Biotechnology: Class 11 — Chapter 5: Cellular Processes

• Receptor: A protein located on the cell surface, inside the cytoplasm, or in the nucleus. A cell can only respond to a signal if it possesses the corresponding receptor.
• Ligand: A specific chemical messenger that binds to a receptor, inducing conformational changes that initiate an intracellular message relay system.

Depending on the proximity of the sender and recipient cells, signaling is classified into three major categories:

• Anabolic pathways: Endergonic processes that consume energy to synthesize larger, complex molecules from smaller ones. Formula: $$\text{Useful energy} + \text{Small molecules} \rightarrow \text{Complex molecules}$$ Examples include the synthesis of glucose, proteins, lipids, and DNA.
• Catabolic pathways: Exergonic processes that break down larger molecules, releasing energy to produce ATP and reducing equivalents. Formula: $$\text{Fuel (carbohydrate, protein, fats)} \rightarrow \text{CO}_2 + \text{H}_2\text{O} + \text{Useful energy}$$

• Carbohydrate Metabolism: Glucose is metabolized to pyruvate via glycolysis. Under aerobic conditions, pyruvate enters the mitochondria, converts to acetyl CoA, and is oxidized to $\text{CO}_2$ and $\text{H}_2\text{O}$ via the citric acid cycle. Under anaerobic conditions, pyruvate is reduced to lactic acid.
• Lipid Metabolism: Long-chain fatty acids from diet or synthesized from acetyl CoA are oxidized to acetyl CoA via the $\beta$-oxidation pathway, or esterified with glycerol to form triacylglycerols (fats) stored in adipose tissue.
• Amino Acid Metabolism: Non-essential amino acids are synthesized via transamination (transfer of amino nitrogen to a carbon skeleton). Essential amino acids must be obtained from diet. Excess amino nitrogen is excreted as urea via deamination, while remaining carbon skeletons are oxidized or used in gluconeogenesis.

A universal cytosolic catalytic pathway converting one molecule of glucose (6C) into two molecules of pyruvate (3C) through ten sequential enzymatic steps:

1. Phosphorylation of Glucose: Glucose is phosphorylated to glucose-6-phosphate by hexokinase (uses 1 ATP; irreversible; allosterically inhibited by its product).
2. Isomerization: Glucose-6-phosphate is converted to fructose-6-phosphate by phosphoglucose isomerase.
3. Second Phosphorylation: Fructose-6-phosphate is phosphorylated to fructose-1,6-bisphosphate by phosphofructokinase (PFK) (uses 1 ATP; irreversible; major regulatory site).
4. Cleavage: Fructose-1,6-bisphosphate is cleaved by aldolase into two triose phosphates: glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP).
5. Triose Isomerization: DHAP is reversibly converted to GAP by triose phosphate isomerase.
6. Oxidation & Phosphorylation: GAP is oxidized to 1,3-bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase (produces 1 NADH + $\text{H}^+$ per GAP; uses inorganic phosphate $\text{P}_i$).
7. First Substrate-Level Phosphorylation: Phosphate is transferred from 1,3-bisphosphoglycerate to ADP by phosphoglycerate kinase, yielding ATP and 3-phosphoglycerate.
8. Isomerization: 3-phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerate mutase.
9. Dehydration: 2-phosphoglycerate is dehydrated to phosphoenolpyruvate (PEP) by enolase (requires $\text{Mg}^{2+}$ or $\text{Mn}^{2+}$).
10. Second Substrate-Level Phosphorylation: PEP is converted to pyruvate by pyruvate kinase, yielding ATP (irreversible; regulatory step).

Net Glycolytic Reaction:

Fates of Pyruvate:

• Homolactic Fermentation (Anaerobic in muscles/RBCs): Pyruvate is reduced to lactate by lactate dehydrogenase, regenerating $\text{NAD}^+$: $$\text{Glucose} + 2\text{ADP} + 2\text{P}_i \rightarrow 2\text{Lactate} + 2\text{ATP} + 2\text{H}_2\text{O} + 2\text{H}^+$$
• Alcoholic Fermentation (Anaerobic in yeast): Pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase (requires TPP), then reduced to ethanol by alcohol dehydrogenase, regenerating $\text{NAD}^+$.
• Aerobic Oxidation: Pyruvate is transported into the mitochondrial matrix and converted to acetyl CoA by the pyruvate dehydrogenase complex (PDH): $$\text{Pyruvate} + \text{CoA} + \text{NAD}^+ \rightarrow \text{Acetyl CoA} + \text{CO}_2 + \text{NADH} + \text{H}^+$$

An 8-step cyclic pathway in the mitochondrial matrix that oxidizes acetyl CoA to release $\text{CO}_2$ and generate reducing equivalents ($\text{NADH}$, $\text{FADH}_2$) and $\text{GTP}$:

1. Condensation: Acetyl CoA (2C) condenses with oxaloacetate (4C) to form citrate (6C), catalyzed by citrate synthase.
2. Isomerization: Citrate is isomerized to isocitrate via cis-aconitate by aconitase.
3. First Oxidative Decarboxylation: Isocitrate is oxidized and decarboxylated to $\alpha$-ketoglutarate (5C) by isocitrate dehydrogenase, producing $\text{NADH} + \text{H}^+$ and releasing $\text{CO}_2$.
4. Second Oxidative Decarboxylation: $\alpha$-ketoglutarate undergoes oxidative decarboxylation to succinyl CoA (4C) by the $\alpha$-ketoglutarate dehydrogenase complex, producing $\text{NADH} + \text{H}^+$ and releasing $\text{CO}_2$.
5. Substrate-Level Phosphorylation: Succinyl CoA is converted to succinate by succinyl-CoA synthetase, generating $\text{GTP}$ from $\text{GDP} + \text{P}_i$.
6. Oxidation: Succinate is oxidized to fumarate by succinate dehydrogenase (the only membrane-bound enzyme of the cycle, containing FAD and Fe-S), producing $\text{FADH}_2$.
7. Hydration: Fumarate is hydrated to L-malate by fumarase.
8. Regeneration: L-malate is oxidized to oxaloacetate by malate dehydrogenase, producing $\text{NADH} + \text{H}^+$.

Overall Reaction of the TCA Cycle:

Located in the inner mitochondrial membrane, the ETC transfers electrons from $\text{NADH}$ and $\text{FADH}_2$ through four large protein complexes to oxygen, pumping protons to generate a proton gradient used by ATP synthase to synthesize ATP:

• Complex I (NADH-Q-oxidoreductase): Accepts electrons from cytosolic/matrix $\text{NADH}$ and transfers them to coenzyme Q (ubiquinone), pumping protons into the intermembrane space.
• Complex II (Succinate-Q-reductase): Accepts electrons from succinate via $\text{FADH}_2$ and transfers them to coenzyme Q. Does not pump protons.
• Complex III (Q-cytochrome C oxidoreductase): Transfers electrons from reduced coenzyme Q to cytochrome c, pumping protons.
• Complex IV (Cytochrome C oxidase): Transfers electrons from cytochrome c to $\text{O}_2$ (the final electron acceptor), reducing it to $\text{H}_2\text{O}$ and pumping protons.
• ATP Synthase: Uses the proton motive force (pmf) generated by the pH and electrical gradient to synthesize ATP as protons flow back into the matrix.
• ATP Yield: Oxidation of 1 $\text{NADH}$ yields 2.5 ATP; oxidation of 1 $\text{FADH}_2$ yields 1.5 ATP.

The process by which solar energy is captured and converted into chemical energy:

1. Light Reactions (Photochemical Phase)

Occurs in the thylakoid membranes of chloroplasts, utilizing two photosystems:

• Photosystem I (PSI): Reaction center chlorophyll a responds to 700 nm ($P_{700}$).
• Photosystem II (PSII): Reaction center chlorophyll a responds to 680 nm ($P_{680}$).
• Non-cyclic Photophosphorylation: PSII absorbs light $\rightarrow$ excited electron goes to pheophytin $\rightarrow$ plastoquinone (PQ) $\rightarrow$ cytochrome $b_6f$ complex (pumps protons) $\rightarrow$ plastocyanin (PC) $\rightarrow$ PSI. Light excitation of PSI transfers electrons to ferredoxin (Fd) $\rightarrow$ ferredoxin-NADP reductase (FNR) reduces $\text{NADP}^+$ to $\text{NADPH} + \text{H}^+$. Electrons lost from PSII are replenished by photolysis of water at the oxygen-evolving complex: $$2\text{H}_2\text{O} \rightarrow 4\text{H}^+ + 4e^- + \text{O}_2$$
• Cyclic Photophosphorylation: Occurs when only PSI is active. Electrons from excited $P_{700}$ flow through $A_0 \rightarrow A_1 \rightarrow \text{Fe-S} \rightarrow \text{Fd} \rightarrow \text{Cyt } b_6f \rightarrow \text{PC} \rightarrow P_{700}$. Generates a proton gradient for ATP synthesis, but produces no $\text{NADPH}$ or $\text{O}_2$.

2. Dark Reactions (Calvin Cycle / C3 Pathway)

Occurs in the stroma of chloroplasts in three distinct stages:

• Stage I: Carbon Fixation: $3\text{ CO}_2$ molecules condense with three 5-carbon acceptors (Ribulose 1,5-bisphosphate, RuBP) to form six molecules of 3-phosphoglycerate (PGA), catalyzed by the bifunctional enzyme RuBisCO.
• Stage II: Reduction: Six PGA are phosphorylated using 6 ATP to form 1,3-bisphosphoglycerate, which is then reduced using 6 $\text{NADPH}$ to form six molecules of glyceraldehyde-3-phosphate (GAP). One GAP molecule exits the cycle for starch/sucrose synthesis.
• Stage III: Regeneration of RuBP: The remaining five GAP molecules (15C) are rearranged into three molecules of ribulose-5-phosphate, which are then phosphorylated using 3 ATP to regenerate three RuBP molecules.

3. C4 Pathway (Hatch-Slack Pathway)

An evolutionary adaptation in plants like maize, sorghum, and sugarcane to minimize photorespiration, utilizing Kranz anatomy (mesophyll and bundle sheath cells):

• Mesophyll Cells: Atmospheric $\text{CO}_2$ is converted to bicarbonate and fixed onto phosphoenolpyruvate (PEP, 3C) by PEP carboxylase (which lacks oxygenase activity) to form oxaloacetate (OAA, 4C). OAA is reduced to malate and transported to bundle sheath cells.
• Bundle Sheath Cells: Malate is decarboxylated to release $\text{CO}_2$ (which enters the Calvin cycle via RuBisCO) and pyruvate (3C). Pyruvate is transported back to mesophyll cells, where it is phosphorylated to regenerate PEP.

A typical eukaryotic cell cycle takes approximately 24 hours and is divided into two major phases:

1. Interphase (Resting Phase): Characterized by cell growth and DNA replication. Divided into:
   • G1 Phase (Gap 1): Cell is metabolically active and grows continuously, accumulating RNA, proteins, and organelles, but does not replicate its DNA.
   • S Phase (Synthesis): DNA replication occurs in the nucleus. DNA content doubles ($2C \rightarrow 4C$), but chromosome number remains unchanged ($2n$). Centrioles duplicate in the cytoplasm.
   • G2 Phase (Gap 2): Synthesis of proteins (e.g., tubulin) and organelles continues, preparing the cell for mitosis.
   • G0 Phase (Quiescent Stage): Cells exit the G1 phase to enter an inactive, non-dividing, differentiated state (e.g., heart cells, neurons).
2. Mitotic (M) Phase: The phase of actual cell division, consisting of:
   • Karyokinesis (Nuclear Division): Divided into four stages:
     1. Prophase: Chromatin condenses into distinct chromosomes. Centrioles move to opposite poles, initiating mitotic spindle assembly. Nucleolus, nuclear envelope, Golgi, and ER disappear.
     2. Metaphase: Nuclear envelope is completely gone. Chromosomes align at the equator (metaphase plate). Spindle fibers attach to the kinetochores on the centromere of each chromosome.
     3. Anaphase: Centromeres split, and sister chromatids are pulled toward opposite poles.
     4. Telophase: Chromosomes reach opposite poles and decondense. Nuclear envelope reforms, and nucleolus, Golgi, and ER reappear.
   • Cytokinesis (Cytoplasmic Division):
     • Animal Cells: A contractile ring of actin filaments forms a furrow in the plasma membrane, which deepens and divides the cytoplasm.
     • Plant Cells: Due to the rigid cell wall, division occurs via the formation of a precursor called the cell plate from the center outward.

A specialized double-division process that reduces the chromosome number by half ($2n \rightarrow n$), producing four genetically diverse haploid gametes:

• Meiosis I (Reductional Division): Separates homologous chromosomes.
  • Prophase I: The longest and most complex phase, divided into five substages:
    1. Leptotene: Chromosomes become visible as thin threads.
    2. Zygotene: Homologous chromosomes pair up (synapsis), forming a synaptonemal complex. The paired structure is called a bivalent or tetrad.
    3. Pachytene: Recombination nodules appear. Crossing over occurs between non-sister chromatids of homologous chromosomes, facilitating genetic recombination.
    4. Diplotene: Synaptonemal complex dissolves; homologous chromosomes separate except at crossover sites called chiasmata.
    5. Diakinesis: Chiasmata undergo terminalization. Chromosomes fully condense, spindle assembly completes, and the nuclear envelope breaks down.
  • Metaphase I: Bivalents align on the equatorial plate; spindle fibers attach to homologous chromosomes.
  • Anaphase I: Homologous chromosomes separate and move to opposite poles, but sister chromatids remain attached at their centromeres.
  • Telophase I & Cytokinesis: Nuclear membrane reforms, followed by cytoplasmic division, yielding two haploid cells.
  • Interkinesis: A short-lived gap phase between Meiosis I and II with no DNA replication.
• Meiosis II (Equational Division): Separates sister chromatids, resembling a normal mitotic division (Prophase II, Metaphase II, Anaphase II, Telophase II), resulting in four haploid daughter cells.

Stem cells are unspecialized cells capable of dividing to produce one daughter cell that remains a stem cell and another that differentiates. Their differentiation potential is termed cell potency:

Embryonic stem cells (ESCs) were historically the primary source of pluripotent cells, but their extraction requires the destruction of embryos, presenting severe ethical constraints.

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Engineering Methodology & Mechanism: In 2006, Shinya Yamanaka developed iPSC technology, which reprograms fully differentiated adult somatic cells back into a pluripotent state. This is achieved by introducing and expressing a specific cocktail of transcription factors (Yamanaka factors). iPSCs bypass embryonic ethical issues and allow the generation of patient-matched pluripotent cells, eliminating the risk of transplant rejection.

Cell migration is a highly integrated, cyclical process involving four major steps:

1. Extension (Polarization): The cell establishes a front-rear axis in response to directional cues (chemical, electrical, or mechanical). Actin filaments polymerize and branch at the leading edge, pushing the plasma membrane forward to form protrusions (e.g., pseudopodia).
2. Adhesion: The newly formed protrusions attach to the extracellular matrix (substratum) via specialized adhesion complexes.
3. Translocation: The cell body contracts and slides forward, driven by actomyosin contractile forces.
4. De-adhesion: Adhesion complexes at the rear of the cell disassemble (regulated by proteases and phosphatases), allowing the trailing edge to retract.

• Gastrulation & Organogenesis: Massive, coordinated cell migration forms the three primary germ layers and subsequent organs during embryonic development.
• Homeostasis & Wound Healing: Tissue repair requires the migration of fibroblasts and epithelial cells. Inflammation involves the migration of immune cells from lymph nodes through circulation to the site of injury.
• Metastasis: Pathological migration where cancer cells lose cell-cell adhesion, acquire invasive properties, and migrate from the primary tumor site to colonize distant organs.