Cytoskeleton and Molecular Motors

Cytoskeleton

• The cytoskeleton is a system of protein filaments providing structure and mechanical support to the cell.

• It prevents the cell from collapsing and helps maintain its shape.

• Three major classes of cytoskeletal fibers:

  • Microtubules: Made of tubulin protein.

  • Microfilaments: Made of actin protein.

  • Intermediate filaments: A family of proteins.

• The subunits for microtubules and microfilaments are globular proteins, while intermediate filaments are made of long, helical proteins woven together.

• Cytoskeletal filaments are made of repeating subunits that self-assemble into chains without additional help when in high enough concentration.

• Actin and tubulin filaments are polarized, possessing a plus end (where monomers are added) and a minus end (where monomers are lost).

• Assembly and disassembly of filaments are regulated by regulatory proteins, which control their interaction with other cell components.

• Filaments act as structural girders and railways for motor proteins to move cargo or affect cellular shape changes.

Cytoskeletal Components Overview

• Microtubules: Largest diameter filaments, hollow cylinders, polymers of tubulin.

• Intermediate Filaments: Average size (10 nm diameter), made of helical proteins.

• Microfilaments: Smallest filaments, made of actin.

• Cytoskeletal components consist of:

  • Free soluble monomers or subunits.

  • Filamentous polymer.

• Polymerization is reversible, involving non-covalent protein-protein interactions.

• Signals from inside and outside the cell can rapidly change the dynamics of cytoskeletal components by disassembling them in one location and reassembling them in another.

• Actin and tubulin are regulated similarly, while regulation of intermediate filaments is less understood.

• Monomer subunits bind nucleotide triphosphates (NTPs), with hydrolysis regulating the process.

  • Binding to triphosphate (active configuration) results in higher affinity for other subunits, favoring polymerization.

  • Binding to diphosphate (inactive configuration) results in lower affinity, favoring depolymerization.

  • Tubulin binds GTP, while actin binds ATP.

• NTP-bound monomers tend to add at the plus end, while NTP is hydrolyzed to NDP over time, especially toward the minus end.

• The length of the filament depends on the relative rates of addition at the plus end and loss at the minus end.

  • Faster addition lengthens the filament.

  • Faster loss shortens the filament.

Actin Filaments

• Actin cytoskeletal network is dispersed throughout the cell.

• A high concentration of filamentous actin underlies the plasma membrane, providing structural support (cortical actin network or cortex).

• Most actin filaments are in different directions relative to each other, but there are some parallel actin filaments (e.g., microvilli in absorptive cells).

• Other actin-based structures include:

  • Contractile ring of dividing cells.

  • Structures that move vesicles.

• Actin monomer: A globular protein (g-actin) asymmetrical in construction, with its N-terminus and C-terminus on the same side, defining the plus end.

• Filamentous actin (f-actin): Two chains of g-actin wound around each other in a helical structure.

• Actin is typically the most abundant protein in the cytosol and one of the most highly conserved eukaryotic proteins.

• Averages about 40 kilodaltons in size.
  *Addition mainly occurs at the plus end by ATP-bound monomers, with loss at the minus end of ADP-bound monomers.

• Dynamic Instability in Actin

  • Addition at one end and subtraction at the other end at the same rate

  • Filament remains the same length, subunits are in constant turnover

• Treadmilling: Addition and subtraction happen at the same rate in actin filaments, leading to constant turnover of subunits without changing the filament length.

  • Dynamic instability refers to turnover within the filaments.
    *Movement of filaments is achieved by treadmilling.
    *Extends at the plus end, constricts at the minus end.
    *Constantly walking the tread the length of the treadmill, the treadmill never changes, but you move along it

• Traffic Jam Analogy of Actin Dynamics

  • The back of the traffic jam extends (plus end), while the front is the minus end

  • The whole system migrates down the road, away from the initial site of the accident

  • Just like actin dynamics

Regulation of Actin Polymerization and Depolymerization

• Proteins that facilitate the initial polymerization (nucleation).

• Proteins (e.g., thymosins) that bind to monomers in solution, preventing them from binding into the filament, therefore inhibiting polymerization or promoting net depolymerization by reducing available monomers.

• Proteins that bind to the ends of existing filaments:

  • Capping at the plus end to prevent polymerization.

  • Capping at the minus end to prevent depolymerization.

  • Tropomodulin binds at and stabilizes the + end, preventing further addition of monomers.

• Proteins (e.g., profilin) that facilitate the addition at the plus end, promoting extension.

• Proteins that bind to the filament and cause an increased rate of ATP hydrolysis, increasing the rate of depolymerization.

  • Example: Cofilin accelerates the rate of depolymerization.

• Proteins (e.g., Gelsolin) that sever the filaments in the middle, converting from the gel-like state to the sol-like state, which increases the number of filaments.

• Proteins that cross-link the filaments:

  • At different angles for stability.

  • In parallel to form bundles (e.g., microvilli).

• Proteins that allow filaments to interact with membrane structures (vesicles, plasma membrane).

Rho Family GTPases

• A family of GTPases (closely related to the RAS protein) that serve as molecular switches to turn on regulatory functions.

• Includes Rho, RAC, and Cdc42.

  • Row: Regulates actin bundling, causes actin filaments to bundle in parallel, makes structures like stress fibers

  • RAC: Regulates actin polymerization and facilitates actin polymerization, sheet like projections when cell is crawling, cause membrane ruffles

  • Cdc42: Can activate BOTH simultaneously, tube like projections

    • Filopodium is a tube like extension, get little extensions called micro spikes

Microtubules and Tubulin Dynamics

• Tubulin-based cytoskeleton forms rigid microtubules that radiate from a central point.

• They are important in eukaryotic cells, and make up the basis of the mitotic spindle and the central core of cilia and flagella.

• The minus end points toward the radiating point, and the plus end points toward the periphery.

• The microtubule subunit (monomer) is a heterodimer of alpha and beta tubulin, and they're part of a larger family of proteins (50 kilodalton).

• Microtubule Dynamics In Vitro

  • Tubulin only hydrolyzes one GTP

  • Alpha end points toward the minus, beta points toward the plus

  • Take 13 protofilaments and form cylinder

    • Extend by adding to the ends of the microtubule itself

• Protofilament: Alpha end points toward the minus, and beta points toward the plus.

• 13 protofilments make a cylinder, which has a hollow lumen.

Microtubule Dynamics In Vivo

• Minus ends are anchored to cell structure, not available for tubulin to fall off, all cell dynamics happen at the + end

• Addition at the plus end and loss at the minus end.

• There is rapid addition as long as there is GTP monomers with the plus end (GTP Cap)

  • At a certain point, the end will hyrdolyze at a certain point and catch up, GTP turns to GDP and it all falls apart

  • Pool can exchange GDP, reestablish the cap and then the microtubule can extend again

• If the microtubules are simply assembled in vitro, You would get addition at the plus end and loss at the minus end

• The technical term for what happens when those monomers fall off is catastrophe, it all basically collapses at the plus end as one step

• Later, the GTP cap gets re-established and you can grow

• In microtubules:

  • Either have addition or loss

  • Do not tend to have a loss and addition at the same time; whereas simultaneously, actin has both at once at each end

Regulation of Microtubule Monomers

• The cells regulate whether microtubules will be lengthening or shrinking, by:

  • Monomer sequester proteins

    • Staphmin binds the alpha-beta tubulin subunit to prevent incorporation into cell, and this allows GTP hydrolysis

  • Increase rate of loss of monomers

    • Kinesin 13 is a protein that does this

  • Cut microtubules in the middle

    • Katanin, same roots as katana which is a sliced, like a sword through the middle of fibers

  • Stabilize ends of microtubules and interact with structures, membranes etc

  • Prevent depolymerization and prevents the microtubule from disassembling

• MTOC (Microtubule Organizing Center):

  • In animal cells, it usually is a centrosome, each non-dividing cells has 1

    • Prior to the division, the centrosome will duplicate, 2 centrosomes become the centromeres of the the mitotic spindle

  • Animal Centrosome = Tubulin

  • Composed of 2 centrioles at right angles

  • Surrounded by and electron dense (not well characterized) matrix

  • Each centriole is made of 9 fibrils, and each fibril made of 3 microtubules

    • Each centriole = 27 microtubules -> together it's 54

• Gamma Tubulin - Contained within the MTOC nucleating sites and initiate alpha-beta tubulin subunit

Molecular Motors

• Cytoskeletal Fibers Can Also Serve as Roadways.
  *Molecular motors are a series of shape changes that allow for directional motion and move cellular components, organelles, or vesicles from one part of the cytosol to another.

• The cell needs that movement to be unidirectional, in order to be useful.
  *The directional movement uses ATP hydrolysis to irreversible affect one conformation change.

Myosins

• Molecular motors associated with actin.

• Conventional type 2 myosins are the normal motor proteins that move stuff on actin.

  • 6 polypeptide chaisn as the type 2 myosins

  • Main motor part is the pair of heavy chains.

  • There are 2 pairs of light chains that are associated near the N terminus, helps bind the ATP and the globular head

  • Long coiled coil alpha helical region to form the tail

  • Head will bind, hydrolyze ATP, cause movement -> ATPase Activity

  • Tail associated with other molecules of myosin/proteins -> higher order structure

• Myosin can form a filament of actin.
  *Muscle cells make up basic unit of contraction = sarcomere, has myosin that is held in place.

  *   Myosin walks along, pulls the actin.
  

  • Non-muscle cells, actin filament held in place; Myosin walkas along and moves actins

• Myosin walks from - to +; Cannot go backwards

• In muscle cells, there is one set of molecules parallel from the head, and the other side is opposite of the direction

  • Leads to fixed myosin molecules, right pulls one way, and left pulls into another

    • Contractile unit -> Sarcomere

  *Myosin Molecules in an organized, fixed sacromere arrangement with a fixed position + Heads
  * Myosin wants to walk to the right and one wants to the left

Steps of Sarcomere Walking

*   ATP binds and releases myosin
*   Head moves forward
*   Grab/Pull, Releases, and ADP releases to get back to the initial state

Microtubule Motors

• Microtubules are directional, oriented so the (-) end points toward the center of the cell and the (+) end is at the periphery of the cell

• So there should be both plus end directed motors and minus end directed motors

• Kinesins are the motors from minus to plus, after kinetic, moves from cell body to synapse

• Each one has two heavy chains and two light chains

  • The heavy chains ahve a globular ATP binding head, and an alpha helical coil-coiled structure holds 2 heavy chains together

    • Light chains are now on the tail end, which is where those molecules ahve a motor protein grabs on
      *These globular heads ahve tubulin and will walk along tubulin, from minus to plus

• Have an ATP leading head and ATP lagging head; ATP hydrolyzes the ATP to LADP and takes a new step forward
  * Cargo is then targeted to adapter proteins
  *Dyneins are a type of microtubule motors, plus to minus, that dyne from dynamic - move with speed and direction

  • Heavy Chains = Globular Head; Long Alpha-Helical Shape

  • C-terminus = Bunch of Light chains

    • Dynein -> vesicle interactions and back from + to - end from the cell

Intermediate Filaments

• Large family of related helical proteins.

• They Are not essential for the actual structure of the cytosol but they are important for providing mechanical strength to cells, not essential but they are important mechanical properties
  *assemble via coiled coil interactions to be like threads in cotton (N+ and C- terminus)
  *Mainly mesenchymal origin.

  • Support cells in the nervous system, gilial cells, also epithelial intermediate cells (keratin), intermediate filaments help axons (neurofilament proteins)
    *Regulates polymerization/depolymerization
    *Do not need to memorize all, you should know 1 in the three classes.

Intermediate Filaments & Mechanical Strength

• Provides Mechanical Strength to the Cell - Tensile Strength, Resistance to - Stretching, Compression Resistant.

• Not Regulated Like Tubulin + Actin Filaments
  Nuclear Lamins are intermediate filaments that provide mechanical strength to nuclear membrane (mutations can lead to pre-mature aging + chromosome instability).