Thegoaloftheproposedresearchistogenerategeneticallyencodedbioluminescenttagsforlive-cell luminescence-basedphotoactivatedlocalizationmicroscopy(L-PALM).Thisrevolutionarymodeof superresolutionimagingwillmaintainallofthebenefitsoffluorescencePALM(fPALM)butwilleliminatethe needforexcitationlight.fPALMislargelyunsuitableforimaginglivecellsbecauseitrequireshighexcitation intensitiesthatleadtophototoxicity.Becauseluminescencegenerateslightwithouttheneedforexternal excitation,L-PALMwillnotsufferfromthislimitation.Thelatestgenerationofgeneticallyencoded bioluminescentlabelsarewellsuitedforwidefieldmicroscopyofsubcellularstructures,butarestill approximately1000-foldtoodimtobeusedforsingle-moleculelocalizationonpracticaltimescales.Toremedy thisdeficiency,thisstudyisdesignedtoproducebioluminescentprobeswithphotonoutputrates sufficienttolocalize~100,000moleculesinoneminute.Togeneratethisincreasedoutput,luciferaseswill firstbecoupledtoourbrightestfluorescentproteinstomaximizeluminescencequantumyieldviatheFrster resonanceenergytransfermechanism.Oncemaximaloutputisachievedinthisfirststep,theluciferase portionofthefusionwillthenbesubjectedtostructure-guideddirectedevolutiontargetedatlowering oxyluciferinbindingaffinityandthusincreasingthecatalyticrateoftheenzyme.Suchalterationsarepredicted toreducetheluminescencequantumyieldoftheluciferase,butenergytransfertoafluorescentproteinwill rescuetheluminescence,allowingmuchfasterenzymestobeengineeredwiththisstrategy.Tobeusefulfor live-cellL-PALM,bioluminescentprobesmustalsobecapableofswitchingonandoffcontrollablyto preventsignaloverlapbetweenindividualmoleculesineachimageframe.Twoindependentmechanismsfor producingswitchablelightoutputwillbepursuedinthisproject:(1)optimizationofenergytransferbetween luciferasesandphotoswitchablefluorescentproteins,followedbydirectedevolutiontoincreaselightoutput andimproveswitchingkinetics;?(2)insertionoflight-modulateddomainsintosplitluciferasesinorderto allostericallycontrolenzymeactivity.Throughouttheproject,heavyemphasiswillbeplacedonRosetta-based structure-guidedcomputationaldesignforgeneratingnovelluciferase-fluorescentproteinfusiontopologies, alteringluciferaseactivesiteenvironments,andengineeringallosterically-regulatedluciferases.Directed evolutionwithimage-basedscreeningwillthenbetheprimaryapproachforimprovingthepropertiesofprobes underdevelopmentineachaim.Theendproductsofthisprojectwillbeasetofgeneticallyencoded bioluminescentprobeswithbrightnessandphotoswitchingpropertiessuitableforthedevelopmentofL- PALMmethodologies.BeyondtheirultimateutilityforL-PALMimaging,manyoftheprobescreatedinthe courseofthisprojectwillbethebrightestandhighest-performingbioluminescenttagsyetdeveloped,andas suchwillhighlyusefulinnumerousotherlive-cellandwhole-organismimagingapplications.
The goal of the proposed research is to develop the technologies necessary to allow high-precision imaging of single molecules inside living cells without causing damage. The tools developed in this study will be made widely available to the biological and biomedical research community where they will be highly useful for studying basic biological and disease processes in living cells across a very broad range of medically relevant areas such as cancer, neurodegenerative disease, and aging.
