Cell Biology I - Lecture 12: Cell Division

General Overview of Cell Division

• Definition and Basic Process: Cell division generates two daughter cells following DNA replication. The process consists of two primary stages:
      * Segregation: The mechanical segregation of cellular contents, specifically sister chromatids, occurs during mitosis.
      * Physical Separation: The actual physical separation of the daughter cells occurs during cytokinesis.

Historical Context and Importance of Segregation Fidelity

• Evolution of Observation: Stages of cell division were identified as early as $1887$ by Flemming. By $1890$, researchers including Von Hansemann were documenting the importance of chromosome segregation fidelity.
• The Aneuploidy Hypothesis: Proposed by Theodor Boveri in $1914$ in his work "Zur Frage der Entstehung maligner Tumoren."
      * Boveri's Hypothesis (Verbatim): "Die Urzelle des Tumors ist nach meiner Hypothese eine Zelle mit einem bestimmten, unrichtig kombinierten Chromosomenbestand. Dieser ist die Ursache für die Wucherungstendenz, die auf alle Abkömmlinge der Urzelle übergeht."
      * Concept: Aneuploidy (an incorrect combination of chromosomes) is the root cause of the proliferative tendencies in tumor cells, which is inherited by all descendants of that original tumor cell.

Anatomy of the Mitotic Spindle

• Structural Components: The mitotic spindle is a complex apparatus designed to move chromosomes. Key structures include:
      * Centrosome/Spindle Pole: Two centrosomes are replicated and move to opposite poles to organize the spindle.
      * Centromere: The chromosomal region where sister chromatids are joined.
      * Kinetochore: A protein structure on the centromere to which microtubules attach.
• Microtubule Types:
      * Astral Microtubules: Radiate from the centrosome towards the cell cortex, helping to position the spindle.
      * Kinetochore Microtubules: Attach directly to the kinetochores of the chromosomes.
      * Interpolar Microtubules: Overlap in the center of the spindle (spindle midzone) and are cross-linked by motor proteins.
• Polarity: Centrosomes represent the minus ends ($-$) of microtubules, while the plus ends ($+$) extend outward toward the chromosomes or the cell periphery.

Prophase: Condensation and Maturation

• Chromosome Preparation: Replicated chromosomes, each consisting of two closely associated sister chromatids, begin to condense.
      * Cohesin: Deposited during S-phase; it keeps sister chromatids physically together along their length.
      * Condensin: Recruited during prophase; it uses condensin rings to compact chromatin into highly condensed, discrete chromosomes.
• Centrosome Separation and Maturation:
      * The mitotic spindle begins to assemble outside the nucleus as centrosomes move apart.
      * Centrosome Maturation: Duplicated centrosomes recruit additional microtubule-organizing material, specifically $\gamma$-tubulin.
• Mitotic Kinases: Activities are regulated by specific kinases:
      * Aurora A: Localizes to centrosomes; drives mitotic entry, changes in microtubule dynamics, centrosome separation, and maturation.
      * Aurora B: A "chromosomal passenger" that localizes to the inner centromere in early mitosis and the spindle midzone later. It drives kinetochore attachment and cytokinesis.
      * Polo-Like Kinase 1 ($Plk1$): Localizes to centrosomes, kinetochores, and the spindle midzone. It drives mitotic entry, centrosome maturation, sister chromatid resolution, and cytokinesis.

Dynamics of Microtubules in Mitosis

• Instability: As cells enter mitosis, microtubules become significantly more unstable.
• Catastrophe Frequency: The primary parameter change is a marked increase in the catastrophe frequency (the transition from growth to shrinkage).
• Result: This leads to a population of more numerous, but shorter, microtubules emanating from the centrosomes compared to interphase.
• Spatial Regulation: Microtubule stability is not uniform. The stabilizer $XMAP215$ localizes specifically to spindle microtubules rather than astral microtubules.

Nuclear Envelope Disassembly (NEBD) and Mitotic Variation

• NEBD Mechanism: M-Cdk phosphorylates nuclear lamins and nuclear pore complex ($NPC$) proteins, triggering the disassembly of the nuclear lamina and $NPCs$. Inner nuclear membrane proteins retract into the Endoplasmic Reticulum ($ER$).
• Types of Mitosis:
      * Open Mitosis: Occurs in vertebrate somatic cells where the nuclear envelope completely disassembles.
      * Closed Mitosis: Occurs in organisms like yeast; the nuclear envelope remains intact, and an intranuclear spindle forms from spindle pole bodies.
      * Semi-open/Semi-closed Mitosis: Occurs in $C.\,elegans$ and $Drosophila$ embryos where the nuclear envelope is only perforated at the spindle poles.
• Chromosome Repulsion: The protein $Ki-67$ coats mitotic chromosomes, acting as a highly charged "brush" to keep individual chromosomes from clumping together after NEBD.

Prometaphase and Spindle Assembly Factors

• Prometaphase Initiation: Starts abruptly with NEBD, allowing cytoplasmic microtubules access to the chromatin. Chromosomes attach to microtubules and undergo active motion.
• Microtubule Motors:
      * Eg5 ($Kinesin-5$): A homotetrameric motor that cross-links anti-parallel microtubules and pushes spindle poles apart.
      * Kinesin-14 family: Minus-end directed motors that counteract the separation forces of $Eg5$.
      * Dynein: Cortically-localized dynein centers the spindle; cytoplasmic dynein (in complex with $NuMA$) focuses the spindle poles.
      * Chromokinesins ($Xklp1$, Kinesin-4, 10 families): Localized on chromosomes; they push chromosome arms away from the poles, a phenomenon often called the "polar wind."
• Clinical Relevance: Inhibition of $Eg5$ (e.g., by the drug Monastrol) results in monopolar spindles. Such inhibitors are in clinical trials as anti-cancer agents to avoid the neurotoxicity associated with Taxol.

Kinetochore Architecture and Search-and-Capture

• Kinetochore Function: Aligns chromosomes on the metaphase plate, monitors attachment, and is required for segregation in anaphase.
• Structural Layers:
      * CCAN (Constitutive Centromere Associated Network): Localizes to the centromere throughout the cell cycle; provides the structural platform.
      * KMN Network: Comprises the $KNL1$, $MIS12$, and $NDC80$ complexes; forms the core microtubule-binding site.
• Microtubule Binding: Subunits $NDC80$ and $NUF2$ (of the extended $NDC80$ complex) contain Calponin-homology domains that bind to microtubules.
• Chromosomal Search and Capture: Facilitated by motors such as Dynein and $CENP-E$ ($Kinesin$). Loss of $CENP-E$ (via RNAi) leads to wholesale chromosome gain or loss (aneuploidy).

Tension Sensing and Error Correction

• Tension Model: Only chromosomes properly attached to both poles (bi-orientation) experience tension. The cell uses this tension to monitor and stabilize correct attachments.
• Aurora B Kinase Function:
      * When tension is low, $Aurora B$ (localized at the inner centromere) phosphorylates $NDC80$, inhibiting its ability to bind microtubules and destabilizing the incorrect attachment.
      * When tension is high, $NDC80$ is physically pulled away from $Aurora B$. This spatial separation allows protein phosphatase 1 ($PP1$) to dephosphorylate $NDC80$, stabilizing the microtubule interaction.

Chromatin-Driven Spindle Assembly and Ran-GTP

• Bypassing Centrosomes: DNA-coated beads in Xenopus egg extracts can form bipolar spindles without centrosomes or kinetochores. This follows a process of nucleation, coalescence, and bipolarity dictated by plus-end and minus-end motors.
• Ran-GTP Gradient:
      * In the nucleus, $Ran-GTP$ releases cargo from import receptors. In mitosis, after NEBD, $RCC1$ (bound to chromatin) generates a local high concentration of $Ran-GTP$ near chromosomes.
      * This gradient promotes the local release of Spindle Assembly Factors ($SAFs$) like $TPX2$ from importins.
• HAUS/Augmin Complex: Stimulates microtubule nucleation within the spindle, regulated by the $Ran-GTP$ gradient.

Metaphase and the Spindle Assembly Checkpoint (SAC)

• Metaphase State: Chromosomes are aligned at the spindle equator, and sister chromatids are attached to opposite poles.
• The Checkpoint Mechanism:
      * Unattached kinetochores catalyze the formation of the Mitotic Checkpoint Complex ($MCC$), which includes $Cdc20$, $Bub3$, $Mad2$, and $Mad3/BubR1$.
      * The $MCC$ blocks the activation of the Anaphase-Promoting Complex/Cyclosome ($APC/C$).
• The Mad2 Template (Prion) Model: Unattached kinetochores use $Mad1$/$C-Mad2$ complexes to convert inactive "Open-$Mad2$ ($O-Mad2$)" into active "Closed-$Mad2$ ($C-Mad2$)" which binds and inhibits $Cdc20$.
• Transition to Anaphase: Once all kinetochores are attached, the checkpoint is satisfied. $APC/C$ ubiquitylates substrates:
      * Securin: Its degradation releases the protease Separase.
      * Separase: Cleaves the Kleisin subunit of the Cohesin ring, allowing sister chromatids to separate.
      * Cyclin B1: Its degradation leads to $Cdk1$ inactivation and mitotic exit.

Anaphase and Telophase

• Anaphase Stages:
      * Anaphase A: Movement of separated sister chromatids toward the spindle poles, driven by microtubule plus-end depolymerization and biased diffusion (mediated by the $Ndc80$ complex).
      * Anaphase B: Physical separation of the spindle poles themselves.
• Microtubule Flux: Observed via fluorescence speckle microscopy; microtubules move toward the poles while depolymerizing.
• Telophase Process:
      * Daughter chromosomes arrive at poles and decondense.
      * Nuclear Reassembly: $NPC$ assembly initiates by prepore deposition on chromatin. $ER$ tubules attract to the chromatin surface, flatten into sheets, and fuse. The nuclear lamina then reforms.

Cytokinesis and Abscission

• The Contractile Ring: An actomyosin structure (Actin and Myosin II) that forms the cleavage furrow. Actin filaments are anchored at the cell cortex.
• Ring Dynamics: Unlike larger structures that might dilute as they shrink, the concentration of contractile proteins (Myosin II, Septin, Anillin) remains constant during ring closure. The ring shortens at a constant rate dictated by actin filament depolymerization.
• Cleavage Plane Specification:
      * The furrow always forms halfway between the poles, perpendicular to the spindle.
      * Rappaport Experiment ($1961$): Demonstrated that even in the absence of a complete spindle, overlapping astral microtubules from two different spindles can induce a cleavage furrow.
• RhoA Signaling: The target for cytokinetic signaling.
      * RhoGEF activates $RhoA$ ($GDP \rightarrow GTP$).
      * Active RhoA stimulates Formin (for actin filament formation) and Rock (Rho-activated kinase).
      * Rock inhibits myosin phosphatase and phosphorylates regulatory myosin light-chains, leading to Myosin II activation.
• Abscission: The final cut. The remaining connection is the midbody. Abscission requires severing midbody microtubules, contraction of helical filaments, and membrane delivery/fusion involving $Rabs$, the Exocyst, $SNAREs$, and the $ESCRT$ complex.