Membrane Transport

What are the components of a cell membrane?

Cell membranes contain a phospholipid bilayer, cholesterol, and carbohydrates

What are the characteristics of the phospholipid bilayer?

1. Polar head groups facing out and nonpolar tails facing in

2. Cis double bond allows for membrane fluidity

3. Nonrandom composition

What is the function of cholesterol?

Decreases fluidity and permeability of cell membrane

What is the glycocalyx and what is its purpose?

Carbohydrate-rich zone on cell surface that offers protection, helps in cell signaling, and affects ion concentration at cell surface

What are the five main types of phospholipids present in cell membranes?

Phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, sphingomyelin, sphingosine

What is the lipid monolayer model?

1. Earliest cell membrane model

2. Determined using Langmuir trough

What is a Langmuir trough?

A wooden rectangular box with water on one side and oil on the other. The lipid layer is slowly poured into the oil side and the barrier is moved until the layer is a single cell in thickness. The surface area of the rectangle can then be measured.

How was the lipid bilayer model formed?

1. A known number of RBC ghosts were formed

2. The total surface area of the ghosts was calculated using the geometry of a disk

3. Lipids were extracted from ghosts using organic solvents

4. Surface area of lipids was calculated using Langmuir trough

5. Monolayer SA was twice that of ghost SA, meaning the ghosts must have a lipid bilayer

6. These results were replicated across multiple species

What mistakes were made when forming the lipid bilayer model?

1. RBCs are biconcave, not disks (underestimated ghost SA)

2. Centrifuges did not fully extract lipids (underestimated monolayer SA)

3. These two mistakes cancelled each other out

What is the sandwich model?

Membrane proteins coat cells and create protein-lined pores. It was hypothesized that heavy metals used in imaging were binding to these proteins

What were the problems with the sandwich model?

1. Didn’t allow for membrane fluidity

2. The “railroad track” structure seen in imaging was still present with artificial membranes made w/o proteins

3. Different protein: lipid ratios found in different membranes

4. Many proteins known to be associated with membranes contained hydrophobic regions

How was the unit membrane model developed?

Electron microscopy determined “railroad track” structure seen was consistent across a variety of cells, meaning this membrane type must be universal

What is the fluid mosaic model?

1. Lipid bilayer w/ hydrophilic and hydrophobic parts

2. Proteins can interact w/ surface through transient polar contacts

3. Proteins can either be partially or totally embedded in lipid bilayer

What is the lipid raft model?

1. Lipid rafts are formed in the membrane through preferential association of sphingolipids, cholesterol, and proteins

2. Proteins can be grouped together for signaling

3. Held together with weak interactions

How are RBC ghosts formed?

Red blood cells are placed in a hypotonic solution, causing them to burst. The cells are then washed and resealed to leave behind only the cell membrane.

Why are RBCs ideal for studying cell membranes?

Red blood cells are simple, containing no membrane-bound organelles. They are also readily available.

How would one create a leaky RBC ghost?

After lysing, wash only. Do not reseal.

How would one create an inside-out vesicle?

After lysing, sonicate ghosts and shear without magnesium

How would one create a right-side-out vesicle?

After lysing, sonicate ghosts and shear with magnesium

Describe the steps in freeze-fracture microscopy.

1. Freeze cells in liquid nitrogen

2. Use a sharp knife to carefully cut along hydrophobic region of membrane

3. Spray with gold or platinum and image

4. Any bumps bigger than phospholipid heads are proteins. Adding a protease can confirm.

Describe cell fusion

1. Fuse together two cells from two different species in glycerol to form heterocaryon

2. Add fluorescently-conjugated antibodies to detect specific proteins

3. Incubate at biological temperature.

4. After incubation, mixing of signals will occur if proteins are mobile

Describe the FRAP method

Fluorescence Recovery After Photobleaching measures the recovery rate of fluorescence in a photobleached area. If the area recovers fluorescence, unbleached proteins are migrating into that area.

Describe the FLIP method

Fluorescence Loss In Photobleaching measures the loss of fluorescence in one area after photobleaching in a different area. If the measured area loses fluorescence, then bleached proteins are migrating into that area.