Brief history of single-molecule Imaging and Manipulation 1976: - - PowerPoint PPT Presentation

In vitro mechanics of molecular motors

Pasquale Bianco

PhysioLab, BIO, University of Florence, Italy Muscle Biophysics Summer School , 2018, Budapest

Brief history of single-molecule Imaging and Manipulation

1986: J. Spudich, T. Yanagida, in vitro motility assay 1991: J.Spudich, T.Yanagida, J.Molloy, single myosin mechanics 1994: K.Svoboda, S. Block, single kinesin mechanics 1998: Kinosita, F1F0 ATPase stepping kinetics 1997: W.E. Moerner, GFP blinking 1996: C.Bustamante, D.Bensimon, DNA overstretch (B-S) transition 1994: T.Yanagida, single ATP turnover in myosin 2001: J.Liphardt, C.Bustamante, RNA hairpin mechanics 1998: J. Fernandez, genetic polyprotein mechanics 1997: M.Kellermayer, M.Rief, L.Tskhovrebova, mechanical unfolding of titin 1996: T.Ha, S.Weiss, single pair FRET 2004: J.Fernandez, single-protein refolding 1976: Fluorescence image of single antibody molecule

Methods of mechanical manipulation

magnetic field magnetic bead photon field moveable micropipette latex bead Stokes drag

Optical tweezers Flow field Magnetic tweezers

Δz cantilever bending = F/k laser beam deflection

Glass micropipette Microfabricated cantilevers AFM

microfabricated silicon cantilever reference beam pedestal pulled glass micropipette Δx

Cantilever methods Force field methods

Molecular manipulators are picotensiometers

Photon field Cantilever

F = KΔx F = KΔx

Δx ≈ nanometer scale K ≈ 0.1 - 10 pN/nm F ≈ picoNewton scale

Virtual spring

60 80 100 120 140 0.22 0.24 0.26 0.28 0.3 0.32 0.34

Extension (µm) Force (pN)

Δx ΔF

How to manipulate individual molecules?

• 1. Atomic force microscope

laser cantilever photodetector molecule

Δx

End-to-end length

Force = kΔx Single Molecule Force Spectroscopy

How the Optical Tweezers works

F F

Microscope objective Scattering force Gradient force

Laser Refractile microbead EQUILIBRIUM Refractile microsphere Incoming light beam P1 P2 ΔP F=ΔP/Δt

Photon field

F =KΔx

Virtual spring

Δx ≈ nanometer scale K ≈ 0.15 pN/nm F ≈ picoNewton scale

Direct Force Measurement

Measuring the change of light momentum

Objective: partially filled with incoming laser beam Objective: partially filled with incoming laser beam Outgoing laser beam: Integrated intensity And position monitored Outgoing laser beam: Integrated intensity And position monitored

Smith et al, Science 271, 795, 1996.

Microscope objective Refractile microbead Laser 2 Laser 1 Microscope objective

In the Dual-beam optical tweezers utilizing counter-propagating beams, DLOT, two microscope objectives face each other and focus two separate laser beams to the same

• spot. Since the scattering force due to reflection is approximately the same for each laser,

these forces cancel and the axial trap stability is greatly enhanced. Dual-beam optical tweezers are therefore able to generate higher trapping forces for a given laser power and can be constructed with lower NA microscope objectives. Red lines represent light reflected at the surface

The DLOT enhances the axial trapping stability

Direct measurement of the angular intensity distribution of the laser as it enters and leaves the trap, determines the change in the momentum flux of the light beam, which is equal to the externally applied force on the particle; the force calibration becomes independent of particle’s size, shape, refractive index, viscosity of the medium, etc… n1 = refractive index of the medium θ = angular deflection of the beam RL = focal length of the lens ΔX = linear distance of the angular deflection

ΔX/ RL = n1 sinθ Ftrap = (n1W/c) sin(θ)

c = speed of light

Ftrap = (W/c)*(ΔX/RL)

The force calibration depends on the value of ΔX

W = intensity of the laser

Viscous drag forces are compared to light-momentum sensor output

(Smith, S.B., Y. Cui, and C. Bustamante, Optical-trap force transducer that operates by direct measurement of light momentum. Methods Enzymol, 2003. 361: p. 134-62) Bead diameter (µm)

A light dynamometer!

Stokes law Ff = −6πηrv

Stokes’ force (pN) force (pN)

Trapping laser 1 Trapping laser 2 Bright field illumination Fluorescence emission Fluorescence excitation

Working range: Force 0-200 pN, resolution ∼0.3 pN; Movement 0-75.000 nm, resolution ∼0.3 nm; rise time ≤ 2 ms

Dual-beam counter-propagating optical tweezers

Copper jackets X-Y-Z nanopositioner X-Y micropositioner for temperature control

DLOT implementations:

1. Temperature control in the range 4-45 °C. 2. Integration of the fast nano-positioner into a micro-positioner to provide centimeter movement for transport of particles in a multi-compartment chamber. 3. Development of a fast force and length feedback (force steps complete within 2 ms). 4. Measurement of Intracellular Calcium Signal.

+

• bjective
• bjective

thermocouple circulation fluids circulation fluids Laser 2 Laser 1 copper jackets for temperature control flow chamber

Detail of the copper jacket (Mao et al. (2005), Biophys. J.89:1308_1316)

Temperature control in the range 4-40 ° C A) The temperature in the chamber, measured by a miniaturized thermocouple recovered the set value within 7-8 s B) Power spectrum of force fluctuations for an optically trapped polystyrene bead of 3.28 µm diameter: gray trace, bath on; black trace, bath off. Acquisition time, 15 s at 15 kHz.

Characteristics of the temperature control

DLOT Driven output (+/- piezo movement)

) ( piezo

x

Σ

x Δ

command x k F Δ = * k Trap stiffness

• +
• Piezo position

x

) (light

x Length

) ( piezo

x

+

• )

(bead

x

) ( ) ( bead piezo

x x L − = Force

Force and length clamp mode

increase in force

Schematics of force-driven reactions

molecule ~ 10 nm microbead ~ 1 µm

How to grab individual molecules?

Design of molecular handles

Handling individual molecules I.

• 2. Sequence-specific antibodies

bead Ab molecule Titin’s I-band segment

• 1. Non-specific adsorption

AFM cantilever Surface Layer of molecules

• 3. Non-covalent tags

cantilever tip streptavidin biotin myosin subfragment-1 actin filament

Biotin/streptavidin

Streptavidin dimer binds 4 biotins (tetrameric protein purified from Streptomyces avidinii) Biotin: vitamin derivative Binding specific and strong, Kd~10-14

His-tag/Ni-NTA

Electrostatic interaction Strength controlled with Hisn length Works under denaturing conditions

GST-tag

Glutathion-S-transferase Conjugated to protein of interest Binds glutathion specifically, strongly

Handling individual molecules II.

• 4. Covalent cross-linkers
• 5. Au-S bond
• 6. Photoreactive cross-linkers

EDC: 1-ethyl-3-(3-Dimethylaminopropyl)carbodiimide Carboxy- and amino-reactive

Specific surface chemistries

Au Protein with terminal vicinal cysteines

S

Gold-coated AFM cantilever Gold surface SH groups (usually vicinal cysteines) Covalent bond Non-specific Photoreactive N3-group UV illumination

Handling individual molecules III.

• 7. DNA handles

Molecular dimensions Can be made specific via cloning techniques Provides mechanical fingerprint

• 8. Recombinant polyprotein
• 9. Functionalized carbon nanotube

High aspect ratio High Young modulus Chemical activation difficult Genetic polymer of known protein domain (titin I27) Repetitive sawtooth force pattern Provides mechanical fingerprint I27 Protein of interest

A synthetic nanomachine based

• n the fast myosin isoform of

skeletal muscle

Pertici et. al. (2018) Nature Communication, in press

A B

+

• +
• Biological motility is due to motor proteins that use the free energy of the

hydrolysis of ATP to generate force and reciprocal displacement between the motor and polar filamentous structures (tracks) formed by the polymerisation of the globular proteins actin and tubulin.

Motors function as porters, when they carry intracellular cargoes walking along their tracks Porters are processive motors: they walk long distances along their track. This is possible for a single motor because it is a dimer with a duty ratio ≥0.5. Motors function as rowers when they are fixed to a substratus and powers the sliding of the track Rowers are organised in array to generate steady force and sliding by cyclic interaction with their track. The duty ratio is <<0.5 and reduces with increase in sliding velocity

Background

In each half-sarcomere, myosin motors are mechanically coupled by their attachment to the thick filament and this collective motor, not accessible to investigations using single molecule mechanics, is the functional unit that accounts for the power output of the striated muscle. Cell studies cannot give the details of the motor coupling mechanism, being complicated by the large ensemble of motor proteins and filaments and by the hardly distinguishable role of cytoskeleton proteins.

thick (myosin) filament thin (actin) filament myosin II motor

(H.E. Huxley, J. Biophys. Biochem. Cytol., 1957)

Single molecule studies on purified proteins suffer from the intrinsic limit that they cannot detect the function emerging from the motor ensemble and its architecture in the half-sarcomere.

Myosin Heavy Chain ELC RLC LMM HMM S1 S2

95 nm

From cell to molecule measurement

FORCE TRANSDUCER LENGTH TRANSDUCER

Optical trap moveable micropipette Latex bead molecule Laser focus

Dual laser optical tweezers (DLOT) Actin filament Gelsolin Myosin II motors Core of the

• ptical fibre

Actin filament Gelsolin Polystyrene bead

A myosin II nanomachine

Proteins assembly in the flow chamber

HMM fragments of myosin II from rabbit psoas muscle

+ end

• + end
• end

Fluorescent image of bead-tailed actin (BTA) (Suzuki et al. Biophys. J., 1996)

→| |←

4 µm Etched single-mode optical fibre functionalized with nitrocellulose

4 µm

HMM LMM

AFM imaging of the myosin arrangement

Control on MICA

0.1 µm 1 nm

AFM imaging of the myosin arrangement

Tip of the etched fibre

Before HMM deposition With HMM

µm µm µm µm 200 nm 1 nm 1 µm 10 nm

AFM imaging of the myosin arrangement

Lateral surface of the etched fibre

Before HMM deposition With HMM

µm µm µm nm 0.2 µm 1 nm 0.2 µm 0.4 nm

Number of HMM available for actin interaction determined from rigor rupture events

1 2

The number of ruptures saturates at a value of 8.2 ± 1.2 with 100 µg/ml HMM.

Mechanical responses in 2 mM ATP (23°C)

Force rises to F0 in position clamp and then, following the switch to force clamp (arrow), up to five F-V points (for a shortening distance up to 3 µm) are determined.

1 2 3

F0 is consistently recovered independent

• f the amount of actin sliding.

Mechanical responses in 2 mM ATP (23°C)

Initial force rise (A) and force recovery following a release (B) can be fitted by a single exponential with τ ~ 0.15 s. 1 2 3

A B A B

Force-velocity relation and power in 2 mM ATP

• F0 has a gaussian distribution centered at 15.45 ± 0.41 pN
• F-V relation fitted with Hill’s equation: a/F0 = 0.23 ± 0.15, V0 = 3.40 ± 0.45 µm/s
• Pmax (at ~0.3 F0) is 5.4 aW

Mechano-kinetic model

Simulation of the mechanical output of the half-sarcomere of fast mammalian muscle

The kinetic scheme is fitted to the mechanics and energetics of the half-sarcomere of the mammalian fast skeletal muscle at 23°C (circles, Ranatunga, J Physiol. 1984; Woledge et al, Energetics aspects of muscle contraction 1985) and then applied to the synthetic machine, with the following constraints:

• - - The series compliance is raised to that of

the trap, 3.7 nm/pN ⋅⋅⋅⋅⋅⋅ ⋅⋅⋅⋅⋅⋅ The random orientation of the motors reduces the force to 55% of that of correctly

• riented motors (Ishijima et al., Biophys J.

1996)

• rat EDL at 23°C (N = 294 motors)

hs model simulation

• - Cs 3.7 nm/pN

.... Cs 3.7 nm/pN & random

Model simulation of the mechanical output of the machine

The machine responses are best fitted with a number of available motors N = 16. In comparison to the predicted value, the observed V0 (5.1 µm/s) is 50% lower, likely due to a depressant effect of random motor orientation, solving the debate whether also V0 is affected by correct myosin orientation or not.

• - - - experimental value

Model predictions

The model predicts that the number of attached motors decreases in proportion to the load, in agreement with mechanical and X-ray diffraction measurements on intact muscle fibres (Piazzesi et al., Cell 2007). According to a duty ratio of ~ 0.3 for the isometric contraction in vivo, the number of motors attached (Na) and contributing to isometric force is between 5 and 6. The average isometric force per motor is 2.8 pN, which indicates a force per correctly-oriented motor of ~ 6 pN (Ishijima et al., Biophys J. 1996).

Model predictions

The efficiency calculated for 32 heads (0.16), once corrected for the effect of the random

• rientation, is (0.16/0.55 ~) 0.3, similar to that reported for fast mammalian muscle.

The efficiency (ε) increases with N up to a saturating value attained for N ≥ 32. with 16 heads → 480 ATP/s with ΔGATP = 110 zJ → Ė = (110 zJ · 480/s ~) 53 aW εr = 5.4/53 ~ 0.1 ε = 0.1/0.55 ~ 0.18 (< observed in situ) At ~ 0.3 F0, the maximum power is 5.4 aW, and the predicted ATPase rate is ~ 30/s per myosin head.

Model predictions

N = 16 N = 32 Only for N ≥ 32 the number of actin-attached motors remains above 3 (the minimum number that satisfies the condition that at least one motor is attached at any time, Uyeda et al 1990) whatever is the load. This explains the reduced efficiency of the nanomachine with N = 16.

Summary

It has been possible to demostrate that a one dimensional synthetic machine made by less than ten HMM molecules from fast skeletal muscle generates steady force and shortening in the presence of physiological [ATP], delivering a maximum power of 5.4 aW at 1/3 F0. In this way muscle mechanics and energetics are recapitulated at the molecular level, providing the structural constraints for the efficiency and power of skeletal muscle:

• 1. A minimum number of 32 motors (16 HMM)
• 2. An architecture which simply requires that motors are anchored through their rod to a common mechanical

ground without any specific geometry such as that of the myosin molecules on the thick filament. The machine output can be simulated by a kinetic model derived from the performance of mammalian muscle. The nanomachine has been implemented with the possibility to assemble it independently of Ca2+ concentration for future studies with the regulated thin filament. Starting from purified proteins, the nanomachine allows the mechanics and energetics of the collective myosin II motor to be studied in the absence of cytoskeleton and regulatory proteins, the effects of which can then be selectively tested with different degrees of reconstitution. This machine opens new possibilities for the investigation of muscle diseases related to mutations in sarcomeric proteins and testing small molecule effectors.

Alternative approach to the support for the biomachine

The motor proteins are randomly dispersed on the flat -etched lateral surface

• f quartz square rod functionalized

etched quartz rod

Gelsolin Actin filament Polystyrene bead Dual laser optical tweezers (DLOT)

myosin motors

200-400 µm >500 µm 1-3 mm >15 mm 70 µm 1-5 µm 5- 15 µm 20-50 µm

Square quartz rod etched down to 1 µm

SEM image

2 µm

AFM imaging of the myosin arrangement Lateral surface of etched square rods

0.1 µm 4 nm 0.1 µm 0.1 µm 0.1 µm 0.1 µm