Lecture 2 The plasma membrane

Plasma membranes: 1. Lipid composition of cell membranes 2. Singer and Nicolson fluid mosaic model 3. Proteins associated with the plasma membrane can be integral or peripheral 4. Functions of membrane proteins 5. Microdomain organization of the plasma membrane 6. FRAP: A method for measuring diffusibility in the membrane

Cell membranes surround all cellular compartments Functions 1. compartmentalization 2. scaffolding for biochemical activities (e.g. energy production) 3. selective transport 4. reception of stimuli from the environment 5. interactions with neighboring cells (e.g. adhesion)

Composition of cell membranes Observation by electron microscopy (1960s) revealed an electron-dense bilayer separated by a clear layer (lipids) 2

Structure of phospholipids in cell membranes hydrophillic head choline phosphate glycerol non-polar hydrophobic tail images from Nature Education website

Cell membranes are bilayers of amphipathic lipids

The chemical nature of membrane lipids allow them to self-assemble into membranes

The chemical nature of membrane lipids allow them to self-assemble into membranes Detergents dissociate plasma membranes

The cell membrane is a dynamic structure that is constantly renewed ER Membrane is made in the ER and added to the plasma membrane by vesicle fusion. When vesicles fuse to the plasma membrane their lipids are incorporated into it. Endocytosis brings membrane back into the cell.

The chemical synapse: An extremely dynamic membrane region

Intracellular membranes Membranes that form organelles have the same basic plasma membrane structure. However, the specific composition of lipids and proteins differs significantly between organelles and from cell to cell

Cell membranes contain many chemically distinct kinds of lipids with different properties

Different membranes in a cell contain different complements of lipids van Meer G, Voelker DR, Feigenson GW (2008). Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol. 9(2):112-24.

How do different lipids end up in different membranes? ER Selective transport: Lipids made in the ER or incorporated from the outside are trafficked to different destinations in a cell.

How do different lipids end up in different membranes? PI3kinase Lipids can be phosphorylated or dephosphorylated to endow their head groups with different chemical properties

How do different lipids end up in different membranes? Lipids can be cleaved by lipases

How do different lipids end up in different membranes? Flippases, floppases and scramblases are enzymes that use energy from ATP to exchange lipids from one leaflet to another Clark, MR (2011). Flippin' lipids. Nature Immunology 12: 373 375.

How do different lipids end up in different intracellular membranes? Newly formed lipids are transported to specific locations Lipids can be covalently modified; for example, they can be phosphorylated, dephosphorylated or cleaved Lipids can be flipped from one leaflet to another PI3kinas

Lipids and proteins in the membrane can diffuse laterally-- fluid mosaic Singer and Nicolson model

Singer and Nicolson (1972) Science

Temperature affects the fluidity of the lipid bilayer Above the transition temperature, the molecules of the lipid bilayer are free to move. Below the transition temperature, the movement of molecules is reduced and restricted.

Membranes contain many associated proteins and carbohydrates Singer and Nicolson model 1972

Proteins can be associated with membranes in several different ways Transmembrane proteins Peripheral proteins Lipid - anchored proteins

Integral membrane proteins can be recognized from their amino acid sequences Transmembrane domains are often alpha helices consisting of hydrophobic amino acid residues Regions of hydrophobicity can be visualize in a Kyte-Doolittle plot

Proteins can be covalently conjugated to lipids Lipid modifications regulate cell signaling by targeting intracellular proteins to cell membrane 16

An experimental approach for deducing the nature of protein association with plasma membrane How are proteins associated with red blood cell membranes?

Studying proteins of the plasma membrane Solubilize the membrane and run proteins on a gel

Studying proteins of the plasma membrane Add trypsin to digest proteins on outside of cell

Studying proteins of the plasma membrane Permeabilize and add trypsin to digest proteins on inside and outside of cell

Studying proteins of the plasma membrane

Membranes contain many associated proteins and carbohydrates Singer and Nicolson model 1972

Functions of Membrane Proteins image from Nature Education website

Functions of Plasma Membrane Proteins - Receptors Signal Receptors recognize specific signals from the outside to promote transcription, proliferation, survival, death, changes in cell shape, migration, etc

Functions of Plasma Membrane Proteins - Attachment Receptors on the surface of cells can be used to attach to the extracellular matrix or to other cells

Functions of Plasma Membrane Proteins - Transport

The plasma membrane is organized into domains Distribution of proteins within the plasma membrane can change to accommodate the functional demands of the cell

Proteins move within the lipid bilayer Depending on the cell type, the specific protein and environmental conditions, integral membrane proteins can exhibit several types of mobility.

The structure of the plasma membrane consists of discrete microdomains that segregate functions into subcompartments yeast cell microdomains Both lipids and proteins are segregated into microdomains

Cholesterol- and sphingolipid-rich lipid rafts are microdomains that are thought to concentrate signaling proteins From the inner life of the cell

Surveying membrane localization in yeast Tagged Proteins Lipid reporters Most, if not all, proteins localize in non-uniform patches of cell membranes Patchwork organization of the yeast plasma membrane into numerous coexisting domains. Spira F, Mueller NS, Beck G, von Olshausen P, Beig J, Wedlich-Söldner R. Nat Cell Biol. 2012 Apr 29;14(6):640-8.

What factors regulate protein diffusion in the endoplasmic reticulum? Is diffusibility affected by folding status? Is diffusibility affected by sugar modifications? Nehls et al. (2000). Dynamics and retention of misfolded proteins in native ER membranes. Nature Cell Biology 2:288.

FRAP-Fluorescence Recovery After Photobleaching A method for measuring protein movement within plasma membranes

FRAP-Fluorescence Recovery After Photobleaching A technique that can be used to determine the diffusibility of proteins in the membrane

Figure 3: How quickly do proteins move around in the plasma membrane? Patchwork organization of the yeast plasma membrane into numerous coexisting domains. Spira, F., Mueller, N.S., Beck,G.S., von Olshausen, P., Beig, J. and R. Wedlich-Söldner (2012). Nature Cell Biology 14, 640-680.

What factors regulate protein diffusion in the endoplasmic reticulum? Is diffusibility affected by folding status? Is diffusibility affected by sugar modifications? Nehls et al. (2000). Dynamics and retention of misfolded proteins in native ER membranes. Nature Cell Biology 2:288.

What factors regulate protein diffusion in the endoplasmic reticulum? VSVG-GFP: An integral membrane protein in the ER that is fused to GFP and expressed in COS cells

Is diffusibility affected by folding status? VSVG folds properly at 32ºC, but is misfolded at 40ºC Nehls et al. (2000). Dynamics and retention of misfolded proteins in native ER membranes. Nature Cell Biology 2:288.

Is diffusibility affected by sugar modifications? Tunicamycin is a drug that prevents sugar synthesis Nehls et al. (2000). Dynamics and retention of misfolded proteins in native ER membranes. Nature Cell Biology 2:288.

What factors regulate protein diffusion in the endoplasmic reticulum? The diffusibility of VSVG in the ER is affected by sugars but not by folding. Nehls et al. (2000). Dynamics and retention of misfolded proteins in native ER membranes. Nature Cell Biology 2:288.

Plasma membrane summary: 1. Cell membranes are bilayers of amphipathic lipids 2. The lipid composition of membranes varies 3. Membrane fluidity is regulated by temperature 5. Three types of membrane proteins: 1. Integral, 2. Peripheral, 3. Lipid-anchored 4. Membrane proteins function in signaling, adhesion, and transport 5. The membrane is organized into microdomains 6. Techniques for studying membrane proteins: proteolysis/protection assay, FRAP