Membranes: Structure and DynamicsPart 2

Cell BiologySummer 2015Dr. Newton

ObjectivesKnow: • Plasma membrane structure and function• Structure and function of specific components of a

membrane• The Fluid Mosaic Model• How eicosanoids are produced• Membrane asymmetry and the role of flippases• The effects of lipids & sterols on membrane fluidity• About lipid rafts• About the glycocalyx• The role of spectrin and ankyrinin the red blood cells • Clinical Correlations

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Fluid Mosaic Model

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Membranes are viewed as two-dimensional fluids in which proteins are inserted into lipid bilayers.

• Membranes contain several major classes of lipids▫ Phospholipids

⚫ Glycerol-based phosphoglycerides⚫ Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine

⚫ Sphingolipids⚫ Sphingomyelin

▫ Glycolipids – no phosphate !!⚫ Cerebrosides

⚫ Neutral - single uncharged sugar⚫ Other neutral glycolipids (e.g., A, B, and O blood group antigens), which

contain many sugar moieties⚫ Gangliosides

⚫ 1 or more negatively charged sialic acids (eg N-acetylneuraminic acid)▫ Sterols

⚫ Cholesterol

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The Lipid Bilayer

The Lipid Bilayer

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Phosphatidylcholine

• Comprised of a heterogeneous mixture of phospholipids▫ Most common type is phosphatidylcholine

• Contains amphipathic molecules▫ Hydrophilic heads face water whereas

hydrophobic tails are repelled•Fluidity depends on its composition:▫Unsaturated (cis) phospholipids⚫More fluid; Fewer hydrophobic forces due to kink in tail

▫Cholesterol⚫ Less fluid at normal physiological temperatures

Figure 10-12 Molecular Biology of the Cell (© Garland Science 2008)

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Figure 10-3 Molecular Biology of the Cell (© Garland Science 2008)

Four Major Phospholipids 7

Sphingosine8

SphingolipidGlycolipids

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Cholesterols

Table 10-1 Molecular Biology of the Cell (© Garland Science 2008)

Membrane Lipid Composition Variations

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Movement of Lipids in a bilayer• Transverse diffusion - rare

▫ Catalyzed by phospholipid translocators (flippases) in the plasma membrane

• Lateral diffusion ▫ very rapid

• Rotation –super rapid

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Flippases

• Some are members of the ABC Transporter superfamily ▫ABC transporters: transmembrane proteins that utilize ATP to move various substrates across membranes.

• Flippase removes phosphatidylethanolamine and phosphatidylserine from the outer-monolayer of the cell membrane and uses the energy from ATP hydrolysis to transport them into the inner membrane.

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• Many lipids are distributed unequally between the two monolayers▫ Phosphatidylcholine and sphingomyelin more abundant in the

outer-monolayer (extracellular side)▫ Phosphatidylserine, phosphatidylethanolamine and

phosphatidylinositol are more abundant in the inner-monolayer (cytolpasmic side)⚫(Some Enter Innermembrane)⚫Signal transduction (inositol forms 2nd messengers)

• Asymmetry in part caused by phospholipid translocators (flippases)

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Asymmetry of the Lipid Bilayer

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• New membrane is formed in the sER▫ Exported to other membranes by vesicle budding and fusion

• Orientation of the bilayer relative to the cytosol is preserved during vesicular transport process

• Membranes have a distinctive “inside” and “outside” face▫ Glycolipids primarily located on non-cytosolic face

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Asymmetry of the Lipid Bilayer

▫ Negatively charged phosphatidylserine (blue) facing cytoplasm ▫ Glycolipids are found on the external surface of all plasma

membranes

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Asymmetry of the Lipid Bilayer

▫ Negatively charged phosphatidylserine (blue) facing cytoplasm

▫ Glycolipids are found on the external surface of all plasma membranes

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Asymmetry of the Lipid Bilayer

Lipid Rafts

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• Small, specialized areas in membranes where certain types of lipids of are concentrated.

• Because the lipid bilayer is somewhat thicker in the rafts, membrane proteins accumulate, such as those involved in cell signaling (aka microdomains).

The Lipid Bilayer is Fluid• Evidence from fluorescence recovery after photobleaching

(FRAP)▫ A cell surface containing fluorescently labeled lipid is exposed

to a laser beam▫ The laser beam causes a small area to become bleached▫ A few seconds later, fluorescent-labeled lipids fill the bleached

area

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FRAP

http://zeiss-campus.magnet.fsu.edu/articles/livecellimaging/techniques.html

In this example the movement of green fluorescent protein (GFP) within the cytosol is being assayed by measuring the kinetics and extent of GFP fluorescence recovery.

Step 1: Record fluorescence baseline values of fluorescence for a given region of interest (roi).

Step 2: Photobleach within the roi so that fluorescence values are that of background.

Step 3: Image the roi at regular intervals to calculate the halftime of GFP fluorescence recovery.

Step 4: Once the fluorescence stabilizes, take several more images to establish the extent of recovery.

FRAP

http://zeiss-campus.magnet.fsu.edu/articles/livecellimaging/techniques.html

Step 1: Record fluorescence baseline values of fluorescence for a given region of interest (roi).

Step 2: Photobleach within the roi so that fluorescence values are that of background.

Step 3: Image the roi at regular intervals to calculate the halftime of GFP fluorescence recovery.

Step 4: Once the fluorescence stabilizes, take several more images to establish the extent of recovery.

Information gathered from the images is used to calculate the halftime of recovery as well as the immobile fraction of GFP

FRAP

FRAP is one of many methods used by cell biologists that uses fluorescence microscopy. Fluorescence microscopy is a powerful tool and is often used for the studying the following:o Intracellular distribution and movement of macromolecules

and organelles.o Cellular functions such as exo- and endocytosis, signal

transduction and the generation of transmembrane potentials.

o Rate and extent of enzyme function.o Protein-to-protein interactions.o Dynamics of macromolecule dissociation and diffusion.o In vivo movements of single molecules.o Movement of specific molecules within the membrane

The Lipid Bilayer is Fluid

• Usually, proteins and other molecules are free to diffuse laterally throughout the membrane

• Demonstrated by fusing mouse and human cells and monitoring distribution of proteins over time

• The cell can restrict movement to certain domains if needed.

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The Lipid Bilayer is Fluid

Proteins self-assembled into aggregates

Proteins tethered by interactions with assemblies of macromolecules outside the cell…

…or inside the cell

Proteins can interact with proteins on the surface of another cell

Restricted diffusion24

Protein diffusion in membrane of gut epithelial cells are restricted by tight junctions

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Restricted diffusion

Membrane molecules can be restricted to a particular membrane domain by tight junctions

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Polarized intestinal epithelial cell

Restricted diffusion

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Cell fusion experiments

Mobility of membrane proteins

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Membrane proteins vary in their mobility

•Experimental evidence of protein mobility▫Cell fusion experiments (next slide)▫Freeze-fracture experiments of membrane exposed

to an electric field•Lipid rafts can be enriched for membrane

factors•Experimental evidence of restricted mobility▫Photobleaching▫Distinct membrane domains

•Mechanisms of restricting protein mobility1. Tight junctions2. Anchoring proteins3. Polarized cells: cells in which membrane proteins

are restricted to a specific part of the cell membrane

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Membrane proteins: The “Mosaic” part of the model• The membrane consists of a mosaic of proteins and lipid rafts: ▫ Evidence from freeze-fracture microscopy

⚫Frozen sample is fractured by a sharp blow along the plane between the bilayer, giving rise to a P (protoplasmic) face and an E (exterior) face by EM⚫Abundance of integral proteins on the P face

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PE

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Membrane proteins

TransmembraneAnchored Lipid-

linkedProtein-linked

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Ways in which membrane proteins associate with the membrane

Hydrophobic amino acids form the alpha helix in contact with the hydrophobic tails of phospholipids

About 20 amino acids are required to span the membrane

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Polypeptide usually crosses the lipid bilayer as an alpha-helix

Solubilization of membrane by detergent

A detergent micelle

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solubilized by detergent

Solubilization and purification of membrane

proteins in detergent

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Experiment demonstrating the mixing of plasma

membrane protein on mouse-human cells

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Peripheral and Integral Proteins

•Peripheral Membrane proteins▫Associate through electrostatic forces and H-bonding▫Lack discreet hydrophobic segments

• Integral membrane proteins ▫Singlepass or multipass proteins▫Hydrophobic transmembrane segments▫Some are lipid-anchored proteins

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Solubilization of Integral Membrane Proteins by Detergents

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Lipid-anchored proteins

• Covalently-linked to lipids in the membrane• Proteins bound to the inner surface of the PM are

linked either to a FA or an isoprene derivative (prenyl group such as farnesyl (C15))

• Lipid-anchored proteins attached to the external surface of the plasma membrane are linked to glycosylphosphatidyl-inositol (GPI).▫GPI-anchored proteins are made in ER as single-pass

proteins, cleaved, then linked to GPI▫GPI-anchored proteins are found in lipid rafts and can

be released from the membrane by phospholipase C • Clinical Correlation: ▫The prion protein, present in neuronal membranes,

provides an example of a protein attached to the membrane through a GPI anchor.

▫Altered pathogenic conformation of prion protein found in mad cow disease and Creutzfeldt-Jakob disease

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Figure 10-27 Molecular Biology of the Cell (© Garland Science 2008)

Single-pass transmembrane protein

Key points

• Oligosaccharide chains and disulfide bonds are on the extracellular side

• Alpha-helix transmembrane domain

• The sulfhydryl groups in the cytosolic domain of the protein do not normally form disulfide bonds because the reducing environment in the cytosol maintains these groups in their reduced (-SH) form

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Lipid-anchored proteins

• Some proteins (eg Thy-1) are inserted into the outer leaflet of the PM by GPI anchors on the C terminus.▫ These proteins are

glycosylated and exposed on the cell surface.

• Other proteins are anchored in the inner leaflet by covalently attached lipids.

• They are translated on free cytosolic ribosomes and modified by myristic (C14) or palmitic (C16) acid or to prenyl groups

• Many of these proteins (including Src and Ras) play roles in signal transduction.

Made in ERM

ade in cytosol

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Membrane proteins have variety of functions

Many membrane proteins are glycosylated• N-linked glycosylation

▫ Asparagine• O-linked glycosylation

▫ Serine, Threonine, ▫ Hydroxylysine,

Hydroxyproline• Most common sugars

(galactose, mannose, N-acetylglucosamine, and sialic acid)

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Carbohydrates and membranes• Usually branched oligosaccharides (<15 monomers)• Some covalently bonded to lipids, forming glycolipids• Most covalently bonded to proteins, forming glycoproteins• Significant variability from

▫ species to species ▫ between individuals of the same species ▫ among cells within the same individual

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GlycocalyxThe surface oligosaccharide side chains of glycolipids and integral membrane glycoproteins (proteoglycans) contribute to the glycocalyx in many cells

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Figure 10-28a Molecular Biology of the Cell (© Garland Science 2008)

GlycocalyxThe carbohydrate groups on the cell surface are important recognition sites of membrane receptors such as those involved in binding extracellular signal molecules, antibody-antigen reactions and intercellular adhesion.

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Figure 10-28a Molecular Biology of the Cell (© Garland Science 2008)

glycocalyx of a lymphocyte

Erythrocyte Plasma Membrane

• Has a specialized, spectrin- based cytoskeleton that helps maintain the RBC structural integrity and biconcave shape.

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• Most widely studied membrane structure• No internal membranes• Most erythrocyte membrane proteins are peripheral proteins

bound to the cytosolic side.

Actin

Erythrocyte Plasma Membrane

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• Key membrane proteins: ▫ Band 3 (anion exchange protein – Cl¯ and bicarbonate)▫ Glycophorin: external negative charge that repels other cells

▫Spectrin: provides scaffold for linking proteins on membrane

▫Ankyrin-1: anchors band 3 to spectrin▫Band 4.1: links glycophorin to actin and spectrin▫Actin

Erythrocyte Plasma Membrane

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● Spectrin:○ A long, thin, flexible rod ○ ~ 25% of the membrane-associated protein mass in RBC. ○ It is the principal component of the erythrocyte cytoskeleton

Erythrocyte Plasma Membrane● Glycophorin: provides an external negative charge that repels

other cells○ Heavily-glycosylated on extracellular portion

● Band 3: Anion exchange protein (Cl¯ and bicarbonate)○ Forms dimers

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From: Structure of the Plasma Membrane The Cell: A Molecular Approach. 2nd edition. Cooper GM. 2000.

Figure 10-41a Molecular Biology of the Cell (© Garland Science 2008)

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The Spectrin-Based Cytoskeleton

Clinical Connection #1A 9-month-old infant is brought to the pediatrician because of jaundice, lethargy, and easy fatigability. The parents of the child are immigrants of northern European origin. Physical examination reveals pallor, mild jaundice and palpable splenomegaly. Laboratory results indicate microcytic anemia with small, rounded, dark red blood cells lacking central pallor (spherocytes).

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1) What is the most likely diagnosis for this infant?

2) What is the molecular pathogenesis of this disorder?

Laboratory results indicate microcytic anemia with small, rounded, dark red blood cells lacking central pallor (spherocytes).● Microcytic anemia is a

generic term for any type of anemia characterized by small red blood cells.

● Spherocytes are almost spherical in shape. They are not biconcave like a normal red blood cell

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normal

normal

spherocyte

Clinical Connection #1

• The infant has Hereditary Spherocytosis▫ Most commonly caused by autosomal dominant mutations in

the Ankyrin-1 or the Spectrin genes.⚫~50% of cases are caused by mutations in the Ankyrin gene

▫ Can also result from inherited defects in the Band 3 or Protein 4.2 genes.

• Mechanism:▫ The mutation results in uncoupling of the lipid bilayer from the

underlying cytoskeleton. ▫ This causes the spherocyte formation. ▫ These abnormal cells are more rigid and cannot traverse the

spleen. ▫ The cells ultimately succumb to extravascular hemolysis.

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Clinical Connection #1

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● Pathogenesis : the primary defect in hereditary spherocytosis is a deficiency of membrane surface area. Decreased surface area may be produced by 2 different mechanisms:○ defects of spectrin, ankyrin, or protein 4.2 lead to reduced density of the

membrane skeleton, destabilizing the overlying lipid bilayer and releasing band 3-containing microvesicles.

○ defects of band 3 lead to band 3 deficiency and loss of its lipid-stabilizing effect. This results in the loss of band 3-free microvesicles.

HS: Additional Info (Optional)

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HS: Additional Info (Optional)● Both pathways result in membrane loss, decreased surface area, and formation of

spherocytes with decreased deformability. ○ These deformed erythrocytes become trapped in the hostile environment of the

spleen where splenic conditioning inflicts further membrane damage, amplifying the cycle of membrane injury.

○ The splenic environment is hostile to erythrocytes. Low pH, low levels of glucose and ATP, and high local concentrations of toxic free radicals produced by adjacent phagocytes all contribute to membrane damage.

Production of EicosanoidsThe lipid component of the membrane releases arachidonic acid

(Ω-6),which → to the formation of eicosanoids via the following processes:1. In response to physical injury or inflammatory response,

enzymes catalyze the breakdown of membrane lipid to arachidonic acid

2. Arachidonic acid may be converted to straight-chain eicosanoids called leukotrienes by lipoxygenase, or …

3. To cyclic eicosanoids called prostaglandins, prostacyclin and thromboxane by cyclooxygenases

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Arachidonic Acid is a Substrate for Lipoxygenase and Cyclooxygenase

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Inhibited by NSAIDS

NSAIDS: nonsteroidal anti-inflammatory drugs: aspirin, ibuprofen, naproxen.

Production of EicosanoidsCyclooxygenases COX-1 and COX-2 are isozymes:

■ Catalyze the same reaction but differ in AA sequence■ Kinetics and regulatory properties may differ.

● COX-1 produces eicosanoids used in many normal physiological process (the “good COX”)

● COX-2 produces eicosanoids used in inflammatory response (the “bad COX”)

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COX-1 and COX-2 Isozymes● COX-1: “housekeeping enzyme”, constitutively expressed ● COX-2 synthesis is induced in special cells

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selectiveArachidonic acid

Physiologic Prostaglandins

Pathologic Prostaglandins

COX-1 COX-2

COX-1 and COX-2 Isozymes● Prostaglandin synthesis by COX-1 results in mucus production

(important for stomach), regulation of kidney and water excretion

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COX-1 COX-2

Physiologic Prostaglandins

Pathologic Prostaglandins

● Cox-2 makes prostaglandins for signaling pain, inflammation.

● COX-2 production is stimulated by cytokines and growth factors.

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Clinical Correlation: COX & NSAID

● Aspirin (acetylsalicylic acid): a nonsteroidal anti-inflammatory drug (NSAID) that irreversibly inhibits cyclooxygenase is used clinically to ameliorate effects of myocardial infarction, inhibit platelet aggregation, ↓ pain, ↓ fever, and as a general anti-inflammatory agent.

● Ibuprofen and naproxen are NSAIDs that reversibly inhibit cyclooxygenase and are used clinically to ↓ pain, treat rheumatoid arthritis, and to treat osteoarthritis

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Remember: ObjectivesKnow: • Plasma membrane structure and function• Structure and function of specific components of a

membrane• The Fluid Mosaic Model• How eicosanoids are produced• Membrane asymmetry and the role of flippases• The effects of lipids & sterols on membrane fluidity• About lipid rafts• About the glycocalyx• The role of spectrin and ankyrinin the red blood cells • Clinical Correlations

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Objectives• Be familiar with the structure and function of the following

components of a membrane:▫ Lipids⚫Phospholipids: Phosphatidylcholine, sphingomyelin,

phosphatidylethanolamine & phosphatidylserine⚫Cholesterol

▫ Proteins⚫Integral, peripheral, and lipid anchored membrane

proteins▫ Carbohydrates⚫Glycolipids: cerebrosides and gangliosides⚫Glycoproteins⚫Both glycolipids and glycoproteins found facing on the

extracellular side of the bilayer

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