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Panel of Foundation Studies
Cell Biology
Dr Rebecca K Y LeeSchool of Biomedical Sciences
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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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Lipids in the Plasma Membrane (2)
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
Biochemistry, Van Holde
Galactosylceramide
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Substitutions arefound on the hydroxyl
group here!
Biochemistry, Van Holde
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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
Lipids in the Plasma Membrane (3)
Textbook of Biochemistry with Clinical Correlations
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
Textbook of Biochemistry with Clinical Correlations
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)
Textbook of Biochemistry with Clinical Correlations
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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
Textbook of Biochemistry with Clinical Correlations
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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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Textbook of Biochemistry with Clinical Correlations
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.
Modified from: The Cell: A Molecular Approach
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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.
Modified from: The Cell: A Molecular Approach
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
The Cell: A Molecular Approach
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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
The Cell: A Molecular Approach
Glucose Transport by
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Connective tissueAnd blood supply
The Cell: A Molecular Approach
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.