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Transcript
G2020–October2015
Cellsunderthemicroscope–partII
Nanotomy,T1D,electronmicroscopy
Lecturer:BenN.G.Giepmans
Department:CellBiology,UMCG
Contact:www.cellbiology.nl
Last update: 2015-09-07 BNGG
Cellsunderthemicroscope–partIINanotomy–T1D–EM
Preparation
Reading
 Ravellietal.SREP01804(2013)accessiblevia:
http://www.nature.com/srep/2013/130508/srep01804/full/srep01804.html
 You also can study the Dutch summary in the Nederlands tijdschrift voor diabeteologie
(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
Importantsourcesofinformation
Inadditiontothismanualandthelistedbooks:http://www.ronaldschulte.nl/
Assignments1‐3havebeenmodifiedfromDrs.D.Opstelten&N.A.Bos,ManualforCellBiology
Location: Combizaal311multipurposeroom,situatedonthe3rdfloorofbuilding3214,onthe
Ant.Deusinglaan1
NB:Althoughtheassignmentsmustbecompletedduringthepractical,itisessentialtoreadthem
inadvanceforfullcomprehension.
Afterthisworkshop,studentsshouldbeableto:
1. recognize and interpret electron microscopic images, with regard to tissue
characteristics,celltypes,organelles,andmacromolecularcomplexes
2. explain how functional information about cell function, e.g. regarding secretion, can be
determinedusingnanotomy
3. describe the structure of the cell, cell organelles and macromolecular complexes and
nametheirfunctions
4. usetheinformationfrom1‐3forpathophysiologicalanalysis,inthisworkshopprimarily
withregardtotype1diabetes
2
Cellsunderthemicroscope–partIINanotomy–T1D–EM
Assignment1:Electronmicroscopystructure/function
Acellcontainsorganellesthatareessentialforitsfunction.Dependingoncellularfunction,one
typeofcellwillhaveahighernumberofcertainorganellesthanothers.Tocheckifyouknowthe
variouscellorganelles,examinethefollowingschematicdrawingofacell(anexocrinecell).
a. Identifythevariouscellorganellesbyplacingtherightnumberattherightline
b. Statethemainfunction(s)oftheorganelleinthetable
Fig.1.Source:LaboratoryManualofHistology,Pappas.(W.C.Brown,1990)
Structure
Function
Structure
Function
1.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
8.microfilaments
15. rough endoplasmic reticulum
16.secretiondrops
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Cellsunderthemicroscope–partIINanotomy–T1D–EM
Assignment2:Studyelectronmicroscopy
IntransmissionEM(TEM),ahighvoltagegeneratedbetweenaheated
cathode (incandescent filament) and an anode produces a beam of
electrons.Oneormorecondenserlensesfocusthisbeamontotheplane
of focus of the objective lenses, where an ultrathin specimen section
which can be irradiated has been placed. The objective lenses create a
magnifiedimageoftheobjectwhichisshownonscreenorcapturedby
camera.AstandardTEMprovidesmagnificationofupto300,000times,
with a resolution of ∼ 2 nm, of specimen sections which are ∼ 60 nm
thick,withamaximumdiameterof3mm.
ThebiologicalmaterialpresentintheultrathinsectionmainlycomprisesC,H,NandOanddoes
not scatter electrons sufficiently to provide an image, which is why the specimen must be
stained with heavy metals, which do scatter electrons. The most common contrast medium is
osmiumtetroxide(OsO4),whichbindsparticularlyeasilytodoublebondsoflipids,fixingthem
bycreatingcross‐links,makingmembranesvisible.
In scanning EM (SEM), the electron
beam is focused by the condenser
and objective lenses in the same
manner as in TEM. Here, too, the
specimenisplacedinthefocalpoint.
The primary electron beam is not
stationary,likeinTEM,butscansitin
a grid‐like fashion. The electron
beamscansthespecimensurfaceline
for line, releasing secondary
electrons (SE2) in the sample or
having the electrons reflected
(backscatter electrons). These are
both used to create an image of the specimen surface. If the specimen section isultrathin,the
electronswillofcoursepassthroughit.Byplacingadetectorunderneath,aTEMimagecanbe
transformedintoanSEMimage.ThisisknownasSTEM:scanningtransmissionEM.
Alsoread:EssentialCellBiology,Albertsetal.,(Garland,4thed.2013)page11.
4
Cellsunderthemicroscope–partIINanotomy–T1D–EM
Assignment3:Studyingmembranetransport
Membranesformcompartments.Theplasmamembraneistheboundarybetweenthecytoplasm
andtheextracellularside.Thisboundaryisdynamic,withvarioustransportprocessesallowing
substancestopassinandoutofthecell.Someprocessesareexemplifiedusingglucose‐induced
insulinsecretion(Fig.2).Notethatvariousmoleculesandsubstancescanbetransportedacross
themembraneinregulatedfashion.Inthefigurebelow,indicatewhattypeoftransportprocess
isinvolved(encircleone).
Fig.2.Insulinsecretionin
beta cellscausedbythe
increasingbloodsugar
levels.
UptakeofglucosebyGLUT2
andglycolytic
phosphorylationofglucose
causestheATP:ADPratioto
rise.Thisinactivatesthe
potassiumchannelwhich
depolarizesthemembrane
sothatavoltage‐dependent
calciumchannelopens.The
increaseinthecalcium
concentrationleadstothe
releaseofinsulin
Source:www.betacell.org
1.GlucoseuptakebyGLUT2isprimarilydependenton:
Exocytosis/Concentration/Voltage‐dependentinflux/Effluxinhibition
2.TheATP‐sensitiveK+pumpisatypeof:
Exocytosis/Concentration/Voltage‐dependentinflux/Effluxinhibition
3.Thevoltage‐gatedcalciumpumpisatypeof:
Exocytosis/Concentration/Voltage‐dependentinflux/Effluxinhibition
4.Insulinsecretionisatypeof:
Exocytosis/Concentration/Voltage‐dependentinflux/Effluxinhibition
5
Cellsunderthemicroscope–partIINanotomy–T1D–EM
Assignment4:Fromtissuetomolecularcomplexes
Microscopy allows studying samples at different magnifications. Figs.1and2showedmodels.
Figure3isahistologicaldepictionoftheisletsofLangerhans.Intheassignmentbelow,wewill
lookatasectionofasingleislet.
1.DrawascalebarInFigures1,2and3,indicatingtheestimateddimensions.Areyouableto
givevarious‐sizedscalesinFig.2?Ifyoucannot,returntoitaftercompletingAssignment5.
Fig. 3. The beta cells grouped in the islets of Langerhans in the pancreas. The rest of the
pancreas consists of exocrine tissue where digestive enzymes are produced. The endocrine
tissue–theisletsofLangerhans–producesotherhormonesbesidesinsulin.
Source:www.bu.edu/histology/p/10401loa.htm
Furtherreading:FunctionalHistology,Kerr(Mosby,2nded.2009)p404‐407.
6
Cellsunderthemicroscope–partIINanotomy–T1D–EM
Assignment5:Nanotomy:EMoftissues,
cells,organellesandmacromolecules
Type 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 that
causes the disease is also unknown. Finding alternatives for insulin therapy and making
advances in etiology of T1D benefits from a full structural and functional insight into Islets of
Langerhans. 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 and
macromoleculesinGoogle‐Earth‐likefashion.Here,nanotomyhasbeenusedinananimalmodel
forType1diabetes:Ravellietal.(2013);www.nature.com/srep/2013/130508/srep01804/full/srep01804.html.
StudytheultrastructureofanisletofLangerhans.Gotowww.nanotomy.nlandclickonthe
largestislet(grey).ThisdatasetcanbestudiedinthesamewayyouviewalandscapeinGoogle
Earth. Click on the IIP icon at top left for extra instructions if necessary. Go through the
annotations and answer the questions below. The numbers correspond to theannotations:1A
refersto1,forexample.Ifyouplaceyourcursoron'Supracellular',forexample,asubmenuwill
appear, including A, Islet. Before you begin, drag the menu at bottom left up a bit and the
scalewillappear.
1.IsletsofLangerhansinrecent‐onsettype1diabetes(rat)
Judge what the largest structure is that you can recognize. And if you zoom in, what is the
smallest?Whatarethedimensions?
Onceagain:Theannotationmenucanbedraggedtoallowthescaletoshow.
Largest:
Sizeisapproximately:
Smallest:
Sizeisapproximately:
1A.NametheclearestdifferenceswhichdistinguishtheisletsofLangerhansfromtheexocrine
pancreas.
Whatarethevariousfunctions?
1B.Whichcellisinthecapillary?
1C.Whichtwotypesofcelldoyourecognizeinthevein?Whatarethemostobviousdifferences?
7
Cellsunderthemicroscope–partIINanotomy–T1D–EM
1D.Thecentroacinarlumenbelongstothe:
a. endocrinepancreasandcontainsenzymes
b. endocrinepancreasandcontainshormones
c. exocrinepancreasandcontainsenzymes
d. exocrinepancreasandcontainshormones
1E. Depicted here is a cross‐section of a bundle of unmyelinated axons. The bundle was
discovered more or less by chance: the electrons cause the axons to be slightly lighter. These
containroundtubulesandlight‐greyfilaments.Howmanyaxonsdoyouseehere?
1F.Insomeexocrinecellsthenucleusisnotvisible.Why?
2A. The exocrine cell contains a lot of rough ER for protein synthesis. The content will be
secretedwithcytoplasmicvesiclesandeventuallyendupinthe:
a. blood
b. digestivetract
2B.Thealphacellproducesglucagon,whichisvisibleinthedarkvesicles.Glucagonensuresthat
thebloodsugarlevels:
a. increase
b. decrease
2C. The depicted beta cell is in bad shape: the rat has diabetes. Later you will compare the
differences with a healthy rat. Do you recognize the various organelles? Only a few granules
withhormonesarevisible,inparticulartothebottomleftofthenucleus.Thecrystal‐likeshape
istypicalandisevenmorepronouncedinhumanbetacells.Whichhormoneisit?
2D.Somatostatin‐producingdeltacellsalsoformpartoftheislets,althoughtheycompriseonly
afewpercent.Wecandistinguishvariouscelltypesthankstothedifferentgranulestructures.
Howcanwedifferentiatebetweensomatostatingranulesandglucagonorinsulin?
2E. Is the centroacinar cell important for protein production, or the structure of the efferent
ducts?Howcanyouseethis?
2F.Theindicatedcell,knownasapericyte,separatesthehormone‐producing……….....pancreas
fromtheenzymeorproenzyme‐producing……………....pancreas.
2G.Inflammatorycellsarepresentbecausethereisanongoingimmuneresponsetotheisletsin
therat.Whattypeofleukocyteisvisiblehere?Howcanyouseethis?
8
Cellsunderthemicroscope–partIINanotomy–T1D–EM
2H.Whatistheapproximatesizeofthiserythrocyte?
2I.Whatistypicalofthenucleusofamonocyte?
2J.Thisphagocyteis(a)passiveor(b)active,because:
2K. The granulocytes in the blood cell practical were spherical / round. These clearly are not.
Explainthedifference?
2L.Theleukocytehasblackspotsonit.Whatistheapproximatesizeofthese?Whatcouldthey
be?
2M. The small platelets clearly have a more heterogeneous content than the adjacent
erythrocytes.Howmanyplateletsarevisibleinthisvein?
3A.TheroughERisimportantamongotherthingsfor:
Theblackspotsmeasureapproximately...........nm.Theseare................ontheinside/outsideof
theroughER.
3B.Amitochondrioniseasilyrecognizedby:
3C.Thecellnucleuscontains:
Severaltypescanbedistinguishedandtheseare:
Withregardtofunction,thisreflectstheprocessthatwecall:
3D.TheGolgiapparatuscanbenano‐anatomicallydistinguishedfromtheERbecauseit:
TheGolgiapparatusisimportantforsuchthingsas:
4A.Zymogengranulescontainenzymesandproenzymesinthe.................cells.
9
Cellsunderthemicroscope–partIINanotomy–T1D–EM
4B.Insulinisproducedbythe…………...cells.
4C./4D./4E. Exosomes are secreted granules. Using the various stages, is it possible to form a
pictureofexosomerelease?Yes/No
Inwhatwaydoesexosomereleasedifferfromvesiclefusionin,forinstance,insulinsecretion?
4F.Glucagonisproducedbythe……….....cells.
4G.Somatostatinisproducedbythe……………..cells.
5.Structure/functionofvesicles
5A.Densebodiesareknownassuchbecause:
5B.Lysosomesplayanimportantrolein:
5C.Whattypeofcellisthiswhichisswarmingwithcaveolae?
5D.Theseareaboutthesmallestvesiclesinexistence.Whatistheirdiameter?
5E.Andwhatisthediameterofthelipiddroplets?
5F.Whichtwocharacteristicsallowthistoberecognizedasanearlyendosome?
5G.Clathrin‐coatedpitsarecharacteristicallyinvolvedin(a)endocytosisor(b)exocytosis.
5H.Ifthemulti‐vesicularbodiesfusewiththeplasmamembrane,itisconceivable
that:
6A.Crystaearetypicalof:
6B.Whichatomisaccumulatedinthismembranousmass?
6C.Doyourecognizethecells?Thefenestraeenable:
6D.Thebasementmembranedepictedhereisbetweentwotypesofcells,whichare:
6E.Here,thebasementmembraneformspartofacomplexstructure.Thisisstillthediabeticrat.
Morphologicallyspeaking,bothcellscontainingnucleiclearlyappeartobeleukocytes.However,
theseareindifferentlocations.Theleukocyteontheleftisinthe....,whiletheotherclearlyis
not.Explainwhatmaybegoingon.
10
Cellsunderthemicroscope–partIINanotomy–T1D–EM
7. Macromolecules are just barely discernible at these image settings. There are certain
characteristicswhichallowthevariousmacromoleculestoberecognized.
7A. How many nuclear pores can you distinguish in the ENTIRE cross‐section of the nuclear
membrane?
7B.Thisisthetipofthenucleuswherenuclearporescanalsobedistinguished.Howmanyare
there?
Basedon7A.and7B.,sketcha3Dreconstructionofasinglenuclearpore.
7C.Polysomescomprise:
7D.Sketchamodelofasinglepolysomewith5ribosomes.Ifpossible,indicatethe5’UTRand3
‘UTRandsketchadiagramofproproteins.
7E.Desmosomesarespecializedcell‐cellcontactpoints,whichinparticularareimportantfor
(a)tissuestrengthor(b)formingabarrier.
7F.Tightjunctionsarespecializedcell‐cellcontactpoints,whichinparticularareimportantfor
formingabarrier.Otherthanindesmosomes,thereisnomajorconcentrationofintermediate
filamentsonthecytoplasmicside.Whichbarrierhasbeencreatedhere?
7G./H.Collagenis(a)cytoplasmicor(b)extracellularandservesparticularlyto:
7I./7J.Centriolesareoftenfoundperinuclearlyandaremainlymadeupof:
7K.Everycellhasapairofcentrioles.Givearoughestimateofhowmanycentriolesshouldbe
visibleinthisdataset.Explain.
11
Cellsunderthemicroscope–partIINanotomy–T1D–EM
Assignment6:Isletsduringtype1diabetes
Following this introduction to the EM of cells, organelles and macromolecules, the focus will
nowturntotheeffectoftype1diabetesintheratmodel.Returntothehomepage(nanotomy.nl)
andcompareDataset1(control)withDataset5(diabetes).
1.Whatisthebloodsugarlevelofthehealthyanimal?Andthatoftheanimalwithdiabetes?
2.Thisiscausedbyadeficitin:
3.Thisiscausedbythebreakdownofbetacells.Insulitisclearlyexists,sinceDataset5shows
manymore:
4.Thebeta‐celldestructionisclearlyrecognizableduetothefollowingcharacteristics(nameat
least3):
5.Peoplewithdiabeteswillbenefitfromthefollowingtreatment:
6.Toomuchtreatmentleadsto....andcanbecompensatedby:
Comatosepatientsbenefitfrom:
Two stages have now been shown. Time permitting, knowledge can be further tested by
studyingtheotherstages.Thiscanalsobedoneathome.
12
Cellsunderthemicroscope–partIINanotomy–T1D–EM
Assignment7:Testingnewlyacquiredknowledgeona
healthyislet(Dataset1)
Distinguish9differenttypesofcells.Whichcharacteristicscanbeusedindoingso?
1.
2.
3.
4.
5.
6.
7.
8.
9.
Sketch4differentorganellesandindicatetheircharacteristics.
Sketch4differentmacromoleculesandmacromolecularcomplexes,andnameafunction.
‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐endofpractical‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
13