H. M. Nussenzveig, UFRJ

On 15 AUGUST 1932 Niels Bohr delivered a lecture entitled “Light and Life”

to the International Congress on Light Therapy in Copenhagen.

hmoyses@globo.com N. Bohr, Nature 131, 421 (1933)

Q.: Is QM relevant to biology?

(revisited)

Bohr’s “Light and Life” Revisited Phys. Scr. 90 (2015) 118001

IBCCF, 19/10/15

Biological Insights

“Nothing in biology makes sense except in the light of evolution” Theodosius Dobzhanski (1973)

*Comment: 1 photon is detected, but it takes ~ 3 to 5 photons for a visual sensation

Threshold of hearing: air displacements ~ 1/10 of an atomic diameter.

Evolutionary advantages: early detection of a predator in the jungle.

Teleology: Living organisms have a program:

preservation and the self-generation of individuals

Bohr 1932

Bohr 1932 Genesis 1:28

Timeline Q.: Is life (order increase) consistent with the 2nd law of thermodynamics?

“The general struggle for existence of animate beings is not a struggle

for raw materials – these, for organisms, are air, water and soil, all

abundantly available – nor for energy which exists in plenty in any body

in the form of heat, but a struggle for [negative] entropy, which

becomes available through the transition of energy from the hot sun to

the cold earth” (Boltzmann 1875) negative entropy ≅ order

Schrödinger 1943 “What is Life?”: explains genetic stability in inheritance by QM, modeling gene as

“aperiodic crystal” (today’s biopolymer), acknowledging Delbrück, who was influenced by Bohr’s lecture

Alan Turing 1952: The riddle of morphogenesis. Assumed the

existence of “morphogens”, diffusing through tissue and

chemically reacting to create patterns. Introduced

reaction-diffusion equations

Feb. 28 1953, 12 noon, pub:

“We have discovered the secret

of life” (Francis Crick)

Was it? Mechanism of gene replication, followed

by unraveling of universal genetic code,

t-RNA (hypothesized by Crick) & m-RNA

Of comparable importance: discovery

of the regulation of gene transcription by

Jacob and Monod, in 1961

The double helix

Peter Mitchel, 1966: chemiosmotic hypothesis, the universal mechanism

of energy production in cells – the most counter-intuitive idea in biology since Darwin (L. Orgel)

º

The origin of life

DNA (info) RNA (messenger)

protein (catalyst)

RNA world : RNA stores info, can act as catalyst (ribozymes)

and regulator (riboswitches, RNA interference)

• Chemical origin (ingredients?)

• Energetic origin (mechanism?)

Most accepted hypothesis for energetic origin

Lost City hydrothermal field

Deep ocean alkaline hydrothermal vents, with microstructure of

cell-sized micropores, walls lined with catalytic Fe-S, behave as

natural flow reactors, powered by thermal and electrochemical

gradients. Within pores, H and CO2 accumulate, react and yield

acetyl phosphate, ATP analogue precursor of RNA world.

Thermophoresis

From Prokaryotes to Eukaryotes

The first remnants of terrestrial life are traces of Prokaryotes

(unicellular organisms without a nucleus) ≈ 3.8 Gy ago.

Prokaryotes include Bacteria and Archaea (initially classified

as bacteria; now recognized as a separate domain of life,

different from bacteria in several important aspects). Still the

dominant life form on Earth in terms of biomass.

CHEMICAL Origin of Life Precursors for all building blocks in the chemistry of life,

nucleic acids (RNA), amino acids (proteins), and lipids

(membranes), could have arisen at ≈ the same time from HCN

deposited by comets and SO4, with the help of UV light.

Cyanobacteria

–3.5 Gy X modern

LUCA (Last Universal Common Ancestor)

150 mV / 5 nm ≈ 30 MV/m!

≈ lightning bolt!

Must have had a common ancestor (LUCA) with the 2 universal

features of all life: Genetic Code and use of chemiosmosis to

generate ENERGY. Chemiosmosis is the employment of proton

pumping across a membrane to generate an emf (used for ATP).

The emergence of the eukaryotic (nucleated) cells is regarded as

the most important event in the evolution of complex life. It is

generally accepted that Eukaryotes originated from a unique

encounter between bacteria and archaea, establishing an

endosymbiotic relationship, that gave rise to mitochondria, the

powerhouse of all eukaryotic cells. Mitochondria preserve many

prokaryotic features.

The presence of independent power sources within cells allowed a

huge expansion (by factors of order 104) of the genome. A further

endosymbiosis with cyanobacteria gave rise to chloroplasts.

NYTimes, 05/06

Loki

03/16

Lokiarchaeota

Sunlight and Life From ≈ 2.5 Gy ago, Cyanobacteria oxygenated the atmosphere, leading to oxygenic photosynthesis

and cellular respiration, through which all life on Earth is now maintained by sunlight.

Photosynthesis yields nutrients (Calvin cycle)

and returns O2 to the atmosphere (main source).

Cellular respiration breaks down nutrients,

restores the H+ gradient and generates ATP. Both

chloroplasts and mitochondria employ

chemiosmosis. Alkaline hydrothermal vents

bathed by an acidic ocean provided a natural

precursor of chemiosmosis. The greenhouse

effect helped to lower the average Earth surface

temperature to a comfortable level.

ATP = life’s energy currency. Our daily power

consumption is ≈ that of a 100 W light bulb, but

per gram this is 104 that of the Sun, not like a

candle, more like a rocket launcher!

Is Quantum Mechanics Relevant to Biology?

“The quantum of action has offered a clue to … the intrinsic stability of atoms” Bohr 1932

✓ The stability of matter is a consequence of QM (with the Pauli Principle).

QM explains the structure of matter as well as chemical processes.

What about biological interactions? Do they also require QM explanation, like covalent bonds?

No! They are typically much weaker: van der Waals-London, H bonds, ionic (shielded by H2O),

hydrophobic, … This is required for flexibility: protein folding, biochemical reactions, catalysis

Does Quantum Coherence Play a Role?

In green sulphur bacteria, excitons produced

by solar photons are transferred to reaction

center with ≈ 100% efficiency, believed to be

assisted by quantum coherence. However,

experiments employ lasers, not sunlight.

Migratory robins apparently orient themselves

by Earth’s magnetic field. It is assumed that

Evolutionarily unlikely: quantum coherence is very fragile, fast destroyed by environmental noise

Most investigated candidates: light harvesting in photosynthesis and bird magnetoreception

light induces a singlet spin state in a pair of molecules in the bird’s

eye, the evolution of which depends on magnetic inclination. However,

nothing is known about the molecules and the biological circuitry involved.

Roger Penrose + Stuart Hameroff theory of consciousness: brain is a quantum computer, wave function

collapse is a quantum gravity effect, QM processing is performed by microtubules (cytoskeleton fibers

that conduct neuron signals). [Neither a quantum computer nor a quantum gravity theory exist yet!].

Max Tegmark critique: brain is a warm, wet environment. Computed typical decoherence time scales:

~ 10–13 – 10–20 s, to be compared with typical neuron firing time scales ~ 10–3 – 10–1s PRE 61, 4194 (2000)

QM, Brain & Mind

“…the interaction between the measuring instrument and the object… exhibits a close analogy to the

fact that the mental content is invariably altered when the attention is concentrated on… it”

The mature human brain is the most complex system known in the universe.

86 billion neurons, each connected to ~ 10,000 others, 85 billion non-neuronal

cells (more connections than the number of stars in our galaxy). Each neuron

has a complex structure; many different types. Highly parallel and distributed

operation. Signals get processed and reprocessed in both directions among

layers (reentry). Organized in interconnected modules. Dynamical system: the

strength of synapses (connections) gets reorganized continually. Computer

analogies are totally misleading.

(Bohr 1932)

Complex system: a large driven conglomerate of nonlinear interacting elements. One of its

characteristic properties is self-organized criticality, the spontaneous appearance (with no need

of tuning a control parameter) of bifurcations, with long-range spatial/temporal autocorrelations

and scale-free (power-law) decay, with fractal exponents. They give rise to unanticipated

collective emergent properties and to bursts of activity known as avalanches.

Typically, the critical state represents the boundary of a disorder/order

transition. Experiments employing functional magnetic resonance imaging

of the human brain in the resting state have revealed a large-scale

spatiotemporal organization into distinct functional networks, associated

with cognitive, visual, auditory and motor cortical areas. Remarkably,

employing experimental data on the neuroanatomical connectivity of the

human brain, a recent dynamical model has shown that the empirical data

can be matched if and only if the activation threshold is set exactly at the

critical state. From an evolutionary point of view, this allows maintaining

enough order to ensure coherent functioning, while allowing a degree of

disorder and flexibility to respond to a varying environment.

Neuronal avalanches have also been detected. PNAS 106, 15921 (2009)

Chialvo+al PRL 110 (2013)

Consciousness

How reliable is consciousness?

Illusions

• We do not see the blind spot in our visual field: our brain fills it up.

“Manipulation of the visual perspective, in combination with the receipt of correlated

multisensory information from the body was sufficient to trigger the illusion that another person’s

body or an artificial body was one’s own. This effect was so strong that people could experience

being in another person’s body* when facing their own body and shaking hands with it.” *Even of different sex V. I. Petkova, H. H. Ehrsson, PLoS one 3, e3832

(2008)

If I Were You: Perceptual Illusion of Body Swapping

V. Ramachandran., Nature 377, 489 (1995)

• Phantom limbs are felt by many amputees, often with strong pain. By

using a “mirror box” that shows an image of the intact side apparently

connected with the missing limb, Ramachandran performed a “virtual

amputation” of the phantom limb.

“…recognition of the limitation of mechanical concepts in atomic physics would… seem suited to

conciliate the apparently contrasting viewpoints of physiology and psychology”. (Bohr 1932)

• The image on our retina is inverted; the brain rights it up.

Conscious or Unconscious? Henri Poincaré on his discovery of automorphic functions

The recordable cerebral activity (readiness-potential, RP) that precedes a freely voluntary, fully

endogenous motor act was directly compared with the reportable time (W) for appearance of the

subjective experience of ‘wanting’ or intending to act. The onset of cerebral activity clearly

preceded by at least several hundred milliseconds the reported time of conscious intention to act. It

is concluded that cerebral initiation of a spontaneous, freely voluntary act can begin unconsciously,

that is, before there is any (at least recallable) subjective awareness that a ‘decision’ to act has

already been initiated cerebrally. B. Libet & al., Brain 106, 62 (1983)

“… the freedom of the will is to be considered as a feature of conscious life … that not only

evade(s) a causal mechanical description but resist(s) a physical analysis”.

Free Will?

(Bohr 1932)

In human freedom in the philosophical sense I am definitely a disbeliever. Everybody acts not only

under external compulsion but also in accordance with inner necessity. Schopenhauer's saying, that

"a man can do as he will, but not will as he will," has been an inspiration to me since my youth.

A. Einstein, The World As I See It

Tolstoy, War and Peace

Apparently “free” choices can be predicted in advance Soon, PNAS 110 (2013) Science 5/22/15

Most of the brain’s operations are unconscious

Prevailing current view among neuroscientists:

Consciousness and free will are emergent properties of our brain.

Non-invasive experimental techniques combining functional magnetic resonance imaging,

electroencephalography and magnetoencephalography, allow monitoring brain activity with millimetric

and millisecond space-time resolution. They confirmed that our consciousness lags the external world.

In one experiment, words were flashed on a screen while viewers were distracted. Viewers sometimes

reported seeing them, sometimes not. Brain waves were recorded for both conscious and unconscious

trials. In both cases, the activations of the visual cortex were identical. However, for consciously

perceived words only, the activity wave is amplified and peaks around 300 ms,

A theoretical interpretation of these findings, proposed by Dehaene and Changeux, regards

consciousness just as brain-wide, synchronized, global information sharing. It makes relevant

information globally accessible and reportable to others, an evolutionary advantage.

Ignition: Eureka!

A decision to move a finger can be predicted w/ > 80% accuracy 700 ms

before awareness, Neuron 69, 548 (2011).

By implanting chips reading the activity of ≈ 100 neurons simultaneously,

a brain-machine interface allowed a quadriplegic to grasp a glass before

he became aware of his intention.

Science 348, 906 (22 May 2015)

Light as a Tool for Biology: Optical Tweezers

The father of

• Microsphere levitation

• Atom trapping

• Atom cooling

• Optical tweezers and biological applications (1968)

Invented by Arthur Ashkin (1968) at Bell Labs.

In optical tweezers, the gradient force of a strongly focused laser beam overcomes its opposing

radiation pressure and pulls a neutral particle towards the focus. For biological applications, a

microscope objective is employed to focus the beam and the trapped object lies in a water-filled

sample chamber. Infrared light, in a transparency window for water, is employed to avoid cell

damage, allowing in-vivo manipulation.

For quantitative force measurements, transparent microspheres trapped by the beam are employed

to pull on the object, acting as elastic spring-like transducers for small displacements from

equilibrium. Typical forces range from fractions of 1 pN to hundreds of pN, just right for biological

applications

OT transverse stiffness (spring constant) must be calibrated. Absolute calibration achieved in 2014.

Absolute calibration achieved at UFRJ COPEA OT Lab

Debye (1909) exact (scalar) integral representation of focused beam as superposition of plane waves

in all directions within given solid angle, extended by Richards and Wolf (1959) to EM. Mie scattering

(1908) of each plane wave by microsphere. Must include all optical aberrations of the system.

Ingredients

No fitting parameters

MDSA+ agrees with experiment within error bars

OK for all a, all d, all polarizations

“… we should doubtless kill an animal if we tried to carry the investigation of its organs so far that

we could tell the part played by the single atoms in vital functions”. (Bohr 1932)

Prediction is very difficult, specially about the future (attributed to Niels Bohr)

Some of the most important applications of optical tweezers in single-molecule cell biology are to

the study of motor proteins, marvelous molecular machines that directly convert chemical energy

into mechanical work. Within cells, they take part in unidirectional cargo transportation along

cytoskeleton fibers, energy generation, DNA replication and transcription, and numerous other

fundamental tasks. How can they be so smart?

Current application: Casimir forces (femtonewtons!)

Single-molecule cell biology

Proteins are Maxwell Demons

DS =DQ

T=

W

T= k ln 2

Leo Szilard’s 1-molecule gas

L. Szilard, Z. Physik 53, 840 (1929). J. C. Maxwell, 1871

Szilard proposed that the compensatory entropy increase arises from the measurement process

required to detect on which side the molecule is located. That is not the correct explanation.

The Demon Exorcised: Landauer’s Principle Landauer, R. Irreversibility and heat generation in the computing process. IBM J.Res. Develop. 5, 183–191 (1961).

The compensatory entropy arises from the need to erase the demon’s memory:

Erasure of 1 bit of information requires an entropy increase DS ³ kln2

First experimental verification of Landauer’s Principle (using OT!): A. Bérut et al., Nature 483, 187 (2012)

Smoluchowski proposed automating Maxwell’s demon by attaching a spring to the trap door

M. v. Smoluchowski, Phys. Z. 13, 1069 (1912).

Brownian Ratchet

A brownian ratchet rectifies Brownian fluctuations, generating unidirectionality.

Motor proteins work by rectifying Brownian fluctuations!

An automatic Maxwell demon

Life would not exist without Brownian fluctuations

Linear Motors Transport of loads along cytoskeleton

Actina Actina

Motor Domains

(heads, ‘feet’) Cargo Receptor

Actin Filament

Microtubule

Force: ~ 5 pN Step size: ~ 8 nm

Kinesin

In neurons, kinesin can carry vesicles containing neurotransmitters along the axon,

for transmission across the synapse to another neuron (brain signaling connection)

Length ≈ 60 nm

Kinesin and Optical Tweezers

Darwin in action:

• Step size = tubulin dimer spacing

• 1 step per ATP hydrolysis

Optical Tweezers have allowed study

of single-molecule interactions in cells

ººº

Myosin V walks over actin filaments

M. L. Walker et al., Nature 405 (2000) 807

Brownian fluctuations

N. Kodera et al, Nature 468 (2010) 72, AFM movies

Boyer: Rotary ATP Catalysis FoF1

H. Noji et al., Nature 386 (1997) 299

Brownian ratchet

Reversible: Efficiency ≈ 100%!

One 120 rotation per ATP

hydrolysed

The motor of life

Tether pulling by optical tweezers

B. Pontes, N. B. Viana, L. T. Salgado, M. Farina, V. Moura Neto, and HMN,

Bioph. J. 101, 43 (2011)

Mechanobiology Mechanical forces play a crucial role in cell shape control and migration, in cell division and in

differentiation. Complementing the traditional view based on morphogen gradients and genetics, it

has been found that forces play a crucial role in tissue growth and development, the process of

morphogenesis. Proteins within the cell can be released by forces, migrate to the nucleus and

activate growth-inducing genes.

Microspheres, trapped by optical tweezers and attached to a cell membrane, are employed to pull on

the cell, extracting a cylindrical tube known as a membrane tether. The tether radius R, typically of

the order of 50 to 100 nanometers, can be measured by electron microscopy, and the steady-state

force F exerted by the microsphere after extraction follows from the tweezers calibration. According

to a theory developed by Helfrich and Canham, the elastic constants of the cell membrane can be

determined from these two parameters. These constants, surface tension σ and bending stiffness κ,

are influenced both by the membrane and by its support, the cortical cytoskeleton.

Tunneling Nanotubes (TNT)

A. Rustom et al., Science 303 (2004) 1007

Membrane nanotubes are also spontaneously extended by cells and connected to

other distant cells. They have become known as TNT (tunneling nanotubes), because they allow

selective transfer of molecules and vesicles from one cell to another

By pulling on them with optical tweezers, their elastic constants have been determined in our lab. It

was found that, by extended pulling, they are first deformed to a V shape, after which a new branch

emerges from the apex (V-Y bifurcation). Similar branching is observed in TNT networks (Fig. C).

1st Observation of TNT’s at our Lab

After 24hrs: TNT’s!

Ca Flux Triggers Lamellipodium and Dendritic Cell Motion

First demonstration of signal transmission by non-neuronal cells to

distant cells through physically connected network

Membrane Spreading Occurs Following Calcium Flux Induced by Mechanical Stimulation.

Time-lapse imaging of DC stimulated with a microinjection tip (marked by the red arrow). Calcium flux is overlaid in

green on the DIC image. Veil extension is seen several minutes after the initial flux in several cells, most notably in the

cell in the lower middle portion of the frame. Note that the cell in the upper right does not flux calcium and does not

show subsequent membrane veil extension. Time is indicated in hr:min:s.

Watkins & Salter Immunity 23, 309 (2005

TNT

TNT can act to rescue endangered cells or transfer

an apoptosis signal. They are opportunistically

employed by pathogens as conduits for cell

invasion. The fact that this happens for a wide

variety of pathogens, such as bacteria, virus

(including HIV) and prions, indicates that TNT’s

have an ancient evolutionary origin. It has been

shown that TNT’s exist in vivo. They are also

involved in cancer cell pathogenesis and invasion.

TNT : long-range cell-to-cell communication system

Gerdes & Carvalho

20, 470 (2008)

Gousset 2009 Sowinski 2008

Önfelt, 2006

Bacteria Virus Prions

Bacteria also communicate

via nanotubes and transfer

antibiotic resistance this way. Dubey, Cell 144, 590 (2011)

The title of Monod’s book aptly synthesizes the complementary roles of order and chaos

in life. Order is embedded in the genome, the blueprint for cell function and inheritance. It is also

stored in the molecules on which living organisms feed, produced by capturing sunlight. It is

apparent in the structure of the marvelous molecular machines that orchestrate cell metabolism.

Chaos, manifested in fluctuations, affects the genome through random mutations and

Mendel’s laws. It is the source of Brownian motion, allowing proteins to behave as Maxwell demons.

Chance may have played a role in the origin of eukaryotes, possibly a single event in life’s history.

Chaos also plays an important role for the generation of diversity in the immune system, allowing it

to respond to an enormous diversity of antigens.

It is crucial, when applying these concepts to biology, to constantly keep in mind

Darwin’s theory of evolution. Thermal ratchets have been called “Darwin’s motors” : they work by

natural selection of favorable Brownian fluctuations.

When he gave his lecture, Bohr could not have foreseen the amazing technological

advances that biology would undergo over the following eighty years. However, his paper is a prime

example of the precept that finding the right answers is less important than asking the right

questions. As he stated when revisiting it, “Life will always be a wonder, but what changes is the

balance between the feeling of wonder and the courage to try to understand it”.

Life Blends Chaos and Order

Le Hasard et la Nécessité

Light and Life: Wonders, Not Miracles

Stevin’s Epitaph

(Symmetry and conservation laws!)

Lab Team: LPO-UFRJ Marcos Farina, Yareni Ayala, Rafael Dutra, Vivaldo Moura Neto, Nathan Bessa Viana, HMN, Bruno Pontes. P. A. Maia Neto

Diney Ether, Luis Pires

Rotary Motor Proteins

Flagelar bacterian motor FoF1 ATPase

The “motor of life”

Supplies energy to all living organisms

Reversible: Efficiency > 80%!

Paul Boyer & John Walker: Chemistry Nobel Prize1997

Applications to Cell Biology: Membrane Nanotubes Phospholipid bilayer fluid, but differs from a

soap film: no resistance to stress, but resists

curvature, which expands average

phospholipid spacing on one side and

compresses it on the other.

Torque t = kc = k R , c = curvature

k= bending m odul us

U = 12

0

L

ò kc2ds = 12 k ds

R20

L

ò

Extension to curved surface

Mean curvature:

H º1

2

1

R1

+1

R 2

æ

èçö

ø÷

Potential energy:

U = 12 k 2H( )

A

ò2

dA

extended over membrane area A

The corresponding potential

elastic energy of the membrane

is associated with its bending

modulus κ.

Analogue of Hooke’s law

for bending of a rod:

Standard (empty) tether pulling theory Powers et al., Phys. Rev. E 65, 041901 (2002), Derényi et al., Phys. Rev. Lett. 88, 238101 (2002)

Assumptions:

1. Pure membrane (empty) tether

2. Force applied at a single point

Analogy:

Growth of soap film catenoid

pulled between two rings

However: for a soap film, only surface tension, so that equilibrium shape minimizes area infinitely

thin wire, but this has ∞ curvature, opposed by membrane bending modulus. Compromise between

surface tension and bending stiffness leads to cylindrical tube (nanotube) as equilibrium shape.

Helfrich-Canham Free Energy

Mean curvature: H º1

2

1

R1

+1

R2

æ

èçö

ø÷=

1

2R for cylindrical tube, radius R

Potential curvature energy: U = 1

2k 2H( )

A

ò2

dA = 1

2

k

R2´ 2pRL for tube of length L

F =

k

2R2+ s

æ

èçö

ø÷2pRL - FLTotal free energy:

Surface tension Applied force

¶F ¶R = ¶F ¶L = 0Þ F = 2p 2ks , R = k 2sAt equilibrium:

∴ Measuring equilibrium F and R determines membrane elastic parameters κ, σ

s =F

4pR

Result: force × displacement curve in tether pulling

1–3–Rising portion

4–Sudden drop (signals

formation of nanotube)

Plateau (signals

existence of a

membrane reservoir

5–Equilibrium tether force F

B. Pontes, N. B. Viana, L. T. Salgado, M. Farina, V. Moura Neto, and HMN, Bioph. J. 101, 43 (2011)

13% drop

Standard theory prediction How to account for the much larger observed drop?

The bead contact with the cell is not a point, but a

circular patch of radius Rp.The maximum force it

can support is Fmax = 2πσRp. Pulling > collapses

the membrane onto the nanotube of radius R. With

M. Dogterom + al, PRL 94, 068101 (2005)

s =F

4pR⇒

Fm ax

F=

1

2

Rp

R

For Rp >> R, this yields a large drop.

Force Barrier Theory

Cell Cytoskeleton and Tether Extraction B. Pontes, N. B. Viana, L. T. Salgado, M. Farina, V. Moura Neto, and HMN, Bioph. J. 101, 43 (2011)

Aim – tether extraction by OT from 3T3 fibroblasts, analysing force × deformation

curve: dynamics, structure and elastic properties of membrane/cytoskeleton

Findings of previous study with the same cells [D. Raucher, M. P. Sheetz et al., Cell 100 (2000) 221]:

1 – Agrees with conventional wisdom: tethers are formed by pure membrane

(no cytoskeleton inside)

2 – Maximum force Fmax ~ 10 pN ; tether force F0 ~ 7 pN.

Our results contradict 1 and 2. Trouble with referees!

1 – Tethers contain F-actin 2 cell groups: normal (control) and treated with Cytochalasin D (cyto D), that disrupts F-actin

Cells stained with phaloidin-FITC, marker of polymerized actin that fluoresces green

Conclusion: tethers contain F-actin, even after cyto D treatment

Measurement of patch radius

Rp = asinqMeasurement of tether radius

Measured by SEM (Scanning Electron Microscopy)

RcytoD >> Rcontrol (effect of actin disruption)

Determination of Membrane Elastic Parameters

Comments: 1) Results based on Helfrich-

Canham without actin (possible changes)

2) Cyto D changes as expected [for κ,

proportionality to (average tube radius)3

Test of force barrier theory

Growing Importance of Cell Membrane/Cytoskeleton Interaction Trends in Cell Biology October 2012, Vol. 22, No. 10

There is mounting evidence that the plasma membrane is highly dynamic and organized in a complex

manner.The cortical cytoskeleton is proving to be a particularly important regulator of cellular

organization, modulating the mobility of proteins and lipids in the membrane, facilitating their

segregation, and influencing their clustering. This organization plays a critical role in receptor-mediated

signaling. K.Jaqaman & S. Grinstein, 515

The tension in the membrane physically affects cell functions and recent studies have highlighted that

this physical signal orchestrates complex aspects of trafficking and motility. Despite its undeniable

importance, little is known about the mechanisms by which membrane tension regulates cell functions or

stimulates signals. N. C. Gauthier, T. A. Masters, and M. P.Sheetz, 527

Conclusions on Cell Membrane Tethers

• Contrary to conventional wisdom, they contain F-actin

• Previous force measurements were wrong by large factors. Reliable calibration essential

• The force barrier theory is OK, and provides new technique for radius measurement

• The force X displacement curve yields information on membrane–CSK interaction

• Recent extension (in progress) to other cell types finds correlations between elastic

properties and specialized cell functions (e. g., phagocytosis)

Applications to Cell Biology: Single-Molecule Studies

Motor Proteins are marvelous molecular machines, that directly convert chemical

energy into mechanical work. In cells they act in: catalysis, transporting cargoes,

energy generation, DNA replication, transcription, cell division, motility, transduction,…

How can they be so smart?

Brownian Motion

In 1827, botanist Robert Brown observed in a

microscope a suspension of pollen grains dancing

in water and thought they were “the elementary

molecules of organic bodies” – the life force. After

reproducing it with dust grains, he changed his

mind. But his first idea was correct: life as we

know it would not exist without Brownian motion!

Proteins are Maxwell Demons Jacques Monod, Le Hasard et la Nécéssité (1970))

Maxwell’s Demon

DS =DQ

T=

W

T= k ln 2

Leo Szilard’s 1-molecule gas

L. Szilard, Z. Physik 53, 840 (1929).

J. C. Maxwell, 1871

Szilard proposed that the compensatory entropy increase arises from the measurement process

required to detect on which side the molecule is located. That is not the correct explanation.

The Demon Exorcised: Landauer’s Principle Landauer, R. Irreversibility and heat generation in the computing process. IBM J.Res. Develop. 5, 183–191 (1961).

The compensatory entropy arises from the need to erase the demon’s memory:

Erasure of 1 bit of information requires an entropy increase DS ³ kln2

First experimental verification of Landauer’s Principle (using OT!): A. Bérut et al., Nature 483, 187 (2012)

Smoluchowski proposed automating Maxwell’s demon by attaching a spring to the trap door

M. v. Smoluchowski, Phys. Z. 13, 1069 (1912).

What a piece of work is a man! Hamlet, Act II, Sc.ii

There is grandeur in this view of life, with its several

powers, having been originally breathed into a few forms

or into one; and that, whilst this planet has gone cycling

on according to the fixed law of gravity, from so simple a

beginning endless forms most beautiful and most

wonderful have been, and are being, evolved.

Darwin, The Origin of Species

The RNA Revolution Before Project ENCODE

o Crick’s “Central Dogma”: Path of Info: DNA mRNA Protein

o Gene linear DNA sequence encoding a single protein

o ncRNA = Non-protein-coding “Junk” DNA (~97% of human genome!)

After ENCODE

• “Central Dogma” violated (already by prions).

• “Junk” DNA is transcribed into new kinds of RNA, ncRNA, many of which

form networks that play a crucial role in the regulation of gene expression.

• Our number of coding genes is only 2 to 3X that of worms, but our

difference in complexity arises from the regulation mechanisms.

DNA contains the instruments of the orchestra and some musical passages;

RNA’s play the role of conductors. They can combine, in many different ways,

pieces all along the DNA to produce different “genes”.

Control is exerted, e. g., by double-stranded RNA’s or micro-RNA’s

and by mechanisms of interference or gene silencing (2006 Nobel). This

revolution, over the past 5 years, may be compared with that of Quantum

Mechanics (RNA - the ‘dark matter of biology’).

Very recent results have also greatly strengthened the “RNA World”

hypothesis for the origin of life (RNA playing the double role of “program”

and “enzyme”). In RNA interference, evolution has made great use of the AT–

GC (or GU) base pairing code in small pieces of single-stranded siRNA.

Conclusion: Regulation plays THE central role in the cell.

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RNA Interference

G.M. Cooper & R. E. Hausman, The Cell _ A Molecular Approach

Conclusion: Chaos, Order and Life Le Hasard et la Nécéssité

• Chaos: Plus ça change, plus c’est la même chose.

• Order: Permanence sub specie aeternitatis (clockwork).

• Life: Plus ça change, plus c’est différent!

• Living organisms make use of two kinds of information (order) in their dynamical evolution, at the border order/chaos: staying at this border allows them to combine robustness with adaptation. Cells are complex adaptive systems. Self-organized critical state.

• Requirements of natural selection:

• (I) Reproduce fast in optimal conditions;

• (ii) Survive (rare) extremal conditions.

• Two kinds of order:

• 1) Stored in the genome;

• 2) Stored in the molecules on which they feed (origin: the Sun).

• Chaos (producing fluctuations) plays an important role, when combined with natural selection :

• 1) In genome mutations;

• 2) In rectification, by proteins, of favorable brownian fluctuations: mechanism of Maxwell’s demon.

• Boltzmann wanted to become “the Darwin of matter”. The Brownian ratchet mechanism of protein function may be regarded as “natural selection of favorable fluctuations”.

Sunlight and Life

Sunlight maintains average Earth surface at ~ +14°C, with greenhouse effect (without, –19°C)

“What an organism feeds upon is negative entropy” (E. Schödinger, 1947)

order, information

Sunlight is the source of this order, through photosynthesis

Sunlight is the source of life

Darwin and the Eye

“… the absorption of a single light quantum* by…retinal partitions is sufficient for a sight impression.”

(Bohr 1932)

“Nothing in biology makes sense except in the light of evolution” Theodosius Dobzhanski (1973)

*Comment: 1 photon is detected, but it takes ~ 5 photons for a visual sensation

Threshold of hearing: air vibrations ~ 1/10 of an atomic diameter.

Evolutionary advantages: early detection of a predator in the jungle. ✓Living organisms have a program: “the self-preservation and the self-generation of individuals”

(Bohr 1932)

QM & Biology

“…the quantum of action has offered a clue to our understanding of the intrinsic stability of atoms” (Bohr 1932)

✓ The stability of matter is a consequence of QM (with the Pauli Principle).

Besides stability, permanence: genetic inheritance over many generations. How?

“We believe a gene – or perhaps the whole chromosome fiber – to be an aperiodic solid” (Schrödinger 1947)

(Before Watson & Crick). ✓ “Aperiodic solid” Biopolymer.

What about biological interactions? Do they also require QM explanation, like covalent bonds?

No! They are typically much weaker: van der Waals-London, H bonds, ionic (shielded by H2O),

hydrophobic, … This is required for flexibility: protein folding, biochemical reactions, catalysis

Does Quantum Coherence Play a Role?

Possibly (still not settled), again in connection with light: in photosynthesis.

G. Fleming+ al., Nature 434, 625 (2005)

Transfer of excitation energy from pigment antenna

molecule to reaction center, observed by 2D

femtosecond photon-echo spectroscopy, is not

stepwise down energy ladder, but by coherent

oscillations among delocalized Frenkel excitons.

However, no entanglement among chromophores

has been observed so far.

QM, Brain & Mind

“…the interaction between the measuring instrument and the object… exhibits a close analogy to the

fact that the mental content is invariably altered when the attention is concentrated on… it” (Bohr 1932)

The mature human brain is the most complex system known in the universe.

86 billion neurons, each connected to ~ 10,000 others, 85 billion non-neuronal

cells (more connections than the number of stars in our galaxy). Each neuron

has a complex structure; many different types. Highly parallel and distributed

operation. Signals get processed and reprocessed in both directions among

layers (reentry). Organized in interconnected modules. Dynamical system

– strength of synapses (connections) gets reorganized continually.

Computer analogies totally misleading.

Roger Penrose + Stuart Hameroff theory of consciousness: brain is a quantum computer, wave function

collapse is a quantum gravity effect, QM processing is performed by microtubules (cytoskeleton fibers

that conduct neuron signals).

Max Tegmark critique: brain is a warm, wet environment. Computed typical decoherence time scales:

~ 10–13 – 10–20 s, to be compared with typical neuron firing time scales ~ 10–3 – 10–1s PRE 61, 4194 (2000)

The resting brain is in a self-organized critical state Chialvo et al., PRL 110, 178101 (26 April 2013)

The model consists of a network of interconnected nodes (i.e., the connectome),together with a dynamical rule.

Difference in the correlations

between the model using the

connectome and a randomized

version. The dashed horizontal

line indicates the level of p<0:001,

The spatial organization of

the human brain RSN (left

column)emerges

spontaneously in the model

near Tc.

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SM = Sensory-Motor

EC = Executive Control

Global Neuronal Workspace Model Associative perceptual, motor, attention, memory, and value areas interconnect to form a higher-level unified

space where information is broadly shared and broadcasted back to lower-level processors by “ignition” of a large-

scale prefronto-parietal network. The GNW is characterized by its massive connectivity, made possibly by thick

layers II/III with large pyramidal cells sending long-distance cortico-cortical axons

Light as a Tool for Biology “… we should doubtless kill an animal if we tried to carry the investigation of its organs so far that

we could tell the part played by the single atoms in vital functions”. (Bohr 1932)

"We never experiment with just one electron or atom or (small) molecule. In thought-experiments

we sometimes assume that we do; this invariably entails ridiculous consequences”. E. Schrödinger, Brit. J. Phil. Sci 3, 233

(1952)

Single-molecule cell biology was made possible by

Optical Tweezers

The father of

• Microsphere levitation

• Atom trapping

• Atom cooling

• Optical tweezers and biological applications

“Blue Sky” research (à la Lord Rayleigh)

Invented by Arthur Ashkin (1968) at Bell Labs.

Prediction is very difficult, specially about the future (attributed to Niels Bohr)

Typical Set-Up Optical traps for m-sized neutral particles (e.g., microspheres)

Radiation pressure pushes, gradient force pulls.To trap, must win tug of force over pressure

Requires highly focused laser beam

Nd:YAG laser (1.064 m) favored for cell biology: no damage for in-vivo use (up to few

hundred mW) because transparency window for H2O.

Force range < 1pN to 100’s of pN on a m-sized object – just right for biological applications.

Can detect ~ 0.3 nm displacements in real time (ms).

Manipulate transparent microspheres as handles & force transducers (the Glass Bead Game)

OT transverse stiffness (spring constant) must be calibrated

OT Features

UFRJ OT Lab

Kinesin Moves Hand Over Hand Brownian Ratchet: diffusive

search for next site

(Indirect) Force Calibration

Comparison with drag force: Move fluid chamber past sphere, measure escape velocity v and use Stokes’ law:

Drag force = v = x

Alternative: power spectrum of Brownian (oscillator) fluctuations

Neuman & Block, Rev. Sci. Instrum. 75 (2004) 2787 Radiation Pressure on Perfect Mirror

Momentum per photon = h/c

p per photon = 2 h/c

Laser beam power = P

# of photons/sec = P/h

Force = (# of photons/sec) × p

Radiation pressure force on perfect mirror = 2P/c

Dimensionless force efficiency factor defined by

n1 = medium refractive index

Absolute (1st-principles) calibration theoretical derivation of force.

Usually a ~ λ = laser wavelength, neither Rayleigh nor GO (geometrical optics).

Ashkin’s GO (WKB) single-ray forces

P = laser power, n1 = medium refractive index

R = reflectivity, T = transmissivity, = angle of incidence

A. Ashkin, Biophys. J. 61, 569 (1992)

Attempts at EM Theory

GLMT = Generalized Lorenz-Mie Theory

(Gouesbet+al, series of papers over 20 y)

Represents highly focused illuminating laser beam as Gaussian

including 5th-order non-paraxial corrections in powers of /(beam waist).

However, it does not account for effects of diffraction by the objective

GLMT is highly referenced but wrong

So, 20y after Ashkin’s invention, no 1st-principles theory existed

Debye (1909) exact (scalar) integral representation of

focused beam as superposition of plane waves in all

directions within given solid angle.

Mie-Debye (MD) First-Principles Theory(2000) P. A. Maia Neto & HMN, Europhys. Lett. 50 (2000) 702.

where the unit vectors û span the solid angle Ω. EM extension:

Incident beam Gaussian before the objective, but diffracted by it.

where is from Abbe sine condition and ê(θ,φ) is the

rotated (circular) polarization unit vector.

Mie scattering (1908) of each plane wave by microsphere.

High accuracy model (accuracy comparable to QED).

Dimensionless force efficiency factor defined by

Axial efficiency is easiest to evaluate

B. Richards and E. Wolf, Proc. R. Soc. London, Ser. A 253, 358(1959).

j(P) = A exp ikuiR( )W

òò dWu

Axial Stiffness and Oscillations At focus z = 0, ka >> 1, find asymptotic approximation of the exact result:

Fabry-Perot interference oscillations from two edges of sphere

GO hyperbola is average over

interference oscillations (error shown in inset) Detection of predicted oscillations employing

supercontinuum light source and H2O droplets Guillon + al Opt. Express 16 (2008) 7655.

A. Mazoli, P. A. Maia Neto & H. M. Nussenzveig, Proc. Roy. Soc. Lond. A 459 (2003), 3021-3041 MD Extension to Transverse Stiffness

Checking the result in GO Asymptotic Limit

Nontrivial calculation: includes new asymptotic WKB approximation to rotation matrix elements.

Average is over Mie quasiperiod. Result: recovery of Ashkin’s single-ray WKB formula integrated

over incident angular distribution in laser beam.

MD Transverse Theory Disagrees with 1994 Experiment

Reason: Interface Spherical Aberration

The focal region gets degraded ⇒ trap stiffness decreases with height above slide

Spherical aberration is incorporated into MD theory by inserting a phase factor for the path difference

(aberration function). Result: MDSA Theory, a candidate for absolute calibration.

All parameters in the theory are measurable: none adjustable.

Experimental Tests of MDSA (2004-07) N. B. Viana, M.S. Rocha, O. N. Mesquita (UFMG), A. Mazolli, P. A. Maia Neto, HMN (UFRJ); APL (2006), PRE (2007)

2 very ≠ setups: 1064 nm, overfilled objective (UFRJ), 832 nm, underfilled objective (UFMG);

Every parameter measured by 2 ≠ methods. Tests performed independently,reproducibly at months intervals

h – a = 2.7 m

h – a = 13.2 m

MDSA predicts no stable trapping if a< 0.52 m;

confirmed by scattering of data and bead escape

Large discrepancies: only for a < /2

Multiple Equilibria and “Jumps”

UFMG

UFRJ

Balance Sheet for MDSA

•Agreement OK for a > /2

•OK for ≠ setups

•OK for height dependence

•Predicts trapping thresholds

•Predicts multiple equilibria

•Predicts “jumps”

Final Step: MDSA+ (2012) R. S. Dutra, N. B. Viana, P. A. Maia Neto & HMN, APL100, 131115 (2012)

Inclusion of all primary optical system aberrations in MDSA shows that only relevant one is astigmatism

The astigmatism parameters

are measured with the setup

and introduced into MDSA.

Astigmatism coefficient is

~ 60% from objective and

~ 40% from rest of system.

Stiffness/(unit power) X height d reveals no

stable trapping for d > 3 m, as is observed

(contrary to MDSA)

a = 0.376 m

axial optical potential

d = 3 m

MDSA overestimates

stiffness by

factor >4.

Spatial phase

modulator

astigmatism

correction

might achieve

nanonewton

forces

MDSA+ agrees

with experiment

within error bars OK for all a, all d, all polarizations

No fitting

parameters

Implications of absolute calibration of optical tweezers

•Reliable measurements in circumstances where indirect calibration is difficult

•Predicts trapping stability in special circumstances

•Allows Improved design and trapping range (by aberration compensation)

•Extends the domain of applicability:

•Usually stated force range: ~ 1pN to tens of pN

•Possible force range: a few fN to several nN (overlap with AFM)

Application under way: measurement of Casimir forces

1st Observation of TNT’s at our Lab Cultured U-87 MG human glioblastoma cells

After 24hrs: TNT’s!

Structure and Elastic properties of TNT’s B. Pontes, N. B. Viana, L. Campanati, M. Farina, V. Moura Neto & HMN, Eur. Biophys. J. 37 (2007) 121

Average U87 TNT Radius

Hard to measure directly (below limit of resolution) so electron microscopy

Result: <R> = 48 ± 6 nm

Formation

By separation of initially connected cells

F-Actin Content (Fluorescence)

A - DIC; B- Fluorescence with phaloidin-FITC staining: F- actin inside TNT’s

Immunofluorescence for tubulin: negative

F-Actin Content (Field Emission Microscopy)

Cell membranes washed out with detergent. TNT are filled with F-actin.

Their implantation within cell looks similar to that of filopodia.

Sensitivity to mechanical stress (rupture) is apparent.

Membrane-Cytoskeleton Interaction: Tether Pulling

F-actin

Tubulin

Nucleus

Tether pulling by optical tweezers

B. Pontes, N. B. Viana, L. T. Salgado, M. Farina, V. Moura Neto,

and HMN, Bioph. J. 101, 43 (2011)

Tunneling Nanotubes (TNT)

A. Rustom et al., Science 303 (2004) 1007

TNTs are membrane nanotubes that connect distant cells. Observed by Rustom+al

In cultured rat adrenal gland PC12 tumor cells (also normal human kidney cells).

Cells are connected via one (A) or several (B) TNT’s, straight, [rarely branched (C)],

extended above the substrate (D) and continuously connecting cell membranes (F).

They are sensitive to stress (rupture). Immunostaining shows they contain F-actin

but no tubulin (microtubules). Diameters: 50-200 nm; lengths up to many μm

Vesicle Transfer under Stress

Observed only under stress (no CO2)

Distress signal? Call for apoptosis?

Arkwright et al, Cell Research 20 (2010) 72

confirmation

Features of V–Y Bifurcation

s » 4´10-5 N/m

k » 2´10-19

JFirst determination of surface

tension and bending rigidity

of TNT

Possible “zipper”mechanism

of Y formation (in vesicles) Lobovkina + al, PRL 97 (2006) 188105

Discussion of Bifurcation Results

over

0

»1.15

consistent with almost point-like attachment of

microsphere to TNT.

No plateau after drop indicates lack of

membrane reservoir for TNT.

V–Y Bifurcation

Assume for R0 average from electron

microscopy, ~ 50 nm, and take F0 just

beyond dip, ~ 25 pN. Then, estimates

assuming steady-state equations yield

s » 4´10-5 N/m

k » 2´10-19

Jwhich are reasonable values

I–D Bifurcation

Likelihood that 2 TNT’s (possibly entwined) are involved.

If so, F includes force needed for gradual separation of the two TNT’s.

For ® 0, over

0

®1.13.

Calcium flux signals propagate through TNT’s

S.C. Watkins + R. D. Salter, Immunity 23 (2005 309

Dendritic (immune) Cells DC stimulated by microinjection tip (red arrow) with bacterial

antigen respond by transmitting Ca flux (false colors) to neighboring cells through TNT’s

TNT connections can range up to 100 m

Left: fluorescent image; Right: DIC image with time lapse in seconds

Ca2+ is an essential transmitter of information in the organism

Bacteria and Beads Surfing along Thin NT

Önfelt et al., Journal of Immunology, 2006, 177: 8476

HIV Spreads between T Cells Along ICN

HIV-1 Gag protein fused with GFP

moves along NT from infected ( )

to uninfected ( ) Jurkat T cells

Sowinski, S., et al. 2008. Nat. Cell Biol. 10, 1038

This increases infectivity by

several orders of magnitude

Viral Cytonemes (TNT) N. M. Sherer + al, Nature Cell Biol. 9 (2007) 310

MLV virus surfs along TNT’s a–e

from infected to non-infected cell

TNT is pulled from non-infected to

Infected (green arrow) while virus

surfs in opposite direction (red arrow)

(retrograde flow)

Model for virus spread

“Filopodial” formation: extended from cell to cell, like original TNT

Prions Propagate from Gut to Brain via TNT’s

Marked vesicle containing prions

penetrates into neural cell through TNT’s

Gousset, K. et al. Prions hijack tunnelling nanotubes for intercellular spread. Nature Cell Biol. 8 Feb 2009

TNT’s Exist In Vivo

(2) H. R. Chinnery + al., J. Immunol. 180 (2008) 5579

Transparency of mouse cornea allowed direct observation. In inflamed corneas,

dendritic cells were found to be connected by TNT’s. Inflammation or exposure to

bacterial toxins increases the number of TNT’s. Conjecture: they may serve to

transfer antigen/receptor complexes between widely separated dendritic cells.

(1) Already found in Drosophila embryo (cytonemes)

“… the freedom of the will is to be considered as a feature of conscious life … that not only

evade(s) a causal mechanical description but resist(s) a physical analysis”. (Bohr 1932)

Free Will

Roger Sperry and Michael Gazzaniga’s experiments with split-brain patients (with

two conscious minds) have shown that the two hemispheres have very different

capabilities. The right brain is a realist, does holistic processing. The left brain is

the “interpreter”, that continuously tries to interpret and make sense out of what

goes on or has gone on inside the brain, even if it has to invent an explanation,

e. g., with illusions.

“The recordable cerebral activity (readiness-potential, RP) that precedes a freely voluntary, fully

endogenous motor act was directly compared with the reportable time (W) for appearance of the

subjective experience of 'wanting' or intending to act. The onset of cerebral activity clearly preceded

by at least several hundred milliseconds (about 300) the reported time of conscious intention to act….

It is concluded that cerebral initiation of a spontaneous, freely voluntary act can begin unconsciously,

that is, before there is any (at least recallable) subjective awareness that a 'decision' to act has

already been initiated cerebrally.” B. Libet & al., Brain 106, 62 (1983)

In human freedom in the philosophical sense I am definitely a disbeliever. Everybody acts not only

under external compulsion but also in accordance with inner necessity. Schopenhauer's saying, that

"a man can do as he will, but not will as he will," has been an inspiration to me since my youth.

A. Einstein, The World As I See It

Prevailing current view among neuroscientists

Consciousness and free will are emergent properties of our brain.

S. Dehaene & J. P. Changeux, Neuron 70, 200 (2011)