Lecture 5/4

Structural Support and Stereocilia in the Outer Segment

• Definition and Nature of Stereocilia:     * Stereocilia in the context of rod and cone cells are specialized membrane protrusions that play a strictly structural role.     * Distinction from Cilia: Despite the name, stereocilia are not microtubule-based structures. They are actin-based structures.     * Similarity to Filipodia: They are functionally and structurally similar to filipodia, consisting of little projections that stick out from the cell.     * Internal Composition: They contain parallel bundles of actin filaments which create the protrusion of the membrane.

• Comparative Examples of Stereocilia:     * In the hair cells of the inner ear, stereocilia are used to detect fluid movement. They bend in response to physical stimuli, allowing for the conversion of sound waves into neurological signals.     * In the eye, stereocilia do not have a sensory role; their function is purely to provide support for the outer segment.

• Structural Mechanism in Photoreceptors:     * Cage-like Function: They create a surrounding cage around the region where the connecting cilium resides.     * Prevention of Movement: This cage stabilizes the narrow junction between the inner and outer segment, preventing the bulky outer segment from swaying or moving from side to side within the eye.

• Molecular Composition and Anchoring:     * Cross-sectional Anatomy: Looking top-down, the backbone of the outer segment contains microtubules. Surrounding this are the stereocilia, which appear as circles in cross-section.     * Actin Bundling Proteins: The actin filaments within stereocilia are held in tight parallel bundles by the protein fimbrin.     * Cadherin-like Proteins: Specifically, protocadherin 15 is used to create adherence-type junctions. These junctions occur within the same cell (between the stereocilia membrane and the outer segment disc membranes).     * Inter-connectivity: Protocadherin 15 is present in both the discs and the stereocilia, anchoring them together. They also form lateral connections between individual stereocilia for reinforced support.

Photoreceptor Turnover and the Role of the RPE

• Radiation Damage and Need for Renewal:     * The primary purpose of cone and rod outer segments is to absorb light, which is a form of radiation.     * Cumulative exposure to radiation leads to the steady damage of cellular components and proteins.     * To compensate for this damage, there is a high rate of turnover within the outer segment structures.

• The Process of Disc Renewal:     * New discs are continually synthesized at the base of the outer segment.     * As new discs are added, older discs progress upward (distally) away from the base.     * Proteins and membranes are transported outward through intraflagellar transport towards the distal tip.

• Turnover Statistics and Metabolic Cost:     * Approximately $\frac{1}{10}$ (one-tenth) of the discs in the outer segment are lost every day.     * The entire outer segment of every rod and cone cell must be completely replaced every $10$ days.     * This is a highly energy-intensive process requiring significant activity in the Endoplasmic Reticulum (ER):         * Smooth ER: Responsible for synthesizing the massive amount of membrane required for the discs.         * Rough ER: Responsible for synthesizing the specialized opsin and support proteins.

• Phagocytosis by Retinal Pigmented Epithelial (RPE) Cells:     * Location: RPE cells are situated behind the retina (behind the rods and cones).     * Function: If the sloughed-off discs were not cleared, they would accumulate as cellular debris and impair vision. RPE cells perform phagocytosis to ingest the older discs.     * Breakdown and Recycling: Phagocytic vesicles containing ingested discs fuse with lysosomes. The components are broken down and reused by the RPE cells.

Anatomy of Visual Signaling

• The Light Path:     * Light enters the eye and is focused toward the fovea (the center of vision). Light also hits the peripheral regions of the retina.     * Signal Transmission Hierarchy:         1. Rod and Cone cells receive signal.         2. Connecting neurons (e.g., bipolar cells) receive impulses from photoreceptors.         3. Bipolar cells signal the ganglial cells.         4. Ganglial cells send axons into the optic nerve.         5. The optic nerve transmits signals to the brain for interpretation.

• Photoreceptor Distribution:     * Cone Cells: Concentrated at the fovea. They provide high-acuity color vision. Color vision is better for objects directly in front of the viewer.     * Rod Cells: More abundant in the peripheral retina. They are absent from the very center of the fovea. In low-light conditions, vision is often better slightly off-center because rods take over.

Types of Opsin Proteins and Color Detection

• Rod Cells and Rhodopsin:     * Rods express rhodopsin, a G-protein coupled receptor (GPCR).     * Rods cannot differentiate between wavelengths; they only detect the intensity and presence of light (black and white vision).

• Cone Cells and Color Opsins:     * S-opsin: Detects short wavelengths (blue spectrum).     * M-opsin: Detects mid-range wavelengths (green spectrum).     * L-opsin: Detects long wavelengths (red spectrum).     * Subtle Variations: The difference between M-opsin and L-opsin is caused by only $3$ specific amino acid changes in the protein backbone. These changes shift the protein's interaction with light.     * Color Perception: Wavelengths between specific spectra (like yellow) activate multiple cone types simultaneously. Yellow light effectively looks the same to the brain as a mixture of red and green light.

The Biochemical Mechanism of Phototransduction

• The Retinal Molecule:     * Opsins and Rhodopsins are GPCRs associated with a molecule called retinol (vitamin A derivative).     * 11-cis Retinol: The kinked, inactive form synthesized by the cell. When bound, the GPCR is in the "off" state.     * All-trans Retinol: The linear, active form. When light of the correct wavelength hits the 11-cis retinal, it undergoes photoisomerization into all-trans retinal.

• G-Protein Coupled Receptor (GPCR) Signaling:     * Activated opsin triggers a trimeric G-protein (Transducin).     * The complex consists of alpha ($\alpha$) and beta-gamma ($\beta\gamma$) subunits.     * The activated receptor acts as a Guanine nucleotide Exchange Factor (GEF), removing GDP from the alpha subunit and replacing it with GTP.     * The alpha subunit then activates the downstream effector protein.

• Phosphodiesterase (PDE) and cGMP:     * The primary target of the activated alpha subunit is the enzyme Phosphodiesterase (PDE).     * Dark State: In the dark, there are high levels of cyclic GMP (cGMP) produced by guanylyl cyclase. cGMP acts as a ligand for sodium channels in the membrane.     * Light State: Activated PDE breaks down cGMP into GMP.

Electrical Polarization of Photoreceptors

• Depolarization in the Dark:     * Rod and cone cells are unique because they are depolarized in their resting (dark) state.     * Ligand-gated sodium channels are kept open by the binding of cGMP on the cytosolic side. Sodium flows into the cell, maintaining a depolarized potential.     * In this state, the cells constantly release neurotransmitters to the bipolar cells.

• Polarization in the Light:     * When light hits, PDE reduces the concentration of cGMP.     * Without cGMP, the sodium channels close.     * Sodium-potassium pumps ($Na^+/K^+\text{ pumps}$) and exchangers continue to pump ions, causing the cell to become polarized (hyperpolarized).     * The change in polarization reduces or eliminates the release of neurotransmitters.

• Signal Intensity:     * The response is scalable. Bright light closes more channels, causing greater polarization and a greater reduction in neurotransmitters. Dim light causes only a partial reduction.

Signal Reseting and Recovery

• Shutting Down the Signal:     * RGS Protein: Acts as a GAP (GTPase Activating Protein) to help hydrolyze GTP to GDP on the alpha subunit, shutting down the trimeric G-protein.     * GPCR Kinase (GRK): Adds three phosphates to the activated opsin protein.     * Arrestin: Binds to the phosphorylated opsin to prevent further G-protein activation.

• Refreshing Retinal:     * The GPCR must be internalized (endocytosis) to remove the all-trans retinal and replace it with a fresh 11-cis retinal molecule.     * This recovery process is incredibly fast, allowing the eye to detect rapid changes in light, such as a strobe light.

Questions & Discussion

• Question: Is it also the alpha subunit that is activated in opsin signaling?     * Response: Yes, the primary G-protein functions like other GPCRs where the alpha subunit activates downstream targets (like PDE), but all subunits ($\alpha$ and $\beta\gamma$) are generally involved in the signaling cascade.

• Question: Would light be considered a ligand in this case?     * Response: Light effectively acts as the initial tactile step that triggers the change in the retinal molecule, which then activates the receptor, similar to how a ligand would.

• Question: Are there organisms with better vision than humans?     * Response: Yes, organisms with more than three types of opsin genes have a much broader range of color vision than humans.