1. Lecture to Sensory Genomics Class, 10/21/09, Richard Payne Introduction to eye types Introduction to ciliate and microvillar photoreceptor types Phototransduction in Drosophila photoreceptors involves Ca2+ influx from the Extracellular space This Ca influx occurs through the high Ca permeability of the trp channel. Phototransduction in Limulus ventral photoreceptors involves release of calcium from internal stores, not influx. Why the difference – are different families of trp channels being used? Work shown performed in my lab from Kyrill Ukhanov (post doc), Youjun Wang (grad student), Jay Demas, Oluwaseyi Motajo (undergrad students).

2. Not every animal has eyes designed like ours. Do they have different photoreceptor types? Yes, there are different photoreceptor types, but not rigidly linked to eye design

3. Invertebrates have both simple (single lens) and compound eyes – do they have the same photoreceptor types as ours? No – both the compund fly eye and the squid simple eye shown here have “Microvillar “rather than our “Ciliate” photoreceptors.

4. What are “ciliate” and “microvillar” photoreceptors?

5. 10.1 Anatomy of the human eye. Section – see over

6. 10.4 Structure of the retina.

7. The vertebrate rod photoreceptor is a “ciliate” photoreceptor cilium It hyperpolarizes upon illumination Due to closure of cGMP-activated Na+ - channels. Because the cGMP –activated channels also let through Ca 2+ ions, the [Ca 2+ ] in the cell falls upon illumination. The reduction in cGMP is achieved Via a biochemical cascade that is linked via a G-protein to the visual pigment, Rhodopsin.

8. Compound and other eyes of many invertebrate species (including at least one chordate, Amphioxus) use a different kind of photoreceptor: The “microvillar” or “rhabdomeric” photoreceptor

9. By William Harman, Steven Barrett, Cameron Wright, and Michael Wilcox

10. nature.com homepage Jump to main content Jump to navigation Login My account E-alert sign up Register Subscribe Publications A-Z index Browse by subject Search Advanced searchAdvanced search Journal homeJournal home > Archive > insight > Full text > Figure 2ArchiveinsightFull text FIGURE 2. Photoreceptor structure. From the following article: Visual transduction in Drosophila Roger C. Hardie and Padinjat Raghu Nature 413, 186-193(13 September 2001) doi:10.1038/35093002 BACK TO ARTICLE In Drosophila, as in most invertebrate photoreceptors, the photoreceptive membrane is organized into tightly packed, tubular microvilli, each 1–2 m long and 60 nm in diameter, together forming a 100- m-long rhabdomere. At the base of the microvilli a system of submicrovillar cisternae (SMC) have often been presumed to represent smooth endoplasmic reticulum Ca 2+ stores endowed with Ins(1,4,5)P 3 receptors. However, the SMC may have a more important role in phosphoinositide turnover. Vertebrate rod outer segments (ROS) contain stacks of membranous discs ( 1,000) and are connected to the cell body by a narrow cilium. In both cases the overall structure serves to maximize absorption of light by forming a cylindrical light-guiding structure with a high density of rhodopsin-containing membrane. Inset shows electron micrograph of one rhabdomere (courtesy of A. Polyanovsky; scale bar, 1 m). Next figureNext figure | Previous figure | Figures & Tables indexPrevious figureFigures & Tables index BACK TO ARTICLE Top Nature ISSN: 0028-0836 EISSN: 1476-4687 About NPG Contact NPG Nature jobs.com Privacy policy Legal notice Accessibility statement RSS web feeds Help © 2007 Nature Publishing Group© 2007 Nature Publishing Group – partner of AGORA, HINARI, CrossRef and COUNTERAGORA, HINARI, CrossRef and COUNTER Actin-filled microvilli The ommatidia contain microvillar photoreceptors rhodopsin

11. Unlike rods, Drosophila photoreceptors depolarize upon illumination

12. Hardie, R. C. J Exp Biol 2001;204:3403-3409 The light-induced depolarization of Drosophila photoreceptors results from the opening of Na+ channels, in the microvillar membrane, allowing Na + ions into the cell. Opposite to rods Na +

13. Hardie 1996; In Drosophila photoreceptors, unlike in rods, there is a light-induced increase in intracellular [Ca 2+ ]. The Ca 2+ comes through the light-sensitive channels along with Na + Hardie, 1996

14. The current model of Drosophila phototransduction is confined to a single microvillus, containing 10-30 channels. After Hardie et al 2002 No role for IP3R or ER calcium stores in excitation – confirmed by genetic knockout of the IP3R

15. So how does Ca 2+ enter the Drosophila photoreceptor? Drosophila photoreceptors two channel proteins in their microvilli that together constitute the “light-sensitive conductance” Trp and trpl Trp;trpl double mutants are blind – no ability to generate a light-induced inward current. So these are the “light-activated” channels trp has a much higher Ca 2+ selectivity than trpl A third putative channel protein, trpgamma, is also expressed in the photoreceptors – role unknown. Trp, trpl and trpgamma are all members of the trpC gene family

16. trp mutants lack the TRP channel protein and have a transient light- activated current and receptor potential. The trp phenotype can be mimicked by blocking trp channels in a wild-type (WT) fly with extracellular lanthanum ions. In trp mutants, the remaining transient response is carried by the trpl channels.

17. The light-induced current flowing into WT flies shows a positive, calcium-sensitive “reversal potential” due to a large calcium permeability. Responses of trp mutants show a more negative reversal potential, with less calcium sensitivity, consistent with the elimination of the calcium-permeable trp channel.

18. An Aspartate at position 621 is largely responsible for the Ca 2+ selectivity of trp

19. Copyright ©2007 Society for Neuroscience Liu, C. H. et al. J. Neurosci. 2007;27:604-615 Figure 10. TRPC pore sequences and putative structure of the dTRP pore

20. The current model of Drosophila phototransduction is confined to a single microvillus After Hardie et al 2002 No role for IP3R or ER calcium stores in excitation – confirmed by genetic knockout of the IP3R

21. Is this the model phototransduction scheme for all microvillar photoreceptors? More-or -less, yes But the differences are significant

22. The Limulus Microvillar photoreceptors are also found in simple eyes without lenses, such as the “ventral eye” in the horseshoe crab, Limulus (After Calman & Chamberlain, 1982)

23. Microscopic structure of the photoreceptor The ventral photoreceptor 1  m 10  m

24. Ventral nerve Amplifier Light 0.1 S 10 mV Voltage Electrode 0.1 S Light 10 nA Current Measuring the light induced electrical responses of Limulus ventral eye photoreceptors shows that, as in Drosophila, they depolarize in response to light due to an influx of Na+ through ion channels in the plasma membrane

25. Ventral nerve Laser Ca signal Photomultiplier (PMT) Pressure Injection of Ca-sensitive fluorescent dye Electrode Beam splitter Fluorescence Calibration Intense Light 1 S Ca concentrations within the Limulus photoreceptors rise upon illumination, as in Drosophila Measuring fluorescent Ca signals with confocal microscopy

26. Removal of extracellular calcium shows that the source of the light- induced calcium increase is different in Drosophila and Limulus photoreceptors Hardie 1996; Hardie, 1996

27. Instead of Ca 2+ influx, Ca 2+ release from intracellular stores by inositol (1,4,5) trisphosphate occurs in in Limulus ventral photoreceptors This Ca 2+ release stimulates the electrical response

28. PLC Microvillus IP 3 DAG h Rh   GqGq IP 3 R Extracellular spaceCytoplasm Ca ? TRPC ? Na Model of the photo-transduction cascade in Limulus ventral photoreceptors – Ca2+ is released by IP3 from intracellular stores, and does not flow through the light-sensitive channels

29. Compare with the current model of Drosophila phototransduction Ca 2+ enters through the light-sensitive channels After Hardie et al 2002 No role for IP3R or ER calcium stores in excitation – confirmed by genetic knockout of the IP3R

30. But what about Limulus ventral photoreceptors other invertebrate photoreceptors that exhibit little or no light-induced Ca 2+ influx? Do they: a)Express a trpl ortholog only? b) Express a trp ortholog, but with a mutated pore region (no D621) c) Express a trpC channel that is different from either trp or trpl? If (c), then does this use of a different channel have any correlation with, or impact on the evolution of different eye designs in invertebrate species.

31. Phylogenetic questions: What trp channel orthologs are present in invertebrate genomes other than Drosophila, what is their phylogeny and how conserved are their putative pore regions? For example, a trpC - like gene transcript can be cloned from ventral eye tissue, but it only has only 36% identity with Drosophila trp and has a dissimilar pore region (Bandyophadyay and Payne 2004). What is the phylogenetic relationship between this and the Drosophila channel? Functional questions: How many other microvillar photoreceptors show little or no light-induced Ca 2+ influx? What is the role of calcium influx vs. calcium release in phototransduction? What constraints and advantages accompany each mechanism? Questions