4. Connections to the brain 3. retina 1. lens and optics 2. photoreception event 5. A master switch that controls differentiation 6. Time frame for evolution of the major features 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 1

4. Connections to the brain 3. retina 1. lens and optics 2. photoreception event 5. A master switch that controls differentiation 6. Time frame for evolution of the major features 2 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

“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 other across 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 4

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 mol/liter) x (5.5 x 10 4 g/mol) = 2.2 x 10 2 g/l = 220 g/l. The cell is ~22% protein! 5

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” 6 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 single smooth 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 7

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 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 10 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 Biology 11 Enzymes Like Fig. 26-8

cytosol The usual GPCR pathway nucleus kinase phosphorylated protein cAMP Ca 2+ intracellular messenger receptor tsqi G protein enzymechannel effector membrane from Lecture 12 12

The GPCR pathway in a photoreceptor channel receptor tsqi G protein enzymechannel effector cytosol intracellular messenger Ca 2+ cAMP cGMP 13 membrane

GTPGDP + P i Effector: enzyme or channel outside inside Neurotransmitter or hormone binds to receptor  activates G 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 14

Photon isomerizes retinal bound to rhodopsin Special aspects of the G protein-coupled receptor pathway in photoreception How fast? < 100 ms How far? < 1  m GTP activates G protein     Effector is an enzyme GDP + P i h Although the components are not membrane-bound, the membranes effectively restrict their motion In rods and cones, these proteins lack lipid tails 15 cytosol between disks, or between folds Like Fig. 26-7

Intracellular messengers bind to proteins kinases phosphorylated protein A few ion channels (olfactory system, retina) Ca 2+ and Expanding on a previous lecture, we said... intracellular messenger Ca 2+ cAMP cGMP Cyclic nucleotide (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

cAMP ATP Effector enzyme “cyclase” Breakdown enzyme “phosphodiesterase” Inhibited by caffeine uninteresting cGMP GTP Enzyme “cyclase” Breakdown enzyme “phosphodiesterase” uninteresting The effector for Gt like a previous Lecture A paralog expressed elsewhere in the body is inhibited by Viagra channel receptor tsqi G protein enzymechannel effector intracellular messenger Ca 2+ cAMP cGMP “Viagra... may cause a perception of bluish haze or increased light sensitivity in some patients.” 18

Rods and Cones have cGMP-activated Na + /Ca 2+ Channels Excised “inside-out” patch allows access to the inside surface of the membrane no cGMP no channel openings +cGMP* closed open like a previous Lecture receptor qi G protein channel ts enzymechannel effector intracellular messenger Ca 2+ cAMP cGMP 19

Light Response of the Photoreceptor Cell The vertebrate photoreceptor functions electrophysiologically opposite to most neurons 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 21

The Phototransduction Cascade: 1. Amplification2. 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 ~10 10 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

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 [Ca 2+ ] > ~75 nM. (2) Ca 2+ influx through the tonically open cation channel sets the cytosolic level of Ca 2+ to ~ 500 nM. (3) When the cation channel closes upon light stimulation, Ca 2+ continues to be pumped out via the usual processes, lowering cytosolic Ca 2+ to ~50 nM and activating guanylate cyclase 24 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 25 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 26

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. 27

28 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, Sperry’s “chemoaffinity” in the retinotectal system: a 21st Century view A Normal A PA P Retina Tectum Cell bodies Growth cones A PA P C Inactivate Ephrin A5 B Confined overexpression of Ephrin A2 A PA 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! 30

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 Estimate: several hundred thousand yr from primitive eyespot to fisheye with lens Selective advantages of the intermediate steps are summarized here: Time frame for evolution of the major structural features 31

32 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 Ca 2+ Increased cyclic GMP synthesis Channel opening Visual excitation is followed by Recovery and Adaptation The role of Ca 2+ in adaptation also appears to be important, but this process is not understood in molecular detail yet. 33