Force-dependent polymorphism in type IVpili reveals hidden epitopesNicolas Biaisa,1, Dustin L. Higashib, Jasna Brujićc, Magdalene Sob, and Michael P. Sheetza

aDepartment of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, NY 10027; bDepartment of Immunobiology and the BIO5Institute, University of Arizona, 1657 East Helen Street, Tucson, AZ 85719; and cDepartment of Physics, New York University, 4 Washington Place, NewYork, NY 10003

Edited by Douglas C. Rees, Caltech/Howard Hughes Medical Institute, Pasadena, CA, and approved May 11, 2010 (received for review October 1, 2009)

Through evolution, nature has produced exquisite nanometricstructures, with features unrealized in the most advanced man-made devices. Type IV pili (Tfp) represent such a structure: 6-nm-wide retractable filamentous appendages found in many bacteria,including human pathogens. Whereas the structure of Neisseriagonorrhoeae Tfp has been defined by conventional structural tech-niques, it remains difficult to explain thewide spectrumof functionsassociated with Tfp. Here we uncover a previously undescribedforce-induced quaternary structure of the N. gonorrhoeae Tfp. Byusing a combination of optical andmagnetic tweezers, atomic forcemicroscopy, and molecular combing to apply forces on purifiedTfp, we demonstrate that Tfp subjected to approximately 100 pNof force will transition into a new conformation. The new structureis roughly 3 times longer and 40% narrower than the original struc-ture. Upon release of the force, the Tfp fiber regains its originalform, indicating a reversible transition. Equally important, we showthat the force-induced conformation exposes hidden epitopespreviously buried in the Tfp fiber. We postulate that this transitionprovides a means for N. gonorrhoeae to maintain attachment to itshost while withstanding intermittent forces encountered in theenvironment. Our findings demonstrate the need to reassess ourunderstanding of Tfp dynamics and functions. They could alsoexplain the structural diversity of other helical polymers while pre-sentingauniquemechanism for polymer elongation andexemplify-ing the extreme structural plasticity of biological polymers.

force polymorphism ∣ alternate immunogenic properties

Helical protein filaments are found throughout nature (e.g.,filamentous actin, microtubules, flagella, and pili) and serve

a multitude of functions. Structural modifications in the proteinmonomer can lead to changes at the macromolecular level, influ-encing the assembly, disassembly, and function of these filaments(1, 2). Whereas studies on force-induced secondary and tertiarystructural changes are well documented (3–5), quaternary struc-tural changes on well-defined macromolecular assemblies havereceived less attention (6–8). The full structural diversity ofhelical protein filaments has proven difficult to assess becausethe variety of their molecular arrangements have often eludedobservation and exceeded expectations (9, 10). A large numberof these polymers have mechanical and structural roles in biologyand thus may be subject to physical stress. Structural changeslinked to mechanical stress have mainly been studied in bacterialflagella and P-pili, but such changes may be more common thanpreviously thought (11–14).

Type IV pili (Tfp) are retractable helical filamentous appen-dages found in many bacteria, including human pathogens(15, 16). Despite recent findings regarding the Neisseria gonor-rhoeae Tfp quaternary structure (16, 17), it remains difficult toexplain the wide spectrum of functions associated with Tfp(18), including: twitching motility (19), DNA uptake (20), humancell infectivity (21, 22), and immunogenic properties (23).Because the Tfp retraction motor is one of the strongest mole-cular motor known to date (24) and certain pilin monomersare thought to be affected by force (25), we hypothesized that

force could extend the repertoire of Tfp structures and functions.Here we use the N. gonorrhoeae Tfp for exploring force-inducedstructural changes in helical filaments.

Results and DiscussionN. gonorrhoea Tfp Undergoes Reversible Force-Induced Polymorph-ism. We have previously shown, by using optical tweezers beadassays, that a single N. gonorrhoeae Tfp can sustain forces inthe range of 100 pN (24). A typical Tfp retraction event consistsof a transient tensile force (lasting up to a few seconds) with asubsequent and rapid release of force (24, 26). This abruptrelease of force has been interpreted as a breakage event (24),a severing of the connection between the Tfp and the bead inthe laser trap. Closer examination of recordings from those ex-periments (19) revealed bead return speeds too slow to be com-patible with a free release/breakage event. (A small back-of-the-envelope calculation of a free release in the optical tweezers leadsto a speed of at least 10;000 μm∕s to compare with the speed ofaround 5 μm∕s measured.) Rather, it suggested the persistence ofa Tfp tether between the bacterium and the force apparatus (19).On the other hand, it is interesting to note that the speed of theseelongations (5 μm∕s) is 5–10 times greater than the Tfp elonga-tion caused by polymerization previously recorded (0.5–1 μm∕s)(27, 28).

We hypothesized that the force release profile is the signatureof a structural change in the Tfp filament itself. We thereforeexplored the effect of force on purified N. gonorrhoeae Tfpfilaments to assure that we would not measure the propertiesof the attachment of the Tfp to the bacterial wall or be hinderedby the elongation and retraction cycles of the Tfp. In our initialexperiments, a Tfp was tethered between a silica bead and anelastic hydrogel pillar (26). Force was applied to the Tfp by usingoptical tweezers to pull on the bead (Fig. 1A and Movie S1). Lowforces (typically 10–20 pN) applied to the bead were transmittedthrough the Tfp to the pillar, causing a deflection of the elasticpillar. Higher forces (typically around 50 pN) applied forextended periods of time resulted in a sudden release of force,returning the pillar to its resting state and increasing the distancebetween the bead and the pillar (Fig. 1 B and C). After the pillarreturned to its resting state, we progressively increased thedistance of the bead from the pillar. We observed a second dis-placement event that confirmed the persistence of the Tfp tetherand verified that the loss of force was not caused by a breakageevent. Importantly, the Tfp tether was now longer than theoriginal unperturbed fiber (Fig. 1C). Application of force on

Author contributions: N.B., M.S., and M.P.S. designed research; N.B. performed research;N.B., D.L.H., J.B., M.S., andM.P.S. contributed new reagents/analytic tools; N.B., D.L.H., andJ.B. analyzed data; and N.B., D.L.H., J.B., M.S., and M.P.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: nb2200@columbia.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0911328107/-/DCSupplemental.

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the transitioned Tfp during the second displacement led to anincrease in the bead/pillar distance that suggests elastic propertiesof the transitioned Tfp.

Next, the force that had been applied to the bead/Tfp wasremoved for over 5 min and then reapplied. Upon reapplicationof small forces, the initial distance between the bead and thepillar was restored. With increased forces, we again detectedthe extension of the Tfp, demonstrating the reversibility of thisstructural transition (Movie S1). To visualize the structuralchange in Tfp during the transition, we conducted similar experi-ments with Tfp stained with carboxytetramethylrhodamine(TAMRA) succinimidyl ester (see Materials and Methods fordetails). By using magnetic tweezers (29), we applied forces toa magnetic bead attached to a labeled Tfp filament linkingtwo elastic pillars in tandem (Fig. 1D and Movie S2). In thisexperimental setup, one pillar was in contact with the magneticbead and the second pillar to part of the Tfp attached to the bead.This configuration enabled us to follow the fluorescence of Tfpwithout being hindered by the effects of Brownian motion. Con-tacts between the bead, the pillars, and the Tfp were confirmed byapplying tensile forces to the bead and monitoring for the deflec-tion of both pillars. Lower forces (typically 20–30 pN) thusresulted in the displacement of both pillars whereas the Tfp fluor-escent signal remained constant. Upon application of higherforces (around 100 pN), the pillar attached to the Tfp returnedto its resting state with a concomitant decline in Tfp fluorescence(Fig. 1E and Movie S2). This decrease in fluorescence was con-sistent with an increase in Tfp fiber length. In addition, the longerstretch-transitioned Tfp became subject to Brownian motion. Aswith unlabeled Tfp, this transition was reversible: Relaxation ofthe force for a few minutes led to the restoration of both theinitial fluorescence and the mechanical contact between thetwo pillars (Movie S2). Finally, the entire process could be re-peated by reapplying high forces to the Tfp. These results confirmthe existence of a previously undescribed, force-induced quatern-ary structure that can be adopted by N. gonorrhoeae Tfp.

Atomic Force Microscopy (AFM) Characterization of the Force-InducedTfp Transition. We next characterized this force-induced Tfp tran-sition with greater temporal and spatial resolution by using AFMin the force spectroscopy mode (Fig. 2A) (5). Tfp were subjectedto force-extension studies with the AFM while operating at phy-siologically relevant speeds (∼1 μm∕s) (24). Force-extension ex-periments on single polyproteins have shown well-defined forcepeaks associated with the strength of the mechanical transitionstate and precise spacing in length because of the unravelingof the amino acids into a linear polypeptide chain for each mono-mer. This unraveling has previously been modeled by the worm-like chain model, which assumes an entropic elasticity. In the caseof stretching of Tfp, we observed a qualitatively different forceresponse potentially because of the supramolecular structuralorganization within each Tfp fiber. Namely, we identified longextensions with no measurable force (below the 10-pN resolutionof our apparatus) and a sudden nonlinear increase in the forcefollowed by a linear force response from ∼20 pN up to a well-defined transition at 100� 20 pN (Fig. 2 B and C). Beyond thistransition, the force on the Tfp relaxed to zero (or at least belowthe 10-pN resolution of ourAFM)and theTfp continued to extend(Fig. 2B, traces 1–4). In rarer cases, further transitions occurredafter the first one (Fig. 2B, trace 5). This pattern is in agreementwith our optical and magnetic tweezers experiments in which wepredominantly observe a single peak of force with little or no forcefollowing the transition. This characteristic force profile is compa-tible with a sudden transition to a longer configuration of the Tfp.We tested the reversibility of the transition by pulling multipletimes on the same Tfp. We first confirmed that we had pickedup a Tfp: The cantilever was moved away from the surface untilone or more peaks were detected (the cantilever position wastypically set between 0.5 and 1 μm away from the surface). Fromthis initial point, the Tfp was further extended and force peakswere recorded (Fig. 2D, trace 1). The cantilever was then returnedto its initial position. After 3 min, the Tfp was again extended.The presence of peaks in the subsequent force measurementconfirmed the reversible nature of the conformational change

Fig. 1. N. gonorrhoea Tfp undergoes reversible force-induced polymorphism. (A) Schematic of the optical tweezers experimental design. (B) Movie framesillustrating the position of the elastic pillar and silica bead before and after pilus transition. (Scale bar: 1 μm.) (C) Time course of the distance between the centerof the bead and the center of the pillar and of the deflection of the pillar (time 0 ¼ time of the Tfp transition). The displacement of the pillar represents aforce of 45 pN. (D) Schematic of the magnetic tweezers experimental design. (E) Successive fluorescent images of a fluorescently labeled Tfp before transition(Left), in transition (Center), and after recovery (Right). (Scale bar: 1 μm.)

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(Fig. 2D, trace 2). Interestingly, extension away from the surface of“picked-up” Tfp led to more frequent observation of multiplepeaks. These results suggest that the transition need not invokethe entire Tfp but can consist of multiple transitions of regionswithin the Tfp.

The AFM data presented here are similar to previous experi-ments performed on Pseudomonas aeruginosa Tfp (30). In thoseexperiments, the Tfp tested were not purified and were stillattached to the bacterium body leading to the hypothesis thatthe AFM data could be caused by a component of the bacterialwall. Nevertheless, the AFM force traces in that study showedpredominantly a single peak of force, with rarer instances ofmultiple peaks in one trace, nearly identical to our findings withpurified N. gonorrhoeae Tfp. These AFM data could not beexplained with the known elastic properties of P. aeruginosaTfp. A force-induced transition model of P. aeruginosa Tfp wouldprovide an explanation for this apparent paradox and hint at acommon nanomechanical behavior of Tfp across species. A forcetransition, which exhibits linear elasticity, could be assigned to a“nanospring” behavior, analogous to that reported in α-helicalmacrostructures of ankyrin repeats (31). This behavior is thoughtto originate from the stacking energy between these repeats, and,in the case of Tfp, it may originate from the stacking energy of theα-helices in the center of the fiber (17).

Beyond the existence of a force-induced transition betweentwo structures of the Tfp, some particular features of the transi-tion are of special interest. The ability of a single Tfp to undergomultiple transitions makes the Tfp a very good candidate modelfor the design of nanothreads with great adhesive strength (32). Italso indicates that the Tfp fiber is able to buffer force fluctuationsthrough changes in its length, potentially enabling N. gonorrhoeaeto remain attached to its host in the presence of high transienthydrodynamic forces (such as those originating from urinationor mucous flow) that would otherwise break those attachments.In this context, it is interesting to note that the stall force of theTfp retraction motor is about 100 pN (24). The consistent natureof our observations on the transitional forces of Tfp along with

the previously reported stall forces of the Tfp retractile motorseems to exemplify an adaptation or coevolution between theN. gonorrhoeae Tfp motor and the physical properties of theN. gonorrhoeae Tfp filament. This coevolution is reminiscent ofthe optimization of the mechanical properties of Escherichia colitype I fimbriae in order to maintain adhesion under differentfluid flow conditions (8).

Extended Tfp Conformation Reveals Hidden Epitopes. To furthercharacterize the structural change of the Tfp, given the transientnature of the force-induced Tfp transition, we next locked the Tfpfilament in its “stretched” or extended state. By using a modifiedversion of molecular combing (33) (Fig. 3A and Movie S3), wewere able to apply surface tension forces on Tfp. An estimateof the force thus applied on Tfp is available (33) (∼1 nN), andit is a force sufficient to cause the Tfp polymorphic transition.Indeed, after the action of the drying meniscus, the Tfp wereirreversibly fixed on the cover glass,most of them in their stretchedconfiguration. We determined whether the stretched Tfp fiberwas recognized by the SM1 monoclonal antibody. The SM1mAb recognizes the conserved epitope EYYLN in the pilinmono-mer. In the native Tfp fiber, the SM1 EYYLN epitope was pre-dicted to be buried (17). We found that the SM1 mAb boundto only the exposed ends of unstretched Tfp filaments, as reported(34) (Fig. 3 B–D). In contrast, the SM1 mAb bound the length ofstretched Tfp (Fig. 3 B–D). Thus, force-induced stretch exposedresidues in Tfp that were hidden in the unstretched form.

To estimate the fractional length change upon stretch, Tfpwere stained with the SM1 mAb in combination with eitherTAMRA or pAB127 (Pan127), a polyclonal antibody that recog-nized native purified Tfp. As predicted, the TAMRA and Pan127signals were observed along the length of the stretched andunstretched Tfp fiber. By using TAMRA or Pan127, we estab-lished a correspondence between fluorescent signals and Tfplengths. Signals per unit of length of stretched and unstretchedTfp fibers revealed that stretched fibers were approximately 3times longer (having 3 times less fluorescence) than unstretched

Fig. 2. AFM characterization of the force-induced Tfp transition. (A) Schematic of the AFM experimental design. (B) Typical example of force-extension curvesshowing the transition occurring at ∼100 pN. Insets are zooms on the force peaks for four curves. (C) Force histogram of the force associated with the Tfptransition event averaging 100� 20 pN (standard deviation, n ¼ 1;210). (D) The reversibility of the force-induced transition is demonstrated by the observationof multiple transition events in two consecutive trajectories separated by a time Δt ¼ 3 min.

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fibers [550� 25 arbitrary units (a.u.) vs. 1;800� 100 a:u: forTAMRA fluorescence signal and 2;500� 100 a:u: vs. 7;300�250 a:u: for Pan127 fluorescence signal; standard errors andn ¼ 60 in all cases; see Fig. S1A and Materials and Methodsfor details]. In any given sample, not all Tfp were stretched;unstretched fibers were used as an internal control (Fig. 3E). Mo-lecular combing was also used to stretch Tfp on EM slot grids.Images of stretched samples also revealed a mixture of extendedand nonextended Tfp (Fig. 4 A and B). The stretched Tfp were

approximately 40% narrower in diameter than unstretched fibers(3.5� 0.7 nm vs. 6.0� 0.7 nm for evaluation “by eye” of the dia-meter and 3.9� 0.6 nm vs. 6.2� 0.5 nm for evaluation via plotprofile; standard deviations are indicated for 100 measurementsover 20 different micrographs in each case; see Fig. S1B andMaterials and Methods for details), indicating that they hadonly about 1∕3 of the mass per length of the unstretched fibers(Fig. 4 A and B). Rare instances of partially transitioned Tfp (thefraction that has not undergone transition corresponds to less

Fig. 3. Extended Tfp conformation reveals hiddenepitopes. (A) A schematic of the molecular combing ex-perimental design. (B) Close-up of fluorescent imagesof stretched or unstretched TAMRA-prestained puri-fied Tfp. The green signal indicates the SM1 epitope;the red signal (TAMRA) labels the exterior of the Tfpfiber. (Scale bar: 1 μm.) (C) Close-up of fluorescentimages of stretched or unstretched purified Tfp. Thegreen signal indicates the SM1 epitope; the red signal(Pan127 antibody) labels the exterior of the Tfp fiber.(Scale bar: 1 μm.) (D) Fluorescent images of TAMRA-prestained purified Tfp processed by molecular comb-ing. The red signal is against an exposed region of Tfp(TAMRA), whereas the green signal indicates the SM1epitope. (Scale bar: 5 μm.) (E) Dual fluorescent imagesof the SM1 epitope (Green) and the exposed epitopePan127 (Red) of purified Tfp processed by molecularcombing. (Scale bar: 1 μm.)

Fig. 4. Stretched Tfp are 40% narrower thanunstretched Tfp. (A) Electron micrograph ofpurified Tfp processed by molecular combing.Note the smaller diameter of the stretchedTfp. Direction of stretch indicated by thedouble-headed arrow. (Scale bar: 50 nm.)(B) Electron micrograph of purified Tfp pro-cessed by molecular combing. Note the smal-ler diameter of the stretched Tfp. Direction ofstretch indicated by the double-headed ar-row. (Scale bar: 50 nm.) (C) Plots of averagedintensities across a line drawn perpendicu-larly to the direction of a stretched Tfp atthe level of either the star or the circle inB. Those plots, along with B, present an exam-ple of partially transitioned Tfp.

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than 1% of the total length of observed pili) can be found(Fig. 4 B and C).

ConclusionIn summary, we have established that N. gonorrhoeae Tfp canundergo a reversible force-induced transition into a uniquequaternary structure that is longer and narrower than the restingstate not subjected to force (Fig. S2 and Movie S4). In itsextended form, hidden epitopes in the fiber become exposed.Our findings open an unforeseen chapter for the study of thedynamics of the Tfp structure as well as of its immunogenic prop-erties. We predict that, besides explaining previously observedstructural diversity in the Tfp (35), force-induced structuralchanges ofN, gonorrhoeae Tfp will prove a useful model in under-standing the structural diversity of other helical polymers (36, 37).Finally, we postulate that the exact structural origin of the presenttransition may, in the future, facilitate the design of usefulbiomimetic nanostructures.

Materials and MethodsPili Preparation. Purified Tfp were prepared in a similar manner as previouslypublished (17). One or two plates of WT MS11 N. gonorrhoeae grown for16–20 h on (GonoCoccal Broth) agar plates were resuspended in 1 mL of50 mM CHES [2-(cyclohexylamino)ethanesulfonic acid from Sigma] pH 9.5.The suspension was vortexed for 2 min and the bacteria bodies were spundown at 18;000 × g for 5 min. The supernatant was collected and spun downat 100;000 × g for 1.5 h. The pellet was resuspended in 1 mL 50 mM CHES pH9.5. In the case of prestained Tfp, the bacteria were resuspended in 1 mL oflabelling buffer (50 mM KPO4. pH 8.0∕5 mM MgCl2∕25 μM EDTA), vortexed,and spun down at 18;000 × g for 5 min. About 40 μL of a 10 mg∕mL solutionof TAMRA succinimidyl ester in DMSO was added to the supernatant andallowed to react at room temperature for 1.5 h. The mixture was then spundown at 100;000 × g for 1.5 h. The pellet was washed 5 times with 50 mMCHES pH 9.5 and finally resuspended in 1 mL 50 mM CHES pH 9.5.

Optical Microscopy. All the optical microscopy was performed on conven-tional inverted microscopes (either Olympus IX 81 or Zeiss Axiovert 100).The images were analyzed by using ImageJ software [National Institutesof Health (NIH)]. The measurements of the intensity per unit of length ofthe Tfp were performed by using a home-written plug-in on ImageJ follow-ing the procedure outlined in Fig. S1A: The fluorescence signal over a rectan-gle of set width and length along Tfp fiber was recorded. The fluorescencesignal over a similar rectangle parallel to the first one and away from the Tfpwas also measured to normalize for background noise. The fluorescencesignal per unit of length of the Tfp was the difference between thesetwo measurements. The measure was repeated for both stretched andunstretched Tfp.

Optical Tweezers. The optical tweezers system consisted of a neodymium-doped yttrium aluminum garnet-neodymium (Nd:YAG) laser (2 W) mountedon a Zeiss Axiovert 100. Purified Tfp were put into contact overnight with1.5 μm carboxylated silica beads at a concentration allowing on average asingle Tfp to bind per bead. The beads were then allowed to settle ontoa bed of hydrogel micropillars prepared as presented elsewhere (26). Thecoverslip was then scanned in order to find the following condition: a beadstill agitated by Brownian motion but remaining in the vicinity of a pillar(Fig. 1A). We verified the connection between the bead and the pillar byapplying force on the bead by using optical tweezers.

Magnetic Tweezers. The magnetic tweezers setup has been presented else-where (29) and was mounted on an Olympus IX81 inverted scope. TAMRA

stained purified Tfp were put into contact overnight with 1 μm MyOnecarboxylated magnetic beads (Invitrogen) at a concentration allowing onaverage a single Tfp to bind per bead. The beads were then allowed to settleonto a bed of hydrogel micropillars prepared as presented elsewhere (26).The surface was then scanned for the following configuration: a bead, withone Tfp attached to it, stuck to one pillar with the Tfp attached to a neigh-boring pillar (Fig. 1D). We could apply forces on the magnetic bead by usingthe magnetic tweezers.

AFM. The details of the custom-made AFM have been described elsewhere(5). Each cantilever used in our experiments (Si3N4 Veeco MLCT-AUHW)was individually calibrated by using the equipartition theorem, giving riseto spring constants between ∼20 and ∼100 pNnm−1. We worked at a dilutedconcentration of purified Tfp to prevent multiple Tfp from binding at thesame time. No force peak has been recorded when a Tfp preparation froma nonpiliated strain was used. The force-extension curves were recorded andanalyzed by using IGOR 6.0 software (Wavemetrics).

Molecular Combing. Cleaned cover glasses were incubated in a solution ofpurified Tfp in 50 mM CHES pH 9.5 at a dilution between 1∕1;000 and1∕10;000 from an original purified Tfp solution for 15 min at the bottomof the well of a six-well plate. After incubation, cover glasses were main-tained vertically, with the bottom end placed on a lint-free Kimwipes tissueto remove excess liquid and allow slow drainage by gravity and capillarityaction. The receding meniscus on the cover glass exerted forces on Tfp, caus-ing most of them to be stretched. A number of Tfp remained in their nativestate likely because of a prior close contact with the cover glass, thereforeresisting the combing action. The samples processed by combing werereferred to as “stretched” samples. “Unstretched” samples were obtainedby incubating cover glass with the same diluted solution of Tfp for15 min, followed by three washes with no drying of the cover glass.

Immunostaining. Either stretched or unstretched samples on cover glass werefixed with 3.7% formaldehyde in PBS, blocked with a solution of 0.2% fishgelatin in PBS, and incubated with either a monoclonal antibody against theSM1 domain of pilin or the polyclonal antibody pAb127 (Pan127) generatedagainst purified Tfp, or a combination of both antibodies. Samples incubatedwith the SM1 antibodywere stained with an Alexa 488-conjugated secondaryanti-mouse antibody. Samples incubated with the Pan127 antibody werestained with an Alexa 568-conjugated secondary anti-rabbit antibody.

EM. A 1 × 2 mm formvar/carbon coated slot grid (Electron MicroscopySciences) was allowed to float on top of 10 μL of a solution of purifiedTfp for 5 min. The grid was then held vertically with tweezers and drainedfrom the bottom end with a lint-free Kimwipes tissue to remove excess liquidand allow slow drainage by gravity and capillary action. The grid was thenfloated on top of a 10-μL droplet of a solution of 3.7% formaldehyde in PBSto fix the sample. The grid was then floated on top of a 10-μL droplet of a 2%solution of uranyl acetate for negative staining. The excess stain solution wasremoved and the sample imaged by using a JEOL transmission electronmicroscope. The images were analyzed by using ImageJ software (NIH).The diameter of the Tfp was either evaluated by eye and measured witha line selection or a plot of averaged intensities across a 50-pixel-wide lineperpendicular to the direction of the fiber was created and the diameterwas the width at midheight of the resulting peak.

ACKNOWLEDGMENTS. The authors thank Simon Moore for critical reading ofthe manuscript and the rest of the Sheetz lab members for their technicalsupport. N.B. and M.S. acknowledge the award of NIH Grant AI079030.J.B. holds a Career Award at the Scientific Interface from the BurroughsWellcome Fund.

1. Mitchison T, Kirschner M (1984) Dynamic instability of microtubule growth. Nature312(5991):237–242.

2. Oda T, Iwasa M, Aihara T, Maeda Y, Narita A (2009) The nature of the globular- tofibrous-actin transition. Nature 457(7228):441–445.

3. del Rio A, et al. (2009) Stretching single talin rod molecules activates vinculin binding.Science 323(5914):638–641.

4. Zlatanova J, Lindsay SM, Leuba SH (2000) Single molecule force spectroscopy inbiology using the atomic force microscope. Prog Biophys Mol Bio 74(1–2):37–61.

5. Oberhauser AF, Marszalek PE, Erickson HP, Fernandez JM (1998) The molecularelasticity of the extracellular matrix protein tenascin. Nature 393(6681):181–185.

6. Miller E, Garcia T, Hultgren S, Oberhauser AF (2006) The mechanical propertiesof E. coli type 1 pili measured by atomic force microscopy techniques. Biophys J91(10):3848–3856.

7. Andersson M, Axner O, Almqvist F, Uhlin BE, Fällman E (2008) Physical propertiesof biopolymers assessed by optical tweezers: Analysis of folding and refolding ofbacterial pili. ChemPhysChem 9(2):221–235.

8. Forero M, Yakovenko O, Sokurenko EV, Thomas WE, Vogel V (2006) Uncoilingmechanics of Escherichia coli type I fimbriae are optimized for catch bonds. PLoS Biol4(9):e298.

9. Galkin VE, et al. (2008) Divergence of quaternary structures among bacterial flagellarfilaments. Science 320(5874):382–385.

10. Kueh HY, Mitchison TJ (2009) Structural plasticity in actin and tubulin polymerdynamics. Science 325(5943):960–963.

11. Esther Bullit, Makowski L (1995) Structural polymorphism of bacterial adhesion pili.Nature 373:164–167.

11362 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0911328107 Biais et al.

Dow

nloa

ded

by g

uest

on

Janu

ary

5, 2

020

12. Hotani H (1982) Micro-video study of moving bacterial flagellar filaments: III Cyclictransformation induced by mechanical force. J Mol Biol 156(4):791–806.

13. Brown AEX, Litvinov RI, Discher DE, Purohit PK, Weisel JW (2009) Multiscale mechanicsof fibrin polymer: Gel stretching with protein unfolding and loss of water. Science325(5941):741–744.

14. DeRosier DJ, Tilney LG, Bonder EM, Frankl P (1982) A change in twist of actin providesthe force for the extension of the acrosomal process in Limulus sperm: The false-discharge reaction. J Cell Biol 93(2):324–337.

15. Mattick JS (2002) Type IV pili and twitching motility. Annu Rev Microbiol 56(1):289–314.

16. Hansen JK, Forest KT (2006) Type IV pilin structures: Insights on shared architecture,fiber assembly, receptor binding and type II secretion. J Mol Microbiol Biotechnol11(3–5):192–207.

17. Craig L, et al. (2006) Type IV pilus structure by cryo-electron microscopy and crystal-lography: Implications for pilus assembly and functions. Mol Cell 23(5):651–662.

18. Craig L, Li J (2008) Type IV pili: Paradoxes in form and function. Curr Opin Struct Biol18(2):267–277.

19. Merz AJ, So M, Sheetz MP (2000) Pilus retraction powers bacterial twitching motility.Nature 407(6800):98–102.

20. Chen I, Christie PJ, Dubnau D (2005) The ins and outs of DNA transfer in bacteria.Science 310(5753):1456–1460.

21. Merz AJ, So M (2000) Interactions of pathogenic Neisseriae with epithelial cellmembranes. Annu Revi Cell Dev Biol 16(1):423–457.

22. Howie HL, Glogauer M, So M (2005) The N. gonorrhoeae type IV pilus stimulatesmechanosensitive pathways and cytoprotection through a pilT-dependent mechan-ism. PLoS Biol 3(4):e100.

23. Tramont EC, Boslego JW (1985) Pilus vaccines. Vaccine 3(1):3–10.24. Maier B, Potter L, So M, Seifert HS, Sheetz MP (2002) Single pilus motor forces exceed

100 pN. Proc Natl Acad Sci USA 99(25):16012–16017.

25. Helaine S, Deyer DH, Nassif X, Pelicic V, Forest KT (2007) 3D structure/function analysisof PilX reveals how minor pilins can modulate the virulence properties of type IV pili.Proc Natl Acad Sci USA 104(40):15888–15893.

26. Biais N, Ladoux B, Higashi D, So M, Sheetz M (2008) Cooperative retraction of bundledtype IV pili enables nanonewton force generation. PLoS Biol 6(4):e87.

27. Maier B, KoomeyM, Sheetz MP (2004) A force-dependent switch reverses type IV pilusretraction. Proc Natl Acad Sci USA 101(30):10961–10966.

28. Clausen M, Koomey M, Maier B (2009) Dynamics of type IV pili is controlled byswitching between multiple states. Biophys J 96(3):1169–1177.

29. Tanase M, Biais N, Sheetz M, YuLi Wang, Dennis ED (2007) Magnetic tweezers in cellbiology. Methods in Cell Biology, (Academic, New York), Vol 83, pp 473–493.

30. Touhami A, Jericho MH, Boyd JM, Beveridge TJ (2006) Nanoscale characterizationand determination of adhesion forces of Pseudomonas aeruginosa pili by using atomicforce microscopy. J Bacteriol 188(2):370–377.

31. Lee G, et al. (2006) Nanospring behaviour of ankyrin repeats. Nature 440(7081):246–249.

32. Smith BL, et al. (1999) Molecular mechanistic origin of the toughness of naturaladhesives, fibres and composites. Nature 399(6738):761–763.

33. Bensimon A, et al. (1994) Alignment and sensitive detection of DNA by a movinginterface. Science 265(5181):2096–2098.

34. Forest KT, et al. (1996) Assembly and antigenicity of the Neisseria gonorrhoeae pilusmapped with antibodies. Infect Immun 64(2):644–652.

35. Swanson J, Kraus SJ, Gotschlich EC (1971) Studies on gonococcus infection: I. Piliand zones of adhesion: Their relation to gonococcal growth patterns. J Exp Med134(4):886–906.

36. Wang YA, Yu X, Silverman PM, Harris RL, Egelman EH (2009) The structure of F-pili.J Mol Biol 385(1):22–29.

37. Wang YA, Yu X, Ng SYM, Jarrell KF, Egelman EH (2008) The structure of an archaealpilus. J Mol Biol 381(2):456–466.

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