Cytosol & Cytoskeleton (Comprehensive Study Notes)
Page 1 – Introductory Information
• Topic: Cellular Organelles and Functions – focus on Cytosol & Cytoskeleton (Part 1).
• Lecturer: Alex R. B. Thomsen, BSc, MSc, PhD.
Page 2 – Generalized Animal Cell (Visual Overview)
Listed organelles/structures normally found in a typical animal cell:
• Centrioles
• Lysosome
• Endosome
• Clathrin-coated vesicle & clathrin-coated pit
• Cytosol
• Nuclear envelope, nucleolus, chromatin
• Rough endoplasmic reticulum (RER) with ribosomes
• Smooth endoplasmic reticulum (SER)
• Golgi apparatus
• Peroxisome
• Mitochondrion
• Cytoskeletal elements
• Cell membrane (plasma membrane)
Page 3 – Cytosol Components
• Water ≈ 70\% (pH 7.0\text{–}7.4).
• Ions: K^+, Na^+, Cl^-, Mg^{2+}, Ca^{2+}, HCO_3^-.
• Small organic molecules: amino acids, carbohydrates, lipids, nucleic acids, inositol phosphates, etc.
• Proteins: structural, enzymes, signalling molecules, degradation machinery, etc.
Page 4 – Cytosol Functions
• Signal transduction – second messengers, protein phosphorylation, protein–protein interactions.
• mRNA translation / protein biosynthesis (free ribosomes).
• Major biochemical pathways: pentose-phosphate pathway, glycolysis, gluconeogenesis.
• Proteasomal degradation (ubiquitin–proteasome system).
• Host environment for cytoskeleton: microfilaments, microtubules, intermediate filaments.
Page 5 – Cytosol: “A Crowded Place”
• Illustration (David Goodsell) shows molecular crowding; macromolecules occupy most volume leaving restricted free water.
• Biological consequence: macromolecular crowding affects diffusion rates, reaction kinetics, and protein folding.
Page 6 – Cryo-Electron Tomography: Cytosol–ER Interface
Depicted elements (rotated +90^{\circ} view):
• Free ribosomes vs. ER-bound ribosomes
• Endoplasmic reticulum membrane (E)
• Cdc48 (AAA-ATPase, involved in protein quality control)
• 26S proteasome complexes
• Highlights dynamic exchange between cytosolic translation, ER translocation, and cytosolic degradation.
Page 7 – Cryo-ET: Cytosol–Nuclear Envelope Interface
Color key:
• White = nuclear envelope
• Magenta = nuclear pore complexes
• Green = microtubules
• Red = actin & intermediate filaments
• Light blue = large ribosomal subunit
• Light yellow = small ribosomal subunit
• Golden yellow = nuclear interior density
• Demonstrates spatial coordination of cytoskeletal tracks with nucleo-cytoplasmic transport.
Page 8 – Cytoskeletal Components
Microfilaments (Actin)
Microtubules (Tubulin)
Intermediate filaments
Numerous accessory & regulatory proteins unique to each filament system
Page 9 – Cytoskeletal Functions
• Maintain cell shape & mechanical resistance.
• Generate whole-cell movement (crawling, swimming).
• Contraction (muscle & non-muscle).
• Rapid shape change (e.g., lamellipodia, cytokinesis).
• Provide structural integrity & spatial organization of organelles.
• Enable intracellular transport of vesicles/organelles along tracks.
Page 10 – Actin: Core Biochemistry
• Monomer: G-actin (globular).
• Polymer: F-actin (filament). Features:
– Polarized: plus (barbed, fast-growing) end vs. minus (pointed, disassembling) end.
– Requires ATP and Mg^{2+} for polymerization.
• Microfilament diameter: \approx 8\,\text{nm} (termed “thin filament” in muscle).
• Capping proteins can stabilize both ends; isoform expression is tissue specific.
Page 11 – Major Actin Isoforms (Microfilaments)
• \alpha-skeletal – skeletal muscle
• \alpha-cardiac – cardiac muscle
• \alpha-vascular – vascular smooth muscle
• \gamma-enteric – visceral (gut) smooth muscle
• \beta-cytoplasmic – non-muscle cells
• \gamma-cytoplasmic – non-muscle cells
Page 12 – Sarcomere & Striated Muscle Architecture
Hierarchy: muscle fasciculus → muscle fiber → myofibril → sarcomere → myofilaments.
Labelled EM regions:
• Z disk, A band, I band, H band, M line.
• Thin filaments (actin) anchored at Z disk.
• Thick filaments (myosin II) centered at M line.
• SR (sarcoplasmic reticulum) stores Ca^{2+} for contraction.
Page 13 – Actin Filament Polymerization & Accessory Proteins
• G-actin ⇌ F-actin; hydrolysis of bound ATP influences dynamics.
• Regulatory proteins:
– Tropomyosin (stabilizing coiled-coil along groove).
– Troponin complex (TnT, TnI, TnC) controls Ca^{2+} response.
– CapZ (barbed-end cap at Z disk).
– Tropomodulin (pointed-end cap).
– \alpha-actinin (cross-links actin at Z disk forming lattice).
Page 14 – Myosin II Thick Filaments
• Bipolar filament: tails face center, heads project outward.
• Central bare zone length \approx 160\,\text{nm} (no heads).
• Filament width \approx 14\,\text{nm}.
• Individual myosin head (motor domain) ≈ 10.7\,\text{nm} long; full head-neck ≈ 15\,\text{nm}.
• Giant elastic proteins: titin (links Z disk ↔ M line), nebulin (specifies actin length).
Page 15 – Cross-Bridge Cycle (Energy-Dependent Sliding)
Step 1 – Myosin head binds ATP → dissociates from actin.
Step 2 – ATP hydrolysis (forms ADP + Pi) → head cocks into high-energy state. Step 3 – Head binds new actin site, releases Pi → power stroke; thin filament slides toward M line.
Step 4 – Release of ADP leaves rigor state; binding of new ATP restarts cycle.
Reaction is reversible and repeats as long as ATP and Ca^{2+} are available.
Page 16 – Supplementary Video
• YouTube link (registered 2004) visually animates actin-myosin interaction: https://www.youtube.com/watch?v=zQocsLRm7_A
Page 17 – Sarcomere Length Change
• Relaxed: wider I band and H zone.
• Contracted: I band & H zone narrow; A band remains constant.
• Thin filaments slide past thick filaments, drawing Z disks closer.
Page 18 – Ca^{2+}-Mediated Regulation in Striated Muscle
• Depolarization → Ca^{2+} released from SR.
• Ca^{2+} binds TnC subunit of troponin → tropomyosin shifts, exposing myosin-binding sites on actin → contraction proceeds.
Page 19 – Cardiac Muscle Contraction
• Cells linked by intercalated disks containing:
– Fasciae adherentes (anchoring actin)
– Desmosomes (maculae adherentes)
– Gap junctions (electrical coupling)
• Abundant mitochondria power continuous rhythmic contraction.
• Coordinated contraction yields synchronous heartbeat.
Page 20 – Smooth Muscle Architecture & Contraction
• No sarcomeres; thin & thick filaments criss-cross cell.
• Dense bodies (cytoplasmic & membrane-attached) act like Z disks anchoring actin and intermediate filaments.
• Mechanical junctions couple neighboring cells.
Page 21 – Smooth Muscle Molecular Mechanism
• Ca^{2+} enters cytosol → binds calmodulin → activates myosin light-chain kinase (MLCK).
• MLCK phosphorylates regulatory light chains on myosin II → enables actin binding.
• Myosin self-assembles into bipolar filaments (≈ 15 \text{–} 20 molecules).
• Contraction terminates when phosphatase removes phosphate.
• Requires ATP; generates slow, tonic force.
Page 22 – Actin Contraction in Non-Muscle Cells
Examples:
• Cytokinesis: contractile ring with actin + myosin II.
• Focal adhesions: integrin-based membrane sites where actin bundles (stress fibers) anchor via adapters (vinculin, talin, \alpha-actinin).
Page 23 – Roles of Actin in Non-Muscle Cells
• Governs cell shape, motility, endocytosis, and trafficking.
• Nearly all functions involve physical linkage to plasma membrane.
• Numerous actin-binding proteins (profilin, cofilin, Arp2/3, formins, etc.) fine-tune polymerization dynamics.
Page 24 – Actin-Dependent Cell Shape (Illustrated)
• (A) Microvilli support
• (B) Lamellipodia
• (C) Filopodia
• (D) Contractile bundles / stress fibers.
Page 25 – Spectrin-Based Membrane Cytoskeleton (Terminal Web)
• Lattice of spectrin tetramers linked to short actin filaments.
• Anchoring proteins: ankyrin, adducin.
• Connects to membrane channels (e.g., anion exchanger, band 3).
• Confers elasticity to erythrocytes & polarized epithelial surfaces.
Page 26 – Spectrin Gene Superfamily
• Spectrin I (αIβI) – erythrocyte specific.
• Spectrin II (αIIβII) – ubiquitous in non-erythroid cells.
• α-actinin – cross-linker in muscle Z disk & focal adhesions.
• Dystrophin – skeletal muscle; mutation causes Duchenne/Becker muscular dystrophies.
Page 27 – Microvillus Core
• Bundle of \sim 20 parallel actin filaments, plus ends at tip, minus ends anchored to terminal web via spectrin.
• Function: increase surface area to enhance absorption/secretion (e.g., intestinal epithelium).
Page 28 – Actin in Cell Migration
Sequential events:
Polymerization at leading edge → lamellipodium protrusion.
New focal adhesions form via integrin–ECM binding.
Cell body translocates; stress fiber contraction retracts trailing edge.
Adhesions disassemble at rear.
• Key proteins: \alpha-actinin, vinculin, talin, Arp2/3 complex.
Page 29 – Myosin Family & Vesicular Transport
• > 40 human myosin genes.
• Directionality: Myosin I & V move cargo toward actin plus end; Myosin VI moves toward minus end (unique reversal).
• Cargo: secretory vesicles, endosomes, melanosomes, mRNA particles.
Page 30 – Overall Summary
• Cytosol = intracellular space between plasma membrane and organelles/nucleus.
• Performs numerous parallel tasks: signalling, protein synthesis, metabolism, proteolysis.
• Cytoskeleton (actin, intermediate filaments, microtubules) organizes cytosol.
• Actin filaments drive contraction, shape modulation, motility, and cargo transport via myosin motors.