Cell Bio Module 6: Cell Cycle Control, Mitosis, and Stem Cell

Art of Microscopy and Module Introduction

• Chapter Opener Micrograph: A fluorescence micrograph of a newt lung cell captured during mitosis.
• Staining Details:
  • Centrosomes: Magenta.
  • Microtubules: Green.
  • Chromosomes: Blue.
  • Keratin intermediate filaments: Red.
• Origin of Work: Lab of Dr. Alexey Khodjakov at the Wadsworth Center, Department of Health, New York.
• Equipment and History: The image was taken in the year $2000$ using a modified Nikon TE200 regular fluorescence microscope. It is not a confocal image.
• Recognition: Won first prize in the annual Olympus-Nature Light microscopy imaging competition in $2000$.

Overview of the Cell Cycle

• Definition: The process where cells reproduce by duplicating their content and dividing into two. The time required is the cell cycle duration.
• Cell-Specific Durations:
  • Fly embryo cells: Approximately $8\,\text{min}$ (extremely fast).
  • Mammalian liver cells: Greater than $1\,\text{year}$ (divide infrequently).
• Major Phases:
  • Interphase: The longest portion of the cycle where regulatory complex processes occur.
  • M Phase (Mitotic Phase): A relatively short phase where structural changes and division occur.
• General Structure of Interphase:
  • Gap 1 ($G_1$): The first growth phase. The cell monitors the environment (nutrients, etc.) to ensure conditions are suitable for DNA replication.
  • Synthesis ($S$) Phase: The DNA synthesis phase where DNA is replicated.
  • Gap 2 ($G_2$): A safety gap to ensure chromosomes/DNA have been duplicated properly before entering mitosis. This prevents detrimental effects on daughter cells.
• Quiescent Stage ($G_0$):
  • Cells can exit the cycle after mitosis to enter a non-dividing, quiescent state.
  • Cells can re-enter the cycle upon specific stimuli.
  • Neurons: Permanently in $G_0$ (amitotic).

Detailed Stages of Mitosis and Cytokinesis

• Interphase (Pre-Mitosis): Chromosome replication (S-phase) and duplication of the microtubule organizing center (MTOC) occur.
• Prophase:
  • Migration of two MTOCs (spindle poles) to opposite cell poles.
  • Chromosome condensation begins (becoming visible).
  • Nuclear envelope begins to disappear.
• Prometaphase:
  • Nuclear envelope breaks down.
  • Chromosomes are captured by microtubules.
  • Kinetochores assemble on the centromere.
  • Cohesin is shed from chromosome arms.
• Metaphase:
  • Chromosomes align at the center (metaphase plate).
  • Sister chromatids attach to spindle fibers from opposite poles (bi-orientation).
• Anaphase:
  • Sister chromatids are pulled apart toward opposite poles.
  • Spindle fibers elongate the spindle.
• Telophase:
  • Chromosomes decondense at opposite poles.
  • Nuclear envelopes reform; nucleoli reappear.
  • Mitotic spindle breaks down.
  • Assembly of the contractile ring (actin and myosin).
• Cytokinesis:
  • In animal cells, a cleavage furrow forms due to actin-myosin contraction, pinching the cells apart.
  • In plant cells, a cell plate (precursor to the cell wall) separates daughter cells.

The Mitotic Apparatus and Microtubule Classes

• Organization: Forming a football-shaped spindle in the center with tufts at the ends.
• Three Classes of Microtubules:
  • Kinetochore Microtubules: Interact directly with the chromosomes at the kinetochore region.
  • Polar Microtubules: Do not interact with chromosomes; they interact with each other in the center of the spindle.
  • Astral Microtubules: Form tufts at the ends (spindle poles) and interact with the cell cortex/plasma membrane.
• Bi-orientation: Essential for ensuring each daughter cell receives exactly one copy of every chromosome. Failure leads to chromosome aneuploidy (e.g., Down's syndrome/Trisomy 21).

Microtubule Attachment to Centromeres

• The Kinetochore: A large protein complex structure on the centromeric DNA where microtubules attach.
• Structure Layers:
  • Inner Kinetochore Protein Complex: Associates with centromeric DNA.
  • Outer Kinetochore Protein Complex: Associated with the plus ($+$) end of microtubules.
  • Corona: A region outside the outer kinetochore.
• Molecular Components:
  • $CENP-A$: A specific variant of Histone 3 ($H3$) that marks the kinetochore region for the mitotic apparatus.
  • Ndc80 Complex: A rigid, sleeve-like multiprotein complex that connects the microtubule to the inner kinetochore. It serves as a recruitment site for motor proteins.
  • Chromosome Passenger Complex (CPC): Contains Aurora B kinase, which regulates bi-orientation by phosphorylating kinetochore proteins.
• Mechanism of Bi-orientation Regulation:
  • If tension is low (improper attachment), Aurora B kinase phosphorylates Ndc80, weakening the microtubule-kinetochore association.
  • If bi-orientation occurs, tension pulls the kinetochore away from the CPC. Aurora B can no longer reach the Ndc80 complex to phosphorylate it, resulting in a strong, stable attachment.

Chromosome Capture and Congression

• Capture Models:
  • End Capture: The plus end of a growing microtubule directly contacts the kinetochore by chance.
  • Side Capture: Kinetochore proteins interact with the sides of a microtubule; motor proteins then slide the chromosome to the end.
• Motor Proteins in Congression:
  • Dynein-Dynactin: Associated with one kinetochore; walks toward the minus ($-$) end of the microtubule to pull the chromosome toward the pole.
  • Kinesin-7 ($CENP-E$): Holds onto the growing microtubule during congression.
  • Kinesin-13: Stimulates microtubule disassembly at the plus ($+$) end on the shortening side.
  • Kinesin-4 (Chromokinesin): Associated with chromosome arms, pushing them toward the center of the spindle.
• Bi-directional Oscillations: A "tug of war" where one side shortens (Kinesin-13) while the other elongates (Kinesin-7).

Microtubule Behavior during Anaphase A and B

• Metaphase Treadmilling: Kinetochore microtubules maintain constant length through equal rates of addition ($+$ end) and subtraction ($-$ end).
• Anaphase A: Chromosomes move toward poles.
  • Powered by rapid shrinking of kinetochore microtubules (‘loss’ at the $+$ end) via Kinesin-13.
  • Disassembly also occurs at the minus ($-$) end in the spindle poles.
• Anaphase B: Spindle poles separate and the cell elongates.
  • Polar Microtubule Force: Kinesin-5 (bipolar, plus-end directed) attaches to antiparallel polar microtubules and pushes them apart.
  • Astral Microtubule Force: Dynein motors anchored to the plasma membrane walk toward the minus ($-$) end of astral microtubules, pulling the poles toward the cell periphery.

The Cell Cycle Control System (Checkpoints)

• G1 Checkpoint: Asks if the cell is big enough, the environment is favorable, and if DNA is damaged.
• G2 Checkpoint: Asks if all DNA is replicated properly and if the environment is favorable.
• Mitosis (Spindle) Checkpoint: Asks if all chromosomes are aligned on the spindle.
• Experimental Proof: Cell fusion experiments creating a heterokaryon (mitotic cell fused with interphase cell) showed that mitotic factors induce chromosome condensation in the interphase nucleus.

Biochemical Controllers: Cyclins and Cdks

• Mitosis Promoting Factor (MPF): A dimer of Cyclin and Cyclin-dependent kinase (Cdk).
• Cdk Properties: A serine/threonine kinase that phosphorylates target proteins. Cdk levels remain constant through the cycle, but activity changes based on cyclin presence.
• Cyclin Properties: Synthesized and degraded (via proteasomes) in a cyclic manner.
• APC/C (Anaphase-Promoting Complex/Cyclosome): A ubiquitin ligase that targets proteins (like securin or mitotic cyclins) for degradation to allow transition from metaphase to anaphase.

Molecular Regulation of Cycle Transitions

• G1 to S Transition (RB and E2F):
  • Retinoblastoma (RB) protein normally binds and inhibits E2F (a transcription factor).
  • Cyclin D/Cdk4 or Cyclin D/Cdk6 phosphorylate RB, causing it to release E2F.
  • E2F induces the expression of S-phase genes (e.g., Cyclin E, DNA replication components).
  • Retinoblastoma Cancer: Caused by mutant/absent RB, leading to constitutive E2F activity and unregulated cell growth.
• S-Phase Initiation (Sic1):
  • Sic1 is an inhibitor of S-phase cyclin-Cdk dimers.
  • G1 cyclins phosphorylate Sic1, signaling it for ubiquitination by SCF and degradation by the proteasome.
  • Released S-phase cyclin-Cdks phosphorylate pre-replication complexes at origins of replication to initiate synthesis.
• Specific Checkpoint Signaling:
  • Un-replicated DNA (G2): ATR activates Chk1, which inhibits Cdc25c (prevents M-phase entry).
  • Spindle Assembly (M): Mad2 inhibits the proteasome from degrading securin, preventing anaphase.
  • Chromosome Segregation (Late M): Inhibition of Cdc14 prevents mitotic cyclin degradation.
  • DNA Damage (Guardian of the Genome): ATM/ATR kinases activate p53 (a transcription factor). p53 induces $p21^{CIP}$, which binds and inhibits Cyclin-Cdk dimers to stall the cycle.

Stem Cell Biology

• Medical Context: Degenerative diseases (e.g., heart disease, cancer, stroke, Alzheimer's) are leading causes of death in developed nations. Stem cells offer potential cures.
• Nobel Prize 2012:
  • John Gurdon: Pioneered Somatic Cell Nuclear Transfer ($SCNT$) in frogs.
  • Shinya Yamanaka: Created Induced Pluripotent Stem Cells (IPSCs) by expressing four transcription factors: $OCT3/4$, $Sox2$, $klf4$, and $c-MYC$.
• Properties of Stem Cells:
  • Self-renewal: Asymmetric division produces another stem cell and a progenitor cell.
  • Differentiation: Capability to become specialized cell types.
• Stem Cell Potency:
  • Unipotent: Form only one type of differentiated cell.
  • Multipotent: Form multiple differentiated cells (e.g., hematopoietic stem cells).
  • Pluripotent: Form all ~$200$ cell types in the human body (e.g., ESCs, IPSCs).
  • Totipotent: Form all cell types plus specialized tissues like the placenta.

Sources of Adult and Embryonic Stem Cells

• Adult Stem Cells (ASCs):
  • Bone Marrow: Hematopoietic stem cells for lymphoid (B/T cells) and myeloid (RBCs, platelets, etc.) lineages.
  • Intestinal Crypts: $LGR5+$ stem cells (Leucine Rich G-Protein Coupled Receptor $5$) and "reserve" $+4$ stem cells replace the epithelium every $3$-$5$ days. Paneth cells provide antimicrobial defense.
  • Embryonic Carcinoma (EC) Cells: Derived from germline tumors (Teratomas [benign] or Teratocarcinomas [malignant]). Highly plastic; can differentiate into neurons with retinoic acid ($10\,\text{mM}$ for $28\,\text{days}$).
• Embryonic Stem Cells (ESCs):
  • Derived from the inner cell mass (ICM) of a blastocyst.
  • Required growth on irradiated feeder cells (fibroblasts) or specific cytokines.
  • Technical Controversy: Contamination from feeder cells.
  • Ethical Controversy: Isolation necessitates destruction of the blastocyst.
• Tests for Pluripotency:
  • Formation of all tissues during normal development.
  • Injection into immunodeficient mice results in teratomas containing all three germ layers (ectoderm, mesoderm, endoderm).
  • GFP Chimera: Labeling ESCs with GFP and injecting them into a blastocyst leads to fluorescent tissues throughout the offspring body.

Advantages and Challenges of Therapeutic Applications

• ESC Status:
  • Advantages: Pluripotent.
  • Disadvantages: Teratoma formation, inefficient establishment ($1/10$ embryos), transplant rejection (foreign genome), genome instability.
  • In vivo success: Limited; a single reported success in populating mouse liver with GFP-hepatocytes.
• ASC Status:
  • Advantages: Less fastidious growth, multiply indefinitely, autologous transplants (no rejection).
  • In vivo success: Used for spinal cord injuries, heart tissue regeneration, diabetes, Crohn's, and Parkinson's disease.

Animal and Human Cloning

• Somatic Cell Nuclear Transfer ($SCNT$): Removing the nucleus from an egg cell (enucleation) and inserting a diploid nucleus from a donor adult cell.
• Cloning History:
  • Dolly the Sheep (1996): First mammal cloned from an adult cell.
  • CC (Copycat): First cloned pet cat.
  • Snuppy: Afghan hound survivor from $123$ surrogate mothers.
  • Extinct Species Work: Woolly Mammoth Cloning Project using CRISPR to create a hybrid with an Asian elephant.
• Inefficiency and Health Concerns:
  • Low Efficiency: Dolly required $277$ attempts; CC required $188$ embryos for one live birth.
  • Health Issues: Dolly suffered early-onset arthritis and cancer, dying early (suggesting she might have inherited the "biological age" of the $6$-year-old donor sheep).
• Types of Human Cloning:
  • Reproductive Cloning: Live-birth cloning (no evidence of successfully being done in humans; historical claims by Clonaid and South Korean researchers were hoaxes or retractions).
  • Therapeutic Cloning: Using $SCNT$ to generate patient-specific ESCs to differentiate into needed tissues (avoids rejection but involves ethical blastocyst destruction).

Bioethics in Stem Cell and Cloning Research

• Key Questions:
  • What is the moral status of a human embryo (person vs. property)?
  • Who owns the surplus frozen embryos from infertility treatments?
  • Scientific Freedom vs. Stewardship: Should experiments be performed just because they are technically possible?