Smas - Plasma Membrane

8/22/2019 Smas - Plasma Membrane

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Membrane Structure and Composition

Plasma Membrane 1

Dr. Cynthia [email protected]

Dept. of Biochemistry & Cancer BiologyOffice:Rm 401D BHS; Lab: Rm 466 BHS

383-4527, 383-4131

Your questions are welcome at any time!

Wednesday 8/22/201210-12 p.m.


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References and Readings:

Biochemistry (Stryer 7th ed.) Chapter 12 (relevant portions)

Harpers Illustrated Biochemistry, 29th ed. Chapter 40.

Optional: Molecular & Cellular Biology (Lodish 4th ed, 2000.) Chapter 5

Available free on-line through NCBI/PubMed.

http://www.ncbi.nlm.nih.gov/books/NBK21475/

(searchable only, cannot browse it)

Plasma Membrane 1

Also note the 5th edition of Stryer is also free through

NCBI/Books but is only searchable, not browsable

http://www.ncbi.nlm.nih.gov/books/NBK21154/
http://www.ncbi.nlm.nih.gov/books/NBK21475/http://www.ncbi.nlm.nih.gov/books/NBK21154/http://www.ncbi.nlm.nih.gov/books/NBK21475/


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Plasma Membrane 1Learning Objectives

1. Describe 3 common types of membrane lipids and discussamphipathic nature of each. Indicate how membrane lipid

structure facilitates self-assembly of the lipid bilayer.

2. Explain what types of molecules can pass directly throughthe lipid core of the membrane and which cannot and why.

3. Discuss the distinguishing features of integral and peripheralmembrane proteins, using glycophorin as an example.

4. Explain how lipids can serve to anchor some proteins in theplasma membrane.


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5. Explain the impact of fatty acid length and saturation, andthe effects of cholesterol on membrane fluidity as reflectedby the melting temperature (Tm).

6. Discuss one example of asymmetry of membrane lipids andof membrane proteins. For example, the impact ofexoplasmic phosphatidylserine on cell destruction orthe asymmetric nature of glycosylation of membrane proteins.

7. Discuss the types of interactions among the key proteincomponents of the RBC membrane. Describe theircontribution to its strength and flexibility in health & disease, for example in

heriditary spherocytosis.

Plasma Membrane 1Learning Objectives (cont.)


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The Cell & Intracellular Organellesare Surrounded by a Membrane Bilayer

Plants

(A composite eukaryotic cell)

Source: Molecular Cell Biology, Lodish et. a. 4th ed, W.H. Freeman, 2000


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Regulate nutrient & ion transport into the cell

Regulate transport ofwaste out of the cell

Maintain correct chemical conditions in the cell

Provide a site for lipid-based chemical reactions

Interact with other cells or the ECM

Detect & transduce signals from environment to cell

Functions of the Plasma Membrane

Signal Transduction


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Lipids & Many Membrane Proteins areLaterally Mobile in the Plane of the Membrane

The Fluid Mosaic Model

Source: Addison Wesley Longman, Inc.

Plasma Membrane/Cell Membrane/ Cell Surface


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A.The Basic Architecture of theMembrane

1. Types of membrane lipids

2. Membrane proteins

B. Membrane FluidityC. Membrane Asymmetry

The Plasma Membrane - TopicsFluid Mosaic



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Biological Membranes Have 2 MainComponents- Lipids & Proteins

Lipids: Form a permeability barrier Define the basic architecture

Proteins:

Define the unique functions of membranes Determine selective permeability Transporters, channels, junctions Energy uptake, signal transduction

Protein to lipid ratio can vary with the cell type.This can be related to the function of that cell type.


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Water-insoluble biomolecules

Highly soluble in organic solvents

Great variety of structures

Fuel and energy storage

Signaling

Membrane components

Lipids


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1. Phospholipids

2. Glycolipids

3. Cholesterol

3 Common Types of Membrane Lipids

Membrane lipids have an amphipathicnature.

2-loves the hydrophilic aqueous environment andthe hydrophobic non-aqueous environment

Hydrophilic (polar) head groupHydrophobic (non-polar) acyl side chains (tails)


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Phospholipids

Basic Structure & Examples

You do not need to know details of structure

Why is it amphipathic?


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A Phospholipid is Composedof 4 Groups

16-18 C

1C

2C

3C

Hydrophobic Hydrophilic

Phospholipids are the Major Typeof Membrane Lipid

Source: Biochemistry, Stryer 5th ed. W.H. Freeman


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Phosphatidate (Diacylglycerol 3-phosphate)Example of a Simple Phosphoglyceride

GlycerolBackbone

Phosphatehead group

at C3 of

glycerol

C1 & C2

Amphipathic: Hydrophobic HydrophilicSource: Biochemistry, Stryer 5th ed. W.H. Freeman

Side chains


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Commonly Occurring Membrane Phospholipids(Phosphoglycerides)



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Glycolipids are sugar-containing lipids

A sugartakes the place of the phosphate group

Sphingosine

Membrane Lipids Can AlsoInclude Carbohydrate Moieties


Very enriched - where?


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Cholesterol is Commonin the Plasma Membranes of Animals

4 LinkedHydrocarbon

Rings

Hydrophilic Hydrophobic

A lipid based on a steroid typestructure



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In Aqueous Media Phospholipids and GlycolipidsReadily Form a Bilayer Sheet

Two faces of the plasma membrane (leaflets):

Exoplasmic- toward the extracellular environmentCytoplasmic- toward the intracellular environment


Cross Section

Polar Head Groups Hydrophobic Interior Polar Head

Groups


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Micelles Solubilization and purification ofmembrane proteins, LDL and bileparticles are mixed micelles

Liposomes * Used in functional study

of membrane proteins

* Important for drug deliveryand therapeutics.

Two Additional Structures Satisfythe 2-loves of Amphipathic Lipids

= drug

NOTE: Cut-away views half of a micelle and half of a liposome is shown

= membraneprotein

+ =

Or

TM


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Hydrophobic interactions by exclusion of water drivesformation of energetically stable structures.

Van der Waals attractive forces between hydrocarbon tailsfavor close packing.

Electrostatic and H-bond attractions between the polarhead groups and water molecules also involved.

Membrane formation is due to the amphipathic nature ofmembrane lipids.

Why is this Bilayer SheetEnergetically Favored and Stable?


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The lipid bilayer membrane is a cooperative

structure that is formed & maintained by multiple

noncovalent interactions.

The role of lipids in the plasma membrane are to definethe basic bilayer architecture which acts to form a

permeability barrier for the cell.

In summary:


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B. Membrane Fluidity

C. Membrane Asymmetry

The Plasma MembraneFluid Mosaic



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The Concept ofSelective

Permeability

Some molecules can passdirectly through lipid core.

If they cannot, membraneproteins must serve as

structures for passage. Channels, transporters,

junctions, pumps.

Lipid Bilayers Selectively Maintain DifferencesIn ECF and ICF Concentration

Source:Molecular Cell Biology, Lodish et. a. 4th ed, W.H. Freeman, 2000

Simple

diffusio

n

Category Examples

Membrane

*

*Depends on cellular/organ setting


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Define the unique functions of membranes

Determine selective permeability

Examples - transporters, channels, junctions

Function in energy uptake, signal transduction

Today, I will present basic concepts regarding membrane proteins.Next time, several specific membrane proteins will be discussed in detail.

The PROTEINSof the membrane determine itsfunctional complexity for each specific cell type.

Proteins Carry Out Most Membrane Processes


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eripheral- More loosely associated with the membranemoved with mild conditions (salt/pH)

Do not enter or span the hydrophobic core

Integral- More tightly associated with the membrane Removed only with harsh conditions (detergents).

Enters or spans the hydrophobic core (Single-pass or multiple-pass)

Membrane Proteins are TypicallyClassified as Either Peripheral or Integral

EXOPLASMIC

CYTOPLASMIC

OUTER

(LEAFLETS)

INNER


Yellow = IntegralBlue = Peripheral


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-Helix -Barrel


Specific Secondary Structures EnablePolar Proteins to Happily Exist in the

Hydrophobic Lipid Core

(A single -helix is circled in blue)

Most commonly used in higher organisms Often utilized by bacteria


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Why is the hydrophobic -helixperfectly suited to span the lipid core?

The peptide bond is polar Proteins contain charged AA

But the membrane core is hydrophobic! An -helix can function to span the core

A sequence of 20-25 AA, rich inhydrophobic/nonpolar AA with an

-helical structure often servesas a transmembrane segment.

The membrane spanning -helix

Membrane Proteins Often Have an -HelicalStructure to Span the Lipid Bilayer


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Example - Glycophorin Uses This Type of-Helix to Span the Membrane

Hydrophobic AA arenot restricted tomembrane regionsbut a 20-25 AA stretch

suggests a possibletransmembranedomain.

Hydrophobicor Neutral

(+)

(-) Charged

The erythrocyte integral membrane glycoprotein glycophorin

is the basis for the MN blood group

Charged

Sugar

(glycoprotein)


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Transmembrane -Helices Can BePredicted from Hydropathy Plots


Hydrophob

icityIndex

Alanine

Valine

Isoleucine Leucine

Methionine

Phenylalanine

Tyrosine

Tryptophan

Transmembrane - helicesmay likely include:

Multiple -helices predicted:

Which AA areunlikely in a

membrane-spanning region?

+4+3+2+10-1-2

-3 50 100 150 200

1 2 3 4

Inde

x

+4+3+2+10-1-2

-3 50 100 150 200

1 2 3 4

Amino Acid


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Schematic Representations - Membrane Proteins

Source: Biochemistry, Stryer 5th ed. W.H. Freeman; Source: Addison Wesley Longman, Inc.


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Prostaglandin H Synthase:An Example of an Integral* Membrane ProteinThat Enters but Does Not Span the Membrane


*The hydrophobic side chains of this -helicalstructure allow the protein to be tightly

associated (integral) with plasma membrane

*


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Farnesylation

Palmitoylation

Myristoylation

GPI Anchor (Glycosylphosphatidylinositol)

Certain Proteins Rely on Specific Lipid Structuresfor Association with the Plasma Membrane

Source: Molecular Biolo o the CellAlberts, et.al. 3rd ed.

Lipid Anchorsare covalently

attached to specific proteins.

Lipid anchors are hydrophobic and canembed in the hydrophobic core of the

plasma membrane, to anchor (localize)

the protein at the membrane.

Types of lipid anchors(structures)

BOAT

ANCHOR

DOCK

ANCHORS

Protein

Thy-1,Ras, Src,or others

Lipid Core

of the

Plasma

Membrane


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Alterations and Defects in Membrane ProteinsUnderlie Many Human Diseases

Cancer: Alterations of membrane protein and/or lipid arekey to metastasis and invasion of tumor cells asthey spread throughout the body.

Diabetes: Defective insulin signaling, defective functionof glucose transporters (More in next lecture)

Heart Disease: Defective cell-cell communication (example connexins in arrhythmias,

will be discussed by other instructors)


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Reconstitution isOften Used to Study

Function ofMembrane Proteins

Why?Membrane proteins need to be present in

a membrane bilayer, and withcorrect topology to function properly

(even for lab studies)

They will denature otherwise



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Manipulation of Membrane Protein Functionin Disease Treatment

PRILOSEC GASTRIC H+/K+ ATPasePROZAC Na+-coupled serotonin transporterVast numbers of other membrane proteins . . .

Membrane proteins are highly useful drug targets:

The more details we know about membrane protein structureand function the better we can:

Make designer drugs targeting protein active sites

Predict how mutations in a gene for a membraneprotein may impact protein function

In general, the more we know about a protein, thebetter we can treat disease


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Membrane FluidityWhy Is It Important?

Influences arrangement of proteins and lipids

Foster assembly/disassembly of protein subunits andsignaling complexes in the membrane

Changes membrane permeability

Excessive fluidity leads to membrane destruction

Altering fluidity can alter membrane and/or cell function

Many Studies of Membrane Fluidity areConducted in Experimental Settings

Keep in Mind - Biological Membranes are Much More Complex !

M b Fl idit


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Membrane FluidityEffects of Fatty Acid Composition

Desaturated:

Saturated:


Kink disrupts tight packingof hydrophobic side chains

Allows tight packingof hydrophobic side chains


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Tm: Phase Transition Temperature(a.k.a. Melting Temperature)


A Distinct Melting Temperature (Tm) is Noted forSimple (Non-Biological) Membranes

Shorter Acyl Chain Desaturated

Longer Acyl Chain Saturated

M

embraneFluidit

y

Need less heat energy todisrupt the membrane

Need more heat energy todisrupt the membrane

Tm higher

Tm lower

Curve will shift left if: Curve will shift right if:

Phasetransition

Lessfluid

Morefluid


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Fatty Acyl Chain Length Effect on MembraneTransition Temperature (Melting Temperature)

Regulation of membrane fluidity by alteration of fatty acyl chainlength and saturation is employed by bacteria.


*

*


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Cholesterol is the Key Determinant ofMembrane Fluidity in Animals

Disrupts regular interactionsof fatty acyl side chains

Makes membranes less likely toundergo phase transition

Source: Molecular Biology of the Cell. Alberts, et.al. 3rd ed.1944 Garland

Cholesterol acts as a buffer against changes to membrane fluidity

Cell membranes need to have a correct degree of fluidity for function.

This requires the right amount of cholesterol in the cell membrane.

The Effects of Cholesterol on Membrane Fluidity


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Above Tm: Decreases fluidity

The Effects of Cholesterol on Membrane FluidityOutcome: Maintains Proper Tm

Below Tm: Increases fluidity* Below the melting temperature, the membrane

is gel-like or more solid

* The lipid side chains are tightly packed and orderly

* Introducing a kinked structure disrupts this, increases fluidity

* Above the melting temperature, the membrane is more fluid-like

* The lipid side chains are disorganized and moving

* Cholesterol acts to limit/restrict the overall free movement of the lipid sidechains due to its planar shape (steroid nucleus), thus making it less fluid


M b Li id Hi hl M bil


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Why is lipid flip-flop so very rare?

1 sec

LATER

AL

very rare

rapid

rapidrapid

rapid

Membrane Lipids are Highly Mobilein the Plane of the Membrane


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The size of the molecule

Interactions with other molecules

Temperature

Lipid composition of the membrane

The composition of the protein

Membrane Proteins Also Have Lateral Mobility

This depends on:

How Do We Know Fluidity of Membrane Proteins?


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How Do We Know Fluidity of Membrane Proteins?

FRAPMethod:

F luorescence

R ecoveryA fterP hotobleaching


Done on live cells -in real time.

Uses an antibody specific for the membrane protein you want to study.(Can also use other types of tools such eGFP fusion proteins . . .)

The antibody also has a fluorescent label linked to it (to allow us to see if).

Location of the protein under study shown by fluorescence (green) signal(fluorescence microscopy used).

Signal in any area of membrane can be obliterated (bleached by a laser).

The recovery of signal in bleached area (green) for your protein is observed.

This measurement shows rate of movement of this protein in the membrane.

Note: Only 1 molecule of a membrane proteinis shown, it is really in many on surface.Picture not drawn to scale.

FRAP Experimental Set up:


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Visualization

Quantification

Time

Recovered

Recovered

FRAP Experimental Set up:

Cells (membrane protein) + Labeled Antibody ( ) =

Then -


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Asymmetry is Key to Function of MembranesAll Biological Membranes Are Asymmetric

The exoplasmic and cytoplasmic surfaces have different

proteins and different enzymatic and other activities.

An asymmetric distribution occurs for both membranelipids & proteins.

This asymmetry is key to proper function.

What are some examples of this asymmetry for membranelipids & proteins?


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Membrane Asymmetry - Each Leaflet of theMembrane Has A Distinct Lipid Composition

The choline -containing phospholipids are mostly exoplasmic. The amino-phospholipids are mostly cytoplasmic. Flippases, floppases & scramblases can impact lipid

asymmetry.

Amino-containing

Fatty acyl side chains alsoshow leaflet enrichment.

RBC: Cytoplasmic leafletenriched in unsaturatedfatty acyl chains.

Choline-containing


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Altered Distribution of Membrane LipidsCan Target Cells for Destruction

Platelet activation and aggregation:

Cascade of thrombin activation and protein C pathways Activation of alternative complement pathways

Recognition and removal of cells:

RBCs: Exposed phosphatidyl serine causes macrophagerecognition and destruction of these RBCs in the spleen.

Apoptosis (programmed cell death): Cells undergoing apoptosis expose phosphatidyl serine on

their exoplasmic leaflet. This triggers macrophage recognitionand destruction of apoptotic cells.

Exposure of phosphatidyl serine on the exoplasmic leafletoccurs in physiological and pathologic states.


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Every Membrane Protein Has a Specific &Consistent Topology

Determined at time of their synthesis in the ER.

Maintained during their journey to the (plasma) membrane.

A protein requires proper topology for function.

They dont flip flop

Cytosolic

Exoplasmic


Glycosylation Sites are One Example


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Glycosylation Sites are One Example

of Asymmetry for Some Membrane Proteins

Source:

Molecular Biology of the Cell. Alberts. 3rd ed.1944 Garland

Some membrane proteins are glycosylated, occurs at sites in theirexoplasmic portion.

Complex sugar groups added in ER& Golgi by successive reactions.

(many types, much variety )

Can be N-linked (asparagine) orO- linked (serine, threonine).

Occurs only in the lumen of ER &Golgi.

Can confer specificity & function.

Example: Blood Group Antigens


Remember Glycophorin ?

= glycosylation sites

C f


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Each Cell Type Has a Unique Repertoire of(Asymmetrically distributed)

Plasma Membrane Proteins

A. Erythrocytes

B. Retinal rod cells

C. Muscle (SR)

SDS-PAGE ANALYSIS(Protein Gel Electrophoresis)

Separates proteins by size

SIZ

EMW


What is your interpretationof this

SDS-PAGE?

Higher MW

Lower MW


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A Variety of Membrane Protein InteractionsGives Strength & Flexibility

to the Fluid Mosaic



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The RBC is a Key Example of the RelationshipBetween Membrane Proteins and Disease

Biconcave shape facilitates gas exchange.

Flexible shape for travel through tight spots (capillaries).

The functionality of the RBC is closely tied to plasmamembrane integrity. Defects in RBC membrane proteins are

often indicated by clearly visible alterations in RBC morphology.

Th R d Bl d C ll Pl M b i


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The Red Blood Cell Plasma Membrane isOne of the Most Thoroughly Studied

SDS-PAGE analysis

of RBC membrane proteins

Creation of RBC ghosts to

study membrane function


*Why?*

Major Proteins of


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Major Proteins ofthe RBC

Plasma Membrane

Integral:Glycophorins (A,B,C)

single-pass, glycoproteinbasis of MN blood group

Anion exchange protein

multi-passexchange ofchoride for bicarbonate

Also called band 3

Inward tension is created via cytoskeleton/membrane protein interaction,this determines cell shape and flexibility.

Peripheral:

Spectrin (, form dimer)

Ankyrin

Band 4.1

Actin

G-3-P dehydrogenase

Tropomyosin

Proper Protein Interactions at the Membrane


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Clinical Example - Hereditary Spherocytosis

* Mutations in genes for spectrin,ankyrin (or other genes) leads to ...

* Weakened interaction of peripheral

and integral membrane proteins

* Cytoskeletal architecture altered

* Detected by osmotic fragility test

* Autosomal dominant (1/5000).

* Spherocytic cells subject todestruction in the spleen - anemia.

pAre Key to Biconcave Shape/Function.

Due to defects in some RBC membrane proteins,

H. S. RBCs lack flexibility and clog upsplenic sinusoids, & are destroyed.

Which blood smear is normal RBC morphology&

which is abnormal (H.S.), why?

K C t


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The plasma membrane is a selective permeability barrierthat is essential for proper cell function.

Proteins and amphipathic lipids are the two maincomponents of the bilayer membrane.

Membranes form spontaneously and are stable.

The lipids of the membrane define the basic architecture.

The proteins impart specific functions.

The lipids and proteins of the plasma membrane areanddynamic (fluid) and the asymmetrically distributed.

Plasma membrane proteins can interact with extracellularsignals (hormones, other cells, the extracellular matrix, other)

and the cytoskeleton.

Key Concepts


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In my next lecture:

How does the plasma membrane function in

communication between cell and environment?

Smas - Plasma Membrane

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