6. Time frame for evolution of the major features
1. lens and
optics
2. photoreception
event
5. A master switch
that controls
differentiation
BBE/CNS 150 Lecture 14
Wednesday, October 30, 2013
Vision 1: Phototransduction and the Retina
and
Evolution of the Eye
Henry Lester
Chapter 26, co-written by Markus Meister
4. Connections
to the brain
3. retina
1
6. Time frame for evolution of the major features
1. lens and
optics
2. photoreception
event
5. A master switch
that controls
differentiation
BBE/CNS 150 Lecture 14
Wednesday, October 30, 2013
Vision 1: Phototransduction and the Retina
and
Evolution of the Eye
Henry Lester
Chapter 26, co-written by Markus Meister
4. Connections
to the brain
3. retina
2
“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
Example: globin genes
orthologs & paralogs
Hemoglobin paralogs in the mouse genome
mouse e vs b
orthologs resemble each other
across species (mouse b vs human b)
paralogs resemble each other,
distant or
closely,
within
a species
Hemoglobin
paralogs
in the
human
genome
human e vs b
gG vs gA
chromosome
7
Myr BP
Myr BP
4
1. Lens and optics
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.
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!
5
Pax-6, a transcription factor with orthologs in many species
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
Pax-6 (vertebrates)
Ey (Drosophila)
Existing proteins have been
used for an additional
functions.
Presence of multiple
sufficient gene regulatory
mechanisms can underlie
“gene sharing”
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.
Crystallin
6
The aperture mechanism: controlled by smooth muscles
blocker: atropine from Atropa belladonna
nerve from brain;
single
muscarinic synapse
smooth muscle cell
Contracts and thickens:
leads to smaller pupil
inextensible fibers
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.
7
2. The photoreception event
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.
8
The photoreceptor cells receive light from “the back”
Rhodopsin
Free-floating discs
Rhodopsin
hn
hn
Like Figs.
26-5, 26-7
9
There are 4 opsin paralogs in the human genome.
Each opsin interacts distinctly with retinal, producing a distinct absorption spectrum.
Absorption spectra of cone pigments
Blue-
greenabsorbing
red-
Mutations that change the spectrum
Like Fig. 26-8, 26-9
10
Detection of light by retinal bound to opsin
Enzymes
From Darnell et al., Mol. Cell Biology
Like Fig. 26-8
11
from Lecture 12
outside
membrane
receptor
b g

G protein
i q s t

b g
inside
effector
channel enzyme
The usual GPCR pathway
intracellular
messenger
Ca2+ cAMP
cytosol
kinase
phosphorylated
protein
nucleus
12
membrane
receptor
G protein
i q s t
cytosol
effector
channel enzyme
The GPCR pathway in a
photoreceptor
intracellular
messenger
cAMP
Ca2+ cGMP
channel
13
like previous lectures
Beginning of the G Protein-Coupled Receptor Pathway
How far?
How fast?
100 ms to 10 s Probably less 1 mm
Neurotransmitter or hormone
binds to receptor
activates
G protein
Effector:
enzyme or channel
outside
inside
b g

GTP

GDP + Pi
b g
14
Special aspects of the G protein-coupled receptor pathway in photoreception
Photon isomerizes
retinal bound to rhodopsin
hn
activates
G protein
How fast?
< 100 ms
How far?
< 1 mm
Effector is an enzyme
In rods and cones, these proteins lack lipid tails
b g


GTP
GDP + Pi
cytosol between disks,
or
between folds
Although the components
are not membrane-bound,
the membranes effectively
restrict their motion
Like Fig. 26-7
15
Expanding on a previous lecture, we said . . .
intracellular
Intracellular messengers bind to proteins messenger
cAMP
Ca2+ cGMP
kinases
A few ion channels
(olfactory system, retina)
phosphorylated
protein
NH2
N
N
Ca2+
and
O
O
O
P
-O
O
N
N
H
H
OH
Cyclic nucleotide
cyclic AMP (cAMP)
(cAMP
or cGMP)
16
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.
17
receptor
like a previous Lecture
G protein
i q s t
Effector enzyme
“cyclase”
effector
channel enzyme
cAMP
ATP
Breakdown enzyme
“phosphodiesterase”
uninteresting
Inhibited by caffeine
intracellular
messenger
cAMP
Ca2+ cGMP
channel
Enzyme
“cyclase”
cGMP
GTP
A paralog expressed
elsewhere in the body is
inhibited by Viagra
Breakdown enzyme
“phosphodiesterase”
The effector for Gt
“Viagra . . . may cause a
perception of bluish haze or
increased light sensitivity in
some patients.”
uninteresting
18
like a previous Lecture
receptor
Rods and Cones have
cGMP-activated Na+/Ca2+ Channels
G protein
i q s t
effector
channel enzyme
Excised
“inside-out” patch
allows access
to the inside surface
of the membrane
+cGMP*
intracellular
messenger
cAMP
Ca2+ cGMP
channel
no cGMP
no channel openings
open
+cGMP*
closed
19
Light Response of the Photoreceptor Cell
The vertebrate photoreceptor functions electrophysiologically opposite to most
neurons.
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.
20
The “ribbon synapse” facilitates the tonic high rate of transmitter release
Photoreceptor to horizontal cell synapse
21
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.
22
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.
23
The Phototransduction Cascade:
(1. Amplification)
2. Adaptive/homeostatic mechanisms
2a. Transducin hydrolyses GTP to GDP and thus inactivates itself.
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
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
24
3. Neurons of the retina
Rod
Cone
Glutamate is the major transmitter;
Some neurons make
dopamine & acetylcholine.
Inhibitory neurons release GABA.
Many paralogs to genes expressed
elsewhere:
Channels, receptors, transporters.
Synapses of outer plexiform layer
Horizontal cells
Bipolar cells
Synapses of inner plexiform layer
Ganglion cell is unique in firing impulses
optic nerve
Like Fig. 26-2
25
A previous Lecture
4. Connections to the brain
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 postulated a
“chemoaffinity” between
the nerves and their target
cells.
Sperry also conducted the “Split brain”
experiments that form the basis for
modern ideas about the distinct
specialties of the two hemispheres.
26
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.
Horseshoe crabs
(Limulus polyphemus)
27
Discussed in a previous lecture
Sperry’s “chemoaffinity”
in the retinotectal system:
a 21st Century view
Ephrins:
cell-surface proteins that
can induce growth cone
collapse.
A Normal
Growth cones
Cell bodies
A
tyrosine
kinase
receptors
Retina
P
A
Tectum
B Confined overexpression of Ephrin A2
P
peptide
ligands for
these
receptors
Eph kinases and Ephrins
are distributed in gradients
in the retina and tectum.
A
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
P
A
P
A
P
C Inactivate Ephrin A5
A
P
28
5. Master switches for eye development?
Pax-6 / Ey functions when expressed at various locations in Drosophila
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!
30
6. Time frame for evolution of the major structural features
1
2
3
4
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
5
6
7
8
31
Henry Lester will not have “office” hours this Friday
BBE/CNS 150
End of Lecture 14
32
Visual excitation is followed by Recovery and Adaptation
Light
Cyclic GMP
hydrolysis
Channel
Closure
Lowered
cytosolic
Ca2+
Increased
cyclic GMP
synthesis
Channel
opening
Dark
State
The role of Ca2+ in adaptation also appears to be important, but this
process is not understood in molecular detail yet.
33