Exam 4: Lecture 5

Question 1

Photoreceptor Cells In Humans

Answer 1

• rods: responsible for detecting objects and motion

- cones: responsible for detecting color

Question 2

Opsins

Answer 2

• proteins expressed in rods and cones
• belong to superfamily of proteins containing several transmembrane domains
• capable of capturing photons of light and in turn initiate transduction of light into electrical signals
• number of different opsin genes in human genome and each will encode opsin protein that can capture light of particular wavelengths
• contain extracellular domain, seven transmembrane segments and an intracellular domain.

Question 3

Opsins in Cones

Answer 3

• capture light in red, blue, and green wavelengths

- each cone receptor will express only one type of opsin molecule

Question 4

Ratio of Cone Cell Colors

Answer 4

• not 1:1:1
• always more red cones than green cones than blue cones
• stochastic ratio of cone cell but they’re positioned randomly

Question 5

Photoreceptor Domains

Answer 5

• structurally divided into domains

- region of cell responsible for capturing light is outer segment which is made of hundreds of membrane stacks

Question 6

Opsin Protein and Capturing Light

Answer 6

• light capturing protein opsin located in sacks of outer segment
• after translation opsin protein folded within ER, is modified by addition of sugar residues within Golgi and targeted to outer segment.
• needs cofactor to capture light
• bound to chromophore molecule called retinal.

Question 7

Rhodopsin

Answer 7

• opsin-retinal complex
• can absorb photons of different light wavelengths
• loss of individual opsin genes can lead to various forms of color blindness

Question 8

Phototransduction

Answer 8

• outer segments contain entire machinery required to transduce photons of light into electrical signals
• includes opsin proteins and attached chromophores, set of membrane associated and cytoplasmic factors and several ion channels
• capture of light by rhodopsin triggers opening and closing of channels changing electrical potential across membrane
• change in potential communicated to neurons in brain
• brain reconstructs electrical signals into image

Question 9

Photoreceptors in low light

Answer 9

• rhodopsin molecules not interacting with downstream phototransduction machinery
• downstream sodium channels are open allowing Na+ into cell
• balanced by movement of K+ out of cell va K channels
• more K+ exported than Na+ imported
• gives 40 mV voltage difference across membrane

Question 10

Photoreceptors Exposed to Light

Answer 10

• rhodopsin molecules activated and interact with phototransduction machinery
• cyclic GMP binds and closes Na channel thus blocking Na+ to enter cell
• K+ still allowed to exit cell
• causes a -70 mV difference across membrane

Question 11

Rhodopsin Genes in Drosophila

Answer 11

-insect retina=simple nervous system with stereotyped developmental pattern and limited number of cell types

Question 12

Charles Zuker and Joseph O’Tousa

Answer 12

• coned first invertebrae rhodopsin (Rh1)
• absorbs light in orange spectrum
• essentially used as motion detector

Question 13

Charles Zuker, Karl Fryxell, and Craig Montell

Answer 13

• cloned Rh2, Rh3, and Rh4 rhodopsin genes

- absorb light in ultraviolet spectrum

Question 14

Claude Desplan and Steve Britt

Answer 14

• cloned Rh5 and Rh6 rhodopsins
• absorb blue and green light
• have shown that flies can see in color
• don’t see well in red, but rather well in blue and green

Question 15

Structure of Drosophila Retina

Answer 15

• compound eye contains approximately 800 identical unit eyes (ommatida) packaged into hexagonal array
• each ommatidium contains ~20 cells divided into 8 photoreceptors (4 cones, 8 pigment cells)
• photoreceptor cells contain rhodopsin protein and are responsible for capturing and transducing light
• cone cells secret overlying lens and pigment cells optically insulate each ommatidium from its neighbors
• interspersed between ommatidia are mechanosensory bristles used to sense changes in air velocity and detect presence of foreign objects

Question 16

Photoreceptors of Fly Retina

Answer 16

• each ommatidium contains 8 photoreceptor (R) cells
• you will only see 7 in any one given plane
• R1-R6 cells extend entire depth of retina and R7 sits on top of R8.
• R7 is distal R8 is proximal
• occupy stereotyped positions within ommatidium and are arranged as asymmetric trapezoid

Question 17

Rhabdomeres

Answer 17

• part of insect photoreceptors
• homologous to rod outer segments of vertebrate photoreceptors
• contain all phototransduction machinery including rhodopsin proteins

Question 18

Rh1 protein

Answer 18

• expressed in six outer photoreceptors
• absorbs light in orange spectrum
• required for motion detection

Question 19

Rh2

Answer 19

• not expressed in photoreceptors of compound eye

- found within photoreceptors of three simple eyes (ocelli) that sit at vertex of fly head

Question 20

Rh3 and Rh4

Answer 20

• expressed in non-overlapping sets of R7 cells
• 70% of ommatids will have R7 cell that expresses R3
• remaining 30% of ommatidia will have R7 cell that contains Rh4 protein
• capture light in ultraviolet spectrum

Question 21

Rh5 and Rh6

Answer 21

• expressed in non-overlapping sets of R8 cells
• 70% of ommatidia will have R8 cell that expresses Rh5
• remaining 30% of ommatidia have R8 cell that contains Rh6 protein

Question 22

Pattern of Rhodopsin Expression

Answer 22

• if ommatidium contains R7 cell that expresses Rh3, then R8 cell will express Rh5
• if ommatidium contains R7 cell that expresses Rh4 then R8 cell will express Rh6

Question 23

Color Blindness

Answer 23

-loss of individual rhodopsin genes can lead to various forms of color blindness

Question 24

Pseudo-Isochromatic Plate (PIP) Test

Answer 24

-used to determine if patient is color blind

Question 25

Retinal Degeneration

Answer 25

• other phenotype
• followed by an expansion of overlying retinal pigment epithelium
• called retinis pigmentosa
• characterized by pigmentation of eye
• decreases field of view due to invasion of retinal pigment epithelium ultimately leading to blindness

Question 26

Loss of Rh1 in Flies

Answer 26

• leads to retinal degenration of R1-R cells

- rhabdomeres completely lost in such mutants

Question 27

Sevenless Pathway and R7 Development (Experiment)

Answer 27

• R7 cell expresses rhodopsins that can absorb light from ultraviolet spectrum
• Donald Ready set up T maze and let flies chose different paths
• end of one path, normal visible light
• end of other path, ultraviolet light
• wild type flies choose ultraviolet light 100% of time
• interested in genes controlloing light prefrence
• introduced random mutations and allowed them to choose again
• most mutants went to ultraviolet indicating that flies were not affected in ability to discriminate between light types
• few mutant flies chose visible light thus indicating they are insensitive to UV light (can’t detect it)

Question 28

Thoughts on Light Experiement

Answer 28

• thought these mutations would be in genes that encoded members of phototransduction machinery
• noticed that R7 cell was completely missing
• called mutant sevenless

Question 29

Sevenless RTK Pathway

Answer 29

• after discovery that sevenless mutation resulted in failure of R7 cell to develop, large number of research groups set out to clone sevenless gene and determine which protein is encoded
• Utpal Baneriee, Gerald Rubin and Ernst Hafen determined sevenless protein was a RTK
• wanted to identify all cytoplasmic and nuclear proteins downstream of receptor
• did screnes similar to Don Rady
• over decade identified Ras, Raf, MEK, MAPK and several TFs
• each case, loss of these genes leads to loss of R7
• each of these genes subsequently shown to be expressed in R7 cell

Question 30

Bride of Sevenless (Boss) Ligand

Answer 30

• Larry Zipursky identified mutation like sevenless that resulted in loss of R7 cell
• cloned gene and determined expression pattern
• unlike other pathway members of this gene not expressed in R7 cell
• expressed in R8 cell
• suggests that this gene is actually a ligand called Boss since ligand is bound to sevenless receptor
• comparison of sevenless expression indicated that it is expressed in not just R7 cell but also R3, R4 and the four cone cells
• only presumptive R7 cell actually becomes an R7. Why?
• of sevenless expressing cells the only one that is contact with R8 cell is presumptive R7
• since only cell that becomes R7 suggests that Boss ligand is tethered and functions through justacrine signaling
• biochemically proven