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Panel of Foundation Studies

Cell Biology

Dr Rebecca K Y LeeSchool of Biomedical Sciences

([email protected])

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Cell membrane and membrane transport

Cytoskeleton and cell movement

Cell organelles

Endocytic and secretory pathways

Cell junction and cell adhesion

Cell cycle and cell death

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Cell Biology

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PFOS-011/12

Cell Membrane &Membrane Transport

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To describe different classes of lipids and proteins and howthey interact to form the cell membrane

To describe the functions of plasma membrane

To understand the importance of selective permeability in cellmembrane

To understand various mechanisms that cells use to transportsubstances across the plasma membrane

To differentiate and give examples of simple diffusion,facilitated diffusion, primary active transport and secondaryactive transport

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Learning Outcomes

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Schematic Structure of aBiological Membrane

Textbook of Biochemistry with Clinical Correlations

Biology, Campbell Reece

1. Lipids + proteins

2. Membrane asymmetry

3. Dynamic structure permits cell movement

4. Selectively permeable

5. Signal transduction,Cell-cell recognition,Maintain cell shape,Cell locomotion

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6Textbook of Biochemistry with Clinical Correlations

Chemical Composition of Membranes

Proteins + Lipids

Amount varies greatly betweendifferent types of membranes

Example: myelin Insulators Few metabolic functions Lipids > proteins

Example: mitochondria Membranes that surroundmetabolic factories

Relatively rich in proteincontent

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A) Glycerophospholipids

Most abundant

4 components: Fatty acids

Glycerol

Phosphate

Alcohol

Medical Physiology

Biochemistry, Stryer

Lipids in the Plasma Membrane (1)

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Biochemistry, Van Holde

B) Sphingolipids

Basis: sphingosine

Ceramide: sphingosine + fatty acid

Optional: phosphate group + alcohol(e.g. serine), or carbohydrates

Glycosphingolipids: sphingolipids thatcontain carbohydrates

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Substitutions are

found on the hydroxylgroup here!

For example


Galactosylceramide

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Substitutions arefound on the hydroxyl

group here!


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C) Cholesterol

Bulky, rigid structure compared with other hydrophobicmembrane components, e.g. fatty acids

Bacterial cells do not have cholesterol, neither inmitochondria too



Cholesterol carbohydratesPeripheralproteins

Cholesterol

molecule

Hydrophobicfatty acid

chain

Hydrophilic polar head Peripheral proteins

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12Structure of some common membrane lipidsMedical Physiology

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Amphipathic

Glycerophospholipids & sphingolipids

Hydrophilic head

Hydrophobic tail

Held together by hydrophobic interactions

Amphipathic Lipid Bilayer

Hydrophilic head

Hydrophobic tail

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e.g. erythrocyte membrane

Outer layer: spingomyelin

Inner layer:phosphatidylethanolamine

Membrane Asymmetry

Phosphatidylethanolamine

Phosphatidylserine

Phosphatidylcholine

Sphingomyelin

Total phospholipid

Outside

Inside


Percentageoftotal

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(a) Rapid rotational diffusion

Rotation around the FA chains

(b) Very slow transverse (flip-flop) exchange

Thermodynamic constraints

(c) Rapid lateral diffusion

Mobility of Lipid Components in Membranes

(a) (b) (c)


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Hydrophobic interactions Proteins are free to move laterally

Degree of fluidity increases with:

Increasing temperature

Shorter FA chain

Increasing no. of double bonds

Less cholesterol

The Fluid Mosaic Model

http://lhs2.lps.org/staff/sputnam/Ent801/Lab2.htm

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A)Proteolipids

Presents in many membranes, e.g. myelin, >50% of the proteincomponent

B) Glycoproteins Carbohydrates covalently attached to proteins

Sugars include glucose, galactose, mannose, fucose, N-acetylgalactosamine, N-acetylglucosamine

Integral protein: span the thickness of the plasmamembrane

Peripheral proteins: attached to either inner / outersurfaces of the plasma membrane

Proteins in the Plasma Membrane

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Extracellularside

Cytosolicside

Integral membrane proteins Peripheral membrane proteins

Integral Membrane Proteins areImmersed in Lipid Bilayer


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Serve as ligand-binding receptor

Serve as adhesion molecules

Cell-matrix adhesion molecules

e.g. integrin (please refer to the lecture cell junctions)

Cell-cell adhesion molecules

e.g. cadherin

Serve as enzymes, e.g. Na

+

/K

+

-ATPase

Allow transport of substances across the membrane

Functions of Membrane Proteins

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Summary

The major components of plasma membrane areand

The lipid molecules are molecules, they haveboth hydrophilic head and hydrophobic tail

Membranes are held together by interactions The lipid bilayer is a fluid-like structure, with

fluidity regulated by the no. of in the FA &cholesterol content

The proteins & lipids are free to move but no or

little flip-flopping is allowed The components of membranes with lipids &

proteins are oriented: the twofaces are different

Lipidbilayer

Biology, Campbell Reece

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Paracellular transport: through tight junctions between epithelial cells

Transcellular transport: through apical & basolateral membrane

Transcytosis: endocytosis & exocytosis

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Paracellular / Transcellular Transportand Transcytosis

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Plasma membrane is a semi-permeable membrane: highlyimpermeable to ions & polarmolecules

Diffusion of gases occur rapidly &depend entirely on concentrationgradient

Water diffuses readily throughbiological membranes via gaps inthe hydrophobic environment

Overview of TransportMechanisms GASES/SMALL

HYDROPHOBIC

MOLECULES

SMALLUNCHARGED

POLARMOLECULES

LARGEUNCHARGED

POLARMOLECULES

IONS

O2CO2N2benzene

H2Oureaglycerol

glucosesucrose

H+, Na

+

HCO3-, K+

Ca2+

, Cl-

Mg2+

synthetic

lipid bilayer

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Passive transport

Simple diffusion

Transport of molecules from high to low concentrationthrough the plasma membrane (non-selective)

Facilitated diffusion

Transport of molecules from high to

low concentration Channels / transporters

Movement of Molecules across Membranes

Rateofmoveme

nt

Throughamembrane

Concentration of solute

Redrawn from:Textbook of Biochemistry with Clinical Correlations

Facilitated diffusion

Diffusion

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Active transport

Primary active transport

Consumes ATP directly to drive the transport Pumps

Secondary active transport

Coupled transport due to the movement of other molecules

down their respective electrochemical gradients Transporters

e.g. Na+/Glucose transporter

Movement of Molecules across Membranes

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Exterior

Cytosol

Membrane Transport Proteins

Channels: facilitated diffusion

Transporters (carriers): facilitated diffusion or secondaryactive transport

Pumps: primary active transport using energy of ATPhydrolysis

* Gradients are indicated by triangles with the tip pointing toward lower concentration Molecular Cell Biology

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(1) Ion Channels

Ion channels are selective, depends on:

Charge

Size

Rapid transport

Can be gated / non-gated

Non-gated channel: leak channels (pores)

Always open

Ions pass through them continuously

Example:

K+ leak channel:responsible for theresting membranepotential

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Can be gated / non-gated Gated channels: have gates that can open & close the channel

Voltage-gated channels: controlled by voltage

Ligand-gated channels: controlled by ligand-binding

Mechanically gated: controlled by mechanical stress (e.g. shear force)

(1) Ion Channels

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Voltage-gated Ion Channels

Sodium channel

4 transmembrane domains

Each has 6 transmembrane helices

http://stke.sciencemag.org/content/sigtrans/vol2004/issue253/images/large/2532004re15F2.jpeg

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Voltage-gated Ion Channels

Sodium channel

4 transmembrane domains

Each has 6 transmembrane

helices

Potassium channel

1 transmembrane domain

Each has 6 transmembrane

helices

P: pore Medical Cell Biology

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Na+ channel blockers

Example:

Tetrodotoxin: from puffer fish

Saxitoxin:

from algae / shellfish poisoning

Specific in blocking Na+ channels

At low dose, paralytic effects observed in patientsintoxicated with these toxins

Can cause death due to respiratoryfailure

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Toxins that Block the Na+ Channels

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Transporters:

Bind the substrate(s) to be transported

Undergo conformational change

Transfer the bound solute across the membrane

* The substrate-binding site is sequentially accessible on one side ofthe bilayer and then on the other

* unlike channel proteins, which forms a direct connection betweencytosol and extracellular compartment

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(2) Transporters

lipidbilayer

Modified from Molecular Biology of the Cell

Tansporter Channel

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Different Types of Transporters

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Uniporter

Symporter

Antiporter

Uniporter: moves a singleparticle down its concentrationgradient (by facillitated diffusion)

Cotransporter: move more than onekind of particles (molecules or ions)by secondary active transport

Symporter: particles move in samedirection

Antiporter: particles move in differentdirection

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Uniporter: Glucose Transporter

(a) (b) (c)

(c) Glucose is released into thecytosol, followed by thereturn of the transporter toits original conformation.

The Cell: A Molecular Approach

(a) The glucose-binding sitefaces the outside of thecell.

(b) Binding of glucose inducesa conformational changeand the transporter facesthe inside of the cell.

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Directly use energy obtained from hydrolysis of ATP to moveparticles across the membrane against electrochemicalgradient (primary active transport)

Classified as P-, V-, and F-type ATPases & the

ABC transporters

a) P-type ATPase

Phosphorylated / dephosphorylated during transport

e.g. Na+/K+ ATPase, Ca2+ & H+/K+ ATPase

b) V-type (vacuolar) ATPase

Vesicles e.g. lysosomes, endosomes

Acidification

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(3) Pumps / Transport ATPase

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(3) Pumps / Transport ATPase

35http://www.bioetch.com/mitochondria-atpase-p-64.html

F-type ATPasec) F-type ATPase

F1F0ATPase

In mitochondrial inner membrane

Synthesizing ATP from ADP & phosphate

d) ABC transporters ATP-binding cassette (ABC) transporter

e.g. P-glyprotein, cystic fibrosistransmembrane conductance regulator (CFTR)

* Different from other ATPase, which use the energyobtained from ATP hydrolysis to drive the movement ofions against the concentration gradient across the plasmamembrane, F0F1 ATPase helps to synthesize ATP

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Some selectedfree ions

Concentration (mM)

Intracellular Extracellular

Na+ 5-15 145

K + 140 5

Ca2+ 0.0001 2.5-5

Cl - 4 110

Extracellular and Intracellular IonConcentrations are maintained by Ion Pumps

Modified from: The Cell: A Molecular Approach

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P-Type ATPase: Na+/K+ ATPase (1)

Na+: 140 mmol/L

K+: 145 mmol/L

K+: 5 mmol/L

Na+: 10 mmol/L

Extracellular:

Intracellular:

3 Na+ bind to sites exposed insidethe cell.

The binding of Na+ stimulatesATP-dependent phosphorylationof the pump.

Phosphorylation exposes the Na+

binding sites to the cell surfaceso that Na+ is released outsidethe cell.


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At the same time, 2 K+ bind tohigh-affinity sites exposed on thecell surface.

The binding of K+ stimulatesdephosphorylation of the pump.

The pump then returns to itsoriginal conformation, releasingK+ inside the cell.


P-Type ATPase: Na+/K+ ATPase (2)

Na+: 140 mmol/L

K+: 145 mmol/L

K+: 5 mmol/L

Na+: 10 mmol/L

Extracellular:

Intracellular:

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Propagation of electric signals in nerve & muscle

Secondary active transport: utilization of anelectrochemical gradient of Na+ for the active transport of

other molecules

To maintain osmotic balance & cell volume

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Importance of Na+/K+ ATPase


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Energy NOT derived from ATP hydrolysis

From the coupled transport of a second molecule in theenergetically favorable direction

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Secondary Active Transport Drivenby Ion Gradient

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Intestinallumen


Glucose Transport by

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Connective tissueAnd blood supply


Glucose Transport byIntestinal Epithelial Cells

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Located in the plasma membrane (pumps Ca2+ out of thecells) and in the ER (pumps Ca2+ into the ER lumen) tomaintain low intracellular concentration

Enable cells sensitive to small increases in intracellular levels

Important in muscle contraction

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P-Type ATPase: Ca2+ Pump

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Someselectedfree ions

Concentration (mM)

Intracellular Extracellular

Na+ 5-15 145

K + 140 5

Ca2+ 0.0001 2.5-5

Cl- 4 110

ER: Endoplasmic reticulum

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Cl- channel

H+/K+

ATPase

K+ channel

Parietal cell~ pH7 ~ pH1

Cl-/HCO3-

exchanger

Intracellular:

K+: 140mMNa+: 5-15mMCl-: 4mM

Extracellular:K+: 5mM

Na+

: 145mMCl-: 110mMVanders Human Physiology

P-Type ATPase: H+/K+ ATPase

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Consist of: Two transmembrane domains

Two cytosolic ATP-binding domains

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ABC transporters

NH2

ATP binding domains

Oligosaccharidechains

NH2

ATP binding domains

Oligosaccharidechains

R domain

P-glycoproteinCystic Fibrosis transmembrane

conductance regulator

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Expression of Pgp found in normal tissues including liver,blood-brain barrier

Function unclear, involved in protection against toxicnatural products

Over-expression of Pgp inmultidrug-resistance(MDR) cancer cells

Efflux pump for

hydrophobic drugs

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P-glycoprotein (P-gp)

P-gp: P-glycoprotein

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http://www.nature.com/nrc/journal/v2/n6/pdf/nrc823.pdf

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Cystic Fibrosis TransmembraneConductance Regulator (CFTR)

http://journals.cambridge.org/download.php?file=%2FERM%2FERM3_07%2FS1462399401002551a.pdf&code=cc455181bc5f93369a01ec5fab104dcd

ATP-binding domain

Transmembranedomain-1

Transmembranedomain-2

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Found in the epithelial cells of many organs including lungs and skin

Function: as a Cl- transport protein, ATP binding to the CFTR isrequired for opening

Cl- moves out of the epithelial cell to the covering mucus in lung

Cystic fibrosis: lethal, autosomal recessive disease Mutations in the CFTRgene

Characteristic manifestations: salty sweat,thick mucus secretions obstruct smallairways, lead to recurrent bacterial

infections Reduced Cl- permeability impairing fluid &

electrolyte secretion, leading to luminaldehydration

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Cystic Fibrosis TransmembraneConductance Regulator (CFTR)

http://www.genemedresearch.ox.ac.uk/cysticfibrosis/protein.html

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Summary Mechanisms for Transporting Ions andSmall Molecules across Cell Membranes

PropertySimple

DiffusionFacilitatedDiffusion

Primary ActiveTransport

SecondaryActive

Transport

Requires specificprotein

Solute transportedagainst its gradient

Coupled to ATPhydrolysis

Driven bymovement of a

cotransported iondown its gradient

Examples ofmoleculestransported

Modified from Molecular Cell Biology

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Summary

Hydrophobic and small molecules pass the plasma membraneby

Polar molecules and ions can be transported faster across themembrane by facilitated transporter: and

Molecules can be transported against the concentrationgradient by primary and secondary active transport

Four types of primary active transporters:a) , e.g. F

1F

0ATPase

b) , e.g. H+ pump in lysosomesc) , e.g. Na+/K+ ATPase, Ca2+ ATPase, H+/K+ ATPased) , e.g. P-glycoprotein, CFTR

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Amphipathic

Referring to a molecule or structure that has both a hydrophobic and ahydrophilic part

Cadherins

A family of dimeric cell-adhesion molecules that aggregate in adherens

junctions and desmosomes and mediate Ca2+

dependent cell-cell interactions

Hydrophilic

Interacting effectively with water

Hydrophobic

Not interacting effectively with water; in general, poorly soluble or insoluble inwater

Integral membrane protein

Any protein that containe one or more hydrophobic segments embedded within

the core of the phospholipid bilayer; also called transmembrane protein50

Glossary (1)

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Integrins

A large family of heterodimeric transmembrane proteins that function asadhesion receptors, promoting cell-matrix adhesion.

Ligand

Any molecule, other than an enzyme substrate, that binds tightly and

specifically to a macromolecule, usually a protein, forming a macromolecule-ligand complex.

Myelin

Stacked specialized cell membrane that forms an insulating layer aroundvertebrate axons and increases the speed of impulse conduction.

Peripheral membrane protein

Any protein that associates with the cytosolic or exoplasmic face of amembrane but does not enter the hydrophobic core of the phospholipid bilayer

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Glossary (2)

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References

1. Medical Physiology: A Cellullar and Molecular Approach. WalterF Boron, and Emile L Boupaep, 2nd edition. Chapter 2. P.9-47.

2. Molecular Cell Biology. Harvey F Lodish, 6th

edition. Chapter10-11. P.409-478.

3. The Cell: A Molecular Approach. Geoffrey M Cooper and RobertE Hausman, 4th edition. Chapter 13. P. 529-568.

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