# Lecture 21: Mitosis and Cytokinesis

## Cell Biology

---

## Learning Objectives

By the end of this lecture, students will be able to:

1. Describe the stages of mitosis and the key events in each stage
2. Explain the structure and function of the mitotic spindle
3. Describe how chromosomes attach to the spindle and achieve bi-orientation
4. Explain the spindle assembly checkpoint and anaphase triggering mechanism
5. Describe the mechanism of cytokinesis and its regulation

---

## Lecture Content

### I. Mitotic Entry

Entry into mitosis is triggered by activation of **Cyclin B-CDK1 (MPF)**. During G2, cyclin B steadily accumulates, but CDK1 is held in an inactive state by inhibitory phosphorylation on Thr-14 and Tyr-15, catalyzed by Wee1 kinase. At the G2/M boundary, **Cdc25** phosphatase is activated and removes these inhibitory phosphorylations, unleashing CDK1 activity. A powerful **positive feedback** loop accelerates this transition: active CDK1 phosphorylates and further activates Cdc25, while simultaneously phosphorylating and inhibiting Wee1. This creates a **bistable switch** that produces a rapid, irreversible burst of CDK1 activation, ensuring an abrupt and decisive entry into mitosis.

The activated CDK1 then phosphorylates numerous **CDK1 substrates** to drive the dramatic reorganization of the cell that characterizes mitosis. Phosphorylation of nuclear lamins triggers nuclear envelope breakdown. Phosphorylation of condensins promotes chromosome condensation. Golgi matrix proteins such as GM130 are phosphorylated, leading to Golgi fragmentation. Microtubule-associated proteins are modified to increase microtubule dynamics, and centrosomal proteins are phosphorylated to drive centrosome maturation and separation.

### II. Stages of Mitosis

During **prophase**, chromosomes begin to condense as condensin I and II compact the chromatin. Cohesin is partially removed from chromosome arms by the protein Wapl, but centromeric cohesin is protected by the Shugoshin-PP2A complex. Each chromosome at this stage consists of two sister chromatids joined at the centromere. The centrosomes separate and begin to form the mitotic spindle.

**Prometaphase** begins with the breakdown of the nuclear envelope, which involves disassembly of nuclear pore complexes, phosphorylation-driven lamin depolymerization, and absorption of nuclear membranes into the ER. With the nuclear envelope gone, microtubules gain access to chromosomes. **Kinetochores** assemble on centromeric chromatin, built on a foundation of CENP-A nucleosomes. The inner kinetochore consists of CENP-A, CENP-C, and the constitutive centromere-associated network (CCAN), while the outer kinetochore contains the KMN network (KNL1, Mis12, and Ndc80 complexes), which directly binds microtubules. Dynamic microtubules probe the cytoplasm in a search-and-capture process, attaching to kinetochores as chromosomes congress toward the spindle equator.

At **metaphase**, all chromosomes have aligned at the **metaphase plate**, equidistant from both spindle poles. Each sister chromatid pair has achieved **bi-orientation (amphitelic attachment)**, meaning the two kinetochores are connected to microtubules emanating from opposite poles. Once the spindle assembly checkpoint is satisfied, anaphase can proceed.

**Anaphase** occurs in two mechanistically distinct phases. During **anaphase A**, sister chromatids separate and move toward opposite poles. This is triggered by the enzyme **separase**, which cleaves the Scc1/Rad21 subunit of **cohesin**, dissolving the molecular glue holding sisters together. Kinetochore microtubules shorten through depolymerization at both the kinetochore and pole ends, pulling chromatids poleward. During **anaphase B**, the spindle poles themselves move apart. Kinesin-5 (Eg5) slides antiparallel interpolar microtubules relative to each other, pushing the poles apart, while dynein anchored at the cell cortex pulls on astral microtubules to draw the poles outward.

In **telophase**, chromosomes decondense and the nuclear envelope reassembles around each set of chromosomes. Lamin dephosphorylation by PP1 and PP2A allows the lamina to reform, and nuclear pores are reassembled. CDK1 is inactivated as cyclin B is destroyed by the APC/C ubiquitin ligase.

<image>Stages of mitosis. Panel A: Sequential diagrams of a cell progressing through prophase (condensing chromosomes, separating centrosomes), prometaphase (nuclear envelope breakdown, kinetochore-microtubule attachments forming), metaphase (chromosomes aligned at the metaphase plate with bi-oriented attachments), anaphase A (sister chromatids separating, moving poleward) and anaphase B (poles moving apart), and telophase (chromosomes decondensing, nuclear envelopes reforming). Panel B: Kinetochore structure — centromeric DNA with CENP-A nucleosomes, inner kinetochore (CCAN), outer kinetochore (KMN network with Ndc80 binding microtubule plus ends). Panel C: Types of kinetochore-microtubule attachments — amphitelic (correct, bi-oriented), syntelic (both kinetochores to same pole — incorrect), merotelic (one kinetochore to both poles — incorrect), monotelic (one kinetochore unattached).</image>

### III. The Mitotic Spindle

The mitotic spindle is a bipolar structure of microtubules organized by two centrosomes, which serve as the spindle poles. **Three classes of spindle microtubules** work together to build and operate the spindle. **Kinetochore microtubules (K-fibers)** have their plus ends attached to kinetochores and are responsible for pulling chromosomes. In human cells, approximately 20 to 30 microtubules comprise each K-fiber. **Interpolar microtubules** emanate from both poles and overlap in the spindle midzone. Kinesin-5 (Eg5) cross-links antiparallel interpolar microtubules and slides them apart, providing structural support and driving anaphase B elongation. **Astral microtubules** radiate outward from the poles toward the cell cortex, where they interact with cortical dynein to position the spindle within the cell. Astral microtubules are particularly important for spindle orientation, which determines the plane of cell division.

**Spindle assembly** relies on two cooperating pathways. In the **centrosome-dependent pathway**, microtubules nucleated from centrosomal gamma-TuRC grow outward and capture kinetochores through a search-and-capture mechanism. In the **chromatin-dependent pathway**, Ran-GTP generated near chromosomes by the chromatin-bound Ran-GEF RCC1 releases spindle assembly factors such as TPX2 and NuMA from importin-mediated inhibition, promoting local microtubule nucleation and stabilization near chromosomes. Both pathways cooperate in most cells, but the chromatin pathway is essential in cells that lack centrosomes, such as oocytes.

### IV. The Spindle Assembly Checkpoint (SAC)

The spindle assembly checkpoint is a surveillance mechanism that ensures all kinetochores are properly attached to spindle microtubules before anaphase begins. **Unattached kinetochores generate the "wait" signal** by recruiting the **Mad1-Mad2** complex. Mad2 undergoes a conformational change from an open to a closed form and binds **Cdc20**, forming the **Mitotic Checkpoint Complex (MCC)**, which consists of Mad2, BubR1, Bub3, and Cdc20. The MCC inhibits the **Anaphase-Promoting Complex/Cyclosome (APC/C)**, the E3 ubiquitin ligase that serves as the master trigger of anaphase.

When the APC/C is inhibited by the SAC, anaphase cannot proceed. However, once all kinetochores are properly attached and under tension, Mad1-Mad2 is stripped from kinetochores by dynein-mediated transport to the spindle poles, the MCC disassembles, and Cdc20 is freed. The now-active **APC/C-Cdc20** ubiquitinates two critical substrates. First, it ubiquitinates **securin**, targeting it for proteasomal degradation and releasing **separase**, which cleaves cohesin to trigger sister chromatid separation and anaphase A. Second, it ubiquitinates **cyclin B**, leading to CDK1 inactivation and mitotic exit.

**Error correction** is mediated by Aurora B kinase, which resides at the inner centromere. Aurora B detects the lack of tension at incorrect kinetochore-microtubule attachments, such as syntelic or merotelic configurations. It phosphorylates Ndc80 and other kinetochore substrates, destabilizing these incorrect attachments. When correct bi-oriented attachments are established, the tension generated pulls kinetochore substrates away from Aurora B, preventing their phosphorylation and allowing stable microtubule binding.

<image>The spindle assembly checkpoint and anaphase triggering. Panel A: Unattached kinetochore with Mad1-Mad2 complex generating the MCC (Mad2-BubR1-Bub3-Cdc20), which inhibits APC/C-Cdc20. The cell is arrested in metaphase. Panel B: All kinetochores attached with bi-oriented tension — Mad1-Mad2 stripped off (dynein-mediated), MCC disassembles, APC/C-Cdc20 becomes active. Panel C: APC/C-Cdc20 ubiquitinates securin (degraded by proteasome) freeing separase, which cleaves the Scc1 subunit of cohesin between sister chromatids. Simultaneously, APC/C-Cdc20 ubiquitinates cyclin B, leading to CDK1 inactivation and mitotic exit. Panel D: Aurora B error correction — Aurora B kinase (red) at the inner centromere; syntelic attachment (no tension) allows Aurora B to phosphorylate Ndc80, destabilizing the attachment; correct amphitelic attachment (tension) stretches the kinetochore away from Aurora B, allowing stable microtubule binding.</image>

### V. Cytokinesis

Cytokinesis is the physical division of the cytoplasm into two daughter cells, and it begins during anaphase. The **cleavage plane** is determined by the position of the mitotic spindle. During anaphase, the **central spindle (spindle midzone)** forms between the separating chromosomes, consisting of bundled antiparallel microtubules stabilized by PRC1 and the centralspindlin complex (MKLP1 plus MgcRacGAP). The central spindle and astral microtubules together signal to the cell cortex to specify where the **contractile ring** should assemble.

The key signaling event is **RhoA activation** at the equatorial cortex. Centralspindlin recruits ECT2, a RhoGEF, which activates RhoA specifically at the equatorial cortex. Active RhoA in turn activates formins to drive actin polymerization and ROCK to stimulate myosin II activity. The resulting **contractile ring**, composed of actin and myosin II assembled at the equatorial cortex, constricts progressively to create a **cleavage furrow**. As the furrow ingresses, it narrows to form the **midbody**, a dense structure at the intercellular bridge connecting the two nascent daughter cells.

**Abscission**, the final severing of the intercellular bridge, is mediated by the **ESCRT-III** complex, which carries out membrane scission in a mechanism analogous to its role in multivesicular body formation and viral budding. **Aurora B** kinase at the midbody monitors for trapped chromatin in the bridge and delays abscission if any is detected, functioning as an abscission checkpoint. The microtubule-severing enzyme spastin helps clear microtubules from the bridge to facilitate the final cut.

### VI. Special Cases and Clinical Relevance

Several variations on standard mitotic division have important biological and clinical significance. **Meiosis** differs from mitosis in that it involves two successive divisions, with homologous recombination and a reductional division occurring in meiosis I. **Asymmetric division** produces daughter cells of different size or fate and is particularly important for stem cells, where the orientation of the division plane relative to the niche determines whether division is symmetric or asymmetric. Cortical polarity cues involving Par proteins and the LGN-NuMA-dynein complex orient the spindle to achieve the appropriate division mode.

**Polyploidy** arises when cytokinesis fails, producing tetraploid cells that can subsequently undergo abnormal divisions leading to aneuploidy and chromosomal instability. Some tissues, including hepatocytes, cardiomyocytes, and megakaryocytes, are normally polyploid. **Anti-mitotic drugs**, including taxanes and vinca alkaloids, arrest cells in mitosis by activating the SAC, as discussed in Lecture 17. Newer therapeutic targets under investigation include inhibitors of kinesin-5 (Eg5), Aurora kinases, and Plk1. **Aneuploidy** is a significant cause of human disease, with trisomy 21 (Down syndrome) being the most common aneuploidy compatible with survival. Errors in meiosis I, especially in oocytes, are the most frequent source of aneuploidy.

---