Mod 3: BIO 302 Cell Biology: Membranes and Cell Motility

Membrane Structure, Functions, and Specialized Compartments

General Definition and Overview: Membranes serve as critical barriers between the cell and its external environment. In eukaryotic organisms, membranes also function as compartments to create specialized organelles.
Key Objectives of Membrane Biology:
- Define membrane properties that confer specific biological functions.
- Describe the maintenance of cell homeostasis through membrane functions.
- Analyze the passage of chemicals and macromolecules through the bilayer.
- Determine the role of membranes in the function of other organelles.
- Explain the Fluid Mosaic properties of the membrane.
Specific Membrane Locations and Faces:
- Plasma Membrane: The outer boundary of the cell.
- Organelle Membranes: Includes the Endoplasmic Reticulum (ER), Single membrane of the peroxisome, Inner and Outer mitochondrial membranes (separated by the intermembrane space and cristae), and Inner, Outer, and Thylakoid membranes of chloroplasts.
- Bilayer Structure: Consists of an Exoplasmic face (or Ectoplasmic face, facing the cell exterior or organelle lumen) and a Cytosolic face (or Protoplasmic face, closest to the cytosol).
Primary Membrane Functions:
1. Compartmentalization: Allows for specialized activities within specific regions.
2. Cellular Diversity: Separates different cell types and allows for diverse functional specializations.
3. DNA Protection: Nuclear membranes protect genetic material.
4. Chemical Reaction Spot: Provides a localized environment or scaffold for biochemical reactions to occur.
5. Enzymatic Protection: Keeps a cell's own proteases from damaging the rest of the cellular contents.
6. Scaffolding: Provides anchoring points for attachment.
7. Selective Permeation: Facilitates the exchange of specific molecules while preventing unrestricted access.
8. Transportation: Utilizes specialized proteins for the import and export of materials.
9. Signal Transduction: Receptors recognize extracellular ligands and transmit signals internally.
10. Cellular Contact: Mediates interaction and nutrient exchange between neighboring cells for homeostasis.
11. Energy Transduction: Converts chemical or solar energy into charge potentials to drive cell functions.

Lipid Composition and the Basic Units of Membrane Structure

Amphipathic Nature: The basic unit of the membrane is the phospholipid, which is amphipathic (possesses both a polar/hydrophilic head group and a non-polar/hydrophobic hydrocarbon tail).
- Polar Head Group: Attracted to aqueous environments inside and outside the cell.
- Lipid Composition: The makeup of lipids directly determines the membrane's function.
Hydrocarbon Tail Variations and Packing:
- Single-tail Phospholipid: Straight chain with a cone shape, allowing for circular groupings and spherical shapes.
- Double-tail Phospholipid: Typically contains one straight chain and one kinked chain (due to double bonds), allowing for a cylinder shape and flat groupings.
Saturation and Fluidity:
- Unsaturation (Double Bonds): Creates a "kink" in the tail. Results in a More Fluid membrane and a Lower Melting Point.
- Branched Chains: Increase fluidity and lower the melting point.
- Saturated Chains: Result in a Less Fluid (Gel Phase) membrane and a Higher Melting Point.
Leaflet Asymmetry and Curvature:
- Concave Head Groups: Associated with lipids like PE (Phosphatidylethanolamine), PA (Phosphatidic Acid), and Ceramide.
- Linear/Cylindrical Head Groups: Associated with PC (Phosphatidylcholine) and SM (Sphingomyelin).
- Convex Head Groups: Associated with PI (Phosphatidylinositol), GM (Gangliosides), PS (Phosphatidylserine), and PIP2 (Phosphatidylinositol 4,5-bisphosphate).
- High proportions of unsaturated acyl chains in the inner leaflet can favor convex bending, which is essential for processes like endocytosis.

The Fluid Mosaic Model and Molecular Techniques

The Fluid Mosaic Model: Characterizes the membrane as a dynamic structure where constituents are in constant motion.
- Molecular Movements: Includes flexing of hydrocarbon tails, rotation of lipid moieties, lateral movement within a leaflet, and the rarer "flipping" (transverse diffusion) of lipids from one leaflet to another.
- Evidence of Fluidity: Shown through membrane ruffling during movement, membrane pulling during ctenophore egg division, and the fusion of sperm and egg membranes.
Immunofluorescence: A technique where an antibody (Ab) is raised against a protein of interest and tagged with a fluorescent dye (fluorochrome), allowing visualization via microscopy.
Fluorescence Recovery after Photobleaching (FRAP):
- Proteins are labeled with fluorescent dye.
- A localized area is irradiated with a laser to "bleach" the fluorescence.
- The area is monitored for the return of fluorescence, which occurs due to the lateral migration of surrounding unbleached proteins.
Cell Fusion Experiments: Fusing a mouse cell (H-2 protein) and a human cell (HLA protein). Initially, proteins are separated on halves of the fused cell. Over several hours, or when warmed, those proteins disperse throughout the entire cell, proving membrane mobility.

Membrane Lipid Components and Carbohydrate Linkages

Three Main Lipid Classes:
1. Phospholipids: Composed of an R group, Phosphate ( $PO_4$ ), and a Lipid. Examples include Phosphoglycerides and Sphingomyelin.
2. Cholesterol: Contains a polar OH group; inserts into the membrane to regulate fluidity. In phospholipids with unsaturated tails, cholesterol decreases fluidity; in sphingolipid-rich domains, it may break tight packing and increase fluidity.
3. Glycolipids: Composed of a Sugar and a Lipid (e.g., Cerebroside).
Specific Lipid Composition by Tissue (Source: Alberts):
- Liver: 17% Cholesterol, 7% PE, 4% PS, 24% PC, 19% SM, 7% Glycolipids.
- Red Blood Cell: 23% Cholesterol, 18% PE, 7% PS, 17% PC, 18% SM, 3% Glycolipids.
- Mitochondria: 3% Cholesterol, 25% PE, 2% PS, 39% PC, 0% SM, Trace Glycolipids.
Membrane Carbohydrates:
- Typically covalently linked to lipids or proteins on the outer leaflet.
- N-glycosidic bond: Linkage with the amino acid Asparagine.
- O-glycosidic bond: Linkage with Threonine or Serine.
- Glycolipids and Blood Groups: Oligosaccharides on the glycolipids are unique to each blood group. The immune system raises antibodies against unfamiliar oligosaccharide markers.
Lipid Rafts: Complex structures involving cholesterol, proteins, phospholipids, and glycolipids. They cluster protein complexes (like receptors) in close proximity to facilitate efficient signal transduction.
Liposomes: Synthetic spherical bilipid compartments.
- Uses: Recombination experiments (introducing DNA), delivering insoluble chemicals, and drug delivery for chemotherapy.

Membrane Proteins and Spanning domains

Classes of Membrane Proteins:
1. Integral Proteins: Transmembrane proteins that penetrate the bilayer. They have both extracellular and cytoplasmic domains. They cannot be easily released and often form channels.
2. Peripheral Proteins: Located entirely outside the bilayer on either side. Associated with the membrane via interactions with lipid head groups or other proteins.
3. Lipid-Anchored Proteins:
  - GPI Anchor: Glycosylphosphatidylinositol attaches the protein to an inositol phospholipid via a carbohydrate.
  - Myristilation: Myrstyl anchor via an amide link between amino groups and fatty acids.
  - Farnesylation: Farnesyl anchor via a thioether linkage with cysteine.
Predicting Transmembrane Domains:
- Usually consists of an Alpha helix of 20-30 predominantly non-polar (hydrophobic) R groups.
- Hydropathy Plot: Predicts the hydrophobicity of peptide sections; high values indicate likely transmembrane segments.
- Specific Attractions: Positive charge amino acids (Lysine/Lys, Arginine/Arg) often interact with negative charge phosphatidylserine.

Ion Gradients and Transport Mechanisms

Standard Concentrations (Table 15-1):
- Squid Axon: Cell $K^+$ : $400\text{ mM}$ , Blood $K^+$ : $20\text{ mM}$ ; Cell $Na^+$ : $50\text{ mM}$ , Blood $Na^+$ : $440\text{ mM}$ ; Cell $Cl^-$ : $40-150\text{ mM}$ , Blood $Cl^-$ : $560\text{ mM}$ .
- Mammalian Cell: Cell $K^+$ : $139\text{ mM}$ , Blood $K^+$ : $4\text{ mM}$ ; Cell $Na^+$ : $12\text{ mM}$ , Blood $Na^+$ : $145\text{ mM}$ ; Cell $Cl^-$ : $4\text{ mM}$ , Blood $Cl^-$ : $116\text{ mM}$ .
Transport Types:
1. Simple Diffusion: Spontaneous, passive movement down an Electrochemical Gradient (concentration + charge). High permeability for $O_2$, NO, $CO_2$, and steroids. Low permeability for sugars, ions, and proteins.
2. Channels: Passive diffusion of ions. Includes Ungated Leak Channels, Voltage-Gated, Ligand-Gated, and Gap Junction Channels.
3. Facilitated Diffusion: Protein-mediated (uniporters) passive transport. Faster than passive diffusion, specific to molecules (e.g., GLUT1 for glucose), and limited by uniporter number.
4. Active Transport: Energetically unfavorable movement against a gradient; requires coupling with ATP hydrolysis, photo absorbance, or electron flow.
5. Cotransport (Secondary Active Transport): Movement against a gradient is driven by the movement of another ion down its gradient.
  - Symporter: Both molecules move in the same direction (e.g., Glucose/Na+ Symporter).
  - Antiporter: Molecules move in opposite directions (e.g., $Cl^-/HCO_3^-$ antiporter).

Specialized Transporters and Channels

Aquaporins: Specialized leak channels for water ( $H_2O$ ).
- Structure: Pore contains a hydrophobic residue-lined channel with a constriction of aromatic R groups. Asparagine (Asn76, Asn192) amido groups block proton ( $H^+$ ) permeation.
- AQP-1: Forms tetrameric assemblies.
- AQP-5: Primarily associated with tear production.
- AQP-0: Likely a gated aquaporin in the eye.
Voltage-Gated $K^+$ Channel: Six transmembrane domains per subunit. The H5-loop lines the channel; the S4 domain senses voltage changes to initiate a conformational opening.
Ligand-Gated $Na^+$ Channel (Acetylcholine Receptor): Five separate proteins with 4 transmembrane domains each. The M-2 subdomain (containing negatively charged Aspartic acid and Glutamic acid) lines the pore to attract Na+ and repel Cl-.
$Na^+/K^+ \text{ - ATPase}$ Pump:
1. E1 state: 3 intracellular $Na^+$ bind.
2. ATP hydrolysis phosphorylates the pump, changing it to E2 state.
3. $Na^+$ ions are released to the exterior; 2 $K^+$ ions bind.
4. Dephosphorylation returns the pump to E1 state, releasing $K^+$ inside.

Cellular Interactions and the Extracellular Matrix (ECM)

Extracellular Matrix (ECM): An organized structure of proteins and carbohydrates providing physical support, cushioning, and protection. Produced by fibroblasts.
Key ECM Proteins:
- Collagen: Most abundant protein (25% of body). Fibrous, high strength, proline and glycine rich. Bundled via covalent disulfide bridging.
- Proteoglycans: Protein-polysaccharide complexes with GAGs (glycosaminoglycans). Acidic sulfate groups bind cations and water to resist compression/provide cushion.
- Fibronectin: Dimeric protein with binding domains for heparin, collagen, and fibrin. Vital for cell migration.
- Laminin: Cross-shaped glycoprotein; binds cell receptors, proteoglycans, and collagen (Type IV in basal lamina).
Basal Lamina: A specialized sheet-like layer of ECM underlying epithelial/endothelial cells and surrounding muscle/fat cells for structural support and filtering.
Glycocalyx: A layer of oligosaccharide chains on the outer leaflet of intestinal lumen cells; serves as a barrier and coordinates cellular interactions.

Cell Adhesion and Junctional Complexes

Integrins: Major transmembrane proteins (alpha and beta subunits) mediating cell-to-ECM attachment.
- Focal Adhesions: Complexes of integrins anchored to the actin cytoskeleton through Vinculin, Actinin, and Talin. Paxillin facilitates signaling to the nucleus.
- Hemidesmosomes: Disk-shaped adhesion points anchoring cells to the basal lamina using integrins, BP180, and Plectin.
Non-Junctional Adhesion:
- Selectins: Calcium-dependent; bind oligosaccharides on other cells via a lectin binding domain (e.g., leukocyte trapping).
- Ig-like CAMs: Immunoglobulin-like proteins; calcium-independent binding.
- Cadherins: Calcium-dependent glycoproteins that join similar cells together (tissue-specific).
Junctional Adhesion:
1. Tight Junctions (Zonula Occludens): Barrier formed by Claudin, Occludin, and JAMS. Maintains blood-brain barrier and cell polarity.
2. Adherens Junctions (Adhesion Belts): Use Cadherin (extracellular) and Catenins (cytoplasmic) linked to the actin cytoskeleton.
3. Desmosomes (Macula Adherens): Disk-shaped, high-stress adhesion (skin, heart) using Desmogleins and Desmocollins; anchored to intermediate filaments via Plakoglobin and Desmoplakin.
4. Gap Junctions: Communicating channels made of Connexons (each composed of 6 Connexin subunits). Each Connexin has 4 alpha helix spanning domains.

The Cytoskeleton and Cell Motility

Three Primary Elements:
1. Microtubules: Rigid tubes of alpha and beta tubulin heterodimers. 13 protofilaments form a cylinder.
  - Polarity: Plus end (+, Beta subunit, fast-growing); Minus end (-, Alpha subunit, slow-growing).
  - Dynamic Instability: GTP hydrolysis to GDP in the Beta subunit causes the structure to close and eventually fall apart.
2. Microfilaments (Actin): Helical monomers of G-actin polymerizing into F-actin. Requires ATP. Polarized with a plus and minus end.
3. Intermediate Filaments: Ropelike fibers (Keratin, Lamin, Neurofilaments). Assembled from tetramers; no polarity due to reverse orientation of dimers.
Motor Proteins:
- Kinesin: Tetramer (2 heavy, 2 light chains). Moves anterograde (cell body toward axon/+ end).
- Dynein: Moves retrograde (axon toward cell body/- end).
Cilia and Flagella: Utilize a 9+2 arrangement of microtubules.
- Includes central sheath, radial spokes, and Nexin bridges linking doublets.
- A Tubule is a complete microtubule; B Tubule is partial.
- Dynein arms provide the sliding force for movement.
Muscle Contraction (The Sarcomere):
- Structure: Z-line, I-band, A-band, H-zone.
- Capping/Anchoring: Cap Z, Tropomodulin, Titin.
- Mechanism: ATP-driven sliding of actin against myosin. Calcium ( $Ca^{2+}$ ) binds to Troponin, causing Tropomyosin to shift and expose myosin-binding sites on actin.
Cell Crawling and Actin Polymerization:
- Arp2/3 Complex: Nucleates branched actin polymerization; activated by WASP.
- Treadmilling: Simultaneous polymerization at the ATP-bound plus end and dissociation at the ADP-bound minus end (enhanced by Cofilin).
- Example: Listeria monocytogenes: Secretes the protein ActA to hijack host actin, forming "actin tails" for propulsion through the cytoplasm and into adjacent cells.