Cell Bio Module 6: Cell Cycle Regulation, Mitosis, Stem Cells, and Cloning

Micrography and Visualizing the Cell Cycle

• The Art of Microscopy: A definitive example of the beauty of cellular processes is found in the fluorescence micrograph of a newt lung cell during mitosis.

  • Staining Details:

    • Centrosomes: Stained in magenta.

    • Microtubules: Stained in green.

    • Chromosomes: Stained in blue.

    • Keratin Intermediate Filaments: Stained in red.

  • Historical Context: The image was captured in the year $2000$ by Dr. Alexey Khodjakov at the Wadsworth Center, New York Department of Health. It was taken using a modified Nikon TE200 regular fluorescence microscope (not a confocal microscope) and won first prize in the annual Olympus-Nature Light microscopy imaging competition.

Overview of the Eukaryotic Cell Cycle and Phases

• Definition: The cell cycle is the process by which cells reproduce by duplicating their contents and dividing in two.

• Duration Variations: The timing is cell-specific:

  • Fly Embryo Cells: Approximately $8\,\text{min}$, representing extremely rapid division.

  • Mammalian Liver Cells: Greater than $1\,\text{year}$, as these cells divide infrequently.

• Major Phases:

  • Interphase: The longest portion of the cycle, where the cell grows and prepares for division. It is subdivided into $G_1$, $S$, and $G_2$.

  • Mitotic (M) Phase: A relatively short phase where nuclear and cytoplasmic division (cytokinesis) occurs.

• $G_0$ (Quiescent) Phase: An offshoot of the cycle where cells enter a resting state. They are not actively dividing but can re-enter the cycle upon receiving specific stimuli.

  • Permanent $G_0$: Neurons are "amitotic," meaning they stay in $G_0$ permanently and never re-enter the cell cycle.

Interphase Sub-phases and Growth

• $G_1$ (Gap Phase 1): Considered the first growth phase. Features include:

  • Cell enlargement after exiting mitosis.

  • Monitoring the environment (nutrients, safety) to ensure conditions are favorable for DNA replication.

• $S$ Phase (Synthesis Phase): The period during which DNA is replicated (DNA synthesis).

• $G_2$ Phase (Gap Phase 2): Acts as a "safety gap."

  • Ensures DNA replication is complete and accurate.

  • Prevents detrimental consequences for daughter cells that would arise from incomplete or damaged DNA duplication.

Detailed Stages of Mitosis and Cytokinesis

• Prophase:

  • Chromosomes condense and become visible.

  • Centrosomes (microtubule organizing centers) migrate toward opposite poles.

  • The mitotic spindle begins to form.

• Prometaphase:

  • The nuclear envelope breaks down.

  • Microtubules attach to chromosomes at the kinetochores.

  • Cohesin is shed from chromosome arms.

• Metaphase:

  • Chromosomes align at the "metaphase plate" (center of the cell).

  • Bi-orientation is established: each sister chromatid is attached to spindle fibers from opposite poles.

• Anaphase:

  • Sister chromatids are pulled apart toward opposite poles.

  • The spindle elongates to further separate the genetic material.

• Telophase:

  • Chromosomes arrive at poles and begin to de-condense.

  • The nuclear envelope reforms around the new nuclei.

  • The mitotic spindle breaks down.

• Cytokinesis:

  • Animal Cells: A contractile ring composed of actin and myosin (similar to muscle structure) contracts to create a cleavage furrow, pinching the cells apart.

  • Plant Cells: A cell plate (precursor to the cell wall) forms to separate the daughter cells.

Molecular Structure of the Mitotic Apparatus

• The Mitotic Apparatus: Observed during metaphase, consisting of the football-shaped spindle and tufts of microtubules called astral microtubules.

• Three Classes of Microtubules: All are structurally identical microtubules but serve distinct functions:

  • Kinetochore Microtubules (Pink): Directly interact with and bind to the chromosomes.

  • Polar Microtubules (Dark Purple/Blue): Do not touch chromosomes; they overlap and interact with each other in the center of the spindle.

  • Astral Microtubules (Yellow): Form tufts at the ends of the apparatus, anchoring it and assisting in elongation.

• Importance of Bi-orientation: Each sister chromatid pair must attach to microtubules from opposite poles. Failure leads to Chromosome Aneuploidy.

  • Example: Down's Syndrome is caused by trisomy of chromosome $21$ due to unequal distribution during gamete development.

Mechanisms of DNA-Microtubule Attachment

• Kinetochore Regions: Large protein complexes that assemble on the centromere DNA.

  • Inner Kinetochore: Associated directly with centromeric DNA, which is wrapped around histones (nucleosomes).

  • Outer Kinetochore: Interacts with the plus (+$) ends of microtubules.\n * **Corona:** A region outside the outer kinetochore that microtubules must traverse.\n* **Key Proteins of the Kinetochore:**\n * **CENP-A:** A specific variant of Histone 3 (H3) that marks the kinetochore region for the mitotic apparatus.\n * **Ndc80 Complex:** A rigid, sleeve-like multiprotein complex that connects the microtubule to the inner kinetochore. It is the recruitment site for motor proteins.\n\n# Regulation of Bi-Orientation via Aurora B Kinase\n\n* **The Chromosome Passenger Complex (CPC):** Contains **Aurora B Kinase**.\n* **Under Low Tension (Incorrect Orientation):**\n * Aurora B kinase is positioned to phosphorylate the outer kinetochore proteins (like Ndc80).\n * Phosphorylation results in a weak association between the microtubule and the kinetochore.\n* **Under High Tension (Bi-Orientation):**\n * Mechanical tension pulls the outer kinetochore complex away from the inner kinetochore (and thus away from Aurora B).\n * Aurora B can no longer phosphorylate Ndc80.\n * Unphosphorylated Ndc80 forms a very strong, stable association with the microtubule.\n\n# Chromosome Capture and Congression\n\n* **Model: Search and Capture**\n 1. **End Capture:** A growing microtubule's end directly hits the kinetochore region.\n 2. **Side Capture:** If a microtubule misses the end-on target, kinetochore proteins interact with the side of the microtubule. \n* **Motor Protein Movement:**\n * **Dynein-Dynactin:** Moves the chromosome toward the minus (-) end (the spindle pole).\n * **Kinesin-7 (CENP-E):** Helps hold onto growing microtubules during bi-orientation.\n* **Congression (Metaphase Alignment):** Chromosomes undergo bi-directional oscillations. \n * **Shortening Side:** Kinesin-13 stimulates disassembly at the plus (+) end; Dynein pulls toward the pole.\n * **Lengthening Side:** Kinesin-7 maintains the connection as microtubules polymerize.\n * **Kinesin-4 (Chromokinesin):** Associated with chromosome arms, pushing them away from the poles toward the center.\n\n# Forces in Anaphase A and B\n\n* **Treadmilling in Metaphase:** Microtubules maintain constant length because the rate of subunit addition at the plus (+$) end equals the subtraction at the minus ($-) end. Subunits appear to move toward the poles.\n* **Anaphase A (Chromosome Movement):**\n * Powered by rapid microtubule shortening.\n * **Kinesin-13** acts at both the plus (+$) and minus ($-) ends to accelerate disassembly, pulling chromosomes to the poles.\n* **Anaphase B (Spindle/Cell Elongation):**\n * **Polar Microtubule Action:** Dual-head **Kinesin-5** motors walk toward the plus (+) ends of overlapping polar microtubules, pushing them apart.\n * **Astral Microtubule Action:** **Dynein** motors anchored to the plasma membrane walk toward the minus (-) ends of astral microtubules, pulling the poles toward the cell periphery.\n\n# The Biochemical Engine of the Cell Cycle Control System\n\n* **Experimental Evidence:** Fusing a mitotic cell with an interphase cell (G_1$,$S$, or$G_2) creates a **Heterokaryon**. The interphase chromatin begins to condense, proving that a cytoplasmic factor induces mitosis.\n* **Mitosis Promoting Factor (MPF):** A dimer consisting of:\n 1. **Cyclin-Dependent Kinase (Cdk):** A serine/threonine kinase whose levels remain constant.\n 2. **Cyclin:** A regulatory protein whose levels cycle via synthesis and degradation.\n* **Cyclin Dynamics:** Specific cyclins trigger specific phases. After they perform their role, they are tagged with **Ubiquitin** and degraded by **Proteasomes**.\n* **APC/C (Anaphase-Promoting Complex/Cyclosome):** A ubiquitin ligase that targets mitotic cyclins and anaphase inhibitors (securin) for degradation to allow the exit from mitosis.\n\n# Phase-Specific Regulation: G1 to S Transition\n\n* **Retinoblastoma (RB) and E2F Pathway:**\n 1. In early G_1, the **RB protein** binds to the **E2F Transcription Factor**, keeping it inactive.\n 2. As environment signals trigger growth, **Cyclin D/Cdk4** or **Cyclin D/Cdk6** accumulate.\n 3. These dimers phosphorylate RB.\n 4. Phosphorylated RB releases E2F, which enters the nucleus to initiate transcription of genes for S-phase (DNA polymerase, etc.) and **Cyclin E**.\n 5. **Cyclin E/Cdk2** further phosphorylates RB, creating a positive feedback loop.\n* **Retinoblastoma Disease:** A pediatric eye cancer discovered in children under 3\,\text{years}$old. A mutation in the$RB gene means E2F is always active, leading to uncontrolled cell division and tumor formation.\n\n# Initiation of S-Phase and DNA Replication\n\n* **Sic1 Inhibitor:** S$-phase cyclin-Cdks are made in$G_1 but are kept inactive by the inhibitor protein **Sic1**.\n* **Activation:** G_1 cyclins phosphorylate Sic1. This tags Sic1 for ubiquitination by the **SCF complex**, leading to its degradation by the proteasome.\n* **Replication Origins:** \n * Pre-replication complexes assemble in early/mid G_1 but remain inactive.\n * Active S-phase cyclin-Cdks phosphorylate origin components, causing the DNA to unwind and replication to initiate.\n\n# Cell Cycle Checkpoints and the Guardian of the Genome\n\n* **The Checkpoint Network:**\n * **Un-replicated DNA Checkpoint (G2):** Mediated by **ATR** and **Chk1**, which inhibit **Cdc25c** to prevent entry into mitosis.\n * **Spindle Assembly Checkpoint (M):** Mediated by **Mad2**, which inhibits the proteasome from degrading **Securin** (keeping sister chromatids together) until all chromosomes are aligned.\n * **Chromosome Segregation Checkpoint (Late M):** Inhibits **Cdc14** if chromosomes are not delivered correctly to poles.\n* **p53 (DNA Damage Checkpoints):** Often called the "Guardian of the Genome."\n * There are 4$DNA damage checkpoints ($G_1$, just before$S$, during$S$, and$G_2).\n * When DNA damage is detected, **ATM/R** stabilizes **p53**.\n * p53$acts as a transcription factor to induce **$p21\,CIP**.\n * p21\,CIP$is a powerful Cdk inhibitor that halts the cell cycle to allow for DNA repair. Mutations in$p53 are heavily implicated in many cancers.\n\n# Stem Cell Biology: Potency and Properties\n\n* **Two Core Properties:**\n 1. **Self-Renewal:** Asymmetric division where one cell remains a stem cell and the other becomes a progenitor.\n 2. **Differentiation:** The ability to develop into specialized cell types.\n* **Levels of Potency:**\n * **Unipotent:** Forms only one differentiated cell type.\n * **Multipotent:** Forms multiple types (e.g., Hematopoietic stem cells).\n * **Pluripotent:** Can form all 200 cell types of the body (e.g., ESCs).\n * **Totipotent:** Can form all body cells plus extra-embryonic tissues like the placenta.\n* **Nobel Prize 2012:**\n * **John Gurdon:** Pioneered Somatic Cell Nuclear Transfer (SCNT).\n * **Shinya Yamanaka:** Discovered that expressing four genes (**OCT3/4, SOX2, KLF4, c-MYC**) can turn adult cells into **Induced Pluripotent Stem Cells (iPSCs)**.\n\n# Sources of Stem Cells\n\n* **Adult Stem Cells (ASCs):**\n * **Bone Marrow:** Hematopoietic stem cells produce lymphoid (T and B cells) and myeloid (RBCs, platelets, etc.) lineages.\n * **Intestinal Crypts:** LGR5+$stem cells replace the epithelium every$3\text{--}5\,\text{days}$.$+4 cells serve as a reserve. \n* **Embryonic Carcinoma (EC) Cells:** Derived from germline tumors (sperm or egg). \n * **Teratoma:** Benign tumor; can contain hair and teeth.\n * **Teratocarcinoma:** Malignant.\n* **Embryonic Stem Cells (ESCs):** Derived from the **Inner Cell Mass** of a blastocyst.\n * Isolated cells require "feeder layers" of irradiated fibroblasts or specific cytokines to grow.\n * **Controversies:** Destruction of the blastocyst (ethical) and potential fibroblast contamination (scientific).\n\n# Somatic Cell Nuclear Transfer (SCNT) and Cloning\n\n* **Process:**\n 1. An egg cell is **enucleated** (nucleus removed).\n 2. A nucleus from an adult donor cell (diploid) is inserted into the egg.\n 3. The egg is stimulated to develop.\n* **Cloning History:**\n * **Dolly the Sheep (1996$):** The first mammal cloned from an adult cell. It took$277 attempts.\n * **CopyCat (CC):** The first cloned pet cat.\n * **Snuppy:** An Afghan hound; the lone survivor from 123 surrogate mothers.\n * **Idaho Gem:** A cloned mule (mules are normally sterile).\n * **Primate Cloning (2017$$):** Chinese scientists successfully cloned monkeys, reigniting human cloning debates.

• Woolly Mammoth Project: Using CRISPR technology to insert mammoth genes into the Asian elephant genome (a hybrid approach).

Reproductive vs. Therapeutic Cloning

• Reproductive Cloning (Live-Birth Cloning):

  • Extremely inefficient: Only a fraction of embryos reach the blastocyst stage or result in live births.

  • Health Concerns: Dolly died early of arthritis and cancer. Cloned mice often have shorter lifespans.

• Therapeutic Cloning:

  • Generating ESCs from a patient's own somatic cells using SCNT.

  • Advantage: Allows for autologous transplants, eliminating the risk of transplant rejection because the DNA matches the patient.

  • Disadvantage: Still requires the creation and destruction of a blastocyst, posing an ethical dilemma.

Bioethical Considerations

• Moral Status of the Embryo: Is it a person or property?

• Surplus Embryos: Thousands of embryos are frozen from infertility treatments; the question remains of who owns them and how long they can be stored.

• Scientific Stewardship: The debate over whether experiments should be conducted simply because they are technologically possible.