Cell Biology - Ch. 7

Front 1

5 Functions of Membranes

Back 1

1) Boundary and permeability barrier.

2) Organization and localization of function.

3) Transport processes.

4) Signal detection.

5) Cell-to-Cell communication.

Front 2

5 Functions of Membranes

Visual

Back 2

Front 3

5 Roles of Membranes

Back 3

1) Define the boundaries of the cell and define its compartments – Permeability Barriers.

2) Serve as a loci (centers) of specific functions.

3) Possess transport proteins that facilitate and regulate the movement of substances [into and out of the cell and its compartment].

4) Contains receptor [proteins] required for the detection of external signal.

5) Provide mechanisms for cell-to-cell communication.

.

Front 4

Membranes: Defining Boundaries

Back 4

Physical separation.

Desirable substances IN and undesirable substances OUT.

Hydrophobic interior – permeability barrier for hydrophilic ions and molecules.

Plasma Membrane - Permeability barrier of the cell.

Intracellular membranes – Compartmentalize functions.

Front 5

Membranes: Sites of Specific Function

Back 5

Site of specific function - Special molecules and proteins embedded within membranes.

Membrane (Bio) Markers – protein or enzyme that associate with specific membranes e.g. Glucose phosphate – ER membrane protein.

Front 6

Membrane: Transport Regulation (Part 1)

Back 6

Concentration Gradients – molecule with no net charge.

Electrochemical potential – sum of its concentration gradient and the charge gradient across the membrane facilitate the movement of ions.

Front 7

Membrane: Transport Regulation (Part 2)

Back 7

Simple diffusion – water, oxygen, alcohol.

Facilitated diffusion – polar molecules e.g. sugars and amino acids moved across the membrane by transport proteins’.

Facilitated Water Movement – Aquaporins in Kidney cells.

Front 8

Membrane: Transport Regulation (Part 3)

Back 8

Active transport – against concentration gradient.

Direct active transport – requires

ATP for ‘uphill’.

Indirect active transport – of the

solute to ‘downhill’.

Front 9

Membrane: Transport Regulation (Part 4)

Back 9

Large Protein Transport

Endocytosis – into the cell –

intracellular vesicles facilitate the

movement of proteins.

Exocytosis – out of the cell – ER

proteins or cytosolic proteins can be

imported into membrane bound

organelles such as lysosomes,

peroxisomes, mitochondria.

Front 10

Membrane: Signal Detection (Part 1)

Back 10

Electrical or chemical signals on the outer surface of cell

Signal Transduction – mechanisms that detect and direct specific signal in and out of the cell.

Front 11

Membrane: Signal Detection (Part 2)

Back 11

Chemical signal transduction – Hormone based

Estrogen – a polar steroid enter into

target cell and interact – nonpolar

molecule.

Some molecules need specific

regulator protein.

Androgen – Androgen receptor.

Progesterone – Progesterone

Receptor.

Hormones – ligands – binds to

receptor and activate series of

internal chemical signal – second

messenger.

Front 12

Specific Transporters

Back 12

Glucose and Amino acid transporters.

Nerve Cell – Na+ and K+ ion transporters (Channels).

Muscle cell – Ca ++channels.

Chloroplast – Phosphate Ions transporters.

Mitochondria – Aerobic respiration intermediate transporters.

Front 13

Signal Detection

Back 13

Insulin receptor – on muscle and liver cells.

Immune cell receptors.

Light - sensing phytochromereceptors in plants.

Membrane receptors.

Hormone receptors.

Front 14

Cell Adhesion Mediators (Part 1)

Back 14

Intercellular communication by gap junctions in animal cells.

Intracellular communication by plasmodesmeta in plant cells.

Front 15

Cell Adhesion Mediators (Part 2)

Back 15

Intercellular communication by Cell to cell contact.

Cadherins – membrane proteins

extracellular domains – bind

calcium ionsand stimulate adhesion

between similar cells in a tissue.

Pathogenic organisms – Listeria

and Shigella use adhesive

membrane proteinsto invade.

Front 16

Cell Adhesion Mediators (Part 3)

Back 16

Adhesive Junctions: holds cell together.

Tight Junctions: Form seals to block passage of fluids between cells.

Ankyrin – Membrane proteins –

point of attachment to cell

cytoskeleton.

Front 17

Important Functions of Biomembranes

Back 17

Compartmentalization

Localization of function

Transport

Signal detection

Intercellular communication

Front 18

Fluid Mosaic Model of Membrane

Back 18

Different membranes.

Composition of ‘material’ differ.

Structure varies.

Fluid mosaic model evolved.

- Two fluid layers of lipids.

- Proteins localized within and on

the lipid bilayer.

- Specific orientation.

Front 19

Fluid Mosaic Timeline Visual

Back 19

Front 20

Lipid Monolayer (Part 1)

(Experimental Perspective)

Back 20

Overton: Lipids are important components – used cells of plant root hair (lipid soluble, water insoluble) - Lipophilic.

- lipids ‘coat’ on the cell surface.

- ‘coat’ a mixture of cholesterol and

lecithin.

Front 21

Lipid Monolayer (Part 2)

(Experimental Perspective)

Back 21

Langmuir: properties of purified phospholipids by dissolving in benzeneand layering on water.

.

One molecule thick.

Phospholipids are amphipathic

molecules.

Front 22

Lipid Bilayer

(Experimental Perspective)

Back 22

Gorter and Grendel: Lipids are important components – Extract lipids from known number of Erythrocytes - RBC.

Lipid film on water – lipid area twice

the estimated total surface area of

erythrocytes

Conclusion: Plasma membrane

consists of two layers of lipids –

LIPID BILAYER.

Wrong estimation: underestimated

1/3 of the surface and lipids. Also

didn't account membrane proteins.

Front 23

Membrane Containing Protein

(Experimental Perspective)

Back 23

Davson and Danielli: studied Surface tension, Solute permeability, Electrical resistance of membranes.

Based on observations: movements of ions, hydrophilic solutes.

Importance of proteins in

membrane structure.

Protein-lipid-protein Sandwich.

Front 24

Common Underlying Structures

(Experimental Perspective)

Back 24

Robertson: Used electron microscope.

All cellular membranes share a common underlying structure – Unit membrane.

Front 25

Fluid Mosaic Model

(Experimental Perspective)

Back 25

Singer and Nicolson: A membrane consists of a mosaic of proteins in a fluid lipid bilayer.

Mosaic of proteins.

Embedded in, attached to –fluid

lipid bilayer.

Front 26

Membrane Proteins:

Integral Membrane Proteins

Back 26

Integral Membrane Proteins – embedded within the lipid bilayerby affinity of hydrophobic segment of the proteins for the hydrophobic interior of the bilayer.

Front 27

Membrane Proteins:

Peripheral Proteins

Back 27

Peripheral Proteins – hydrophilic and located on the surface – linked noncovalently to the polar head groups of phospholipids.

Front 28

Membrane Proteins:

Lipid-Anchored Proteins

Back 28

Lipid-anchored Proteins – hydrophilic proteins – on membrane surface and covalently attached to embedded lipid molecules.

Front 29

Membrane Protein Visual

Back 29

Front 30

Transmembrane Segments

(Experimental Perspective)

Back 30

Unwin and Henderson: Most membrane contains transmembrane segments – bacteriorhodopsin – light absorbing molecule of Retinal (Halobacterium).

Seven transmembrane segments: anchor hydrophobic regions of proteins in lipid bilayer and align them.

Front 31

Recent Findings

Back 31

Membranes are NOT homogenous, freely mixing structures.

Lipids and proteins are ordered

within membranes.

Outer membrane monolayer.

Ordering occurs in dynamic

microdomains call LIPID RAFTS. –

Contains lot more cholesterol,

sphingolipids (e.g. shingomylin) and

less phosphatidylcholine.

Front 32

Recent Findings

Back 32

Serve as organizing centers for the assembly of signaling molecules.

Front 33

Protein, Lipid, Carbohydrate Ratios

Back 33

Front 34

Singer and Nicolson:

Fluid Mosaic Model Visual

Back 34

Front 35

The Fluid Part of the Model:

Phospholipid

Back 35

Phospholipid

- Glycerol based Phosphoglycerides.

- Sphingosine based Sphingolipids.

Front 36

The Fluid Part of the Model:

Glycolipids

Back 36

Glycolipids

- Carbohydrategroup attached to

lipids.

- Glycerolas well as sphingosine

based.

Front 37

The Fluid Part of the Model:

Sterols

Back 37

Sterols

Animal Cell – cholesterol.

Plant Cell - phytosterol.

Front 38

Glycolipids:

Cerebrosidesand Gangliosides

(Part 1)

Back 38

Glycolipids - Glycosphingolipids

Cerebrosides – Neutral glycolipids –

single uncharged sugar attached as

head group.

Gangliosides – One or more

negatively charged sialic acid

residues.

Front 39

Glycolipids:Cerebrosidesand Gangliosides(Part 2)

Back 39

Prominent in brain and nerve cell membranes.

Ganliosides on the surface – function as antigens.

Example – Cell surface markers

(Glycosphingolipids) for ABO blood

group.

Front 40

Tay-Sachs Disease

Back 40

Tay-Sachs Disease – absence of acetylhexosaminidasein lysosome.

– Accumulation of ganglioside in

brain and nerve cells

Front 41

Sterols

Back 41

Mitochondria and chloroplast inner membrane - do not have sterols.

Prokaryotes do not produce sterols.

Prokaryotes produce - Hopanoids.

Front 42

3 Major Lipid Membrane Classes:

Phospholipid

Back 42

Front 43

Schematic Diagram: (Part 1)

Phospholipids (top)

Sphingolipid (bottom)

Back 43

Front 44

Schematic Diagram:

Glycolipids

Back 44

Front 45

Schematic Diagrams:

Sterols

Back 45

Front 46

Schematic Diagram: (Part 2)

Phospholipids

Back 46

Front 47

Schematic Diagram: (Part 3)Sphingolipids

Back 47

Front 48

Cholesterol - The Steroid

Back 48

Third important membrane lipidAbundant in plasma membrane of mammalian cellsBasic structure –four membrane ringAmphipathic –due to the presence of a hydroxyl group.

Front 49

Sterol Substitutes in Bacteria and Cyanobacteria

Back 49

Rigid.

Strong hydrophobic.

Short hydrophilic side chain.

Abundant in petroleum deposits – Membrane component of ancient prokaryotes.

Sterols are not found in bacterium but found in MYCOPLASMA species.

Front 50

Different Membrane Compositions

Visual

Back 50

Front 51

Lipid Analysis via Thin Layer Chromatography (TLC)

Back 51

Stationary Phase:

Thin layer of silicic acid on glass or

metal plate.

Mobile Phase:

Mixture of appropriate solvents.

Chloroform, Methanol, Water

Front 52

Lipid Separation via Polarity

Back 52

Their Relative Affinities (based on polarity) for the stationary or mobile phase.

Phospholipids (more polar) interact with silicic acid more strongly – slow their movement.

Cholesterol (less polar) do not adhere strongly with silicic acid – move faster.

Front 53

Thin Layer Chromatography (TLC)

Visual

Back 53

Front 54

Fatty Acids and Membrane Function

Back 54

Fatty acids – components of all membrane lipids except the sterol.

Long Hydrocarbon tails from hydrophobic barriers.

Chain of fatty acids determine the thickness of bilayer.

Front 55

Common Fatty Acid Structures in Membranes

Visual

Back 55

Front 56

Membrane Asymmetry (Part 1)

Back 56

Membranes contains different kind of lipids.

Lipids are randomly distributed in TWO MONOLAYERS.

Variety of cell types – different lipids are unequally distributed between two monolayers – Membrane Asymmetry.

Front 57

Membrane Asymmetry (Part 2)

Back 57

Differences - both in the kinds of lipids present and in the degree of unsaturation of the fatty acids in the phopholipids molecules.

Glycolipids – in animal membrane are mostly restricted to the OUTER of the two monolayer – facilitate signaling events.

Front 58

Membrane Asymmetry (Part 3)

Back 58

Phospholipids – prominent in the INNER monolayer – transmit signals from plasma membrane to interior of the cell.

Phosphatidylethanolamine.

Phosphotidylinositol.

Phosphotidylserine.

Phosphotidylcholine.

Front 59

Freeze Fracture: Membrane Proteins

Back 59

Freeze the sample – Membranes.

Slice through a diamond Knife.

- Opens two layers.

- Nonpolar lipid bilayer is the path

of least resistance.

- Split the lipid bilayer into its inner

and other monolayers.

Front 60

Freeze Fracture

Visual

Back 60

Front 61

Phospholipid Movement within Membrane

Back 61

Phospholipid Molecules exhibit three kinds of movement in a membrane.

Rotation along long axis.

Lateral diffusion by exchanging place with neighboring molecule.

Transverse diffusion OR Flip-Flop.

Phospholipid translocators or Flippase.

Front 62

Recovery from Photobleaching

Visual

Back 62

Front 63

Fluid State: Proper Membrane Function

Back 63

Transition Temperature causes Phase Transition.

Composition of Fatty acids affect membrane fluidity.

Sterols also affect fluid state.

Organisms can regulate membrane fluidity.

Front 64

MP of Fatty Acids

vs.

Chain Length and # of Double Bonds

Visual

Back 64

Front 65

Packing of Saturated Fatty Acids

Back 65

Saturated fatty acids pack well together.

Front 66

Packing of Saturated and Unsaturated Fatty Acids

Back 66

Lipids with a mixture of saturated and unsaturated fatty acids don't pack well together in the membrane.

Front 67

Cholesterol Orientation within Bilayer

Back 67

Front 68

Separation of Proteins (Part 1)

Back 68

Isolation of Membrane proteins.

SDS - Polyacrylamide Gel electrophoresis.

SDS – sodium dodecyl sulfate (detergent).

Polyacrylamide – matrix.

Front 69

Separation of Proteins (Part 2)

Back 69

Separation based on charge.

Molecular Weight markers to follow progress of electrophoresis.

Gel Stained with coomassie brilliant blue.

Front 70

SDS-PAGE Electrophoresis Gel

Back 70

Front 71

Freeze Fracture of Membrane Protein

Visual

Back 71

Front 72

Freeze Fracture of

Bilayer w/w-out Protein

Visual

Back 72

Front 73

Hydropathy Analysis

of Membrane Protein

Visual

Back 73

Front 74

Application of Techniques to Study Membrane Proteins

Visual

Back 74

Front 75

Main Classes of Membrane Proteins

Back 75

Front 76

Membrane Proteins (Part 1)

Back 76

Integral Membrane proteins – embedded within the lipid bilayer by affinity of hydrophobic segment of the proteins for the hydrophobic interior of the bilayer.

Front 77

Membrane Proteins (Part 2)

Back 77

Peripheral Proteins – hydrophilic and located on the surface – linked noncovalently to the polar head groups of phospholipids.

Lipid-anchored Proteins – hydrophilic proteins – on membrane surface and covalently attached to embedded lipid molecules.

Front 78

Three Main Tyles of Membrane Proteins

Back 78

Based on the conditions required to extract membrane proteins – Nature of their association with the lipid bilayer.

Integral Membrane Proteins.

Peripheral Proteins.

Lipid-Anchored Proteins.

Front 79

Integral Membrane Proteins (Part 1)

Back 79

Three parts - two hydrophilic and one hydrophobic.

Amphipathic.

Hydrophobic embedded in lipid bilayer.

Front 80

Integral Membrane Protein (Part 2)

Back 80

Integral Monotopic proteins.

Transmembrane proteins.

- Single pass.

- Multipass.

- Multisubunit multipass.

Front 81

Integral Membranes Protein (Part 3)

Back 81

20 –30 AA with hydrophobic R group.

Helical conformation hydrophobic domains.

Sheets or barrel.

Examples Porins, glycoporeins, band 3 protein, anion exchange proteins.

Front 82

Structure of Two Membrane Proteins

Visual

Back 82

Front 83

Peripheral Membrane Proteins

Back 83

Lack hydrophobic domains.

Bound to membrane (polar heads) surface by (noncovalent) hydrogen bonding and electrostatic forces.

Easily removed from membranes by changing pH and ionic strengths.

Examples – Spectrin, ankyrin, band

4.1 in erythrocytes.

Front 84

Erythrocyte Plasma Membrane

Visual

Back 84

Front 85

Lipid-Anchored Membrane Proteins (Part 1)

Back 85

Fatty Acid Anchored Proteins.

Synthesized in cytosol.

Covalently attached to saturated fatty acid.

Usually Myristic (14 carbon) acid or Palmitic (16 carbon) acid.

Front 86

Lipid-Anchored Membrane Proteins

(Part 2)

Back 86

Prenylated Anchored Membrane Proteins

•Synthesized as soluble cytosol

proteins.

•Covalently attached to saturated

fatty acid.

•attached to Isoprenyl (5 carbon)

group.

Front 87

Lipid-Anchored Membrane Proteins(Part 3)

Back 87

Usually in the form of Farnesly(15 carbon) group or 20 carbon Geranylgeranylgroup.

GPI anchor –

Glycosyl-phophatidyl-inositol.

Front 88

N-Linked and O-Linked Glycosylation

Visual

Back 88

Front 89

Carbs found in Glycoproteins

Visual

Back 89

Front 90

Carbohydrate Group

Visual

Back 90

Front 91

Cell Fusion Experiment

Back 91

David Frye and Michael Edidin.

Fluorescent tagged Antibodies – Anti-mouse Green (Fluorescein) and Anti-human Red (Rhodamine) colored.

Sendai Virus Infection which fuses eukaryotic cells.

Conclusion – Lateral diffusion of protein by fluid bilayer of plasma membrane.

Front 92

Cell Fusion Experiment

Visual

Back 92

Front 93

Mobility of Membrane Proteins

Visual

Back 93

Front 94

Labeling Membrane Proteins Inside and Out of Cell

Visual

Back 94

Front 95

Membrane Proteins:

Variety of Functions

(Part 1)

Back 95

Enzymes

Electron Transport Proteins

Transport Proteins – Sugar, Amino Acids

Channel proteins –Na, K, Cl etc.

Front 96

Membrane Proteins:Variety of Functions(Part 2)

Back 96

Transport ATPases are Channel Proteins.

Receptors.

Autophagy – digest own organelles.

Structural Roles – Stabilizing and Shaping.

Front 97

Integral Membrane Protein:

Hydropathy Plot

Back 97

Front 98

Heat Absorption of Membranes with Various Protein Concentrations

Visual

Back 98

Front 99

Heat Absorption of Normal Membrane

Back 99

Front 100

Heat Absorption of Membrane Enriched with Oleate or Stearate

Visual

Back 100

Front 101

Glycocalyx of Intestinal Epithelial Cells

Visual

Back 101