viewing guide

M O L E C U L A R B I O L O G Y O F

THE CELLF O U R T H E D I T I O N

Artistic and Scientific Direction by Peter WalterNarrated by Julie Theriot

Bruce AlbertsAlexander Johnson

Julian LewisMartin Raff

Keith RobertsPeter Walter

Cell BiologyInteractive

Viewing Guide

· Introduction· Table of Contents· Complete Set of Scripts

Production Design and Development by Michael Morales

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INTRODUCTION

Welcome to Cell Biology Interactive

New biological imaging and computer technologies have changed theways in which we learn about and appreciate the behavior and innerworkings of living cells. We now include Cell Biology Interactive with everycopy of Molecular Biology of the Cell. This CD-ROM disk contains over 90video clips, animations, molecular structures, and high-resolution micro-graphs that were selected to complement the individual chapters. Nearlyall items on the disk are accompanied by a short narration that introducesand explains key concepts. Our intent is to provide students and instruc-tors with an opportunity to observe living cells and molecules in action,since words and static images simply cannot do justice to the remarkabledynamics of the cellular and molecular world.

One cannot watch cells crawling, dividing, segregating their chromo-somes, or rearranging their membranes without a feeling of surprise andcuriosity about the mechanisms that underlie these processes. We hopethat Cell Biology Interactive can provide a valuable tool for motivating stu-dents to master the topics covered in the text. We also hope that instruc-tors can use these visual resources in the classroom to illuminate, not onlythe course material, but also the beauty and wonder of this microcosm.We designed animations to bring to life some of the more complicated fig-ures in the book (and also for everyone’s enjoyment). Many of the videosprovide visual demonstrations of topics that can be difficult to appreciate,such as membrane fluidity, and the high-resolution micrographs allowstudents to explore some magnificent cell images in detail. We have alsocreated three-dimensional models of some of the most interestingmolecules, presented in short and truly interactive tutorials that invitefurther exploration.

The contents of this CD-ROM represent the work of numerous labsaround the world that provided video clips from original research, anima-tion segments, micrographs, and molecular data. We are deeply indebtedto the scientists who generously made this material available to us.

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TABLE OF CONTENTS

CHAPTER 1 CELLS AND GENOMES

1.1 V Keratocyte Dance1.2 V Crawling Amoeba1.3 V Swimming Eutreptiella1.4 EM Plant Cells

CHAPTER 2 CELL CHEMISTRY AND BIOSYNTHESIS

2.1 A Analogy of Enzyme Catalysis2.2 A Brownian Motion2.3 M Molecular Structures Introduction2.4 M Amino Acids2.5 M Nucleotides2.6 M Lipids2.7 M Sugars

CHAPTER 3 PROTEINS

3.1 M a Helix3.2 M b Sheet3.3 M Disulfide Bridges3.4 M Proline Kinks3.5 M Coiled-Coil3.6 M SH2 Domain3.7 M Lysozyme3.8 M Oligomeric Proteins3.9 M Aspartate Transcarbamylase3.10 A EF-Tu3.11 A The ‘Safe Crackers’

CHAPTER 4 DNA AND CHROMOSOMES

4.1 M DNA Structure4.2 EM Liver Cell:View 1

CHAPTER 5 DNA REPLICATION, REPAIR,AND RECOMBINATION

5.1 A DNA Helicase5.2 M DNA Polymerase5.3 M Sliding Clamp5.4 A DNA Replication Fork5.5 A Cross-Strand Exchange

CHAPTER 6 HOW CELLS READ THE GENOME: FROM DNA TO PROTEIN

6.1 M RNA Structure6.2 M RNA Polymerase II6.3 M tRNA6.4 A mRNA Translation6.5 A Ribosome Elongation Cycle6.6 A Ribosome Ratchet

Key to symbols:A = animation,V = video, M = molecular model (Chime), EM = cell image (magnifier)

4CHAPTER 7 CONTROL OF GENE EXPRESSION

7.1 M Homeodomain7.2 M Zinc Finger Domain7.3 M Leucine Zipper7.4 M TATA-Binding Protein

CHAPTER 8 MANIPULATING PROTEINS, DNA,AND RNA

8.1 A Polymerase Chain Reaction8.2 V Anatomy of a PDB File

CHAPTER 9 VISUALIZING CELLS

9.1 A Lazer Tweezers9.2 EM Liver Cell:View 29.3 EM Liver Cell:View 39.4 EM Liver Cell:View 4

CHAPTER 10 MEMBRANE STRUCTURE

10.1 V Fluidity of the Lipid Bilayer10.2 M Lipids and Lipid Bilayer10.3 V Membrane Disruption by Detergent10.4 V Membrane Effects in a Red Blood Cell10.5 M Bacteriorhodopsin10.6 V FRAP

CHAPTER 11 MEMBRANE TRANSPORT OF SMALL MOLECULES AND THE ELECTRICALPROPERTIES OF MEMBRANES

11.1 M Potassium Channel11.2 V Hair Cells

CHAPTER 12 INTRACELLULAR COMPARTMENTS AND PROTEIN SORTING

12.1 V Nuclear Import12.2 V ER Tubules12.3 A Ribosome/ER Translocator12.4 EM Freeze Fracture of Yeast Cell12.5 EM Liver Cell:View 5

CHAPTER 13 INTRACELLULAR VESICULAR TRAFFIC

13.1 A Clathrin13.2 V Secretory Pathway13.3 V Exocytotic Transport13.4 V Endosome Fusion13.5 V Phagocytosis13.6 EM Pancreas:View 113.7 EM Pancreas:View 2

CHAPTER 14 ENERGY CONVERSION: MITOCHONDRIA AND CHLOROPLASTS

14.1 A ATP Synthase—A Molecular Turbine14.2 V ATP Synthase—Disco14.3 M Photosynthetic Reaction Center14.4 V Mitochondrial Fission and Fusion14.5 A Tomograph of Mitochondrion

CHAPTER 15 CELL COMMUNICATION

15.1 V Calcium Signaling15.2 V Chemotaxis of Neutrophils


15.3 V Neutrophil Chase15.4 A cAMP Signaling15.5 A G-protein Signaling15.6 A Inositol Phosphate Signaling15.7 M Src15.8 M Ras15.9 M Calmodulin15.10 M Growth Hormone Receptor

CHAPTER 16 THE CYTOSKELETON

16.1 V Dynamic Instability of Microtubules16.2 V Microtubule and ER Dynamics16.3 V Microtubule Dynamics in Vivo16.4 V Beating Heart Cell16.5 V Organelle Movement on Microtubules16.6 A Myosin16.7 A Kinesin16.8 V Crawling Actin16.9 EM Heart Tissue16.10 EM Heart Muscle Cell

CHAPTER 17 THE CELL CYCLE AND PROGRAMMED CELL DEATH

17.1 V Apoptosis17.2 M p53-DNA Complex17.3 M Cdk2

CHAPTER 18 THE MECHANICS OF CELL DIVISION

18.1 V Plant Cell Division18.2 V Animal Cell Division18.3 V Mitotic Spindles in a Fly Embryo18.4 V Mitotic Spindle18.5 EM Mitotic Chromosomes

CHAPTER 19 CELL JUNCTIONS, CELL ADHESION,AND THE EXTRACELLULAR MATRIX

19.1 V Adhesion Junctions Between Cells19.2 V Rolling Leukocytes19.3 EM Junction Between Two Muscle Cells

CHAPTER 20 GERM CELLS AND FERTILIZATION

20.1 V Calcium Wave During Fertilization20.2 V Sea Urchin Fertilization

CHAPTER 21 DEVELOPMENT OF MULTICELLULAR ORGANISMS

21.1 V Developing Egg Cells21.2 V Gastrulation21.3 V Drosophila Development21.4 V Early Zebrafish Development21.5 V Neurite Outgrowth

CHAPTER 22 HISTOLOGY:THE LIVES AND DEATHS OF CELLS IN TISSUES

22.1 V Wound Healing22.2 V Megakaryocyte22.3 EM Liver Cell:View 622.4 EM Liver Cells (Sinusoid Space)22.5 EM Endothelial Cell in Liver

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CHAPTER 23 CANCER

23.1 V Breast Cancer Cells

CHAPTER 24 THE ADAPTIVE IMMUNE SYSTEM

24.1 M Antibodies24.2 M MHC Class I24.3 M MHC Class II24.4 V Lymphocyte Homing24.5 V Killer T-Cell

CHAPTER 25 PATHOGENS, INFECTION,AND INNATE IMMUNITY

25.1 M Hemagglutinin25.2 V Listeria Parasites

APPENDIX

A.1 PDF Viewing GuideA.2 V The AuthorsA.3 V Credits

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1.1 KERATOCYTE DANCE

Keratocytes, found on the scales of fish, are specialized for very rapidmotility in order to heal scratches.

Video editing and concept:Justin Reichman

Music:Freudenhaus Audio Productions (www.fapsf.com)

1.2 CRAWLING AMOEBA

This single-celled amoeba crawls around by using actin polymerization topush out pseudopods, or false feet, to explore new territory. At the sametime, organelles move in complex patterns within the cell.

Reproduced by copyright permission of CELLS alive!, CDROM and Video Library,1998–2001.

1.3 SWIMMING EUTREPTIELLA

Some cells use rather peculiar ways to move, such as this eutreptiella flag-ellate, which uses both flagella and pronounced cell shape changes toswim.

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CELLS alive!www.cellsalive.com

Mark S. CooperUniversity of Washington

Richard E.TriemerRutgers, State University of New Jersey

1.4 PLANT CELLS

Show me:

· plasma membranes and cell wall· vacuoles· chloroplasts· thylakoids

2.1 ANALOGY OF ENZYME CATALYSIS

Envision a molecule, here symbolized by the ball, that, in principle, canreact in four different ways.

To undergo any of these reactions, the molecule must overcome an activa-tion energy barrier that is of a characteristic height for each of the possiblereactions.

This can be achieved, for example, by putting more energy into the system,such as when heat is added. In the example shown here, the molecule willenter many different reaction paths by overcoming similar activationenergy barriers.

An enzyme, in contrast, reduces the activation energy barrier of only onespecific reaction path. An enzyme therefore allows reactions to proceed atnormal temperatures and directs them into one desired pathway.

Animation:N8 Digital (www.N8digital.com)

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Doug BrayThe University of Lethbridge, Canada

Brian Oates and Cyprien LomasThe University of British Columbia

Original illustrations and storyboard:Nigel Orme and Christopher Thorpe

2.2 BROWNIAN MOTION

Powered by thermal energy, molecules are constantly in motion—calledBrownian motion—which allows them to diffuse through cells in a randomwalk. Our simulation shows the degree to which a small sugar moleculeon the left and a larger protein on the right explore the interior space of acell, here shown as a cube with a 10 micrometer side. The animationrepresents one second in real time.

Note that the paths of the molecules in the second simulation are differentfrom those in the first, thus showing the randomness of Brownian motion.

Final composition:Blink Studio Ltd. (www.blink.uk.com)

2.3 MOLECULAR STRUCTURES INTRODUCTION

2.3.1The molecular structures you are about to explore allow a variety of inter-active possibilities. You can zoom in and zoom out, if you hold down theshift key, then hold down the mouse button, and drag the cursor up anddown the screen. Go ahead and try it.

2.3.2You can also grab a structure and rotate it in three dimensions, simply byholding down the mouse button and dragging the cursor over themolecule. Try it.

2.3.3You can stop movie playback at any time in midstream by clicking on thepause button. Try it.

Molecular modelling and animation:Timothy DriscollMolvisions (www.molvisions.com)

2.4 AMINO ACIDS


Source:Klotho: Biochemical Compounds Declarative Database(www.ibc.wustl.edu/klotho/)

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Julian LewisImperial Cancer Research Fund

PDB ID source: public domain

PDB ID source: Klotho

2.5 NUCLEOTIDES



2.6 LIPIDS


Source:Lipidat Database(www.lipidat.chemistry.ohio-state.edu/)

2.7 SUGARS


Sources:Klotho: Biochemical Compounds Declarative Database(www.ibc.wustl.edu/klotho/)

NYU MathMol Library of 3D Molecular Structures(www.nyu.edu/pages/mathmol/library/)

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PDB ID source and number *: Compilationof ribonucleotides and deoxyribonucleotides(Klotho); Nucleoside diphosphate kinaseisoform B (1BE4)

PDB ID source: Lipidat

PDB ID source: Monosaccharides (Klotho);Fructose (NYU MathMol)

* PDB ID numbers with an asterisk are from The RCSB Protein Data Bank (http://www.rcsb.org/pdb). Source: H.M. Bergman, J.Westbrook, Z. Feng, G.Gilliland,T.N. Bhat, H.Weissig, I.N. Shindyalov, P.E. Bourne.The protein data bank. Nucleic Acids Research, 28: 235–242, 2000.

3.1 a HELIX

3.1.1The a helix is one of the most common secondary structures in proteins.Amino acid side chains project outwards from the polypeptide backbonethat forms the core of the helix. The chain is stabilized in this conforma-tion by hydrogen bonds between the backbone amino group of oneamino acid and the backbone carbonyl group of another amino acid thatis four positions away. These interactions do not involve side chains. Thusmany different sequences can adopt an a helical structure.

3.1.2a helices are regular cylindrical structures. Amino acid side chains projectoutwards from the peptide backbone that forms the core of the helix. Onefull turn occurs every 3.6 residues and extends the length of the helix byapproximately 0.5 nm.

3.1.3Secondary structures are often represented in cartoon form to clarify theunderlying structure of a protein. In this representation, the twisted 3Dribbon follows the path of the peptide backbone.


Source:Glactone (www.chemistry.gsu.edu/glactone)

3.2 b SHEET

3.2.1The b sheet is another common secondary structure. In contrast to ana helix, it is formed by hydrogen bonds between backbone atoms on adja-cent regions of the peptide backbone, called b strands. These interactionsdo not involve side chains. Thus, many different sequences can form a bsheet.

3.2.2A b sheet is a regular and rigid structure often represented as a series offlattened arrows. Each arrow points towards the protein’s C-terminus. Inthe example shown here the two middle strands run parallel—that is, inthe same direction—whereas the peripheral strands are antiparallel.

3.2.3The amino acid side chains from each strand alternately extend aboveand below the sheet, thereby allowing each side to have distinct proper-ties from the other. b sheets are usually twisted and not completely flat.


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PDB ID source: Glactone

PDB ID number *: 1PGB

3.3 DISULFIDE BRIDGES

3.3.1Disulfide bonds stabilize the structure of many proteins by formingintramolecular bridges. In this example, five disulfide bonds “zip up” thecenter of a protease inhibitor. Most extracellular proteins contain disul-fide bonds.

3.3.2Disulfide bonds are formed by oxidation of two cysteine residues. In thisreaction, the hydrogen atoms are removed from their sulfur atoms toallow formation of the sulfur–sulfur bond.


3.4 PROLINE KINKS

3.4.1Because of the geometry of its side chain, proline cannot readily fit intothe regular structure of an a helix. Prolines are therefore often found inloops at the ends of a helices, acting as “helix breakers.”

3.4.2Of the twenty naturally occurring amino acids, proline is the only one thathas a cyclic side chain, forming a five-membered ring that includes thebackbone nitrogen. This geometry severely limits the flexibility of thebackbone.

3.4.3Because of this restricted conformational flexibility, many prolineresidues introduce sharp kinks into the path of a polypeptide backbone.



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PDB ID number *: Bovine a-chymotrypsin-eglin c complex (1ACB); L-proline (Klotho);Gal4 complex with DNA (1D66)

PDB ID number *: 1BI6

3.5 COILED-COIL

3.5.1In a typical coiled-coil two a helices wrap around each other to form astable structure. One side of each helix contains mostly aliphatic aminoacids, such as leucines and valines, while the other side contains mostlypolar residues. Helices containing distinct hydrophobic and polar sidesare called amphipathic. In a coiled-coil, two amphipathic helices arealigned so their hydrophobic sides snuggle tightly together in the center,with their polar faces exposed to the solvent.

3.5.2A triple coiled-coil is another stable structure formed by a helices. In thiscase, three amphipathic helices twist around a central axis. Thehydrophobic sides of all three helices face the center of the coil, creatinga stable hydrophobic core.

3.5.3Coiled-coils are often found in elongated, fibrous proteins. A triple coiled-coil is the major structural theme in fibrinogen, a protein involved inblood clotting. The fibrous nature of this protein is intimately related to itsability to form clots.


3.6 SH2 DOMAIN

3.6.1The SH2 domain of the tyrosine kinase and oncogene Src is used hereto demonstrate the different ways in which a protein structure can bedisplayed. The backbone view shows the path of the polypeptide chain.The chain is colored blue at its C terminus.

3.6.2The ribbons view accents a helices and b sheets. These secondary struc-ture elements determine the fold of most polypeptide chains. b strandsare shown as arrows pointing from the N- to the C-terminus, and a helicesare shown as twisted cylinders.

3.6.3In a wireframe presentation, the covalent bonds between all of the atomsin the polypeptide are shown as sticks.

3.6.4A spacefill view depicts each atom in the polypeptide as a solid sphere.The radius of the sphere represents the van der Waals radius of the atom.The coloring scheme follows the same rainbow spectrum used beforewith the N terminus red and the C terminus blue.

3.6.5In this spacefill view different atoms in the polypeptide chain are coloredaccording to element. By convention, carbon is colored gray, nitrogenblue, oxygen red, and sulfur yellow.


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PDB ID number *: GCN4 leucine zipper(2ZTA);Trimeric coiled-coil domain of chicken cartilage matrix protein (1AQ5);Native chicken fibrinogen (1EI3)

PDB ID number *: 1SHA

3.7 LYSOZYME

3.7.1Lysozyme is a small enzyme that binds to polysaccharide chains andbreaks them apart by hydrolysis. It has two structural domains. Onedomain is composed mostly of a helices, while the other domain is com-posed mostly of b strands. The interface between the two domains formsa cleft in which the substrate binds. The structure shown here containsone of the products of the hydrolysis reaction.

3.7.2Lysozyme acts as a catalyst by adding a molecule of water to the bondbetween two sugars, breaking the bond. This reaction is catalyzed by twostrategically positioned amino acid side chains in the enzyme’s active site:glutamate 35 and aspartate 52. The highlighted group on the reactionproduct shown here would have formed the bond cleaved in the reaction.


3.8 OLIGOMERIC PROTEINS

3.8.1Many proteins are composed of multiple polypeptide chains, or subunits.The Cro repressor, for example, is a homodimer formed of two identicalsubunits. The subunits join in a head-to-head fashion as two small bsheets—one from each subunit—zipper up and form a larger b sheet.

3.8.2The enzyme neuraminidase is composed of four identical subunitsarranged in a square. Each pair of two subunits is held together in head-to-tail fashion, by repeated use of the same binding interaction. Thisbecomes clear when the polypeptide chains are colored in a rainbowpattern, so that the same regions of each subunit have the same colors. Allsubunits adhere to each other through contacts between the orange andlight-blue regions.

3.8.3Hemoglobin is a tetrameric protein that transports oxygen. It is composedof two a subunits and two closely related b subunits. Oxygen binds toheme groups in the protein, which are shown in red. Each subunit cansense whether neighboring subunits contain bound oxygen. The proteinsubunits therefore communicate with one another through the interfacesthat hold them together.

3.8.4The tumor suppressor protein p53 is a tetramer of four identical subunits.Each p53 subunit contains a simple tetramerization domain composed ofa single b strand connected to an a helix. The tetrameric form of p53assembles as a dimer of dimers. Two copies of p53 interact via b strands,forming a two-stranded b sheet. Two such dimers interact via their ahelices to form the tetrameric assembly.


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PDB ID number *: Cro repressor proteinfrom bacteriophage lambda (5CRO);Neuraminidase of influenza virus (1NN2);Deoxy human hemoglobin (1A3N); p53tetramerization domain (1C26)

PDB ID number *: 1LSZ

3.9 ASPARTATE TRANSCARBAMYLASE

3.9.1Aspartate transcarbamylase, or ATCase for short, is a well-studied exam-ple of allosteric regulation. ATCase catalyzes one of the early reactions inpyrimidine biosynthesis. This huge enzyme complex is composed of 12subunits. Six are regulatory subunits that form a belt around the center ofthe complex. The remaining six subunits are arranged as two catalytictrimers, each positioned on one end of the enzyme.

3.9.2ATCase alternates between two conformational states: an inactive tenseor T state and a catalytically active relaxed or R state. ATCase is inactivewhen the inhibitor cytosine triphosphate is bound to its regulatory sub-units. Binding of the two substrates, carbamylphosphate and aspartate, tothe catalytic subunits switches the enzyme into the active R state.

3.9.3The conformational change in ATCase from T state to R state involves adrastic change in the interactions between catalytic subunits. In the T state,glutamate 239 from a subunit of one catalytic trimer interacts with lysine164 and tyrosine 165 from an adjacent subunit of the opposing catalytictrimer. With the transition to the R state, these subunit-subunit interac-tions are lost; the glutamate now interacts with the lysine and tyrosinefrom its own subunit. These atomic-level changes result in large move-ments of the subunits relative to one another.

3.9.4In each catalytic subunit of ATCase, the region of subunit–subunit inter-action, our glutamate/lysine/tyrosine trio, is very close to the enzyme’sactive site. These conformational changes that affect the subunit–subunitinterface in turn affect the active site residues. In the active R state, theactive site side chains nestle up to the substrate to promote substratebinding and catalysis. In contrast, in the inactive T state, the active siteside chains are dispersed.


3.10 EF-TU

Elongation factor Tu has three domains, which are compactly arranged inits GTP-bound state. Here we show the surface of the protein with each ofits domains in a different color.

Regions of all three protein domains contribute to the tRNA binding site.

An important dynamic element in the structure of elongation factor Tu isthe switch helix.

As GTP is hydrolyzed and the gamma phosphate is released, the switchhelix rearranges.

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PDB ID number*: Aspartate transcarbamy-lase complex with CTP (5AT1); Compilationof aspartate transcarbamylase complex withCTP & aspartate transcarbamylase complexwith PAM, MAL, and CTP (5AT1 & 8AT1)

This in turn leads to a major structural rearrangement of the three proteindomains, which disrupts the tRNA binding site.

Thus upon GTP hydrolysis, the tRNA is released from elongation factorTu.

Animation:Graham JohnsonFivth Element (www.fivth.com)

3.11 THE ‘SAFE CRACKERS’

Individual proteins often collaborate as subunits of large protein assem-blies, in which their individual activities may be coordinated.

Energetically favorable changes in substrates bound to one or moresubunits, such as the hydrolysis of ATP, lead to orderly movementsthroughout the protein complex that accomplish a specific task.


4.1 DNA STRUCTURE

4.1.1Two DNA strands intertwine to form a double helix. Each strand has abackbone composed of phosphates and sugars to which the bases areattached. The bases form the core of the double helix, while thesugar/phosphate backbones are on the outside. The two grooves betweenthe backbones are called the major and minor groove based on their sizes.Most protein–DNA contacts are made in the major grove, because theminor groove is too narrow.

4.1.2The DNA backbone is assembled from repeating deoxyribose sugar unitsthat are linked through phosphate groups. Each phosphate carries a neg-ative charge, making the entire DNA backbone highly charged and polar.

4.1.3A cyclic base is attached to each sugar. The bases are planar and extendout perpendicular to the path of the backbone. Pyrimidine bases are com-posed of one ring and purine bases of two rings. Adjacent bases arealigned so that their planar rings stack on top of one another. Base stack-ing contributes significantly to the stability of the double helix.

4.1.4In a double helix, each base on one strand is paired to a base on the otherstrand that lies in the same plane. In these base pairing interactions,guanine always pairs with cytosine, and thymine with adenine.

4.1.5A GC pair is stabilized by three hydrogen bonds formed between aminoand carbonyl groups that project from the bases.

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PDB ID number *: 132D


4.1.6In contrast, an AT pair is stabilized by two hydrogen bonds.

4.1.7The specificity of base pairing ensures that the two strands are comple-mentary.


4.2 LIVER CELL: VIEW 1

Show me:

· nuclear envelope· nuclear pore· euchromatin· heterochromatin

5.1 DNA HELICASE

Helicases separate nucleic acid duplexes into their component strandsusing energy from ATP hydrolysis.

The crystal structure of this DNA helicase from bacteriophage T7, revealsan hexagonal arrangement of six identical subunits. Surprisingly, the ringis not sixfold symmetric, but is slightly squished.

A model for the mechanism of how the enzyme might work explains thisstructural asymmetry. Of the six potential ATP binding sites, two oppos-ing ones bind ATP tightly, two are more likely to bind ADP and phosphate,and two are empty. These three states may interconvert in a coordinatefashion as ATP is hydrolyzed, creating a ripple effect that continuouslyruns around the ring.

Because of these conformational changes, the loops that extend into thecenter hole of the ring—that are proposed to bind DNA—oscillate up anddown, as seen in this cross section. The oscillating loops might pull a DNAstrand through the central hole, thus unwinding the double helix in theprocess.

A frontal view shows the full dynamics of this fascinating proteinmachine.


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Dale B.Wigley and Martin R. SingletonImperial Cancer Research Fund

Tom EllenbergerHarvard Medical School

Michael R. SawayaUniversity of California at Los Angeles



5.2 DNA POLYMERASE

5.2.1DNA polymerase adds nucleotides to a growing DNA strand, guided bythe sequence of the template strand. Incoming nucleotides are added tothe free 3¢ hydroxyl group that is presented at the end of the growingstrand. The growing DNA strand is therefore assembled in the 5¢ to 3¢direction.

5.2.2The 3¢ end of the growing strand is positioned in the active site of DNApolymerase. Catalysis is carried out by two highly-conserved aspartateresidues and several crucial magnesium ions. The correct incomingnucleotide, deoxyCTP in this case, is selected by the next unpaired baseon the template strand.

5.2.3The incoming nucleotide is added to the growing strand. A new covalentphosphodiester bond is formed that extends the length of the growingstrand by one nucleotide. The polymerase moves down the DNA to thenext unpaired base on the template strand, and the catalytic process isrepeated.


5.3 SLIDING CLAMP

5.3.1Sliding clamps allow DNA polymerases to remain attached to their DNAtemplate. In this way, DNA polymerase can synthesize long stretches ofDNA efficiently without falling off the template DNA. The multi-subunitclamp forms a ring that encircles the DNA helix; its structure thus relatesto its function in a most intuitive way.


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PBD ID number *: 1QE4

PDB ID number *: Human DNA polymerase beta complexed with gappedDNA (1BPX); Compilation of human DNApolymerase beta complexed with gappedDNA & human DNA polymerase betacomplexed with gapped DNA and ddCTP(1BPX & 1BPY)

5.4 DNA REPLICATION FORK

In a replication fork, two DNA polymerases collaborate to copy the leading-strand template and the lagging-strand template DNA.

In this picture, the DNA polymerase that produces the lagging strand hasjust finished an Okazaki fragment.

The clamp that keeps the lower DNA polymerase attached to the laggingstrand dissociates, and the DNA polymerase temporarily releases the lag-ging-strand template DNA.

As the DNA helicase continues to unwind the parental DNA, the primasebecomes activated and synthesizes a short RNA primer on the growinglagging strand.

The DNA polymerase binds to the DNA again and becomes locked in bythe clamp.

The polymerase uses the RNA primer to begin a short copy of the laggingstrand-template DNA.

The polymerase stalls when it reaches the RNA primer of the precedingOkazaki fragment, and the entire cycle repeats.


Music:Christopher Thorpe

5.5 CROSS-STRAND EXCHANGE

Cross-strand exchange is an important intermediate structure in generalDNA recombination.

The characteristic junction is formed when two homologous double-stranded DNA molecules reciprocally exchange DNA strands.

This junction can be visualized directly in the electron microscope.

An important aspect of recombination is that the junction point is notfixed after it has formed, but can migrate up and down the DNA as shownin this animation.

In the cell, this junction is formed and stabilized by a specific protein,which can be seen here in the background.

Introductory animation:Allison Bruce

Reproduced by permission of Professor David Rice, Krebs Institute

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Electron micrography:David DresslerUniversity of Oxford

Huntington PotterUniversity of South Florida

Molecular animation:David A.WallerAstbury Centre for Structural MolecularBiology, University of Leeds

David Rice, Peter Artymiuk, John Rafferty,and David HargreavesKrebs Institute, University of Sheffield

6.1 RNA STRUCTURE

6.1.1Like DNA, RNA strands also pair to form a double helix. The pairednucleotide bases are packed in the middle of an RNA helix, surrounded bythe backbone. The RNA helix has a different geometry than a standardDNA helix. For example, the RNA helix has a significantly narrower anddeeper major groove.

6.1.2The backbone is composed of repeating ribose sugars and phosphategroups. Unlike the 2¢ deoxyribose used in DNA, ribose has a hydroxylgroup attached to the 2¢ carbon. This ‘extra’ hydroxyl group influences thesecondary structure. It is too bulky to allow RNA to fold like DNA, whichis the primary reason why an RNA helix has a different structure.

6.1.3Base pairing between strands is similar to that in DNA, except that adeninepairs with uracil instead of thymine. Thymine is exclusively used in DNA.The A-U pair also has two hydrogen bonds.

6.1.4RNA strands often fold into complex structures. In this stem-loop struc-ture, also called an RNA hairpin, a single-stranded RNA molecule foldsback onto itself. The stem at the bottom is a classical RNA helix. Bases inthe single-stranded loop, in contrast, are either exposed or engaged innonstandard interactions.


6.2 RNA POLYMERASE II

6.2.1Eukaryotic RNA polymerase II transcribes all messenger RNA moleculesin the cell. It is a huge complex of ten different protein subunits. Theactive site of the enzyme lies at the interface between the two largest sub-units. In this structure a short stretch of a DNA-RNA hetero-duplex wasco-crystallized. New nucleotides would be continually added to the 3¢hydroxyl group of the RNA shown in red.

6.2.2In the active site of RNA polymerase II, a single-stranded DNA template istranscribed into a complementary RNA transcript. The initial product is aDNA:RNA hybrid from which the newly synthesized RNA strand isstripped off. It leaves the polymerase via an exit groove on the protein’ssurface.

6.2.3A pore on the back side of RNA polymerase II extends from the proteinsurface all the way to the active site. The nucleotide triphosphates used tobuild the growing RNA transcript enter the polymerase through this pore.


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PDB ID number *: RNA duplex containing apurine-rich strand (1RRR); RNA tetraloop(1AFX)

PDB ID number *: 1I6H

6.3 tRNA

6.3.1All tRNAs have a characteristic L-shape, with an amino acid attached tothe 3¢ end at the tip of the shorter arm. The anticodon loop is positionedat the opposing end of the molecule and contains the anticodon basetriplet. Interactions between the equally conserved D and T loops areimportant in maintaining the tRNA’s shape.

6.3.2The amino acid, a phenylalanine in this case, is covalently attached to aconserved sequence, CCA, that is common to the 3¢ terminus of all tRNAs.

6.3.3The anticodon is comprised of three nucleotides complementary to thecodon in the mRNA. The bases are exposed, and are thus freely accessiblefor basepairing during protein synthesis. In this example, the anticodonsequence is GAA, which would match a UUC codon on a messenger RNA,specifying phenylalanine.

6.3.4The charging of tRNAs with the correct amino acids is carried out byaminoacyl-tRNA synthetases. As revealed in the cut-away view, thecomplex of phenylalanine-tRNA with its cognate synthetase shows anextensive contact surface that includes recognition sites for the anticodonbase triplet. The tRNA’s CCA end is deeply buried in the enzyme.


6.4 mRNA TRANSLATION

To extend a growing polypeptide chain the ribosome must select thecorrect amino acids that are specified by the messenger RNA.

An aminoacyl-tRNA bound to elongation factor Tu, EF-Tu for short, entersthe free A site on the ribosome. Elongation factor Tu hydrolyzes its boundGTP and releases the aminoacyl-tRNA.

If the anticodon of the charged tRNA does not match the codon in themessenger RNA, the tRNA is rejected.

Elongation factor Tu leaves the ribosome and the process of trial and errorrepeats until the correct tRNA is identified.

The correct tRNA remains bound for a long enough time to allow elonga-tion factor Tu to leave first, and is then committed to be used in proteinsynthesis.

The ribosome undergoes a dramatic conformational change that catalyzesthe formation of the new peptide bond. Elongation factor G binds to theribosome. Hydrolysis of GTP by elongation factor G switches the ribo-some back to the state in which it can accept the next incoming tRNA.


21

PDB ID number *: Phe-tRNA, elongationfactor Ef-Tu: Gdpnp ternary complex(1TTT);Yeast aspartyl-tRNA synthetase(1ASY)


6.5 RIBOSOME ELONGATION CYCLE

Models of the structure of the ribosome and the position of bound tRNAshave been used in this animation to display the ballet of orchestratedmovements underlying translation elongation.

In this view we see the ribosome from the top.

Removal of the small 30S ribosomal subunit from the model reveals themRNA and a peptidyl tRNA in the P site at the interface between the tworibosomal subunits.

Elongation factor Tu brings in the next aminoacyl tRNA which snugglesinto the acceptor or A site if it matches the codon specified by the mRNA.

Peptide bond formation takes place.

Elongation factor G pushes the now empty tRNA into the exit or E site.

And the cycle repeats.

Animation:Amy Heagle WhitingHoward Hughes Medical InstituteHealth Research Incorporated at the Wadsworth Center, State University ofNew York at Albany

Final composition:Graham JohnsonFivth Element (www.fivth.com)

Funded, in part, by NIGMS and NCRR, National Institutes of Health

6.6 RIBOSOME RATCHET

Comparison of two states of a bacterial ribosome, either with the initiatorfMet-tRNA bound or with elongation factor EF-G bound, reveals thesignificant conformational changes that the ribosome is thought toundergo during each elongation cycle. The ratchet-like rearrangements atthe interface between the two ribosomal subunits may help move themRNA and tRNAs through the ribosome during protein synthesis.

The models shown here are a computer reconstruction made from manythousands of images of single ribosomes in vitreous ice that wereobserved with an electron microscope.




22

Joachim Frank and Rajendra K.AgrawalHoward Hughes Medical InstituteHealth Research Incorporated at theWadsworth Center, State University of NewYork at Albany

Joachim Frank and Rajendra K.AgrawalHoward Hughes Medical InstituteHealth Research Incorporated at theWadsworth Center, State University of NewYork at Albany

7.1 HOMEODOMAIN

7.1.1Homeodomains are found in many transcription regulatory proteins andmediate their binding to DNA. A single homeodomain consists of threeoverlapping a helices packed together by hydrophobic forces. Helix 2 andhelix 3 comprise the DNA-binding element, a helix-turn-helix motif.

7.1.2Amino acids in the recognition helix make important, sequence-specificcontacts with bases in the DNA major groove.

7.1.3Three side chains from the recognition helix form hydrogen bonds withbases in the DNA. A hydrogen bond is a strong, noncovalent interactionthat forms when two neighboring electronegative atoms, like oxygen andnitrogen, share a single hydrogen.

7.1.4In addition to the contacts between the recognition helix and the bases inthe DNA major groove, an arginine residue from a flexible loop of theprotein contacts bases in the minor groove.


7.2 ZINC FINGER DOMAIN

7.2.1Zinc finger domains are structural motifs used by a large class of DNAbinding proteins. They use centrally coordinated zinc atoms as crucialstructural elements.

7.2.2A single zinc finger domain is only large enough to bind a few bases ofDNA. As a result, zinc fingers are often found in tandem repeats as part ofa larger DNA-binding region.

7.2.3The helical region of each zinc finger rests in the major groove of the DNAhelix. Basic side chains project out from the helix and contact bases in theDNA. The identities of these side chains determine the precise DNAsequence recognized by each zinc finger. Assembling different zinc fingermotifs allows precise control over the sequence specificity of the protein.The specific contacts made between protein and DNA are hydrogenbonds.


23

PBD ID number *: 1APL

PDB ID number *: Zinc finger DNA-bindingdomain (1ZNF); Zif268-DNA complex(1ZAA)

7.3 LEUCINE ZIPPER

7.3.1A leucine zipper domain is comprised of two long, intertwined a helices.Hydrophobic side chains extend out from each helix into the space sharedbetween them. Many of these hydrophobic side chains are leucines, giv-ing this domain its name. A spacefilling view reveals the tight packing ofside chains between the leucine zipper helices; this makes the domainespecially stable.

7.3.2Extensions of the two leucine zipper helices straddle the DNA majorgroove. Side chains from both helices extend into the groove to contactDNA bases.

7.3.3The specific interactions between side chains and bases are hydrogenbonds. In this example, an arginine residue makes two contacts with aguanine base.


7.4 TATA-BINDING PROTEIN

7.4.1Eukaryotic transcription begins when RNA polymerase II binds to thepromoter region of a gene. A crucial part of this initiation process is therecognition and binding of the TATA sequence, a short stretch of DNA richin thymine and adenine nucleotides. The subunit of RNA polymerase IIthat binds to the TATA sequence is called the TATA-binding protein.

7.4.2The TATA-binding protein binds to DNA using an eight-stranded betasheet that rests atop the DNA helix like a saddle. Two protein loops drapedown the sides of the DNA like stirrups.

7.4.3Binding of the TATA-binding protein introduces a severe kink in the DNAbackbone. This kink dramatically bends the DNA helix by nearly ninetydegrees and is thought to provide a signal to assemble the rest of thetranscription complex at the initiation site.


24

PDB ID number *: 1YSA

PDB ID number *:TATA element ternarycomplex (1VOL); Compilation of TATA ele-ment ternary complex & Gal4 complex withDNA (1VOL & 1D66)

8.1 POLYMERASE CHAIN REACTION

The polymerase chain reaction, or PCR, amplifies a specific DNA frag-ment from a complex mixture.

First, the mixture is heated to separate the DNA strands. Two differentspecific oligonucleotide primers are added that are complementary toshort sequence stretches on either side of the desired fragment. Afterlowering the temperature, the primers hybridize to the DNA where theybind specifically to the ends of the desired target sequence. A heat stableDNA polymerase and nucleotide triphosphates are added. The poly-merase extends the primers and synthesizes new complementary DNAstrands. At the end of this first cycle, two double-stranded DNA moleculesare produced that contain the target sequence.

This cycle of events is repeated. The mixture is again heated to melt thedouble-stranded DNA. The primers are hybridized and the DNA poly-merase synthesizes new complementary strands.

At the end of the second cycle, four doubled-stranded DNA molecules areproduced that contain the target sequence. In the third cycle, the mixtureis heated, the primers are hybridized and DNA polymerase synthesizesnew complementary strands. At the end of the third cycle, eight double-stranded DNA molecules are produced that contain the target sequence.Two of these molecules are precisely the length of the target sequence.In future cycles this population increases exponentially.

Cycle 4—heating, hybridization, DNA synthesis.

At the end of the fifth cycle there are 22 double-stranded DNA fragmentsof the correct length and 10 longer ones.

Cycle 6, 10, 15, 20 . . .

After 30 cycles there are over 1 billion fragments of the correct length butonly 60 longer ones. The product therefore consists of essentially puretarget sequence.


8.2 ANATOMY OF A PDB FILE

Three dimensional structures of macromolecules—that have been deter-mined by NMR or x-ray crystallography—are archived in protein databasefiles, or PDB files for short.

Each PDB file begins with a description of the molecule, credits to theauthors who solved the structure and experimental details of the analysis.Next, the file lists the amino acid sequence of the protein.

The heart of the PDB file defines the precise position of each atom of thestructure in three-dimensional space. Each atom is described in a separ-ate line. The first few columns define the atom as part of a particularamino acid in the sequence. The later columns list a set of x, y, and z

25

Original illustrations, storyboard and music:Christopher Thorpe

Graham JohnsonFivth Element (www.fivth.com)

coordinates that precisely locate the atom in the structure. Programs suchas Rasmol or Chime, used on this CD, directly read PDB files and use thecoordinates to build three dimensional models on your computer screen.PDB files are stored in publicly accessible databases and can be readilydownloaded from the Internet.

9.1 LASER TWEEZERS

The light of a laser beam that is focused into a cone through a microscopeobjective exerts small forces that can trap particles with high refractiveindices near the focal point. This experimental set-up is called ‘lasertweezers.’ It can be used to move small particles, including cells andorganelles.



Show me:

· rough endoplasmic reticulum· smooth endoplasmic reticulum· regions of continuities between rough and smooth endoplasmic

reticulum· mitochondria

26



Christopher Thorpe


Show me:

· plasma membranes· tight junction· rough endoplasmic reticulum· glycogen granules


Show me:

· Golgi apparatus· plasma membranes· lysosome

10.1 FLUIDITY OF THE LIPID BILAYER

To demonstrate the fluidity of the lipid bilayer, a piece of the plasmamembrane of this neuronal cell is pulled out with laser tweezers. Remark-ably, moving this membrane tubule rapidly back and forth does notrupture the plasma membrane, which flows quickly to adapt to themechanical distortion.


27





Steven M. BlockStanford University

10.2 LIPIDS AND LIPID BILAYER

10.2.1Phospholipids contain a head group, choline in this case, that is attachedvia a phosphate group to a 3-carbon glycerol backbone. Two fatty acidtails are attached to the remaining two carbons of the glycerol.

10.2.2The head groups and the phosphate are polar, that is, they prefer to be inan aqueous environment.

10.2.3In contrast the fatty acid tails are hydrophobic, that is, they are repelledfrom water. The fatty acid tails on phospholipids can be saturated, with nodouble bonds, or unsaturated, with one or more double bonds. Thedouble bonds are usually in the cis-configuration, which introduces sharpkinks. When forming a bilayer, unsaturated fatty acid tails pack loosely,which allows the bilayer to remain fluid. If there were no double bonds,bilayers would solidify to a consistency resembling bacon grease.

10.2.4Cholesterol is another lipid component of most cell membranes. It has ahydroxyl group, a tiny polar head group so to speak, attached to a rigidhydrophobic tail. Cholesterol can fill gaps between phospholipids andthus stabilizes the bilayer.

10.2.5In a lipid bilayer, lipids arrange themselves so that their polar heads areexposed to water and their hydrophobic tails are sandwiched in themiddle. In this model, water molecules are shown in red.


Source:Beckman InstituteThe Theoretical Biophysics GroupUniversity of Illinois Urbana-Champaign

H. Heller, M. Schaefer, K. Schulten. Molecular dynamics simulation of a bilayer of200 lipids in the gel and in the liquid-crystal phases. Journal of Physical Chemistry97:8343–8360, 1993.

Lipidat Database(www.lipidat.chemistry.ohio-state.edu/)

28

PDB ID source: Compact lipid moleculestructure (Beckman Institute); Compilationof saturated and unsaturated fatty acids andcholesterol (Lipidat)

10.3 MEMBRANE DISRUPTION BY DETERGENT

When detergent is added to this red blood cell, its membrane ruptures,and the cytosol spills out.

10.4 MEMBRANE EFFECTS IN A RED BLOOD CELL

Red blood cells must deform when they squeeze through small bloodvessels.

In this experiment a red blood cell is pushed and deformed with lasertweezers. It quickly springs back to its original shape because it has anextremely tough cytoskeleton to which the plasma membrane is anchored.

When the cell is placed in high-salt solution, however, the shape changesdramatically. Driven by the difference in osmotic pressure, water rushesout of the cell causing spikelike protrusions to form as the cell collapses.

10.5 BACTERIORHODOPSIN

10.5.1Bacteriorhodopsin is an abundant light-driven proton pump found in themembrane of Halobacter halobium, a purple archeon that lives in saltmarshes in the San Francisco Bay Area. Bacteriorhodopsin is a multipassmembrane protein that traverses the plasma membrane of the cell withseven long a helices. The helices surround a chromophore, retinal, that iscovalently attached to the polypeptide chain and gives the protein andcells their characteristic purple color.

10.5.2Retinal is a long, unsaturated hydrocarbon chain that is covalentlyattached to a lysine side chain of the protein. When retinal absorbs aphoton of light, one of its double bonds isomerizes from a trans to a cisconfiguration, thus changing the shape of the molecule. The change inretinal’s shape causes conformational rearrangements in the surroundingprotein.

29


Henry Bourne and John SedatUniversity of California at San Francisco

Orion WeinerHarvard Medical School


PDB ID number *: Compilation of Bacteri-orhodopsin Br state intermediate & Bacteri-orhodopsin M state intermediate (1C8R &1C8S)

10.5.3The light-induced isomerization of retinal is the key event in protonpumping. In the excited state, retinal is positioned so that it can transfer aproton to an aspartate side chain, aspartate 85, that is positioned towardsthe extracellular side of the protein. Aspartate 85 quickly hands off theproton to the extracellular space via a bucket brigade of water molecules.The now negatively-charged retinal takes up a proton from another aspar-tate, aspartate 96; this one is positioned towards the cytosolic face of theprotein. Upon re-protonation, the retinal returns to the ground state.Aspartate 96 replenishes its lost proton from the cytosol, and the cycle canrepeat. The net result: for each photon absorbed, one proton is pumpedout of the cell.


10.6 FRAP

The lateral mobility of membrane proteins can be measured in living cellsby FRAP, which stands for fluorescence recovery after photobleaching.

For this purpose, membrane proteins are often expressed as fusion pro-teins with the green fluorescent protein GFP and observed with a fluores-cence microscope.

A selected area of the cell is then bleached with a strong, computer contr-olled beam of laser light.

Those membrane proteins that are not anchored and therefore can diffusein the plane of the membrane, quickly exchange places with their neigh-bors and fill back in the bleached area.

From the rate of this fluorescence recovery, the diffusion constant of theprotein can be calculated.

Here, GFP is fused to a membrane protein that lies in the membranenetwork of the endoplasmic reticulum.

After bleaching, we observe quick recovery of the fluorescence, showingthat the protein is very mobile in the plane of the membrane.

The same experiment can be repeated using a protein that is firmlyanchored and not free to diffuse. Here, we observe GFP fused to a proteinof the inner nuclear membrane that binds tightly to the meshwork of thenuclear lamina.

After photobleaching, no fluorescence recovery can be seen over the sametime frame.


Video reproduced from:The Journal of Cell Biology 138:1193–1206, Figure 4B, 1997. © The RockefellerUniversity Press.

30

Jennifer Lippincott-SchwartzNICHD, National Institutes of Health

11.1 POTASSIUM CHANNEL

11.1.1The bacterial potassium channel is a multipass transmembrane proteinin the plasma membrane. It is built from four identical subunits that arearranged symmetrically. A pore in the center of the protein allows selec-tive passage of potassium ions across the membrane.

11.1.2Four rigid protein loops, one contributed by each subunit, form a selec-tivity filter at the narrowest part of the pore. This structure is responsiblefor the channel’s high degree of selectivity for potassium ions over sodiumions.

11.1.3In the selectivity filter, carbonyl groups line the walls of the pore. Thesecarbonyl groups are spaced precisely to interact with an unsolvatedpotassium ion, balancing the energy required to remove its hydrationshell. Passage of a sodium ion through the channel is energeticallyunfavorable because the sodium is too small for optimal interaction withthe carbonyl groups.


11.2 HAIR CELLS

The stereocilia that project from hair cells vibrate in response to soundwaves.

Here the bundle of stereocilia projecting from a single hair cell is pushedwith laser tweezers to simulate this movement. Movement opens stress-activated ion channels in the plasma membrane, leading to membranedepolarization. This is translated into the perception of sound.

Moving an individual stereocilium demonstrates the flexible attachmentof these structures to the cell body.


31

PDB ID number *: 1BL8


12.1 NUCLEAR IMPORT

Nuclear import and export can be directly visualized in living cells thatexpress the green fluorescent protein GFP fused to the gene regulatoryprotein NF-AT.

NF-AT is normally localized in the cytosol, and excluded from the nucleus.But when the cytosolic calcium concentration is raised, NF-AT migratesto the nucleus.

This is done here experimentally by adding an ionophore that allowscalcium to enter the cells from the medium.

Upon removal of the ionophore, calcium levels return to normal andNF-AT is exported from the nucleus.

Readdition of the ionophore triggers reimport of NF-AT.

Final composition:Allison Bruce

12.2 ER TUBULES

The endoplasmic reticulum is a highly dynamic network of interconnectedtubules that spans the cytosol of a eukaryotic cell—like a spider’s web.

The network is continually reorganizing with some connections beingbroken while new ones are being formed.

Motor proteins moving along microtubules can pull out sections of endo-plasmic reticulum membranes to form extended tubules that then fuse toform a network.

32

Frank McKeonHarvard Medical School

Futoshi Shibasaki The Tokyo Metropolitan Institute of MedicalScience

Roydon PriceHarvard University

Annie YangHarvard Medical School

Part I:Jennifer Lippincott-SchwartzNICHD, National Institutes of Health

Part II:Ron D. ValeHoward Hughes Medical InstituteUniversity of California at San Francisco

12.3 RIBOSOME/ER TRANSLOCATOR

This model derived from an electron microscopic reconstruction, shows ayeast ribosome with the protein translocator of the endoplasmic reticu-lum attached.

The hole in the center of the translocator lines up precisely with the endof the tunnel in the large ribosomal subunit through which the newlysynthesized polypeptide chain, shown as the string of beads, exits theribosome.

This is best seen in this cross section, in which the front half of the ribo-some is removed from the model.




12.4 FREEZE FRACTURE OF YEAST CELL

Show me:

· outer nuclear membrane· inner nuclear membrane· nuclear pore complexes

33

Günter BlobelHoward Hughes Medical InstituteThe Rockefeller University

Joachim Frank Howard Hughes Medical InstituteHealth Research Incorporated at theWadsworth Center, State University of NewYork at Albany




Show me:

· nucleus· rough endoplasmic reticulum· mitochondria· nucleolus

13.1 CLATHRIN

Individual clathrin molecules can be seen in the electron microscope asthree-legged structures, called triskelions. Each triskelion contains threeheavy chains and three light chains.

When a clathrin coat forms on a membrane, the globular domains thatmake up the tips of the heavy chains bind to adaptins, which interact withcargo membrane proteins.

The assembly of a clathrin coat can occur spontaneously as numerousindividual triskelions come together, interact through their leg domains,and ultimately form a closed cage.

Animation:Allison Bruce

E. ter Haar, A. Musacchio, S.C. Harrison, and T. Kirchhausen. Atomic structure ofclathrin: a b-propeller terminal domain joins an a-zig-zag linker. Cell 95:563–573,1998.

A. Musacchio, C.J. Smith, A.M. Roseman, S.C. Harrison,T. Kirchhausen, B.M. Pearse.Functional organization of clathrin in coats: combining electron cryomicroscopy andx-ray crystallography. Mol Cell 3:761–770, 1999.

34

Tomás KirchhausenHarvard Medical School



13.2 SECRETORY PATHWAY

Fluorescently labeled membrane proteins start their journey to the plasmamembrane after synthesis in the endoplasmic reticulum.

They are first dispersed throughout the extensive membrane network ofthe endoplasmic reticulum from where they move to exit sites that formin random locations in the membrane network. At each of these sites,the membrane proteins are concentrated and packaged into transportvesicles. Clusters of the transport vesicles fuse to form transport interme-diates.

At the next stage, transport intermediates move along microtubule tracksto the Golgi apparatus near the center of the cell. The membrane proteinsexit the Golgi apparatus. They move in transport vesicles that are nowpulled outward on microtubules, which deliver them to the plasmamembrane.

Each time a Golgi-derived vesicle fuses with the plasma membrane, itscontent proteins disperse.

Video reproduced from:The Journal of Cell Biology 143:1485–1503, Figure 1A, 1998. © The RockefellerUniversity Press.

13.3 EXOCYTOTIC TRANSPORT

Passenger proteins exiting the Golgi apparatus on the way to the cell sur-face are often packaged into tubular transport vesicles of significant size.

Such tubular vesicles can branch and fragment before they fuse with theplasma membrane.

The transport vesicles move along microtubules which are stained herewith a red fluorescent dye.

The green cell in the corner does not contain fluorescent microtubules.


35



Patrick Keller and Kai SimonsEuropean Molecular Biology Laboratory

13.4 ENDOSOME FUSION

In these cells, fluorescent Rab5 protein has been overexpressed. Rab5binds to endosomes and promotes their fusion with one another, therebyincreasing the steady-state size of individual endosomal compartments.

Individual membrane fusion events can be observed at higher magnif-ication.


13.5 PHAGOCYTOSIS

Phagocytosis allows cells to take up large particles, such as these yeastcells that are being engulfed by the slime mold Dictostelium.


Video reproduced from:M. Maniak, R. Rauchenberger, R. Albrecht, J. Murphy, and G. Gerisch. Coronininvolved in phagocytosis. Cell, 83:91–924. © 1995, with permission from ElsevierScience.

13.6 PANCREAS: VIEW 1

Show me:

· cell outlines· nuclei· secretory vesicles· microvilli

36



Markus ManiakUniversity of Kassel, Germany

Philip D. Stahl, Alejandro Barbieri andRichard RobertsWashington University School of Medicinein St. Louis

13.7 PANCREAS: VIEW 2

Show me:

· secretory vesicles· pancreatic duct· tight junctions· centrioles

14.1 ATP SYNTHASE—A MOLECULAR TURBINE

ATP synthase is a molecular machine that works like a turbine to convertthe energy stored in a proton gradient into chemical energy stored in thebond energy of ATP.

The flow of protons down their electrochemical gradient drives a rotorthat lies in the membrane. It is thought that protons flow through an entryopen to one side of the membrane and bind to rotor subunits. Only proto-nated subunits can then rotate into the membrane, away from the staticchannel assembly. Once the protonated subunits have completed analmost full circle, and have returned to the static subunits, an exit channelallows them to leave to the other side of the membrane. In this way, theenergy stored in the proton gradient is converted into mechanical, rota-tional energy.

The rotational energy is transmitted via a shaft attached to the rotor thatpenetrates deep into the center of the characteristic lollipop head, the F1ATPase, which catalyzes the formation of ATP.

The F1 ATPase portion of ATP synthase has been crystallized.

Its molecular structure shows that the position of the central shaft influ-ences the conformation and arrangement of the surrounding subunits. Itis these changes that drive the synthesis of ATP from ADP. In this animatedmodel, different conformational states are lined up as a temporalsequence as they would occur during rotation of the central shaft.

Like any enzyme, ATP synthase can work in either direction. If the con-centration of ATP is high and the proton gradient low, ATP synthase willrun in reverse, hydrolyzing ATP as it pumps protons across the mem-brane.

To show the rotation of the central shaft, a short fluorescent actin filamentwas experimentally attached to it. Single filaments attached to single F1ATPases can be visualized in the microscope.

37



Video:Masasuke YoshidaTokyo Institute of Technology

When ATP is added, the filament starts spinning, directly demonstratingthe mechanical properties of this remarkable molecular machine.


14.2 ATP SYNTHASE—DISCO

Subunits:Center (gamma subunit): Toyoki AmanoLeft (beta subunit 1): Hiroyuki NojiRight (beta subunit 2): Satoshi P. TsunodaBack (beta subunit 3): Masaki Shibata

Dance direction:Nagatsuta Bon-Odori

Camera work and production:Hiroyuki Noji

14.3 PHOTOSYNTHETIC REACTION CENTER

14.3.1The bacterial photosynthetic reaction center is a large complex of fourprotein subunits. Three subunits, called the H, L, and M subunits, containhydrophobic a helices that span the membrane and anchor the complex.The fourth subunit is a cytochrome that is peripherally attached.

14.3.2Energy transfer through the reaction center involves pigment moleculesthat are organized in the interior of the protein complex. Excited electronsgenerated after absorption of light move from centrally located chloro-phylls to pheophytins. From there they move to a quinone, which is thenreleased from the reaction center to feed the electrons into the electrontransport chain. Electrons lost from the chlorophylls are replaced througha conduit of heme groups found in the cytochrome subunit.


38

PDB ID number *: 1DXR

14.4 MITOCHONDRIAL FISSION AND FUSION

Dynamic properties of the mitochondrial network can be seen in thisliving yeast cell which expresses the green fluorescent protein GFP fusedto a mitochondrial signal sequence.

Membrane fusion and fission events constantly reshape the organelle.

Using a confocal microscope, an optical slice containing only the top focalplane of the cell is recorded in this movie; the remainder of the network isout of focus.

14.5 TOMOGRAPH OF MITOCHONDRION

A mitochondrion contained in a one-half micrometer thick section ofchicken brain is viewed with a high voltage electron microscope. Whenthe section is tilted in the microscope, it can be viewed from many differ-ent angles, and a large amount of three-dimensional detail becomesapparent. Images from such a series of tilted views can be used to calcu-late a three-dimensional reconstruction, or tomogram, of the mitochon-drion.

The tomogram of the same tissue slice is shown here as a series of stackedimages. The movie steps through the images one by one, from the bottomof the stack, to the top, and back. This allows us to trace individual mem-branes in three-dimensions.

To create a three-dimensional model, membranes in an individual slice ofthe tomogram are traced. In this case the inner membrane is traced inlight blue, where it parallels the outer membrane, and traced in yellow,where it folds into the cristae that protrude into the mitochondrial interi-or. The tracings from all sections are then modeled as three-dimensionalsurfaces, and displayed as a three-dimensional model by a computerprogram. Such a model can now be viewed from any angle.

In this view, only four cristae are shown and the others are omitted. Thecristae are colored differently and show the variety of shapes and connec-tions to the inner membrane in a single mitochondrion.

The model also shows the reconstitution of the outer mitochondrialmembrane, represented in dark blue, as well as two fragments of endo-plasmic reticulum. Regions of such close proximity between the twoorganelles are quite frequently seen in cells. Note that there is no conti-nuity between the mitochondrial and endoplasmic reticulum mem-branes. Lipids are thought to be shuttled between the two organelles byspecial carrier proteins that operate in this gap.


39

Terrence G. FreySan Diego State University

Guy PerkinsUniversity of California at San Diego

Gustavo PesceHoward Hughes Medical InstituteUniversity of California at San Francisco

Peter WalterHoward Hughes Medical InstituteUniversity of California at San Francisco

15.1 CALCIUM SIGNALING

In this experiment, glial cells from the rat brain are grown in cell culture.

Calcium concentrations are visualized with a fluorescent dye thatbecomes brighter when calcium ions are present. In the presence of smallamounts of a neurotransmitter, individual cells light up randomly as ionchannels open up and allow calcium ions to enter the cell.

Occasionally, calcium waves are transmitted to adjacent cells through gapjunctions at regions where the cells contact each other.

15.2 CHEMOTAXIS OF NEUTROPHILS

These human neutrophils, taken from the blood of a graduate student, aremobile cells that will quickly migrate to sites of injury to help fight infec-tion. They are attracted there by chemical signals that are released byother cells of the immune system or by invading microbes.

In this experiment tiny amounts of chemoattractant are released from amicropipette. When neutrophils sense these compounds they polarizeand move towards the source. When the source of the chemoattractant ismoved, the neutrophil immediately sends out a new protrusion, and itscell body reorients towards the new location.

40

Henry Bourne and John SedatUniversity of California at San Francisco

Orion WeinerHarvard Medical School

Ann H. Cornell-BellViatech Imaging

Steven FinkbeinerGladstone Institute of Neurological Diseaseat the University of California, San Francisco

Mark S. CooperUniversity of Washington

Stephen J. SmithStanford University School of Medicine

15.3 NEUTROPHIL CHASE

Neutrophils are white blood cells that hunt and kill bacteria. In this spreada neutrophil is seen in the midst of red blood cells. A Staphylococcusaureus bacterium has been added. The bacterium releases a chemoat-tractant that is sensed by the neutrophil. The neutrophil becomes polar-ized, and starts chasing the bacterium which, powered by its flagella,swims in a random path, seemingly avoiding its predator. Eventually, theneutrophil catches up with the bacterium and engulfs it by phagocytosis.

Digital capture:Tom StosselBrigham and Women's Hospital, Harvard Medical School

Final composition:N8 Digital (www.N8digital.com)


15.4 cAMP SIGNALING

Adenylyl cyclase is a membrane-bound enzyme whose catalytic domainis activated by the GTP-bound form of the stimulatory G protein alphasubunit (or G-alpha-s for short).

Activated adenylyl cyclase converts ATP to cyclic AMP which then acts asa second messenger that relays the signal from the G-protein-coupledreceptor to other components in the cell.

In most animal cells, cyclic AMP activates cyclic-AMP-dependent proteinkinase (or PKA). In the inactive state PKA consists of a complex of twocatalytic subunits and two regulatory subunits. The binding of cyclic AMPto the regulatory subunits alters their conformation and liberates thecatalytic subunits which are now active and phosphorylate specific targetproteins.

In some endocrine cells, for example, the activated PKA catalytic subunitsenter the nucleus, where they phosphorylate a transcription factor calledCREB. Phosphorylated CREB then recruits a CREB-binding protein. Thiscomplex activates transcription after binding to specific regulatoryregions that are present in the promoters of appropriate target genes.

Animation:Thomas DallmanBioveo (www.bioveo.com)

41

David RogerVanderbilt University

Original illustrations:Nigel Orme

15.5 G-PROTEIN SIGNALING

Many G-protein-coupled receptors have large extracellular ligand-bindingdomains.

When an appropriate protein ligand binds to this domain, the receptorundergoes a conformational change that is transmitted into the cytosol,where the receptor activates trimeric GTP-binding proteins (or G proteinsfor short).

As their name implies, trimeric G proteins are composed of three proteinsubunits called alpha, beta, and gamma subunits. Both the alpha andgamma subunits have covalently attached lipid tails that anchor them inthe plasma membrane.

In the absence of a signal, the alpha subunit has a GDP bound, and theG protein is inactive. Activated receptors associate with inactive G pro-teins and, through conformational changes, weaken the affinity of theG proteins for GDP.

GTP, which is abundant in the cytosol, then readily binds to the emptysites. GTP binding causes the alpha subunit to undergo a further confor-mational change, which dissociates the G protein trimer into two activatedcomponents: a GTP-bound alpha subunit, and a beta–gamma complex.

Both of these components can regulate the activity of target proteins inthe plasma membrane, as shown here for a GTP-bound alpha subunit.The activated target proteins then relay the signal to other components inthe signaling cascade.

Eventually, GTP hydrolysis occurs on the alpha subunit. This step is oftenaccelerated by the binding of another protein, called a regulator of G-protein signaling (or RGS). The inactivated, GDP-bound alpha subunitre-associates with the beta–gamma complex, thereby turning off otherdownstream events.

As long as the signaling receptor remains stimulated, G-protein complexescan be activated over and over. Upon prolonged stimulus, however, thesignaling receptors are turned off, even if their activating ligands remainpresent.

In this case, a receptor kinase phosphorylates the cytosolic portions of theactivated receptors. Once a receptor has been phosphorylated in this way,it binds with high affinity to an arrestin, which inactivates the receptor bypreventing its interaction with G proteins.

Arrestins also act as adaptors, and recruit receptors to clathrin-coatedpits, from which the receptors are taken up into cells and degraded.


42


15.6 INOSITOL PHOSPHATE SIGNALING

Phosphatidylinositol-4,5-bisphosphate (or PIP2) is a key signalingmolecule. For example, the differentiation of a B cell into an antibody-producing plasma cell is triggered when an activated B cell receptorrecruits PI3-kinase, which binds to a phosphotyrosine on the receptorcomplex.

PI3-kinase phosphorylates PIP2 in the plasma membrane to form PI3,4,5-trisphosphate (or PIP3). Intracellular signaling proteins then bind to PIP3.

In activated B cells, PIP3 recruits both the cytoplasmic tyrosine kinaseBTK and phospholipase C to the plasma membrane. This brings these twocomponents into close proximity, so that BTK can activate phospholipaseC by phosphorylation.

Activated phospholipase C then splits additional PIP2 molecules into twofragments: soluble IP3 and diacylglycerol, and both of these fragmentsrelay the signal onwards. IP3 diffuses through the cytosol and binds tocalcium channels in the endoplasmic reticulum membrane.

Upon IP3 binding, channels open releasing calcium ions into the cytosol.This rise in the cytosolic calcium concentration causes protein kinase C tomove to the plasma membrane, where it is activated in turn. Proteinkinase C activation requires both binding to diacylglycerol and the contin-ued presence of calcium ions.

Protein kinase C then phosphorylates its target proteins that ultimatelyactivate the transcriptional program leading to B cell differentiation.


15.7 Src

15.7.1The Src protein kinase acts as a molecular signal integration device. Srcconsists of three clearly demarcated domains: an N-terminal SH3domain, an SH2 domain, and a tyrosine kinase domain. In addition, alinker and the short C-terminal tail play regulatory roles.

15.7.2To be an active protein kinase, the active site of the Src kinase domainmust first become accessible to substrate proteins. In the inactive state,the active site is blocked by an activation loop. A phosphate group istransferred from ATP to the tyrosine in the activation loop. This phos-phorylation leads to a rearrangement of the loop so that other substrateproteins can bind and become phosphorylated.

15.7.3Src is kept inactive by an interaction of its SH2 domain with the C-terminaltail peptide. A phosphorylated tyrosine on its C-terminal tail is buried ina deep binding pocket in the SH2 domain. To activate Src, this phosphategroup is removed by specific phosphatases. Upon dephosphorylation ofthe tyrosine, the C-terminal tail is released from the SH2 domain. The Srckinase then phosphorylates its activation loop and becomes active.

43

PDB ID number *: Human tyrosine-proteinkinase C-Src (2SRC); Phosphotyrosinerecognition domain SH2 of v-src (1SHA);Hiv-1 Nef protein (1EFN)


15.7.4A third condition must be met for the activation of Src. The activator pro-tein Nef must bind to the SH3 domain. A proline-rich region of Nef bindsto a groove on the surface of the SH3 domain and displaces the Src linkerpeptide.


15.8 Ras

15.8.1The Ras protein is a representative example of the large family of GTPasesthat functions as molecular switches. The nucleotide-binding site of Rasis formed by several conserved protein loops that cluster at one end of theprotein. In its inactive state, Ras is bound tightly to GDP.

15.8.2As a molecular switch, Ras can toggle between two conformational statesdepending on whether GDP or GTP is bound. Two regions, called switch1 and switch 2, change conformation dramatically. The change in confor-mational state allows other proteins to distinguish active Ras from inactiveRas. Active, GTP-bound Ras binds to, and activates, downstream targetproteins in the cell signaling pathways.

15.8.3A space-filling model shows that the conformational changes betweenthe GDP and GTP bound forms of Ras spread over the whole surface of theprotein. The two switch regions move the most.

15.8.4Ras hydrolyzes GTP to switch off; that is, to convert from the GTP-boundstate to the GDP-bound state. This hydrolysis reaction requires the actionof a Ras GTPase activating protein, or Ras-GAP for short. Ras-GAP bindstightly to Ras burying the bound GTP. It inserts an arginine side chaindirectly into the active site. The arginine, together with threonine andglutamine side chains of Ras itself, promotes the hydrolysis of GTP.


44

PDB ID number *: Compilation of c-H-Rasp21 protein catalytic domain complex withGDP & structure of p21-Ras complexedwith GTP at 100K (4Q21 & 1QRA); Ras-Rasgap complex (1WQ1)

15.9 CALMODULIN

15.9.1Calmodulin is a dumbbell-shaped protein formed by a single polypeptidechain. Its N-terminal and C-terminal globular domains are separated byan extended central helix. Each globular domain contains two high-affinity calcium-binding sites.

15.9.2Binding of four calcium ions induces major allosteric changes in calmod-ulin. Most notably, the two globular domains rotate relative to each other.These conformational changes enable calmodulin to bind to targetproteins and regulate their activity.

15.9.3Reminiscent of a boa grabbing its prey, calcium-bound calmodulin cap-tures helical peptides on target proteins by wrapping tightly around them.To make this possible, the central helix of calmodulin breaks into twohelices now connected by a flexible loop. Although the calcium ionsremain tightly bound during this remarkable reaction, they are not shownin the animated part of this movie.


Source:Intermediate structures provided by Eric Martz(www.umass.edu/microbio/rasmol/) and calculated by the Yale UniversityMorph Server, Mark Gerstein and Werner Krebs (bioinfo.mbb.yale.edu/)

15.10 GROWTH HORMONE RECEPTOR

15.10.1Human growth hormone receptor is a dimer of two identical subunits.Only the extracellular domains are shown. Its ligand, human growthhormone, binds in a cleft between the two subunits to activate the recep-tor. The lack of symmetry in this binding interaction is remarkable. Whilethe receptor is a twofold symmetrical structure with two identical subunits,the growth hormone is a single chain asymmetric protein that binds as amonomer. Thus, the interfaces between each receptor subunit and thehormone are completely different.


45

PDB ID number *: Calcium-free calmodulin(1CFD); Compilation of calcium-freecalmodulin & calcium-bound calmodulin(1CFD & 1OSA); Compilation of calcium-bound calmodulin & calcium-boundcalmodulin complexed with rabbit skeletalmyosin light chain kinase (1OSA & 2BBM)

PDB ID number *: 3HHR

16.1 DYNAMIC INSTABILITY OF MICROTUBULESMicrotubules continually grow from this centrosome added to a cellextract. Quite suddenly however, some microtubules stop growing andthen shrink back rapidly, a behavior called dynamic instability.


16.2 MICROTUBULE AND ER DYNAMICSGoverned by the principles of dynamic instability, microtubules const-antly extend into the leading edge of a migrating cell and retract again.

Superimposed on the dynamic microtubule cytoskeleton (shown here inred), the membrane network of the endoplasmic reticulum (shown herein green) exhibits its own dynamic behavior as tubes are extended bymotor proteins on the microtubule tracks.

Video reproduced from:C.M.Waterman-Storer and E.D. Salmon. Endoplasmic reticulum tubes aredistributed in living cells by three distinct microtubule dependent mechanisms.Current Biology 8:798–806. © 1998, with permission from Elsevier Science.

16.3 MICROTUBULE DYNAMICS IN VIVOEB1 is a protein that binds to the GTP-tubulin cap at the growing ends ofmicrotubules.

Cells expressing an GFP-EB1 fusion protein reveal the spectaculardynamics of the microtubule cytoskeleton.

Note that many but not all microtubules in this cell grow from the centro-some.

Only the ends of growing microtubules are visible in this experiment;those that are static or shrinking have lost their GTP-tubulin caps and donot bind EB-1.

In contrast, when all microtubules are labeled with GFP-tubulin, the trueextent of the microtubule cytoskeleton emerges.

Both growing and shrinking microtubules can be observed.


46

Yuko Mimori-KiyosueKAN Research Institute

Shoichiro TsukitaFaculty of Medicine, Kyoto University

Timothy MitchisonHarvard Medical School

Clare M.Waterman-StorerThe Scripps Research Institute

Edward D. (Ted) SalmonUniversity of North Carolina at Chapel Hill

16.4 BEATING HEART CELL

Single heart muscle cells spontaneously contract when grown in cellculture. This cell is grown on a flexible rubber substratum. Each time thecell contracts, it pulls on the substratum which becomes wrinkled.

Although individual heart cells can beat with their own rhythms, they arecoordinated in an intact heart so that all cells beat synchronously.

Reproduced from:CELLebration, 1995, edited by Rachel Fink, produced and distributed by SinauerAssociates, Inc., by copyright permission of Barbara Danowski.

16.5 ORGANELLE MOVEMENT ONMICROTUBULES

In this experiment a cell homogenate containing many differentorganelles is added to microtubules.

Motor proteins are normally attached to the organelles. When ATP isadded as a fuel for the motor proteins, some organelles bind micro-tubules, and are moved along the tracks by their motors.

Most kinesin motors move towards the plus end of microtubules. Dyneinmotors always move in the opposite direction. Both motors are used totransport organelles, and occasionally a single organelle, which musthave both types of motor attached, can be seen to switch directions.

The bi-directional traffic observed here is reminiscent of that in an intactcell.

47

Barbara DanowskiUnion College

Kyoko Imanaka-YoshidaMie University

Jean Sanger and Joseph SangerUniversity of Pennsylvania School ofMedicine

Nira Pollack University of California at San Francisco

Ron D.ValeHoward Hughes Medical InstituteUniversity of California at San Francisco

16.6 MYOSIN

Muscle myosin is a dimer with two identical motor heads that act inde-pendently. Each myosin head has a catalytic core and an attached leverarm. A coiled-coil rod ties the two heads together, and tethers them to thethick filament seen on top. The helical actin filament is shown at thebottom.

In the beginning of the movie, the myosin heads contain bound ADP andphosphate, and have weak affinity for actin.

Once one of the heads docks properly onto an actin subunit, phosphate isreleased. Phosphate release strengthens the binding of the myosin headto actin, and also triggers the force-generating power stroke that movesthe actin filament. ADP then dissociates, and ATP binds to the emptynucleotide binding site, causing the myosin head to detach from the actinfilament.

On the detached head, ATP is hydrolyzed, which re-cocks the lever armback to its pre-stroke state. Thus, like a spring, the arm stores the energyreleased by ATP hydrolysis, and the cycle can repeat.

The actin filament does not slide back after being released by the motorhead, because there are many other myosin molecules also attached to it,holding it under tension.

The swing of the lever arm can be directly observed on single myosinmolecules, here visualized by high-speed atomic force microscopy.


Animation reproduced with permission from Vale & Milligan, Science 288:88–95,Supplemental Movie 1. © 2000 American Association for the Advancement ofScience.

16.7 KINESIN

The motor protein kinesin is a dimer with two identical motor heads.Each head consists of a catalytic core and a neck linker. In the cell,kinesins pull organelles along microtubule tracks. The organelle attachesto the other end of the long coiled-coil that holds the two motor headstogether. The organelle is not shown here.

In solution, both kinesin heads contain tightly bound ADP, and moverandomly, driven by Brownian motion. When one of the two kinesinheads encounters a microtubule, it binds tightly. Microtubule bindingcauses ADP to be released from the attached head. ATP then rapidlyenters the empty nucleotide binding site.

This nucleotide exchange triggers the neck linker to zipper onto the cat-alytic core. This action throws the second head forward, and brings it nearthe next binding site on the microtubule.

The attached trailing head hydrolyzes the ATP, and releases phosphate. Asthe neck linker unzippers from the trailing head, the leading headexchanges its nucleotide, and zippers its neck linker onto the catalyticcore, and the cycle repeats.

48

Ron D.ValeHoward Hughes Medical InstituteUniversity of California at San Francisco

Ron MilliganThe Scripps Research Institute

Part I:Ron D.ValeHoward Hughes Medical InstituteUniversity of California at San Francisco

Ron MilliganThe Scripps Research Institute

Part II:Toshio AndoKanazawa University, Japan

In this way, kinesin dimers move processively, step-by-step, along themicrotubule.


Animation reproduced with permission from Vale & Milligan, Science 288:88–95,Supplemental Movie 1. © 2000 American Association for the Advancement ofScience.

16.8 CRAWLING ACTIN

Myosin motors can be attached to the surface of a glass slide. Fluorescentactin filaments will bind to the motor domains of the attached myosins.When ATP is added, the myosin motors move the actin filaments.

This rapid movement can be observed in a fluorescence microscope asthe actin filaments appear to crawl across the slide.


16.9 HEART TISSUE

Show me:

· endothelial cell surrounding blood vessel· smooth muscle cell· budding/fusing transcytotic vesicles· junctions between endothelial cells

49



James SpudichStanford University School of Medicine

16.10 HEART MUSCLE CELL

Show me:

· mitochondria· M-line· thin filaments (actin)· ribosomes

17.1 APOPTOSIS

Programmed cell death or apoptosis has been induced in these culturedcells.

Cell death is characterized by blebbing of the plasma membrane andfragmentation of the nuclei.

Suddenly, cells lose all attachment to the substratum that they have beengrowing on and shrivel up without lysing.

The mechanism of apoptosis involves many tightly controlled steps, threeof which are visualized here by different staining techniques.

One initial event is the sudden release of cytochrome c from mitochon-dria into the cytosol. This event has been visualized here using fluores-cently labeled cytochrome c. Initially the green/yellow staining is restrictedto a reticular pattern which then suddenly disperses as the mitochondriarelease their content proteins into the cytosol.

At a later step, the lipid asymmetry of the plasma membrane breaks down.In normal cells, phosphatidyl serine is only found on the cytosolic side ofthe plasma membrane, but when cells undergo apoptosis, it becomesexposed on the outside of the cell. This event has been visualized here byadding a red fluorescent protein to the media which specifically binds tophosphatidyl serine head groups as they become exposed. In an intactorganism, exposure of phosphatidyl serine on the cell surface labels thedead cell and its remnants to be rapidly consumed by other cells, such asmacrophages.

Finally—although apoptosing cells don’t lyse—their plasma membranesdo become permeable to small molecules. This event has been visualizedhere by adding a dye to the media that fluoresces blue when it can entercells and bind to DNA.

All three of these events can be observed simultaneously.



50

Gerard EvanUniversity of California at San FranciscoCancer Center

Joshua Goldstein La Jolla Institute for Allergy and InfectiousDiseases



17.2 p53-DNA COMPLEX

17.2.1p53 is a tumor suppressor protein that prevents cells from dividing inap-propriately. Loss of p53 function is associated with many forms of cancer.The DNA binding domain of p53 is folded as a b barrel. It exerts its func-tion by binding to DNA as a negative transcriptional regulator.

17.2.2The p53-DNA interface is complex. It involves several loops and a helixthat extends from the b barrel core. Residues from one loop and the helixbind in the DNA major groove. Arginine 248 from another loop makesextensive contacts with the DNA backbone and, indirectly through watermolecules, with bases in the minor groove. Mutations in arginine 248 arecommonly found in tumor cells. Such mutations disrupt the ability of p53to bind DNA.

17.2.3Loop 2 does not bind to DNA directly but is essential for correctly posi-tioning arginine 248 on the DNA. Three cysteines and a histidine fromboth loop 2 and loop 3 cooperate to sequester a zinc ion, forming the rigidheart of a zinc-finger motif. Mutations that disrupt interactions in thismotif are also common in tumor cells.


17.3 Cdk2

17.3.1Cyclin-dependent kinases, or Cdks for short, are crucial regulatory pro-teins in the cell cycle. When activated, these kinases transfer phosphategroups from ATP to serine and threonine side chains on target proteins.When inactive, the active site of Cdks is sterically occluded by a loop,often referred to as the T loop.

17.3.2As their name suggests, cyclin-dependent kinases are activated by cyclins.Cyclin binding to Cdk pulls the T loop away from the active site andexposes the bound ATP, allowing it access to target proteins. Thus a Cdkcan phosphorylate target proteins only when it is in a cyclin-Cdk complex.

17.3.3A third protein called a Cdk-activating kinase is required for full activationof Cdk. This kinase adds a phosphate group to a crucial threonine in the Tloop, thereby enabling Cdk to bind to and phosphorylate its target proteins.

17.3.4Target peptides bind to the active site of the cyclin-Cdk complex so thatthe target serine or threonine side chains are precisely positioned withrespect to the g phosphate of the bound ATP.

17.3.5Cdk inhibitor proteins, or CKIs, help regulate the rise and fall of cyclin-Cdk activity. Some inhibitors—like the one shown here—bind directly at

51

PDB ID number *: Human Cyclin-dependent kinase 2 (1HCK); CyclinA/Cyclin-dependent kinase 2 complex(1FIN); Phosphorylated cyclin-dependentkinase 2 bound to cyclin A (1JST);Phosphorylated Cdk2–cyclin A substratepeptide complex (1QMZ); p27/cyclinA/Cdk2 complex (1JSU)

PDB ID number *: 1TSR

the kinase active site and block kinase activity by interfering with ATPbinding.


18.1 PLANT CELL DIVISION

As this plant cell, taken from a lily, prepares for division, the chromo-somes first condense. Next, the mitotic spindle lines them up in the cen-ter of the cell.

At the metaphase to anaphase transition, the sister chromatids of everychromosome pair separate suddenly, in striking synchrony. The chromo-somes are pulled along the microtubules of the spindle to opposite endsof the cell.

After chromosome separation, membrane vesicles line up in the centerand fuse with each other to form the new plasma membranes that sepa-rate the two daughter cells.

At telophase, the chromosomes decondense in the newly formed nuclei.

Final composition:Thomas DallmanBioveo (www.bioveo.com)

18.2 ANIMAL CELL DIVISION

Differential interference contrast microscopy is used here to visualizemitotic events in a lung cell grown in tissue culture.

Individual chromosomes become visible as the replicated chromatinstarts to condense.

The two chromatids in each chromosome remain paired as the chromo-somes become aligned on the metaphase plate.

The chromatids then separate and get pulled by the mitotic spindle intothe two nascent daughter cells.

The chromatin decondenses as the two new nuclei form and cytokinesiscontinues to constrict the remaining cytoplasmic bridge until the twodaughter cells become separated.


Video reproduced from:The Journal of Cell Biology 122:859–875, 1993. © The Rockefeller University Press

52

Andrew S. Bajer University of Oregon

Edward D. (Ted) Salmon and Victoria SkeenUniversity of North Carolina at Chapel Hill

Robert SkibbensLehigh University

18.3 MITOTIC SPINDLES IN A FLY EMBRYO

In an early Drosophila embryo, nuclei divide rapidly and in perfect syn-chrony. In this experiment, both DNA and tubulin are visualized withdifferent fluorescent dyes.

After the mitotic spindle has assembled, the microtubules—shown ingreen—start pulling the blue chromosomes to either pole.

The chromosomes decondense and fill the newly formed round nuclei.

In preparation for the next round of mitosis, the centrosomes duplicateand migrate to opposite poles of each nucleus where they form newmitotic spindles and the process repeats.

The whole embryo rhythmically contracts with each division cycle.

18.4 MITOTIC SPINDLE

The mitotic spindle of a dividing human cell is reconstructed here in itsfull beauty from multiple optical sections that were recorded with a fluo-rescent microscope. Microtubules are stained in green, DNA is stained inblue, and the kinetochores—where microtubules attach to the DNA—arestained in pink.


18.5 MITOTIC CHROMOSOMES

Show me:

· microtubules· kineticore

53

William SullivanUniversity of California at Santa Cruz

Claudio E. Sunkel,Tatiana Moutinho-Santos,Paula Sampaio, Isabel Amorim andMadalena CostaInstitute of Biologia Molecular and CellBiology, University of Porto, Portugal

Kevin F. SullivanThe Scripps Research Institute



19.1 ADHESION JUNCTIONS BETWEEN CELLS

These epithelial cells express green fluorescent cadherin. They are grownat low density, so that isolated cells can be observed. Initially, labeledcadherin is diffusely distributed over the whole cell surface.

As cells crawl around and touch each other, cadherin becomes concen-trated as it forms the adhesion junctions that link adjacent cells.

Eventually, as the cell density increases further, the cells become com-pletely surrounded by neighbors and form a tightly packed sheet ofepithelial cells.


19.2 ROLLING LEUKOCYTES

Leukocytes are white blood cells that help fight infection. At sites of injury,infection, or inflammation, cytokines are released and stimulate endothe-lial cells that line adjacent blood vessels.

The endothelial cells then express surface proteins, called selectins.Selectins bind to carbohydrates displayed on the membrane of the leuko-cytes, causing them to stick to the walls of the blood vessels. This bindinginteraction is of sufficiently low affinity that the leukocytes can literallyroll along the vessel walls in search for points to exit the vessel. There, theyadhere tightly, and squeeze between endothelial cells—without disrupt-ing the vessel walls—then crawl out of the blood vessel into the adjacentconnective tissue.

Here, leukocyte rolling is observed directly in an anaesthetized mouse.The up and down movement of the frame is due to the mouse’s breathing.Two blood vessels are shown: the upper one is an artery—with blood flow-ing from right to left . The lower one is a vein—with blood flowing fromleft to right. Leukocytes only adhere to the surface of veins; they do notcrawl out of arteries.

Some leukocytes are firmly attached and are in the process of crawlingthrough the vessel walls, whereas others have already left the vessel andare seen in the surrounding connective tissue.

When the blood flow is stopped temporarily by gently clamping the ves-sels, we can appreciate how densely both vessels are filled with red blood

54

Stephen J. SmithStanford University School of Medicine

Cynthia AdamsFinch University of Health Sciences andChicago Medical School

Yih-Tai ChenCellomics, Inc.

W. James NelsonStanford University School of Medicine

Marko Salmi and Sami TohkaMediCity Research Laboratory, University ofTurku, Finland.

cells. Red blood cells do not interact with the vessel walls and move so fastunder normal flow that we cannot see them. When the blood flow isrestored, some of the leukocytes continue rolling, whereas all noninter-acting cells are immediately washed away by the shear.

Animation:Blink Studio Ltd. (www.blink.uk.com)

19.3 JUNCTION BETWEEN TWO MUSCLE CELLS

Show me:

· plasma membranes· desmosomes· Z disks· transverse tubules and sarcoplasmic reticulum

20.1 CALCIUM WAVE DURING FERTILIZATION

When a sperm cell fuses with this sea urchin egg cell, calcium ions beginrushing into the cell at the site of fusion.

In these experiments, calcium concentrations are visualized and measuredwith a fluorescent dye that becomes increasingly brighter the more calci-um is present.

Brightness is then translated into a color scale, and, in this three-dimen-sional display, into peak heights, where red and high peaks represent thehighest calcium concentrations.

A second rise of the calcium concentration can be observed after fertil-ization. It occurs during the movements of the sperm and egg pronucleito meet and fuse near the center of the egg.

Final composition:Allison Bruce

55



Part I:Carolyn A. LarabellLawrence Berkeley National Laboratory

Jeff HardinUniversity of Wisconsin, Madison

Part II:Michael WhitakerUniversity of Newcastle Upon Tyne

Isabelle GillotUniversity of Nice-Sophia Antipolis

20.2 SEA URCHIN FERTILIZATION

A sea urchin egg during fertilization is visualized here simultaneously byphase contrast microscopy and by fluorescence microscopy. The eggcontains a fluorescent dye that becomes brighter in the presence of calci-um ions.

When a sperm cell fuses with the egg, the fluorescence image shows awave of calcium ions that sweeps through the cytosol, starting from theinitial point of sperm–egg fusion. Following the path of the calcium wave,we see a membrane, called the fertilization envelope, rising from the cellsurface. The fertilization envelope protects the fertilized egg from the out-side environment, and prevents the entry of additional sperm. The rise incytosolic calcium triggers an elevation of the fertilization envelopethrough the process of exocytosis.

Exocytosis releases hydrolytic enzymes stored in vesicles. Action of thereleased hydrolases causes a swelling of material surrounding the cell,which in turn elevates the fertilization envelope.

Exocytosis can be visualized directly in this system. For this purpose, theplasma membrane is labeled with a fluorescent dye, seen on the right.Each time a vesicle fuses, it leaves a depression in the plasma membranewhich, in the optical sections shown, appears as a ring of increased fluo-rescent staining.

On the left, differential interference contrast microscopy is used to directlyview the exocytic vesicles that underlie the plasma membrane. The vesi-cles are visible here, because they are densely packed with protein andconsequently have a different refractive index from the surroundingmaterial. Each time a vesicle exocytoses, it disperses its contents and dis-appears from the image. This effect is best seen when we step back andforth between adjacent frames of the movie.

Final composition:N8 Digital (www.N8digital.com)

21.1 DEVELOPING EGG CELLS

This frog egg cell has been fertilized and starts dividing. The first cell divi-sions occur very rapidly. Cells divide every thirty minutes.

This timing is very precise. Egg cells that have been fertilized at the sametime divide and develop in almost perfect synchrony.

After a day or two, embryonic development is completed and tadpoleshatch from the eggs.

56

Mark TerasakiUniversity of Connecticut Health Center

From "From Egg to Tadpole" Jeremy Pickett-Heaps and Julianne Pickett-HeapsCytographics (www.cytographics.com)

21.2 GASTRULATION

During gastrulation, cells of this developing frog embryo rearrange in adramatic ballet of orchestrated cell movements. In a continuous motion,cells from the outer layer of the embryo sweep towards the vegetal poleand start invaginating, forming a deep cavity in the interior.

The paths of the cells and the topology of these rearrangements are bestseen in this animation of an embryo that has been sliced open. The differ-ent cell layers that are formed in this way have very different fates.

Cells that line the newly formed cavity, called the endoderm, develop intothe lining of the gut and many internal organs such as liver, pancreas, andlung. Cells in the middle layer, called the mesoderm, give rise to muscleand connective tissue. Cells remaining on the outside, called the ectoderm,go on to form the outer layer of the skin.

21.3 DROSOPHILA DEVELOPMENT

During development, a Drosophila embryo undergoes many complexmorphological changes. We first see migration of pole cells from the pos-terior end. These cells are destined to become the germ cells of the fly.A crest develops which separates a region that will develop into the head,mouth parts, and fore gut. At this stage, the future tail end of the body isfolded over on the dorsal side. Body segments then become defined.

The first three segments will give rise to the head and mouth parts, thenext three to the thorax, and the remaining ones to the abdomen. Even-tually, the rear end of the embryo will retract back onto the ventral sideand straighten out the embryo. Development to this stage takes about 10hours.

We can appreciate the complexity of these events by morphing a series ofindividual scanning electron micrographs into a continuous temporalsequence: migration of pole cells; development of various surface inden-tations, including openings to the air ducts, or tracheal tubes; segmenta-tion, and tail retraction.

A similar sequence viewed from the top—or the dorsal side. Pole cellsmigrate and then move into the interior as the hind gut invaginates. Therear end is temporarily folded over onto the dorsal side and eventuallystarts retracting to straighten out the embryo.

Early in development when seen from the bottom—or ventral side—adeep groove forms during gastrulation, as mesodermal cells migrateinward, where they become the precursor cells for many internal organs.The groove then seals off as the cells that remain exterior zipper up.


57

From "From Egg to Tadpole" Jeremy Pickett-Heaps and Julianne Pickett-HeapsCytographics (www.cytographics.com)

Thomas C. KaufmanHoward Hughes Medical InstituteIndiana University, Bloomington

SEM:Rudi TurnerIndiana University, Bloomington

Morphing:Michael Kaufmann, Jeffrey Giacoletti andChris MacriIndiana University, Bloomington

21.4 EARLY ZEBRAFISH DEVELOPMENT

The first divisions of a zebrafish egg occur synchronously about every 30minutes and create a mass of cells sitting on top of a enormous yolk.

This blastoderm then begins to spread as a continuous sheath over theyolk in a movement called epiboly.

During epiboly, some cells from the external layer tug into the interior ofthe embryo. They will eventually form the lining of the gut.

The first somite border forms, and the head process and tail bud becomevisible.

As more somites form, the tail bud continues to move ventrally, and weclearly see the eye develop.

At the 16 somite stage, or 17 hours into development, the yolk has beencompletely internalized into the developing gut cavity.


Video reproduced from:R.O. Karlstrom and D.A. Kane, Development 123:461. © 1996 The Company ofBiologists Ltd.

21.5 NEURITE OUTGROWTH

Neuronal precursor cells, taken from the hippocampus of an embryonicrodent brain, differentiate in culture and send out long extensions, calledneurites, that could later become dendrites or axons.

These neurites are pulled out of the cell body by growth cones that cancrawl independently over the substratum. Occasionally, a growth conereleases from the substratum and the neurite retracts.

New growth cones can grow from the sides of existing neurites, formingbranches.


58

Rolf O. KarlstromUniversity of Massachusetts at Amherst

Donald A. KaneUniversity of Rochester, New York

Frank B. Gertler and Lorene LanierMassachusetts Institute of Technology

22.1 WOUND HEALING

Fibroblasts grown in vitro in a culture dish form a confluent monolayer ofcells. Cells in a monolayer are relatively static; contacting each otherinhibits their migration.

Such cell layers can be wounded experimentally by scratching them witha needle.

In such an experiment, we can observe that the fibroblasts at the edge ofthe wound become migratory and quickly move to repair the gap.

Such cell migration is important for wound repair in an intact organism.

Animation:Blink Studio Ltd. (www.blink.uk.com)

22.2 MEGAKARYOCYTE

Megakaryocytes are the precursor cells from which blood platelets derive.These gigantic cells undergo an elaborate fragmentation process thatpinches off portions of the cell’s cytoplasm. These fragments are theplatelets, which are then swept away in the blood stream. Platelets areimportant for blood coagulation at sites of injury.


Video reproduced from:The Journal of Cell Biology 147:1299–1312, 1999. © The Rockefeller UniversityPress.


Show me:

· nuclear envelope· nuclear pore complex· ribosomes bound to outer nuclear membrane· rough endoplasmic reticulum

59

Sheryl Denker and Diane BarberUniversity of California at San Francisco

Joseph E. Italiano, Jr.Brigham and Women's Hospital andHarvard Medical School



22.4 LIVER CELLS (SINUSOID SPACE)

Show me:

· microvilli of hepatocytes· white blood cell (neutrophil)· endothelial cells· nucleus of neutrophil

22.5 ENDOTHELIAL CELL IN LIVER

Show me:

· outline of endothelial cell· outline of surrounding liver cells· secretory vesicles with condensed content proteins

23.1 BREAST CANCER CELLS

Normal human breast epithelial cells can be grown in cell culture. Theyform structures that resemble the little sacs of cells from which the mam-mary gland is built. Cells assemble into a well organized, polarizedepithelium that forms a closed sphere with an internal lumen. In themammary gland, this space would be connected to ducts, and the cellswould secrete milk into it.

By contrast, these human breast cancer cells grown under the same con-ditions, divide aggressively and in an uncontrolled fashion. They are alsomore migratory and grow into disorganized clumps which would formtumors in the body.


60





Mina J. Bissell, Karen Schmeichel, Hong Liuand Tony HansenLawrence Berkeley Laboratories

24.1 ANTIBODIES

24.1.1Antibodies of the immunoglobulin G class are Y-shaped glycoproteinsthat circulate in the blood stream. They bind to and inactivate foreignmolecules—the antigens—and mark them for destruction. Each IgGmolecule consists of two light chains and two heavy chains. The heavychains have carbohydrates attached. The regions of the antibody thatbind to antigens are located at the very tips of the two arms.

24.1.2Each arm of the antibody is composed of four domains. Two are called thevariable domains, contributed by the heavy and light chains, and hencecalled VH and VL. The variable domains are attached to two constantdomains, again one each from the heavy and light chains, and hencecalled CH and CL.

24.1.3Variable and constant domains share a similar structure, called the Ig fold.Each domain consists of a pair of beta sheets, one with three strands andone with four. A single covalent disulfide bridge holds the two sheetstogether, which results in a rigid and very stable domain.

24.1.4As their name implies, the variable domains vary in amino acid sequencefrom one antibody molecule to another, thus providing the vast diversityin structure required by the immune system. The antigen binding site inthe variable domains is composed of hypervariable loops that are espe-cially susceptible to sequence variations. Sequence variations in thehypervariable loops are responsible for the specificity of antibodies toparticular antigens.

24.1.5Antigens bind to the tip of each antibody arm, generally two moleculesper antibody. Most antigens bind to an antibody via a large contact surface,providing a tight and highly specific association.


24.2 MHC CLASS I

24.2.1Class I Major Histocompatibility Complex proteins display short pep-tides, or antigens, derived from normal cell proteins. Peptide-loadedMHC proteins are located on the cell surface where they can be examinedby passing T cells of the immune system. The MHC complex has twosubunits. The smaller subunit, b2 microglobulin, resembles animmunoglobulin domain. The larger a subunit also has an immunoglob-ulin-like domain which is linked to a head domain containing the antigen-binding groove.

24.2.2The antigen-binding groove in the MHC head domain is built from twowalls composed of long a helices that rest on a floor composed of an eightstranded b sheet. The peptide on display fits snugly between the helices inthe groove.

61

PDB ID number *: Compilation ofimmunoglobin G1 & immunoglobin Fc andfragment B of Protein A complex (2IG2 &1FC2); FabD1.3-lysozyme complex (1FDL)

PDB ID number *: MHC class I molecule(1A1M); Human T-cell receptor, viral peptideand Hla-A 0201 complex (1AO7)

24.2.3The peptide backbone is bound at both ends by highly conserved regionsof the MHC protein. Some peptide side chains extend downwards intospecific binding pockets in the groove, while other peptide side chainsproject upwards where they can be recognized by T cells.

24.2.4MHC class I proteins display their bound peptides on the cell surface forimmune surveillance. Immune cells, called cytotoxic or killer T cells, forexample, express T-cell receptors that bind to the MHC head domain andthe bound peptide. If the cell expressing the MHC protein displays apeptide foreign to the immune system, the T cell is activated by thisreceptor-MHC interaction. The activated T cell then proceeds to destroythe abnormal cell. Cut-away views of this peptide-bound MHC proteincomplexed with a T-cell receptor reveal the exquisite precision with whichthe interacting surfaces fit together.


24.3 MHC CLASS II

24.3.1MHC class II proteins have an overall similar structure to MHC class I pro-teins, although their subunit structure is distinct. MHC class II proteinsare composed of two subunits that contribute to the structure of the headdomain containing the antigen-binding groove.

24.3.2As a rule, MHC class II proteins bind longer peptides than MHC class Iproteins. As seen in this comparison, the different shape of the antigen-binding groove allows the ends of the peptide to stick out. MHC class IIproteins display peptides on the surfaces of specialized antigen-present-ing cells and activate a different class of immune cells, called helper Tcells.


24.4 LYMPHOCYTE HOMING

To visualize lymphocyte homing to a site of injury, a zebrafish larva wasanaesthetized and its fin pierced with a needle to introduce a smallwound.

A vein is seen at the bottom of the frame.

Because the fin is very thin and transparent, we can watch directly as lym-phocytes crawl out of the blood vessel and migrate towards the wound.

They are attracted there by chemicals released from damaged cells, invad-ing bacteria, and other lymphocytes.

62

Michael Redd and Paul MartinUniversity College London

PDB ID number *: Compilation of MHCclass I molecule & MHC class II/superantigencomplex (1A1M & 2SEB)

In a zoomed out view we can appreciate that lymphocyte invasion isrestricted to the wounded area.

The static cells that are dispersed in the connective tissue are fibroblasts.

In these movies, 60 minutes of real time are compressed into 15 seconds.


24.5 KILLER T-CELL

Cytotoxic lymphocytes, also called killer T-cells, bind tightly to their targetcells and then release toxic compounds by exocytosis into the cleftbetween the two cells.

Here, a killer T-cell has attached to a fibroblast and proceeds to attack it.The fibroblast quickly retracts and rounds up.

The movie is too short to tell whether it has actually been killed or willrecover.


25.1 HEMAGGLUTININ

25.1.1Hemagglutinin is a membrane fusion protein expressed on the surface ofinfluenza viruses. By mediating the fusion of viral and cellular membranesduring infection, it allows the viral genome to enter the cell. During thefusion reaction, hemagglutinin inserts a hydrophobic fusion peptide intothe host-cell membrane and thus transiently becomes an integral mem-brane protein in the two lipid bilayers. The transmembrane helix thatanchors hemagglutinin in the viral membrane is omitted from thisstructure.

25.1.2The fusion reaction is triggered by low pH which the virus encountersafter up-take into endosomes of host cells. This change in pH leads to amassive structural change, including the formation of a long a helix in thecore of the protein shown here. The fusion peptide, that was previouslytucked away in the protein’s stalk, is now displayed prominently at the tipof the helix, ready to slip into the host-cell membrane. The fusion peptidehad to be removed from the protein to allow crystallization.

25.1.3On the viral surface, hemagglutinin is a complex of three identical sub-units. It is likely that the concerted action of a small number of hemag-glutinin trimers is required to trigger a membrane fusion event.


63

James Bear and Frank B. GertlerMassachusetts Institute of Technology

PDB ID number *: Hemagglutinin (1HGF);Compilation of hemagglutinin & structure ofinfluenza hemagglutinin (1HGF & 1HTM)

25.2 LISTERIA PARASITES

This mammalian cell has been infected with pathogenic Listeria monocy-togenes. These bacteria move throughout the cytosol by recruiting hostcell actin which polymerizes and pushes them forward, producing acomet’s tail in their wake.

Whenever a bacterium is pushed into the plasma membrane, it creates atemporary protrusion and is then bounced back to continue its randompath.

If we look closely, we can see a bacterium divide inside the host cell.Immediately after separation, the two daughter cells assemble their ownactin tails and start moving about.

These bacteria can also form actin comet tails and move in cell extracts.Here, the bacteria are expressing the green fluorescent protein, and actinis labeled red with a fluorescent dye.

The dynamics of the actin tails, that propel the bacteria through thecytosol, can be modeled, based on known biochemical and physicalproperties of actin and actin filaments.


Video reproduced by permission from Nature Reviews Molecular Cell Biology1:110–119. © 2000 Macmillan Magazines Ltd.

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Part I:Julie A.TheriotStanford University School of Medicine

Daniel A. PortnoyUniversity of California at Berkeley

Part II:Julie A.TheriotStanford University School of Medicine

Frederick S. SooStanford University

Part III:Jonathan B. AlbertsUniversity of Washington, Seattle

GENERAL CREDITSArtistic and scientific direction:


Narrated by:Julie TheriotStanford University School of Medicine

Production design and development:Michael MoralesGarland Science Publishing

Interface design:Blink Studio Ltd. (www.blink.uk.com)

N8 Digital (www.N8digital.com)

Josef PuseduSun Microsystems

Interface programming:N8 Digital (www.N8digital.com)

Timothy DriscollMolvisions (www.molvsions.com)

Integration New Media (www.IntegrationNewMedia.com)

Molecular modeling and animations in Chime:Timothy DriscollMolvisions (www.molvsions.com)

Animation and video production:Blink Studio Ltd. (www.blink.uk.com)

N8 Digital (www.N8digital.com)

Graham JohnsonFivth Element (www.fivth.com)

Allison Bruce

Thomas DallmanBioveo (www.bioveo.com)

Amy Heagle WhitingHealth Research Incorporated at the Wadsworth Center, State University of New York at Albany

Michael KusieSyhart

Michael MoralesGarland Science Publishing


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High-resolution electron micrographs:Doug BrayThe University of Lethbridge, Canada


Content review:Karen K. BerndDavidson College

Internet research:Anthony L. DePassLong Island University

Audio recording and engineering:Freudenhaus Audio Productions (www.fapsf.com)

Original music:Freudenhaus Audio Productions (www.fapsf.com)

Christopher Thorpe

Licensed music and sound effects:CSS Music (www.cssmusic.com)

Sounddogs (www.sounddogs.com)

Nutritional consultant:Patricia CalderaKirkham /12th

Production Assistants:Angela Bennett, Kirsten Jenner, Kate Harper, Rebecca Law, Kim FongGarland Science Publishing

Marketing Executives:Nasreen Arain and Adam SendroffGarland Science Publishing

Executive Producers:Denise Schanck and Sarah GibbsGarland Science Publishing

Copyright 2002 by Garland Science Publishing

66

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