Cytoskeleton and Microtubules Comprehensive Notes

The Cytoskeleton

The cytoskeleton consists of three main protein polymers:

  • Actin (Microfilaments)
  • Microtubules
  • Intermediate filaments

These components are crucial for:

  • Internal cell organization
  • Cargo transport
  • Cell shape and integrity
  • Cell division
  • Cell motility
  • Communication with the cell exterior

This lecture series builds upon Year 1 LF104: Molecules, Cells and Organisms and the following sources:

  • Chapter 17, 18 & 20 Lodish et al. (9th) Molecular Cell Biology
  • Chapter 16 and 19 Alberts et al. (7th) Molecular Biology of the Cell
  • Chapters 34-40 Pollard and Earnshaw (4th) Cell Biology

Lecture Topics

  • Lecture 14: Microtubules, MAPs, and Motors
  • Lecture 15: Microfilaments, Muscles, and Myosins
  • Lecture 16: Cell Motility and Chemotaxis
  • Lecture 17: Intermediate filaments and force resistance

Lecture 14: Microtubules, MAPs, and Motors

  • Introduction to the three major cytoskeletal systems
  • Mechanics of microtubule assembly and dynamic instability
  • Microtubule Associated Proteins (MAPs) such as EB-1 and γ-tubulin
  • Kinesin and Dynein motor proteins in organelle organization, vesicle trafficking, and chromosome segregation
  • Complex behavior: bi-directional transport of vesicles

Movement and Organization Scales

The cytoskeleton facilitates movement and organization across different scales:

  • Protein (1 nm)
  • Cell (1 mm)
  • Human (1 m)
  • Country (1000 km)

These sizes are expressed on a logarithmic scale (10n meters).

Cytoskeleton Responsibilities

The cytoskeleton is responsible for cellular and body mechanics including:

  • Organization of organelles
  • Chromosome segregation
  • Protein and RNA transport
  • Cell division
  • Cell motility and chemotaxis
  • Maintaining cell integrity
  • Food mastication (chewing)
  • Digestion
  • Blood circulation
  • Communication (talking)
  • Reproduction (giving birth)
  • Body movement (walking)

Cytoskeletal Networks

The cytoskeletal networks are made from protein polymers:

  • Microfilaments:
    • 7-9 nm
    • Actin
    • Red
  • Microtubules:
    • 25 nm
    • αβ-Tubulin dimer
    • Green
  • Intermediate filaments:
    • 10 nm
    • Various
    • Blue (Desmin)

Properties of Cytoskeletal Filaments

Microfilaments (Actin):
  • Binds ATP.
  • Forms rigid gels, networks, and linear bundles.
  • Regulated assembly from a large number of locations.
  • Highly dynamic.
  • Polarized.
  • Tracks for myosins.
  • Contractile machinery and network at the cell cortex.
Microtubules (αβ-tubulin):
  • αβ-tubulin binds GTP.
  • Rigid and not easily bent.
  • Regulated assembly from a small number of locations.
  • Highly dynamic.
  • Polarized.
  • Tracks for kinesins and dyneins.
  • Organization and long-range transport of organelles.
Intermediate Filaments:
  • IF subunits don't bind a nucleotide.
  • Great tensile strength.
  • Assembled onto pre-existing filaments.
  • Less dynamic.
  • Unpolarized.
  • No motors.
  • Cell and tissue integrity.

Microtubule Organization

  • Each microtubule in the network grows out from a focus (the MTOC) at the nuclear periphery.
  • Microtubules are polymers of a dimer containing one molecule of α-tubulin and one molecule of β-tubulin.
  • Both α-tubulin and β-tubulin subunits of the free α,β-tubulin dimer bind GTP.
  • GTP bound α,β-tubulin dimer is incorporated into existing microtubules by binding to an exposed α,β-tubulin dimer at the end of the polymer.
  • Each protofilament has alternating α-tubulin and β-tubulin subunits.
  • Incorporation of the dimer causes hydrolysis of GTP bound β-tubulin to GDP bound β-tubulin.
  • α-tubulin remains bound to GTP.
  • The α-tubulin and β-tubulin subunits also make side contacts with other α-tubulin and β-tubulin subunits in adjacent protofilaments to make sheets of (usually) 13 parallel protofilaments which zipper together to form a microtubule, leaving a hole down the center of the tubule called the lumen.

Microtubule Structure

  • α-tubulin and β-tubulin heterodimer (= microtubule subunit)
  • Protofilament
  • Lumen
  • Plus end
  • Minus end

GTP Hydrolysis and Dynamic Instability

  • GTP bound β-tubulin (T form) undergoes slow hydrolysis to GDP, so that GDP bound β-tubulin (D form) is the predominant form in the lattice.
  • At the plus end, polymerization is much faster than hydrolysis, so polymerizing microtubules have a GTP cap.
  • Polymerization is slower than hydrolysis at the minus end, so the minus end is always in the D form.

Dynamic Instability

  • Rapid growth with GTP-capped end.
  • Accidental loss of GTP cap leads to catastrophe.
  • Rapid shrinkage.
  • Regain of GTP cap leads to rescue.
  • Cycle repeats.

Microtubule Plus Ends

The plus ends of interphase microtubules are directed towards the cell cortex where they probe the inner face of the plasma membrane (PM). Microtubule plus tips frequently pause at the plasma membrane, allowing proteins that track the plus tips of MTs to interact with proteins at the PM. These interactions determine the length of the pause. In this manner, MTs can direct and maintain cell shape or determine where new sites of cell growth will occur. Microtubules can also grow along existing MTs and that single microtubules grow outwards more slowly than shrinking microtubules contract away from the PM.

Microtubule Dynamics

  • Elongating filaments are straight.
  • Shrinking filaments have curved or frayed ends.
  • Microtubule assembly is favored when the microtubule is straight.
  • Hydrolysis of β-tubulin bound GTP to GDP causes a conformational change in the α,β-tubulin dimer, which tenses the MT lattice.
  • The protofilaments splay outwards when depolymerizing but are prevented from doing so when the microtubule is polymerizing by the presence of the GTP-tubulin cap.
  • Cap dissociation promotes explosive (catastrophic) depolymerization.
  • Microtubule associated proteins (MAPs) and some motors shift the equilibrium by promoting either assembly or disassembly.

Regulation of Microtubule Dynamics

The cycle of microtubule polymerization and depolymerization is essential for the dynamic instability of microtubules observed in vivo. The rate of microtubule polymerization, pausing, depolymerization, and rescue can be modulated by microtubule associated proteins (MAPs) and motors.

EB-1:GFP Fusion Protein

An EB-1:GFP fusion protein expressed in a mammalian cell only binds the growing ends of microtubules. Many other proteins can surf the growing microtubule tip by binding EB-1. EB-1 (End Binding protein-1) is a plus-end MT binding protein.

Drugs that Affect Microtubule Stability

  • Nocodazole: Binds to the α,β-tubulin dimer to prevent its addition to microtubules. Addition of Nocodazole prevents microtubule polymerization and causes microtubules to disassemble.
  • Colcemid: Is structurally unrelated to Nocodazole, but has the same effect.
  • Both are used frequently in biological research.

Microtubule Organizing Centers (MTOCs)

  • Microtubules are nucleated throughout the cytoplasm, but are especially concentrated around the nucleus.
  • When colcemid is added to cells injected with fluorescent tubulin, microtubules disassemble.
  • When colcemid is removed, microtubules can be seen to grow from a microtubule organizing center (MTOC). In animal cells, this is the centrosome.
  • The minus ends of microtubules are at the MTOC near the nucleus, and the plus ends spread out towards the cell periphery.

Centrosome

The major microtubule organizing center (MTOC) in animal cells is the centrosome, which is composed of two centrioles surrounded by pericentriolar material (centrosome matrix) to which γ-tubulin ring complexes (γ-TURCs) are associated. The γ-TURCs are responsible for nucleation of microtubules. Recruitment of γ-TURCs to the centrosome explains the nucleation pattern of microtubules.

γ-Tubulin Ring Complex (γ-TURC)

  • The γ-tubulin ring complex (γ-TURC) is composed of a ring of 13 subunits of γ-tubulin onto which α,β-tubulin dimers bind.
  • In addition, γ-TURC contains several other accessory proteins which are also highly conserved in eukaryotes.
  • An electron micrograph of the minus end of a microtubule shows a cap-like structure that is the γ-TURC, which prevents depolymerization from the minus end of the MT.

Tubulin Gene Family

  • α and β tubulin
    • α/β heterodimers form microtubules.
    • Major constituents of microtubules.
    • Found in all eukaryotes.
  • γ tubulin
    • Major component of γ-tubulin ring complex (γ-TURC) recruited to microtubule organizing centers (MTOCs) - usually the centrosome.
    • Found in all eukaryotes.
  • δ and ε tubulin
    • Components of centrioles and basal bodies.
    • Found only in some eukaryotes and in protozoa such as Paramecium and Chlamydomonas (green algae).
  • ζ and η tubulin
    • Found only in some species.
    • Specialized function.
  • FtsZ
    • Bacterial tubulin relative that forms polymers for cytokinesis.

Cytoskeleton Functions Revisited

The cytoskeleton is responsible for cellular and body mechanics including:

  • Organization of organelles
  • Chromosome segregation
  • Protein and RNA transport
  • Cell division
  • Cell motility and chemotaxis
  • Maintaining cell integrity
  • Food mastication
  • Digestion
  • Blood circulation
  • Communication
  • Reproduction
  • Body movement

Kinesin and Dynein Motors

Kinesin and Dynein motors are responsible for numerous microtubule-dependent transport events in eukaryotic cells. The organization of the ER and Golgi depends on the orientation of microtubules and motor activity. Kinesin motors generally direct organelles (e.g., ER) and vesicles towards the plasma membrane (e.g., early endosomes and secretory vesicles for exocytosis). By contrast, Dynein directs both organelles (e.g., Golgi) and vesicles away from the plasma membrane (e.g., late endosomes for endocytosis, lysosomes, and ER-Golgi intermediate compartment (ERGIC)). Some organelles (e.g., mitochondria) and vesicles (e.g., pigment granules) bind both Kinesin and Dynein.

Microtubule Motor Proteins

Kinesin:
  • Almost exclusively plus-end directed motors (except kinesin-14 which is minus-end directed).
  • 14 structurally related classes.
  • Use ATP to generate force.
Dynein:
  • Exclusively minus-end directed motor.
  • Part of a very large Dynein-Dynactin complex.
  • Structurally unrelated to kinesins.
  • Use ATP to generate force.

Organelle Transport

  • Kinesin-1 (conventional) (+)
  • Kinesin-2 (heterotrimeric) (-)
  • Kinesin-5 (bipolar)
  • Kinesin-13 (Kinl) (+/-) end disassembly
  • Sliding (-)

Kinesin Structure

  • Head (MT-binding sites)
  • Light chain
  • Stalk (Coiled-coil)
  • Tail
  • Linker

Mechanics of Kinesin Movement

The single dimer of kinesin can move processively along the microtubule because the action of the two heads are coordinated, and one of the two heads is always bound. Thus, an individual kinesin molecule can transport a cargo such as a vesicle or mitochondrion a long way - for example, along an axon.

The mechanics of kinesin movement:

  1. Forward motor binds β-tubulin, releasing ADP.
  2. Forward head binds ATP.
  3. Conformational change in neck linker causes rear head to swing forward.
  4. New forward head releases ADP, trailing head hydrolyzes ATP and releases Pi.

Dynein-Based Organelle Transport

Dynein moves exclusively towards the minus end of microtubules and is a much faster motor than kinesin. It cannot bind cargo itself. This is mediated by an 11 subunit complex called the Dynactin complex which contains Dynactin and other proteins including Arp1 (an actin related protein) which forms a mini filament that is responsible for cargo binding. Dynactin also has a microtubule binding domain to ensure processivity of the Dynein-Dynactin complex, when the Dynein motor heads lose contact with the microtubule.

Dynein Motor Structure

The Dynein heavy chain (motor) contains an N-terminus tail which binds the Dynactin complex (to bind cargo) and a C- terminus composed of 6 AAA ATPase domains which are arranged in a wheel. The C-terminal ATPase domain closes the wheel by forming contacts with the first AAA domain (red) which is the major ATPase that generates the conformational change which alters the position of the tail relative to the ATPase wheel. A stalk region in between AAA domains 4 and 5 binds the microtubule. The two conformations of the dynein motor can be seen by electron microscopy. The stalk domain (which contains the MT binding site) protrudes from between the fourth and fifth ATPase domain. Dynein is an extremely fast motor (100 times faster than the best kinesin).

Bi-Directional Organelle Transport

Organelles can exhibit bi-directional transport, moving in both directions along microtubules.

Regulated Melanosome Movement

Regulated melanosome movement in fish pigment cells; these giant cells are responsible for changes in skin coloration and contain large pigment granules that can change their location in response to hormonal or neuronal stimulation. The second messenger in these signaling pathways is cAMP. The pigment granules aggregate or disperse depending on the concentration of cAMP in the cell (through the action of cAMP-dependent protein kinase). Kinesin drives the melanosomes to the plasma membrane when cAMP increases and Dynein moves them back when cAMP decreases.

  • Kinesin drives the melanosomes to the plasma membrane when cAMP increases.
  • Dynein moves them back when cAMP decreases.

Bi-Directional Transport in Neurons

  • Cell body
  • Axon
  • Neuropeptide Y-GFP
  • Retrograde transport
  • Anterograde transport
Kymographic Analysis
  • Distance
  • Time
  • Retrograde movement
  • Pause
  • Anterograde movement

Organelle Components

  • Organelle
  • Dynein
  • Dynactin complex
  • Kinesin