Panel of Foundation Studies

Cell Biology

Dr Rebecca K Y Lee School of Biomedical Sciences (rebec.lee@cuhk.edu.hk)

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Cell Biology

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

PFOS-011/12 Cell Membrane & Membrane Transport

Learning Outcomes

To describe different classes of lipids and proteins and how they interact to form the cell membrane To describe the functions of plasma membrane To understand the importance of selective permeability in cell membrane To understand various mechanisms that cells use to transport substances across the plasma membrane To differentiate and give examples of simple diffusion, facilitated diffusion, primary active transport and secondary active transport
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Schematic Structure of a Biological Membrane

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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Textbook of Biochemistry with Clinical Correlations

Biology, Campbell Reece

Chemical Composition of Membranes

Proteins + Lipids

Amount varies greatly between different types of membranes Example: myelin

Insulators Few metabolic functions Lipids > proteins Membranes that surround metabolic factories Relatively rich in protein content
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Textbook of Biochemistry with Clinical Correlations

Example: mitochondria

Lipids in the Plasma Membrane (1)

A) Glycerophospholipids

Most abundant 4 components:

Fatty acids Glycerol Phosphate Alcohol

Medical Physiology

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Biochemistry, Stryer

Lipids in the Plasma Membrane (2)

B) Sphingolipids

Basis: sphingosine Ceramide: sphingosine + fatty acid Optional: phosphate group + alcohol (e.g. serine), or carbohydrates Glycosphingolipids: sphingolipids that contain carbohydrates

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Biochemistry, Van Holde

For example

Substitutions are found on the hydroxyl group here!

Galactosylceramide

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Biochemistry, Van Holde

Substitutions are found on the hydroxyl group here!

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Biochemistry, Van Holde

Lipids in the Plasma Membrane (3)

C) Cholesterol

Bulky, rigid structure compared with other hydrophobic membrane components, e.g. fatty acids Bacterial cells do not have cholesterol, neither in mitochondria too
Cholesterol
Cholesterol molecule Peripheral proteins carbohydrates

Hydrophobic fatty acid chain

Textbook of Biochemistry with Clinical Correlations

Hydrophilic polar head

Peripheral proteins

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Medical Physiology

Structure of some common membrane lipids

Amphipathic Lipid Bilayer

Amphipathic

Hydrophilic head

Glycerophospholipids & sphingolipids

Hydrophilic head Hydrophobic tail

Hydrophobic tail

Held together by hydrophobic interactions

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Membrane Asymmetry
Total phospholipid

e.g. erythrocyte membrane

Outside

Outer layer: spingomyelin Inner layer: phosphatidylethanolamine

Percentage of total

Sphingomyelin Phosphatidylcholine

Phosphatidylserine Phosphatidylethanolamine

Inside 14
Textbook of Biochemistry with Clinical Correlations

Mobility of Lipid Components in Membranes

(a) Rapid rotational diffusion

Rotation around the FA chains

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

Thermodynamic constraints

(c) Rapid lateral diffusion

(a) (b) (c)

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Textbook of Biochemistry with Clinical Correlations

The Fluid Mosaic Model

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

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

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Proteins in the Plasma Membrane

A) Proteolipids

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

B) Glycoproteins

Carbohydrates covalently attached to proteins Sugars include glucose, galactose, mannose, fucose, Nacetylgalactosamine, N-acetylglucosamine

Integral protein: span the thickness of the plasma membrane Peripheral proteins: attached to either inner / outer surfaces of the plasma membrane

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Integral Membrane Proteins are Immersed in Lipid Bilayer

Integral membrane proteins
Extracellular side

Peripheral membrane proteins

Cytosolic side

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Textbook of Biochemistry with Clinical Correlations

Functions of Membrane Proteins

Serve as ligand-binding receptor Serve as adhesion molecules

Cell-matrix adhesion molecules

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

Cell-cell adhesion molecules

Serve as enzymes, e.g. Na+/K+-ATPase Allow transport of substances across the membrane

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Summary

The major components of plasma membrane are and The lipid molecules are molecules, they have both 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 Lipid bilayer The components of membranes with lipids & proteins are oriented: the two faces are different
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Biology, Campbell Reece

Paracellular / Transcellular Transport and Transcytosis

Paracellular transport: through tight junctions between epithelial cells Transcellular transport: through apical & basolateral membrane Transcytosis: endocytosis & exocytosis

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Overview of Transport Mechanisms

GASES/SMALL HYDROPHOBIC MOLECULES

Plasma membrane is a semipermeable membrane: highly impermeable to ions & polar molecules Diffusion of gases occur rapidly & depend entirely on concentration gradient Water diffuses readily through biological membranes via gaps in the hydrophobic environment

O2 CO2 N2 benzene H2O urea glycerol

SMALL UNCHARGED POLAR MOLECULES LARGE UNCHARGED POLAR MOLECULES

glucose sucrose

IONS

H , Na + HCO3 , K 2+ Ca , Cl 2+ Mg

synthetic 22 lipid bilayer

Movement of Molecules across Membranes

Passive transport

Simple diffusion

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

Facilitated diffusion
Rate of movement Through a membrane

Transport of molecules from high to low concentration Channels / transporters

Facilitated diffusion

Diffusion 23 Concentration of solute

Redrawn from:Textbook of Biochemistry with Clinical Correlations

Movement of Molecules across Membranes

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

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Membrane Transport Proteins

Channels: facilitated diffusion Transporters (carriers): facilitated diffusion or secondary active transport Pumps: primary active transport using energy of ATP hydrolysis

Exterior

Cytosol

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

(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 the resting membrane potential

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(1) Ion Channels

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)

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Voltage-gated Ion Channels

Sodium channel

4 transmembrane domains Each has 6 transmembrane helices

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

Voltage-gated Ion Channels

Sodium channel

Potassium channel

4 transmembrane domains Each has 6 transmembrane helices

1 transmembrane domain Each has 6 transmembrane helices

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P: pore

Medical Cell Biology

Toxins that Block the Na+ Channels

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 patients intoxicated with these toxins Can cause death due to respiratory failure

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(2) Transporters

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 of the bilayer and then on the other * unlike channel proteins, which forms a direct connection between cytosol and extracellular compartment

lipid bilayer

Tansporter

Channel

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Modified from Molecular Biology of the Cell

Different Types of Transporters

Uniporter: moves a single particle down its concentration gradient (by facillitated diffusion) Cotransporter: move more than one kind of particles (molecules or ions) by secondary active transport

Uniporter

Symporter: particles move in same direction Antiporter: particles move in different direction

Symporter

Antiporter

32 32
Textbook of Biochemistry with Clinical Correlations

Uniporter: Glucose Transporter

(a) (b) (c)

(a) The glucose-binding site faces the outside of the cell.

(b) Binding of glucose induces a conformational change and the transporter faces the inside of the cell.

(c) Glucose is released into the cytosol, followed by the return of the transporter to its original conformation.
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The Cell: A Molecular Approach

(3) Pumps / Transport ATPase

Directly use energy obtained from hydrolysis of ATP to move particles across the membrane against electrochemical gradient (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

F-type ATPase

c) F-type ATPase

F1F0ATPase In mitochondrial inner membrane Synthesizing ATP from ADP & phosphate

* Different from other ATPase, which use the energy obtained from ATP hydrolysis to drive the movement of ions against the concentration gradient across the plasma membrane, F0F1 ATPase helps to synthesize ATP

d) ABC transporters

ATP-binding cassette (ABC) transporter e.g. P-glyprotein, cystic fibrosis transmembrane conductance regulator (CFTR)
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http://www.bioetch.com/mitochondria-atpase-p-64.html

Extracellular and Intracellular Ion Concentrations are maintained by Ion Pumps

Some selected free ions Na+ K+ Ca2+ ClConcentration (mM) Intracellular Extracellular 5-15 140 0.0001 4 145 5 2.5-5 110

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Modified from: The Cell: A Molecular Approach

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

Extracellular:

Na+: 140 mmol/L

K+: 5 mmol/L

3 Na+ bind to sites exposed inside the cell.

The binding of Na+ stimulates ATP-dependent phosphorylation of the pump.

Phosphorylation exposes the Na+ binding sites to the cell surface so that Na+ is released outside the cell.

Intracellular:

K+: 145 mmol/L

Na+: 10 mmol/L

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Modified from: The Cell: A Molecular Approach

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

Extracellular:

Na+: 140 mmol/L

K+: 5 mmol/L

At the same time, 2 K+ bind to high-affinity sites exposed on the cell surface.

The binding of K+ stimulates dephosphorylation of the pump.

The pump then returns to its original conformation, releasing K+ inside the cell.

Intracellular:

K+: 145 mmol/L

Na+: 10 mmol/L

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Modified from: The Cell: A Molecular Approach

Importance of Na+/K+ ATPase

Propagation of electric signals in nerve & muscle Secondary active transport: utilization of an electrochemical gradient of Na+ for the active transport of other molecules To maintain osmotic balance & cell volume

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The Cell: A Molecular Approach

Secondary Active Transport Driven by Ion Gradient

Energy NOT derived from ATP hydrolysis From the coupled transport of a second molecule in the energetically favorable direction

Intestinal lumen

40
The Cell: A Molecular Approach

Glucose Transport by Intestinal Epithelial Cells

Connective tissue And blood supply

41
The Cell: A Molecular Approach

P-Type ATPase: Ca2+ Pump

Located in the plasma membrane (pumps Ca2+ out of the cells) and in the ER (pumps Ca2+ into the ER lumen) to maintain low intracellular concentration Enable cells sensitive to small increases in intracellular levels Important in muscle contraction
Some selected free ions Na+ K+ Ca2+ ClConcentration (mM) Intracellular Extracellular 5-15 140 0.0001 4 145 5 2.5-5 110
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ER: Endoplasmic reticulum

P-Type ATPase: H+/K+ ATPase

Parietal cell ~ pH7 ~ pH1

K+ channel

Cl-/HCO3exchanger Intracellular: K+: 140mM Na+: 5-15mM Cl-: 4mM Extracellular: K+: 5mM Na+: 145mM Cl-: 110mM

H+/K+ ATPase

Cl- channel

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Vanders Human Physiology

ABC transporters

Consist of:

Two transmembrane domains Two cytosolic ATP-binding domains

P-glycoprotein
Oligosaccharide chains

Cystic Fibrosis transmembrane conductance regulator

Oligosaccharide chains

NH2

ATP binding domains

NH2

R domain ATP binding domains

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P-glycoprotein (P-gp)

Expression of Pgp found in normal tissues including liver, blood-brain barrier Function unclear, involved in protection against toxic natural products Over-expression of Pgp in multidrug-resistance (MDR) cancer cells Efflux pump for hydrophobic drugs

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P-gp: P-glycoprotein

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

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Transmembrane domain-1 Transmembrane domain-2

ATP-binding domain

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

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Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Found in the epithelial cells of many organs including lungs and skin Function: as a Cl- transport protein, ATP binding to the CFTR is required for opening Cl- moves out of the epithelial cell to the covering mucus in lung Cystic fibrosis: lethal, autosomal recessive disease Mutations in the CFTR gene Characteristic manifestations: salty sweat, thick mucus secretions obstruct small airways, lead to recurrent bacterial infections Reduced Cl- permeability impairing fluid & electrolyte secretion, leading to luminal dehydration
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http://www.genemedresearch.ox.ac.uk/cysticfibrosis/protein.html

Summary Mechanisms for Transporting Ions and Small Molecules across Cell Membranes

Property
Requires specific protein Solute transported against its gradient Coupled to ATP hydrolysis Driven by movement of a cotransported ion down its gradient Examples of molecules transported

Simple Diffusion

Facilitated Diffusion

Primary Active Transport

Secondary Active Transport

Modified from Molecular Cell Biology

Summary

Hydrophobic and small molecules pass the plasma membrane by Polar molecules and ions can be transported faster across the membrane by facilitated transporter: and Molecules can be transported against the concentration gradient by primary and secondary active transport Four types of primary active transporters: a) , e.g. F1F0ATPase b) , e.g. H+ pump in lysosomes c) , e.g. Na+/K+ ATPase, Ca2+ ATPase, H+/K+ ATPase d) , e.g. P-glycoprotein, CFTR
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Glossary (1)
Amphipathic Referring to a molecule or structure that has both a hydrophobic and a hydrophilic 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 in water Integral membrane protein Any protein that containe one or more hydrophobic segments embedded within 50 the core of the phospholipid bilayer; also called transmembrane protein

Glossary (2)
Integrins A large family of heterodimeric transmembrane proteins that function as adhesion 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 macromoleculeligand complex. Myelin Stacked specialized cell membrane that forms an insulating layer around vertebrate axons and increases the speed of impulse conduction. Peripheral membrane protein Any protein that associates with the cytosolic or exoplasmic face of a membrane but does not enter the hydrophobic core of the phospholipid bilayer
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References
1. Medical Physiology: A Cellullar and Molecular Approach. Walter F Boron, and Emile L Boupaep, 2nd edition. Chapter 2. P.9-47. 2. Molecular Cell Biology. Harvey F Lodish, 6th edition. Chapter 10-11. P.409-478. 3. The Cell: A Molecular Approach. Geoffrey M Cooper and Robert E Hausman, 4th edition. Chapter 13. P. 529-568.

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