Cell Organization and Movement II: Microtubules and Intermediate Filaments

Microtubule and Intermediate Filaments

18.1 Microtubule Structure and Organization

• αβ-Tubulin:
  • Assembles into dynamically unstable and polarized microtubules.
  • Microtubules have (+) and (–) ends.
  • Walls consist of 13, 13+10, and 13+10+10 protofilaments.
• GTP Hydrolysis: Assembled tubulin hydrolyzes GTP.
• MAPs (Microtubule-Associated Proteins): Mediate the:
  • Assembly
  • Dynamics
  • Function of microtubules.
• MTOCs (Microtubule-Organizing Centers):
  • All microtubules are nucleated from MTOCs.
  • Many microtubules remain anchored by their (–) ends.
• Centrosome MTOCs: Consist of:
  • Two centrioles
  • Surrounding pericentriolar material

Physical Properties and Functions

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

Tubulin Dimers and Microtubule Organization

• α-tubulin: GTP is never hydrolyzed and is nonexchangeable.
• β-tubulin: GDP is exchangeable with GTP, which can be hydrolyzed.
• Subunit Addition: Occurs preferentially at the (+) end where β-tubulin monomers are exposed.

Singlet, Doublet, and Triplet Microtubules

• Singlet Microtubule: 13 protofilaments; most cytoplasmic MTs.
• Doublet Microtubule: Additional wall of 10 protofilaments forms a second tubule (B) in cilia/flagellar outer doublets.
• Triplet Microtubule: Two 10-protofilament walls (B) and (C) on the 13-protofilament (A) microtubule in centriole and basal body microtubule organizing centers.

Microtubule Organizing Centers (MTOCs)

• Interphase Cell: Centrosome MTOC.
• Mitotic Cell: MTOC during cell division.
• Neuron: MTOC for neuronal microtubules.
• Cilium/Flagellum: MTOC at the base.

Structure of Centrosomes

• Centrioles: Mother and daughter centrioles, each with nine linked triplet microtubules.
  • Mother centriole has distinctive distal appendages (blue spheres).
• Pericentriolar Material: Contains γTuRC (γ-tubulin ring complex) MT nucleating structures.

γ-Tubulin Ring Complex (γ-TuRC)

• (a) In Vitro Assembly: Microtubules assembled in vitro (green) with a γ-TuRC (red) at the (–) end.
• (b) Model: γ-TuRC nucleates microtubule assembly forming a template for the MT (–) end.

18.2 Microtubule Dynamics

• Dynamic Instability: Individual microtubule (+) ends exhibit dynamic instability with alternating periods of growth and rapid disassembly (catastrophe), depending on GTP-cap or GDP-cap status.
• Energy Storage: Assembling microtubules store energy derived from GTP hydrolysis in the microtubule lattice.
• Work: Microtubules can do work when disassembling.
• Searching and Capturing: Dynamically unstable microtubules can “search” the cytoplasm and “capture” targeted structures or organelles.

Microtubule Growth

• Microtubules grow preferentially at the (+) end.

Dynamic Instability of Microtubules in Vitro

• Microtubule length changes over time due to assembly, disassembly, catastrophe, and rescue events.

GTP-β-tubulin Cap

• Dynamic instability depends on the presence or absence of a GTP-β-tubulin cap.
• Assembly: Favored with GTP-β-tubulin.
• Disassembly: Favored when GDP-β-tubulin is exposed after GTP hydrolysis and loss of the GTP cap.
• Catastrophe: Rapid disassembly.
• Rescue: Switch back to assembly.

Microtubule Growth from MTOC

• Microtubules grow from the MTOC.

18.3 Regulation of Microtubule Structure and Dynamics

• Side-Binding MAPs: Stabilize microtubules.
• (+) End-Binding +TIPs:
  • Alter microtubule dynamic properties.
  • Attach cell components to the (+) end.
• Destabilizing Proteins:
  • Kinesin-13 family of proteins
  • Op18/stathmin
  • Enhance catastrophe frequency.

Spacing of Microtubules

• Depends on the length of the projection domains of microtubule-associated proteins.
• Side associations with several monomers along protofilaments stabilize MTs and dampen dynamic instability.

+TIP Protein EB1

• Associates dynamically with the (+) ends of microtubules.
• Cell Staining: Antibodies to tubulin (red) and the EB1 +TIP protein (green) are enriched on MT (+) ends.
• EB1 and XMAP215: Promote microtubule growth by enhancing polymerization at the (+) end.
• CLASPs: Reduce the frequency of catastrophes.

Proteins That Destabilize Microtubule Ends

• Kinesin-13: Enhances the disassembly of either a (+)/(–)-MT end; ATPase activity dissociates Kinesin-13 from the αβ-tubulin dimer.
• (b) Op18/stathmin: Binds selectively to two dimers in curved protofilaments and enhances their dissociation from a MT end.

18.4 Kinesins and Dyneins: Microtubule-Based Motor Proteins

• Kinesin Motor Superfamily: (+) end motor that:
  • Transports organelles
  • Slides antiparallel microtubules past each other.
• Kinesin-1: Highly processive motor because it coordinates ATP hydrolysis by its two heads so that one head is always firmly bound to a microtubule.
• Cytoplasmic Dynein: (–) end motor that associates with the dynactin complex and cargo adapters to transport cargo.
• Tubulin Post-translational Modifications:
  • Stabilize microtubules
  • Regulate ability to interact with motors.

Kinesin-1

• Powers vesicle movement down axons toward the (+) ends of microtubules.
• Attaches to a vesicle surface receptor and transports vesicles from the (−) end to the (+) end of a stationary microtubule.

Kinesin Superfamily

• Kinesin-1: (+) end-directed microtubule motor involved in organelle transport.
• Kinesin-2: (+) end-directed vesicle transport with two closely related but nonidentical heavy chains and a third cargo-binding subunit.
• Kinesin-5 Family: (+) end-directed motor with four heavy chains assembled in a bipolar configuration that can slide antiparallel microtubules past each other.
• Kinesin-13 Family: Motor domain in the middle of the heavy chain has no motor activity; destabilizes microtubule ends for disassembly.

Kinesin-1 Walking Mechanism

• Step 1: Leading head binds ATP.
• Step 2: ATP binding induces a conformational change causing the linker to swing forward and dock into the head. This motion swings the former trailing head to become the leading head.
• Step 3: New leading head finds a binding site on the microtubule 16 nm ahead of its previous site.
• Step 4: Leading head releases ADP, and coordinately the trailing head hydrolyzes ATP to ADP + Pi. Pi is released, and the linker becomes undocked.

Kinesin-1 Regulation

• Regulated by a head-to-tail interaction where the head folds back and interacts with the tail, inhibiting ATPase activity.

Myosin and Kinesin

• Similar catalytic core structures and similar myosin-II and kinesin-1 lever arms, showing convergent structural evolution of the ATP-binding cores of myosin and kinesin heads.

Cytoplasmic Dynein

• (–) end motor consisting of two large (>500 kDa), two intermediate, and two small subunits (two-headed).

Dynein Power Stroke

• Force-generation mechanism: ATP-dependent change in the position of the linker causes movement of the microtubule-binding stalk.
• The first AAA repeat may be the only one involved in converting ATP hydrolysis energy into mechanical work.

Dynactin Complex

• Links dynein to cargo.

Organelle Transport

• Kinesins and dyneins cooperate in the transport of organelles throughout the cell.
  • Cytoplasmic Dyneins (red): Mediate retrograde transport of organelles toward MT(–) ends of microtubules at the centrally-located MTOC.
  • Kinesins (purple): Mediate anterograde transport toward MT(+) ends at the cell periphery.
• Motors reaching the end of a MT get carried back on organelles moved in the reverse direction by the other motor type.
• Most organelles have one or more microtubule-based motors associated with them.

Pigment Granule Movement

• Movement of pigment granules in frog melanophores depends on cAMP levels.
• Low cAMP: Dispersed melanosomes.
• High cAMP: Aggregated melanosomes.

18.5 Cilia and Flagella: Microtubule-Based Surface Structures

• Structure: Cilia/flagella are cell-surface projections with a central pair of singlet MTs and nine outer doublet MTs.
• Axonemal Dynein: Motors attached to the A tubule on one doublet produce force on the B tubule of another to bend cilia and flagella.
• Intraflagellar Transport (IFT): System transports material to the tip by kinesin-2 activity and from the tip back to the base by cytoplasmic dynein activity.
• Primary Cilium: A nonmotile primary cilium on most cells functions as a signal antenna.

Cilia and Flagella Organization

• Detailed structural organization including:
  • Plasma membrane
  • Outer-arm dynein
  • Nexin
  • Central pair of singlet microtubules
  • Inner-arm dynein
  • Axoneme
  • Transitional zone
  • Basal body
  • Radial spoke head
  • Doublet microtubule (A tubule and B tubule)

18.7 Intermediate Filaments

• Characteristics:
  • Nonpolar fibrous filaments composed of five classes of IF proteins.
  • Four IF classes show tissue-specific expression and functions.
  • Class V lamins underlie and support the membrane structure of all eukaryotic nuclei.
  • Lamins interact with chromosomes inside the nucleus and, through connecting proteins, with the cytoskeleton in the cytoplasm.
  • IF defects cause human diseases.
• Subunits are evolutionarily related but much more heterogeneous and often expressed in a tissue-dependent manner.
• Provide great tensile strength (hair and nails consist primarily of the intermediate filaments of dead cells).
• No intrinsic polarity like microfilaments and microtubules.
• Subunits do not bind a nucleotide.
• No IF motors are known.
• Dynamic subunit exchange occurs, but IFs are much more stable than microfilaments and microtubules because the exchange rate is much slower.

Structure and Assembly

• Intermediate filaments are assembled from subunit dimers. The human genome has 70 IF genes encoding proteins in at least five subfamilies.
  • (a) IF proteins: Conserved coiled-coil core domain; Subfamily-specific globular heads and tails; Form parallel dimers through coiled-coil core domains.
  • (b) Tetramer filament subunit: Antiparallel, staggered, side-by-side aggregation of two identical dimers.
  • (c) Protofibril: End-to-end and laterally associated tetramers.
  • Mature filament consists of four protofibrils with the globular domains forming beaded clusters on the surface.
  • Comparison of vimentin and lamin A structures - lamin protein has a nuclear localization sequence that targets it to the nucleus.
• No known IF nucleating, sequestering, capping, or filament-severing proteins.

Major Classes of Intermediate Filaments in Mammals

Epidermolysis Bullosa Simplex

• Transgenic mice carrying a mutant keratin gene exhibit blistering similar to that in the human disease epidermolysis bullosa simplex.

18.8 Coordination and Cooperation Between Cytoskeletal Elements

• IFs are linked to adhesion junctions for cell stability and cross-linked by associated proteins to MFs and MTs for functions.
• Cdc42 G-protein coordinates MF and MT activities during cell migration.
• Neuron growth cone migration involves interplay between MFs and dynamic MTs.

Plectin Cross-Links

• Gold-labeled antibody identifies plectin cross-links between intermediate filaments and microtubules.
  • Intermediate filament–associated proteins (IFAPs), including plakins, attach intermediate filaments to other structures.
  • Intermediate filaments (blue) are cross-linked to microtubules (red) by plectin plakins (green), that are stained with gold-labeled antibodies to plectin (yellow).

Cdc42 Regulation

Independent Cdc42 regulation of microfilaments and microtubules to polarize a migrating cell:

• Active Cdc42·GTP at the front of the cell leads to Rac and WASP activation, which stimulate assembly of a microfilament-based leading edge.
• Cdc42·GTP binds the Par6 polarity factor, leading to the capture of microtubule (+) ends and recruits and activates dynein-dynactin complexes at the cell leading edge.
• Leading-edge dynein pulls on microtubules to locate the centrosome on the side of the nucleus toward the front of the cell.
• MT orientation organizes the secretory pathway, which delivers secretory vesicles carrying adhesion molecules, including integrins, along microtubules for insertion into the plasma membrane at the front of the cell.