Membrane Transport

Learning objectives

• Understand basic structure of cell membrane

  • Components

  • Organization

  • Fluidity/motion

• Understand and explain the fundamental experiments that led to our current understanding of the structure of cell membranes

Fundamental biological discoveries

• End up in the textbooks

• Apply to most/all cells

• Hold up over time

• Are replicated and built upon, even in specialized systems

Earliest model: Lipid monolayer

• Oil forms a lipid monolayer at the air-water interface

• Langmuir trough

  • Water-filled trough with movable barrier

  • Known amount of oil is added

  • Barrier slides, increasing area covered by oil until layer is one molecule thick

  • Forms droplets when overextended

RBCs

• Size

• Simplicity

  • No internal membranes

• Easy to collect

RBC “ghosts”

• Place RBCs in hypotonic solution

• Cell swells and bursts

• Wash and reseal ghosts

• End product is cell membrane with nothing inside

  • Phospholipids

  • Proteins

  • Carbohydrates

Lipid bilayer model

• Known number of RBCs turned into ghosts

• Calculate total surface area

• Extract lipids from ghosts with organic solvent

• Poured gently into Langmuir trough

• Area of monolayer/Area of RBC = 2

• RBC must have a lipid bilayer

• Made several mistakes that canceled out

  • Underestimated surface areas of both monolayer and RBCs

Arrangement of phospholipids

• Polar heads are arranged on the outside of the cell with nonpolar layer inside

• Energetically favorable for nonpolar layer to form sealed spherical bilayer

Visualizing with electron microscopy

• Has resolution needed for nanometer level resolution of ultrastructure

• Saw “railroad track” structure in 1950s-60s after staining with heavy metals

• About 8 nm apart

• Robertson, 1959

  • Railroad track structure consistent across a variety of cells

  • Called “Unit Membrane”

What else is in the cell membrane?

• Surface tension was lower than pure oil-water interface

• Something else must be present to reduce surface tension

  • Proteins

Sandwich model

• Was wrong

• Danielli and Davidson, 1935

• Membrane proteins on top of lipid bilayer with protein-lined pores

• Robertson thought EM images were consistent with this model

• Hypothesized heavy metals were binding to proteins

Problems with sandwich model

• Didn’t allow for fluidity of membrane

• Artificial membranes with no proteins still showed “railroad track” structure

• Different ratios of protein:lipid found in different membranes

• Many proteins known to be associated with membranes were known to have large hydrophobic sections

How are proteins arranged in the cell membrane?

• Biochemical approach

  • Create ghosts without resealing

    • Creates leaky ghost

  • Sonicate and shear with Mg

    • Reseals small and right-side out

  • Sonicate and shear without Mg

    • Reseals small and inside-out

  • Add a membrane impermeant enzyme that adds radioactivity to all proteins it contacts

  • Analyze samples by SDS-Page

  • Results

    • Some proteins get labeled on all sides of membrane

      • Must span the membrane

    • Some proteins get labeled on one side of membrane or the other

      • Must be peripheral proteins

• Microscopy approach

  • Freeze RBCs in liquid nitrogen

  • Use a knife edge to cut

    • Fracture propagates along hydrophobic interface, separating the two monolayers

  • Spray gold or platinum on inner leaflet, then use bleach to dissolve biological material

  • Bumps bigger than phospholipid head groups are visible

    • These are proteins

Do proteins move in the cell membrane?

• Microscopy approach

  • Take a cell from one species and a cell from a different species

    • Can be done in living cells

  • Add glycerol to medium to encourage cells to fuse

    • Forms heterocaryon

  • Add fluorescently-conjugated antibodies

    • At time = 0, should have each color on original half of fused cell

    • After incubation, if motion is occurring, signals should mix

    • At lower temperatures, mixing should not occur

• FRAP

  • Fluorescence recovery after photobleaching

  • Bleach tag with laser beam in a certain area

  • Watch to see how quickly fluorescence recovers

  • Slope shows motion of protein

• Why are some proteins less mobile?

  • Proteins may aggregate

  • Might attach to ECM

  • Might attach to cytoskeleton

  • Might interact with another cell

• FLIP

  • Fluorescence loss in photobleaching

  • Bleach one area, measure in a different area

  • If area loses signal, bleached cells are migrating into the area

How do we account for motion in the membrane?

• Fluid mosaic model

  • Lipid bilayer with hydrophilic and hydrophobic parts

  • Proteins can interact with surface through transient polar contacts

  • A lot of proteins are partially or totally embedded in the lipid bilayer

• Synthesized experimental facts

  • Permeability and transport studies that predicted enzyme-like transmembrane proteins

  • EM pictures like freeze-fracture identifying membrane proteins

What is in the cell membrane?

• Phospholipids

  • Polar head group

  • Nonpolar tails

    • Cis-double bond

      • Allows for movement

  • Phosphatidylethanolamine

  • Phosphatidylserine

  • Phosphatidylcholine

  • Sphingomyelin

  • Sphingosine

  • Head groups can:

    • Affect membrane protein functions

    • Promote recruitment/concentration of cytosolic proteins

  • Not randomly distributed

    • Lipid composition for each monolayer is different in many membranes

• Cholesterol

  • Decreases mobility of hydrocarbon chains

    • Lipid bilayer is less deformable

    • Lipid bilayer is less permeable to small water-soluble molecules

• Carbohydrates

  • Oligosaccharide chains covalently bound to proteins and lipids

  • Glycocalyx

    • Carbohydrate-rich zone on cell surface

    • Detected by staining w/ ruthenium red or lectins

    • Contains some secreted glycoproteins and proteoglycans that adsorb onto cell surface

    • Functions:

      • Protect against mechanical and chemical damage

Problems with fluid mosaic model

• Assumes a uniform lipid bilayer randomly studded with floating proteins

Lipid raft model

• Preferential association b/t sphingolipids, cholesterol, and specific proteins bestows cell membranes lateral segregation potential

  • Cholesterol allows lipid rafts to form by decreasing fluidity

  • Held together with weak bonds

• Confers functional signaling properties to cell membranes

• Acts as another way to anchor a protein in the membrane

  • Helps to organize proteins in the membrane

    • Concentrate proteins for transport in small vesicles

    • Help proteins work together to convert extracellular signals to intracellular signals

• Domain-induced budding

  • Proteins associate with rafts to various extents

  • Clustering is induced

  • Scaffolded raft-associated proteins form a raft cluster