Lecture Notes: Pharmacy, Cytoskeleton (Actin, Intermediate Filaments, Microtubules)

• Introduction to Cytoskeleton- History: Initially viewed as static, now known to be dynamic.

  • Role in diseases, including cancer.

• Components of the Cytoskeleton- Microfilaments (Actin Filaments/F-actin): Smallest.

  • Intermediate Filaments: Intermediate in size.

  • Microtubules: Largest; critical due to drugs targeting them for cancer treatment.

Comparison of Cytoskeletal Elements

• Comparison Table (F-actin, Microtubules, Intermediate Filaments)- Building blocks: ATP for microfilaments, GTP for microtubules.

  • Dynamics: Microfilaments and microtubules are highly dynamic; intermediate filaments are less so.

  • Intermediate filaments: Act like ropes, found in tissues undergoing tensile strength.

Microfilaments (F-Actin)

• Building Blocks: G-actin (globular actin).

• Location in Cells:- Polarized cells: Around the outside edge/cell cortex, microvilli (structural support).

  • Cell movement: Critical for all cells, especially cancer cells (metastasis).

• Functions- Microvilli structure.

  • Cell movement.

  • Stress fibers (hooked up to integrin receptors).

  • Phagocytosis.

  • Vesicle movement.

  • Contractile ring at the end of mitosis.

• F-Actin & G-Actin Relationship- F-actin: Double helix.

  • G-actin: Building block for Factin, requires ATP and magnesium.

• Polarity of F-Actin- Polarity first noted using electron microscope with myosin S1.

  • Myosin S1: Head part of myosin that binds directly to actin.

  • Ends: Pointed end (minus end) and barbed end (plus end).

• G-Actin and F-Actin in a Test Tube:- Nucleation: Initial slow binding.

  • Elongation: Rapid formation of filament.

  • Steady State: Mass of filament stays constant, adding and subtracting at the same time.

• Critical Concentration (Cc):- Concentration of G-actin where there is no net change in length at that end.

  • Calculated for both ends: Plus end CC = 0.12 micromolar, Minus end CC = 0.6 micromolar.- Net addition of G-actin to the plus end is five times greater than at the minus end.

• Treadmilling:- Description of what happens at steady state; a net increase at one end and a loss at the other.

• Drugs Affecting F-Actin- Cytochalasin D:- Inhibits F-actin polymerization.
  - Used as a research tool, not in cancer treatment.

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  • Phalloidin:- Promotes polymerization (found in death cap mushroom--angel of death mushroom--very poisonous).

    • Used as a fluorescent label (rhodamine-labeled phalloidin) to bind to F-actin better than any monoclonal antibody.

F-Actin in Cells

• Treadmilling: Does not occur in cells due to controlling proteins.

• Proteins in Cells That Control G-Actin and F-Actin Relationship- Thymosin Beta 4:- A sequestering protein: Sequesters or captures G-actin to increase the G-actin pool.

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  • Profilin:- ATP/ADP Exchanger: Recharges G-actin with ATP which facilitates binding (ATP is the energy currency of the cell and used to for G-actin bind to Factin).

  • Cofilin:- A Severing Protein: Cuts off/severs G-actin at the minus end and then depolarizes it to G-actin and then add it to the plus end.

  • Capping Proteins:- Cap off to block one of the ends of Factin; no addition or subtraction or polymerization, depolarization can occur.

    • Example) CapZ and Tropomodulin.

  • Formin:- A Facilitator Protein: Facilitates nucleation. It is a dimer and can bind to G-actin which helps in nucleation.

• Optical Tweezers Microscope:- For biomechanical properties: Measure shift steps and the power used by F-actin with the relationship to myosin S1 heads.

Role of Listeria Monocytogenes

• A bacterium that causes food poisoning and death.- Recent outbreak (02/02/2025) in supplemental shakes.

  • Can cross the placental barrier.

  • Cause eye problems.

• Mechanism of Action (Listeria organizes Factin to move around--it is not motile until this happens).- Listeria has a nucleating protein: ActA.- ActA Facilitates actin assembly.
  - G-actin adds to Actin filaments.
  - Severing proteins: Cofilin cuts off filaments and then depolarizing it.
  - Has all capping proteins.

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  • Moves forward when G-actin is added.

• Eukaryotic Cell- Has ARP2/3--does similar as listeria with ACTA nucleating protein.

Factin in Eukaryotic Cells

• Similar Function: Moves endocytotic vesicle; similar to moving a bacteria.

• Role in phagocytosis- Initial stage: Viruses/bacteria cause immune reactions; antibodies bind (opsonization) and then they get pulled in like little mini comets.

  • Phagocytosis blocked with cytochalasin D: It's F-actin mediated.

• Moving Cell Adhesion- Cell moves from left to right due to Factin filaments.

  • Cell leaves components behind such as focal adhesion plaque.

• Facts about Diaphanous Gene- Figures in inherited hearing problems.

  • Codes for: Formin-like molecules.

  • Mechanotransducer: Translates sound waves--hair cells.

  • With age: inherited hearing loss diseases occur (age 20-30).

Miscellaneous Facts on F-Actin

• Aging and Autophagy of Facts on F-Actin.

• Study on Drosophila Melanogaster: flies live longer on a low protein diet.

• Conclusion:- Brain aging in fruit flies by suppressing F-actin and restoring autophagy.

  • Rapamycin can do this--extend fly lifespan--lower the amounts of F-actin and the autophagy is effective. F-actin prevents Autophagy from working.

Intermediate Filaments

• Toughest cytoskeletal element.

• Arrangement: Monomer, dimer, etc.

• Different Intermediate Filaments Shown- Acidic and basic keratins.

  • Desmin and vimentin-interesting itself.

• Neurofilaments: Important for long Axons.

• Lamins: Important for the structure of the nucleus and is tied to intermediate filaments and microtubules and are important for cell division.

Disease (Epidermolysis Bullosa Simplex)

• Problem: Defect in keratin filaments in skin.

• Shearing stress: Causes cells to shear in half.- See picture shown (ugly).

• Basement membrane: Does not exist b/c regenerated layer is sheared.

Story about Development and Drug Discovery related to Epidermolysis Bullosa: Chemical Defense

• US Army's interest in skin blistering agents like mustard gas.

• Use of engineered skin (Epiderm) for drug discovery.

• Development of topical skin protectant for mustard gas.

• Environment was unique: Alarms due to the fact people would need to leave by a certain time, the military setting.

Modern Day Drugs for Epidermolysis Bullosa

• Current Treatments include: a company formed generated engineer skin; Columbia University developed a technology which they originally called before it was even dealt developed Orselv, which got FDA approved for EB.

• Dystrophic Epidermolysis Bullosa- See the same result as above with blistering b/c a defect and actually collagen seven.

• Current Gene Therapy includes- BIJUVAK restores type seven collagen.- Costs: 2. 5 ml cost $26,000.

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Intermediate Filaments and Disease

• Lamins:- Important for structure of the nucleus. Plays huge role with diseases in Nucleus (plays a huge role in Mitosis also).- The trigger that causes the nucleus to dissolve entirely during Mitosis is something to do with phosphorylation.

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Microtubules

• If Had to Pick Pharmaceutically Import One, Choose Microtubules b/c of Drugs to Treat Cancer.

• Overview: Have cell structure. See with fluorescent dye over chromosomes.- Non-polarized, Polarized cells: Radiate a star-like arrangement.

  • Axon and Microtubules: are structurally linked, can sometimes also see them using flagella or Silia.

  • Key: Like Factin, they are Dynamic. They are building block like Alpha and Beta Tubulin.

Alpha & Beta Tubulin Facts

• Alpha- non-exchangeable GTP

• Beta- Dyamic!

  • Exchangeable GTP when functional unit wants to bind to Microtubule: Requires ATP

• Microtubule itself: Comprised of 13 protofilaments.

• Exhibit Treadmilling- The Plus End and Minus End: Alpha and Beta need a dimer where will be changed on length not both change each other each other etc.

  • GTP Cap Stability: Stabilzes them; event of hydrolization to deep polymerize is to hydrolize.

• Facts on molecular movement:- Serve as important tracks for molecular motors; Move vesicle to move anything in the cell.

  • Melanosomes in frog example.

Microtubule Associated Proteins

• Maps!- tenaciously to microtubules.

  • Control many functions with them- Stabilixing proteins can act like maps.

    • Microtubule destabilizing proteins can also like maps.

• Motor Proteins will be coming up.- Continine: Is a severing protein but used only in braniac, and has some ability to be age specific.

Famed Chemotherapy Drugs

• Colchichine has been around for a while used to treat gout (de-polymerization).

• Taxol Taxatir series:- Promo Polymerization:

  • Also both Colchichine Derivative and taxoles work by dying from the microtubules during mitsos and trigger aboptosis after this event to prevent cancer as well.

Motility of Vesicles with Molecules

• What are molecular Motors? (Motor Proteins.)

• Radioactively tagged amino acids show how this functions when they bind axon.

• Anteregrade- Motor Proteins: Kinesin 1, 2 and 3

• Retrograde- Dyne

Kinesin

• One and Two there- Only Moves in ONE direction and has to use the energy there to keep sliding.

• Diennen: Uses atp- Works in retrograde direction and binds moleculars for transport and cellular function.

• Cartagener Syndrome: (Defective Diennen): Defective Diennen where lung defects mucus in lug- very serious problem happens do to that defect.