Microfluidics pushes forward microscopy analysis of actin dynamics.

2011·1 MB·English

by  Jegou, Antoine

PERSPECTIVE
BioArchitecture1:6,271–276;November/December2011;G2011LandesBioscience
Microfluidics pushes forward microscopy analysis of actin dynamics
Antoine Jégou, Marie-France Carlier* and Guillaume Romet-Lemonne
CytoskeletonDynamicsandMotilityGroup;Laboratoired’EnzymologieetBiochimieStructurales;CentredeRecherchedeGif;CNRS;Gif-sur-Yvette,France
Actin filaments, an essential part of and briefly outline promising develop-
the cytoskeleton, drive various cell ments of this technique.
processes, during which they elongate,
disassemble and form different architec-
tures.Overthepast30years,thestudyof Power and Limitations of Bulk
actin dynamics has relied mainly on bulk Solution Studies of Actin
© 2012 Lasolutinon medasuremeents,swhic hBrevealeid osAcssemibley Dynnamiccs e.
thekineticsandthermodynamicsofactin
self-assemblyatbarbedandpointedends, Since 1981, the change in fluorescence
its control by ATP hydrolysis and its of pyrenyl-labeled actin,1 and to a lesser
regulation by proteins binding either extent of NBD-labeled actin,2 has proven
monomeric actin or filament ends and instrumental in the quantitative analysis
sides. These measurements provide ofactinself-assemblyparametersatbarbed
Do not distribute.
quantitative information on the averaged and pointed ends. The size of the nucleus
behavior of a homogeneous population (a trimer) was derived from theanalysis of
of filaments. They have been comple- spontaneousassemblycurves;3-5theassem-
mented by light microscopy observations bly and disassembly rate parameters at
of stabilized individual filaments, pro- barbed and pointed ends were derived
viding information inaccessible using from seeded assembly assays using spec-
averaging methods, such as mechanical trin-actin seeds and gelsolin-actin seeds,
properties or length distributions. In the and dilution-induced depolymerization
past ten years, the improvement of light assays. These methods were powerful, in
microscopy techniques has allowed bio- addition to standard sedimentation and
physicists to monitor the dynamics of other biochemical assays, to quantitatively
individual actin filaments, thus giving characterize the activities of G-actin
access to the length fluctuations of sequesterers and of filament capping,
filaments or the mechanism of processive severing, stabilizing or destabilizing fac-
assembly by formins. Recently, in order tors.6Bulksolutionmeasurementsactually
Keywords:actinassemblydynamics,single
to solve some of the problems linked measure the reactivity of filament ends.
filament, TIRF microscopy, microfluidics
to these observations, such as the need On the other hand, these averaging
to immobilize filaments on a coverslip, methods were blind to the length distri-
Abbreviations: F-actin, filamentous actin;
we have used microfluidics as a tool to bution of filaments. How many nuclei
G-actin, globular (monomeric) actin;
improve the observation, manipulation were formed, and how the number of
TIRFM, Total Internal Reflection
andanalysisofindividualactinfilaments. filaments is affected by fragmentation and
Fluorescence (or evanescent wave)
This microfluidic method allowed us to reannealing reactions was derived from
Microscopy
rapidly switch filaments from polymeriz- kinetic modeling, not directly measured.5
Submitted: 12/28/11 ing to depolymerizing conditions, and Bulk solution studies provide no informa-
derive the molecular mechanism of ATP tion on fluctuations in length and con-
Revised: 01/03/12
hydrolysis on a single filament from formations of filaments in solution, or
Accepted: 01/04/12
the kinetic analysis of its nucleotide- on any heterogeneity in dynamics of the
http://dx.doi.org/10.4161/bioa.1.6.19338 dependent disassembly rate. Here, we filaments that compose the population,
discuss how this work sets the basis for whichcouldresultfrompossiblestructural
*Correspondenceto:Marie-FranceCarlier;
Email:[email protected] future experiments on actin dynamics, changes or cooperative binding of some
www.landesbioscience.com BioArchitecture 271
regulators. Finally, reactions like filament limitations. Single filament techniques, commercially available microfluidics app-
branching appear in bulk solution as the whether performed in TIRFM or epifluo- aratus allow a fast and reliable control of
autocatalytic generation of ends by a rescence microscopy, often rely on the pressures and flow rates in various ranges.
molecular mechanism that can be spe- anchoring of filaments to the microscope It is therefore possible to vary the flows in
cified, but ignoring the branched struc- coverslip via side-binding proteins.13 the flow-cell at will, and in real time.
ture. Bulk solution methods evidently do In this situation, the filaments interact Microfluidics offers many features that
not allow to monitor processive assembly strongly with the surface, and this con- overcome the technical drawbacks of
by formins. Quantifying all the reactions strainthasbeensuspectedtocauseartifacts standard light microscopy. The artifacts
thatregulatefilamentassemblyatthelevel in the observed dynamics.14 In particular, that may arise from the flow of fluid, in
of individual filaments is important since changes in structure of the filament particular the resulting drag force on the
theseprocessesareessentialaspectsoftheir linked to binding of regulators like ADF/ filament, can be circumscribed easily—as
function in vivo. cofilin or tropomyosin, or to filament well as used as a tool in further studies.
branching cannot be considered to occur In the following, we list improvements
Light Microscopy Live Imaging of with the same freedom as in a 3D and resulting achievements brought by
Individual Filaments: New Insights environment. To minimize this problem, this method.
and Limitations of TIRFM the density of anchoring sites can be Because the filaments are aligned par-
reduced, but the filaments are then very allel to the coverslip by the flow, a single
Bulk measurements have often been com- mobile which can make their analysis attachment point at the pointed end
plemented with epifluorescence (or elec- cumbersomeandinaccurate.Thefrequent (spectrin-actin seeds) or at the barbed
©tron) mic2roscop0y techn1iques,2which hLave ause onf medthylcelleulosesto c onBfine thie oend (sgelsolcin-actiineseeds)nis suffcicienteand .
first provided images of individual fila- filamentina2Dgeometry,whileavoiding theconstraintsinfilamentstructurelinked
ments, stabilized by regulatory proteins, anchorage, has an impact on filaments to several side-attachment points are
drugs, or by the presence of unlabeled (e.g., bundling) due to excluded volume avoided.
actin monomers. This has brought interactions and possible charge screening. Protein interactions might be affected
information on the mechanical properties Molecular confinement has non-trivial by the flow, either because of the flow
of the filament in various ATP hydrolysis effects.17 The influence of methylcellulose velocity of the proteins in solution, or
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states and in the presence of various on the interaction of actin filaments with because of the tension applied to the
stabilizing or destabilizing proteins.7-10 regulatory proteins should be considered anchored filaments. To circumvent this
The branched filament structure was with caution. These effects are difficult potential problem and appreciate its
generated by WASP proteins with the to test. extent, control assays were performed.
Arp2/3 complex,11 or their fragmentation Changing the composition of the For example, filament dynamics were
and reannealing were visualized.12 medium in which filaments are observed monitored comparatively in a strong con-
Overthepastdecade,theimprovements is possible using an open flow cell,18 stant flow and at a very low flow rate,
of microscopy techniques, and Total however the often long dead time (typi- except during the acquisition of images
Internal Reflection Microscopy (TIRFM) cally of the order of one minute19) needed (when a strong flow facilitates the imag-
in particular, have enabled the observa- to switch to the new conditions precludes ing of the filaments). We verified that
tion of the dynamics of individual actin kinetic studies. a large range of flow rates, up to a few
filaments in real time.13 It has become As we shall see, a standard fluorescence mm/s on average in the chamber, can be
possible to monitor the elongation of microscope can be fitted out with micro- used without affecting actin assembly/
filaments at their barbed and pointed fluidics to go beyond these limits. disassembly dynamics. Higher flow rates,
ends individually,14 and to verify that the which we have not tested, may affect
methodprovidedassemblyrateparameters Assets of Microfluidics for the protein interactions or filament structure,
identical to those derived from solution Observation of Single Actin and this could be studied using a similar
studies. Filament severing by ADF/cofi- Filaments setup (see below).
lin15 and processive assembly by formin16 An essential feature offered by micro-
are typical examples of novel information The microfluidic setup we have used to fluidics is the possibility to rapidly change
provided by TIRF microscopy. In addi- observe single actin filaments20 is very the medium to which the filaments are
tion, the observation of individual fila- simple and similar to setups used for the exposed while continuously monitoring
ments should also offer the possibility to study of individual DNA polymers.22 them, by switching the flow rate of the
monitor different subpopulations of fila- Actin filaments are grown from spectrin- incoming solutions from different inlets.
ments, for instance gelsolin-capped and actin seeds, which are adsorbed on the Rapid kinetics can thus be performed on
non-capped, a situation similar to what coverslip. The filaments fluctuate away single filaments like in a stopped-flow
takes place in living cells, where different from the coverslip surface when the flow apparatus.Thisflexibilityinthecontrolof
filament structures coexist. rate is low, while they are maintained filament history also allows the creation
Nonetheless,insightderivedfromsingle close to the surface and aligned with the of various assays where specific filament
filament observations suffers from various flow when it is faster (Fig.1). Today’s compositions are achieved. For example,
272 BioArchitecture Volume1Issue6
© 2012 Landes Bioscience.
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Figure1.Schematicprincipleofthemicrofluidicssetupusedforthestudyofindividualactinfilaments.(A)AstructuredPolyDimethylSiloxane(PDMS)
blockandacoverslipareassembledtocreateaflowcell,withtypically2or3inletsandoneoutlet.Eachinletisconnectedtoareservoir(notshown)in
whichthepressureisregulatedinordertocontroltheflowinthecorrespondingchannel.Thechamberheightisinthe10–100mmrange.(B)Theentry
channelsmergeintoonechannel.Filamentsinafieldlocatedafewmillimetersdownstreamofthejunctionareexposedtotheincomingsolutionwith
thestrongestflow.Theactinfilamentsaregrownfromspectrin-actinseedsanchoredtothecoverslipsurface.Iftheflowrateishighenough,the
filamentsalignwiththeflow,andremaininthevicinityofthecoverslip.(C)TIRFMimagesoftwodifferentactinfilaments,roughly10mmlong,exposed
toamoderateorastrongflowofsolutionintheflowcell.Ineachseries,thetimeintervalbetweenconsecutiveimagesis1sec.
an artificial solid ADP-Pi-actin cap can The accumulation of statistically rele- solution measurements provided the same
be built to mimic vectorial Pi release.20 vant data at the individual filament level global slow rate constant of Pi release but
One can also build filaments with is made considerably easier using micro- could not discriminate, due to averaged
an embedded non-fluorescent segment, fluidics because a field of view typically measurements, between a vectorial and a
hereby getting insight into the dynamics contains a hundred parallel filaments, random mechanism.21
of non-labeled actin, which so far could which are exposed to identical conditions The same microfluidics setup was used
not be observed under the microscope.23 andhavethesamehistory.Takentogether, to monitor filament disassembly and
The continuous flow of fresh medium these features contribute to making the analyze in deeper detail the recently
in the flow cell ensures that reactions data accurate, and reliable. reported “dynamic stabilization” of actin
aremonitored atconstantprotein concen- In our recent work,20 the performance filaments.19 In this study, the spatial and
tration (in contrast with uncontrolled of microfluidics-assisted microscopy has temporal resolution provided by micro-
changes in G/F-actin ratio with time in revealed the molecular mechanism of fluidicswascrucialtodeterminethemole-
standard microscopy assays). This impro- inorganic phosphate release in actin fila- cular mechanism responsible for pauses
vement allows quantitative interpretation ments. The kinetics of change in the during the depolymerization of filaments
of the collected data. depolymerizing rate of actin filament andruleoutastabilizingstructuralchange
Thealignmentofthefilamentswiththe barbed-ends, rapidly switched from an of the polymer upon aging.23 How the
flow, giving them a nearly straight con- assembly to a disassembly regime, actually random nature of reactions on individual
tour, makes the measurement of filament reflects the ADP-Pi profile in a growing actin filaments are used, modified, enhan-
length and position of the end straightfor- filament. The data provide clear evidence ced or limited by actin regulatory proteins
ward and accurate. for a random mechanism, while bulk will thus be easily addressed using the
www.landesbioscience.com BioArchitecture 273
microfluidics approach combined with was done for barbed ends. The idea that (with free or bound barbed ends for
fluorescence microscopy. The method thestructure andreactivityofthefilament instance), react to signal-mimicking cues,
opens promising perspectives into the is determined by the protein bound to or to changes in the steady-state of
molecular mechanisms by which the its end, like formin27 or gelsolin,28 can be filament assembly. In other words, biomi-
specificity and efficiency of actin-based easily addressed by analyzing the effect metics should experience new develop-
processes are achieved. of either ADF, tropomyosin, myosin or ments using microfluidics. Alternatively,
Analysis of length fluctuations of fila- other filament side-binding protein on micropatterning can be used to anchor
ments has been proposed to provide actin assembly dynamics when the fila- proteins of interest in specific regions, so
insight into the mechanism of ATP ments are anchored by various end- that growing filaments could interact
cleavage in actin filaments.24 Such mea- binding proteins. with them as they fluctuate in the vicinity
surements have been difficult to achieve Comparison between various effectors of the surface. In that frame of mind, one
so far, and should be facilitated in the can be performed using micropatterned could take advantage of the microfluidic
future by the improved accuracy provided chambers, so as to bind different anchor- flows to orient the filaments (Fig.2A) in
by the microfluidics setup. As we discuss ing proteins in different spatially defined order to control the interaction of grow-
in the following, the present simple setup regions,thusmimickingacellularenviron- ing filaments with different functionalized
can be modified and made more sophisti- ment in which different filament arrays microdomains of the coverslip. Whether
catedtobroadentherangeofapplications, co-exist and turnover in a coordinated filament branching by WASP proteins
in particular addressing the mechanism of fashion. Additionally, the complexity of with Arp2/3 complex occurs via barbed
regulators of actin dynamics. the medium flowing over the filaments end branching or side branching mech-
© 2012 Lacan bne variedd at weill, tosinve stigBate hoiw oanismscancsimiilarley benaddrecssed ein a .
Future Developments the observed filaments, in a defined state straightforward fashion.
of the Technique
The first results obtained by implement-
ing microfluidics in TIRF microscopy to
monitor filament dynamics open a large
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variety of perspectives on issues that can
easily be addressed, by combining the
technologicalrefinementsofmicrofluidics,
that have been developed in other
fields,22,25 with the biochemical tools that
proved instrumental in solution studies
of actin.
For instance, the filaments grown from
spectrin-actin seeds anchored to the glass
coverslip have a free barbed end and a
stabilized pointed end. This configuration
can be used to characterize the kinetic
and thermodynamic aspects of interac-
tion of filament barbed ends, in various
bound nucleotide states, with a large
number of barbed end regulators like
capping protein, formins or barbed end
trackers like VASP. As an example, we
characterized the interaction of ADP-
and ADP-Pi bound barbed ends with
profilin,toquantifyitsimpactonassembly
dynamics.20
In order to study the dynamics of the
Figure2.Usingtheflowtoorientandbendthefilaments.(A)Inaregionclosetothejunctionof
pointed end, one can anchor filaments
theentrychannels,thefilamentswillhavedifferentorientations,dependingonthedominant
from stabilized barbed ends, while their
incomingflow.Thisprovidesanotherpossibilityforthemanipulationofthefilaments.Itcouldbe
pointed ends are free to elongate and usedtoorientthefilamentstowardprotein-coatedpatterns,forexample.Inaddition,thespectrin-
depolymerize. This can be achieved by actinseedshaverandomorientations,andthefilamentsbendneartheseedwhentheyalignwith
anchoring biotinylated gelsolin to a strep- theflow.Thisbendingcanbemodulatedbychangingtheorientationofthefilaments.(B)Afixed
tavidin-coated surface.26 The dynamics of obstacle(e.g.,aPDMSpillar)willdeformtheflowlines.Thiscanalsobeusedtobendactin
filaments,witharadiusofcurvaturethatwillbeclosetotheobstacleradius.
the pointed ends can thus be analyzed as
274 BioArchitecture Volume1Issue6
One limitation of the microfluidics direction of the flow (Fig.2A). When the beyond). The gradient of force along the
setup presented in Figure1 is the time flow is fast, the filaments are bent over a filament, resulting from the flow, can be
taken for the solutions to transit from very small region, and appear straight.20 used as a tool to analyze how proteins
the reservoirs to the flow cell. A serious When the flow rate is lower, or when like ADF or myosin bind more or less
drawback results when the protein solu- polymers are stiffer (e.g., actin bundles, or well (in a gradient) along filaments
tion undergoes chemical reactions after microtubules) the curvature is lower and maintained under a variable tension.
mixing. For example, maintaining a high detectable. Alternatively, fixed obstacles Quantitative correlations could then be
concentration of G-actin in high ionic placed downstream from the filament established between protein binding and
strength polymerizing conditions is ham- anchor would bend the filament as it local tension.
pered by the spontaneous nucleation of follows the deviated flow lines (Fig.2B). Alternatively, a quasi-constant tension
filaments. This limitation could be over- Flow-induced bending of filaments could might be applied along the filament by
come by mixing reagents (G-actin and be used to study the impact of local attaching small beads to the filament in
salts in this case) rapidly enough at the filamentcurvature ontheaffinityofactin- a region downstream the flow. In this
entranceoftheflowcell,ordirectlywithin bindingproteins,oronfilamentsevering31 setup,thedragforceappliedbytheflowto
the flow cell. Different types of rapid and branching.32 the bead largely predominates over the
mixing devices (e.g., using multiple adja- The flowing fluid exerts a friction force variable weak force applied along the
cent flows of solutions to establish a rapid along the contour of the filament. This filament. The ability to perform measure-
mix) have been developed.25 Another force is maximal at the anchoring point, ments for tens of filaments in parallel,
possibility is to establish controlled gradi- decreases progressively along the filament, represents an important advantage of
©ents t o i2nvestig0ate diff1erent 2protein coLn- aand isnnull adt the gerowinsg end . TBhis forcie othe msicrofcluidicis apeproacnh as ocpposeed to .
centrations simultaneously. straightens the filament, and puts it under tweezers techniques in which only one
mechanical tension. In the range of flow filament is manipulated.
Application to Mechanical Studies rates that we have used (up to a few tens
of microliters per minute, for a flow cell Concluding Remarks
We found that over a large range of flow cross-section of 600 (cid:1) 40 microns), this
rates,thefilamentsarealignedclosetothe force is smaller than one pico-Newton Adding microfluidics to fluorescence
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surface with no interference of the flow and does not provoke the detachment of microscopy enhances the control of bio-
with assembly dynamics. On the other thespectrin-actinseedsfromthecoverslip. chemical and mechanical conditions when
hand, the flowing fluid could be used as However, these flow rates are sufficient monitoring the dynamics of individual
a tool to exert a significant mechanical to greatly reduce the lateral fluctuations actin filaments, while eliminating potent-
force on the filaments. Then the method of the filaments. The microfluidics ial sources of artifacts. The resulting
would become instrumental to address approach hereby offers a way to study improved reliability and accuracy, in
important issues regarding the interplay filament fluctuations, which may have space and time, opens broad perspectives
between the mechanical and biochemical physiological relevance, in a way that for future studies in the actin field.
properties of actin filaments, under tensile differs from classical setups used to These perspectives will certainly be
strengths similar to those experienced in manipulate individual filaments by hold- expanded by further developments of
living cells. Recent reports actually point ing then from both ends, such as optical the method, inspired by forthcoming
to a modulation of the structure of the or magnetic tweezers. biological issues.
filament29 and to the regulation of fila- Moresignificantforcescouldbeapplied
ment side-binding by mechanical con- to filaments either by increasing the flow Acknowledgments
straints.30,31 A few examples of possible rate, or by anchoring the filaments a few G.R.L. acknowledges support from the
assays follow. micrometers above the coverslip surface Human Frontier Science Program (grant
When adsorbing on the glass surface, (for example, by growing the filaments RGY0067/2008). M.F.C. acknowledges
spectrin-actinseedsorshortfilamentsused from micro-beadsanchored tothebottom support from ANR-PCV program, the
as seeds orient randomly. In the presence of the flow cell23), where the local flow Ligue Nationale contre le Cancer (équipe
of G-actin in the flow, filaments initially velocity is higher. Actin filaments could labelisée) a EU 241548 FP7 grant and an
grow in the direction imposed by the thus be put under significant mechanical ERCadvancedgrant(ERC2009-249982-
seeds, before bending and aligning in the tension (in the pico-Newton range and Forcefulactin).
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