4. Connections to the brain

3. retina

1. lens andoptics 2. photoreception

event5. A master switch

that controls differentiation

6. Time frame for evolution of the major features

BBE/CNS 150 Lecture 14 Wednesday, October 30, 2013Vision 1: Phototransduction and the RetinaandEvolution of the Eye Henry LesterChapter 26, co-written by Markus Meister 1

4. Connections to the brain

3. retina

1. lens andoptics 2. photoreception

event5. A master switch

that controls differentiation

6. Time frame for evolution of the major features

2

BBE/CNS 150 Lecture 14 Wednesday, October 30, 2013Vision 1: Phototransduction and the RetinaandEvolution of the Eye Henry LesterChapter 26, co-written by Markus Meister

“Nothing in biology makes sense except in the light of evolution”Theodosius Dobzhansky

All modern biological processes evolved from related processes.

Every modern gene evolved from other genes.

Every gene has an ortholog in related species, and

most genes have paralogs in the same species.

Because all vertebrate eyes are quite similar,

the hunt for orthologs is straightforward and successful in most cases.

That two organisms share many orthologs is powerful evidence for the view

that those organisms are descended from a common ancestor—a central

aspect of evolution.3

Myr BP

Hemoglobin paralogs in the human genome

Myr BP

Hemoglobin paralogs in the mouse genome

chromosome 7

orthologs resemble each otheracross species (mouse vs human )

human vs mouse vs

paralogs resemble each other, distant or closely, within a species

G vs A

Example: globin genes

orthologs & paralogs

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The lens has an index of refraction greater than water,

because it contains a high concentration of protein.

Many proteins different serve this purpose have been used in various animals.

Some of these proteins, termed crystallins, are also enzymes

that perform metabolic functions in other tissues.

Apparently the only requirement is that the protein have good solubility and no

attached groups (such as vitamins) that might absorb light.

1. Lens and optics

from Lecture 1 How much is 4 mM protein?

A typical protein has 500 amino acid residues.

An average residue has a molecular mass of 110.

Therefore the average protein has a molecular mass of 55,000.

( 4 x 10-3 mol/liter) x (5.5 x 104 g/mol) = 2.2 x 102 g/l = 220 g/l.

The cell is ~22% protein!

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Pax-6 orthologs occur in phyla as diverse as as mammals, insects, and molluscs.

Many genes, including crystallins, have acquired a “Pax-6 responsive element”Pax-6 contains a homeo domain & another-DNA binding domain

Crystallin

Pax-6, a transcription factor with orthologs in many species

Pax-6 (vertebrates)Ey (Drosophila) Presence of multiple

sufficient gene regulatory mechanisms can underlie

“gene sharing”

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Existing proteins have been used for an additional functions. Which way were they adopted? Probably the use in the lens came second. Evidently several distinct transcription factors can “share” activation of a given gene.

The aperture mechanism: controlled by smooth muscles

singlesmooth muscle cell

inextensible fibers

Contracts and thickens: leads to smaller pupil

nerve from brain; muscarinic synapse

Innervated smooth muscles control:

diameter of blood vessels, peristaltic activity of the intestinal tract,

diameter of the bladder neck

In each case, the nervous system has evolved circuits that

(1) extract and integrate information from sensors and

(2) employ smooth muscles in a homeostatic loop.

blocker: atropine from Atropa belladonna

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Photoreceptor organs have evolved independently at least 40 times, each

time responding to the visible spectrum and near-UV.

How do we explain the use of a limited part of the spectrum?

Infrared light is not sufficiently energetic to provoke photochemistry such

as cis-trans isomerization.

Shorter-wavelength ultraviolet light is too energetic and would destroy

organic molecules.

2. The photoreception event

8

h

9

h

Free-floating discs

Rhodopsin

Rhodopsin

The photoreceptor cells receive light from “the back”

Like Figs. 26-5, 26-7

Each opsin interacts distinctly with retinal, producing a distinct absorption spectrum.

There are 4 opsin paralogs in the human genome.

Absorption spectra of cone pigments

Blue- green- red-absorbing

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Mutations that change the spectrum

Like Fig. 26-8, 26-9

Detection of light by retinal bound to opsin

From Darnell et al., Mol. Cell Biology11

Enzymes

Like Fig. 26-8

cytosol

The usual GPCR pathway

nucleus

kinase

phosphorylatedprotein

cAMPCa2+

intracellularmessenger

receptor

tsqiG protein

enzymechannel effector

membrane

from Lecture 12outside

inside

outside

inside

12

The GPCR pathway in a photoreceptor

channel

receptor

tsqiG protein

enzymechannel effector

cytosol

intracellularmessenger

Ca2+ cAMPcGMP

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membrane

GTP GDP + Pi

Effector: enzyme or channel

outside

inside

Neurotransmitter or hormonebinds to receptor

activatesG protein

Beginning of the G Protein-Coupled Receptor Pathway

How fast?100 ms to 10 s

How far?Probably less 1 m

like previous lectures

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Photon isomerizesretinal bound to rhodopsin

Special aspects of the G protein-coupled receptor pathway in photoreception

How fast?< 100 ms

How far?< 1 m

GTP

activatesG protein

Effector is an enzyme

GDP + Pi

h

Although the components are not membrane-bound, the membranes effectively

restrict their motion

In rods and cones, these proteins lack lipid tails

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cytosol between disks,or

between folds

Like Fig. 26-7

Intracellular messengers bind to proteins

kinases

phosphorylatedprotein

A few ion channels(olfactory system,

retina)

N

NN

N

NH2

O

OHO

HH

O

P-O

O

cyclic AMP (cAMP)

Ca2+ and

Expanding on a previous lecture, we said . . .intracellularmessenger

Ca2+ cAMPcGMP

Cyclic nucleotide(cAMP or cGMP)

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Cyclic GMP is the second messenger for phototransduction

High cyclic GMP keeps the plasma membrane

depolarized and keeps glutamate release at the

terminal high.

Increased Hydrolysis of cGMP reduces cGMP concentration, resulting in closing of a cation channel in the outer segment

membrane and transient hyperpolarization of the entire plasma

membrane.

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cAMP ATP

Effector enzyme“cyclase”

Breakdown enzyme“phosphodiesterase”

Inhibited by caffeine uninteresting

cGMP GTP

Enzyme“cyclase”

Breakdown enzyme“phosphodiesterase”

uninterestingThe effector for Gt

like a previous Lecture

A paralog expressed elsewhere in the body is

inhibited by Viagra

channel

receptor

tsqiG protein

enzymechannel effector

intracellularmessenger

Ca2+ cAMPcGMP

“Viagra . . . may cause a perception of bluish haze or increased light sensitivity in

some patients.”

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Rods and Cones have cGMP-activated Na+/Ca2+ Channels

Excised “inside-out” patch

allows access to the inside surface

of the membrane

no cGMP no channel openings

+cGMP*

+cGMP*

closed

open

like a previous Lecture receptor

qiG protein

channel

ts

enzymechannel effector

intracellularmessenger

Ca2+ cAMPcGMP

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Light Response of the Photoreceptor Cell

The vertebrate photoreceptor functions electrophysiologically opposite to most neurons.

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1. Rhodopsin absorbs light

2. Cation channels close in the plasma membrane of the outer segment, which hyperpolarizes the entire cell

. 3. The hyperpolarization relays visual information to the synaptic terminal,

where it slows ongoing release of the transmitter glutamate.

The “ribbon synapse” facilitates the tonic high rate of transmitter release

Photoreceptor to horizontal cell synapse

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The Phototransduction Cascade:1. Amplification 2. Adaptative/homeostatic mechanisms

1. When fully dark-adapted, many species can detect ~1 photon per photoreceptor cell

2. When fully light-adapted, many species can accurately analyze light at intensities ~1010 fold brighter

Many adaptive and homeostatic mechanisms underlie these phenomena.

Note: it is incorrect to explain that your favorite process (memory, learning, addiction) occurs “because of” homeostasis or adaptation.

Homeostasis and adaptation are not, by themselves, mechanisms.

There are homeostatic and adaptive mechanisms.

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The Phototransduction Cascade:1. Amplification (2. Adaptive/homeostasic mechanisms)

1a. When the rod is dark adapted, the activated Receptor (O*) can activate 500 transducin proteins.

1b. The phosphodiesterase has a turnover number of 4200/sec, near the diffusion limit for catalysis.

1c. Each millisecond that the cGMP-dependent cation channel in the rod outer segment plasma membrane is open,10,000 ions flow through it.

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2a. Transducin hydrolyses GTP to GDP and thus inactivates itself.

The Phototransduction Cascade:(1. Amplification) 2. Adaptive/homeostatic mechanisms

3c. Guanylate cyclase must synthesize new cGMP from GTP(1) Guanylate cyclase is partially inhibited by [Ca2+] > ~75 nM.(2) Ca2+ influx through the tonically open cation channel sets the cytosolic level of Ca2+ to ~ 500 nM.(3) When the cation channel closes upon light stimulation, Ca2+ continues to be pumped out via the usual processes, lowering cytosolic Ca2+ to ~50 nM and activating guanylate cyclase

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2b. The activated receptor (O* or R*) must also be deactivated.(1) Rhodopsin kinase phosphorylates the carboxyl tail of the receptor(2) The phosphorylation permits binding of the inhibitory protein, arrestin

Rod

Cone

Synapses of outer plexiform layer

3. Neurons of the retina

Glutamate is the major transmitter; Some neurons make

dopamine & acetylcholine.Inhibitory neurons release GABA.

Many paralogs to genes expressed elsewhere:

Channels, receptors, transporters.

Ganglion cell is unique in firing impulses

optic nerve

Synapses of inner plexiform layer

Bipolar cells

Horizontal cells

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Like Fig. 26-2

Roger Sperry’s Nobel prize-winning experiments (1948) (goldfish): After he cut the optic nerve, individual fibers grew back to their original destination in

the brain.

Sperry also conducted the “Split brain” experiments that form the basis for

modern ideas about the distinct specialties of the two hemispheres.

4. Connections to the brain

Sperry postulated a “chemoaffinity” between

the nerves and their target cells.

A previous Lecture

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Horseshoe crabs(Limulus polyphemus)

Maps may be unique to nervous systems,

but

visual maps arose at least 500 Myr ago.

We will discuss visual maps in the next

lectures.

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tyrosine kinase

receptors

peptide ligands for

these receptors

Ephrins:cell-surface proteins that can induce growth cone collapse.

Eph kinases and Ephrins are distributed in gradients in the retina and tectum.

Eph repulsive signaling partially defines Sperry’s “chemoaffinity” that sets up the retinotectal map.

Axons with high Eph kinase expression avoid tectal regions with high

levels of ephrin

Figs 54-13, 54-14

Sperry’s “chemoaffinity”in the retinotectal system:

a 21st Century view

A Normal

A P A PRetina Tectum

Cell bodies Growth cones

A P A P

C Inactivate Ephrin A5

B Confined overexpression of Ephrin A2

A P A P

Discussed in a previous lecture

Pax-6 / Ey functions when expressed at various locations in Drosophila

5. Master switches for eye development?

Little Alberts 8-25 © Garland 29

Eye formation varies enormously among

organisms,

yet even a human Pax-6 ortholog induces an eye in

Ey mutant Drosophila!

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A Pessimistic Estimate Of The Time Required For An Eye To Evolve, D.-E. Nilsson and S. Pelger,

Proceedings of the Royal Society London B, 1994, 256, pp. 53-58.

Estimate: several hundred thousand yr from primitive eyespot to fisheye with lens

Selective advantages of the intermediate steps are summarized here:http://www.pbs.org/wgbh/evolution/library/01/1/l_011_01.html

1 2 3 4

5 86 7

6. Time frame for evolution of the major structural features

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Henry Lester will not have “office” hours this Friday

BBE/CNS 150 End of Lecture 14

Dark State

Light Channel Closure

Cyclic GMP hydrolysis

Lowered cytosolic

Ca2+

Increased cyclic GMP synthesis

Channel opening

Visual excitation is followed by Recovery and Adaptation

The role of Ca2+ in adaptation also appears to be important, but this process is not understood in molecular detail yet.

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