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Development and motility of Dicty actin mutants

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Development and motility of Dicty actin mutants

  1. 1. AndreyDementyev Independentproject Development and motility of Dicty actin mutants (fimbrin, alpha-actinin, and ABP-34) Purpose: 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 experimentwasplannedin5differentsteps: 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 5) Immunofluorescence Hypothesis: 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 proteinsthatwouldbundle actinsfilamentstogether(fimbrin,alpha-actinin,andABP-34),and therefore makingthe processof contractionmuchmore efficientandorganized.Despite this fact, theywouldstill be able todividebecause thereare otherbindingproteins,whose primary
  2. 2. 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 beingdisturbed.Incontrary,shakingsuspensionwouldprovideanunstable environmentfor 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 highly-regulatedmechanism. 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 binding proteins. Since the knownknocked-outproteinare mainlyinvolvedinbundlingof actinnearthe protrudingedge of filopodia,andalsothe peripheryof the cell ingeneral,itwouldbe expectedtosee uneven distributionof actinfluorescence aroundthe cell,if anyatall.
  3. 3. Procedure 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, followedbycentrifuging.HL5mediawasremovedandreplacedwithMCPBstarvationbuffer. 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 thirtyminutes,mediawascarefullyremovedbycapillaryaction,usingfoldedendof Kimwipes. 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
  4. 4. that theywill chemotax atthe same time aswildtype,if theywouldchemotax atall.Therefore, extratime wasgiven,inordernotto be too short,incase mutanttype takeslongertime toreact towardssignalingcAMP. 5) Immunofluorescence.Mutantandwildtype cellswere putonthe coverslipsovernighttobe firmlyattached,before beingtreatedwithchemicalsandwashed.Fourdifferentchemicalswere usedinorderto permeabilize cellmembraneandbindtoactinfilaments,inordertoeventually make themfluoresce underthe lightmicroscope.Tritinwasusedinordertomake holes,by breakingmembrane apart.Formaldehydeandglutaraldehydewere usedtocrosslink membrane proteinsbytheiraminogroups,“mummifying”the cell.AndPhalloidinwasusedto go throughthose poresandbindto actin,preventingitspolymerization.Itsaffectallowstosee fluorescenceinactincytoskeleton. Results Ax2 Plate TKO Plate Ax2 Flask TKO Flask Time (hours) Cell count (cells/mL) Time (hours) Cell Count (cells/mL) Time (hours) Cell count (cell/mL) Time (hours) Cell Count (cell/mL) 0 44000 0 50000 0 110000 0 40000 11.5 96000 17.5 60000 17.5 220000 17.5 60000 23.5 150000 30.75 60000 30.75 620000 30.75 76000 37.5 450000 41 270000 41 1070000 41 116000 47.75 620000 53 930000 53 1830000 53 390000 59.5 1230000 65.33 860000 65.33 3410000 65.33 480000 71 1500000 77.5 1320000 77.5 7500000 77.5 1300000 85 5400000 89.5 1520000 89.5 6250000 89.5 570000 94.5 5400000 101.5 1620000 101.5 7750000 101.5 3630000 107.5 7000000 113.25 1890000 113.25 11100000 113.25 4060000 118.5 12100000 125 4130000 125 9800000 125 2270000 135 11500000 139 4250000 139 9000000 139 7300000 142 9400000 149.25 4330000 146 10700000 149.5 7000000 - - 161 6800000 157.75 11800000 161.25 11800000 - - 172.5 4900000 - - 172.5 3400000 - - 186.5 7500000 - - 186.5 6100000 - - 196 7300000 - - 198.25 10000000 - - 209.25 8100000 - - 211.25 10100000 - - 222.5 7400000 - - - - - - 236 10300000 - - - - Table 1.1 Ax2 and TKO growth in plates and shaking suspension (flasks)
  5. 5. 0 2000000 4000000 6000000 8000000 10000000 12000000 14000000 0 50 100 150 200 250 Cellcount(cells/mL) Time (hours) Figure 1.1 Growth Curve of Ax2 and TKO in plate Ax2 Plate TKO Plate 0 2000000 4000000 6000000 8000000 10000000 12000000 14000000 0 50 100 150 200 250 Cellcount(cells/mL) Time (hours) Figure 1.2 Growth Curve of Ax2 and TKO in flask Ax2 Flask TKO flask
  6. 6. y = 66113e0.0458x y = 68903e0.0307x 1000 10000 100000 1000000 10000000 100000000 0 50 100 150 200 Cellcount(cells/mL) Time (hours) Figure 1.3 Log Graph of Ax2 and TKO in Plate Growth Curve Ax2 Plate TKO Plate Expon. (Ax2 Plate) Expon. (TKO Plate) y = 189504e0.0393x y = 36564e0.0373x 1000 10000 100000 1000000 10000000 100000000 0 50 100 150 200 Cellcount(cells/mL) Time (hours) Figure 1.4 Log Graph of Ax2 and TKO in Flask Growth Curve Ax2 Flask TKO Flask Expon. (Ax2 Flask) Expon. (TKO Flask) Ax2 Plate TKO Plate Ax2 Flask TKO Flask Generation times (hours) 15.13 22.57 17.63 18.58 Table 1.2 Ax2 and TKO growth in plates and shaking suspension (flasks)
  7. 7. -200 -150 -100 -50 0 50 100 -300 -250 -200 -150 -100 -50 0 50 100 150 um um Figure 2.1 AX2 motility in HL5. Rose Plot Track1 Track2 Track3 Track4 Track5 y1 = -0.0834x + 10.464 y2 = 0.0646x + 7.483 y3 = -0.0226x + 7.4309 y4 = -0.0257x + 5.5985 y5 = 0.0491x + 7.1401 -5 0 5 10 15 20 25 30 35 40 0 10 20 30 40 50 60 70 Velocity(um/min) Time (minutes) Figure 2.2 Ax2 time vs velocity in HL5 Track1 Track2 Track3 Track4 Track5
  8. 8. Ax2 (Control) Triple KnockOut Speed(µm/minutes) Persistence Speed(µm/minutes) Persistence Cell 1 7.961 0.229 5.445 0.181 Cell 2 9.483 0.474 6.188 0.110 Cell 3 6.816 0.329 3.701 0.097 Cell 4 4.875 0.207 3.278 0.061 Cell5 8.672 0.330 2.615 0.148 Average 7.561 0.314 4.245 0.119 -80 -60 -40 -20 0 20 40 60 80 -60 -50 -40 -30 -20 -10 0 10 20 30 um um Figure 2.3 TKO motility in HL5. Rose plot Cell1 Cell2 Cell3 Cell4 Cell5 y1 = -0.042x + 6.7042 y2 = 0.0302x + 5.2825 y3 = -0.0406x + 4.9198 y4 = -0.0216x + 3.9278 y5 = -0.0106x + 2.9345 -5 0 5 10 15 20 25 30 0 10 20 30 40 50 60 70 Velocity(um/min) Time (minutes) Figure 2.4 TKO time vs velocity in HL5 Cell1 Cell2 Cell3 Cell4 Cell5 Table 2.1 Ax2 and TKO random motility in HL5. Speed and persistencein one hour
  9. 9. Tracks of 5 Ax2 cells in one hour of motility in HL5 media Tracks of 5 TKO cells in one hour of motility in HL5 media Image 1 Image 2
  10. 10. Image 4 Image 3 TKO fruitingbodiesoveragar Ax2 fruitingbodiesoveragar
  11. 11. -160 -140 -120 -100 -80 -60 -40 -20 0 20 -80 -60 -40 -20 0 20 40 60 um um Figure 3.1 Ax2 under agar. Rose plot Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 y1 = 0.0275x - 5.6859 y2 = -0.0076x + 5.1612 y3 = -0.0374x + 14.291 y4 = -0.0364x + 14.164 y5 = -0.0351x + 13.636 0 2 4 6 8 10 12 14 260 270 280 290 300 310 320 330 Velocity(um/min) Time (minutes) Figure 3.2 Ax2 under agar w/ MCPB. Time vs velocity Cell 1 Cell 2 Cell 3 Cell 4 Cell 5
  12. 12. 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 -200 -180 -160 -140 -120 -100 -80 -60 -40 -20 0 20 -50 0 50 100 150 200 um um Figure 3.3 TKO under agar w/ MCPB. Rose plot Cell 1 Cell 2 "Cell 3" Cell 4 Cell 5 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 5 10 15 20 25 30 0 10 20 30 40 50 60 70 80 Velocity(um/min) Time (minutes) Figure 3.4 TKO under agar w/ MCPB. Time vs. Speed Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Table 3.1 TKO and Ax2 chemotaxis towards cAMP in MCPB
  13. 13. Discussion 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
  14. 14. 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 video(http://homepages.uconn.edu/~mb2225vc/MCB_2225/Cellbio6_files/Cellbio6/Andreys_Ind 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
  15. 15. 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.
  16. 16. Citations Fisher,Paul. “GeneticAnalysisof Phototaxisin Dictyostelium”.Web.05 May 2012. <http://books.google.com/books?id=2nevsljDiCYC&pg=PA545&lpg=PA545&dq=fimbrin+slugs&source=b l&ots=K2nRjPNDaP&sig=N8Y6FabgqkQU5xyOZt3YOgPUgU4&hl=en&sa=X&ei=o8KkT8LPAqre0QGlnv2SB Q&ved=0CE0Q6AEwAQ#v=onepage&q=fimbrin%20slugs&f=false> Kovar,David."ActinFilamentBundlingbyFimbrinIsImportantforEndocytosis,Cytokinesis,and PolarizationinFissionYeast*." Actin FilamentBundling by Fimbrin Is ImportantforEndocytosis, Cytokinesis,and Polarization in Fission Yeast.Web.05 May 2012. <http://www.jbc.org/content/286/30/26964>. 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. <http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013921> 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>.

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