Block I Learning Objectives

Lecture I
Course Overview Know the Basic Characteristics of Living Systems 1. Metabolism = Sum of all chemical processes  breakdown of large molecules into small building blocks and new structural components as well as providing chemical energy for cells 2. Responsiveness Detect and respond to changes in internal or external environmental stimuli, such as electrical signals, hormones, or glandular secretions. 3. Movement (at any structural level)  The body, an organ a cell or cellular component 4. Growth/Differentiation  Increasing the number of size of cells or the material found between the cells causing specialization of cells for a specific function. 5. Reproduction  Formation of new cells or new individuals Understand levels of structural organization – cells, tissues, organs, etc. The levels of structural organization are: 1. Chemical Level: atomic and molecular Organismic Level level 2. Cellular Level: Smallest living unit of the Organ System body Organ Level 3. Tissue Level: Group of cells and the materials surrounding them that work together on Tissue Level one task. 4. Organ Level: Grouping of 2 or more tissue Cellular Level types into a recognizable structure with a specific function. Chemical 5. Organ System: Collection of related organs Level with a common function (sometimes an organ is part of more than one system) 6. Organismic Level: One living individual. Know what we mean by the term homeostasis Homeostasis is a relative constancy of the internal environment, meaning that the physical and chemical parameters of living systems (e.g., temperature, pH, concentrations of important molecules) tend to remain constant over time. This also means that there are mechanisms to keep the system in relative constancy, such as negative feedback loops.

Lecture II
Case Introduction Know the basic structures of the eye. The basic structure of the eye is as follows:

2

-­‐ -­‐

-­‐

-­‐

-­‐

-­‐ -­‐ -­‐

-­‐

Cornea and tear film: The cornea is the transparent film that covers the iris, pupil, and anterior chamber, the major windows of light in the eye. Aqueous humor: A clear gelatinous liquid containing low-protein concentrations supporting the lens, through maintenance of intraocular pressure, nutrition, transportation, and protection of biological Cornea & tear film and environmental elements. Lens: Transparent, biconvex structure that Aqueous humor refracts light to be focused on the retina. It Lens functions (in changing shape) manipulates Cilary muscles the focal distance of the eye so that it Sclera focuses on objects at various distances. Choroid Ciliary Muscle: Ring of striated smooth Vitreous humor muscle that controls accommodation for Retina viewing objects at varying distances and regulates the flow of aqueous humor. It Optic nerve changes the shape of the lens within the eye, not the size of the pupil. Sclera: Opaque, fibrous, protective outer layer of the eye containing collagen and elastic fiber. In humans, the whole sclera is white. Choroid: Vascular layer of the eye consisting of connective tissue, and between the retina and the sclera. Vitreous Humor: Clear gel that fills the space between the lens and the retina of the eyeball. Retina: Light-sensitive tissue lining the inner surface of the eye. They contain photoreceptors (rods and cones) that detect photons of light. There are specific areas of the retina of interest. o Macula (or macular lutea): Central area of the retina capable of clearest, most distinct vision o Fovea: Central part of macular where all the photoreceptors are cones o Blind Spot: Optic nerve head where photoreceptors are absent Optic Nerve (Cranial Nerve II): Transmit visual information from the retina to the brain.

Learn the transparent tissues and be able to name structures photons pass through to reach the photoreceptor. The transparent tissues of the eye are based in the lens and cornea of the human eye. These non-nucleated tissues allow for optimal entrance of light (without opaque particulates such as nuclei obstructing the field of vision). As light makes contact with the retina, it is integrated by a series of cells until it reaches the rods and cones. Photoreceptors detect photons. Other neurons process the signal to detect

3

motion, edges, etc. As seen in the picture, the visual processing begins at the rods and cones, while light encounters the retinal blood vessels and optic nerve first. Know rods and cones are distributed differently in the peripheral and central retina. The rods and cones are distributed differently and are present in much different quantities. Remember that rods are more This allows for the following conclusions to be drawn: 1. Rods are far more numerous than cones. This allows more black and white photoreception than color photoreception. 2. Rods are predominant in the peripheral retina. This explains why color is not typically seen at night or in the periphery. 3. Cones are predominant in the central retina. The most central part of the macular is known as the fovea (where all the photoreceptors are cones). Know that the retina connects via the optic nerve to the brain (LGN) and visual pathway leads to the striate cortex. Remember that the visual pathway extends into the brain. The lateral geniculate interfaces and feeds into the striate cortex. The retinal signals are related to specific parts of the brain. The optic nerve leads from the eye to the lateral geniculate nucleus (LGN). The neuronal layers of the lateral geniculate nucleus correspond to the left and the right eyes. The functional layers correspond to the left and right eyes. If blind at young, the individual is unable to develop the lateral geniculate nucleus. Of the eyes do not work, then the connections will be poorly rebuilt.

Lecture III
Cell Structure and Plasma Membrane

Know the basic structure of a cell. Before knowing about the basic structures of a cell, we should know the cell theory. The cell theory has approximately 4 statements: 1. Cells are the “building blocks” of animals and plants. 2. Cells arise from pre-existing cells. 3. Cells are the smallest unit having all the functions of living systems. 4. Homeostasis of higher levels of biological organization arises form coordinated activity of cells. But now we shall understand the common cell structures:
Cell Structure Plasma Membrane Description The cellular membrane of the body, involved in several functions: 1. Barrier

4

Nucleus Cytoplasm

2. Shipping and Receiving for Cell 3. Security 4. Identification 5. Communication with other cells Contains the genetic material necessary for reproduction and diversity. Everything between the membrane and the nucleus, containing two major aspects: 1. Cytosol: Aqueous intracellular fluid, electrolytes, and other dissolved solutes 2. Organelles: Subcellular structures with specific functions

Recognize membranous and non-membranous organelles. Remember that a membrane encapsulates the membranous organelles. Non-membranous organelles lack this membrane.
-­‐ -­‐ -­‐ -­‐ -­‐ Membranous Nucleus Mitochondrion Endoplasmic Reticulum Lysosome Peroxisome -­‐ -­‐ -­‐ Non-Membranous Ribosome Proteasome Cytoskeleton

Understand plasma membrane structure and composition. The plasma membrane’s functions include being a barrier, shipping and receiving (as well as security) checkpoint, identification, and cellular communication. As the years progressed, the has been much to learn about the view of the plasma membrane. The lipid bilayer composes of three major lipids: 1. Phosphoglycerides (also called glycerophospholipids or simply phospholipids) 2. Sphingolipids 3. Sterols
Type of Lipid Phosphoglycerides Chemical Structure Composition Glycerol backbone and two fatty acyl tails in ester linkage and a polar head group (a phosphate ester of another alcohol like choline, ethanolamine, serine, or inositol) Sphingosine backbone (1 tail) and 1 fatty acid tails in amid and a polar head group, either a carbohydrate or a phosphate ester of another alcohol like choline or ethanolamine. Amphipathic, containing a hydrophilic and hydrophobic portion, containing a steroid ring and a fatty acid tail. Description Major lipid constituent of plasma membrane

Sphingolipids

Second component of plasma membrane, but found particularly in nerve and brain tissues.

Sterols

Wedges into bilayer and has effect on fluidity by interacting with first few hydrocarbon groups on phospholipid tails.

5

Recognize hydrophobic and polar regions of phosphoglycerides. The hydrophobic and polar regions of phosphoglycerides are the fatty acid tails and the alcohols (head), respectively. Such amphipatic properties allow the phospholipids to assemble in a way that minimizes the interactions the hydrophobic tail from water. Understand the terms amphipathic, lipid bilayer, micelle.
Term Amphipathic Illustration Definition Containing both hydrophobic and hydrophilic parts of the molecule, allowing plasticity in terms of hydrophobic and hydrophilic interactions.

Lipid Bilayer

Micelle

Interaction between the hydrophobic tails of the phospholipid as well as the interaction of the hydrophilic heads with water, thus creating a membraneous barrier. It is dependent on lipid composition. The longer the fatty acid chain length, the tighter packing of tails and less fluid. Number of double bonds in fatty acid chain is a contributing agent  more double bonds means looser packing of tails and more fluid. Cholesterol is the mediator of fluidity. The spherical assembly in which the phospholipids fully minimize the hydrophobic interactions with water. The polar head groups are in the exterior, while the nonpolar tail groups are interior.

Lecture IV
Subcellular Organization Undertand basic form and function of the cytoskeleton. The cytoskeleton is an elaborate arrangement of protein fibers to serve several functions: 1. Definition and maintenance of cell shape 2. Provision of mechanical strength 3. Locomotion 4. Chromosome separation in mitosis and meiosis 5. Intracellular transport of organelles The cytoskeleton consists of three types of fibers: 1. Microfilaments 2. Intermediate Filaments 3. Microtubules Distinguish microtubules, intermediate filaments, microfilaments. Microfilaments (or actin filaments) are made of the protein actin, which polymerize to form long, thin fibers. Microfilaments form a band just beneath the plasma membrane, providing mechanical strength, membrane protein linkage for cell surface receptors and cytoplasmic proteins, centrosome anchoring to opposite poles of the

6

cell during mitosis, and pinches diving animal cells apart during cytokinesis. The microfilaments also play a role in locomotion, as cilia, and function as thin filaments in muscle (which interact with thick filaments of myosin) to yield force. They show often in cells as villi mainly because they increase the surface area of cells, which is beneficial for cellular function. Intermediate filaments are large than microfilaments, and are found in several types of cell. Their main function is to provide structural support within the cell itself. It can be made of different proteins, depending on the type of cell. For example, keratins are found in epithelial cell. Nuclear lamins form a meshwork that stabilize the inner membrane of the nuclear envelope. Neurofilaments strengthen the long axons of neurons. Vimentins provide mechanical strength to various cells, especially muscle cells. Microtubules are the largest of the types of cytoskeletal fibers, being 25 nm hollow cylinders made by a ring of 13 “protofilaments”, mainly because they are built from dimers of alpha and beta tubulins (meaning that one of each is required to make a tubulin). It is involved in cellular structure as well as movement. It plays the most prominent role in cellular reproduction. The centrosome is an orthogonal arrangement of two centrioles, but the centriole is a barrel-shaped microtubule structure made from 9 microtubule triplets. Microtubules are dynamic, growing from the polymerization of tubulin dimers and hydrolysis of GTP. Simultaneously, they shrink with the release of tubulin dimers (depolymerization). Thus there are two ends, with the growing end termed the “plus” end and the shrinking end designated as the “minus” end. Microtubules also participate in locomotion. The cells have protein “motors” that move along the microtubule with the input of ATP. There are two groups of microtubule motors: (1) kinesins (which are attracted to the plus end of microtubules) and (2) dyneins (which move toward the minus end). Below is a general overview of the distinguishing elements of microtubules, intermediate filaments, and microfilaments.
Protein Filament Microfilaments Diagram Length (nm) 7 Individual Unit Strand of protein (actin) Function Connect organelles to membranes Influence cell motility and shape Structural Stability

Intermediate Filaments

10

Microtubules

25

Composed of various proteins such as keratins, lamins, neurofilaments, and vimentins depending on function Strands of tubulin

Influence cell structure and shape Motility – organelle movent such as cilia and flagella

7

Understand cilia and flagella.
Type of Structure Flagella Major Function Motility and Locomotion Movement Whip-Like Diagram

Cilia

Sweeping of Particles

Synchronous Beating

Cilia and flagella are built from microtubules. Flagella are whip-like and undulate to move cells. Cilia are hair-like in structure that beat in synchrony. Both types of structures can have three types of arrangement (of fused pairs of microtubules on the outside of a cylinder + unfused microtubules in the center): 9 + 0, 9 + 2, 9 + 4. However, all cells are beginning to exhibit cilia, primary or primordial cilia, in an effort to provide a response to mechanical stimuli. Know the importance of the cilium in photoreceptor development. The outer segment of the photoreceptor is specialized cilium essentially. Consequently, the photoreceptors (the rods and cones) are specialized cilia. The connecting cilium between rod inner and outer segment are 9 + 0 cilia. With a certain mutation and poor development, there can be defects in these cilia that cause such blindness. It was later shown that mutations in a gene called RP-1 are associated with 3% of retinitis pigmentosa cases, and later RP-1 was discovered to be a photoreceptor cilium-associated protein.

Lecture V
Using Membrane Voltage Know the Nernst equation. The Nernst Equation was designed to explain the change in voltage through the intracellular and extracellular concentrations of in terms of permeability of the ion and the intracellular as well as extracellular concentrations of the ion. From this we can calculate the electrical voltage in terms of potassium concentrations by: 𝑉! ≅ −
!" !! !

log

R is the universal gas constant, T is 310 Kelvin, Z is the valence charge of the ion, and F is

! ! !" ! ! !"#

=   −

!" !!

log

! ! !" , ! ! !"#

where

8

96,500 coulombs per mole. For a single ion the Nernst equation describes the theoretical balance between these forces. Understand the principle that ion concentration gradients and ion permeabilities determine PD. We need to remember that the plasma membrane is a barrier between the cytoplasm and extracellular space, and ion concentrations are different when comparing the cytosolic and extracellular environment. In all cells, there is an electrical voltage (change in potential energy) across the plasma membrane. Since there is electrical voltage in all cells, there is also a baseline electrical condition known as a resting membrane potential. It is dependent on two parameters: 1. Transmembrane Ion Gradients (particularly sodium and potassium ions): Ion gradients deveop and maintain steady-state ion gradients for ALL cells. 2. Membrane Permeability of Those Ions: Permeability allows for a rate of movement of these ions.

cyto
Na+

out

Na+
K+

cyto (+)
Na+

out

Na+
K+

ATP

ATP

K+
(-)

K+

Wat we should also know that there is two opposing forces along the membrane: 1. Chemical Force (Potassium Gradient)  Tends to push potassium ions OUT 2. Developed Electrical Force (inside negative)  Tends to ‘pull’ potassium ions IN. In most cells the chemical and electrical forces for potassium ions are nearly in balance. This means that the outwardly-direct potassium ion gradient results in an inside-negative electrical potential (determined mainly by potassium), and is characteristic of cells in their resting state. This leads to an electrical potential difference (PD), which is measured in P [K+] + P [Na+]in + PCl [Cl ]out millivolts (mV). Altering the membrane permeability Vm produce changes inNa can 60 mV log K + in the membrane PK[K ]out + PNa[Na+]out + PCl [Cl ]in potential. Responses are according to changing the change.
+50

VNa

Recognize the Goldman equation. The Goldman equation reflects a more realistic situation where the ions and chloride are involved and the ionic permeabilities can change. This equation takes into account the permeabilities of the ions (rapidly variable) as well as their intracellular and extracellular concentrations (relatively stable). It states the following: 𝑽𝒎 ≅ −𝟔𝟎  𝒎𝑽 𝐥𝐨𝐠 𝑷𝑲
𝑲! 𝒊𝒏 !𝑷𝑵𝒂 𝑵𝒂! 𝒊𝒏 !𝑷𝑪𝒍 𝑪𝒍! 𝒐𝒖𝒕 . 𝑷𝑲 𝑲! 𝒐𝒖𝒕 !𝑷𝑵𝒂 𝑵𝒂! 𝒐𝒖𝒕 !𝑷𝑪𝒍 𝑪𝒍! 𝒊𝒏

Membrane Potential (mV)

0

‘boundary conditions’

50

100

Vm V Cl VK

This confirms the regulation of channel-mediated ion permeability allowing cells to change voltage. Remember that the two parts to manipulation of the potential difference: 1. Establishment and maintenance of sodium and potassium ion gradients 2. Variation in the activity of specific ion channels

9

Know whether light depolarizes or hyperpolarizes the rod photoreceptor. We will need to know that the sodium channels are open in the absence of light, thus allow sodium to enter the cytosol. Thus, we can state that it thus becomes more positive inside the cell with respect to the extracellular environment. Thus, it DEPOLARIZES! We also do know that when light is present, the sodium channels are closed. In the presence of light, sodium channels cannot enter the cytosol. It becomes less positive (and more negative) inside the cell with respect to the environment. Thus, it HYPERPOLARIZES!

Compare a typical cell

Lecture VI
Rhodopsin – a G-protein-coupled receptor Know where rhodopsin is located in rod photoreceptor. Rhodopsin is located in the outermost of the outer segment of the rod photoreceptor. This is determinant of cellular depolarization and hyperpolarization depending on the presence of light. Remember that in the dark, the rod photoreceptor is depolarized, while a light ….to a rod photoreceptor causes the rod photoreceptor to hyperpolarize, and that rhodopsin is the light sensitive molecule in the rod photoreceptor. Understand the similarity between rhodopsin and ligand-activated G-protein coupled receptors. Rhodopsin is a G protein coupled receptor in these that it is an integral membrane protein (or a 7-helix receptor because of its 7 transmembrane alpha helixes). Gprotein coupled receptors utilize trimeric GTP binding proteins to relay signals to effector proteins inside cells. GTP-binds to the same G-alpha subunit which separates from G-beta and G-gamma subunits. IN GPCR activation, the ligand binding causes GTP to exchange for GDP on the Gprotein, causing the alpha subunit to dissociate from the complex and activate signaling pathways that bring about a signaling response. The only difference is mainly the ligand in question. The ligand in the rhodopsin is already present (11-cis-retinal), and is activated when light (in the form of photons) reach 11-cis-retinal. When a phton reaches 11-cis-retinal on rhodopsin, the rhodopsin is activated, causing the change in conformation similar to the GPCR. The light sensitive ligand 11-cis-retinal is covalently bound within the opsin molecule. When the photon is absorbed by the chromophore (ligand) 11-cis-retinal, there is fast isomerization to all-transretinal. In the dark, there is reversion to the cis adaptation. A

10

signaling cascade occurs in either event. The isomerization of all-trans retinal forces a conformation change in the opsin, activating the G-protein transducing, and the G-alpha subunit separates from the G-beta and G-gamma subunits, and G-alpha continues to activate a phosphodiesterase enzyme, leading to the hydrolysis of cyclic GMP. The reduction in cGMP concentration causes close of the sodium channels and hyperpolarization of the photoreceptor cell. Based on this, one can infer the enzyme cascade associated in this sequence.
Activation causes the GDP bound to the alpha subunit to be exchanged with GTP from solution and results in activated alpha dissociating from beta-gamma. Activate transducin-alpha then causes cyclic GMP phosphodiesterase to increase its activity, thereby lowering the concentration of cGMP. Termination occurs when (active) Transducin-GTP is hydrolyzed to Transducin-GDP, a process which is accelerated by a complex containing an RGS (Regulator of G-protein signaling)-protein and the gamma-subunit of the effector, cyclic GMP phosphodiesterase.

Heterotrimeric Transducin is activated by conformational change in rhodopsin due to the adsorption of a photon by rhodopsin's active group 11-cis-retinal

Know light causes cGMP concentration decrease and ion channels closure. Photoreceptor cells are relatively depolarized in the dark and the sodium channels are open. In the presence of light, the sodium channels are closed and there is hyperpolarization. The G-alpha subunit activates the phosphodiesterase enzyme, causing hydrolysis of cyclic GMP. Decreases in the cGMP concentration cause the closure of the sodium channels and hyperpolarization of the photoreceptor cell. In summary, light typically causes cGMP concentrations to decrease and sodium channels to close and consequently hyperpolarization. However, in the dark, cGMP concentrations increase and sodium channels are open and consequently there is depolarization. Know whether light depolarizes or hyperpolarizes the rod photoreceptor. Remember that the absence of light depolarizes the rod photoreceptor because of sodium channel opening, and the presence of light hyperpolarizes the rod receptor due to G-alpha subunit separation, cGMP hydrolysis, and consequence closure of the sodium channels.

Lecture VII
Subcellular Structures - Mitochondria Know the structure of mitochondria. Mitochondria are the power stations for a cell, and are typically round and rod-shaped. They have two membranes, in which the outer membrane is a boundary, and the inner membrane is an inner membrane, with foldings known as “cristae mitochondriales” in order to increase surface area and intermembrane exchange. How are mitochondria organized to be the power station of a cell? Mitochondria are the power stations of a cell, having metabolic pathways in the supply of energy for oxidative phosphorylation by mitochondria. It has two membranes, outer and inner membranes, like a bacterium. However, unlike the typical prokaryote, the inner

11

membrane has folding and projections (the cristae), allowing for increased surface area for exchange and transport. This also allows the intermembrane space to be more spacious, allowing for other metabolic reactions to occur. Understand concepts of glycolysis to the Krebs cycle and the electron transport chain. Outside the mitochondria, glycolysis (an anaerobic reaction) occurs, briefly involving the conversion of glucose (in a series of reactions) to pyruvate. From pyruvate, acetyl coenzyme A (acetyl-coA) is produced and thus entered into the Krebs cycle (which occurs in the interior of the mitochondrion). It does not generate ATP, but generates other intermediates (FADHs, GTP, and NADH) for the electron transport chain, which along the inner membrane. Mitochondria (and chloroplasts) utilize chemiosmotic coupling to harness energy. This is the link between chemical bondforming reactions that generate ATP and membrane transport, and consequently generating an osmotic force. The electron transport chain utilizes the chemiosmotic coupling in the transport of protons and movement of electrons (as well as intermediates from the Krebs Cycle) to cause a shift, which is then harnessed by ATP Synthase to generate ATP. The Electron Transport Chain has to reach four complexes before it is able to reach ATP Synthase: Complex I: NADPH Oxidase, Complex II: Succinate Reductase, Complex III: Cytochrome bc1, Complex IV: Cytochrome Oxidase. In summary, we must recognize the metabolic reactions in the mitochondria. In the cytoplasm, glycolysis produces a net of 2 ATPs per glucose molecule, involved in the conversion of glucose to pyruvate. The pyruvate (taken into the mitochondria) is then converted to acetyl-CoA and utilized in the Krebs cycle, and the products of the Krebs cycle (intermediates such as FADHs, NADH, and GTP) are then utilized in the electron transport chain to produce 34 ATP per glucose (pyruvate) molecule. This confirms the general idea of the mitocondria’s role in the cell. Understand how mitochondria divide. Mitochondria, like bacterium, can simply divide by fission, yielding 2 mitochondria from a mitochondrion. Mitochondria can be moved or can undergo fission where ATP demand is highest. Mitochondria contain some DNA of their own, and have ribosomes and tRNA and can make many of their own proteins. The DNA is found in the matrix in punctuate structures called “nucleoids”. Each nucleoid may contain 4-5 copies of the mitochondrial DNA (mtDNA). Mitochondrial DNA is circular.

12

Know what happens to worn out mitochondria. When mitochondria are worn out, they are broken down by lysosomes in a process called autophagy. Autophagy is mainly a process in which the cell itself digests its own cellular material. This process involves wrapping endoplasmic reticulum around the mitochondrion. Enzymes are recruited into the vesicle, and the inside becomes acidic and mitochondria are consequently digested. Know mitochondria play a role in apoptosis. Apoptosis is a programmed series of events leading to a clean and tidy cellular death. In essence, cytochrome c concentrations increase within the mitochondria, and the opening of the permeability transition pore allows cytochrome C to exit. Cytochrome C (as an enzyme) also activates a protein known as Apaf-1, which activates the caspases, and intracellular proteases that degrade cellular components. Thus, cytochrome c plays a role in signal amplification for an organized destruction of a designated cell.

Lecture VIII
Subcellular Structure – nucleus, ribosomes Know terms related to DNA and RNA structure e.g. nucleotide, nucleoside. Term Definition Nucleotide Combination of a nucleoside and phosphate Nucleoside Combination of base and sugar Nucleic Acid Sequence of nucleotides

Know DNA and RNA bases and base pairing. Nucleic Acid Found in: Number of Bonds Adenine (A) DNA, RNA 2 Thymine (T) DNA 2 Cytosine (C) DNA, RNA 3 Guanine (G) DNA, RNA 3 Uracil (U) RNA 2

Complement Thymine (T), Uracil (U) Adenine (A) Guanine (G) Cytosine (C) Adenine (A)

Remember also that hydrogen bonding stabilize “complementary” interactions between A/T and G/C. Complementary pairing of nucleotides ensures that each ‘strand’ permits regeneration of the other. Understand transcription. Remember the primary base sequence. This sequence permits the storage of information. The “double helix” of DNA contains two complimentary strands, each of which contains: 1. Information to replicate the other strand 2. Information concerning protein structure

13

Transcription is mainly the synthesis of RNA from DNA. It synthesizes a complimentary strand of RNA using the base sequence of DNA as a template. Transcription occurs in the nucleus and yields a copy of DNA known as messenger RNA (mRNA). However, before DNA makes a copy known as pre-mRNA, which contains two types of data (introns and exons). The introns contain data not of interest, and thus removed via a process called splicing. The mature mRNA sequence, containing the exons only, is prepared for translation in the cytoplasm. Understand translation. Translation is the synthesis of protein from RNA. It synthesizes a protein using the base sequence of RNA as a template. It mainly occurs in the cytoplasm, and involves three types of RNA: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). The overall function of this process is to yield a protein. These secreted or membranous proteins can be deposited in the endoplasmic reticulum membrane or lumen, and processed in the Golgi apparatus. Distinguish mRNA, tRNA, rRNA. Type of Function RNA mRNA RNA that encodes chemical model for protein product tRNA Transfer amino acids that mRNA codes for rRNA Enzyme that is the site of protein synthesis in cells

Synthesizing Enzyme RNA Polymerase II RNA Polymerase III RNA Polymerase I

Know how the AA sequence and DNA and RNA sequence relate to each other. Remember by the Central Dogma that DNA (while replicating itself) codes for RNA, and RNA codes for proteins. Thus, we also know that DNA plays a role in the regulation of gene expression, because protein expression requires transcription and translation. Regulation of both processes is important. Remember also that ‘promoter regulation’ is a mechanism for the regulation of gene expression. The promoter is the DNA sequence ‘upstream; to the start of a gene to which RNA polymerase binds. The transcription factors are proteins that bind to the promoter to augment binding of RNA polymerase. Different cells must be positioned in different parts of the body, and these cells are different in their expression. Thus, a possible mutation (no matter how large or small, because it is still a mutation) on the DNA can cause potential changes in function.

14

Lecture IX
Subcellular Structure – Endoplasmic Reticulum Know basic structure of endoplasmic reticulum. The endoplasmic reticulum is an organelle found in all eu karyotic cells. It is an interconnected network of membranous compartments, with vesicles and cisternae. It has multiple functions. It handles hydrophobic and amphiphilic problems to be utilized eventually in the plasma membrane (receptors and transporters). It handles proteins to be secreted from the cell by exocytosis (digestive enzymes). It is also a store for calcium, and producer and storage of glycogen, steroids and some other molecules, and plays a role in phospholipid synthesis. It was hypothesized that the ER was an invagination of the plasma membrane, thus becoming part of the cell’s endomembrane system. The basic membrane structure and composition is similar to the plasma membrane. There are two types: rough endoplasmic reticulum and smooth endoplasmic reticulum. Understand difference between rough ER and smooth ER. Type of Physical Feature Enzymes Produced ER Rough Surface of the rough endoplasmic Lysosomal Enzymes, Secretory reticulum is dotted with ribosomes giving Proteins, and Integral Membrane it a bumpy or rough appearance. They Proteins that stay embedded in the are not really part of the ER, but bind to membrane. the ER to synthesize a protein destined for delivery into the ER lumen. It is continuous with the outer layer of the nuclear envelope. Smooth Surface of the smooth endoplasmic Lipids, steroids, and morphine, and reticulum does not have ribosomes. are involved in the metabolism of carbohydrates and steroids (but not lipids)

Understand general concepts of ER function. It has multiple, general functions. It handles hydrophobic and amphiphilic problems to be utilized eventually in the plasma membrane (receptors and transporters). It handles proteins to be secreted from the cell by exocytosis (digestive enzymes). It is also a store for calcium, and producer and storage of glycogen, steroids and some other molecules, and plays a role in phospholipid synthesis. Understand how proteins enter the ER. The translocon is considered a mechanism to get proteins into the ER. Translocons are docking point for ribosomes to attach to the membrane. It causes a series of steps to get secretory proteins into the ER.

15

1. As the signal sequence emerges from the ribosome, it is recognized and bound by the signal recognition particle (SRP). 2. The SRP escorts the complex to the ER membrane, where it binds to the SRP receptor. 3. The SRP is released, the ribosome binds to a membrane translocation complex of Sec61 proteins, and the signal sequence is inserted into a membrane channel. 4. Translation resumes, and the growing polypeptide chain is translocated across the membrane. 5. Cleavage of the signal sequence by signal peptidase releases the polypeptide into the Targeting proteins to the ER lumen & membrane lumen of the ER.

Targeting proteins to the ER membrane (contd)
Copyright 2008 by Saunders/Elsevier. All rights reserved.

Fig. 20-7

Fig. 20-7

The rough ER is responsible for carbohydrate addition to proteins. This process is known as Copyright 2008 by Saunders/Elsevier. All rights reserved. glycosylation. N-linked glycosylation involves amide group of asparagine residues. O-linked glycosylation involves OH of serine or threonine residues. Most protein made in the ER become N-glycosylated. Some proteins are glycosylated on certain asparagine residues (Nlinked glycosylation) within the ER while translation is ongoing. Oligosaccharide units consisting of 14 sugar residues are added to the growing polypeptide chain. The oligosaccharide is synthesized earlier on a lipid (dolichol) carrier anchored in the ER membrane. Four sugar residues (three glucose and one mannose) are removed while the

16

protein is still within the ER, and the protein is modified further after being transported to the Golgi apparatus.
ER accumulates and stores Because phospholipids are synthesized on the cytosolic side of the ER membrane, calcium are they added only to the cytosolic half of the bilayer. They are then translocated across the membrane by phospholipids flippases, resulting in even growth of Plasma membrane both halves of the phospholipid bilayer. ER calcium ATPase ATP
ADP
Ca2+

ER Store

Know ER is a calcium store. Receptor activation causes release of ER Endoplasmic reticulum accumulates and stores calcium. Receptor calcium activation causes release of the calcium within the endoplasmic reticulum. GPCR activation can release calcium from the ER. The G protein activates phospholipids C (PLC) which generates IP3, a signaling molecule, that diffuses to an IP3 receptor on the receptor. ER Store We can detect cytoplasmic calcium by loading cells with a fluorescent dye like FURA-2 that is sensitive to [Ca2+]. We can measure the fluorescence signal using a spectrophotometer or an epifluorescence. Thus, we can conclude from here that the endoplasmic reticulum pumps calcium ions. One example is the sarcoplasmic reticulum. It is a specialized type of smooth ER found in smooth muscle and striated muscle. Calcium storage and release by the sarcoplasmic reticulum is a critical part of muscle function.
Agonist Plasma membrane receptor ATP ADP
Ca2+

Know that in some RP mutants, rhodopsin is trapped in ER. In individuals with retinitis pigmentosa, rhodopsin is trapped in the endoplasmic reticulum. Faulty proteins can get trapped in the ER. Some mutant cGMP-sensitive rod photoreceptor ion channels seem to get stuck and cannot reach the plasma membrane. Thus, they cannot be expressed when light is present because of their misplacement in another part of the cell.

Lecture X
Subcellular Structure – Golgi apparatus and Secretory Pathway Know what the Golgi apparatus looks like and where it is. The Golgi apparatus is a collection of membrane-bound sacs known as cisternae. By electron microscopy it generally appears as a stack of pancakes near the cell nucleus. It has a cis face and a trans face, with a cis-Golgi point towards the cell nucleus, and trans-Golgi pointing away from the nucleus. It is located in the cytoplasm, and organized by the cytoskeleton. When a package of protein exits the endoplasmic reticulum, it enters the Cis face. The Golgi modifies, sorts and packages the raw proteins, and the packaged protein exits the Trans face. Understand in general terms what the Golgi apparatus does. The Golgi apparatus modifies, sorts, and packages proteins. It also sorts and delivers lipids around the cell. There are various modifications that occur in the Golgi apparatus, as a carbohydrate and lipid factory:

17

-­‐ -­‐ -­‐ -­‐ -­‐ -­‐

Proteolysis (refinement of pro-hormones to hormones) Add polypeptide signal sequence for sorting Carbohydrate factory, involved in glycosylation (addition of carbohydrate groups), synthesis of proteoglycans and glycolipids. Phosphorylation (addition of phosphate groups) Synthesis of sphingolipids Addition of carbohydrate groups to make glycolipids

Understand the concept of the Secretory Membrane System. The secretory membrane system modifies and packages and sorts proteins destined for export from the cell or transfer to specific parts of the cell. The major players are the Golgi apparatus, the endoplasmic reticulum, and carrier vesicles. There are three examples of vesicles (secretory, lysosomal, and exocytotic). Secretory vesicles are cargo for regulated, extracellular release, held in the cell until a signal causes vesicle to fuse with plasma membrane and release contents. Lysosomal vesicles are cargo head towards lysosomes, including lysosomal enzymes and lipid membrane components. Endocytotic vesicles are cargo for extracellular release, moving continuously to fuse with plasma membrane and release contents. The secretory membrane system distributes integral membrane proteins to the apical or basolateral plasma membrane of epithelial cells. It is also involved in the retrieval of resident ER proteins. For example, proteins destined to remain in the lumen of the ER are marked by the sequence Lys-Asp-Glu-Leu (KDEL) at their carboxy terminus. These proteins are exported from the ER to the Golgi in the nonselective bulk flow of proteins through the secretory pathway, but they are recognized by a receptor in the ER-Golgi Intermediate compartment (ERGIC) or the Golgi apparatus and selectively returned to the ER. Know proteins are sorted to specific parts of the cell. Rhodopsin is directed to the rod outer segment. Protein coats facilitate the formation of vesicles that transport materials forward and backwards between the Golgi and ER. In some cells, COPII coats vesicles headed from the ER to the Golgi while COPI coats vesicles headed back to the ER. SNARE proteins on the vesicles are required as the targeting and docking mechanism. Vesicles are routed in a precise manner. In some cells secretory granules form at the trans Golgi apparatus. These vesicles are essential for shipping. Rod photoreceptors sort rhodopsin to

18

the outer segment. Rhodopsin protein synthesis is confined to the rough endoplasmic reticulum of the inner segment. The newly synthesized proteins traverse the inner segment and assemble into the disks of rod outer segments (and the lamellae of cone outer segments). There are fewer cells that are more spectacularly organized than the photoreceptor. Understand how GFP might be used to track proteins. Green fluorescent protein (GFP) a 238 amino acid protein was isolated from a species of jellyfish. Isolation of cDNAs made it possible to express GFP in cells. GFP can be tagged onto the end of a protein as a way to visualize its position in a cell. There are various comparisons of the distribution of GFP and GFP fusion proteins in the rods of transgenic frogs. This allowed determination of the localization signal of rhodopsin in rods resides within the last amino acids of its sequence. Type of GFP GFP GFP-CT44 Description Fills the cytoplasm and nucleoplasm of the inner segment and synapticterminal. Only a small amount is found in the rod outer segment (ROS). Fusion protein of GFP with the last 44 amino acids of Xenopus rhodopsin. It is membrane bound because its cysteins are palmitoylated and it localizes nearly exclusively to the ROS. Fusion protein of GFP with the last 39 amino acids of the C-terminal of the alpha-adrenergic receptor (AAR), a receptor resembling rhodopsin in overall structure. In contrast to GFP-CT44, GFP-AAR localizes predominantly to the synaptic terminal. Fusion protein of GFP, the C-terminal of AAR which has had its last 8 amino acids replaced by the last 8 amino acids of Xenopus rhodopsin. The switch of this small domain changes the distribution of the fusion protein from the synaptic terminal © to the ROS to resemble GFP-CT-44.

GFP-AAR

GFP-AARCC-CT8

Lecture XI
Endocytosis - Phagocytosis Understand concept that cells use vesicles for entry of substances as well as exit. Endocytosis is a shift of material in an inward direction (bringing material into the cell). There are several methods of endocytosis, depending on the size and selectivity. Know the principal mechanisms of endocytosis. Mechanism Target Size Phagocytosis Large Macropinocytosis Small particles (essentially extracellular fluid) Clathrin-Mediated Small Endocytosis

Selectivity Selective Non-Selective Selective

19

Caveolae-Dependent Uptake Nonclathrin/noncavaeolaemediated endocytosis
Phagocytosis

Small Small or Large

Non-Selective Non-Selective

In phagocytosis, the attachment is dependent on the ability of the cell to recognize the particle. The target cell is tagged with proteins called opsonins, which bind to the bacteria complementarily. This prepares it for ingestion by the cell. In ingestion, the receptors of the cell recognize the molecule of the bacterium, and the actin on the acting cell surrounds the target cell and essentially ingests the target. From there, actin gradually moves away from the site after complete ingestion and then the endosome of the cell attaches the phagosome (the vesicle containing the target, damaged cell), spurring maturation. Once the phagosome matures, primary and secondary lysosomes attack the mature phagosome causing complete destruction of the phagosome (and formation of phagolysosome) and its infected contents through self-destruction of the phagolysosome. The materials are essentially broken down into monomers, such as monosaccharides, disaccharides, nucleotides, and lipids.
Macropinocytosis

Macropinocytosis essentially involves the ingestion of extracellular fluid. They are nonselective, and involve actin-driven ruffles of the plasma membrane. The material attained by this mechanism is typically extremely small.
Caveolae-dependent uptake

Caveolae-mediated endocytosis is involved in the ingestion of extracellular substances, such as serum proteins and nutrients. They are nonselective, and the mechanism involve caveolae (“little caves”), specialized regions of the plasma membrane that are rich with cholesterol stabilized by caveolin. They are prevalent in endothelial cells, but absent in nueurons. Microdomains, rich with cholesterol and glycosphingolipids contain lipid rafts that exist independently of caveolin protein. The molecules gather in the raft, and caveolin binds to cholesterol, cholesterol, stabilizes the domain, and forms the cave structure.
Clathrin-mediated endocytosis

Clathrin-mediated endocytosis is a selective ingestion of extracellular substances, involving specialized regions of the plasma membrane called “coated pits”. These pits contain a lattice of clathrin and adaptor proteins and are involved in the uptake of nutrients. The point entry of ligand receptor complexes may be concentrated into the pits. The protein components involved include AP2, Clathrin, and Dynamin.

20

In this mechanism, AP2, clathrin, and other proteins assemble upon the vesicle, forming a coat at the pit. This coated pit is then “collared” by the dynamin and another set proteins and then is detached. After detachment the clathrin and AP2 dissociate from the transport vesicle. Analogy: Dressing your pet and then leashing it for a walk in the park. You put on its coat before you unleash the dog for a walk. But then you realize it is too hot outside so you take the coat off the dog.
Nonclathrin/noncavaeolae-mediated endocytosis

This mechanism is typically nonselective, and accounts for endocytosis that occurs when clathrin and caveolin are disrupted or absent. The mechanism is still uncertain, but it is known that it may involve lipid rafts. It allows for cholera toxin and shigella toxin to enter the cell. Understand which mechanisms are selective. We know that phagocytosis and clathrin-mediated endocytosis are the two selective mechanisms of endocytosis. We can recognize that they are selective because they require certain “tags” and processes involved in order to prepare them for entry into the cell. Know the endosomal membrane system. The endocytosomal membrane system is the system of different membranes suspended in the cytoplasm of the eukaryotic cell. The membranes divide the cell into functional and structural compartments. Such compartmentalization allows for the intracellular specialization of the cell. There are four stages of the endocytosomal membrane system: early endosomes  multivesicular bodies  late endosomes  Lysosomes. These can be sorted by a variety of factors, such as pH. If there is progressive decrease in luminal pH, there may be protein sorting in endosomal compartments. The interaction of cargo molecules with their receptor is pHdependent. At low pH, the molecules break free into the lumen while the receptors stay in the vesicle membrane. Receptors may or may not be recycled. Understand principles of rod outer segment shedding and phagocytosis. In the retina, the rod outer segment (a large molecule) is being ingested via phagocytosis. This is important to eliminate the old photoreceptors while producing newer photoreceptors. The shedding of the rod outer segment and phagocytosis occurs after the onset of light. In the pathologies such as retinitis pigmentosa, there is no replacement, thus these aged photoreceptors eventually die off.

21

Know importance of RPE to photoreceptor function. The retinal pigment epithelium disposes of worn out rod outer segments through phagocytosis, so that newer photoreceptor rods can emerge and improve vision. Rod outer segment shedding and phagocytosis occurs after the onset of light. If these aged rods fail to be eaten via phagocytosis, they may accumulate and distort vision.

Lecture XII
Protein Turnover and Degradation Understand rod disc renewal and role of RPE. Rod disc renewal involves phagocytosis of the older rod discs while the newer rod discs of the photoreceptor are pushed towards the surface. Retinal pigment epithelium disposes of worn out rod outer segments. As photoreceptors age, they ascent to the outer segment of the photoreceptor (which are pointed at the retinal pigment epithelium), and are eventually are ingested. Know the concept that proteins are continuously synthesized and degraded. Different proteins turn over at different rates. The same protein may turn over at different rates in different tissues. It can be massive. The actual pieces are taken away and degraded, and the cell constituents are turned over. It then becomes a balancing act between synthesis and degradation. We can study protein synthesis and degradation through methionine labeling or pulse chase. In methionine labeling, cells with 3H-methionine are incubated and the incorporation into the target protein is measured. The proteins are then separated by electrophoresis. In pulse chase, there are two phases. The pulse phase involves incubation with 3H-methionine to allow incorporation into the target protein. The chase phase involves incubation with nonradioactive methionine and watch disappearance of the labeled target protein. Understand the concept of a protein’s half-life. The half-life of a protein is the amount of time required for the quantity to decay to half its initial value. Know the general principle of protein degradation by lysosomes. Proteins are degraded by proteolysis by both lysosomes and proteasomes. Lysosomes are membrane-bound compartments containing various hydrolases, including proteases. The pH inside of the lysosome is low. The typical proteins degraded in a lysosomal process are long-lived cytosolic proteins or integral membrane proteins in secretory and endosomal vesicles. Lysomes contain greater than 60 different hydrolytic enzymes – proteases (E.g. cathepsins), lipases, phospholipases, glycosidases, and nucleases. Enzyme precursors are tagged with mannose-6-phosphate in the Golgi. Trans Golgi receptors divert the enzyme precursors to endosomes and lysosomes where they are activated. Lysosomes have a proton pump known as vacuolar ATPase (V-ATPase), which shifts protons into lysosomes and endosomes. It utilizes primary active transport to move protons inside. Lysosomes degrade extracellular materials taken up by endocytosis. They also degrade cellular materials shifted

22

in via cytoplasmic vesicles. This accounts for 50-70% of protein turnover. Proteins destined for the lysosome are tagged for destruction with mannose-6-phosphate. Lysosomes typically are involved in autophagy, or the cell’s mechanism for the disposal of large particles. The particles are initially enveloped and sealed to isolate the desired particles, and then the lysosome merges with the sealed vesicle, and the contents are destroyed. Such destruction yields a vesicle of reduced contents known as a residual body. Another process involving lysosomes is known as crinophagy. Crinophagy is a process for the degradation of secretory proteins (namely hormones) that have expired, meaning they are beyond their “shelf-life”. Lysosomes fuse with the aged secretory vesicles and degrade everything inside. Understand what is meant by “ubiquination”. Ubiquination is typically tagging a protein for destruction by proteasomes by attaching a protein called ubiquitin. Some ubiquitin can be tagged once (called monoubiquination) or more than once (through poly-ubiquination). Damaged or unneeded proteins are marked for destruction by the covalent attachment of chains of a small protein, ubiquitin. A large, ATP-dependent complex called a proteasome subsequently degrades Polyubiquitinated proteins. Sidechain amino groups of lysine residues on the target protein are covalently linked to the Cterminus of ubiquitin. The process involves ubiquitin-activating enzyme (E1), ubiquitinconjugating enzyme (E2), and ubiquitinprotein ligase (E3). One ATP molecule is hydrolyzed for every ubiquitin molecule attached to the target protein. Know the general principle of protein degradation by proteasomes. Proteasomes are proteolytic machines are assembled from several different protein subunits. The proteolysis sites are inside of a narrow cylindrical chamber. Proteasomes processes typically degrade short-lived cytosolic proteins and endoplasmic reticulum membrane proteins. They are about half as large as a ribosome. The proteolysis sites line the inside of a narrow cylindrical chamber. Regulatory complexes guard the openings to allow access to only selected, unfolded, polypeptides. In response to signaling cascades or at specific transitions in the cell cycles proteasomes degrade key substrates. They degrade abnormal and misfolded proteins, through unfolding and dismantling into amino acids (the monomers of proteins). Certain proteasomes can

23

degrade antigens. In terms of structural, there is a set pattern of cap-core-cap. The core makes up the central chamber. The core is a 20S proteasome. The 20S proteasome has 28 subunits arranged in four seven-membered rings. The alpha subunits are the tom and bottoms rings, while the beta subunits compose the middle two rings. Proteolytic sites on the beta rings face the inner chamber. Damaged or unneeded proteins are marked for destruction by the covalent attachment of chains of a small protein, ubiquitin. A large, ATP-dependent complex called a proteasome subsequently degrades polyubiquitinated proteins. Side-chain amino groups of lysine residues on the target protein are covalently linked to the C-terminus of ubiquitin. The process involves ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). One ATP molecule is hydrolyzed for every ubiquitin molecule attached to the target protein. Know what is meant by the term Visual Cycle. Turnover of photoreceptor cell constituents occurs at a fast rate. Photoreceptors rely on the retinal pigment epithelium (RPE) to handle disposal of outer segments. Retinal pigment epithelium also handle recycling of the photosensitive ligand all-trans-retinol back to 11-cis retinol. This process is known as the visual cycle. If the visual cycle is faulty, the loss of photoreceptor function can occur. Retinol from the blood moves into the retinal pigment epithelial (RPE) cells and is esterified to form a retinyl ester that can be stored. When needed, retinyl esters are converted to 11-cis retinal which then are shuttled to the rod to bind the protein opsin, forming rhodopsin. Photons isomerize all of 11-cis-retinal to all-trans-retinal which then is released. All-transretinal is converted to all-trans-retinol, which is transported across the interphotoreceptor matrix to the retinal pigment epithelium where it is recycled. In short, the visual cycle is the process by which all-trans-retinol is returned to 11-cis-retinol. This is simply a recycling of the trans-retinol and 11-cis-retinol after its exposure to light.

Lecture XII
Synapse I Know rod photoreceptors form a synapse with bipolar cells. We need to first remember that there is more than one cell, and that cells communicate. The major focus of the photoreceptors is the retinal pigment epithelium, and thus must eliminate older outer segments and recycle all-trans-retinal to 11-cis-retinal. The retina of the cell (the main focus) is a multilayered part of the eye. Photoreceptors detect photons, surely, but neurons are needed to detect motion and fine-tune our perception

24

through horizontal cells (involved in spatial coordination of a signal) and amacrine cells. Through a string a neurons, the impulse eventually goes out of the eye via retinal ganglion cells. There are a lot of rods, and the lot converges to a bipolar cell. This confirms that there is circuitry through the retina. The convergence is to two specific ganglions. Rods converge to bipolar cells, which then converge to amacrine cells, and then finally to a ganglion. One ganglion responds to the presence of light, while the other reacts to the absence of light. The initial electrical signal is grade photoreceptor hyperpolarization. The electrical signal that emerges from the retina is a train of action potentials in retinal ganglion cells. Each action potential is the same, and offers tremendous change in signaling. Know difference between chemical and electrical synapse. To understand the signaling elements, we do need to comprehend the idea of the synapse. What is a synapse? Well, simply put, it is a region where communication occurs between two neurons or between a neuron and a target cell. There are two major types of synapses: chemical synapse and electrical synapse. Chemical synapses utilize neurotransmitters (chemicals that act as a message) to communicate between cells. The presynaptic neuron secretes this chemical into the cleft between the cells. The chemical selectively activates the receptors on the surface of the postsynaptic cell. Activation of the receptor can change (giving a stimulatory or inhibitory effect) the postsynaptic cell. This known form of communication is typically unidirectional. Electrical synapses involve special channels known as gap junctions to conduct electricity between two cells. Changing the voltage in one cell spreads to the adjoining cell. The gap between the cells is smaller than the gap involved in a chemical synapse. It has an acute bidirectional form of communication. The activity in the 2nd cell is les than the first. There is no amplication, mainly because of the effectiveness lost. The electrical synapse has spur excitatory or inhibitory effects. Distinguish presynaptic and postsynaptic mechanisms. We do know that presynaptic cells are involved in the synthesis, packaging, and release of neurotransmitter, and that postsynaptic cells exhibit the receptors for the neurotransmitter and gives off a consequent response in the binding of these neurotransmitters. We do know that these mechanisms involve several steps:

25

12/7/11 2:03:17 PM

Membrane  potential   depolarization  in  the   presynaptic  cell  (may   or  may  not  be  due  to   an  action  potential)  

Explain how the End Plate Potential (EPP) initiates an Action Potential to enable full activation of the muscle fiber. Response  may  be   Fusion  of   the unique characteristics of the NMJ that enables this to be the normal and expected response. r   Diffusion  of   Change  in  open/ Identify excitatory  o Change  in  ion   neutransmitter  (NT)-­‐ neurotransmitter   closed  state  of  ion   Change  in  membrane   inhibitory  depending   permeability  of  the   containing  vesicles   and  binding  to   channels  in  the   potential  in  the   postsynaptic  cell   on   eceptor  and   postsynaptic  cell   and  emptying  into   postsynaptic   postsynaptic  cell   Acetylcholine binds to its receptor, which ris channel   ion   a non-selective membrane   synaptic  cleft   receptors   membrane   cation channel. Once open, both sodium and potassium ions characteristics   flow down their concentration gradients (sodium in, potassium out), but since the inside of the muscle is negative, sodium flux is much greater (driven by both concentration and electrical gradients). Potassium outflow is ‘impeded’ by the negative charge inside the cell, ‘pulling the positive charge of potassium ions back in’. The end result is a local depolarization or End Plate Potential (EPP).

Excitation may involve increasing the sodium channels conductance in the postsynaptic cell or decreasing the potassium or chloride channels conductance the NMJ, i.e.,postsynaptic cell. in the the flow of The magnitude of the End Plate Potential reduces as it flows away from charged particles impeded by internal aspect of of the the potential decays with distance Inhibition may involve increasingis the conductancethe axon, andpotassium channel in the away from the neuromuscular junction. As the end plate potential moves away from the synaptic postsynaptic cell, or increasing thesize. This would not allow sufficient depolarization or activationthe postsynaptic cell. cleft, it decreases in chloride channel conductance in of the total muscle iber, especially if How do we do this? By manipulatingyou consider that many fibers are manythe cells manythese ions! the permeability of inches or even to feet in length.
Adjacent to the active zones of the NMJ, there are voltage-gated sodium and voltage-gated potassium channels. The end place potential magnitude in normal healthy muscle is still large enough when it reaches these to activate them in a sequential fashion, with sodium channels opening first. The resultant sodium influx depolarizes the cell (upstroke of the Action potential), followed by opening of the potassium channels, and the resultant outflow of potassium to repolarize the cell (downstroke of action potential).

Understand the functional role of glutamate at the rod synapse. Glutamate is going to be the neurotransmitter of focus at the retina. In the retina, rod photoreceptor synapses release glutamate. Some other neurotransmitter activate two types of receptors: ionotropic receptorsretrograde transmission from receptors. and metabotropic ‘normal’ anterograde transmission with respect to process and Differentiate
function. Type of Transmission Diagram Anterograde Retrograde

Distinguish ionotropic and metabotropic receptors. Ionotropic receptors contain a neurotransmitter Process Presynaptic neuron communicates with postsynaptic neuron Postsynaptic neuron communicates with or muscle. presynaptic neuron. binding site and an Function Events upstream can support, inhibit, suppress the function or Events downstream can support, inhibit, activity of the one downstream. suppress the function or activity of the one ion channel. It is upstream. simply known as an Several trophic factors are synthesized and released from muscle (and ion channel. Its function is a short-term switch that the activating thefrom the neuron), mainly BDNF and NT 4/5, by binding to tyrosine kinase receptors, ras-MAPK pathway to result in both acute and long-term effects. Acutely, this is done by phosphorylating exocytosis turns on or off the movementsuch asions. or syntagmin, which help mobilize the larger vesicles containing additional of synapsin Metabotropic proteins neurotrophic substances as well as increase acetylcholine release to strengthen synaptic receptors contain a neurotransmitter-binding site communication to ensure end plate potential magnitude is adequate and increase local depolarization coupled to a G protein thatlocal release of calcium from sarcoplasmic reticulum. In a chronic (long-term) state, the trophic and can relay signal to a neuronal for modulation of ongoing protein synthesis separate ion channel. factors are transported back to Increasingsomaproduction of neurotransmitters as well as theor transcription of new proteins. the associated
vesicles and microtubules does this for transport of same and improve neuromuscular junction structure with more integrated or altered shape.

Understand the functional role of vesicles and snare proteins at the synapse. The functional role of vesicles is to envelope the neurotransmitter move into the postsynaptic cell in the synapse. In the retina, the enzyme phosphate activated 14 glutaminase (PAG) converts glutamine to glutamate. Vesicular glutamate transports (vGLUTs) accumulate glutamate in vesicles. These transports are exchange transporters that shift glutamate inward and protons outward. The transporters are driven by the vesicle proton gradient. Remember that the interior is acidic relative to the exterior because of vesicular-ATPase. In photoreceptors the transporter is vGLUT1. The vGLUT is an exchange transporter that shifts glutamate into the vesicle and protons out from the vesicle to the cytoplasm. Vesicles (which are part of the vesicular secretory system) are acidic

26

because of the vesicular proton-ATPase pump. The proton gradient is essentially the driving force. The importance of the snare proteins at the synapse is to utilize the cytoskeleton to dock the vesicle and allow fusion with the membrane in order to secrete the neurotransmitter. V-SNARE (synaptobrevin and T-SNARES (syntaxin and SNAP-25) dock together forming a bone, which eventually allows easing of the vesicle to fuse with the membrane causing the secretion of the neurotransmitter.

Lecture XIV
Synapse II Know rod photoreceptors form a ribbon synapse with bipolar cells. Rod photoreceptors form a ribbon synapse with bipolar cells. Photoreceptors are unusual in the sense that neurotransmitter is not released in a burst driven. These photoreceptors have specialized ribbon synapses. Synaptic ribbons are electron-dense structures approximately 30-50 nanometers in diameter and 2 micrometers long. Synaptic vesicles containing glutamate surround them. They are proposed as a “conveyor belt” to channel synaptic vesicles to the site of release. The shape of this can change temporally, such as in night or in the day. Know difference between vGLUT transporters and EAATs. Remember in synaptic economics, the vesicles are recycled. The glutamate is recaptured by nearby glial cells and the presynaptic cell. This involves essentially the recapture mainly by presynaptic cells. Glutamate uptake occurs via excitatory amino acid transporters (EEATs). The EAAT is able to shift glutamate anion towards using the sodium electrochemical gradient. Protons are also moved inwards and potassium ions moved outwards. It is driven by the sodium gradient from sodium/potassium ATPase. There are several types of transporters within this family. There is a distinction between EAATs and vGLUTs that shift the glutamate into presynaptic vesicles. EAATs serve to terminate the excitatory signal by removal (uptake) of glutamate from the neuronal synapse into neuroglia and neurons. When glutamate is taken up into glial cells, they are converted to glutamine until transported to the presynaptic neuron, and taken up into synaptic vesicles the VGLUTs. Vesicular Glutamate Transporters (vGLUT) pack glutamate into vesicles to be released into the synapse. They are dependent on the proton gradient in the secretory system, and have much less affinity for glutamate relative to EAATs. Cell Glial Transporter GLAST/EAAT1 GLT-1/EAAT2 EAAC1/EAAT3 EAAT4 EAAT5 Location Retinal Muller Cells Cerebellar Bergmann Glia Astrocytes throughout brain Neuronal somata and dendrites Cerebellar Purkinje cells Retinal photoreceptors and bipolar cells

Neuronal

-­‐ -­‐ -­‐ -­‐ -­‐ -­‐

27

Know that mGluR is a G protein-coupled receptor. The transport mGluR is a G protein-coupled receptor. It is also know as a seven-helix receptor because of its seven transmembrane alpha helices. It uss trimeric GTP-binding proteins to relay signals to effector proteins inside the cells  GTP-binds to the Gα subunit which separates from G-βγ  Binding of ligand causes separation of the G subunit! Know why and how light leads to a hyperpolarizing response in OFF bipolar cells.
Less  glutamate  causes  fewer  cation   channels  to  be  in  open  state.   Hyperpolarization  of  bipolar  cell   Signal  relayed  onwards  is   INHIBITORY   Bipolar  cell  turns  OFF  

If the bipolar cell has ionotropic glutamate receptors, less glutamate causes fewer cation channels to be in an open state. The bipolar cell hyperpolarizes. The signal relayed onwards is inhibitory, and the bipolar cell is turned off. OFF bipolar cells have only ionotropic glutamate receptors. Cones connect to ON or OFF bipolar cells. Know why and how light leads to a depolarizing response in ON bipolar cells.
Heterotrimeric  G  proteins   reassemble.   In  the  dark,  one  of  the  G  proteins,  Go,   causes  a  nonselective  cation  channel   to  remain  closed.    In  the  light,  Go  is   absent  and  the  channel  opens.   The  bipolar  cell  switches  from  a   hyperpolarized  to  a  depolarized  state.   The  signal  relayed  onwards  is   excitatory.  

Human rod bipolar cells have metabotropic GLUR6 metabotropic glutamate receptors – less glutamate reduces receptor activation. Heterotrimeric G proteins reassemble. In the dark, one of the G proteins (Go) causes a nonselective cation channel to remain closed. In the light, Go is absent and the channel opens. The bipolar cell switches from a hyperpolarized to depolarized state. The signal relayed onwards is excitatory. This describes an ON bipolar cell. ON bipolar cells mainly have mGLUR6 metabotropic glutamate receptors (but also some ionotropic receptors too). Human rods connect to ON bipolar cells. Recognize terms “Center-Surround” in the context of a ganglion cell receptive field and know difference between ON-center and OFF-center. It is necessary to remember that the rod photoreceptors connect to ON bipolar cells and the cones connect to ON or OFF bipolar cells. Rod photoreceptors form a synapse with bipolar cells. Horizontal cells also may be part of the synapse. Activating a nearby area of the retina can alter the bipolar cell response because horizontal cells, which are stimulated at a distant point, release neurotransmitter that depolarizes the photoreceptor. Horizontal cells are lateral neurons in the retina. They permit one area of the retina to “compare” electrical signals from an adjacent area. If the spot of light reaches the center, it excites the ganglion and causes action potentials to increase. This is important because such firing allows us to see black and white details of depth and contrast in the periphery and more definition about color in the center. On center and off center retinal ganglion cells respond oppositely to light in the center and surround of their receptive fields. A strong response means high frequency of firing, while a weak response is firing at a low frequency. No response means that no action potential is generated.

28

Lecture XV
Recognize the role of the RPE in the Visual Cycle. Remember that the light transduction stars with photoabsorption by rhodopsin. The process of light absorption involves the stereochemical change of 11cis-retinal into all-trans-retinal (because it requires a LOT of energy to change the double bond). Alltrans-retinal is metabolized into all-trans-retinol and transported to the retinal pigment epithelium. In the retinal pigment epithelium, retinol is reisomerized to 11-cis-retinal and then redelivered to the photoreceptors. Here is a more detailed reaction progress (from slides): The visual cycle is a pathway of enzyme reactions responsible for the recycling of retinoids that are used during light detection in the photoreceptor cells. The activation of the photoreceptor pigment rhodopsin by light occurs through the isomerisation of the bound chromophore, 11-cis-retinal, to alltrans-retinal. While the phototransduction cascade continues, the all-trans-retinal is released from rhodopsin, reacts with the membrane lipid phosphatidyl-ethanolamine (PE) and is transported to the cytoplasm by ABCA4. After modification to all-trans-retinol by retinol dehydrogenase (RDH), it is transported to the RPE and trans-isomerised to 11-cisretinal through the actions of LRAT, RPE65 and 11-cis-RDH. After transport back to the photoreceptor cell, 11-cisretinal binds rhodopsin, rendering it sensitive to light. Retinoidbinding proteins IRBP, CRBP and CRALBP are involved in the transport of the hydrophobic retinoids in an aqueous environment. Interruption of the visual cycle by mutations in the RPE65 protein results in a lack of 11-cis-retinal to bind to Rhodopsin. In the absence of active visual pigment, vision is impaired. This has led towards efforts in nutritional strategies towards the limiting the symptoms of retinitis pigmentosa. Remember that retinitis pigmentosa encompasses several inherited diseases with the major symptoms are progressive retinal degeneration and night blindness in young adults. Visual examination may reveal bone spicules that appear on the fundus. Nutritionally, it involves vitamin A and docosahexaenoic acid (DHA). Why does this work? Retinal is just a form of vitamin A (along with retinol and retinoic acid depending on the redox state at the terminal C15). The precursor for this is β-carotene from ainly vegables. Understand in general terms the principles of gene therapy for retinal disease and the factors that make the gene therapy for the eye a reasonable objective. The substantive candidacy of gene therapy for retinal disease is mainly held in three main points: 1. It is an enclosed space for gene delivery. 2. Blood does not immediately disperse deposited meterials. 3. Retinal cells have little or no turnover. The strategy of the treatment involves addressing certain mutations. Some mutations can involve the loss of certain proteins necessary for the eye to function. One form of RP-like retinal disease is caused by a mutation that results in loss of RPE65. The following displays the considerable potential targets for gene therapy: Mutation’s Target proteins Consequence Affecting renewal and -­‐ Rhodopsin

29

shedding of the rod outer segment Affecting the visual transduction cascade Affecting retinal (vitamin A) metabolism

-­‐ -­‐ -­‐ -­‐ -­‐

Peripherin cGMP phosphodiesterase cGMP-gated cation channel (CNCG) Cellular retinaldehyde binding protein (CRalBP) RPE 65

There are several potential targets for gene replacement. Target Affected Proteins Defects in Visual Cycle -­‐ Lecithin retinol acetyltransferase RPE65 -­‐ ABC transporter A4 (ABCA4) -­‐ Retinal dehydrogenase 12 (RDH12) Phototransduction -­‐ Rod cGMP phosphodiesterase (PDE6B) Protein defects Cilium Protein Defects -­‐ Myosin VIIA (MYO7A) -­‐ RPGRIP -­‐ Retinitis pigmentosa GTPase (RPGR) Structural Protein -­‐ Peripherin (RDS/PRPH2) Defects Phagocytosis Defects -­‐ MERTK So far a new research trial will involve a receptor protein called MERTK that is expressed in the retinal pigment epithelium. Humans with the loss of MERTK function have a defect in phagocytosis. AS a result of this defect, debris accumujlates between the photoreceptors and retinal pigment epithelium, resulting in death of photoreceptors and loss of vision. The therapy will introduce a MERTK gene to the RPE.

30

Sign up to vote on this title
UsefulNot useful