Filopodia are active tubular structuresprotruding from the cell surface which allow the cell to sense and interact with the surrounding environment through repetitive elongation-retraction cycles. The mechanical behavior of filo-podia has been studied by measuring the traction forces exerted on external sub-strates.1 These studies have revealed that internal actin flow can transduce a force across the cell surface through transmem-brane linkers like integrins. In addition to the elongation-retraction behavior filo-podia also exhibit a buckling and rota-tional behavior. Filopodial buckling in conjunction with rotation enables th

Bendix, Pól Martin

Leijnse, Natascha

Oddershede, Lene B

Communicative & Integrative Biology

English

PubMed

Natascha Leijnse

Lene B Oddershede

Poul M Bendix

Crossref

Dynamic buckling of actin within filopodia

Filopodia are active tubular structures protruding from the cell surface which allow the cell to sense and interact with the surrounding environment through repetitive elongation-retraction cycles. The mechanical behavior of filopodia has been studied by measuring the traction forces exerted on external substrates.(1) These studies have revealed that internal actin flow can transduce a force across the cell surface through transmembrane linkers like integrins. In addition to the elongation-retraction behavior filopodia also exhibit a buckling and rotational behavior. Filopodial buckling in conjunction with rotation enables the cell to explore a much larger 3-dimensional space and allows for more complex, and possibly stronger, interactions with the external environment.(2) Here we focus on how bending of the filopodial actin dynamically correlates with pulling on an optically trapped microsphere which acts like an external substrate attached to the filopodial tip. There is a clear correlation between presence of actin near the tip and exertion of a traction force, thus demonstrating that the traction force is transduced along the actin shaft inside the filopodium. By extending a filopodium and holding it while measuring the cellular response, we also monitor and analyze the waiting times for the first buckle observed in the fluorescently labeled actin shaft.</p

Copenhagen University Research Information System

u n i ve r s i t y  o f  co pe n h ag e n  Københavns UniversitetDynamic buckling of actin within filopodiaLeijnse, Natascha; Oddershede, Lene B; Bendix, Pól MartinPublished in:Communicative & Integrative BiologyDOI:10.1080/19420889.2015.1022010Publication date:2015Document versionPublisher's PDF, also known as Version of recordCitation for published version (APA):Leijnse, N., Oddershede, L. B., & Bendix, P. M. (2015). Dynamic buckling of actin within filopodia.Communicative & Integrative Biology, 8(2), e1022010. https://doi.org/10.1080/19420889.2015.1022010Download date: 03. Feb. 2020Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=kcib20Download by: [Copenhagen University Library] Date: 15 January 2016, At: 03:00Communicative & Integrative BiologyISSN: (Print) 1942-0889 (Online) Journal homepage: http://www.tandfonline.com/loi/kcib20Dynamic buckling of actin within filopodiaNatascha Leijnse, Lene B Oddershede & Poul M BendixTo cite this article: Natascha Leijnse, Lene B Oddershede & Poul M Bendix (2015) Dynamicbuckling of actin within filopodia , Communicative & Integrative Biology, 8:2, e1022010, DOI:10.1080/19420889.2015.1022010To link to this article:  http://dx.doi.org/10.1080/19420889.2015.1022010© 2015 The Author(s). Published withlicense by Taylor & Francis Group, LLC©Natascha Leijnse, Lene B Oddershede, andPoul M BendixPublished online: 29 Apr 2015.Submit your article to this journal Article views: 157View related articles View Crossmark dataDynamic buckling of actin within filopodiaNatascha Leijnse, Lene B Oddershede, and Poul M Bendix*Niels Bohr Institute; University of Copenhagen; Copenhagen, DenmarkFilopodia are active tubular structuresprotruding from the cell surfacewhich allow the cell to sense and interactwith the surrounding environmentthrough repetitive elongation-retractioncycles. The mechanical behavior of filo-podia has been studied by measuring thetraction forces exerted on external sub-strates.1 These studies have revealed thatinternal actin flow can transduce a forceacross the cell surface through transmem-brane linkers like integrins. In additionto the elongation-retraction behavior filo-podia also exhibit a buckling and rota-tional behavior. Filopodial buckling inconjunction with rotation enables thecell to explore a much larger 3-dimen-sional space and allows for more com-plex, and possibly stronger, interactionswith the external environment.2 Here wefocus on how bending of the filopodialactin dynamically correlates with pullingon an optically trapped microspherewhich acts like an external substrateattached to the filopodial tip. There is aclear correlation between presence ofactin near the tip and exertion of a trac-tion force, thus demonstrating that thetraction force is transduced along theactin shaft inside the filopodium. Byextending a filopodium and holding itwhile measuring the cellular response, wealso monitor and analyze the waitingtimes for the first buckle observed in thefluorescently labeled actin shaft.Actin rich tubular structures like inva-dopodia, filopodia and podosomes are fre-quently observed on the cell surface of anumber of different cell types. Commonto these structures is that they facilitatedirect intercellular communication as wellas cellular interactions with the extracellu-lar matrix. Filopodia arepresent ina largenumber of cell types and are highlydynamic. They are found at the tip of neu-rons and are responsible for steering theneuronal growth by sensing and respond-ing to chemical cues from the environ-ment.3,4 Embryonic cells use filopodia forlong range intercellular communication5aswell as in morphogenetic processes likecompaction of the early embryo which isan essential step in mammalian develop-ment.6 Filopodia also play essential rolesin fibroblasts and endothelial cells duringintercellular interactions and cellularmigration.7 Cancer cells employ filopodiaor filopodia-like structures during migra-tion to facilitate efficient invasion of exter-nal tissue.8,9 Finally, an interestingexample of filopodia function can befound in macrophages which have beenshown to exhibit complex interactionswith foreign objects like bacteria adherenton flat substrates10 or 3 dimensionallytrapped dielectric particles.11Filopodial rotation and bending arefrequently observed,2,3,12 but these phe-nomena are poorly studied and their bio-logical functions are not well understood.Rotational behavior has been detected inmacrophages, neuronal cells and HEK293cells, but it is unclear whether this rota-tional behavior is a generic property offilopodia in all cell types. Detection ofrotational behavior is easier when the filo-podia exhibit a kink large enough to bevisible, e.g., in a confocal microscope andthe studies which have reported rotationalbehavior of filopodia have also reportedsimultaneous buckling. Therefore, rota-tion of the actin shaft (the visible part ofthe actin) within the filopodial membranetube could well be a generic property ofall filopodia which first becomes visible bymicroscopy during buckling. However,buckling and rotation were shown to becoupled, in HEK293 cells, since rotationof the internal actin shaft inducedKeywords: filopodia, actin dynamics, heli-cal buckling, optical trapping, rotation,traction© Natascha Leijnse, Lene B Oddershede, and Poul MBendix*Correspondence to: Poul M Bendix; Email:bendix@nbi.ku.dkSubmitted: 02/13/2015Accepted: 02/17/2015http://dx.doi.org/10.1080/19420889.2015.1022010Addendum to: Helical buckling of actin insideﬁlopodia generates traction”. Leijnse N, Odder-shede LB, & Bendix PM. PNAS 2015, 112(1), 136-141.www.tandfonline.com e1022010-1Communicative & Integrative BiologyCommunicative & Integrative Biology 8:2, e1022010; March/April 2015; Published with license by Taylor & Francis Group, LLCARTICLE ADDENDUMDownloaded by [Copenhagen University Library] at 03:00 15 January 2016 buckling through a friction mechanismbetween the actin shaft and the filopodialmembrane.2 By extending filopodia a fewmicrometers, and simultaneously visualiz-ing the actin, we show here that frequentbuckles appear on the actin shaft as shownin Figure 1A. Initially, the actin appearsstraight, but after a certain waiting time(see Fig. 1B) buckles start to appear asshown in Fig. 1A. Extension of the filopo-dial membrane is accompanied withextension and retraction of the visible partof the actin.2,13 Extension of the actin tothe tip region results in transient pullingevents which are frequently observed insuch experiments.2,13,14 We visualized thefluorescently labeled actin inside filopodiawhile simultaneously measuring the forceexerted by the cell on the optically trappedbead attached to the filopodial membrane.The combined optical tweezers and confo-cal microscope used for this study isdescribed in.15 Quantification of theintensity of actin at the tip region revealeda clear correlation between the presence ofactin and the generation of a force on thetrapped particle. By calibrating the opticaltrap2 we were able to quantify the forcewhile also visualizing and quantifying theactin signal in a region which includes thefilopodial tip. As shown in Figure 1C, theforce (blue curve) correlates strongly withthe actin intensity (green curve) at thetimes when the force exhibits abruptchanges (see arrows). We also note thatoccasional non-correlative behavior can beseen, this seems mainly to occur when theactin is slowly polymerizing toward thetip.Interestingly, there is a correlationbetween buckling and pulling, this is visi-ble in Figure 1A. At t D 340 s the cellpulled on the trapped bead which conse-quently moved toward the cell and at t D480 s we observe a relaxation of the force(bead moves away from the cell) with aconcomitant increase of the curvature ofthe actin shaft (increased buckling). Thisstrongly suggests that bending is notcaused by compressive buckling which hasbeen proposed as a mechanism for helicalbuckling in free filopodia experiencing anaxial compressive tension from the mem-brane.16,17 We held the membrane withthe optically trapped bead and therebyexerted axial tension to the actin shaft justprior to an increase in buckling. Thisintrinsic tendency of the filopodium tobuckle against an external load indicatesthat the structure of the filopodium is notjust a straight rod composed of a parallelbundle of actin filaments. Interestingly,and in accordance with this, recent cryo-genic electron microscopy images haverevealed discontinuous structures of actinin filopodia18,19 which could more easilyfavor a bent structure and lower the bend-ing energy. Actin filaments in HEK293cells are cross-linked by fascin which pro-vides rigidity to the structure, but alsoflexibility through its dynamic associationwith the actin filaments. The off-rateFigure 1. Dynamics of actin inside a ﬁlopodium extended from a HEK293 cell. (A) Fluorescentimages of actin within a ﬁlopodium extended and held by an optically trapped bead. The snapshots are taken at different times during one experiment where the actin exhibits different degreesof bending and pulling. Pulling was detected as a downward movement of the bead (toward thecell and away from the optical trap). The last image to the right is an overlay of all the other images.Scale bar, 5 mm. (B) Statistics of the waiting times for the ﬁrst buckle to occur on ﬁlopodia that areextended by the optical trap, N D 43 buckles. The inset shows the waiting times after which addi-tional buckles occurred (second, third, . . .), N D 52 buckles. Up to 6 buckles could be observed in asingle ﬁlopodium during a measurement period of »10 min. Only clearly resolved and isolatedbuckles were counted and the uncertainty in determining the buckling time from the image serieswas »3 s. (C) Correlation between the force on the trapped bead and the actin intensity at the tipregion. Rapid drops in the force (blue curve) were associated with rapid decreases in the actinintensity (green curve). Yellow arrows denote correlative events at which the force drops rapidlywith associated decrease in actin signal.e1022010-2 Volume 8 Issue 2Communicative & Integrative BiologyDownloaded by [Copenhagen University Library] at 03:00 15 January 2016 of fascin has been measured to be koff D0.12 s¡1and has been shown to allow for agradual and slow deformation of the struc-ture to occur by dynamic cross-linking ofthe actin filaments.20 Moreover, actin sev-ering proteins like cofilin5,18 and gelso-lin21 have been associated withdestabilization of retracting filopodia andmight play a role in the bending of actin.In Ref. 2 it was shown how a physicalmechanism could contribute to bucklingof the actin. The mechanism is based ontorsional twist accumulated within theactin structure during rotation of the actinwithin the filopodial membrane. Frictionbetween the membrane and the actin(from, e.g., transmembrane proteinslinked to actin) causes actin to twist whichsubsequently is converted into actin buck-ling. Moreover, this mechanism canexplain the traction which can occursimultaneously with actin buckling.2The waiting time before onset of thefirst buckle and the periodicity of thebuckles in the actin shaft showed greatvariations as presented in Figure 1B. Weobserved the first buckle on a single shafttypically within ca. 100 s after extendingthe filopodium to »10 mm, only about15% of the first buckles appeared later.Additional buckles on a single shaft werealso frequently observed as shown in theinset of Fig. 1B. Up to 6 buckles could bedetected on the same shaft while keepingthe filopodium extended for »10 min.Filopodia which are extended by anoptically trapped bead at their tip are pre-vented from natural retraction and inte-gration into the lamellipodium as seen infree filopodia. Since free filopodia are fre-quently observed to buckle just prior tointegration into the lamellipodium weanticipate that holding the filopodiumextended will force the cell to perform sev-eral protrusion-retraction cycles for thesame filopodium and hence severalbuckles occur at multiple times during theexperiment.We note that buckling and rotationwere observed regardless of the labelingmethod; rotation and buckling wereobserved both when the actin was labeledwith GFP-Utrophin or Lifeact-GFP. Also,rotation and buckling could be observedwith unlabeled actin in which the mem-brane was fluorescently labeled (via mem-brane fluorophores or transmembraneproteins tagged with GFP).Filopodia exhibit extremely diversemechanical behavior by combining physi-cal and biochemical mechanisms to facili-tate complex interactions with externalobjects. However, much is still to learnabout how physical forces like pulling,rotation and bending act in concert whilebeing controlled or affected by molecularfactors like actin binding proteins, trans-membrane signaling proteins, or myosinmotors. Direct visualization of cellularproteins like actin with parallel detectionof the force, possibly in connection togenetic knock-out of essential compo-nents, will provide an important strategyfor understanding the biomechanical pro-cesses underlying filopodial dynamics.Such future efforts will result in a deeperinsight into the interplay between essentialmolecular components that are importantfor filopodial dynamics.Disclosure of Potential Conflicts of InterestNo potential conflicts of interest weredisclosed.FundingWe acknowledge financial supportfrom the Lundbeck Foundation, the Vil-lum Kann Rasmussen Foundation, andthe Danish National Research Councils(grant DFF – 4002-00099).References1. Chan CE, Odde DJ. Traction dynamics of filopodia oncompliant substrates. Science 2008; 322:1687-91;PMID:190743492. Leijnse N, Oddershede LB, Bendix PM. Helicalbuckling of actin inside filopodia generates traction.Proc Natl Acad Sci USA 2015; 112:136-41;PMID:255353473. Tamada A, Kawase S, Murakami F, Kamiguchi H.Autonomous right-screw rotation of growth conefilopodia drives neurite turning. J Cell Biol 2010;188:429-41; PMID:201239944. Lowery LA, Van Vactor D. The trip of the tip: under-standing the growth cone machinery. Nat Rev Mol CellBio 2009; 10:332-435. Sanders TA, Llagostera E, Barna M. Specialized filopo-dia direct long-range transport of SHH during verte-brate tissue patterning. Nature 2013; 497:628-32;PMID:236243726. Fierro-Gonzalez JC, White MD, Silva JC, Plachta N.Cadherin-dependent filopodia control preimplantationembryo compaction. Nat Cell Biol 2013; 15:1424-33;PMID:242708897. Bornschlogl T. How filopodia pull: what we knowabout the mechanics and dynamics of filopodia. Cyto-skeleton 2013; 70:590-603; PMID:239599228. Arjonen A, Kaukonen R, Ivaska J. Filopodia and adhe-sion in cancer cell motility. Cell Adh Migr 2011;5:421-30; PMID:219755519. Stylli SS, Kaye AH, Lock P. Invadopodia: At the cut-ting edge of tumour invasion. J Clin Neurosci 2008;15:725-37; PMID:1846890110. Moller J, Luhmann T, Chabria M, Hall H, Vogel V.Macrophages lift off surface-bound bacteria using a filo-podium-lamellipodium hook-and-shovel mechanism.Sci Rep 2013; 3: 2884; PMID:2409707911. Kress H, Stelzer EHK, Holzer D, Buss F, Griffiths G,Rohrbach A. Filopodia act as phagocytic tentacles andpull with discrete steps and a load-dependent velocity.Proc Natl Acad Sci USA 2007; 104:11633-8;PMID:1762061812. Zidovska A, Sackmann E. On the mechanical stabiliza-tion of filopodia. Biophys J 2011; 100:1428-37;PMID:2140202413. Bornschlogl T, Romero S, Vestergaard CL, Joanny JF,Nhieu GTV, Bassereau P. Filopodial retraction force isgenerated by cortical actin dynamics and controlled byreversible tethering at the tip. Proc Natl Acad Sci USA2013; 110:18928-33; PMID:2419833314. Farrell B, Qian F, Kolomeisky A, Anvari B, BrownellWE. Measuring forces at the leading edge: a force assayfor cell motility. Integr Biol-Uk 2013; 5:204-1415. Richardson AC, Reihani N, Oddershede LB. Combin-ing confocal microscopy with precise force-measuringoptical tweezers. SPIE Proc 2006; 6326:28-3816. Daniels DR, Turner MS. Islands of conformational sta-bility for filopodia. PloS ONE 2013; 8:e59010;PMID:23555612; http://dx.doi.org/10.1371/journal.pone.005901017. Pronk S, Geissler PL, Fletcher DA. Limits of filopo-dium stability. Phys Rev Letts 2008; 100:25; http://dx.doi.org/10.1103/PhysRevLett.100.25810218. Breitsprecher D, Koestler SA, Chizhov I, Neme-thova M, Mueller J, Goode BL, Small JV, RottnerK, Faix J. Cofilin cooperates with fascin to disas-semble filopodial actin filaments. J Cell Sci 2011;124:3305-18; PMID:21940796; http://dx.doi.org/10.1242/jcs.08693419. Ben-Harush K, Maimon T, Patla I, Villa E, MedaliaO. Visualizing cellular processes at the molecularlevel by cryo-electron tomography. J Cell Sci 2010;123:7-12; PMID:20016061; http://dx.doi.org/10.1242/jcs.06011120. Aratyn YS, Schaus TE, Taylor EW, Borisy GG. Intrin-sic dynamic behavior of fascin in filopodia. Mol BiolCell 2007; 18:3928-40; PMID:17671164; http://dx.doi.org/10.1091/mbc.E07-04-034621. Lu M, Witke W, Kwiatkowski DJ, Kosik KS. Delayedretraction of filopodia in gelsolin null mice. J Cell Biol1997; 138:1279-87; PMID:9298983; http://dx.doi.org/10.1083/jcb.138.6.1279www.tandfonline.com e1022010-3Communicative & Integrative BiologyDownloaded by [Copenhagen University Library] at 03:00 15 January 2016 

CiteSeerX

https://curis.ku.dk/ws/files/153446909/19420889.2015.1022010.pdf

Dynamic buckling of actin within filopodia

Abstract

Similar works

Full text

Available Versions

Crossref

Copenhagen University Research Information System

CiteSeerX