C2006/F2402 '09 -- Outline Of Lecture #3 -- Last update 01/26/2009 10:51 AM

2009 Deborah Mowshowitz, Department of Biological Sciences, Columbia University, New York NY

Handouts:       3A -- Freeze Fracture;  Cell-Cell Junctions &  ECM,
                         3B --  RBC Membrane,  Types of Membrane Proteins
& Role of Anion Exchanger

The video 'The inner life of the cell' -- short version with music -- is at http://www.studiodaily.com/main/searchlist/6850.html


I have started a new page with 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.

Note: Minor changes are sometimes made to the notes after the lecture. (For example, the url given above was added to lecture #2.) The time of the last update is always given at the start of the outline. All changes made after the lecture will be highlighted in blue  so you can find them easily. Anything significant will be noted on the corrections page.


I. Introduction to Membrane Structure
 

    A. The Big Question: What does the structure seen in the EM represent?

    B. Lipid part

1. Amphipathic nature of lipids  -- See Sadava fig. 5.2  -- 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 for EM picture; for fluid mosaic model see Becker fig. 7-5 or Sadava 5.1.

1. Use of freeze fracture/freeze etch 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 5.3 or bottom 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.

c. Freeze fracture vs Freeze etch

(1). Freeze fracture = crack frozen sample open, examine in EM;

(2). Freeze etch = crack open, let some water sublime off to expose deeper layers, then look in EM. For some sample pictures, see Becker figs. 15-16, 15-19 (15-21),  15-25 (15-26), & 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 5.1. Also handout 3B, top right corner.


II. Fluid Mosaic Model of Membrane Structure
 

    A. Fluid Part = Lipid bilayer

1. Formation of Bilayer  --  All amphipathic lipids form bilayers. In cell, all lipids are inserted from one side of the bilayer (side facing the cytoplasm). How do lipids get distributed?

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

3. Two sides of bilayer (leaflet) often have a different lipid composition.

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

1. Peripheral membrane proteins vs. integral membrane proteins

Type of Membrane Protein

Alt. terminology

Protein Removed From Membrane By

Location/Attachment of Protein

Peripheral 

Extrinsic 

salt, pH changes 

On one 1 side of bilayer; non covalently attached to lipid

Integral 

Intrinsic

disrupting lipid bilayer

Goes through bilayer* or Covalently attached to 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 5.1 (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 5.4 and/or Becker fig. 7-19 & 7-21)

a. Single pass vs multipass 

b. Domains -- intracellular, extracellular, transmembrane 

c. Location of carbohydrates -- all in extracellular domain

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

(2). Receptors -- Trap (bind) molecules on outside -- Allow transport of large molecules across membrane (by RME -- receptor mediated endocytosis) or transmission of signals.

(3). Linkers --  physically connect cytoskeleton (inside of cell) to materials on outside of cell (ECM = extracellular matrix) or to next cell.

Some transmembrane proteins act in more than one capacity.

III.  The Red Blood Cell (RBC) Membrane -- The best studied example of a Membrane. 

    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.

    B. RBC membrane proteins -- Structure & Function. See Sadava fig.4-23 (6th ed.) or Becker fig. 7-20 (b) & 15-20 (15-22). (Handout 3B -- middle)

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 

a. Examples

    (1). 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 48.14 or Becker 8-3.

(a). Why is transport of CO2 an issue? Tissues carry out metabolism and generate lots of  CO2 . The CO2 diffuses out of the cells into the blood. However the solubility of CO2 is limited.

(b). 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).

(c). 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, bottom panel.) Carbonic anhydrase catalyzes:

CO2 +  H2O HCO3- + H+

(d). 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.

(e). Physiological Function of Exchanger

i. 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.

ii. 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.

(f). Note on structure -- in picture on handout, anion exchanger looks like a channel allowing simple diffusion of bicarb and Cl- in and out.  Exchanger 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. 

(2). Single pass (glycophorin) -- function of protein not known.

(a). Large amount of (-) charged modified carbohydrate -- sialic acid -- 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.

(c). 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

Membranes of other cells are similar -- contain both single and multipass proteins; anchored and floating proteins. 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)

Note: Becker Chap. 17 goes well beyond what will be covered in this section. References to pictures and diagrams are included FYI. For a summary, see handout 3A, second panel from top.

    A. Major Structural Proteins -- collagen  -- nice picture in Becker fig. 17-13 (12-2) and elastin (diagram in Becker fig. 17-15 (17-4)).

    B. 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 pictures see Becker figs. 17-7 & 17-18 (17-6 & 17-7).

    C. 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.  17-16 (17-5). 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 description

Lots of carbohydrate attached to a protein Core;* Can be 95% carbohydrate

A protein with some Carbohydrate Attached

Are sugar chains branched? 

No

Yes

Length of Sugar Chain

Long

Short

Are sugars repeating?

Yes (repeating disaccharide); & sugars usually modified

No

Type of Carbohydrate

Mucopolysaccharide or GAG (glycosoaminoglycan)
Examples: heparin** , chondroitin sulfate#

Oligosaccharide

Example

See Becker fig. 17-16 (15-5).

Band 3 protein or glycophorin

Location

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

Integral membrane protein (carbohydrates on extracellular domain)

* Multiple proteoglycans can be attached to a core carbohydrate chain (GAG or  mucopolysaccharide)  to form a giant aggregate as shown in Sadava fig. 4-25 (4-26) or Becker fig. 17-16 (17-5). 

** 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. Is used to remove fat particles from blood after a fatty meal.)

#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.

    D. Basal Lamina (see Becker 17-19 (17-8))  -- 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. 17-20 (17-9)

2. Location -- surrounds some cells (skeletal muscle, fat) and underlies some epithelial layers (on basal 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.

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

5. How Connected to cells -- see integrins and hemidesmosomes below.

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

V. Cell-Cell (& Cell-ECM) Connective Structures  -- For Pictures see Sadava 5.7 (5.6), & Becker Ch. 17 -- exact figures listed below.  For comparisons, see table below and/or Becker table 17-1 (17-3).

    A. Basic types of Cell-Cell Junctions -- for overall view, see Becker fig 17-7 (17-17). For the molecular details (FYI only) see fig. 17-3 (17-13). For diagrams, see handout 3A, top.

        1. Gap junctions -- Becker fig. 17-11 (17-21); Sadava  5.7 (5.6) & 15.19 (15.16). 

        2. FYI: Plasmodesmata -- Sadava 15.20 (15.17) or Becker 17-26 (17-25). The plant's equivalent of a gap junction. 

        3. Tight Junctions -- Becker fig. 17-9 (17-19)    

        4. Adhesive (anchoring) junctions  See Becker fig. 17-8 (17-18) & 17-3 (17-13) for molecular details (for your interest only). For a nice EM picture see http://trc.ucdavis.edu/mjguinan/apc100/modules/Integument/_index.html (click on generic desmosome). For diagram of adherens junction see picture from Alberts. Also has nice pictures of desmosomes, gap junctions, etc. Two main types:

            a. Desmosomes = Spot Junctions (with IF)

            b. Adherens junctions = belt junctions (with MF)

    B. Summary Table of Animal Cell-Cell Junctions

Name of Junction

Important Structural Features

Function

Gap Junction

Connexons;  small gap between cells (2-4 nm)

Passage of small molecules and ions (signaling & nutrition)

Tight Junction

Fusion of ridges of membrane -- no gap at ridge.

Water Tight Seal between cells; divide membrane into regions

Adhesive or Anchoring
Junctions

Intracellular Plaques with filaments. Classified by:
    (a) Shape of plaque: spot vs belt
    (b) Type of filaments:
        IF (desmosomes) vs MF (adherens junctions)
Cell/cell protein connectors = cadherins
Larger gap between cells (25-35 nm)

Strength

    C. Cell - ECM junctions.  Connect cytoskeleton to ECM or solid support.  Resemble half of an adhesive junction (adherens junction or desmosome). See handout 3A, 3rd panel from top. (Also Becker Fig. 17-22 (17-11).  For another diagram see http://celljunctions.med.nyu.edu/images/figure1.gif. For a nice EM picture see http://trc.ucdavis.edu/mjguinan/apc100/modules/Integument/skin/hemidesmosome/hemidesmosome2.html

1. Similarities to cell -cell adhesive junctions

a. Transmembrane protein connected (indirectly) to IF or MF on inside of cell.

b. Linker proteins involved -- linker proteins connect IF or MF to transmembrane protein.

2. Differences from cell-cell adhesive junctions

a. Transmembrane protein is an integrin, not a cadherin (See Becker fig. 17-21 (17-10) for a diagram of structure of integrin.) Integrins are a family of proteins with roles in signaling, movement, etc. as well as adhesion.

b. Transmembrane protein connects to ECM. (Not to extracellular domain of protein from another cell. )

3. Two Types.

a. Hemidesmosomes  = "half desmosome";  connect to IF on inside of cell. Connect to basal lamina on outside. Have plaque (thickening visible in EM) formed by linker proteins on inside of cell. Usually found in epithelial cells.

b. Focal Adhesions  = "half adherens junction" --  connect to MF on inside of cell and ECM (can be on a solid support) on outside. Usually found in migrating cells.

At this point, it is a good idea to make a chart for yourself that classifies (or compares and contrasts) all the types of junctions, whether cell-cell or cell-ECM. Also try problems 1-12 & 1-14.

Next time:  What does a real cell look like? Where are the junctions, etc.?