G2020–October2015

Cellsunderthemicroscope–partII

Nanotomy,T1D,electronmicroscopy

Lecturer:BenN.G.GiepmansDepartment:CellBiology,UMCGContact:www.cellbiology.nl Last update: 2015-09-07 BNGG

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PreparationReading Ravellietal.SREP01804(2013)accessiblevia:

http://www.nature.com/srep/2013/130508/srep01804/full/srep01804.html Youalso can study theDutch summary in theNederlands tijdschriftvoordiabeteologie

(Ravellietal.NTD(2013). Forthelabrules,seeCellsunderthemicroscope–part1(September2014) Theassignmentsinthismanual EssentialCellBiology,Albertsetal.,(Garland,4thed.2013).

– Chapters1&15 FunctionalHistology,Kerr(Mosby,2nded.2009)

– Chapters1&17:pp.404‐407 Functionelehistologie[FunctionalHistology,inDutch],Junqueira(Reed,14thed.2014)

– Chapters1,2,3&19:pp.566‐568

Todo Executeassignments1‐6,studytherelatedbackgroundinformation(sources) Assignments5‐6willbediscussed/finishedduringthepracticum Ensureyouareontime

ImportantsourcesofinformationInadditiontothismanualandthelistedbooks:http://www.ronaldschulte.nl/Assignments1‐3havebeenmodifiedfromDrs.D.Opstelten&N.A.Bos,ManualforCellBiologyLocation: Combizaal311multipurposeroom,situatedonthe3rdfloorofbuilding3214,ontheAnt.Deusinglaan1 NB:Althoughtheassignmentsmustbecompletedduringthepractical,itisessentialtoreadtheminadvanceforfullcomprehension.Afterthisworkshop,studentsshouldbeableto:

1. recognize and interpret electron microscopic images, with regard to tissuecharacteristics,celltypes,organelles,andmacromolecularcomplexes

2. explainhowfunctional informationaboutcell function,e.g. regardingsecretion,canbedeterminedusingnanotomy

3. describe the structure of the cell, cell organelles andmacromolecular complexes andnametheirfunctions

4. usetheinformationfrom1‐3forpathophysiologicalanalysis,inthisworkshopprimarilywithregardtotype1diabetes

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Assignment1:Electronmicroscopystructure/functionAcellcontainsorganellesthatareessentialforitsfunction.Dependingoncellularfunction,onetypeofcellwillhaveahighernumberofcertainorganellesthanothers.Tocheckifyouknowthevariouscellorganelles,examinethefollowingschematicdrawingofacell(anexocrinecell).a. Identifythevariouscellorganellesbyplacingtherightnumberattherightlineb. Statethemainfunction(s)oftheorganelleinthetable

Fig.1.Source:LaboratoryManualofHistology,Pappas.(W.C.Brown,1990)Structure Function Structure Function1.centriole 9.microtubules

2.cytosol 10.mitochondria

3.Golgicomplex 11.microvilli

4.nucleus 12.nucleolus

5.nuclearenvelope 13.plasmamembrane

6.nuclearpore 14.ribosomes

7.lysosome 15. rough endoplasmicreticulum

8.microfilaments 16.secretiondrops

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Assignment2:Studyelectronmicroscopy

IntransmissionEM(TEM),ahighvoltagegeneratedbetweenaheatedcathode (incandescent filament) and an anode produces a beam ofelectrons.Oneormorecondenserlensesfocusthisbeamontotheplaneof focus of the objective lenses, where an ultrathin specimen sectionwhich canbe irradiatedhasbeenplaced.Theobjective lenses createamagnifiedimageoftheobjectwhichisshownonscreenorcapturedbycamera.AstandardTEMprovidesmagnificationofupto300,000times,with a resolution of∼2nm, of specimen sectionswhich are∼60nmthick,withamaximumdiameterof3mm.

ThebiologicalmaterialpresentintheultrathinsectionmainlycomprisesC,H,NandOanddoesnot scatter electrons sufficiently to provide an image, which is why the specimen must bestainedwithheavymetals,whichdo scatterelectrons.Themostcommoncontrastmedium isosmiumtetroxide(OsO4),whichbindsparticularlyeasilytodoublebondsoflipids,fixingthembycreatingcross‐links,makingmembranesvisible.In scanningEM (SEM), the electronbeam is focused by the condenserand objective lenses in the samemanner as in TEM. Here, too, thespecimenisplacedinthefocalpoint.The primary electron beam is notstationary,likeinTEM,butscansitina grid‐like fashion. The electronbeamscansthespecimensurfacelinefor line, releasing secondaryelectrons (SE2) in the sample orhaving the electrons reflected(backscatter electrons). These arebothusedtocreatean imageof thespecimensurface. If thespecimensection isultrathin, theelectronswillofcoursepassthroughit.Byplacingadetectorunderneath,aTEMimagecanbetransformedintoanSEMimage.ThisisknownasSTEM:scanningtransmissionEM. Alsoread:EssentialCellBiology,Albertsetal.,(Garland,4thed.2013)page11.

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Assignment3:StudyingmembranetransportMembranesformcompartments.Theplasmamembraneistheboundarybetweenthecytoplasmandtheextracellularside.Thisboundaryisdynamic,withvarioustransportprocessesallowingsubstancestopassinandoutofthecell.Someprocessesareexemplifiedusingglucose‐inducedinsulinsecretion(Fig.2).Notethatvariousmoleculesandsubstancescanbetransportedacrossthemembraneinregulatedfashion.Inthefigurebelow,indicatewhattypeoftransportprocessisinvolved(encircleone).

Fig.2.Insulinsecretioninbeta cellscausedbytheincreasingbloodsugarlevels.UptakeofglucosebyGLUT2andglycolyticphosphorylationofglucosecausestheATP:ADPratiotorise.Thisinactivatesthepotassiumchannelwhichdepolarizesthemembranesothatavoltage‐dependentcalciumchannelopens.TheincreaseinthecalciumconcentrationleadstothereleaseofinsulinSource:www.betacell.org

1.GlucoseuptakebyGLUT2isprimarilydependenton:Exocytosis/Concentration/Voltage‐dependentinflux/Effluxinhibition2.TheATP‐sensitiveK+pumpisatypeof:Exocytosis/Concentration/Voltage‐dependentinflux/Effluxinhibition3.Thevoltage‐gatedcalciumpumpisatypeof:Exocytosis/Concentration/Voltage‐dependentinflux/Effluxinhibition4.Insulinsecretionisatypeof:Exocytosis/Concentration/Voltage‐dependentinflux/Effluxinhibition

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Assignment4:FromtissuetomolecularcomplexesMicroscopyallowsstudyingsamplesatdifferentmagnifications.Figs.1and2showedmodels.Figure3isahistologicaldepictionoftheisletsofLangerhans.Intheassignmentbelow,wewilllookatasectionofasingleislet.1.DrawascalebarInFigures1,2and3,indicatingtheestimateddimensions.Areyouabletogivevarious‐sizedscalesinFig.2?Ifyoucannot,returntoitaftercompletingAssignment5.

Fig.3.Thebeta cellsgrouped in the isletsofLangerhans in thepancreas. The rest of thepancreas consists of exocrine tissue where digestive enzymes are produced. The endocrinetissue–theisletsofLangerhans–producesotherhormonesbesidesinsulin.Source:www.bu.edu/histology/p/10401loa.htmFurtherreading:FunctionalHistology,Kerr(Mosby,2nded.2009)p404‐407.

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Assignment5:Nanotomy:EMoftissues,cells,organellesandmacromoleculesType 1 diabetes (T1D) is an auto‐immune disease results in degradation of the insulin‐producingbetacells(Fig.2),whicharelocatedintheisletsofLangerhansinthepancreas(Fig.3).A cure does not exist; patients depend on lifelong insulin therapy.Moreover, the trigger thatcauses the disease is also unknown. Finding alternatives for insulin therapy and makingadvances inetiologyofT1Dbenefits froma full structuraland functional insight into IsletsofLangerhans. EM can visualize Islet morphology at the highest possible resolution, however,conventionalEMonlyprovidesbiasedsnapshotsandlackscontext.

Nanotomy is an innovation in EM that allows to study tissues, cells, organelles andmacromoleculesinGoogle‐Earth‐likefashion.Here,nanotomyhasbeenusedinananimalmodelforType1diabetes:Ravellietal.(2013);www.nature.com/srep/2013/130508/srep01804/full/srep01804.html. StudytheultrastructureofanisletofLangerhans.Gotowww.nanotomy.nlandclickonthelargestislet(grey).ThisdatasetcanbestudiedinthesamewayyouviewalandscapeinGoogleEarth. Click on the IIP icon at top left for extra instructions if necessary. Go through theannotationsandanswerthequestionsbelow.Thenumberscorrespondto theannotations:1Arefersto1,forexample.Ifyouplaceyourcursoron'Supracellular',forexample,asubmenuwillappear, includingA, Islet.Beforeyoubegin,drag themenuatbottom leftupabitand thescalewillappear.1.IsletsofLangerhansinrecent‐onsettype1diabetes(rat)Judge what the largest structure is that you can recognize. And if you zoom in, what is thesmallest?Whatarethedimensions?Onceagain:Theannotationmenucanbedraggedtoallowthescaletoshow.Largest: Sizeisapproximately:Smallest: Sizeisapproximately:1A.NametheclearestdifferenceswhichdistinguishtheisletsofLangerhansfromtheexocrinepancreas.Whatarethevariousfunctions?1B.Whichcellisinthecapillary?1C.Whichtwotypesofcelldoyourecognizeinthevein?Whatarethemostobviousdifferences?

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1D.Thecentroacinarlumenbelongstothe:a. endocrinepancreasandcontainsenzymesb. endocrinepancreasandcontainshormonesc. exocrinepancreasandcontainsenzymesd. exocrinepancreasandcontainshormones

1E. Depicted here is a cross‐section of a bundle of unmyelinated axons. The bundle wasdiscoveredmoreor lessby chance: theelectrons cause theaxons tobe slightly lighter.Thesecontainroundtubulesandlight‐greyfilaments.Howmanyaxonsdoyouseehere?1F.Insomeexocrinecellsthenucleusisnotvisible.Why?2A. The exocrine cell contains a lot of rough ER for protein synthesis. The content will besecretedwithcytoplasmicvesiclesandeventuallyendupinthe:

a. bloodb. digestivetract

2B.Thealphacellproducesglucagon,whichisvisibleinthedarkvesicles.Glucagonensuresthatthebloodsugarlevels:

a. increaseb. decrease

2C. The depicted beta cell is in bad shape: the rat has diabetes. Later you will compare thedifferenceswith a healthy rat. Do you recognize the various organelles? Only a few granuleswithhormonesarevisible,inparticulartothebottomleftofthenucleus.Thecrystal‐likeshapeistypicalandisevenmorepronouncedinhumanbetacells.Whichhormoneisit?2D.Somatostatin‐producingdeltacellsalsoformpartoftheislets,althoughtheycompriseonlyafewpercent.Wecandistinguishvariouscelltypesthankstothedifferentgranulestructures.Howcanwedifferentiatebetweensomatostatingranulesandglucagonorinsulin?2E. Is the centroacinar cell important for protein production, or the structure of the efferentducts?Howcanyouseethis?2F.Theindicatedcell,knownasapericyte,separatesthehormone‐producing……….....pancreasfromtheenzymeorproenzyme‐producing……………....pancreas.2G.Inflammatorycellsarepresentbecausethereisanongoingimmuneresponsetotheisletsintherat.Whattypeofleukocyteisvisiblehere?Howcanyouseethis?

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2H.Whatistheapproximatesizeofthiserythrocyte?2I.Whatistypicalofthenucleusofamonocyte?2J.Thisphagocyteis(a)passiveor(b)active,because:2K.Thegranulocytes in thebloodcellpracticalwerespherical/round.Theseclearlyarenot.Explainthedifference?2L.Theleukocytehasblackspotsonit.Whatistheapproximatesizeofthese?Whatcouldtheybe?2M. The small platelets clearly have a more heterogeneous content than the adjacenterythrocytes.Howmanyplateletsarevisibleinthisvein?3A.TheroughERisimportantamongotherthingsfor:Theblackspotsmeasureapproximately...........nm.Theseare................ontheinside/outsideoftheroughER.3B.Amitochondrioniseasilyrecognizedby:3C.Thecellnucleuscontains:Severaltypescanbedistinguishedandtheseare:Withregardtofunction,thisreflectstheprocessthatwecall:3D.TheGolgiapparatuscanbenano‐anatomicallydistinguishedfromtheERbecauseit:TheGolgiapparatusisimportantforsuchthingsas:4A.Zymogengranulescontainenzymesandproenzymesinthe.................cells.

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4B.Insulinisproducedbythe…………...cells.4C./4D./4E.Exosomesaresecretedgranules.Using thevariousstages, is itpossible to formapictureofexosomerelease?Yes/NoInwhatwaydoesexosomereleasedifferfromvesiclefusionin,forinstance,insulinsecretion?4F.Glucagonisproducedbythe……….....cells.4G.Somatostatinisproducedbythe……………..cells.5.Structure/functionofvesicles5A.Densebodiesareknownassuchbecause:5B.Lysosomesplayanimportantrolein:5C.Whattypeofcellisthiswhichisswarmingwithcaveolae?5D.Theseareaboutthesmallestvesiclesinexistence.Whatistheirdiameter?5E.Andwhatisthediameterofthelipiddroplets?5F.Whichtwocharacteristicsallowthistoberecognizedasanearlyendosome?5G.Clathrin‐coatedpitsarecharacteristicallyinvolvedin(a)endocytosisor(b)exocytosis.5H.Ifthemulti‐vesicularbodiesfusewiththeplasmamembrane,itisconceivablethat:6A.Crystaearetypicalof:6B.Whichatomisaccumulatedinthismembranousmass?6C.Doyourecognizethecells?Thefenestraeenable:6D.Thebasementmembranedepictedhereisbetweentwotypesofcells,whichare:6E.Here,thebasementmembraneformspartofacomplexstructure.Thisisstillthediabeticrat.Morphologicallyspeaking,bothcellscontainingnucleiclearlyappeartobeleukocytes.However,theseareindifferentlocations.Theleukocyteontheleftisinthe.... ,whiletheotherclearlyisnot.Explainwhatmaybegoingon.

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7. Macromolecules are just barely discernible at these image settings. There are certaincharacteristicswhichallowthevariousmacromoleculestoberecognized.7A. Howmany nuclear pores can you distinguish in the ENTIRE cross‐section of the nuclearmembrane?7B.Thisisthetipofthenucleuswherenuclearporescanalsobedistinguished.Howmanyarethere?Basedon7A.and7B.,sketcha3Dreconstructionofasinglenuclearpore.7C.Polysomescomprise:7D.Sketchamodelofasinglepolysomewith5ribosomes.Ifpossible,indicatethe5’UTRand3‘UTRandsketchadiagramofproproteins.7E.Desmosomesarespecializedcell‐cellcontactpoints,whichinparticularareimportantfor(a)tissuestrengthor(b)formingabarrier.7F.Tightjunctionsarespecializedcell‐cellcontactpoints,whichinparticularareimportantforformingabarrier.Otherthanindesmosomes,thereisnomajorconcentrationof intermediatefilamentsonthecytoplasmicside.Whichbarrierhasbeencreatedhere?7G./H.Collagenis(a)cytoplasmicor(b)extracellularandservesparticularlyto:7I./7J.Centriolesareoftenfoundperinuclearlyandaremainlymadeupof:7K.Everycellhasapairofcentrioles.Givearoughestimateofhowmanycentriolesshouldbevisibleinthisdataset.Explain.

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Assignment6:Isletsduringtype1diabetesFollowing this introduction to the EM of cells, organelles andmacromolecules, the focuswillnowturntotheeffectoftype1diabetesintheratmodel.Returntothehomepage(nanotomy.nl)andcompareDataset1(control)withDataset5(diabetes).1.Whatisthebloodsugarlevelofthehealthyanimal?Andthatoftheanimalwithdiabetes?2.Thisiscausedbyadeficitin:3.This iscausedbythebreakdownofbetacells. Insulitisclearlyexists,sinceDataset5showsmanymore:4.Thebeta‐celldestructionisclearlyrecognizableduetothefollowingcharacteristics(nameatleast3):5.Peoplewithdiabeteswillbenefitfromthefollowingtreatment:6.Toomuchtreatmentleadsto....andcanbecompensatedby:Comatosepatientsbenefitfrom:Two stages have now been shown. Time permitting, knowledge can be further tested bystudyingtheotherstages.Thiscanalsobedoneathome.

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Assignment7:Testingnewlyacquiredknowledgeonahealthyislet(Dataset1)Distinguish9differenttypesofcells.Whichcharacteristicscanbeusedindoingso?1.2.3.4.5.6.7.8.9.Sketch4differentorganellesandindicatetheircharacteristics.Sketch4differentmacromoleculesandmacromolecularcomplexes,andnameafunction.

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