Mitosis & Cytokinesis: M Phase

M Phase: Mitosis & Cytokinesis

Mitosis

• A process of nuclear division where replicated DNA molecules are segregated into two nuclei.
• Typically accompanied by cytokinesis, partitioning the cytoplasm into two separate cells.
• Results in two daughter cells genetically identical to each other and to the mother cell.
• Helps maintain chromosome number and generates new cells for growth and maintenance.
• Mitosis can occur in both haploid and diploid cells.
• Haploid mitotic cells are found in fungi, plant gametophytes, and a few animals (e.g., male bees or drones).
• During mitosis, the cell devotes most of its energy to chromosome segregation.
• Other cellular activities, such as transcription and translation, are largely curtailed.
• The cell becomes relatively unresponsive to external stimuli.
• Mitosis has been studied outside of living cells using extracts from frog eggs.
• These extracts contain essential materials (e.g., histones, tubulin) needed for mitosis.
• When chromatin or nuclei are added to the extract, mitotic chromosomes form and are segregated by a spindle.
• The role of specific proteins in mitosis can be studied through immunodepletion, which removes proteins using antibodies.
• Mitosis is divided into five stages: prophase, prometaphase, metaphase, anaphase, and telophase.
• These stages represent different events within a continuous process.

Prophase (First Stage of Mitosis)

• Duplicated chromosomes are prepared for segregation.
• The mitotic machinery required for chromosome movement is assembled.
• In interphase, chromatin is in a relaxed and extended state (~30 nm fiber) to allow for transcription and replication.
• In mitosis, chromatin undergoes chromosome compaction (condensation) to prepare for segregation.
• This compaction is driven by histones, chromatin looping, and nonhistone scaffolding proteins.
• Histones play the initial role in packaging DNA by forming nucleosomes.
• Nucleosomes further coil into a 30 nm chromatin fiber, which exists both in interphase and mitotic chromosomes.
• While histones help with basic DNA compaction, additional mechanisms are required to achieve the high level of condensation seen in mitosis.
• As chromatin condenses, large loops of chromatin form and attach to a structural scaffold inside the chromosome.
• This scaffold consists of nonhistone proteins that provide additional support.
• The scaffold maintains the chromosome’s overall shape even when histones are removed.
• The looping pattern helps reduce chromosome size and ensures proper organization during mitosis.
• Condensin is a multiprotein complex responsible for chromosome compaction during mitosis.
• Removing condensin prevents normal chromosome condensation.
• Condensin acts as a DNA loop-extruding machine, using ATP energy to actively pull DNA into loops.
• Condensin is activated at the start of mitosis via phosphorylation by cyclin-Cdk complexes, triggering chromosome condensation.
• As chromatin compacts further, mitotic chromosomes appear as distinct, rod-like structures.
• Each mitotic chromosome consists of two identical sister chromatids, which result from DNA replication during interphase.
• These chromatids remain connected until anaphase to ensure equal genetic distribution.
• Before DNA replication, chromosomes are associated with a cohesin complex that binds at multiple points.
• After replication, cohesin holds sister chromatids together through G2 and into mitosis.
• In vertebrates, cohesin is released from chromosomes in two stages:
  • Most cohesin is removed from chromosome arms as chromosomes condense during prophase.
  • Cohesin is fully removed at centromeres during anaphase, allowing sister chromatid separation.
• Polo-like kinase and Aurora B kinase phosphorylate cohesin, leading to its detachment from chromosome arms.
• WAPL Wings Apart-like protein assists in cohesin removal from the arms, loosening chromatid connections.
• A phosphatase at centromeres prevents cohesin removal too early, ensuring chromatids stay together until anaphase.
• During anaphase, the enzyme separase cleaves remaining cohesin at the centromeres, allowing chromatid separation.
• Experimental phosphatase inactivation results in premature chromatid separation, leading to segregation errors.
• Histones initiate DNA compaction, but chromatin looping and scaffold attachment provide higher-order chromosome organization.
• Condensin actively drives chromatin looping, further compacting chromosomes for mitotic segregation.
• Cohesin regulates chromatid cohesion, ensuring proper chromosome distribution before separation.
• The chromosome scaffold provides a structural framework that maintains chromosome integrity even in highly condensed mitotic chromosomes.

Centromeres and Kinetochores

• The centromere is the primary constriction on the mitotic chromosome.
• It contains highly repeated DNA sequences that act as binding sites for centromere-associated proteins.
• The repeated sequences provide a platform for the binding of centromere-associated proteins.
• One of the key proteins that bind to this region is CENP-A (centromeric protein A), a special variant of histone H3.
• CENP-A helps recruit other proteins that form the kinetochore.
• Attaches chromosomes to the mitotic spindle.
• Helps with chromosome movement during mitosis.
• Ensures proper checkpoint signaling to prevent errors in chromosome segregation.
• These repeated sequences are structural docking sites for centromere proteins, necessary for kinetochore formation and chromosome movement.
• The kinetochore is a button-like protein structure located on the outer surface of the centromere.
• It begins assembling at early prophase and consists of 100+ proteins.
• Proteins are recruited to the centromere due to the presence of CENP-A, a histone variant in centromeric nucleosomes.
• The kinetochore is an attachment site for mitotic spindle microtubules.
• The kinetochore houses motor proteins involved in chromosome movement.
• Dynein (Minus-end directed motor) moves chromosomes toward the spindle pole (centrosome).
• Dynein helps pull chromosomes apart during anaphase.
• CENP-E (a type of kinesin, plus-end directed motor) helps attach the kinetochore to microtubules.
• CENP-E ensures proper chromosome alignment at the metaphase plate.
• Kinesin-13 (Microtubule depolymerase) breaks down microtubules at the kinetochore.
• Kinesin-13 helps shorten the spindle fibers to pull chromatids apart.
• The kinetochore plays a role in the mitotic checkpoint, ensuring proper chromosome alignment before separation.
• Before anaphase begins, the kinetochores check whether all chromosomes are properly attached to spindle microtubules.
• If a chromosome is not properly attached, the kinetochore signals to delay anaphase until corrections are made.
• The checkpoint proteins at the kinetochore, Mad2, BubR1, and Bub3, prevent the enzyme separase from cutting the cohesin.
• Once all chromosomes are correctly aligned, the checkpoint is lifted, and anaphase proceeds.

Formation of the Mitotic Spindle

• The centrosome is a microtubule-organizing center.
• At the G2 to mitosis transition, interphase microtubules rapidly disassemble.
• Proteins that stabilize microtubules (MAPs) are inactivated, while destabilizing proteins are activated to allow mitotic spindle formation.
• Centrosome Cycle and Duplication:
  • Parent Cell Starts with One Centrosome (Interphase - G1 Phase): The cell has one centrosome containing two centrioles.
  • Centrioles Begin Duplicating (S Phase of Parent Cell): Each mother centriole grows a new procentriole at a right angle.
  • Centrosome Matures (G2 Phase of Parent Cell): The procentrioles elongate into full-length centrioles.
  • Mitosis Begins (Prophase-Metaphase of Parent Cell): The centrosome splits into two centrosomes, each containing two centrioles.
  • Centrioles Disengage (Anaphase-Telophase of Parent Cell): The two centrioles within each centrosome "disengage"
• Separase enzyme becomes active and cleaves the protein link holding the two centrioles together.
  • Cytokinesis Creates Two Daughter Cells: Each daughter cell inherits one centrosome (which still has two disengaged centrioles).
  • G1 Phase of the Daughter Cells: The disengaged centrioles remain inactive and mature but do not duplicate yet.
  • S Phase of the Daughter Cells: Now, each centriole duplicates, forming new procentrioles at right angles.
• Centrosome duplication is regulated by Cdk2 phosphorylation at the G1-S transition.
• Abnormal centrosome duplication can lead to cancer due to multipolar spindles and incorrect chromosome segregation.
• Aster Formation and Centrosome Separation:
  • Early prophase: Microtubules form a radial "sunburst" aster around each centrosome.
  • Spindle microtubules grow from the minus ends (PCM) outward to their plus ends.
  • Polo-like kinase phosphorylates PCM proteins, stimulating microtubule nucleation.
  • Motor proteins help separate centrosomes and move them toward opposite poles.
  • As centrosomes move apart, spindle microtubules elongate and increase in number.
  • When centrosomes reach opposite ends, they establish the two poles of the bipolar mitotic spindle.
• Some cells lack centrosomes but still form bipolar mitotic spindles (e.g., mouse embryos, plant cells, mutant Drosophila cells).
• Chromosome-dependent spindle formation occurs by microtubule nucleation near chromosomes.
• Microtubules of the mitotic spindle are nucleated near the chromosomes rather than at the poles where centrosomes would normally reside.
• Minus ends of microtubules are clustered at spindle poles by motor proteins.
• Both centrosome-dependent and centrosome-independent pathways function together in most cells.

Dissolution of the Nuclear Envelope and Organelle Partitioning

• Nuclear envelope breakdown (NEB) occurs at the end of prophase, allowing spindle-chromosome interaction.
• NEB is driven by Cyclin B–Cdk1 phosphorylation, which initiates disassembly of nuclear structures.
• Nuclear Pore Complexes disassemble as nucleoporin subcomplexes dissociate into the cytoplasm.
• Nuclear Lamina depolymerizes, breaking the meshwork that supports the nuclear envelope.
• Dynein motor proteins pull on the outer nuclear membrane, creating holes in the envelope.
• Membranes either fragment into vesicles or integrate into the endoplasmic reticulum (ER).
• Some organelles remain intact throughout mitosis:mitochondria, lysosomes, peroxisomes, and plant cell chloroplasts.
• Golgi Complex Behavior: Remains intact throughout mitosis; Breaks into vesicles and tubules (mammals).
• Endoplasmic Reticulum (ER) Behavior: Restructures into extended membrane sheets, localizes to centrosomes ensuring equal distribution.

Prometaphase

• Begins with the dissolution of the nuclear envelope, allowing microtubules to enter the nuclear space.
• Chromosomes, already compacted, remain scattered within the nuclear region.
• The mitotic spindle assembly is completed, setting the stage for chromosome movement.
• Spindle microtubules extend from centrosomes and show dynamic instability (growing and shrinking).
• Microtubules "search" for chromosomes, growing toward chromatin-containing regions.
• Microtubules that contact kinetochores are captured and stabilized.
• The kinetochore initially interacts with the side of a microtubule rather than the tip.
• Motor proteins in the kinetochore move chromosomes along the microtubule wall toward the spindle.
• The kinetochore forms a stable attachment at the plus end of one or more spindle microtubules.
• If a chromosome is attached to microtubules from only one spindle pole, it is considered unstable.
• The unattached sister chromatid captures microtubules from the opposite pole.
• Stable bipolar attachment is achieved, ensuring proper chromosome segregation.
• Some microtubules are nucleated directly near chromatin.
• Tubulin subunits incorporate near chromatin, forming new microtubules.
• Some microtubules emerge from the sides of existing spindle fibers, increasing microtubule density.
• Chromosomes attached to spindle microtubules do not move directly to the metaphase plate, instead, they undergo back-and-forth oscillations, moving poleward and antipoleward.
• Motor proteins on kinetochores and chromosome arms drive chromosome movements.
• Oscillation is a result of a tension mechanism and the dynamic nature of microtubules.
• Each chromosome must attach to spindle fibers from both poles (bipolar attachment).
• The spindle assembly checkpoint ensures that chromosomes are properly attached before allowing progression to metaphase.
• A process called congression directs chromosomes toward the center of the mitotic spindle.
• Forces generated by motor proteins push and pull chromosomes into proper alignment.
• Deficiency in key motor proteins can disrupt chromosome positioning, causing misalignment.
• Microtubules adjust in length to facilitate chromosome alignment.
• The longer microtubules attached to one kinetochore shorten.
• The shorter microtubules attached to the sister kinetochore elongate.
• These changes occur primarily at the plus end while kinetochores remain attached.
• Eventually, each chromosome aligns at the metaphase plate (spindle equator).
• Microtubules from both spindle poles become equivalent in length.

Metaphase

• All chromosomes align at the spindle equator (center of the cell).
• Each chromatid of a chromosome is attached to microtubules from opposite spindle poles via kinetochores.
• The alignment plane is called the metaphase plate.
• Three Types of Microtubules in the Metaphase Spindle:
  • Astral Microtubules radiate outward from the centrosome, help position the spindle within the cell, and determine the plane of cytokinesis.
  • Chromosomal (Kinetochore) Microtubules extend from the centrosome to the kinetochore.
  • Polar (Interpolar) Microtubules extend beyond the chromosomes and overlap with microtubules from the opposite centrosome.
• K-fibers (kinetochore fibers) are specific bundles of microtubules that connect the kinetochore to the spindle pole.
• Metaphase appears as a "pause", but significant activity is occurring at the microtubule level.
• Microtubules exist in a highly dynamic state despite their apparent stability.
• Tubulin subunits are added at the plus end (near the kinetochore) and lost at the minus end (near the pole).
• Kinetochore & Microtubule Dynamics:
  • The kinetochore does not cap the microtubule ends.
  • Net Addition at the Plus End (near the kinetochore).
  • Net Loss at the Minus End (near the spindle pole).
  • This process creates a poleward flux of tubulin subunits along the chromosomal microtubules.
• Role of Motor Proteins in Metaphase:
  • The kinesin-13 family promotes microtubule depolymerization rather than movement.
  • Depolymerization at the poles helps maintain spindle structure and tension.
• If too many subunits are lost at the kinetochore end, the tension between sister chromatids reduces.
• This activates the spindle assembly checkpoint (SAC), preventing anaphase until proper attachment is restored.

Anaphase

• Anaphase begins when sister chromatids of each chromosome split apart.
• The chromatids start moving toward opposite poles of the cell.
• Proper order in the cell cycle is maintained by selective destruction of regulatory proteins.
• Accomplished by SCF and APC multi protein complexes.
• APC contains about a dozen core subunits and requires an adaptor protein to determine which proteins to degrade.
• APC adaptor proteins, Cdc20 and Cdh1, regulate function at different times.
• APC^Cdc20 ubiquitinates securin, an inhibitor that prevents chromatids from separating.
• Securin destruction activates separase, an enzyme that cleaves the Scc1 subunit of cohesin.
• The cleavage of cohesin triggers chromatid separation, marking the beginning of anaphase.
• Proteolytic enzymes cleaved cohesin in cells arrested in metaphase, leading to rapid chromatid separation and anaphase-like movement.
• As Cdc20 is inactivated, Cdh1 takes over APC’s substrate selection.
• APC^Cdh1 completes the degradation of cyclin B, causing a drop in mitotic Cdk1 activity.
• Critical Role of Proteolysis in Cell Cycle Progression: necessary for irreversible cell cycle progression.
• At the onset of anaphase, all chromosomes split synchronously at the metaphase plate.
• Once separated, chromatids are now called chromosomes because they are no longer attached to their sister chromatids.
• Chromosomes migrate toward opposite poles, led by their centromeres, with the arms trailing behind.
• Chromosome movement is slow (~1 µm per minute), ensuring accurate segregation without tangling.
• Tubulin subunits are added to the plus ends of chromosomal microtubules during metaphase to maintain length.
• During anaphase, tubulin subunits are lost from the plus ends, leading to shortening and chromosome movement.
• Tubulin subunits are also removed from the minus ends, continuing the poleward flux of microtubules.
• Anaphase A: Chromosomes move toward opposite spindle poles due to the shortening of chromosomal microtubules.
• Anaphase B: Spindle poles move farther apart, expanding the distance between the two chromosome groups.
• Driven by addition of tubulin subunits to the plus ends of polar microtubules, and simultaneous removal of subunits from chromosomal microtubules.

Forces Required for Chromosome Movements at Anaphase

• Chromosomal microtubule depolymerization is the cause of chromosome movement in anaphase.
• Disassembly of spindle microtubules generates mechanical force to pull chromosomes forward.
• Chromosome movement occurs when attached microtubules depolymerize.
• Chromosomal spindle fibers undergo depolymerization at both their minus and plus ends.
• Moves chromosomes toward the poles via poleward flux.
• Causes microtubules to roll up while towing chromosomes.
• Kinesin-13 acts as an ATP-dependent depolymerase, driving chromosome segregation.
• If either plus-end or minus-end depolymerase is inhibited, chromosome segregation is disrupted.
• The KMN network forms at the outer kinetochore of each chromosome.
• A key structural component is the Ndc80 complex.
• Ndc80 terminal heads move toward the minus end of the microtubule.
• As microtubules shrink, attached chromosomes are pulled toward the spindle poles.

Telophase

• Telophase begins when chromosomes reach their respective poles and start collecting in a mass.
• Daughter cells return to their interphase condition as mitotic structures are dismantled.
• Key cellular events in telophase:
  • Mitotic spindle disassembles
  • Nuclear envelope reforms
  • Chromosomes become more dispersed
• Cytoplasm partitioning begins, leading to the final step of cell division.

Cytokinesis

• Cytokinesis is responsible for dividing the cell into two.
• Mitosis segregates duplicated chromosomes, while cytokinesis splits the cytoplasm to form two daughter cells.
• Begins during anaphase, marked by an indentation in the cell surface.
• The indentation deepens into a cleavage furrow, moving inward toward the center.
• Cytoplasmic vesicles fuse with the cleavage furrow, supplying additional plasma membrane.
• As the furrow advances, it passes through remnants of the mitotic spindle, forming a cytoplasmic bridge.
• Abscission occurs when the cleavage furrow membranes fuse, physically splitting the cell.
• Cytokinesis is driven by a contractile ring beneath the plasma membrane.
• Actin filaments assemble beneath the furrow, formin proteins anchor these to the membrane.
• Short, bipolar myosin II filaments are interspersed among actin filaments in the contractile ring, anti-myosin II antibodies halt.
• In its GTP-bound state, RhoA triggers: actin filament assembly, activation of myosin II motor activity.
• Actin-myosin filaments pull the membrane inward.
• Ray Rappaport’s studies on marine invertebrate eggs spindle poles determine the cleavage site.
• Centralspindlin is a protein complex that regulates cleavage furrow formation.
• Aurora B kinase (a CPC component) plays several roles; Regulates kinetochore-microtubule attachments, ensures abscission does not occur, regulates chromatin decondensation after mitosis.

Mitotic Movements

• Mitosis involves extensive movements of cellular structures at different stages.
• Various motor proteins, primarily microtubule motors (kinesins and cytoplasmic dynein), drive these movements.
• Motor proteins function at different locations within the mitotic spindle, including:
  • Spindle poles
  • Spindle fibers
  • Chromosome kinetochores
  • Chromosome arms
• Prophase → Spindle poles move to opposite ends of the cell.
• Prometaphase → Chromosomes move to the spindle equator.
• Anaphase A → Chromosomes move from the spindle equator to the poles.
• Anaphase B → Spindle elongates, separating the poles further apart.

Spindle Assembly Checkpoint (SAC)

• The spindle assembly checkpoint (SAC) ensures that chromosomes are properly aligned.
• SAC prevents aneuploidy
• If the cell does not have that then that leads to a disorder and it leads to increased risk.
• SAC monitors chromosome attachment to the mitotic spindle for tension
• A protein complex called the Chromosomal Passenger Complex (CPC), which includes Aurora B kinase, senses the missing tension.
• Aurora B kinase sends a "wait" signal, preventing the cell from entering anaphase.
• Mad2 protein is a key component of the SAC complex.
• Inactive Mad2 binds to Mad1 at kinetochores activating it mad2 which recruits and activates more mad2 molucules.
• Active Mad2 binds to Cdc20, a key activator of the Anaphase-Promoting Complex (APC).
• Syntelic Attachment where both chromatids are pulling from same side, they dont attach correctly, Aurora B complex fixed it by phos.