Outline of Lecture 3

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

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 find interesting 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 explore some 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 good ones.

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. For an 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 see Becker 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 (outside of 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 panel on 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 proteins anchored 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 in EM. 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 (or 7-3) 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 inserted from one side of the bilayer of the ER (side facing the cytoplasm). How do lipids get to the other half of the 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 lateral diffusion. 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 to transport.)

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? See handout 3A, bottom panel.

1. Peripheral membrane proteins vs. integral membrane proteins

Type of Protein Removed Membrane Alt. terminology Location/Attachment of Protein From Membrane By Protein

On one 1 side of bilayer; non covalently attached Peripheral Extrinsic salt, pH changes to lipid Goes through bilayer* or Covalently attached to Integral Intrinsic disrupting lipid bilayer lipid 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 this course. 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, each individual 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 all the intracellular domains may cluster together, but the term 'domain' is not usually used for the 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 are transported 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 trapped molecule on outside of cell. Example: Receptor for Acetyl Choline (a neurotransmitter).

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

(3). Connectors -- physically connect cytoskeleton (inside of cell) to materials on outside 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 more than one capacity. Examples: Integrins (connectors & act in signaling).

III. The Red Blood Cell (RBC) Membrane -- The best studied example of a Membrane. For pictures of 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 into vesicles) 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, which supports 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. file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect3.14.html[1/29/2014 10:18:17 AM] Outline of Lecture 3

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

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

(b). Loss of terminal sugars may occur with age and trigger destruction 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 are correlated with resistance to malaria.

b. Example of RBC Multipass -- band 3/anion exchanger -- Catalyzes reversible exchange - - of 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 metabolism and 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) is limited.

(2). Basic idea: Bicarb is much more soluble in plasma than CO2, so lots of bicarb (but not much CO2) can be carried in the blood. Therefore need to covert CO2 to bicarb when want to carry CO2 in blood; need to do reverse to eliminate the 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. (See handout 3B, middle panel.) Carbonic anhydrase catalyzes:

- + CO2 + H2O ↔ HCO3 + H

(4). Role of exchanger: Gases can pass through membranes by diffusion -- CO2 can exit or enter RBC as needed. However bicarb cannot pass through membranes. 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 is converted to bicarb inside the RBC. (Reaction above goes to right.) Then bicarb leaves RBC in exchange for chloride using the anion exchanger.

(b). In lungs, the process is reversed -- bicarb reenters the RBC in exchange for chloride using the anion exchanger. The bicarb is converted 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 happens in the tissue or lung cells. Also note that RBC are always in the blood, inside blood vessels. 'RBC in the lungs' means 'the RBC inside the blood vessels that pass through the lungs.'

Location of RBC

Lungs Tissues

CO2 Bicarb Cl-

(6). Note on structure & terminology -- in picture on handout, anion exchanger looks like a channel allowing simple diffusion of bicarb and Cl- in and out. (It is called 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 are included FYI. For the picture in ppt slide #5 go to http://wiki.pingry.org/u/ap-biology/images/5/52/Image122.gif. The dark purple 'worms' in the picture are adhesive proteins, such as fibronectin. (Alternatively, search Google images for extracellular 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 then secreted -- details of secretion later.

B. What are the major components of the ECM (of animal cells)? Listed on handout 3C, top panel. For a picture of 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 other materials 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 guiding migrating 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 for reference 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, and medical significance of proteoglycans and GAGs.

Proteoglycan (mucoprotein) Glycoprotein General Lots of carbohydrate attached to a A protein with some carbohydrate description protein core;* Can be 95% carbohydrate attached Are sugar No Yes chains branched? Length of Long Short Sugar Chain Are sugars Yes (repeating disaccharide); & sugars No repeating? usually modified Name of Mucopolysaccharide or GAG# Oligosaccharide Carbohydrate (glycosoaminoglycan) Example(s)# See Becker fig. 17-17 (17-16). Band 3 protein or glycophorin Location Extracellular matrix (form gel); Integral membrane protein important in knees and other joints. (carbohydrates on extracellular domain)

* Multiple proteoglycans can be attached to a core carbohydrate chain (GAG or mucopolysaccharide) to form a giant aggregate 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, meaning real 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 or body side). More details of epithelia next time.

3. Terminology -- Also called basement membrane especially in older literature. Has no lipid & is not a real 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 the connective structures?

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