International Review of Cell and Molecular Biology SeriesEditors GEOFFREY H. BOURNE 1949–1988 JAMES F. DANIELLI 1949–1984 KWANGW. JEON 1967– MARTINFRIEDLANDER 1984–1992 JONATHAN JARVIK 1993–1995 Editorial AdvisoryBoard PETER L. BEECH WALLACE F. MARSHALL ROBERT A. BLOODGOOD BRUCE D. MCKEE KEITH BURRIDGE MICHAELMELKONIAN HIROO FUKUDA KEITH E. MOSTOV RAY H. GAVIN ANDREAS OKSCHE MAY GRIFFITH MADDYPARSONS WILLIAM R. JEFFERY TERUO SHIMMEN KEITH LATHAM ALEXEY TOMILIN AcademicPressisanimprintofElsevier 525BStreet,Suite1800,SanDiego,CA92101-4495,USA 225WymanStreet,Waltham,MA02451,USA TheBoulevard,LangfordLane,Kidlington,Oxford,OX51GB,UK 32JamestownRoad,London,NW17BY,UK Radarweg29,POBox211,1000AEAmsterdam,TheNetherlands Firstedition2014 Copyright©2014ElsevierInc.AllRightsReserved. Nopartofthispublicationmaybereproduced,storedinaretrievalsystemortransmittedin anyformorbyanymeanselectronic,mechanical,photocopying,recordingorotherwise withoutthepriorwrittenpermissionofthepublisher. 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BritishLibraryCataloguinginPublicationData AcataloguerecordforthisbookisavailablefromtheBritishLibrary LibraryofCongressCataloging-in-PublicationData AcatalogrecordforthisbookisavailablefromtheLibraryof Congress ISBN:978-0-12-800046-5 ISSN:1937-6448 ForinformationonallAcademicPresspublications visitourwebsiteatstore.elsevier.com PRINTEDANDBOUNDINUSA 14 15 16 10 9 8 7 6 5 4 3 2 1 CONTRIBUTORS WilliamM.AumillerJr. DepartmentofChemistry,PennsylvaniaStateUniversity,UniversityPark,Pennsylvania, USA Aure´lienBancaud CNRSGDR3536,UniversityPierreetMarieCurie-Paris6,Paris;CNRS,LAAS,and Universite´ deToulouse,LAAS,Toulouse,France VictorM.Bolanos-Garcia FacultyofHealthandLifeSciences,DepartmentofBiologicalandMedicalSciences,Oxford BrookesUniversity,Oxford,UnitedKingdom PascalCarrivain CNRSGDR3536,andLPTMCUMR7600,CNRS,Universite´ PierreetMarieCurie- Paris6,Paris,France BradleyW.Davis DepartmentofChemistry,PennsylvaniaStateUniversity,UniversityPark,Pennsylvania, USA AlanR.Denton DepartmentofPhysics,NorthDakotaStateUniversity,Fargo,NorthDakota,USA RonaldHancock LavalUniversityCancerResearchCentre,CRCHUQ-Oncology,Que´bec,Canada,and BiosystemsGroup,BiotechnologyCentre,SilesianUniversityofTechnology,Gliwice, Poland DieterW.Heermann InstituteforTheoreticalPhysics;InterdisciplinaryCenterforScientificComputing(IWR), Heidelberg,Germany;TheJacksonLaboratory,BarHarbor,Maine,USA,andShanghai CenterforBioinformationTechnology(SCBIT),Shanghai,PRChina Se´bastienHuet CNRS,UMR6290,InstitutGe´ne´tiqueetDe´veloppementdeRennes;Universite´Rennes1, Universite´ Europe´ennedeBretagne,Structurefe´de´rativederecherche,Biosit,Faculte´ de Me´decine,Rennes,andCNRSGDR3536,UniversityPierreetMarieCurie-Paris6,Paris, France HansjoergJerabek InstituteforTheoreticalPhysics,andInterdisciplinaryCenterforScientificComputing (IWR),Heidelberg,Germany ChristineD.Keating DepartmentofChemistry,PennsylvaniaStateUniversity,UniversityPark,Pennsylvania, USA xi xii Contributors JunSooKim DepartmentofChemistryandNanoScience,GlobalTop5ResearchProgram,Ewha WomansUniversity,Seoul,RepublicofKorea ChristopheLavelle CNRSGDR3536,UniversityPierreetMarieCurie-Paris6;NationalMuseumofNatural History;CNRSUMR7196,andINSERMU565,Paris,France AndrewMugler FOMInstituteAMOLF,Amsterdam,TheNetherlands ThoruPederson PrograminCellandDevelopmentalDynamics,DepartmentofBiochemistryandMolecular Pharmacology,UniversityofMassachusettsMedicalSchool,Worcester,Massachusetts,USA HubertRanchon CNRSGDR3536,UniversityPierreetMarieCurie-Paris6,Paris;CNRS,LAAS,and Universite´ deToulouse,LAAS,Toulouse,France AngeloRosa ScuolaInternazionaleSuperiorediStudiAvanzati,Trieste,Italy TakahiroSakaue DepartmentofPhysics,GraduateSchoolofSciences,KyushuUniversity,Fukuoka,Japan NaokiSugimoto FrontierInstituteforBiomolecularEngineeringResearch(FIBER)andFacultyofFrontiers ofInnovativeResearchinScienceandTechnology(FIRST),KonanUniversity,Kobe,Japan IgalSzleifer DepartmentofBiomedicalEngineering;DepartmentofChemistry,andChemistryofLife ProcessesInstitute,NorthwesternUniversity,Evanston,Illinois,USA PieterReintenWolde FOMInstituteAMOLF,Amsterdam,TheNetherlands Jean-MarcVictor CNRSGDR3536,andLPTMCUMR7600,CNRS,Universite´ PierreetMarieCurie- Paris6,Paris,France MatthiasWeiss DepartmentofExperimentalPhysicsI,UniversityofBayreuth,Bayreuth,Germany MihoYanagisawa DepartmentofPhysics,GraduateSchoolofSciences,KyushuUniversity,Fukuoka,Japan KenichiYoshikawa FacultyofLifeandMedicalSciences,DoshishaUniversity,Kyotanabe,Kyoto,Japan ChristopheZimmer InstitutPasteur,Unite´ ImagerieetMode´lisation;CNRSURA2582,Paris,France PREFACE Interestinthecellnucleushasbeenheightenedinrecentyears,andtheprin- ciples which determine structure and functions have become considerably clearer during the nearly 30 years since the publication of a volume in this serieswithasimilartheme(Berezney,R.,Jeon,K.W.(Eds.),1995.Nuclear matrix—structural and functional organization. Int. Rev. Cytol. 162). It is noteworthythatthisprogresshasbeenduelargelytoideasfromthefieldsof biophysics,physicalchemistryandpolymer,colloid,andsoft-matterscience, andalsotothedevelopmentof powerfulsimulationmethodsfor modeling the conformations and interactions of macromolecules. In presenting this volume, our hope is to encourage cell and molecular biologiststobecomemorefamiliarwithandunderstandthesenewconcepts and methods, and the crucial contributions which they are making to our perception of the nucleus. They are likely to be essential factors in all the systems in the nucleus and in their activities. It is a great pleasure to thank the authors for their willing and friendly cooperationandforfindingthetimetopreparetheircontributionsinspite of their busy schedules. We also thank workers at Elsevier Academic Press for having produced an excellent volume. RONALD HANCOCK AND KWANG W. JEON July 2013 xiii CHAPTER ONE The Nuclear Physique Thoru Pederson1 PrograminCellandDevelopmentalDynamics,DepartmentofBiochemistryandMolecularPharmacology, UniversityofMassachusettsMedicalSchool,Worcester,Massachusetts,USA 1Correspondingauthor:e-mailaddress:[email protected] Contents 1. Introduction:ABriefHistoryofBiophysics 1 2. TheBiophysicalNucleus 5 2.1 Nuclearstructureatmesoscopicscale 5 2.2 Physicallawsgoverningnuclearfunction 7 2.3 Whatliesaheadandthemethodsthatmaybeinplay 10 Acknowledgments 11 References 11 Abstract Thisvolumebringstogetheranumberofperspectives onhowcertainphysicalphe- nomenacontributetothefunctionaldesignandoperationofthenucleus.Thiscollec- tion could not be more timely, resonating with an increasing awareness of the opportunitiesthatlieattheinterfaceofcellbiologyandthephysicalsciences.Forexam- ple,thiswas amajorthemeinthe2012and2013annualmeetings oftheAmerican SocietyforCellBiology,andonethattheSocietyaimstoemphasizeevenfurthergoing forward.Inaddition,theemergingcanonicalrelevanceofthephysicalsciencestocell biologyhasinrecentsummersmadeamostconspicuousappearanceinthecurriculum (lecturesandintenselabs)ofthefamedPhysiologyCourseattheMarineBiologicalLab- oratoryinWoodsHole.So,muchcreditisduetoRonaldHancockandKwangJeon,the coeditorsofthisvolume,andalltheauthorsforcreatingaworkthatissoaucourant. ItallstartedwiththeBigBang Robertson(2007) 1. INTRODUCTION: A BRIEF HISTORY OF BIOPHYSICS The English word “physics” deerives, as the plural, from the Greek noun “physic” (Fίzik) and has historically been applied to both medicine (thepracticeof“physik”)and,moredurably,naturalphenomena(excluding InternationalReviewofCellandMolecularBiology,Volume307 #2014ElsevierInc. 1 ISSN1937-6448 Allrightsreserved. http://dx.doi.org/10.1016/B978-0-12-800046-5.00001-1 2 ThoruPederson life). The former term is no longer used in medicine except on occasion whenoneseesthataprofessoratsomevenerablemedicalschoolholdsatitle such as “The (insert an august name here, such as Vesalius or Paracelsus) Professor of Physik.” No one can quite be sure when the prefix “bio” was first attached to physics. The combined word does not appear in any of Aristotle’s brilliant essays on either physics or natural history, for example, though his treatise “Movement of Animals” (translated by Farquharson, 1984) makes for fascinating reading, particularly bearing in mind that he wrote more than two millenia before the time of Newton. In themid-eighteenthcentury, theItalian physician andphysicist Luigi Galvani(1737–1798)discoveredthatthelegmuscleofarecentlysacrificed frog could conduct static electricity from a piece of metal, resulting in a twitch.Thisallowedhimtomaketheleapthatelectricityisrelatedtomuscle activityinthelivinganimal.ThesubsequentdiscoverybytheGermanphy- sicianandphysiologistEmilduBois-Reymound(1818–1896)ofwhatcame tobeknownasthenerveactionpotentialpavedthewayforwhatcouldbe called the “pre-biophysics era.” In due course, as the field of biochemistry evolved,aparallellineofthinkingarosethat“mechanical”andindeedeven “engineering” principles were as much at play in living systems as chemistry—a doctrine promoted particularly by the German–American biologist Jacques Loeb (1859–1924). Bythe1920s,thearrivalofbiophysicswasathand,atleastinaformthat took up that name. It was both an approach (particular equipment) and a discipline(avisionofmechanism)andwasdriven,atleasttoaconsiderable degreebyphysiologistscontinuingtobumpintophysicalunderpinningsof biologicalprocesses.Buttherewasasecond,portentousdomainofbiophys- ics in the wind. Back in 1912, the German physicist, Max von Laue (1879–1960), had discovered the diffraction of X-rays by crystalline materials, and subse- quently he and others realized that this diffraction from the lattice would allow the crystal’s structure to be deduced by calculating the pattern back through reciprocal space. In due course, it occurred to several people that such an approach might be applied to more complex molecules than crys- tallineminerals.TheundisputedleaderofthismovementwastheIrishphys- icistJohnDesmondBernal(1901–1971).Hewasaformidablegeniuswho, evenasanundergraduateatCambridge,presentedoneofhisprofessorswith a penetrating mathematical analysis of the 230 crystal space groups, an achievement even more striking given that he had not done well on the mathematics tripos, leading him to turn to the natural sciences curriculum BiophysicalAspectsoftheNucleus 3 (Brown, 2005). Later, having become a leading crystallographer, arguably the leading one, Bernal encouraged his former student Dorothy Crowfoot Hodgkin(1910–1994)totakeupthelargertask(asboththesizeoftheunit cell and magnitude of the achievement, if reached) of tackling biological molecules. By the age of 35, Hodgkin had got the structure of penicillin beforeithadbeendeterminedbychemicalmeans.Thiswasamonumental achievement,andshefolloweditinduecoursebysolvingthestructuresof vitamin B and insulin. 12 Inparallelwiththesetriumphs,therearrivedtheeraofX-raydiffraction analysisofbothcrystallineproteinsand(wetordry)biologicalfibersasinthe case of collagen, wool, and DNA, and these endeavors, in particular in the X-ray crystallography field, increasingly took on the name “biophysics” in manyvenues.Thus,forexample,thelaboratoryatKing’sCollege,London, whereMauriceWilkinsand,later,RosalindFranklinundertookX-raydif- fractionofDNAhadlongbeennamedtheBiophysicsUnitoftheMedical ResearchCouncil.Thetermbiophysicshadbecome,bytheendofthewar, part of the scientific lexicon. The crystallographic axis of biophysics soon reached its first “post-Hodgkin” pinnacle in the DNA work of Wilkins and Franklin at King’s, and of Watson and Crick at the University of Cambridge Department of Physics’ Cavendish Laboratory, and, subse- quently, in the structural solutions of myoglobin and hemoglobin by John KendrewandMaxPerutz,respectively,attheMedicalResearchCouncil’s Laboratory of Molecular Biology in Cambridge. Despite the name of this latterinstitute,itsfoundingwasadirectresultofthebiophysicserathatBernal and Hodgkin had pioneered, and which England so proudly led for more than three decades. Inthe1950s,anotherdimensionofbiophysicscomingtofruitionwasthe study of cellstructure. Phase contrast microscopy,a majoradvance in light microscopy, had been discovered in 1930 by the Dutch physicist Frits Zernike(1888–1966),forwhichhereceivedthe1953NobelPrizeinPhys- ics,unshared.Meanwhile,alsointheearly1930s,theGermanphysicistErnst Ruska(1906–1988)haddevelopedtheprinciplesandprototypesoftheelec- tron microscope, for which he received the 1988 Nobel Prize in Physics, shared with Gerd Binning and Heinrich Rohrer for their invention of thescanningtunnelingmicroscope.Bythetimephase-contrastandelectron microscopyarrived,manycytologists,biochemists,andevenphyscicts(vide infra)weretrying to define“protoplasm.”Rivals seemedready tobet their first-born children on “gel” versus “sol” models of protoplasm. In his very first work with live material, Francis Crick measured the recoil of 4 ThoruPederson intracellularironparticleswhensubjectedtoamagneticfield,asaprobeof therheologicaland,thus,cross-sectionaldensityofcytoplasm(Crick,1950; Crick and Hughes, 1950). I recently reread the first paper (the second is beyond my expertise in the physics required) and would recommend it toallcellbiologistsasamodelofexperimentaleleganceforitstime,aswell as a marvelous example of fine writing. Exceptfortheobviousriskofconfusing(orannoying)readers,thischap- ter might have been titled “Nuclear Physics” and this point reminds us, of course, of another major development in biology that sprang from pure physics, namely, the codiscovery of radioactivity by Marie Curie and Henri Becquerel. The subsequent understanding of this phenomenon (i.e., “nuclear physics,” literally) led to the use of unstable isotopes by the Hungarian chemist George de Hevesy (1885–1966; Nobel Prize in Chemistry,1943)andRudolfScho˝nheimer(1898–1941)astracerstopursue the biosynthesis and flux of molecules in living systems. Later, the stable isotopenitrogen-15wasthekeytotheMeselson–Stahlexperimentdemon- stratingthesemiconservativereplicationofDNA,dubbed“themostbeau- tifulexperimentinbiology”(Holmes,2001).Theentryofradioisotopesto biologicalresearchwaspurebiophysics,althoughtheybecamesuchstandard toolsthattheirusewasnotlabeledasbiophysicsperse.Sotoowastheallied field of radiation biology. Comingtothemodernera(1975–present),biophysicsmightbedefined by what is publishedin theBiophysicalJournal (reminding meof aprofessor who told the class that lipids are defined as substances that are soluble in a lipid solvent). Huge advances have taken place in our ability to measure things such as the diffusion-based transport of molecules in cells—thanks mainlytotheadventoffluorescentdyesandproteins.Morerecently,injust thelastdecade,advanceshavecrackedtheAbbe´ limit((cid:1)200nm)forspatial resolution in diffraction-limited optical microscopy. And meanwhile, the introductionof“optogenetics,”pioneeredbyGeroMiesenbo˝ckandrefined byKarlDeisseroth,hasbroughtnewmeaningto“biophysics.”Thismethod exploits the biophysical properties of opsins as sensors, combined with reportingfluorescentproteins,toallowcellphysiologytobeobservedinreal time.ThispastMay,Iattendedatalkinwhichthespeaker,AdamCohenof Harvard University, showed videos of an action potential running down a neuron and culminating in synaptic transmission. Never did I dream that Iwouldseesuchathinginlivecells,asopposedtoonanoscilloscopescreen. Driving home I pondered: Sic transit gloria biophysica. BiophysicalAspectsoftheNucleus 5 2. THE BIOPHYSICAL NUCLEUS Letusnowturntospecificallythenucleus,thesubjectofthisvolume. Forthepurposesofthisintroductorychapter,threekeyquestionscanbeposed: Whatisthestructureofthenucleusatthemesoscopicscale?Towhatextentdo certain phenomena in the nucleus obey purely physical laws? What are the emergingmethodsthatcantacklethephysicsofthenucleusgoingforward? Beforewemoveon,Iwanttomentionaguidelinethatthebiophysicist DanielBrantontaughtme.Atthe1982meetingoftheAmericanSocietyfor CellBiology,Iwasinchargeoftheprogramandinvitedhimtochaironeof the symposia. In his introductory remarks, he said he had instructed the speakerstoemphasizeanyplacesintheirworkwheretheformationofcova- lentbondshadnotbeenatplay.Ithoughtthiswasamostenablingdeviceat thetime(andhisspeakersindeedobeyed).Itremainsavaluableconstructfor us all, as we delve into any part of biology. 2.1. Nuclear structure at mesoscopic scale Bythetimecytochemicalstudieshadrevealedtheverylargeproportionof thenuclearvolumeoccupiedbyDNA,atleastinmostcells,itbecamelog- icaltoassumethatthephysicalpropertiesofthenucleusderivemostlyfroma veryhighconcentrationoftheseextraordinarilylongpolymers(Thediscov- eryofuninemywasjustaroundthecornerbut,inanycase,noonedoubted thattheDNAmoleculeswereverylong,whetherchromosome-sizeornot.). Later, Thomasand ChristophCremerand colleagues insightfully deduced, fromchromosomebreaksinducedininterphasecellsbyUVirradiation,that eachchromosomeoccupiedadistinctportionofthenuclearvolume,rather thanbeingintertwined(Cremeretal.,1982).Thisandsubsequentworkby themandothersbroughttheconceptofchromosomesterritories(Pederson, 2003,2004)andalso,moresubtly,hintedthatwhilethechromosomeswere not entangled, the amount of interchromosomal space in the nucleus was relatively small. Nonetheless, it was natural for the curious to ponder whethertheinterchromosomespaceitselfpossessedsomekindofstructure. Meanwhile, in the 1960s, Ilya Zbarsky in Moscow was subjecting iso- lated nuclei to escalating concentrations of NaCl and noted that, after the histoneandmuchothernuclearproteinhadbeenextracted,aresidualstruc- ture remained. In a refinement of his work, Berezney and Coffey used DNase and salt extraction to obtain a residual fraction that contained only