Overview of Mitosis and Its Stages

Mitosis is the nuclear division phase of the eukaryotic The Cell Cycle and Its Phases, occurring in the M phase after the genome has already been duplicated during S phase. At the start of mitosis, every chromosome exists as two identical sister chromatids, produced by semiconservative DNA replication and held together along their length by a ring-shaped protein complex called cohesin. The entire purpose of mitosis is to resolve this doubled structure: to separate every pair of sister chromatids and deliver exactly one copy of each chromosome to each of the two forming daughter nuclei. This is distinct from meiosis, which additionally separates homologous chromosome pairs and halves the chromosome number for sexual reproduction — mitosis preserves the chromosome number. Traditionally mitosis is divided into five sequential stages defined by what one can see under a light microscope: prophase, prometaphase, metaphase, anaphase, and telophase. These are not arbitrary labels; each stage corresponds to a distinct, checkpoint-monitored molecular state. Prophase is chromosome condensation and spindle nucleation. Prometaphase is nuclear envelope breakdown and kinetochore capture. Metaphase is the state of stable bi-oriented attachment and equatorial alignment. Anaphase is the irreversible separation event. Telophase is nuclear reconstruction. Mitosis is usually followed immediately by cytokinesis, the physical division of the cytoplasm, though this is mechanistically a separate process driven by an actomyosin contractile ring (in animal cells) rather than by the spindle itself. The defining molecular logic across all five stages is a single protein complex: cohesin. Cohesin loaded during S phase glues sister chromatids together; its stepwise removal — first from chromosome arms in prophase/prometaphase, then completely at the centromere at the anaphase onset — is what converts a static, duplicated chromosome into two independently mobile daughter chromosomes. Layered on top of cohesin removal is the mitotic spindle, a bipolar array of microtubules built from centrosomes (in animal cells) that captures chromosomes at kinetochores, generates the pulling forces of anaphase, and is itself the subject of a dedicated article, The Mitotic Spindle and Its Assembly.

Prophase. Chromatin, diffuse and difficult to distinguish during interphase, condenses into compact rod-shaped structures visible by light microscopy — each chromosome now clearly two sister chromatids joined at a centromere. Condensation is driven by condensin complexes and topoisomerase II, which resolves the tangled, catenated DNA left over from replication. Simultaneously, in the cytoplasm, the two centrosomes (each having duplicated during S phase) begin to separate and nucleate microtubules, forming the two poles of the future spindle. The nucleolus disassembles as rRNA transcription shuts down. Cyclin B–Cdk1 (the master mitotic kinase, "M-phase promoting factor" or MPF) is the trigger for essentially all of these events: it phosphorylates condensin, nuclear lamins, and other substrates to drive chromosome condensation and prepare the nuclear envelope for breakdown.

Prometaphase. Cdk1-mediated phosphorylation of nuclear lamins causes the nuclear lamina to disassemble, and the nuclear envelope breaks into small membrane vesicles — the defining event of prometaphase. This gives spindle microtubules, growing from the two centrosomes, direct access to the condensed chromosomes. Microtubules capture chromosomes via kinetochores, protein complexes (containing CENP-A-marked centromeric chromatin and outer proteins like the Ndc80 complex) assembled at each centromere. Initially, capture is haphazard: chromosomes may attach to only one pole (monotelic), to both poles from one kinetochore (syntelic — an error), or correctly, with sister kinetochores attached to opposite poles (amphitelic/bi-oriented). Error-correction machinery, centered on the kinase Aurora B, destabilizes incorrect attachments so they can be re-tried. Correctly attached chromosomes are pulled and pushed — via kinetochore-fiber dynamics and polar ejection forces — toward the spindle equator.

Metaphase. All chromosomes have achieved stable, bi-oriented attachment and are aligned at the metaphase plate, the imaginary plane equidistant between the two poles. Each pair of sister kinetochores is now under tension, pulled toward opposite poles but held together by centromeric cohesin — this tension is itself part of the error-correction signal, since correct (amphitelic) attachments generate more tension than erroneous ones. The spindle assembly checkpoint (SAC) monitors every kinetochore; as long as even one kinetochore lacks proper microtubule attachment, the checkpoint generates a diffusible "wait" signal (via Mad2 and BubR1 recruited to unattached kinetochores) that inhibits the anaphase-promoting complex/cyclosome (APC/C), delaying anaphase. Metaphase is therefore not a passive resting stage but an active, checkpoint-enforced state of readiness — this checkpoint mechanism is treated in depth in Chromosome Segregation and the Metaphase-to-Anaphase Transition.

Anaphase. Once every kinetochore is properly attached, the SAC is satisfied and APC/C is activated. APC/C is a ubiquitin ligase that targets securin for destruction by the proteasome. Securin normally inhibits the protease separase; its destruction liberates separase, which cleaves the Scc1/RAD21 subunit of centromeric cohesin. This cleavage is the literal, physical trigger of chromatid separation: with the cohesin "glue" cut, sister chromatids — now formally called daughter chromosomes — are pulled apart. Anaphase is conventionally split into two overlapping sub-movements: anaphase A, the shortening of kinetochore microtubules that draws chromosomes poleward, and anaphase B, the elongation of the spindle itself (via sliding of overlapping interpolar microtubules and cortical pulling forces) that pushes the two poles further apart. APC/C simultaneously targets cyclin B for destruction, inactivating Cdk1 — a second, essential event, since falling Cdk1 activity is required to exit mitosis.

Telophase. The separated chromosomes arrive at the spindle poles and begin to decondense. Falling Cdk1 activity allows nuclear lamins to dephosphorylate and reassemble, and nuclear envelope membrane vesicles fuse around each set of chromosomes to reform two intact nuclear envelopes. Nucleoli reappear as rRNA transcription resumes. Mitosis proper ends here; cytokinesis, the physical pinching of the cytoplasm by an actomyosin contractile ring (in animal cells) or by a new cell plate (in plant cells), typically begins during anaphase/telophase and completes the division into two separate daughter cells.

The cohesin cycle underlying this whole sequence is worth visualizing separately, since it is the molecular thread that ties condensation, alignment, and separation together.

Much of what we know about mitotic stages was first established through careful cytology in the late 19th century, then confirmed and mechanistically extended by 20th-century biochemistry.

Walther Flemming's cytological description (1878–1882). Working with salamander (Ambystoma) larval tissues, the German anatomist Walther Flemming used newly available aniline dyes to stain cell nuclei and observed, under the light microscope, that chromatin resolved into thread-like structures during division that split lengthwise before separating to opposite ends of the cell. He coined the term "mitosis" (from the Greek mitos, thread) in his 1882 book Zellsubstanz, Kern und Zelltheilung, and correctly inferred, from fixed-tissue snapshots ordered by inference into a sequence, the stages we now call prophase through telophase. This was purely descriptive light microscopy — but it established, before anyone knew what DNA was, that chromosomes (a term coined slightly later by Waldeyer, 1888) split longitudinally rather than being newly synthesized each division, correctly implying a continuity of hereditary material across cell generations.

Time-lapse and live-cell confirmation. Flemming's static, fixed images left open the question of sequence and timing. This was resolved decisively by 20th-century time-lapse cinemicrography — beginning with the work of researchers like Andrew Bajer and others using phase-contrast and later fluorescence microscopy on living cells (e.g., newt lung cells, chosen for their large, flat, easily-imaged chromosomes) — which filmed mitosis happening in real time, confirming Flemming's inferred stage order and measuring its actual timing: prometaphase-to-metaphase alignment typically takes tens of minutes, while anaphase separation, once triggered, completes within a few minutes.

Mader/Mazia and the discovery of MPF (1971). Yoshio Masui and Clement Markert, working with frog (Xenopus laevis) oocytes, discovered that cytoplasm from a mature (meiotically arrested at metaphase II) oocyte, when injected into an immature oocyte, would induce that immature oocyte to enter M phase — condensing its chromosomes and forming a spindle — even in the absence of new protein synthesis at the injection step. They named the responsible cytoplasmic factor "maturation-promoting factor" (MPF). This was later shown by Manfred Lohka, Marcel Wu, and James Maller (1988) to consist of two components: a kinase (now known as Cdk1) and a regulatory partner, cyclin B (whose periodic synthesis and destruction had been discovered independently by Tim Hunt in 1983, working on sea urchin embryos, for which Hunt shared the 2001 Nobel Prize with Paul Nurse and Leland Hartwell). This body of work established that a single conserved kinase-cyclin complex triggers essentially all of prophase's molecular events — chromosome condensation, nuclear envelope breakdown, spindle formation — across species as distantly related as frogs, sea urchins, and yeast.

Discovery of separase and cohesin cleavage (1990s–2000s). Genetic screens in budding yeast (Saccharomyces cerevisiae) identified mutants that failed to separate sister chromatids properly; cloning the responsible genes identified cohesin subunits (Scc1/Mcd1, Scc3, Smc1, Smc3) and the protease separase (Esp1) and its inhibitor securin (Pds1). Kim Nasmyth's laboratory demonstrated directly, using antibodies against the cohesin subunit Scc1 in budding yeast, that Scc1 is cleaved at a specific site precisely at the metaphase-to-anaphase transition, and that mutating the cleavage site to be protease-resistant blocked chromatid separation entirely — chromosomes remained glued together even as everything else in the cell tried to proceed. This experiment was the definitive proof that cohesin cleavage, not some other mechanism, is the direct trigger of chromatid separation, converting Flemming's century-old cytological description into a precise molecular mechanism.

Example 1 — timing a synchronized cell population. A researcher treats a culture of human HeLa cells with a drug (a "double thymidine block") that arrests cells at the G1/S boundary, then releases the block and takes samples every 15 minutes, staining DNA content by flow cytometry and chromosome morphology by microscopy. She finds: cells begin entering S phase immediately after release; by 7 hours, essentially all cells have 4N DNA content (G2/M); condensed chromosomes first appear (prophase) at 7.5 hours; nuclear-envelope breakdown and metaphase plates are seen at 8 hours; anaphase figures appear at 8.2 hours; and by 8.5 hours two separate nuclei per cell (telophase/early G1) are visible. From this, mitosis itself (prophase through telophase) occupies roughly 1 hour of a roughly 24-hour cell cycle — consistent with the general rule that M phase, though the most visually dramatic stage, is the shortest phase of the cycle, typically under 10% of total cycle time. Within mitosis, anaphase is by far the fastest sub-stage (chromatid separation to poles typically completing in 2–5 minutes), while the alignment process from nuclear envelope breakdown to a stable metaphase plate can take 20–30 minutes — most of mitotic time is spent achieving correct attachment, not executing separation, which reflects the biological priority of accuracy over speed.

Example 2 — diagnosing a segregation error from a karyotype. A prenatal karyotype from chorionic villus sampling shows a fetus with 47 chromosomes: three copies of chromosome 21 (trisomy 21, Down syndrome) instead of the normal two. A genetic counselor is asked to explain, mechanistically, at what stage of division this likely arose, and in which parent. Since the extra chromosome is a whole chromosome 21 (not a partial duplication) and the parents' own karyotypes are normal (46 chromosomes each), the error is a nondisjunction event — a failure of correct chromosome segregation — that occurred during formation of an egg or sperm, i.e., in meiosis, not mitosis (a mitotic error after fertilization would produce a mosaic pattern, with only some cells trisomic, which molecular testing here rules out). Statistically, about 90% of trisomy 21 cases arise from nondisjunction during maternal meiosis I, where the two homologous chromosome 21s fail to separate (rather than sisters failing to separate in meiosis II) — and the risk rises sharply with maternal age, from roughly 1 in 1,250 live births at maternal age 25 to roughly 1 in 100 at maternal age 40, reflecting the fact that human oocytes are arrested in meiotic prophase I for decades and their cohesin — the same protein complex central to mitotic chromatid cohesion — degrades progressively over that time, weakening the physical tether that keeps homologs correctly paired and thereby increasing missegregation risk. This example shows how the mitotic cohesin/separation logic described above extends directly (with modification for homolog vs. sister separation) into meiotic disease mechanism.

Walther Flemming (1843–1905) was a German anatomist and cytologist at the University of Kiel who pioneered the use of aniline dyes to stain cell nuclei, a technique that let him see, for the first time, the fine thread-like structures inside dividing cells. Working painstakingly with fixed and stained tissue sections from salamander larvae — chosen for their large, easily observed cells — Flemming reconstructed the entire sequence of nuclear division from static snapshots, correctly inferring the dynamic order of stages he could never directly watch happen in real time. His 1882 monograph Zellsubstanz, Kern und Zelltheilung ("Cell substance, nucleus, and cell division") laid out this sequence essentially as it is still taught today, and introduced the term "mitosis" itself. Flemming's work predates any knowledge of DNA, chromosomes as carriers of genes, or the biochemistry of cyclins and kinases — yet his careful morphological staging has proven remarkably durable, a testament to how far rigorous observation can carry a science even before its molecular mechanisms are known.

Mitosis's molecular machinery is a major target of cancer chemotherapy: taxanes (paclitaxel/Taxol) stabilize microtubules and prevent the spindle dynamics needed for chromosome alignment, while vinca alkaloids (vincristine, vinblastine) destabilize microtubules — both classes arrest rapidly dividing cancer cells in mitosis and trigger their death, exploiting the fact that tumor cells divide far more often than most normal tissue. Newer targeted agents inhibit mitotic kinases directly, including Aurora kinase inhibitors (targeting the error-correction machinery described above) and Polo-like kinase (PLK1) inhibitors, both in clinical trials for various cancers. Understanding the spindle assembly checkpoint has also illuminated why many cancers show chromosomal instability (CIN) — a weakened or bypassed checkpoint allows cells with missegregated chromosomes to survive and proliferate, generating the aneuploid karyotypes characteristic of most solid tumors. In reproductive medicine, understanding cohesin's slow degradation over the decades-long meiotic arrest of human oocytes has reframed counseling about maternal-age-related aneuploidy risk (as in Example 2 above) and motivates preimplantation genetic testing for aneuploidy (PGT-A) in IVF clinics, which screens embryos for correct chromosome number before transfer. In agriculture, deliberately inducing mitotic errors with colchicine (a microtubule-depolymerizing drug) is a classical and still-used technique for generating polyploid crop varieties (such as seedless watermelon and many ornamental plants) with desirable traits. In basic and clinical cytogenetics, karyotyping itself — arresting cells in metaphase with colchicine, then spreading and staining the condensed chromosomes — remains a standard diagnostic tool for detecting chromosomal abnormalities in prenatal diagnosis, cancer diagnostics, and infertility workups, a direct descendant of the very cytological methods Flemming pioneered.

Ask the tutor: How does the spindle assembly checkpoint actually detect a single unattached kinetochore among dozens of correctly attached ones?