MT-Based Motor Proteins and Discovery of Kinesin Notes

MT-based Motor Proteins and Discovery of Kinesin

Organelle Positioning

• Organelles are positioned within the cell by motor proteins moving along microtubules.

• Microtubules have distinct ends: plus ends and minus ends.

• Examples of organelles include the ER and Golgi apparatus.

• Kinesins and dynein are motor proteins involved in organelle movement.

Axoplasm and Motor-Based Motility

• Axoplasm is an extract system that supports motor-based motility, similar to what occurs in a living giant axon.

• Experiments involve in vitro studies using giant squid axons.

• Shinya Inoue's lab at MBL in Woods Hole utilized video-enhanced DIC (Differential Interference Contrast) microscopy to study these processes.

Action Potential Propagation

• Illustrations show the propagation of an action potential along an axon.

• Sodium (Na+) channels open and close during depolarization and repolarization of the membrane.

Experiment Freezing Vesicles on Microtubules

• Adding AMP-PNP to axoplasm freezes vesicles on microtubules, halting movement.

• This indicates that microtubules are the filaments along which organelles move in both directions.

Gliding Assay

• The gliding assay involves organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon.

Experimental Procedures

• Figure 1: Illustrates experimental conditions:

  • (a) Purified microtubules (MTs).

  • (b) Axoplasm (S2) extract.

  • (c) Control: MTs + ATP release (no AMP-PNP).

  • (d) MTs + AMP-PNP followed by ATP release.

  • (e) MT pellets - AMP-PNP.

  • (f) MT Pellets + AMP-PNP Axoplasm Extracts.

Optic Lobe Extracts Rebinding

• The process involves taking eluted material, diluting it to lower ATP concentration, and rebinding it to MTs +/- AMP-PNP.

• Elution is performed again using salt + ATP.

Chromatography Columns

• Chromatography columns are used in the purification process.

Gel Filtration and SDS-PAGE

• Gel filtration separates proteins by size.

• SDS-PAGE is used to analyze the composition of each fraction.

• The translocator activity corresponds to the ~110 kDa band.

Hydroxyapatite Column

• The hydroxyapatite column is an ion exchange column that separates proteins based on their charge.

Purified Kinesins

• Images of purified kinesins and their motor domains are shown.

Mechanism of Kinesin Walking

• Kinesin walks along microtubules in a hand-over-hand fashion.

• Step 1: HEAD 1 (ADP) binds to the microtubule (MT).

• Step 2: HEAD 1 releases ADP.

• Step 3: HEAD 1 binds ATP, causing the neck linker to zip up and swing HEAD 2 into position.

• Step 4: HEAD 1 hydrolyzes ATP and releases Pi (now HEAD 1 is bound to ADP).

• This process repeats with HEAD 2.

• Coupling conformational changes in the “neck linker” domain to its ATPase cycle allows kinesin to directionally walk along microtubules.

Laser Tweezers (Optical Traps)

• Laser tweezers are used to study kinesin motility.

Optical Trap Principles

• Optical traps work by focusing a laser beam to create a potential well that can trap particles with a different refractive index than the surrounding solution.

• Light is refracted by the bead, and due to the law of conservation of momentum, forces pull the bead into the center of the laser.

Hooke's Law and Force Measurement

• Hooke’s Law: Force = kx, where k is the spring constant and x is the distance.

• Changes in laser light are measured to detect extremely small changes in x (sub-nanometer).

• Forces that can be measured with an optical trap range from 0.1 pN to 100 pN.

Visualizing Kinesin Motility

• Kymographs are used to visualize kinesin motility.

• Motors move directionally along microtubules, and microtubule polarity is important.

Gaussian Analysis and Centroid Determination

• Gaussian analysis of a diffraction-limited spot allows defining the “centroid”.

• By fitting a 2D-Gaussian to the signal, the centroid can be defined with ~1-3nm accuracy if the signal is high enough above background.

Single Molecule Imaging

• Purified kinesin can be made to glow for single-molecule imaging.

• FIONA (Fluorescence Imaging with One Nanometer Accuracy) is a technique used.

• 8 nm steps can be imaged at 50 msec.

Dynein and Retrograde Transport

• Retrograde transport of cargo in axons is mediated by dynein.

• Bidirectional transport of organelles and beads on MTs in axoplasm is observed.

• Dynein activity is required for retrograde transport of beads on MTs in axoplasm.

Dynein vs. Kinesin

• A comparison between dynein and kinesin is made.

Dynactin Complex and Adaptors

• In many organisms, dynein motility requires its association with the dynactin complex via adaptors.

Dynein Stoichiometry

• Quantification of dynein heavy chain stoichiometry reveals double dynein complexes (Urnavicius et al., Nature 2018).

• In vitro, the mean velocity is 1.2 mm/s.

Optical Trapping of Dynein-Dynactin

• Optical trapping is used to study dynein-dynactin interactions.

Step Size Analysis

• Each blip upwards represents a distance of ~8 nm.

• Step size analysis is performed for kinesin and dynein.

Tubulin Structure

• The structure of tubulin is shown, with α-tubulin and β-tubulin subunits.

• The size of the tubulin dimer indicated. 8 nm.

Atomic Force Microscopy (AFM)

• Atomic Force Microscopy (AFM) is used to study molecular structures.

• Hooke’s Law: F = kx, where k is the spring constant of the cantilever arm and x is the distance the arm moved.

• AFM tips can be “functionalized” with antibodies, streptavidin, biotin, etc.

AFM Applications

• AFM can be used to pull on single molecules.

• AFM can be used to scan surfaces coated with molecules to visualize them, including motors like Myosin II on actin filaments.