscar Hertwig. It was described again in 1883, at the level of chromosomes, by the Belgian zoologist Edouard Van Beneden, in Ascaris roundworm eggs. The significance of meiosis for reproduction and inheritance, however, was described only in 1890 by German biologist August Weismann, who noted that two cell divisions were necessary to transform one diploid cell into four haploid cells if the number of chromosomes had to be maintained. In 1911, the American geneticist Thomas Hunt Morgan detected crossovers in meiosis in the fruit fly Drosophila melanogaster, which helped to establish that genetic traits are transmitted on chromosomes.

                              The term "meiosis" is derived from the Greek word μείωσις , meaning 'lessening'. It was introduced to biology by J.B. Farmer and J.E.S. Moore in 1905, using the idiosyncratic rendering "maiosis":

                              We propose to apply the terms Maiosis or Maiotic phase to cover the whole series of nuclear changes included in the two divisions that were designated as Heterotype and Homotype by Flemming. [8]

                              The spelling was changed to "meiosis" by Koernicke (1905) and by Pantel and De Sinety (1906) to follow the usual conventions for transliterating Greek. [9]

                              Meiosis is divided into meiosis I and meiosis II which are further divided into Karyokinesis I and Cytokinesis I and Karyokinesis II and Cytokinesis II respectively. The preparatory steps that lead up to meiosis are identical in pattern and name to interphase of the mitotic cell cycle. [10] Interphase is divided into three phases:

                                : In this very active phase, the cell synthesizes its vast array of proteins, including the enzymes and structural proteins it will need for growth. In G1, each of the chromosomes consists of a single linear molecule of DNA. : The genetic material is replicated each of the cell's chromosomes duplicates to become two identical sister chromatids attached at a centromere. This replication does not change the ploidy of the cell since the centromere number remains the same. The identical sister chromatids have not yet condensed into the densely packaged chromosomes visible with the light microscope. This will take place during prophase I in meiosis. : G2 phase as seen before mitosis is not present in meiosis. Meiotic prophase corresponds most closely to the G2 phase of the mitotic cell cycle.

                              Interphase is followed by meiosis I and then meiosis II. Meiosis I separates replicated homologous chromosomes, each still made up of two sister chromatids, into two daughter cells, thus reducing the chromosome number by half. During meiosis II, sister chromatids decouple and the resultant daughter chromosomes are segregated into four daughter cells. For diploid organisms, the daughter cells resulting from meiosis are haploid and contain only one copy of each chromosome. In some species, cells enter a resting phase known as interkinesis between meiosis I and meiosis II.

                              Meiosis I and II are each divided into prophase, metaphase, anaphase, and telophase stages, similar in purpose to their analogous subphases in the mitotic cell cycle. Therefore, meiosis includes the stages of meiosis I (prophase I, metaphase I, anaphase I, telophase I) and meiosis II (prophase II, metaphase II, anaphase II, telophase II).

                              During meiosis, specific genes are more highly transcribed. [11] [12] In addition to strong meiotic stage-specific expression of mRNA, there are also pervasive translational controls (e.g. selective usage of preformed mRNA), regulating the ultimate meiotic stage-specific protein expression of genes during meiosis. [13] Thus, both transcriptional and translational controls determine the broad restructuring of meiotic cells needed to carry out meiosis.

                              Meiosis I Edit

                              Meiosis I segregates homologous chromosomes, which are joined as tetrads (2n, 4c), producing two haploid cells (n chromosomes, 23 in humans) which each contain chromatid pairs (1n, 2c). Because the ploidy is reduced from diploid to haploid, meiosis I is referred to as a reductional division. Meiosis II is an equational division analogous to mitosis, in which the sister chromatids are segregated, creating four haploid daughter cells (1n, 1c). [14]

                              Prophase I Edit

                              Prophase I is by far the longest phase of meiosis (lasting 13 out of 14 days in mice [15] ). During prophase I, homologous maternal and paternal chromosomes pair, synapse, and exchange genetic information (by homologous recombination), forming at least one crossover per chromosome. [16] These crossovers become visible as chiasmata (plural singular chiasma). [17] This process facilitates stable pairing between homologous chromosomes and hence enables accurate segregation of the chromosomes at the first meiotic division. The paired and replicated chromosomes are called bivalents (two chromosomes) or tetrads (four chromatids), with one chromosome coming from each parent. Prophase I is divided into a series of substages which are named according to the appearance of chromosomes.

                              Leptotene Edit

                              The first stage of prophase I is the leptotene stage, also known as leptonema, from Greek words meaning "thin threads". [18] : 27 In this stage of prophase I, individual chromosomes—each consisting of two replicated sister chromatids—become "individualized" to form visible strands within the nucleus. [18] : 27 [19] : 353 The chromosomes each form a linear array of loops mediated by cohesin, and the lateral elements of the synaptonemal complex assemble forming an "axial element" from which the loops emanate. [20] Recombination is initiated in this stage by the enzyme SPO11 which creates programmed double strand breaks (around 300 per meiosis in mice). [21] This process generates single stranded DNA filaments coated by RAD51 and DMC1 which invade the homologous chromosomes, forming inter-axis bridges, and resulting in the pairing/co-alignment of homologues (to a distance of

                              Zygotene Edit

                              Leptotene is followed by the zygotene stage, also known as zygonema, from Greek words meaning "paired threads", [18] : 27 which in some organisms is also called the bouquet stage because of the way the telomeres cluster at one end of the nucleus. [23] In this stage the homologous chromosomes become much more closely (

                              100 nm) and stably paired (a process called synapsis) mediated by the installation of the transverse and central elements of the synaptonemal complex. [20] Synapsis is thought to occur in a zipper-like fashion starting from a recombination nodule. The paired chromosomes are called bivalent or tetrad chromosomes.

                              Pachytene Edit

                              The pachytene stage ( / ˈ p æ k ɪ t iː n / PAK -i-teen), also known as pachynema, from Greek words meaning "thick threads". [18] : 27 is the stage at which all autosomal chromosomes have synapsed. In this stage homologous recombination, including chromosomal crossover (crossing over), is completed through the repair of the double strand breaks formed in leptotene. [20] Most breaks are repaired without forming crossovers resulting in gene conversion. [24] However, a subset of breaks (at least one per chromosome) form crossovers between non-sister (homologous) chromosomes resulting in the exchange of genetic information. [25] Sex chromosomes, however, are not wholly identical, and only exchange information over a small region of homology called the pseudoautosomal region. [26] The exchange of information between the homologous chromatids results in a recombination of information each chromosome has the complete set of information it had before, and there are no gaps formed as a result of the process. Because the chromosomes cannot be distinguished in the synaptonemal complex, the actual act of crossing over is not perceivable through an ordinary light microscope, and chiasmata are not visible until the next stage.

                              Diplotene Edit

                              During the diplotene stage, also known as diplonema, from Greek words meaning "two threads", [18] : 30 the synaptonemal complex disassembles and homologous chromosomes separate from one another a little. However, the homologous chromosomes of each bivalent remain tightly bound at chiasmata, the regions where crossing-over occurred. The chiasmata remain on the chromosomes until they are severed at the transition to anaphase I to allow homologous chromosomes to move to opposite poles of the cell.

                              In human fetal oogenesis, all developing oocytes develop to this stage and are arrested in prophase I before birth. [27] This suspended state is referred to as the dictyotene stage or dictyate. It lasts until meiosis is resumed to prepare the oocyte for ovulation, which happens at puberty or even later.

                              Diakinesis Edit

                              Chromosomes condense further during the diakinesis stage, from Greek words meaning "moving through". [18] : 30 This is the first point in meiosis where the four parts of the tetrads are actually visible. Sites of crossing over entangle together, effectively overlapping, making chiasmata clearly visible. Other than this observation, the rest of the stage closely resembles prometaphase of mitosis the nucleoli disappear, the nuclear membrane disintegrates into vesicles, and the meiotic spindle begins to form.

                              Meiotic spindle formation Edit

                              Unlike mitotic cells, human and mouse oocytes do not have centrosomes to produce the meiotic spindle. In mice, approximately 80 MicroTubule Organizing Centers (MTOCs) form a sphere in the ooplasm and begin to nucleate microtubules that reach out towards chromosomes, attaching to the chromosomes at the kinetochore. Over time the MTOCs merge until two poles have formed, generating a barrel shaped spindle. [28] In human oocytes spindle microtubule nucleation begins on the chromosomes, forming an aster that eventually expands to surround the chromosomes. [29] Chromosomes then slide along the microtubules towards the equator of the spindle, at which point the chromosome kinetochores form end-on attachments to microtubules. [30]

                              Metaphase I Edit

                              Homologous pairs move together along the metaphase plate: As kinetochore microtubules from both spindle poles attach to their respective kinetochores, the paired homologous chromosomes align along an equatorial plane that bisects the spindle, due to continuous counterbalancing forces exerted on the bivalents by the microtubules emanating from the two kinetochores of homologous chromosomes. This attachment is referred to as a bipolar attachment. The physical basis of the independent assortment of chromosomes is the random orientation of each bivalent along the metaphase plate, with respect to the orientation of the other bivalents along the same equatorial line. [17] The protein complex cohesin holds sister chromatids together from the time of their replication until anaphase. In mitosis, the force of kinetochore microtubules pulling in opposite directions creates tension. The cell senses this tension and does not progress with anaphase until all the chromosomes are properly bi-oriented. In meiosis, establishing tension ordinarily requires at least one crossover per chromosome pair in addition to cohesin between sister chromatids (see Chromosome segregation).

                              Anaphase I Edit

                              Kinetochore microtubules shorten, pulling homologous chromosomes (which each consist of a pair of sister chromatids) to opposite poles. Nonkinetochore microtubules lengthen, pushing the centrosomes farther apart. The cell elongates in preparation for division down the center. [17] Unlike in mitosis, only the cohesin from the chromosome arms is degraded while the cohesin surrounding the centromere remains protected by a protein named Shugoshin (Japanese for "guardian spirit"), what prevents the sister chromatids from separating. [31] This allows the sister chromatids to remain together while homologs are segregated.

                              Telophase I Edit

                              The first meiotic division effectively ends when the chromosomes arrive at the poles. Each daughter cell now has half the number of chromosomes but each chromosome consists of a pair of chromatids. The microtubules that make up the spindle network disappear, and a new nuclear membrane surrounds each haploid set. The chromosomes uncoil back into chromatin. Cytokinesis, the pinching of the cell membrane in animal cells or the formation of the cell wall in plant cells, occurs, completing the creation of two daughter cells. However, cytokinesis does not fully complete resulting in "cytoplasmic bridges" which enable the cytoplasm to be shared between daughter cells until the end of meiosis II. [32] Sister chromatids remain attached during telophase I.

                              Cells may enter a period of rest known as interkinesis or interphase II. No DNA replication occurs during this stage.

                              Meiosis II Edit

                              Meiosis II is the second meiotic division, and usually involves equational segregation, or separation of sister chromatids. Mechanically, the process is similar to mitosis, though its genetic results are fundamentally different. The end result is production of four haploid cells (n chromosomes, 23 in humans) from the two haploid cells (with n chromosomes, each consisting of two sister chromatids) produced in meiosis I. The four main steps of meiosis II are: prophase II, metaphase II, anaphase II, and telophase II.

                              In prophase II, we see the disappearance of the nucleoli and the nuclear envelope again as well as the shortening and thickening of the chromatids. Centrosomes move to the polar regions and arrange spindle fibers for the second meiotic division.

                              In metaphase II, the centromeres contain two kinetochores that attach to spindle fibers from the centrosomes at opposite poles. The new equatorial metaphase plate is rotated by 90 degrees when compared to meiosis I, perpendicular to the previous plate. [33]

                              This is followed by anaphase II, in which the remaining centromeric cohesin, not protected by Shugoshin anymore, is cleaved, allowing the sister chromatids to segregate. The sister chromatids by convention are now called sister chromosomes as they move toward opposing poles. [31]

                              The process ends with telophase II, which is similar to telophase I, and is marked by decondensation and lengthening of the chromosomes and the disassembly of the spindle. Nuclear envelopes re-form and cleavage or cell plate formation eventually produces a total of four daughter cells, each with a haploid set of chromosomes.

                              Meiosis is now complete and ends up with four new daughter cells.

                              The origin and function of meiosis are currently not well understood scientifically, and would provide fundamental insight into the evolution of sexual reproduction in eukaryotes. There is no current consensus among biologists on the questions of how sex in eukaryotes arose in evolution, what basic function sexual reproduction serves, and why it is maintained, given the basic two-fold cost of sex. It is clear that it evolved over 1.2 billion years ago, and that almost all species which are descendants of the original sexually reproducing species are still sexual reproducers, including plants, fungi, and animals.

                              Meiosis is a key event of the sexual cycle in eukaryotes. It is the stage of the life cycle when a cell gives rise to haploid cells (gametes) each having half as many chromosomes as the parental cell. Two such haploid gametes, ordinarily arising from different individual organisms, fuse by the process of fertilization, thus completing the sexual cycle.

                              Meiosis is ubiquitous among eukaryotes. It occurs in single-celled organisms such as yeast, as well as in multicellular organisms, such as humans. Eukaryotes arose from prokaryotes more than 2.2 billion years ago [34] and the earliest eukaryotes were likely single-celled organisms. To understand sex in eukaryotes, it is necessary to understand (1) how meiosis arose in single celled eukaryotes, and (2) the function of meiosis.

                              The new combinations of DNA created during meiosis are a significant source of genetic variation alongside mutation, resulting in new combinations of alleles, which may be beneficial. Meiosis generates gamete genetic diversity in two ways: (1) Law of Independent Assortment. The independent orientation of homologous chromosome pairs along the metaphase plate during metaphase I and orientation of sister chromatids in metaphase II, this is the subsequent separation of homologs and sister chromatids during anaphase I and II, it allows a random and independent distribution of chromosomes to each daughter cell (and ultimately to gametes) [35] and (2) Crossing Over. The physical exchange of homologous chromosomal regions by homologous recombination during prophase I results in new combinations of genetic information within chromosomes. [36]

                              Prophase I arrest Edit

                              Female mammals and birds are born possessing all the oocytes needed for future ovulations, and these oocytes are arrested at the prophase I stage of meiosis. [37] In humans, as an example, oocytes are formed between three and four months of gestation within the fetus and are therefore present at birth. During this prophase I arrested stage (dictyate), which may last for decades, four copies of the genome are present in the oocytes. The arrest of ooctyes at the four genome copy stage was proposed to provide the informational redundancy needed to repair damage in the DNA of the germline. [37] The repair process used appears to involve homologous recombinational repair [37] [38] Prophase I arrested oocytes have a high capability for efficient repair of DNA damages, particularly exogenously induced double-strand breaks. [38] DNA repair capability appears to be a key quality control mechanism in the female germ line and a critical determinant of fertility. [38]

                              In life cycles Edit

                              Meiosis occurs in eukaryotic life cycles involving sexual reproduction, consisting of the constant cyclical process of meiosis and fertilization. This takes place alongside normal mitotic cell division. In multicellular organisms, there is an intermediary step between the diploid and haploid transition where the organism grows. At certain stages of the life cycle, germ cells produce gametes. Somatic cells make up the body of the organism and are not involved in gamete production.

                              Cycling meiosis and fertilization events produces a series of transitions back and forth between alternating haploid and diploid states. The organism phase of the life cycle can occur either during the diploid state (diplontic life cycle), during the haploid state (haplontic life cycle), or both (haplodiplontic life cycle, in which there are two distinct organism phases, one during the haploid state and the other during the diploid state). In this sense there are three types of life cycles that utilize sexual reproduction, differentiated by the location of the organism phase(s). [ citation needed ]

                              In the diplontic life cycle (with pre-gametic meiosis), of which humans are a part, the organism is diploid, grown from a diploid cell called the zygote. The organism's diploid germ-line stem cells undergo meiosis to create haploid gametes (the spermatozoa for males and ova for females), which fertilize to form the zygote. The diploid zygote undergoes repeated cellular division by mitosis to grow into the organism.

                              In the haplontic life cycle (with post-zygotic meiosis), the organism is haploid instead, spawned by the proliferation and differentiation of a single haploid cell called the gamete. Two organisms of opposing sex contribute their haploid gametes to form a diploid zygote. The zygote undergoes meiosis immediately, creating four haploid cells. These cells undergo mitosis to create the organism. Many fungi and many protozoa utilize the haplontic life cycle. [ citation needed ]

                              Finally, in the haplodiplontic life cycle (with sporic or intermediate meiosis), the living organism alternates between haploid and diploid states. Consequently, this cycle is also known as the alternation of generations. The diploid organism's germ-line cells undergo meiosis to produce spores. The spores proliferate by mitosis, growing into a haploid organism. The haploid organism's gamete then combines with another haploid organism's gamete, creating the zygote. The zygote undergoes repeated mitosis and differentiation to become a diploid organism again. The haplodiplontic life cycle can be considered a fusion of the diplontic and haplontic life cycles. [39] [ citation needed ]

                              In plants and animals Edit

                              Meiosis occurs in all animals and plants. The end result, the production of gametes with half the number of chromosomes as the parent cell, is the same, but the detailed process is different. In animals, meiosis produces gametes directly. In land plants and some algae, there is an alternation of generations such that meiosis in the diploid sporophyte generation produces haploid spores. These spores multiply by mitosis, developing into the haploid gametophyte generation, which then gives rise to gametes directly (i.e. without further meiosis). In both animals and plants, the final stage is for the gametes to fuse, restoring the original number of chromosomes. [40]

                              In mammals Edit

                              In females, meiosis occurs in cells known as oocytes (singular: oocyte). Each primary oocyte divides twice in meiosis, unequally in each case. The first division produces a daughter cell, and a much smaller polar body which may or may not undergo a second division. In meiosis II, division of the daughter cell produces a second polar body, and a single haploid cell, which enlarges to become an ovum. Therefore, in females each primary oocyte that undergoes meiosis results in one mature ovum and one or two polar bodies.

                              Note that there are pauses during meiosis in females. Maturing oocytes are arrested in prophase I of meiosis I and lie dormant within a protective shell of somatic cells called the follicle. At the beginning of each menstrual cycle, FSH secretion from the anterior pituitary stimulates a few follicles to mature in a process known as folliculogenesis. During this process, the maturing oocytes resume meiosis and continue until metaphase II of meiosis II, where they are again arrested just before ovulation. If these oocytes are fertilized by sperm, they will resume and complete meiosis. During folliculogenesis in humans, usually one follicle becomes dominant while the others undergo atresia. The process of meiosis in females occurs during oogenesis, and differs from the typical meiosis in that it features a long period of meiotic arrest known as the dictyate stage and lacks the assistance of centrosomes. [41] [42]

                              In males, meiosis occurs during spermatogenesis in the seminiferous tubules of the testicles. Meiosis during spermatogenesis is specific to a type of cell called spermatocytes, which will later mature to become spermatozoa. Meiosis of primordial germ cells happens at the time of puberty, much later than in females. Tissues of the male testis suppress meiosis by degrading retinoic acid, proposed to be a stimulator of meiosis. This is overcome at puberty when cells within seminiferous tubules called Sertoli cells start making their own retinoic acid. Sensitivity to retinoic acid is also adjusted by proteins called nanos and DAZL. [43] [44] Genetic loss-of-function studies on retinoic acid-generating enzymes have shown that retinoic acid is required postnatally to stimulate spermatogonia differentiation which results several days later in spermatocytes undergoing meiosis, however retinoic acid is not required during the time when meiosis initiates. [45]

                              In female mammals, meiosis begins immediately after primordial germ cells migrate to the ovary in the embryo. Some studies suggest that retinoic acid derived from the primitive kidney (mesonephros) stimulates meiosis in embryonic ovarian oogonia and that tissues of the embryonic male testis suppress meiosis by degrading retinoic acid. [46] However, genetic loss-of-function studies on retinoic acid-generating enzymes have shown that retinoic acid is not required for initiation of either female meiosis which occurs during embryogenesis [47] or male meiosis which initiates postnatally. [45]

                              Flagellates Edit

                              While the majority of eukaryotes have a two-divisional meiosis (though sometimes achiasmatic), a very rare form, one-divisional meiosis, occurs in some flagellates (parabasalids and oxymonads) from the gut of the wood-feeding cockroach Cryptocercus. [48]

                              Recombination among the 23 pairs of human chromosomes is responsible for redistributing not just the actual chromosomes, but also pieces of each of them. There is also an estimated 1.6-fold more recombination in females relative to males. In addition, average, female recombination is higher at the centromeres and male recombination is higher at the telomeres. On average, 1 million bp (1 Mb) correspond to 1 cMorgan (cm = 1% recombination frequency). [49] The frequency of cross-overs remain uncertain. In yeast, mouse and human, it has been estimated that ≥200 double-strand breaks (DSBs) are formed per meiotic cell. However, only a subset of DSBs (

                              5–30% depending on the organism), go on to produce crossovers, [50] which would result in only 1-2 cross-overs per human chromosome.

                              Nondisjunction Edit

                              The normal separation of chromosomes in meiosis I or sister chromatids in meiosis II is termed disjunction. When the segregation is not normal, it is called nondisjunction. This results in the production of gametes which have either too many or too few of a particular chromosome, and is a common mechanism for trisomy or monosomy. Nondisjunction can occur in the meiosis I or meiosis II, phases of cellular reproduction, or during mitosis.

                              Most monosomic and trisomic human embryos are not viable, but some aneuploidies can be tolerated, such as trisomy for the smallest chromosome, chromosome 21. Phenotypes of these aneuploidies range from severe developmental disorders to asymptomatic. Medical conditions include but are not limited to:

                                – trisomy of chromosome 21 – trisomy of chromosome 13 – trisomy of chromosome 18 – extra X chromosomes in males – i.e. XXY, XXXY, XXXXY, etc. – lacking of one X chromosome in females – i.e. X0 – an extra X chromosome in females – an extra Y chromosome in males.

                              The probability of nondisjunction in human oocytes increases with increasing maternal age, [51] presumably due to loss of cohesin over time. [52]

                              In order to understand meiosis, a comparison to mitosis is helpful. The table below shows the differences between meiosis and mitosis. [53]

                              Meiosis Mitosis
                              End result Normally four cells, each with half the number of chromosomes as the parent Two cells, having the same number of chromosomes as the parent
                              Function Production of gametes (sex cells) in sexually reproducing eukaryotes with diplont life cycle Cellular reproduction, growth, repair, asexual reproduction
                              Where does it happen? Almost all eukaryotes (animals, plants, fungi, and protists) [54] [48]
                              In gonads, before gametes (in diplontic life cycles)
                              After zygotes (in haplontic)
                              Before spores (in haplodiplontic)
                              All proliferating cells in all eukaryotes
                              Steps Prophase I, Metaphase I, Anaphase I, Telophase I,
                              Prophase II, Metaphase II, Anaphase II, Telophase II
                              Prophase, Prometaphase, Metaphase, Anaphase, Telophase
                              Genetically same as parent? No Yes
                              Crossing over happens? Yes, normally occurs between each pair of homologous chromosomes Very rarely
                              Pairing of homologous chromosomes? Yes No
                              Cytokinesis Occurs in Telophase I and Telophase II Occurs in Telophase
                              Centromeres split Does not occur in Anaphase I, but occurs in Anaphase II Occurs in Anaphase

                              How a cell proceeds to meiotic division in meiotic cell division is not well known. Maturation promoting factor (MPF) seemingly have role in frog Oocyte meiosis. In the fungus S. pombe. there is a role of MeiRNA binding protein for entry to meiotic cell division. [55]

                              It has been suggested that Yeast CEP1 gene product, that binds centromeric region CDE1, may play a role in chromosome pairing during meiosis-I. [56]

                              Meiotic recombination is mediated through double stranded break, which is catalyzed by Spo11 protein. Also Mre11, Sae2 and Exo1 play role in breakage and recombination. After the breakage happen, recombination take place which is typically homologous. The recombination may go through either a double Holliday junction (dHJ) pathway or synthesis-dependent strand annealing (SDSA). (The second one gives to noncrossover product). [57]

                              Seemingly there are checkpoints for meiotic cell division too. In S. pombe, Rad proteins, S. pombe Mek1 (with FHA kinase domain), Cdc25, Cdc2 and unknown factor is thought to form a checkpoint. [58]

                              In vertebrate oogenesis, maintained by cytostatic factor (CSF) has role in switching into meiosis-II. [56]


                              Introduction

                              Cells employ many mechanisms to ensure that their genomes are replicated and segregated with high fidelity every cell cycle [1]. Errors in chromosome segregation result in aneuploidy, which often leads to cell death and is strongly associated with cancer progression [2], [3]. During mitosis the spindle checkpoint monitors kinetochore-microtubule interactions, and only when all sister-chromatid pairs have achieved bi-orientation on the mitotic spindle is anaphase allowed to proceed. This checkpoint inhibits the activity of the anaphase-promoting complex (Cdc20-APC), preventing polyubiquitination and destruction of mitotic regulators such as securin and cyclin, and thereby delays anaphase onset [4], [5].

                              The molecular mechanism of action of the spindle checkpoint remains unclear, although several important findings have been made. First, a single unattached kinetochore is sufficient to activate the checkpoint [6]. Second, all of the checkpoint proteins are recruited to unattached kinetochores, as is their effector Cdc20 [7]–[10]. Third, a sub-set of checkpoint proteins, including Mad2 and BubR1/Mad3, form stable complexes with Cdc20 [11]–[13], which is the critical effector of the spindle checkpoint [14], [15]. Such checkpoint protein complexes are sufficient to inhibit Cdc20-APC activity in vitro [13], [16], [17].

                              Here we focus on the mechanism of recruitment of checkpoint proteins to kinetochores, and their exchange dynamics once recruited. Several fluorescence recovery after photo-bleaching (FRAP) studies have described the dynamics of spindle checkpoint proteins and Cdc20 in vertebrate cells [7]–[10]. These employed either transient transfection of GFP tagged checkpoint constructs, or the production of stable cell lines expressing fusion proteins, and in all cases the cell lines also contained the endogenous wild-type checkpoint protein. This is a major limitation of these studies as it is possible that the GFP fusion proteins do not reflect the behaviour of the wild-type protein. In addition to the possibility that the GFP tag perturbs function, the endogenous protein could out-compete the GFP fusion protein for binding sites on chromosomes. If these were rare and/or stable binding sites, this would have a major influence on the dynamic parameters measured. Vink et al have reconstituted dynamic aspects of Mad2 behaviour in vitro, using Mad1/Mad2 “scaffolds” coupled to beads [18]. These FRAP studies demonstrate that Mad2 behaviour is rather complex: there is a stable kinetochore-bound pool of Mad2, tightly bound to Mad1, and a dynamic Mad2 pool that rapidly exchanges. In kinetochore FRAP experiments, 50�% of Mad2 recovers after the first bleach (the dynamic pool) with a half-time of 6� seconds (see [18] for Tables comparing different kinetic analyses). This dynamic exchange of Mad2 molecules is thought to be critical for Cdc20 interaction and inhibition [19], [20]. As yet, no in vitro work has been reported for BubR1/Mad3, Bub3 or Bub1 dynamics.

                              In fission yeast, Bub1p is necessary for the efficient recruitment of Bub3p and Mad3p to kinetochores, and their targeting is independent of Mad1p and Mad2p [21]. Mutations within the highly conserved N-terminal domain of Bub1p dramatically reduced its own kinetochore targeting, and that of Bub3p, and practically abolished Mad3p kinetochore enrichment [21], [22]. Thus both Bub1p and Mad1p are thought to be kinetochore-based checkpoint scaffolds. Here we demonstrate that Bub1p is a relatively stable component of mitotic kinetochores in fission yeast, and that when ectopically targeted to telomeres it is sufficient to recruit both Bub3p and Mad3p to these ectopic sites on chromosomes.


                              Why Do We See Green?

                              We may grumble about the rain but its one of the reasons Ireland has a vibrant green landscape earning it the name of the Emerald Isle. The North Atlantic drift keeps the climate temperate, offloads a lot of rain and keeps our vegetation varied and lush. Beyond this however I wondered why there are so many shades of green and endeavoured to find out. Why don’t we speak of shades of red or blue? (Fifty shades of grey of course is an entirely a different matter). The answer is science!

                              Visible light is really electromagnetic radiation with wavelengths between infrared and ultraviolet. Objects absorb varying amounts of light radiation and anything which is not absorbed reflects back off the object and is interpreted by our eyes as the different hues of green and other colours. Saturation and intensity also play a role in the different appearances of green. Every photographer knows that the amount of ambient light drastically changes a subject. In certain light conditions the same plant can appear a brighter or a darker shade. The plant itself may not have changed composition but the difference in sunlight or artificial light available will be interpreted differently by our eyes. Try looking at a houseplant in the dark and then again in the light and note the difference. Saturation varies depending on light intensity and the distribution of wavelengths. A colour will appear more saturated if there are fewer wavelengths at a high intensity of light. The colours we see are therefore influenced by a range of various factors.

                              Chlorophyll is essential for photosynthesis (the conversion of light energy to chemical energy which allows plants to survive) and is a key component of plants. Chlorophyll absorbs the red and blue light energy for photosynthesis but absorbs very little of the green light spectrum. Unabsorbed green light is reflected away and interpreted by our eyes as colour. Our retinas have 120 millions rod cells and 6-7 million cones cells. Each cone cell has the ability to detect 100 shades. The cone cells are responsible for colour vision and are most sensitive to wavelengths of light around 550 nanometers (nm) at the centre of our range of vision. This just happens to be where green is situated in the spectrum. In our early evolutionary history humans may have developed higher sensitivity to various shades of green to help us better identify danger or prey through the predominantly green landscape.

                              Plants have more than just chlorophyll in them however and these other molecules such as carotenoid and anthocyanin absorb and reflect light at different wavelengths than chlorophyll does. The reflected light from the other molecules mixes in with reflected light from the chlorophyll and influences the shade of green we perceive. Mostly the intensity of chlorophyll in plants overpowers the reflection of light from these other components and we see a predominantly green colour. There are different varieties of chlorophyll that result in slightly different wavelengths of green light reflecting back. In autumn, leaves of trees produce a lot less chlorophyll and the remaining molecules dominate the colour the leaves bounce back to our eyes appearing as beautiful reds, oranges and browns.

                              So now you know how why there are so many shades of green in Ireland you can enjoy them all the more this St. Patrick’s Day!


                              Tell us about some of the amazing applications being researched, from possible cures for obesity and even autism.

                              Many scientists have looked at how changes in the microbiome affect our risk of disease. There’s now a long laundry list of conditions and disorders that have been linked to changes in the microbiome, from diabetes to arterial sclerosis or colorectal cancer. It’s still unclear in many of these cases whether changes in the community of microbes lead to the conditions or whether it’s the reverse, or both, or neither. But there are many studies that suggest microbes are contributing to the development of these disorders. The reason scientists are so excited about this is that, if it really is an important contributor to these diseases and disorders, then it is a lever upon which we can push in order to improve our health.


                              Depression Essential Reads

                              Why Our Closest Family Relationships Can Lead to Depression

                              New Studies Link Excessive Facebook Use to Depression

                              Clinical experience raises the possibility that just learning to spot and challenge an individual negative thought may not be a very effective means to stop rumination. From my own clinical practice, I have often observed that successfully challenging a single negative thought has little overall impact for people who ruminate, as that thought is almost always followed by a further stream of negative thoughts. It is like catching a single drop of water when being hit by a deluge.

                              Yet there is a paradox to be resolved with respect to rumination. Thinking a lot about personal difficulties, setbacks, and losses isn't necessarily a "bad" behavior. More often than not it is a normal and adaptive response. When any of us experience an unexpected setback—the end of a relationship, becoming unemployed—it is natural enough to try and make sense of what happened by thinking it through and looking at our options. Further, there is an extensive literature in psychology on the value of coming to terms with emotional events by repeatedly thinking these events through (a mental ability known as cognitive and emotional processing).

                              Initially, this literature seems at odds with the research on depressive rumination because it suggests that thinking about upsetting events can be helpful. The best example of this is grieving: Part of the process of accepting a loss involves thinking about and mourning the deceased person.

                              People are likely to think about difficult events that happen to them. Sometimes this seems to be helpful at other times, too much of this thinking might increase the risk of becoming—or staying—depressed. Thus, rather than asking whether repetitive thinking itself is helpful or unhelpful, the more pertinent question may be what factors determine whether repetitive thinking is helpful or unhelpful, and how does such thinking go wrong in depression?

                              We are beginning to get some answers to these questions, and this will be the topic of a future post.


                              Watch the video: Chromosome and Kinetochore (February 2023).