CMMB

Midterm

Topic 1

Lecture 1

• Hook is credited with ‘discovering’ (visualizing) cells for the first time

• Observed cork under a microscope and compared the cells to monks’ dorm cells

Cell theory:

Schwann proposed the first two principles of cell theory:

1. All organisms are composed of one or more cells

2. The cell is a structural unit of life

Virchow proposed a third principle:

1. Cells can only arise by division from a preexisting cell

Since the discovery of DNA, a fourth principle has been added:

1. Cells contain genetic information in the form of DNA, and that information is heritable

• Cells are complex and organized

  • Highly ordered and consistent

• Cells contain genetic information

  • Cells store, use, and transmit genetic information

• Cells acquire and use energy

  • Almost all energy used by life is derived from the sun

• Cells carry out an array of different chemical reactions

  • Almost all chemical changes require enzymes to increase the reaction rate

• Cells are involved in mechanical activities

  • Transporting materials, movement of the whole cell ex

• Cells respond to stimuli

  • Most cells have receptors that allow them to interact with their environment

• Cells are capable of self regulation

  • Cells maintain homeostasis using a highly complex and organized set of molecular tools

• Cells evolve

  • All living organisms evolved from a single common ancestral cell (cells share many features including genetic code, membranes, ribosomes)

• Cells reproduce by division

• Prokaryote: bacteria

• Eukaryote: animals, fungi, plants, protists; have membrane bound organelles**

Covalent bond: a chemical bond in which electron pairs are shared between two atoms

• Number of covalent bonds that an atom can form depends on the number of electrons needed to fill its outermost (valence) shell

Polar molecules:

• Molecules with an uneven distribution of charge because the component atoms have different electronegativities

• Hydrophilic

Nonpolar molecules:

• Molecules whose covalent bonds have a nearly symmetric distribution of charge because the component atoms have approximately the same electronegativities

• Hydrophobic

Noncovalent bond:

• A relatively weak chemical bond based on attractive forces between oppositely charged regions

  Ionic bond (these are relatively weak in a cellular context, ex in the presence of water):

  • Electrostatic interaction that occurs between groups of opposite charges

  Hydrogen bond:

  • Electrostatic interaction between H atom and a second electronegative atom (ex H bonds between strands in the DNA double helix)

  Van der Waals interactions:

  • A weak attractive force due to temporary asymmetries of charge within adjacent atoms or molecules, distant dependent interaction (ex important when interacting proteins have complementary shapes)

Hydrophobic effect:

• The tendency of nonpolar molecules to aggregate in order to minimize their collective interaction with surrounding polar water molecules

• Basis for the formation of lipid bilayer membrane

Lecture 2

• In a eukaryotic cell, the plasma membrane (surrounding the outside of the cell), and organelle membranes are composed of a lipid bilayer

• Cell membranes: phospholipid bilayer

  • Membrane contains a bilayer of phospholipids

  • Polar phosphates face the membrane surfaces

  • Nonpolar fatty acid tails face into the interior of the membrane

  • Phospholipids are amphipathic (hydrophobic component and hydrophilic component)

  • Lipid bilayer prevents random movement of substances in and out of the cell (selective barrier)

• Fatty acids have long, unbranched hydrocarbon chains

• Fatty acids in cells typically have 14-20 carbons

• Fatty acids can be saturated or unsaturated

• Saturated lack double bonds

• Unsaturated:

  • One or more double bonds

  • Introduce a bend into the fatty acid tail

  • Naturally occurring fatty acids have cis double bonds (cis introduces more of a bend into the fatty acid tail compared to trans)

• Membrane lipids:

  • Phosphoglycerides: Are phospholipids

    • Most membrane phospholipids are phosphoglycerides

    • Built on a glycerol

    • Glycerol + 2 fatty acid chains + phosphate group + additional group

    • Often contain one unsaturated and one saturated fatty acid chain

    • Phosphate is negatively charged

    • Overall charge of head groups at physiologic pH:

      • Phosphatidic acid (PA) <— H (Negative)

      • Phosphatidylcholine (PC) <— Choline (Neutral)

      • Phosphatidyl serine (PS) <— Serine (Negative)

      • Phosphatidyl ethanolamine (PE) <— Ethanolamine (Neutral)

      • Phosphatidylinositol (PI) <— Inositol (Negative)

  • Sphingolipids: Some sphingolipids are phospholipids

    • Less abundant membrane lipid

    • Built on sphingosine

    • Example: ceramide is sphingosine + fatty acid

    • Amphipathic

    • Additional groups can be linked to the head group, fatty acid chain can be added to R group (ex phosphorylcholine addition makes sphingomyelin which is a sphingolipid and phospholipid)

    • Tend to have longer and more highly saturated fatty acid chains than phosphoglycerides

    • Roles in signal transduction, membrane structure

  • Cholesterol:

    • Makes up part of the plasma membrane lipids in some animal cells

    • Amphipathic

    • Oriented with the small hydrophilic group facing the membrane surfaces

    • Remainder is embedded in the fatty acid tails of the phospholipids

    • Impairs the movement of the fatty acid tails of the phospholipids

• Membrane Lipid Asymmetry:

  • Asymmetry affects membrane permeability, surface charge, membrane shape and stability

  • Ex PE promotes curvature of the membrane (is ‘cone-shaped’ due to its small head group), PS negative charge interacts with transmembrane proteins, PI has roles in signal transduction

• Membrane Carbohydrates:

  • 10% of membrane carbohydrates are covalently linked to lipids (glycolipids)

  • 90% of membrane carbohydrates are covalently linked to proteins (glycoproteins)

  • All membrane carbohydrates in the plasma membrane face the extracellular space

  • Carbohydrates attached to glycolipids and glycoproteins can have very diverse structures

  • Carbohydrates play important roles as receptors, in sorting membrane proteins and in cell recognition (ex blood group antigens)

• Membrane function:

  • Myelin sheath is composed of multiple layers of plasma membrane, with very little protein

  • Lipid composition can determine the physical state of the membrane, facilitate protein interactions, roles in signal transduction

• Overview of major membrane functions:

  1. Compartmentalization: Membrane compartments in the cell allow specialized activities to occur without impacting one another

  2. Scaffold for biochemical activities: Membrane helps keeps proteins organized and in the right spot so the right reactions can occur in the correct order

  3. Selectively permeable barrier: Prevent the random movement of substances in and out of the cell, but do allow select substances in and out

  4. Solute transport: Specialized machinery, protein channels, pumps that allow solutes to transport in and out

  5. Response to external stimuli: Outer membrane interacts with the environment using receptors that allow it to interact

  6. Cell-cell communication: Cells can recognize and communicate with each other, exchange substances with each other

  7. Energy transduction: Light energy is transformed into chemical energy, mitochondria and ATP ex

Lecture 3

• Increasing the percentage of saturated fatty acids in a membrane decreases the membrane’s fluidity

• Fluidity (viscosity) determines the physical state of the membrane

• Influenced by temperature

• Transition temperature/Melting temperature: Below this temperature the membrane is in a crystalline gel (more solid state), above this temperature the membrane is in a liquid crystalline phase (relatively fluid state)

  • Our cells exist slightly above the transition temperature

• Transition temperature (and thus fluidity) is affected by:

  1. Fatty acid chain saturation:

     • Saturated fatty acids: straight, flexible rod

     • Cis-unsaturated fatty acids: bends at the sites of double bond = increase membrane fluidity

  2. Cholesterol content:

     • Flat, rigid, hydrophobic rings impair the movement of the phospholipid fatty acid tails

     • Eliminates a sharp transition temperature: creates intermediate fluidity

     • Refer to lecture 3 pg 8 graph on D2L: cholesterol acts as a buffer

       

  3. Fatty acid chain length:

     • Shorter fatty acid chains: fewer interactions (van der Waals) = less energy required to break them apart

• A balance of membrane fluidity/rigidity is important for:

  • Maintaining structural organization and mechanical support

  • Enabling interactions (clusters of proteins)

  • Membrane assembly/cell growth/cell division

  • Cell movement, secretion, and endocytosis

• Some cells (ex some fish, plants, bacteria) can alter their lipid composition in response to changing environmental conditions

• In response to colder temperatures:

  1. Desaturated single bonds in fatty acid chains to double bonds

     • Enzyme: desaturase

  2. Change the types of phospholipids that it synthesizes

     • Synthesize more fatty acids with unsaturated bonds and shorter chain lengths

• These two steps decrease transition temperature

  • In response to higher temperatures, the opposite would be true (in order to maintain a balance of membrane fluidity and rigidity)

• Semi-aquatic mammals living in colder latitudes have increased desaturation of their fatty acids

• A membrane may contain hundreds of different proteins

• Proteins are distributed asymmetrically across the two leaflets of the membrane bilayer

• Three classes of membrane proteins:

  • Integral (transmembrane) proteins

  • Peripheral proteins

  • Lipid-anchored proteins

• Integral membrane proteins are usually transmembrane proteins:

  • Contain transmembrane domains

    • Pass through the lipid bilayer once (bitopic) or multiple times (polytopic)

  • Act as receptors, channels, or roles in electron transport

• Transmembrane proteins are amphipathic

  • Transmembrane domains tend to be hydrophobic (form van der Waals interactions with the fatty acids in the bilayer)

  • Portions of the protein at the surfaces tend to be hydrophilic

• Glycophorin A: bitopic membrane protein found in the red blood cells

• Identifying transmembrane domains:

• Peripheral membrane proteins:

  • Associated to the membrane by weak non-covalent bonds ex ionic interactions, hydrogen bonds

  • Some peripheral membrane proteins interact with integral membrane proteins

  • Dynamic: can be recruited to/released from the membrane

  • Roles in signal transduction, mechanical support for the membrane, enzymes

  • Mostly hydrophilic

• Ex: Red blood cell peripheral membrane proteins:

  • Network of proteins that give the cell its shape

  • Major component: spectrin

    • On the internal surface of the membrane

    • Gives the cell shape, flexibility

• Lipid anchored proteins:

  • Covalently linked to a lipid molecule in the bilayer

  1. GPI anchored proteins:

     • Proteins attached to the membrane by a small, complex oligosaccharide linked to PI in the membrane

     • Glycosyl-Phosphatidyllnositol linkage

     • Outer leaflet

     • Roles in cell adhesion, and as receptors

  2. Hydrocarbon chains embedded in the lipid bilayer

     • Usually on the cytoplasmic leaflet

     • Roles in signal transduction

Lecture 4

Phospholipid dynamics:

• Phospholipids can easily move laterally within the same leaflet

• Phospholipids ‘flip-flopping’ to the other leaflet is restricted (transverse diffusion)

  • Why is this thermodynamically unfavourable? Polar head groups of the phospholipid need to pass through the non-polar fatty acid tails

• Enzyme flippase can play a role in establishing membrane asymmetry

Membrane protein dynamics:

1. Random diffusion

2. Immobilized (no movement)

3. Particular direction (motor proteins)

4. Restricted by other integral membrane proteins

5. Restricted by membrane skeleton proteins

6. Restrained by extracellular materials

• Membranes are selectively permeable barriers: Allow the passage of some substances but inhibit the passage of others

• Passive transport:

  • Does not require energy input from the cell

  • Occurs by diffusion (movement from a region of high concentration to low concentration)

• Active transport: Does require energy input, can move substances against a concentration gradient

• Diffusion: The spontaneous process in which a substance moves from an area of higher concentration to one of lower concentration, eventually reaching the same concentration in all areas (equilibrium)

  • The difference in the concentration of a substance between two areas is called the concentration gradient

• Can penetrate the lipid bilayer:

  • Small inorganic solutes, such as O2/CO2/H2O

  • Solutes with high lipid solubility

• Cannot penetrate the lipid bilayer:

  • Ions and polar organic solutes (ex sugars and amino acids)

  • Anything too large

• Osmosis (passive transport): Water moves through a membrane from region of lower solute concentration to a region of higher solute concentration

  • Hypertonic solution: Higher solute concentration outside of the cell

  • Hypotonic solution: Lower solute concentration outside the cell

  • Isotonic solution: Equal solute concentration

• Aquaporin (passive transport):

  • Channel proteins that facilitate the transport of water

  • Allows cells to be more permeable to water than is possible by diffusion through the bilayer

• Ion channels:

  • A transmembrane structure permeable to a specific ion or ions (ex Na, K, Ca, Cl)

  • Most are highly selective

  • Most ion channels are gated: change conformation to be open or closed

• Simple diffusion through a channel — types of ion channels:

  • Voltage-gated channel:

    • Open/closed depends on the difference in ionic charge on either side of the membrane

    • Voltage: difference in charge between two compartments

  • Ligand-gated channel:

    • Open/closed depends on the binding of a specific molecule (a ligand)

    • The ligand is usually not that solute that is passing through the channel

    • Ex neurotransmitter (like acetylcholine) is a ligand that binds to ion channels

  • Mechano-gated channel:

    • Open/closed depends on mechanical forces

    • Ex stretching

• Voltage-gated K+ ion channels:

  • Significant impacts on electrical properties of the membrane:

    • Important for transmitting electrical impulses along axons

    • More than 10 million K+ ions can pass through the channel per second

  • Regulation: Different K+ channels open and close in response to different voltages

• Case study: Use of the illegal drug: 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy), has been associated with many serious medical complications including brain edema (water leaves the blood and enters
  brain cells, causing brain swelling). MDMA has two effects on the body that together can lead to brain edema. Which two?

  • Effect on blood sodium: Decreases blood sodium concentration

  • Effect on hydration: Increases thirst

  • Treatment: Increasing the solute concentration of the blood

    

Facilitative transporter:

• Binding of the solute triggers a conformational change in the transmembrane protein that exposes the solute to the other side

• Exhibit saturation-type kinetics: when the concentration is high, the rate of transport levels off. This is a difference from ion channels:

  • Ion channels: millions of ions/sec

  • Facilitative transporters: 100-1000s of molecules/sec

• Glucose transporter GLUT4 is an example of a facilitative transporter

  • Continued diffusion of glucose in the cell is possible because it becomes phosphorylated and metabolized in the cell

Lecture 5

Channel vs facilitative transporter:

• Channel: Smaller conformational change, open/closed in response to ligand/voltage/mechano

• Facilitative transporter: Larger conformational change, binding of the solute

Active transport:

• Active transport is required to create these steep concentration gradients across the plasma membrane

• Selective transmembrane protein

• Protein undergoes a change in conformation

• Requires energy input

  • Ex hydrolysis of ATP (primary active transport)

  • Ex flow of other substances down their concentration gradient (secondary active transport)

• Primary active transport:

  • Three types: P-type pump, V-type pump, ABC transporter

  • P-type pump:

    • Na+/K+-ATPase is a P-type ion pump

      • ATPase, during active transport becomes phosphorylated

      • Contributes to maintaining the membrane potential (voltage) in cells

      • Per ATP: 3 Na+ pumped OUT, 2K+ pumped INTO the cell

      • Defects in the N+/K+ pump can cause impacts on the endocrine system, hypertension, neuromuscular disorders, seizures, others

    • Step 1:

      • E1 conformation: ion binding sites are accessible on the inside of the cell

      • High affinity for sodium ions

      • ATP is bound

    • Step 2:

      • When ions are bound, the protein closes (occluded E1 state)

    • Step 2-3:

      • Hydrolysis of ATP

      • Pump is phosphorylated

    • Step 3-4:

      • Release of ADP and conformation change to E2

      • Ion binding sites are accessible to the extracellular component

      • Loses affinity for Na+ ions, high affinity for K+ ions

    • Step 5-6:

      • When ions are bound, the protein closes (occluded E2 state)

      • Dephosphorylation

    • Step 7-8:

      • Dephosphorylation returns the protein to E1 conformation

      • Low affinity for K+ ions

      • Due to complex conformational changes, rate of transport is much slower than transport through ion channels (by several orders of magnitude)

  • V-type ion pumps:

    • Utilize ATP energy without becoming phosphorylated themselves

    • Transport H ions across organelles and vacuoles (ex maintain the low pH of lysosomes)

    • Also found in the plasma membrane of some cells (ex roles in maintaining acid-base balance in kidney tubules)

  • ABC transporters:

    • ATP-binding cassette transporters

    • Share a similar structure of ATP-binding domain

    • Mammalian ABC transporters transport ions, lipid, peptides, nucleosides, drugs ex

• Ion gradients are a way to store energy in a cell

• A concentration gradient is a form of stored (potential) energy

• Symporters:

  • Transports two substances in the same direction

  • Also called cotransporter

• Antiporters:

  • Transports two substances in the opposite directions

  • Also called exchanger

• One of the substances is moving along (with) its concentration gradient, providing the energy to move the other substance against its concentration gradient

Na+/glucose cotransporter:

• Transport glucose from the intestinal lumen into epithelial cells

• Na+ ions concentration is low inside cells

• Na+ ions moving down their concentration gradient is used to drive the cotransport of glucose

• Transporting glucose against its concentration gradient

• Primary active transport—Na+/K+ pump establishes the Na+ concentration gradient

Lecture 6

Study protein movements and dynamics within membranes:

• Ex: fluorescence recovery after photobleaching (FRAP)

• Can be used to study membrane dynamics in vivo within the living

Study isolated membrane proteins:

• Ex examine protein size or expression levels (gel electrophoresis)

• In vitro studies within the glass

• Performed outside of their normal biological context

• Need to isolate the protein from the membrane before you can study it

Fluorescence recovery after photobleaching (FRAP):

• Technique to study movement of membrane components (proteins or lipids)

• Step 1: Label membrane component with a fluorescent dye

  • Ex fluorescent antibody that recognizes a particular protein

• Step 2: Photobleach (remove fluorescence) from a portion of the cell

  • ~1um diameter

• Step 3: Monitor reappearance of fluorescence in the previous bleached portion

  • Rate of recovery of fluorescence is a measure of the rate of diffusion of the fluorescently-labeled protein

Isolating membrane proteins - Lyse the cells:

• Step 1: Lyse the cells and collect the plasma membrane

  • Mechanical disruption, freeze/thawing or hypotonic solution

  • Centrifuge the sample to separate into two fractions:

    • Pellet 1: insoluble

    • Supernatant 1: soluble

  • Membranes and membrane-associated proteins are in the pellet

• Step 2: Isolate peripheral membrane proteins using high salt

  • Remember: Peripheral membrane proteins are associated with the membrane through hydrogen bonds and ionic interactions

  • Ions from salt will compete with the charged amino acids of peripheral membrane proteins to disrupt the noncovalent interactions with the membrane

  • Peripheral proteins are released from the membrane

    • Pellet 2: insoluble

    • Supernatant 2: soluble

  • Peripheral membrane proteins are in the supernatant 2

  • Transmembrane proteins are in the pellet 2

• Step 3: Isolate transmembrane proteins with strong detergents

  • Amphipathic: polar end and nonpolar hydrocarbon chain (detergent)

  • Ionic detergents are harsher than non-ionic

  • Remember: transmembrane proteins are embedded in the membrane and interact with lipids by van der Waals interactions

  • Detergents can substitute for phospholipids to stabilize transmembrane proteins and make them soluble in aqueous solution

  • Pellet 3: insoluble

  • Supernatant 3: soluble

  • Transmembrane proteins are in the supernatant 3

  • GPI-anchored lipid proteins are in the pellet 3

• Step 4: Isolate GPI-anchored proteins by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC)

  • GPI-anchored proteins are linked to phosphatidylinositol in the membrane

  • GPI-anchored proteins are usually found in detergent-resistant portions of the membranes that are rich in cholesterol and sphingolipids

  • Pellet 4: insoluble materials

  • Supernatant 4: soluble materials

  • GPI-anchored proteins are found in the supernatant 4

• Overview: A small aliquot of each pellet and supernatant is kept for separate analysis

• Electrophoresis: Separation of charged molecules by migration through an electric field

• Polyacrylamide gel electrophoresis (PAGE): Proteins migrate through a gel matrix made of cross-linked acrylamide polymers

• Before we analyze the proteins’ migration through an acrylamide gel, we need to denature the proteins

Characterization of proteins by SDS-page:

• In order to separate proteins based only on mass # of amino acids, we add SDS to the samples

• SDS is a negatively charged amphipathic detergent:

  • Gives proteins a uniform negative charge

  • Denatures the protein (disrupts protein folding)

  • Repulsion between bound SDS molecules breaks noncovalent bonds (hydrogen, ionic)

  • Protein is unfolded from its native 3D structure

• Protein is mixed with tracking dye and loaded on the polyacrylamide gel

• Electrical current is applied and proteins migrate through the gel to the positive end

• SDS-PAGE: polyacrylamide gel electrophoresis (using SDS to denature the proteins so that they are separated by mass)

• To visualize all proteins in the gel, we can stain with Coomassie blue dye

• To help estimate the size of proteins, we load a protein mixture called a molecular weight marker into the first lane (kDa: kilodalton; a unit often used to measure protein mass, 1kda is ~9 amino acids)

  • We can determine: approximate size of a protein and protein concentration (expression) by analyzing band intensity

  • Thicker bands indicate there is more protein present

Lecture 7

Structure of a neuron:

• All organisms respond to external stimulation

• Neurons (nerve cells) are specialized for communication with other cells in the form of electrical impulses

• In vertebrates, most neurons are part of the central nervous system

• Dendrites receive information

• Axon conducts outgoing information

• Terminal knobs are where impulses are transmitted to the target cell

• Myelin sheath wraps most vertebrate axons

• Nucleus is found in the cell body

Resting potential:

• Membrane potential (membrane voltage): Difference in charge across a membrane

• Resting potential: The membrane potential when a nerve cell is in an unexcited state

• Resting potential of neuron is -70mV

• Negative voltage: inside of cell is negative compared to the outside

• What contributes to the difference in charge across the membrane?

  1. Na+/K+ ATPase pump pumps 3Na+ ions out per 2 K+ ions pumped in

  2. K+ ions are the charged substance with the most permeability in a resting nerve cell

     • Flow out through potassium leak channels (following their concentration gradient, not gated, out)

• Equilibrium: balance is reached between the concentration gradient favouring K+ leaving the cell and the electrical gradient favouring K+ staying in the cell

• Action potential: Changes in membrane potential after a stimulus and is the basis for neural communication

  • Includes depolarization and repolarization phases

  • Takes 5ms in a squid axon

• Depolarization: A decrease in the electrical potential difference across a membrane, more positive

  • A stimulus (ex capsaicin in chili peppers) activates a gated channel, allowing sodium to diffuse in

• If the stimulus results in depolarization above a threshold of -50mv, then voltage-gated sodium channels open

  • If this is reached, an action potential is triggered

• The increased permeability to Na+ ions results in a membrane potential of about 40mV

• Sodium channels spontaneously close after ~1ms

• Repolarization: The depolarization (less negative voltage) triggers the opening of voltage-gated potassium channels

  • Sodium gated channels close

  • Membrane potential goes back to negative (-80mV)

  • Large negative membrane potential causes the voltage-gated potassium channels to close

• Hyperpolarization: Because potassium channels are slow to close (slight dip in the graph at the end)

Propagation of an action potential:

• Nerve impulse: An action potential is propagated along a neuron by triggering action potentials in adjacent portions of the membrane

• Continuous conduction: Occurs in unmyelinated axons

  • Flow of current causes the membrane in the region just ahead to become depolarized

  • The action potential is propagated without any loss in intensity

  • Portion of the membrane that just experienced the action potential will be in a brief refractory period, can only go in one direction (Na+ channels can’t reopen for a few milliseconds after they’ve been activated)

• Saltatory conduction: Occurs in myelinated axons

  • Impulses in myelinated axons are 20x faster than in an unmyelinated axon

  • Myelin prevents the passage of ions across the membrane

  • Most Na+ and K+ channels are found in or near unmyelinated regions called: nodes of Ranvier

  • Action potential at node of Ranvier triggers an action potential at the next node

Lecture 8 (Part 1)

Synaptic transmission:

• Synapse: the specialized junction of a neuron with its target cell

• Presynaptic cell: conducts the impulse towards a synapse (ex neuron)

• Synaptic vesicles: storage for neurotransmitters in the terminal knobs of axons

• Neurotransmitters: chemicals that bind to the postsynaptic cell, transmit signal across the synaptic cleft (red dots)

• Synaptic cleft: space that separates the two cells

• Postsynaptic cell: receives the impulse (ex another neuron, or muscle)

• Turquoise channels below are ion channels

• Depolarization causes voltage gated calcium channels to open in the presynaptic cell

• Calcium diffuses into the cell

• Increased Ca2+ in the cell triggers synaptic vesicles to fuse with the plasma membrane, releasing neurotransmitters (ex acetylcholine) which bind selectively to receptors

• Neurotransmitter is acting as a ligand to open these ion channels, yellow channels below are ligand gated ion channels

a) Influx of positive ions (ex Na+) ‘excites’ the postsynaptic cell (more likely to generate an action potential): depolarized (more positive)

• Nerve impulse may be generated

b) Influx of negative cells (ex Cl-) ions: ‘inhibits’ the postsynaptic cell: hyperpolarization

• Harder for a nerve impulse to be generated

After being released, neurotransmitters have a very short half life

• Enzymes destroy the neurotransmitter in the synaptic cleft (ex acetylcholinesterase hydrolyzes acetylcholine)

• Reuptake of neurotransmitter into the presynaptic cell

• Drugs that interfere with neurotransmitters can have physiological and behavioural effects

  • Ex antidepressants inhibit reuptake of serotonin, cocaine interferes with reuptake of dopamine

Topic 2

Lecture 8 (Part 2)

Endomembrane system

• Cytoplasmic membrane system

• Composed of the cytoplasmic membranes (eukaryotic cells)

• Functionally and structurally interrelated group of membranous cytoplasmic organelles including:

  • Endoplasmic reticulum (ER)

  • Golgi complex

  • Endosomes

  • Lysosomes

  • Vacuoles

• Endomembrane system is a dynamic, integrated network

1. Transport materials from donor compartment to recipient compartment

   • Membrane-bound vesicles shuttle materials between organelles

   • Vesicles bud from the donor compartment

   • Transport in a directional manner with the help of motor proteins and the cytoskeleton network

   • Vesicles fuse with the membrane of the recipient compartment

     • Cargo is released in the destination compartment

     • Vesicle membrane becomes a part of the recipient compartment’s membrane

     • ‘Escaped"‘ resident proteins of the donor compartment can be returned

   • Proteins (secreted proteins, lysosomal enzymes, membrane proteins ex) are directed to the correct destination with sorting signals:

     • Amino acid sequence (that makes up part of the protein)

     • Attached oligosaccharides

   • Signals are recognized by receptors in the membranes of budding vesicles

2. Transport materials out of the cell (Secretory pathway)

   • Examples of biomolecules synthesized in the ER (smooth or rough)

     • Lipids/cholesterol

     • Steroid hormones

     • Secreted proteins

     • Integral membrane proteins

     • Initial glycosolyation of proteins

   • Further modifications occur in the Golgi complex

   • Constitutive or regulated secretion

   • Constitutive secretion:

     • Most cells

     • Materials are continually transported in secretory vesicles from their site of synthesis and secreted

     • Contributes to the formation of the plasma membrane

   • Regulated secretion:

     • Materials are stored in membrane-bound compartments and only released in response to particular stimuli

     • Ex: endocrine cells that release hormones, pancreatic acinar cells that release digestive enzymes, nerve cells that release neurotransmitters

3. Transport materials into the cell (Endocytic pathway)

   • Materials move from the outer surface of the cell to compartments within the cell (endosomes and lysosomes)

   • Endosome:

     • Materials that are taken up are transported to early endosomes for sorting

     • Late endosomes are more acidic than early endosomes

     • Fuse with lysosomes to deliver cargo for degradation

   • Lysosome:

     • Hydrolytic (digestive) enzymes and acidic pH

     • Roles in breakdown of material and organelle turnover

Lecture 9

Autoradiography:

• Following the location of radioactively-labeled materials in a cell

• In particular: pulse-chase experiment can be used to examine a process that takes place over time

• Step 1 Pulse:

  • Radio-labelled amino acids are incorporated in the digestive enzymes being synthesized

    • Exposed to the radio-labelled amino acids for only a short time

• Step 2 Chase:

  • Transfer cells to media with only unlabelled amino acids

    • Enzymes synthesized during this time will not be radio-labeled

Endoplasmic reticulum (ER): a system of membranes and vesicles that encloses the ER lumen (separated from the cytosol)

• Divided into smooth and rough

Rough ER:

• Has ribosomes bound on the cytosolic membrane surface

• Composed of a network of cisternae

• Continuous with the outer membrane of the nuclear envelope

• Extensive in cells with a role in protein secretion

• Ex:

  • Pancreatic acinar cells that secrete hydrolytic enzymes

  • Intestinal cells that secrete mucuproteins

  • Endocrine cells that secrete polypeptide hormones

• Functions:

  • Protein synthesis

  • Addition of sugars is initiated

Smooth ER:

• Lacks ribosomes

• Composed of interconnected curved, tubular membranes

• Continuous with the RER

• Extensive in cell types such as skeletal muscles, kidney tubules, and steroid producing endocrine glands

• Functions include:

  • Synthesis of steroid hormones

  • Synthesis of membrane lipids

  • Detoxification of organic compounds in the liver

  • Sequestering calcium ions in skeletal and cardiac muscle — role in muscle contraction (sarcoplasmic reticulum)

Sites of protein synthesis:

• Free ribosomes:

  • 2/3 of proteins

  • Ribosomes that are not attached to the ER

  • Proteins are released into the cytosol

  • Proteins that remain in the cytosol

  • Peripheral proteins of the cytosolic surface of membranes

  • Proteins transported to the nucleus, mitochondria and chloroplast

• RER ribosomes:

  • 1/3 of proteins

  • Co-translational translocation: Peptides move into the lumen of the ER as it is being synthesized by the ribosome

  • Secreted proteins

  • Integral membrane proteins and soluble proteins that reside in the compartments of the endomembrane system

  • Integral membrane proteins in the plasma membrane

• Both sets of ribosomes are structurally and functionally identical. Their location differs

Co-translation translocation - Synthesis of secreted proteins and soluble proteins that reside in the endomembrane compartments:

• All protein synthesis begins on a free ribosome

• Signal sequence at N-terminal end: 6-15 hydrophobic amino acids

• Signal recognition particle (SRP) binds to the signal sequence and the ribosome

• Polypeptide synthesis is halted temporarily

• SRP directs this complex to the ER membrane by interaction with the SRP receptor

• Ribosome/polypeptide are transferred from the SRP to the translocon: protein pore in the ER membrane

• Contact with the signal sequence displaces the plug

• Translocation through the pore: polypeptide enters the ER lumen

• Upon termination, ribosome is released

• Signal sequence is removed by an enzyme: signal peptidase

• Protein chaperones (ex BiP) aid in protein folding

Co-translation translocation - Synthesis of integral membrane proteins:

• Synthesized by co-translational translocation using the same machinery as secreted proteins (SRP, receptor ex)

• SRP recognizes the hydrophobic transmembrane domain as the signal sequence

• Transmembrane domains do not pass through the pore - instead, they directly enter the lipid bilayer

• As polypeptides pass through the translocon, a gate in the pore opens and allows proteins to partition themselves according to their solubility properties

  • Either in the aqueous pore in the hydrophobic lipid bilayer

• Arginine (R, arg), Lysine (K, lys), Histidine (H, his) are the positively charged amino acids*** (must know)

• Direction of insertion into the bilayer is dependent on the location of the positively charged amino acids relative to the transmembrane domain

• Cytoplasmic leaflet is more abundant with PS and PI phospholipids: negatively charged

• The protein will orient in the membrane such that the positively charged amino acids interact with the relatively negatively charged cytosolic leaflet

• If the positive charges are on the N-terminal side of the transmembrane domain, the translocon will reorient the transmembrane domain

Lecture 10

Glycosylation:

• Majority of proteins produced at the RER become glycosylated (glycoproteins)

• Carbohydrate groups have roles as binding sites

• Aid in proper folding and stabilization

• Sorting/directing proteins to different cellular compartments

• N-linked glycosylation (common)

  • Linkage to asparagine

  • Is initiated in the RER

• O-linked glycosylation

  • Linkage to serine or threonine

  • Occurs in the golgi complex

N-linked glycosylation in the rough ER:

• First seven sugars are transferred one at a time to a lipid: dolichol pyrophosphate, embedded in the ER membrane

• Initial assembly is on the cytosolic side

• Sugars are added by glycosyltransferases

• Dolichol and attached oligosaccharide is flipped across the membrane

• Remaining sugars are attached to dolichol on the cytosolic side

• Flipped across the membrane and attached to the growing oligosaccharide chain

• Completed oligosaccharide is transferred to an asparagine residue of the polypeptide being translated

• Transfer by the enzyme oligosaccharyltransferase to an Asn within the sequence: Asn-X-Ser/Thr (X is not proline)

Quality control for misfolded proteins:

1. Glucosidase I and II remove two glucoses

2. (and 5). Glycoprotein with one glucose is recognized by calpexin (chaperone protein in the ER)

3. Removal of glucose releases protein from chaperon (calpexin releases)

4. Incompletely folded proteins are recognized by UGGT (a conformation sensing enzyme): detects exposed hydrophobic residues. Adds glucose molecule

5. Properly folded proteins exit (step 6 not 5)

6. Improperly folded proteins are degraded in a proteosome in the cytosol (steps 7-8 not 6)

Exiting the ER:

1. Membrane vesicles with enclosed cargo (protein) bud from the ER and travel in the direction of the Golgi

2. Transport vesicles fuse with one another to form larger vesicles in a region called the ERGIC: Endoplasmic Reticulum Golgi Intermediate Compartment

Golgi Complex:

• Golgi compled is composed of cisternae arranged in a stack

• Distinct compartments arranged from the cis face (closest to the ER) to the trans face (exit, furthest from the ER)

• Trans Golgi network (TGN): network of tubules and vesicles

  • Sorting station where proteins are segregated into different types of vesicles (heading to the plasma membrane or other)

• Cis Golgi network (CGN): Interconnected network of tubules

  • Sorted station that distinguishes between proteins that need to be returned to the ER and those that should proceed through the Golgi

Protein modification with the Golgi complex:

• Newly synthesized proteins leaving the ER are sequentially modified

• Ex: modification of N-linked carbohydrate chains

  • Order that sugars are incorporated depends on the location of specific glycosyltransferases (integral membrane proteins in the membrane of the Golgi complex)

  • Glycosylation in the golgi complex can be quite varied

  • O-linked carbohydrates are entirely assembled within the golgi

Lecture 11

Movement of materials through the Golgi complex:

• Model 1: vesicular transport model

  • Golgi cisternae are stable compartments

  • Vesicles carrying cargo bud from one compartment and fuse with the next

  • Evidence:

    • Golgi cisternae have different enzymes

    • Lots of vesicles bud from the edges of Golgi cisternae

• Model 2: cisternal maturation model

  • Cisternae form at the cis face and move towards the trans face, ‘maturing’ as they move

  • Evidence:

    • Drugs blocking vesicle formation at the ER leads to the Golgi complex disappearing

    • Certain large materials (ex collagen) move from cis to trans without ever appearing in smaller vesicles

Current model of vesicle transport:

• Cisternal maturation model of transport through the Golgi complex

• Anterograde transport (forward): from cis to trans

• Retrograde transport (backward): from trans to cis, resident Golgi and ER enzymes

Types of coated vesicles:

• COPII-coated vesicles: Move cargo forward (ER to Golgi complex)

  • These are not required for movement from the cis to trans Golgi

• COPI-coated vesicles: Move cargo backward, from ERGIC/Golgi to ER, from trans to cis Golgi

• Clathrin-coated vesicles: Move materials from the TGN to endosomes, lysosomes, plant vacuoles; also endocytosis

COPII-coated vesicles:

• COPII select and concentrate certain proteins for transport in vesicles: (by interacting with transmembrane proteins that have ‘ER export signals’)

  1. Enzymes destined for the Golgi complex (ex glycosyltransferase)

  2. Proteins involved in vesicle docking and fusion

  3. Protein receptors that bind soluble cargo

1. Sar1 is a COPII coat protein. G protein (molecular switch).

   • Sar1-GDP is recruited by GEF (guanine exchange factor)

2. Sar1-GTP undergoes conformational change so that it inserts into cytoplasmic leaflet (this starts to bend the membrane)

3. Sec23/Sec24 dimer further bends the membrane

   • Sec24 is the primary adaptor protein that interacts with membrane proteins (that have ER export signals)

   • Sec13/Sec31 form an outer structural cage

4. Disassembly is triggered by hydrolysis of GTP bound to Sar1

• Know the names of Sar1 and Sec24***

COPI-coated vesicles:

• COPI coat is made up of a protein complex called coatamer, which forms a thick protein coat directly on the membrane

• Membrane-bending G protein: Arf1 (GTP form bends membrane)

• Retrograde transport of proteins:

  1. Golgi resident enzymes

  2. ER resident proteins (escaped)

• Proteins that reside in the ER contain a retrieval signal

• Soluble ER proteins usually contain the signal: lys-asp-glu-leu (KDEL)

  • Recognized by a KDEL receptor (shuttle between cis Golgi and ER compartments)

• Membrane ER proteins also have a retrieval signal, usually: lys-lys-X-X (X is any amino acid KKXX)

  • KKXX retrieval signal is located on the cytosolic side so it can interact with COPI-coated recycling vesicle

• Each compartment in the endomembrane system may have its own retrieval signal

Vesicle fusion:

• Specific interactions between different membranes

1. Movement of the vesicle toward the specific target compartment

   • Movement mediated by microtubules and motor proteins

2. Tethering vesicles to the target compartment

   • Two types of tethering proteins:

     • Rod-shaped/fibrous (longer)

     • Multiprotein complex (closer)

   • G proteins called Rabs (60+ in humans) help to determine specificity

     • Rabs recruit specific tethering proteins

     • Rabs also interact with motor proteins

3. Docking vesicles to the target compartment SNARE proteins form complexes with another SNARE protein

   • Integral membrane proteins

   • 35+ different proteins in specific compartments

   • v-SNARE: put into transport vesicles during budding

   • t-SNARE: located in the target membrane

   • Form four-stranded bundles

4. Fusion between vesicle and target membrane

   • Interactions between t-SNAREs and v-SNAREs pull lipid bilayers together with enough force to cause fusion

• The ability of a vesicle to fuse to a specific membrane is determined by the specific combination of: Rabs, SNARES, and tethering proteins

• Rab proteins are master regulators of vesicle transport between compartments within cells

Lecture 12

Lysosomes contain hydrolytic enzymes:

• Contains at least 50 hydrolytic enzymes

• Enzymes here have an optimal activity in acidic pH

  • Acid hydrolases

  • pH of lysosome is ~4.6

• pH of lysosome is maintained by a proton pump

Roles of lysosomes:

1. Breakdown of material brought into the cell by endocytosis

   • Ex phagocytic cells in mammals ingest pathogenic microbes

2. Organelle turnover (autophagy)

   • Regulated destruction and replacement of the cell’s organelles

   • Organelle is surrounded by a double-membrane structure: autophagosome

   • Autophagosome fuses with a lysosome: autolysosome

   • Starved cells exhibit increased autophagy

   • Organelle is surrounded by a double-membrane structure

     • Inner autophagosomal membrane: cargo sequestration

     • Outer autophagosomal membrane: fusion with the lysosomal membrane

Sorting and transport of lysosomal enzymes:

• Soluble lysosomal enzymes are recognized by enzymes that add phosphate groups to mannose sugars of N-linked carbohydrate chains

• The phosphorylated mannose (mannose 6-phosphate) residues act as a sorting signal, directing proteins to the lysosome

Targeting lysosomal enzymes to lysosomes:

1. Mannose residues are phosphorylated in Golgi (mannose 6-phosphate)

2. Lysosomal enzymes are incorporated into a clathrin-coated vesicle

   • Clathrin: coat protein that forms structural scaffold

   • GGA Adaptor: connects clathrin to MPRs

   • Mannose 6-phosphate receptor (MPR): transmembrane protein that recognizes and captures proteins with the mannose 6 phosphate signal

   • G-protein: Arf1-GTP, binds to the membrane and initiates formation of the budding vesicle and binding of the other coat proteins

     • Induces membrane curvature when bound to GTP

   • Adaptor: physically links two or more components

   • GGA adaptor has multiple domains:

     • Binds Arf1-GTP

     • Binds clathrin

     • Binds to the cytosolic tails of the MPRs

   • Results in concentrating lysosomal enzymes into clathrin-coated vesicles

3. (Formation of the clathrin-coated vesicle)

4. MPRs separate from the lysosomal enzymes and are returned to the Golgi (step 5)

5. Clathrin coat is disassembled and lysosomal enzymes are delivered to a sorting endosome and on to a lysosome (step 6)

Simplified Steps:

1. Mannose residues are phosphorylated in Golgi (mannose 6-phosphate)

2. Lysosomal enzymes are incorporated into a clathrin-coated vesicle

3. Vesicle formation is complete

4. Clathrin coat is disassembled

5. Vesicle fuses with endosome for sorting

   6a. MPR (receptors) are returned to the Golgi

   6b. Lysosomal enzymes are delivered to the lysosome

Transport of secreted proteins:

• Golgi cisternae move continually toward the TGN, which fragments into vesicles and tubules

• Constitutive secretion may be the ‘default’

Endocytosis:

• Bulk-phase endocytosis:

  • Pinocytosis

  • Non-specific: uptake of extracellular fluids (and any molecules that happen to be present)

• Receptor-mediated endocytosis:

  • Clathrin-mediated

  • Specific molecules binding to receptors on the extracellular surface of the plasma membrane

  • Ex hormones, growth factors, certain nutrients

  • Focus in this course

• Clathrin organization:

  • Each clathrin molecule (triskelion) is composed of three heavy chains and three light chains

  • AP2 complex (adaptor) links cytoplasmic tails of plasma membrane receptors with clathrin

  • Dynamin is a G-protein required for the clathrin-coated vesicle to bud from the membrane

  • Dynamin subunits polymerize to form a ring (step 3)

  • GTP hydrolysis induces a movement in the dynamin ring

  • Vesicle is cleaved and dynamin disassembles

Lecture 13 (Part 1)

Recycling pathway:

• Housekeeping receptors mediate uptake of materials that will be used by the cell (cholesterol, iron, etc)

• Receptors are first transported to an early endosome for sorting

• Ligands dissociate due to acidic pH

• Receptors are concentrated into a recycling compartment of the early endosome

• Vesicles return receptors to the cell surface to be used again

Degradation pathway:

• Signalling receptors bind ligands that affect cellular activities (hormones, growth factors ex)

• First transported to early endosome for sorting, early endosome matures into late endosome

• Late endosome fuses with lysosome for receptor degradation

• Receptor degradation prevents the cell from being further stimulated by the hormone/growth factor

Topic 3

Lecture 13 (Part 2)

Cytoskeleton:

• Network composed of three well-defined filamentous structures:

  • Microtubules

  • Microfilaments (actin filaments)

  • Intermediate filaments

• General functions:

  1. Structural support

  2. Transport of materials (also organelles)

  3. Contraction and motility

  4. Spatial organization

  5. Role in cell division

Microtubule structure and function:

• Each type of cytoskeleton filament is made of protein subunits held together by weak non-covalent bonds

  • Allows rapid assembly and disassembly

• Microtubules: hollow, unbranched, tubular structures made of tubulin

  • Roles in cell support and movement of materials within a cell

  • Can extend across the length or breadth of a cell

• The microtubule is composed of 13 protofilaments aligned side by side to form a tube

• Protofilaments are assembled from dimers of one ⍺-tubulin
  and one β-tubulin

• Protofilament is asymmetric, the microtubule itself has polarity

  • ⍺-tubulin end: negative end

  • β-tubulin: positive end

Assembly of microtubules:

• Centrosome:

  • A type of microtubule-organizing centre which initiates microtubule formation

  • Composed of two centrioles surrounded by pericentriolar material (PCM)

    • PCM: loosely organized fibrous lattice

    • Centrioles: cylinders composed of microtubules

  • When centrosomes replicate, centrioles recruit PCM to form a new centrosome

  • Centrosomes often remain at the centre of the cell’s microtubular network

• Centrosomes are microtubule organizing centers. They dictate:

  • the number of microtubules

  • their polarity

  • the number of protofilaments

  • the time and location of microtubule assembly

  • Not: microtubule stability nor rate of assembly

• New microtubules do not make contact with the centrioles, instead they are initiated in the PCM

• PCM contains ɣ-TuRC (tubulin ring complex):

  • ɣ-tubulin (gamma)

  • Non-tubulin proteins in a ring

• ⍺β-tubulin dimers assemble on the ɣ-tubulin, where only ⍺-tubulin can bind to the ring of ɣ-tubulin

Microtubule dynamics:

• Microtubules in some structures are sensitive to disassembly: mitotic spindle

• Microtubules in some structures are very stable: neurons, cilia, flagella

• Stability is determined by:

  • MAPs: microtubule associated proteins

  • +TIPS, which bind at the + end of growing microtubules

  • Temperature: cold=disassembly

• (Stabilizing) MAPS

  • Increase stability and promotes assembly by linking tubulin dimers together

  • Activity of some MAPs is controlled by the presence of phosphate groups

  • Ex high level of a phosphorylated MAP (called tau) has been associated with the development of Alzheimer’s disease

• GTP is an energy source

  • analogous to ATP

• β-tubulin is a G protein: hydrolyzes GTP to GDP after the dimer is added to the microtubule

  • GTP bound to the β-tubulin subunit is required for microtubule assembly

  • GTP hydrolysis affects microtubule structure

  • GTP is not hydrolyzed by ⍺-tubulin

Lecture 14

1. In a growing microtubule, the top consists of tubulin-GTP dimers in an open sheet

2. Tube closure is associated with hydrolysis of GTP

3. GDP tubulin has a different conformation, introducing mechanical strain

   • MAP stabilize microtubule

4. In the absence of stabilization, protofilaments curl outward and undergo catastrophic shrinkage

+TIPs:

• Bind to the positive end of the microtubule and regulate the rate of growth or shrinkage

• Mediate the attachment to subcellular structures (ex kinetochore of the mitotic chromosome)

• Microtubule polymerization/disassembly can effectively ‘push’ and ‘pull’ material within a cell

Microtubules as structural supports:

• Microtubules provide mechanical support: are stuff enough to resist compression or bending forces

• Help determine the shape of a cell

• Maintains intracellular location of organelles

Microtubules as agents of intracellular motility:

• Transport of membranous vesicles from one membrane compartment to another

• Transport of nonmembrane bound cargo (RNAs, ribosomes, cytoskeletal elements)

• Refer to diagram on pg 14

Microtubule motor proteins:

• Motor proteins: Utilize ATP hydrolysis to generate mechanical forces that move the motor protein and attached cargo along the cytoskeleton

• Cargo examples: membranous vesicles, nonmembrane bound (ribosomes, RNA), organelles (lysosomes, mitochondria), chromosomes, other cytoskeletal filaments

• Three types of motor proteins: microtubule motor proteins (kinesins and dyneins), actin motor proteins (myosins)

• Each type of motor protein moves unidirectionally in a stepwise manner

Kinesin Structure

• Kinesin-related proteins superfamily: Kinesin-1 family

• Tetramer: two heavy chains and two light chains

• Globular head:

  • Binds microtubules

  • ATP hydrolysis

  • Conserved sequences

Kinesin movement:

• Kinesin moves along the microtubule towards the positive end

• Leading head binds one ATP: hydrolysis and release of ADP + Pi = power stroke that swings the trailing head forward

  • Moves the motor 8nm (length of one tubulin dimer)

• Kinesin moves in a hand-over-hand mechanism: at least one head is attached to the microtubule at all times

• Highly processive: capable of moving consiserable distances without falling off

• Speed is proportional to the ATP concentration (to a max speed of 1um/sec)

Dynein structure:

• Dynein is much larger than kinesin

  • Dyein head is ~10x larger than a kinesin head (also faster than kinesin)

  • Two heavy chains + multiple intermediate and light chains

  • Bind to cargo via an adaptor protein (dynactin)

  • Globular head: force generation, ATP binding and hydrolysis

Dynein movement:

• Dynein moves progressively along the microtubule towards minus end

• Roles in:

  • Positioning the spindle and moving chromosomes during mitosis

  • Positioning organelles and moving vesicles

Structure and function of cilia and flagella:

• Cilia and flagella are hairlike organelles that project from various eukaryotic cells

  • Often motile

  • Same structure, different contexts

  • Beware: microvilli are not the same as cilia

Lecture 15

• Motile cilia in multicellular organisms move fluid

  • Ex cilia lining the respiratory tract sweet mucus away from lungs

  • Usually found in large numbers on a cell’s surface

  • Coordinated beating

  • ES: Effective power stroke

  • RS: Recovery stroke

• Flagella have the same structure as cilia, but found in fewer numbers

  • Unicellular alga (eukaryote) moves by an asymmetric waveform

• Cilia/flagella is covered in a membrane that is continuous with the cell’s plasma membrane

• Microtubule organizing center: basal body (ɣ-Turc)

• Axoneme: Core contains microtubules oriented longitudinally

• All microtubules oriented:

  • + at the distal end

  • - near the basal body

Structure of the axoneme:

• 9 peripheral doublet microtubules around a central pair of single microtubules (“9+2 array”)

• Centriole has 9 microtubule triplets, axoneme has 9 microtubule doublets

• Doublets are connected to each other via nexin

• Dynein tails are anchored to one of the tubules in each pair (the ‘A tubule’)

  • Reminder: move towards the - end of the microtubule

Movement of cilia/flagella:

1. Dynenin tails attached to the A tubule and dynein stalks bind to the B tubules

2. Power stroke (conformational change upon ATP hydrolysis)

3. Dynein stalks detach

4. Dyenin stalks reattach (cycle begins again)

• Nexin link limits the extent of movement/sliding

Actin filaments (F-actin) (microfilaments)

• Actin is the most abundant protein in most cells

• Filaments are composed to globular subunits (G-actin)

• Involved in cellular motile processes

• Ex: Movement of vesicles, phagocytosis, cytokinesis

• Provides structural support: shape of cells, support for cellular projections

Structure of actin filaments:

• 

  Actin filaments have polarity:

  • + end barbed

  • - end pointed

• Individual G-actin monomers have directionality and are added to the filament in a particular orientation

• Filament also has directionality (polarity)

• Filament is a double-stranded helix (both strands are oriented in the same direction)

• Ends are named based on binding of a fragment of the myosin motor protein (S1)

Actin filament assembly and disassembly:

• ATP-actin is incorporated into the filament

• After incorporation, actin hydrolyzes it to ADP

• ATP-actin is added to both ends

• Faster addition at the barbed end

• Barbed and pointed ends have different critical concentrations:

  • Minimal concentration of available ATP-actin required to elongate

  • Critical concentration of the barbed end (+) is much lower

1. Preformed actin filament (seed) in the presence of ATP-actin

2. At high ATP-actin concentrations, it will be added to both ends

3. Concentration reaches the critical concentration of the pointed end; addition stops at the pointed end

4. Loss of subunits occurs at the pointed end because ADP-actin dissociates more readily than ATP-actin, but addition continues at the barbed end

5. Relative position of subunits is continually moving: treadmilling

Concrete numbers:

• Assume we’re starting with a high available concentration of actin-ATP, which is decreasing as the subunits get incorporated into the filament

• Initial concentration of actin-ATP (higher than 1.5um)

• Pointed end (-) critical concentration: 1.5um

• Barbed end (+) critical concentration: 0.5um

• As available actin-ATP decreases, the critical concentration of which end will be reached first? Pointed end

• Treadmilling happens when the cell’s available actin-ATP is between 0.1um and 1.5um

• Steady state: when the rate of addition at one end is the same as rate of loss at the other end

  • Occurs at approx 0.3um available actin-ATP

Lecture 16

Actin motor protein - Conventional Myosin (Type II):

• Myosin superfamily:

  • Conventional (type II)

  • Unconventional (type I, types III-XVII)

• Conventional Myosin Type II:

  • Motor (head)

    • Binds the actin filament

    • Binds and hydrolyzes ATP

    • Conserved sequences

  • Neck

    • (or lever arm)

    • Moves during the power stroke

  • Tail

    • Intertwining of the two heavy chains

    • Allows the formation of filaments of myosin

• All myosins (except type VI) move towards the + end (barbed end)

Actin motor protein - Unconventional Myosin (Type V):

• Moves processively along actin filaments

• Moves in a hand-over-hand movement

• Long necks act as swinging arms

• Can take very large steps (~36nm)

• Some myosins (types I, V, VI) can associate with vesicles and organelles (ex myosin type V tail bound to a vesicle via adaptors (including Rab 27a)

Transport by Unconventional Myosins:

• Some vesicles contain both microtubule motors and actin filament motors

• Movement over long distances occurs mostly on microtubules

• Local movement in the outskirts of the cell: actin filaments

Myosin type 2 filaments:

• Myosin II tails allow the protein to form filaments

• In the myosin II filament, tails point towards the center and heads points towards the outside

• Myosin filament:

  • Bipolar: Motor domains are oriented at opposite filament ends

  • Thick: Composed of myosin (in contrast to ‘thin’ filaments that are composed of actin)

Skeletal muscle organization:

• Skeletal muscles are usually anchored to bones

• Muscle fiber: a skeletal muscle cell. Contains multiple nuclei and hundreds of myofibrils

• Myofibrils: composed of repeating contractile units called sarcomeres

• Sarcomeres: contractile unit with a characteristic banding pattern

Sarcomere organization:

• Thick filament (purple): myosin filament

• Thin filament (orange): actin filament

• Z-line: contains proteins important for sarcomere structure stability (one sarcomere is from Z-line to Z-line)

• M-line: dark staining in the center of the sarcomere, contains anchoring proteins

• I bands (light staining) - contain only thin filaments

• H zone: contains only thick filaments

• A band: dark staining (overlap of thick and thin, also includes the H zone)

• Each thick filament is surrounded by 6 thin filaments

• I band and H band decreases in length when the muscle contracts

• A band length does not change when the muscle contracts

• Model: thin filaments slide towards the center of the sarcomere

Molecular basis of contraction:

• Myosin II heads in a thick filament binds to six surrounding actin filaments

• Myosin II is nonprocessive:

  • Only in contact with actin for a fraction of the time

  • Myosin heads are not synchronized

Actin-myosin contraction cycle:

1. ATP binds to myosin head and myosin dissociates from actin

2. ATP hydrolysis, ADP and Pi remain bound to myosin

3. Energized myosin binds actin

4. Release of phosphate triggers conformational change: power stroke - actin moves towards the center of the sarcomere

5. ADP is released

Neuromuscular junction:

• Muscle fibers (cells) within a motor unit are stimulated simultaneously by a single motor neuron

• Neuromuscular junction: Point of contact between motor neuron and muscle fiber; site of transmission of the nerve impulse

• Acetylcholine stimulates an action potential in the muscle cell

Lecture 17

Excitation-contraction coupling:

• Transverse tubules (T-tubules): Membrane folds that propagate an impulse to the interior of a muscle cell

• Sarcoplasmic reticulum: Special smooth ER in muscle cells, stores Ca2+ in lumen (pumped in from the cytosol)

• Arrival of action potential at the SR opens Ca2+ channels, release Ca2+ into the cytoplasm

• Thin filaments contain actin and:

  • Tropomyosin: rod shaped

  • Troponin: globular

  • Absence of Ca2+: tropomyosin blocks myosin-binding sites on actin

  • Presence of Ca2+: Ca2+ binds troponin which moves tropomyosin exposing the myosin-binding site on actin

Actin organization:

• Cell cortex: Actin network on the inner face of the plasma membrane, capable of dynamic remodelling

• Enable cells to crawl/move

• Enable phagocytosis

• Cellular constriction during cell division

• Actin-binding proteins: regulate the assembly, disassembly, and rearrangements of actin networks (more than 100 different proteins)

Actin-binding proteins:

1. Filament nucleating:

   • Slowest step in the formation of an actin filament

   • Proteins can enhance the rate at which actin filaments are formed

   • Arp2/3 complex: Binds to the side of an existing filament (creates branches), remains at the pointed end of the new branch, similar structure to actin monomers

   • Formins: Generate unbranched filaments, stay associated with the barbed end, promote rapid elongation of filaments

2. Monomer-sequestering:

   • Bind to actin-ATP monomers to prevent them from being added to the elongating filament

   • Able to modulate the available monomer pool in certain regions at certain times

   • Ex thymosins

3. End-blocking (capping):

   • Regulate the length of actin filaments

   • Bind at either end

4. Monomer polymerizing:

   • Binds to actin monomers to promote growth of actin filaments

   • Promotes replacement of ADP with ATP on the actin monomers

   • Ex: profilin

5. Depolymerizing:

   • Bind to actin-ADP at the pointed end to promote depolarization

   • Ex: cofilin

6. Cross-linking and bundling:

   • Multiple actin-binding sites, allowing them to alter the 3D organization of the actin filament network

   • Ex: filamin (cross-linking), villin and fimbrin (bundling)

7. Filament-severing:

   • Break an existing filament in two

   • Ex: gelsolin and cofilin

8. Membrane-binding:

   • Actin filaments linked to the plasma membrane

   • Enabling the plasma membrane to protrude outward (cell locomotion) or inwards (phagocytosis)

   • Ex: spectrin family of proteins

• Specific proteins/complexes to know:

  • Arp2/3 (branched filament nucleation)

  • Profilin (monomer polymerizing)

  • Cofilin (depolymerizing)

Cell motility (step 1):

1. Movement is initiated by a protrusion of the cell in the direction of movement (Lamellipodium)

2. A portion of the protusion anchors to the surface below

3. The bulk of the cell is pulled toward the front, over the adhesive contacts

4. Adhesive contacts break, causing retraction of the trailing edge (tail)

• Lamellipodium: the leading edge of a moving cell that extends over the surface, broad and flat

Dynamic actin network at the site of lamellipodium formationL

1. A stimulus is received at the cell surface (ex neutrophil receiving a signal from an infected tissue)

2. Arp2/3 complex at the site of stimulation gets activated

3. Arp2/3 binds the side of an existing filament

4. ATP-actin monomers bind to the Arp2/3 complex, forming a new actin branch

   • Polymerization is promoted by profilin

5. Additional Arp2/3 complexes can bind to the sides of the new filaments, forming additional branches:

   • Older filaments are capped at their barbed ends

6. Newer filaments continue to grow at the barbed end, pushing the membrane of the lamellipodium outward

   • Older capped filaments undergo disassembly promoted by cofilin

Cell motility (step 2):

• Traction forces: When the cell grips the surface (at adhesion points called focal adhesion)

• Focal adhesion: structures in the cell membrane where integrin proteins connect to actin

• Integrin proteins: Transmembrane proteins that mediate the interaction between actin and extracellular components

Cell motility (step 3):

• Contraction forces pull the bulk of the cell forward

• Myosin found near the rear of the lamellipodium

Lecture 18

Intermediate filaments:

• Strong, flexible, stretchy, unbranched fibers

• Only found in animal cells

• Provide mechanical strength to cells

  • Neurons

  • Muscle cells

  • Epithelial cells

• Chemically heterogeneous

  • Encoded by ~70 different genes in humans

  • Five classes (I-V)

• Intermediate filaments don’t have polarity** (both ends are identical)

• New units are added into the middle of an existing filament**

• Bridging (ex: via the protein plectin) to intermediate filaments stabilizes other cytoskeletal elements, increasing cell stability

• Intermediate filaments in neurons: neurofilaments

  • Have sidearms that help to maintain proper spacing

  • Important for determining the axon’s diameter

Final

Lecture 19

Section I: The Plant Cell

• Plant and animal cells diverge from a common unicellular ancestor

  • Plant cells have chloroplasts

• Plant cell structure and function is conserved in many ways

  • Conserved organelles, structures, metabolic processes, genes

  • Many differences between plant, animal, fungi cells

• Plant cells are glued to their neighbours

• ~50 different plant cell types: xylem, phloem, mesophyll (leaf) actively involved in photosynthesis

• Differentiated plant cells: can de-differentiate and form another cell type —> whole plant

  • Differentiated cell —> undifferentiated cell —> new cell type (ex lead mesophyll cell) —> whole plant

  • Individual plant cells can de-differentiate, divide, and form a complete plant

• Totipotency: ability of a cell to divide and form any other cell type, sometimes a complete organism (ex zygote, spore)

  • Important in biotechnology

    • Genetically modified (transgenic) plant

    • All cells in transgenic plant have the transgene (ex canola — most of it is herbicide resistant)

    • After bombardment, cells that contain the transgene are selected and induced to form complete plants with each of their cells containing the transgene

• Movement within plant cells: Lots of cytoplasmic streaming in plant cells

  • Ex root hair

• Organelle movement drives cytoplasmic streaming

  • Ex golgi stacks (100’s in each cell), the plant Golgi stacks move along actin filaments that are associated with ER

  • Move on actin filaments using myosin motors

  • Golgi bodies - punctate structures

  • ER - reticulate structure

• Plant myosin XI — the fastest myosin - takes 35nm steps (one helical rotation)

• Myosin-mediated vesicle movement along actin filaments in Arabidopsis root hair cells

• Chloroplasts move in response to light in leaf cells

  • Dim light: Chloroplasts align perpendicular to the direction of light

  • Bright light: Chloroplasts align parallel to the direction of light

  • Movement is triggered by blue wavelengths of light

Lecture 20

Section II: Plant Cytoskeleton

• Plant cells have microtubules (MT) and actin filaments

  • No intermediate filaments

• Motor proteins: myosin and kinesin

  • No dynein in plant cells

• No centrioles (or centrosomes) in higher plant cells

  • No flagellated or ciliated plant cells

• 4 MT arrays in plant cells

  1. Cortical array - interphase: on inner side of plasma membrane

  2. Pre-prophase band of MTs

  3. Mitotic spindle

  4. Phragmoplast

  • These 4 are found during cell division

Section III: Cell Wall

• All plant cells have a cell wall

  • ~15-30% dry weight of a herbaceous plant

• All have a primary cell wall: extracellular - outside plasma membrane

• Many others also have a secondary cell wall: made after the primary wall (also extracellular)

  • Lies inside of the primary wall (ex xylem cell - dead at maturity)

  • Lignin: a compound that gives wall strength

• Middle lamella: Glues plant cells together

  • Contains pectin

• Plant cells have high internal pressure (turgor pressure)

  • Primary wall prevents cell from bursting

  • Vacuole: high solute concentration (sugar, salts, amino acids ex); osmosis—water moves in

• Leaf: solar panel

• Cellular turgor pressure is important in maintaining leaf shape

Components of the Cell Wall:

• Highly organized structure composed of polysaccharides and proteins

1. Cellulose: bundles of long glucose polymers, glucose subunits bond by B(1,4) linkages

2. Hemicellulose: crosslinks adjacent cellulose microfibrils

   • using H-bonds - weak

   • polymer of glucose and another sugar (ex xyloglucan)

3. Pectins: heterogeneous, branched carbohydrate

4. Proteins: involved in wall stability (ex extensin locks wall into place, expansin loosens wall)

Synthesis of Wall Compounds:

• Cellulose: made by cellulose structure

  • Embedded in plasma membrane

  • Rosettes synthesize cellulose microfibrils

• Cortical MTs guide cellulose synthase movement

• Non-cellulose: hemicellulose and pectin, made in Golgi stacks, protein are made on RER

Lecture 21

• Direction of cellulose microfibrils - determines the direction of cell growth

  • Expansin loosens hemicellulose-cellulose interaction

  • Ex root cell

• Coordinated cell elongation can result in complex plant movements

• Cytokinesis of plant cell: moves centrifugally outward

• Mitosis:

  • Interphase - Non-dividing cell

  • Prophase - Chromosomes begin to condense

  • Metaphase - Condensed chromosomes line up at equator

  • Anaphase - Sister chromatids separate

  • Telophase - New nuclear envelopes begin to form

Pre-prophase band of MTs:

• Transient band of MTs - predicts plane and position of new cell plate

• Forms prior to prophase —> gone by metaphase

• Against plasma membrane —> leaves a “footprint”

Phragmoplast:

• Forms after anaphase

• Helps cell plate development

• Double band of MTs - deliver Golgi derived vesicles to developing cell plate

• Anti-parallel MTs - plus-ends pointing toward each other

• Uses kinesin motor

• Moves laterally

A GFP-MT labelled cell demonstrates the dynamics of three MT arrays during plant cell division - PPB, spindle, and phragmoplast

Plasmodesma(ta):

• Cytoplasmic connections between adjacent cells

• Can move small (ions, sugars) molecules and large (proteins, RNA)

• Connect most cells — plants are supracellular organisms: one big cell

• Protein spokes: regulate large molecule movement

Microinjection Studies:

• Fluorescent dextrans — known molecular weights (0.5kDalton, 5kDalton ex)

• Size exclusion limit (SEL) of PD ~1kDa ~ sugar

• Proteins ~30kDa

  • Some proteins have a signal in peptide sequence which can open up a gate and selectively move that protein in

  • Ex transcription factor: cell-cell movement controls gene expression, move into phloem (long distance transport)

• Actin in plasmodesmata (PD): regulate movement between cells

Section VI - Vacuole:

• Fluid filled compartment bounded by the tonoplast membrane

• Usually 30% of cell volume but can be up to 90%

• Functions:

  1. Storage: ions, organics, sugars, proteins

  2. Digestion: nucleases, proteases

  3. pH and ion homeostasis - decrease pH ex lemon

  4. Defense: accumulate toxic compounds

• 2 types: lytic vacuole and protein storage vacuole

Module 1

Lecture 22

Extracellular Interactions:

• Materials present outside the plasma membrane play an important role in the life of a cell

• Most cells in a multicellular plant or animal are organized into clearly defined tissues

• There are many diverse activities that are regulated by this:

  • Tissue development

  • Wound healing

  • Fighting infection

• Receptor and ligand interactions are common methods of cell interactions

• Direct cell-cell interactions such as in cell-cell contacts

• Cells interact with their extracellular environment

• The epidermis has closely packed cells of epithelial tissue

• The dermis is a type of connective tissue

• Fibroblasts of the dermis have receptors that mediate interactions and transmit message

• Cellular interactions are required for:

  • Intercellular communication

  • Survival

  • Tissue strength

  • Organ function

  • Immune system function

  • Embryonic development

4 different families of integral membrane proteins mediate cell-cell adhesion:

• Selectins

• Immunoglobulin super family

• Members of the Integrin family

• Cadherins

Protein interactions involving the cell surface:

• Homotypic interactions of two L1 molecules through immunoglobulin (Ig) domains

• Heterotypical interactions of IG super family (IGSF) protein with integrin

Cadherins:

• Calcium dependent

• adhesion or transmit signals

• bind a similar cadherin on a neighbouring cell

• Possibly the single most important factor in molding cells into cohesive tissues in the embryo and holding them together in the adult

• Cadherin loss associated with malignancy

• Distributed along cell surfaces or part of intracellular junctions:

  • synapses

  • Adherens junctions

  • Desmosomes

• During embryogenesis:

  • Cells from different ‘germ’ layers display distinctive adhesive properties (ectoderm: outside skin, mesoderm: middle)

  • Selective cell affinities help establish the spatial order of different tissues in the embryo

  • Requires specific molecular interactions: cadherin-cadherin

  • Experiments demonstrated that separated cells redistribute themselves, so each cell adhered to cells of the same type

• Stem cells were induced from differentiated human cells and injected into a developing pig embryo, the human cells successfully integrated into the tissues of the developing pig

  • The human and pig cells must have been able to interact appropriately with their cadherins initially

Immunoglobulin super family (IgSF):

• contain Ig domains that can connect to the integrin family, or connect to another IgSF

• mediate calcium independent adhesion

• many IgSF proteins are ICAMs

  • ICAMs - intracellular adhesion molecules

• Integrins are some of the proteins that acts as receptors for ICAMs

Selectins:

1. E-selectin, present on endothelial cells (blood vessels)

2. P-selectin, present on platelets and endothelial cells

3. L-selectin, present on all types of leukocytes (white blood cells)

• Calcium dependent

• Selectins are a family of membrane glycoproteins that bind to specific oligosaccharide (carbohydrate moiety)

• “Lectin” is a term for a compound that binds to specific carbohydrate group

• Selectins have a small cytoplasmic segment, a single membrane-spanning domain and a large extracellular portion

Lecture 23

Movement of neutrophils from the bloodstream during inflammation:

1. Inflammation activates endothelial cells, which upregulates the selectins and they ___

2. Selectins bind to the carbohydrate residues (PsgI-1) on neutrophil, a phagocytic leukocyte

3. Platelet activating factor on IL-8 on the surface of endothelial cells activates G-protein coupled receptors on the neutrophil and this leads to _____

4. Integrins bind to ICAMs on endothelial surface and a cascade of events results in cytoskeletal rearrangement such that the cell can ___

5. Transendothelial migration

• Cancel calls escape the normal growth control mechanisms and proliferate in an unregulated manner

• Metastasis is the spread of cancer

• One of the most important proteins that reduces metastasis is the presence of ____

How can we visualize cell junctions?

• Electron microscopy uses electrons rather than light

• Very high resolution (visualization) of cellular structures

• Samples are imaged under a vacuum so live cells can’t be imaged

The Junctional Complex:

1. Tight junctions (zonula occluden)

2. Adherens junctions

3. Desmosomes (macula adherens)

Tight junctions:

• At the top of the cell

• Occur between neighbouring epithelial cells

• Prevent solute distribution where different solute concentrations are in adjacent compartments

• They can control:

  1. Gate function - controls the passage of the following between cells (paracellular pathway):

     • Ions

     • proteins

     • blood brain barrier - ions or water can’t pass, but cells of the immune system can pass

     • water - mutations in claudin 1 cause death due to dehydration

  2. Fence function - block diffusion of integral membrane proteins between apical and basolateral membranes of one cell, connect to the actin cytoskeleton and microtubules

• Tight junctions regulate the passage of solutes between cells

  • Form close contacts between cells

• TJ restrict plasma membrane proteins to a particular domain of the membrane

  • Contribute to cell polarity - forming a barrier that blocks proteins

• Adherens junctions and desmosomes increase tissue strength

• Adherens junctions:

  1. Connect the external environment to the actin cytoskeleton

  2. Provide a pathway for signals to be transmitted from the exterior to the cytoplasm and nucleus

  • AJ form a belt (zonula adherens) that encircles the cells near their apical surface in epithelial cells

• Desomosomes - primarily adhesive:

  • Also contain cadherins = desmosomal cadherins

  • The cadherins interact with multiple proteins to form a cytoplasmic plaque on the inner surface of the plasma membranes

    • The intermediate filament cytoskeleton anchors two cells together, this provides strength to a sheet of cells

• Gap junctions are communication channels:

  • Form intercellular channels

  • Transmit small soluble signaling molecules directly through the membrane

  • They are made of connexin proteins: 6 identical connexins from each cell form a transmembrane channel with a central pore called a connexon

  • Two connexons form a gap junction**

  • Gap junctions are molecular “pipelines” that pass through the adjoining plasma membranes and open into the cytoplasm of the adjoining cells

• Hemidesmosomes:

  • Cell matrix attachment in vivo is seen at the basal surface of epithelial cells, anchored to the underlying basement membrane

  • Hemidesmosomes contain a dense cytoplasmic plaque with keratin filaments

  • Keratin filaments are linked to ECM by integrins

• Focal adhesions are discrete sites of cell attachment and are dynamic

  • Cultured cells are anchored to the surface of the dish only at scattered, discrete sites, called focal adhesions

  • Focal adhesions play a key role in cell locomotion

  • Focal adhesions are dynamic structures