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Development and motility of Dicty actin mutants
(fimbrin, alpha-actinin, and ABP-34)
Thisexperimentwasdone onnewlysynthesizedmutantstrandof amoeba Dictyostelium
discoideum,whichlacksthree actin bindingproteins/actincrosslinkingproteins,whichmainlyplaythe
role inactin bundlingatthe protrusionedge of the cell. Thisprojectwasperformedinordertodevelop
an understandingof how these cellsreacttowards harshenvironments:if itrespondstothe cAMPsignal
inorder to come together(if itdoes,thendoesitrespondtothe signal the same wayas Ax2),if it forms
“streams”(if itdoes,thenhowlongthese streamsare and if theyappearany differentfromthe wild
type),and,eventually, slugs(if theyformany,thenhow doestheirmorphologyandmotilitydifferfrom
Ax2),proceedingwithfruitingbodies (if theymake fruitingbodies,thenhow longdoes ittake tomake
them,anddo theylookdifferentlycomparingtothe control cells).Anothergoal of thisexperimentwas
to findoutif the generationtime of thismutantcell type isthe same as for the wildtype Ax2.Finally,
actin cytoskeleton localizationwasobservedunderthe lightmicroscope,inordertosee if the
morphologyof these cellswasanydifferentfromthe control groupof Ax2cells.Therefore,the
1) Growth of TKO and Ax2in twoenvironments:shakingsuspension (flasks),andstationaryplates.
2) Randommotilityof TKOand Ax2in HL5
3) Developmentof TKOandAx2 overagar in MCPB media
4) Response of TKOand Ax2to cAMP underagar
1) a) TKOcells will be able to divideand grow,butit will be slowerin both environmentalconditions
(shaking and stationary) then Ax2cells.
First,wildtype containsanintactactin bindingproteins.Inorderforcellstogrow,theymust
divide.Actin filamentsalongwithmyosinIIformcleavage furrow andconstricta contractile ring
(Lord).Mutants wouldn’tbe able todivide,orwouldn’tdivideasefficientlysince theylack
therefore makingthe processof contractionmuchmore efficientandorganized.Despite this
fact, theywouldstill be able todividebecause thereare otherbindingproteins,whose primary
jobis to handle suchfunctionsof division,suchascofilin.“Immunofluorescencemicroscopy
witha monoclonal antibodyforcofilinrevealedthatthisproteinistemporarilyconcentratedat
the contractile ringduringcytokinesis”(Obinata).
b) Also,both(TKOand Ax2) will grow slowerin moving suspension thanin stationary plates.
Thisway cellscouldattachto the bottomsurface of the dishand undergomitosiswithout
the cellsto growin.
2) TKO will movemuch slowerthen Ax2in randommotility
Due to the lackof bindingandcross-linkingactinproteins,the motility willbe dramatically
affected,since theyplayagreatrole inconnectingactinfilamentsbetweeneachother,making
thembeingdependentoneach other,and,therefore, makingthemworktogetherinone
3) TKO will notdevelop slugsand fruiting bodies,thereforedevelopmentwillbe altered
Since the workingmechanismof actincytoskeletonisdeterioratedatthe unicellularlevel,there
will be noproperorganizationamongcellsinordertoform a slugand especiallyafruitingbody.
4) TKO will havea weakand delayed responseto cAMP signaling,butit will be ableto chemotax
As wasnotedearlier,the properlyworkingmechanismof actincytoskeletonbeingableto work
withitsowncomponentsinunison,andbeingable toproperlyrespondtoanyoutside signaling,will be
damageddue to three differenttypesof actin-bundlingproteins.Therefore,thismechanismwouldnot
workas smoothlyasin Ax2cells,butthe “innate”response tochemotacticagentwill be still present.
5) The morphology of TKOcells will be significantly differentdueto removalof importantactin
Since the knownknocked-outproteinare mainlyinvolvedinbundlingof actinnearthe protrudingedge
of filopodia,andalsothe peripheryof the cell ingeneral,itwouldbe expectedtosee uneven
distributionof actinfluorescence aroundthe cell,if anyatall.
1) Growth. Bothplates forwildtype andmutanttype were trituratedand titered.The initial
concentrationtostart the cell growthwas 5-10 cells/ml forall typesof cells.Cellswouldall start
fromapproximatelyequal concentrationsandthe settingsforbothcell typeswouldbe setto
equal:one setwill be incubatedinplates,while the othersetwouldbe placedinflasks,which
wouldbe locatedonthe shakingsuspension.The concentrationsof eachplate/flaskwill be
takenapproximatelyevery12 hours,until the cell concentrationstopsincreasingovertime,or
will godown,indicatingthatthe cellsstartedtodie outdue to the lack of space andnutrients
available forthe normal proliferation.
2) Motility.The stock platesforbothcell typeswere takenandtiteredforan approximatelyequal
cell count,makingsure that there are at leastfive cellsinthe view tobe counted,andthere are
not toomany cellsforthemto be crowded,since itwouldinterfere withtheirmotility.Cells
were trackedinHL5 mediaforan hour. Five randomcellsfromeachvideowere afterwards
trackedusingimageJ software,average velocity andpersistence werecalculated,andcompared.
Usingthe informationfromthe trackedcells,rose plotgraphswere made usingMicrosoftExcell,
indicatingthe route thateachcell made,where eachroute wassetto start from the same initial
pointof reference “zero”.Alsothe graphfor“velocityovertime”wasmade foreachcell type
and comparedto eachother.
3) Development.Thispartof the experimentrequiredaverydense amountof cells.Itwasmade
sure that there were a lotof cellsinthe plates,andthat theywere inthe logphase. Mediafrom
the stock platesof Ax2and TKO cellswere taken outandtransferredinto15-ml falcontubes,
Afteranotherroundof centrifuging,mediawastakenoutandreplacedbyfreshMCPB inorder
to make sure that there were absolutelynonutrientsleft forthe cells.Thiswouldallow cellsto
chemotax andaggregate duringthe starvation,astheir survival mechanism. These cellswere
put intop60 andwere leftalone tobe settledtothe bottomof the surface (agarose).After
Thiswouldprovide cellswithaharshenvironmentthatwouldforce themtoaggregate together
and eventuallyformslugsandfruitingbodies. The moviesweresetto24 hourseach.
4) cAMP chemotaxisunder agar. Thisexperimentalsorequires adense amountof cellsforeach
cell type.The concentrationwasnot crucial here also,as longas the cellswere inthe logphase,
as inpreviouspartof the experiment. Thiswasasimilarexperiment,exceptthatthe cellswould
be placedunderagarose.Thiswill give asimilarharshenvironment,withnofoodormedia,plus
the weightpressingtop,therefore noslugsorfruitingbodieswere expectedtobe seen.What
was testedhere washowwouldthe cellsreacttocAMP chemotaxicsignal,andtofindoutif the
cellswouldbe able tochemotax,despite the weightbeingpushedonthem.The movie forAx2
was setfor10 hours,while the movie forTKOwassetfor 24 hours,since there isnoguarantee
0 50 100 150 200 250
Figure 1.1 Growth Curve of Ax2 and TKO in
0 50 100 150 200 250
Figure 1.2 Growth Curve of Ax2 and TKO in flask
y = 66113e0.0458x
y = 68903e0.0307x
0 50 100 150 200
Figure 1.3 Log Graph of Ax2 and TKO in Plate
Expon. (Ax2 Plate)
Expon. (TKO Plate)
y = 189504e0.0393x
y = 36564e0.0373x
0 50 100 150 200
Figure 1.4 Log Graph of Ax2 and TKO in Flask
Expon. (Ax2 Flask)
Expon. (TKO Flask)
15.13 22.57 17.63 18.58
Table 1.2 Ax2 and TKO growth in plates and shaking suspension
Ax2 (Control) Triple KnockOut
Speed(µm/minutes) Persistence Speed(µm/minutes) Persistence
Cell 1 2.480 0.768 7.887 0.947
Cell 2 2.899 0.969 3.729 0.702
Cell 3 3.195 0.842 3.625 0.543
Cell 4 3.375 0.956 8.289 0.926
Cell5 3.235 0.712 7.665 0.692
Average 3.037 0.849 6.239 0.762
-50 0 50 100 150 200
Figure 3.3 TKO under agar w/ MCPB. Rose plot
y1 = -0.0999x + 8.5777
y2 = 0.0787x + 1.6049
y3 = -0.0117x + 4.0585
y4 = -0.1202x + 9.9114
y5 = 0.1097x + 5.0877
0 10 20 30 40 50 60 70 80
Figure 3.4 TKO under agar w/ MCPB. Time vs.
Table 3.1 TKO and Ax2 chemotaxis towards cAMP in MCPB
In growth experiment, it tookalmost three weeks to complete the growth of TKO mutants,
while it only tooka weekfor AX2 to plateau both in the flaskand the plate. Even withoutthe
calculations, it was obvious that the mutants had much a larger generation time than the wild type
cells. From the results above, the generation time was calculated by Excel,using the exponential
growth graphs and the followingequation
Nt = N0 · etf
Where N = number of cells at time t
No= number of cells at initial time
T = time in hr and f = generation/hr
The generation time for AX2in stable plate condition and in shaking suspension were 15.13
hours and 17.63 hours, respectively.These results supported my hypothesis (1b). The generation
times forTKO mutant in plate and shaking suspension were respectively 22.57 and 18.58 hours
(Table 1.2). These results partially supported my hypothesis. Overall, the generation time for TKO
was much longer than for Ax2, whichsupported by initial hypothesis (1a). The part that did not
agree with above stated hypothesis was that generation time in flask is much shorter than forthe
plate. This would be suspected in errors that were most definitely made throughout three week
period. Accordingto figure 1.1 and, especially, figure 1.2, there were frequent inconsistencies in
data. Since this experiment was performed by two individuals, each wouldbe responsible for every
other point on the graph. The errors could be done from the way each of us triturated the cells,
when each of us started to dilute the cells concentration for more accurate results, or if one of us
still counted all the cells on the grid without diluting, or counting only portion of the grid,
multiplying it at the end. Since this experiment supported out hypothesis, then it wouldbe
concluded that actin, and, especially, actin binding proteins, play a major role in cytokinesisand cell
growth. It corresponds to one of the experiments, where it was found that “the fission yeast
filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched
filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the
contractile ring” (Kovar).In another article, it was described that “Dictyosteliumfimbrinalso
localizes to certain actin-rich regions of the cell, but a mutant lacking fimbrin has no detectable
defect in cytokinesis and is capable of completing development “(Pringle).Later on in the article he
added that neither “Dictyosteliumα-actininmutants nor mammalian cells microinjected with
antibodies against α-actinin displayed any obvious defectin cytokinesis”.From this article it can be
concluded that neither alpha-actinin nor fimbrin are capable of defecting cytokinesis on their own.
This can probably mean that together they can have permissive effect,a much stronger and more
damaging effectthan they could do by themselves. Itis also important to mention that ABP-34
could have a potential effecton cytokinesis as well. Since it wasn’t known much about such protein,
it could definitely be one of the possibilities. Concerning the shaking vs. stationary environment, the
immobile surface wouldprovide the perfectcondition for the cell proliferation in both wild and
mutant types of cells. This was much more favorablesince there were no distractions or chaos
during their division cycle. Also, data collectedfrom Ax2 and TKO cells, being able to divide and
grow, suggests that presence of fimbrin, alpha-actinin or ABP-34 had a positive effecton cells
proliferation, but its absence didn’t exactly mean that the cells wouldn’t divide. It meant, that it
would take a longer time forthem to grow,meaning that absence of these particular actin binding
proteins does have some inhibition effecton cell proliferation, but it is not necessary for the
division to take place.
In random motility, the results observed werevery obvious,which truly supported given
hypothesis. Both types of cells were recorded foran hour long, and the route of 5 random cells
were taken from each of twoplates. Image 1 and Image 2 were taken right at the end of the movie.
Without even calculating the average velocities of these cells it was clear that TKO cells motility was
very limited comparing to Ax2 cells. The calculateddata showed (Table 2.1) that TKO’s average
velocity (4.245 um/min) was twiceless of Ax2’s (7.561 um/min). There was also the same affecton
persistence. TKO’s average persistence was 0.119, while Ax2’s were 0.314. This shows how the
motility of the mutants is defected, and how disorganized their movement is. This data was
supported by literature, saying that one of our knockedout proteins, including other proteins in “a
set of four coreproteins - profilin, fimbrin/T-plastin, capping protein, and cofilin- crucial for
determining actin tail length, organizing filament architecture, and enabling motility” (Welch).
Another support was also found through the experiments withyeast. “Fimbrins have been
identified in budding yeast, ciliates, slime molds, plants, and a variety of animals. The
Saccharomycescerevisiae fimbrin, Sac6p, localizes to actin cables and patches. A sac6 null mutant
has a mild phenotype on synthetic medium but has more pronounced abnormalities on rich
medium. At 23°C, it is viable witha reduced growth rate (variablein different genetic backgrounds),
and the cells are rounder than wild-type cells” (Pringle). Paradoxically,in the clinic works with lung
fibroblasts, it was suggested that “ACTN4 [alpha-actinin-4] is essential for maintaining normal
spreading, motility,cellular and nuclear cross-sectional area, and contractility of murine lung
fibroblasts by maintaining the balance between transcellular contractility and cell-substratum
adhesion” in a differentkind of way.Surprisingly, whatthey found out was that if expression of
ACTN4 is knockeddown, it will enhance cellular compactionand contractionforce, and increased
cellular and nuclear cross-sectional area. “Interestingly, ACTN4 is phosphorylated upon growth
factorstimulation, and this loosens its interaction with actin” (Pollak).Thiswas very interesting to
find out, since it was though that knockingdown actin binding protein would decrease cell’s
motility. But then again, ACTN4 is only one of the actin binding proteins types. The protein that we
workedon was not exactly specified.
In part of the development experiment it was found out that TKOmutants couldaggregate
and formfruiting bodies just like Ax2 cells, whichdid not support our hypothesis. Accordingto
ependent_Project._Part_1..html) ,it took mutants 16 hours in order to start forming streams, while
Ax2 took9 hours. What is more interesting is that within that 24 hour movie, TKO did not show any
moving slugs. By the end of the movie, those aggregates seemed to looklike slugs, but they did not
move, instead, they began to grow right into whatappeared to be a fruiting body. The movie was
accidentally stopped before finish, but then it was reset fora few more hours, whichstill wasn’t
enough time in order to make sure if those were fruiting bodies coming out or slugs protruding
forwardin order to move. According to Fisher, it could be argued that those were actually slugs. “Of
the known calcium binding proteins that might be relevant, several have been examined genetically
for roles in phototaxis. They are the Calcium-regulated actin binding proteins -the capping/severing
protein severin and the actin crosslinkers alpha-actinin, fimbrin and the 34kDa actin-bundling
protein. Mutants lacking these proteins are unaffected in development and behavior at the
unicellular and multicellular stages, including slug phototaxis and thermotaxis” (Fisher). In the end,
it was clear that fruiting bodies were actually formed (Image 3 and Image 4), which,in most part,
are not very distinguishable from the Ax2 fruiting bodies.
In the next experiment, as was predicted in the above stated hypothesis, TKO cells were
able to chemotax under agar withthe response whichwas delayed (11 hours), comparing to the
controlwild type cells (5 hours). Unfortunately, controlvideo was stopped at 5 hours 23 minutes
from the start of streaming, but it was just enough to see the “stream” forming. Cells that were
responding to the cAMP signal were tracked, streaming into the aggregate, giving very interesting
velocities and persistences. Average persistences for Ax2 and mutants appeared to be the same at
0.849 and 0.762 (Table 3.1), respectively, whichis a very high persistence. On the other hand, the
differences in speed was very notable. Ax2 had the velocity twiceas small (3.037 um/min) as TKO
(0.762 um/min) and twiceas small as its random motility rate (7.561 um/min). This error could be
due to differentmicroscopes used. Different microscope supposed to have different calibrations,
whichcould have been done differently by other people. The microscopeused for Ax2 was not
even the same brand name, as the other microscope that was used for any other of my movies.
Besides the Ax2 data, this experiment gave very impressive result forTKO velocity and persistence,
whichmeans that during chemotaxis these cells woulddouble their speed, as if disregarding those
actin binding proteins. Perhaps ACTN4 was phosphorylated, whichwould lead to its dissociation
with actin filament, and in chain reaction it would enhance its motility and contraction, as was
previously described by Pollak.Also, according to figure 3.4, there weren’t too many points tracked
for Cell 1 and Cell 4 due to a hard visibility when the streaming began. In order to improve this
experiment, it wouldbe better to use a little less density of cells and start tracking them right at the
start when they first get a signal, not at the end, as was done in this case.
Finally, in the immunofluorescence part of the experiment, there were few images made of
Ax2 and TKO. The results were not as striking as they were expected to be. TKOcells did have actin
polymerization on the periphery of the cell, right at the cell cortex. But some of them seemed to lack
actin cytoskeletonat some parts, while most of Ax2 cells had a perfectcircle of actin binding the
outline of the cell. In this case, it couldbe assumed that cell couldnot make actin skeleton in some
parts because it lacked three actin binding protein which are usually found at the protruding edge
of filopodia. In order to improve this experiment, it could be repeated using confocalmicroscope.Its
3D imaging and being able to image the whole cell through could be a key in knowing the exact
morphology of mutant cell type.
Fisher,Paul. “GeneticAnalysisof Phototaxisin Dictyostelium”.Web.05 May 2012.
PolarizationinFissionYeast*." Actin FilamentBundling by Fimbrin Is ImportantforEndocytosis,
Cytokinesis,and Polarization in Fission Yeast.Web.05 May 2012.
Lord, Matthew,EllenLaves,andThomasD. Pollard."CytokinesisDependsonthe MotorDomans of
Myosin-IIinFissionYeastbutNotinBuddingYeast." Www.yale.edu.Anthony Bretscher,6July2005.
Web.3 Feb.2012. <www.yale.edu/pollard_lab/pdf/213.pdf>.
Obinata,T. "Concentrationof Cofilin,aSmall Actin-bindingProtein,atthe Cleavage Furrow during
Cytokinesis."Web.5May. 2012. <http://www.ncbi.nlm.nih.gov/pubmed/7728864>.
Pollak,Martin.“α-Actinin-4Is Essential for Maintaining the Spreading, Motility and Contractility of
Fibroblasts.” Web. 5 May.2012.
Pringle,John."Rolesof aFimbrinandan α-Actinin-likeProteininFissionYeastCell Polarizationand
Cytokinesis."Web.05May 2012. <http://www.molbiolcell.org/content/12/4/1061.full>.
Welch,MD. "DefiningaCore Setof ActinCytoskeletal ProteinsCritical forActin-basedMotilityof
Rickettsia."NationalCenterforBiotechnology Information.U.S.National Libraryof Medicine.Web.05
May 2012. <http://www.ncbi.nlm.nih.gov/pubmed/20478540>.