Outline of Lecture 3

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C2006/F2402 '14 -- Outline Of Lecture #3 -- Last update 01/29/2014 10:10 AM -- Two clarifications were added after the livelectures. They are in blue.

(c) 2014 Deborah Mowshowitz, Department of Biological Sciences, Columbia University, New York NY

Handouts*: 3A -- Freeze Fracture; Types of Membrane Proteins 3B -- RBC Membrane, RBC -- Role of Anion Exchanger 3C -- ECM (Extracellular Matrix)

Pictures (Power Point Slides) shown at the start of the lecture are on Courseworks for registered students.

The main menu page includes a link to the web-sites page. This page has links to web sites that you may findinteresting and/or helpful. The web sites contain animations, explanations, pictures etc. that are relevant to this course. (The list is not complete; I'll add to it as we go.) I will add specific links in the lectures, but you may want to exploresome of the sites on your own. Please let me know if any of the web sites are useful, and/or if you find any other goodones.

I. Introduction to Membrane Structure

A. The Big Question: What does the structure seen in the EM represent? For possibilities, see Becker fig. 7-3. Foran EM picture, see PPt slides, slide #1 or Becker, fig. 7-4.

B. Lipid part

1. Amphipathic nature of lipids -- See Sadava fig. 6.2 -- there are multiple different "two headed" lipids -- each type has a different structure, but each has a hydrophobic end and hydrophilic end.

2. Amphipathic Lipids form a bilayer.

C. Protein part -- where are the proteins (relative to the lipid)? Is it a "unit membrane" or a "fluid mosaic?"

For "unit membrane" See Becker fig. 7-4 or PPT slide #1 for EM picture; for fluid mosaic model seeBecker fig. 7-5 or Sadava fig. 6.1.

1. Use of freeze fracture procedure

a. E vs P faces of bilayer = surfaces you see if you crack bilayer open = inside of bilayer

(1). E face = inside of the monolayer that is closer to extracellular space (outsideof cell)

(2). P face = inside of the monolayer that is closer to protoplasm (inside of cell)

b. What do you see on inside? See Becker fig. 7-16 & 7-17 or Sadava fig. 6.4 or top panelon handout 3A.

(1) Inside is not smooth -- shows proteins go through bilayer (implies "mosaic"model not unit membrane)

(2). More bumps (proteins) on P face than E face -- shows more proteinsanchored on cytoplasmic (protoplasmic) side.

2. Freeze fracture vs Freeze etch

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a. Freeze fracture = crack frozen sample open, examine in EM;

b. Freeze etch = crack open, let some water sublime off to expose deeper layers, then look inEM. For some sample pictures, see Becker figs. 15-15 (15-16), 15-18 (15-19), 15-24 (15-25),& 16-1.

D. Fluid mosaic model -- overview of current idea of how proteins and lipids are arranged. See Becker fig. 7-5 (or7-3) or Sadava fig. 6.1. Also handout 3A, middle of top panel.

II. Fluid Mosaic Model of Membrane Structure

A. Fluid Part = Lipid bilayer

1. Formation of Bilayer -- All amphipathic lipids form bilayers. In cell, virtually all lipids are insertedfrom one side of the bilayer of the ER (side facing the cytoplasm). How do lipids get to the other half ofthe bilayer?

2. Lateral diffusion vs. flip-flop of lipids. See Becker 7-10 & 7-11.

lateral diffusion = movement within plane of membrane -- fast (secs). Animation of lateraldiffusion. Also see Sadava fig. 6.5 or Becker fig. 7-28 & 7-29.

flip-flop = movement from one side of bilayer to the other -- slow (hrs) w/o enzymes. Enzymes(flipases = phospholipid translocators) are needed to speed flip-flop. (More details when we get totransport.)

3. Two sides of a bilayer often have a different lipid composition. (One side = 1/2 of bilayer = a leaflet.)

B. Mosaic Part = Protein. Types of Membrane Proteins -- what do you get if you take a membrane apart? Seehandout 3A, bottom panel.

1. Peripheral membrane proteins vs. integral membrane proteins

Type ofMembraneProtein

Alt. terminology Protein RemovedFrom Membrane By Location/Attachment of Protein

Peripheral Extrinsic salt, pH changes On one 1 side of bilayer; non covalently attachedto lipid

Integral Intrinsic disrupting lipid bilayer Goes through bilayer* or Covalently attached tolipid on one side (Lipid-anchored)**

* A small number of integral proteins do not go all the way through the membrane; they will be largely ignored in thiscourse. For examples see Becker fig. 7-19 (first protein on left) or Sadava fig. 6.1 -- last protein on right.

**Note that lipid-anchored proteins can be considered a type of integral protein or a separate category. See Becker fig.7-19.

2. Transmembrane proteins of plasma membrane (See Sadava fig. 6.3 and/or Becker fig. 7-19 & 7-21)

a. Single pass vs multipass

b. Domains -- intracellular, extracellular, transmembrane. Note: For a multipass protein, eachindividual section or stretch of polypeptide (transmembrane, extracellular, or intracellular) is

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usually considered a separate domain. See answer to problem 1-20. All the extracellular or allthe intracellular domains may cluster together, but the term 'domain' is not usually used forthe entire extracellular (or intracellular) part of the protein.

c. Location of carbohydrates -- all in extracellular domain (all added inside EMS)

d. Anchorage -- Some proteins are anchored to cytoskeleton; some float in lipid bilayer.

e. Types & Functions -- All bridge the membrane but function differs. Can be:

(1). Transport proteins -- Allow transport of small molecules in and out of cells.('pumps' or 'doors'.)

(2). Receptors -- Trap (bind) molecules on outside. Then receptor can facilitate:

(a). Transport -- By invagination of membrane. Trapped large molecules aretransported into cell in a vesicle (by RME -- receptor mediated endocytosis).Example: Receptor for LDL (low density lipoprotein -- a carrier for cholesterol).

(b). Transmission of signals -- relays signals to inside of cell from trappedmolecule on outside of cell. Example: Receptor for Acetyl Choline (aneurotransmitter).

(c). Both -- can facilitate both transmission of signal and internalization ofsignal molecule. Typical Examples: Receptors for growth factors. Specificexample: Receptor for EGF. (EGF = Epidermal Growth Factor)

(3). Connectors -- physically connect cytoskeleton (inside of cell) to materials onoutside of cell (ECM = extracellular matrix) or to next cell. Example: cadherins(connect cells) & integrins (connect cells and ECM). More on this next time.

(4). More than one of the above -- some transmembrane proteins act in morethan one capacity. Examples: Integrins (connectors & act in signaling).

III. The Red Blood Cell (RBC) Membrane -- The best studied example of a Membrane. For picturesof RBC see the PPt slides for lecture #3.

A. Why RBC's

1. Easy to get

2. No internal membranes -- all organelles lost during maturation of human RBC -- see Becker fig. 7-20(a). Only membrane = plasma membrane.

3. Can make 'ghosts' = resealed plasma membranes. Can be resealed (or broken and reformed intovesicles) in either orientation -- "right" or "wrong" side out. See PPt slides, slide #4.

B. RBC membrane proteins -- Structure & Function. See Becker fig. 7-20 (b) & 15-19 (15-20). (Handout 3B --top)

1. Peripheral proteins -- spectrin, ankyrin, (band 4.1), actin. Comprise peripheral cytoskeleton, whichsupports membrane. All cells are thought to have a similar structure under the plasma membrane.

2. Intrinsic proteins -- Two basic kinds -- single pass & multipass.

a. Example of RBC single pass -- glycophorin -- function of protein not known.

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(1). Has large amount of (-) charged modified carbohydrate -- sialic acid.Possible functions:

(a). Neg. charge may cause RBC to repel each other and preventclumping of RBC.

(b). Loss of terminal sugars may occur with age and triggerdestruction of "old" RBC.

(2). Glycophorins make up a gene family; variations in glycophorin A are responsible for MN blood type differences. Variations in glycophorin C arecorrelated with resistance to malaria.

b. Example of RBC Multipass -- band 3/anion exchanger -- Catalyzes reversible exchangeof the anions HCO3

- (bicarb) and Cl- between RBC and plasma. Exchange allows max.transport of CO2 in blood (as bicarb in solution). See Sadava fig. 49.14 or Becker 8-3.

(1). Why is transport of CO2 an issue? Tissues carry out oxidative metabolismand generate lots of CO2 . The CO2 diffuses out of the cells into the blood.However the solubility of CO2 in plasma (cell-free liquid portion of the blood) islimited.

(2). Basic idea: Bicarb is much more soluble in plasma than CO2, so lots ofbicarb (but not much CO2) can be carried in the blood. Therefore need to covertCO2 to bicarb when want to carry CO2 in blood; need to do reverse to eliminatethe CO2 (in lungs).

(3). Role of Carbonic anhydrase: Conversion of CO2 to bicarb (& vice versa)can only occur inside RBC, where the enzyme carbonic anhydrase is. (Seehandout 3B, middle panel.) Carbonic anhydrase catalyzes:

CO2 + H2O ↔ HCO3- + H+

(4). Role of exchanger: Gases can pass through membranes by diffusion -- CO2can exit or enter RBC as needed. However bicarb cannot pass throughmembranes. You need the anion exchanger to get bicarb in and out of RBC.

(5). Physiological Function of Exchanger

(a). Where CO2 is high, as in tissues, CO2 diffuses into RBC and isconverted to bicarb inside the RBC. (Reaction above goes to right.)Then bicarb leaves RBC in exchange for chloride using the anionexchanger.

(b). In lungs, the process is reversed -- bicarb reenters the RBC inexchange for chloride using the anion exchanger. The bicarb isconverted back to CO2 inside the RBC (reaction above goes to left).Then the CO2 diffuses out of the cells and is exhaled.

(c). To be sure you have this straight, fill in the spaces in the table

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below with 'in' or 'out'. For RBC in each location, does the substance(CO2, bicarb or Cl-) go in to the RBC or out of the RBC?Note: In the table, fill in what happens in the RBC, not what happensin the tissue or lung cells. Also note that RBC are always in theblood, inside blood vessels. 'RBC in the lungs' means 'the RBC insidethe blood vessels that pass through the lungs.'

Location of RBC

Lungs TissuesCO2

Bicarb

Cl-

(6). Note on structure & terminology -- in picture on handout, anion exchangerlooks like a channel allowing simple diffusion of bicarb and Cl- in and out. (It iscalled a channel in some earlier literature.) Exchanger is actually more complex -- has moving parts and movement of each ion depends on movement of the other.More details on this & other types of transport proteins next time.

C. Proteins of Other Membranes -- Membranes of other cells are similar. In other membranes:

Find proteins of same protein families as in RBC, as well as entirely different proteins.

Find both intrinsic and peripheral proteins

Intrinsic/integral proteins are both single and multipass proteins; anchored and floating.

Different membrane proteins are found in different cell types.

Try problems 1-2 & 1-3. To review membrane structure, try 1-15 to 1-17 & 1-20.

IV. Extracellular Matrix (ECM) See handout 3C or PPt slide #5.

Note: Becker Chap. 17 goes well beyond what will be covered in this section. References to pictures and diagrams areincluded FYI. For the picture in ppt slide #5 go to http://wiki.pingry.org/u/ap-biology/images/5/52/Image122.gif. Thedark purple 'worms' in the picture are adhesive proteins, such as fibronectin. (Alternatively, search Google images forextracellular matrix. You will see several versions of this picture.)

A. Where do components of the ECM come from? All these components are made inside the cells, and thensecreted -- details of secretion later.

B. What are the major components of the ECM (of animal cells)? Listed on handout 3C, top panel. For a pictureof the ECM, see the bottom panel.

1. Structural proteins. Major ones are

collagen -- nice picture in Becker fig. 17-14 (17-13)

elastin -- diagram in Becker fig. 17-16 (17-15).

2. Adhesive Glyco-Proteins -- fibronectins, laminins, etc. Have multiple binding domains. Connect othermaterials in ECM with each other and/or connect to extracellular domains of transmembrane proteins. For structures

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see Becker figs. 17-18 (17-17) for fibronectin & 17-21 (17-20) for laminin. For role of fibronectin in guidingmigrating cells, see Becker fig. 17-19 (17-18).

3. Proteoglycans -- special type of glycoprotein consisting of lots of carbohydrate attached to a protein core.Provides a gel-like matrix for ECM. See Becker fig. 17-17 (17-16) or handout 3C, bottom panel. Table below is forreference purposes only so you can follow the terminology. See http://themedicalbiochemistrypage.org/glycans.html for a nice web site with a summary of structure, function, andmedical significance of proteoglycans and GAGs.

Proteoglycan (mucoprotein) Glycoprotein

Generaldescription

Lots of carbohydrate attached to aprotein core;* Can be 95% carbohydrate

A protein with some carbohydrateattached

Are sugarchainsbranched?

No Yes

Length ofSugar Chain

Long Short

Are sugarsrepeating?

Yes (repeating disaccharide); & sugarsusually modified

No

Name ofCarbohydrate

Mucopolysaccharide or GAG#(glycosoaminoglycan)

Oligosaccharide

Example(s)# See Becker fig. 17-17 (17-16). Band 3 protein or glycophorin

Location Extracellular matrix (form gel);important in knees and other joints.

Integral membrane protein(carbohydrates on extracellulardomain)

* Multiple proteoglycans can be attached to a core carbohydrate chain (GAG or mucopolysaccharide) to form a giantaggregate as shown on handout 3C or in Sadava fig. 5-22 (5-25) or Becker fig. 17-17 (17-16) or http://www.in-vivo-health.co.uk/image/Proteoglycan%20Aggregate2.jpg

#Name of GAG depends on the sugars in the chain. See figure 17-17 in Becker. Examples: Heparin -- Widely used as an anticoagulant. Inhibits factor required for blood clotting. (Physiological role, meaningreal job in body, may or may not involve inhibition of blood clotting.) Similar to heparan sulfate shown in 17-17. Chrondroitin sulfate -- Often recommended as a dietary supplement (plus glucosamine) in treatment of arthritis.Recent results indicate it may be helpful in a small group of patients but is not a panacea.

C. Connection of ECM to cytoskeleton -- ECM often connected to transmembrane proteins called integrins.Integrins link ECM and cytoskeleton. More details below & next time.

D. Basal Lamina -- see Becker 17-20 (17-19) -- important part of ECM

1. Structure -- Solid layer found in parts of ECM. Main components are networks of laminin & collagen.For structure of laminin, see Becker fig. 17-21 (17-20)

2. Location -- surrounds some cells (skeletal muscle, fat) and underlies some epithelial layers (on basal orbody side). More details of epithelia next time.

3. Terminology -- Also called basement membrane especially in older literature. Has no lipid & is not areal membrane.

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4. Function -- physical barrier, support and/or filter.

5. How Connected to cells -- through integrins.

Next time: Cell-Cell (& Cell-ECM) Connective Structures Then, what does a real cell look like? Where are theconnective structures?