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Atomic force microscopy – looking atmechanosensors on the cell surface
Jurgen J. Heinisch1,*, Peter N. Lipke2,*, Audrey Beaussart3, Sofiane El Kirat Chatel3, Vincent Dupres3,David Alsteens3 and Yves F. Dufrene3,*1Universitat Osnabruck, Fachbereich Biologie/Chemie, AG Genetik, Barbarastr. 11, 49076 Osnabruck, Germany2Department of Biology, Brooklyn College of the City University of New York, New York, NY 11210, USA3Universite Catholique de Louvain, Institute of Condensed Matter and Nanosciences, Croix du Sud 2/18, 1348 Louvain-la-Neuve, Belgium
*Authors for correspondence ([email protected]; [email protected]; [email protected])
Journal of Cell Science 125, 1–7� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.106005
SummaryLiving cells use cell surface proteins, such as mechanosensors, to constantly sense and respond to their environment. However, the way inwhich these proteins respond to mechanical stimuli and assemble into large complexes remains poorly understood at the molecular level. In
the past years, atomic force microscopy (AFM) has revolutionized the way in which biologists analyze cell surface proteins to molecularresolution. In this Commentary, we discuss how the powerful set of advanced AFM techniques (e.g. live-cell imaging and single-moleculemanipulation) can be integrated with the modern tools of molecular genetics (i.e. protein design) to study the localization and molecular
elasticity of individual mechanosensors on the surface of living cells. Although we emphasize recent studies on cell surface proteins fromyeasts, the techniques described are applicable to surface proteins from virtually all organisms, from bacteria to human cells.
Key words: Atomic force microscopy, AFM, Cell adhesion, Cell surface, Cell imaging, Mechanosensing, Microscopy, Single molecule
IntroductionProteins located on the cell surface have pivotal roles in adhesion,
sensing of the environment, signaling, communication, transport,energy transformation, embryonic and tissue development, tumourmetastasis and microbial infection (Sheetz, 2001; Discher et al.,
2005; Vogel and Sheetz, 2006; Lecuit and Lenne, 2007). Thesecomplex functions rely on the assembly and interactions of specificproteins, including mechanosensors and cell adhesion molecules.
Whereas much is known about the molecular biology of suchsurface proteins, their biophysical properties, such as their
mechanics and clustering in the cell membrane, remain poorlyunderstood at the molecular level.
Cells can react to external cues by engaging signal transduction
pathways, which detect environmental changes at the cell surfaceand trigger the appropriate intracellular responses. Frequently,these signaling pathways are initiated by sensor proteins that span
the plasma membrane and ultimately trigger a change in geneexpression and, consequently, in the cell’s proteome (McIntoshet al., 2009; Rodicio and Heinisch, 2010). Such sensor proteins
either bind specific ligands (e.g. hormones, adhesion molecules,etc.) or detect mechanical forces and related physical stimuli.
Hence, mechanosensing and mechanotransduction, which convertthese mechanical forces into biochemical signals, have key roles inregulating processes such as cell growth, differentiation, cell shape
and cell death (Schwartz, 2009; Vogel and Sheetz, 2006; Brownand Discher, 2009).
Cell adhesion molecules also have key roles in mediating
cellular processes such as cell–cell communication (Dalva et al.,2007), tissue development (Morgan et al., 2007), inflammation(Weber et al., 2007), cancer (Gray-Owen and Blumberg, 2006)
and microbial infection (Verstrepen et al., 2004; Kolter andGreenberg, 2006; Telford et al., 2006; Sokurenko et al., 2008).
There is growing evidence that the force-induced deformation of
such proteins has an important role in modulating their cellularfunction. Prominent examples of such processes are ‘catchbonds’, i.e. bonds between receptors and ligands that are
strengthened by tensile mechanical force (Sokurenko et al.,2008), as well as the force-induced exposure of cryptic peptidesequences, as observed in, for example, fibronectin and integrin(Vogel and Sheetz, 2006; Brown and Discher, 2009).
Atomic force microscopy (AFM) makes it possible to observe,manipulate and explore the cell surface at a molecular resolution,and therefore has produced a wealth of new opportunities in cell
biology, including understanding the nanoscale organization anddynamics of cell membranes and cell walls, measuring cellmechanics and cell adhesion, unraveling the molecular elasticityof cellular proteins and the mechanisms by which they assemble
into nanodomains in the membrane (Muller and Dufrene, 2011;Muller et al., 2009). In this Commentary, we explain the basicprinciples of AFM, discuss current strategies for imaging live
cells and for probing single functional proteins on their surfaces,provide a critical evaluation of the potential and limitations of thetechnique, and survey recent discoveries in cell surface biology
that were driven by AFM. Although animal cell studies will becovered, we will especially focus on breakthroughs that havebeen made in yeast cell research, because this is wheresubstantial progress in single-cell surface protein analysis has
recently been made.
Atomic force microscopyGeneral principles
Instead of using an incident beam to visualize a sample – aswould be the case in classical microscopy – AFM senses thesmall forces (in the piconewton range, ,10212 N) that act on the
ARTICLE SERIES: Imaging Commentary 1
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surface of a sample (Binnig et al., 1986; Muller and Dufrene,
2011; Muller et al., 2009). Images of molecules or cells in buffer
or growth medium are made by scanning a sharp tip over the
sample surface and measuring the interaction force between the
tip and the surface (Fig. 1A). The sample is mounted on a
piezoelectric scanner, which ensures three-dimensional
positioning with high accuracy. While the tip is being scanned
across the sample in the x and y directions, the force between the
tip and specimen can be recorded. The sharp tip is attached to a
soft cantilever, and, as the cantilever bends, its deflection is
detected by movement of a laser beam reflected from the tip.
AFM topographic imaging is widely used in the life sciences, and
has provided high-resolution images of biomolecules, membranes
and cells in buffer at unprecedented resolution (Dufrene, 2008a,
Dufrene, 2008b; Engel and Gaub, 2008; Muller and Dufrene,
2011; Muller et al., 2009).
AFM is also widely used to manipulate and analyze single
biomolecules with a method called single-molecule forcespectroscopy (SMFS) (Hinterdorfer and Dufrene, 2006; Engeland Gaub, 2008; Puchner and Gaub, 2009; Muller et al., 2009;
Dufrene et al., 2011). Here, the tip is brought into proximity ofand retracted from the biological sample, and the cantileverdeflection measures the interaction force (Fig. 1B). The force–distance curves that are obtained with this procedure provide key
insights into the localization, elasticity and binding strength ofsingle molecules. As we will discuss below, the manipulation ofsingle molecules on the surface of living cells often requires
labeling the tip with chemical groups or bioligands using specificprotocols (Fig. 1B).
Imaging living cells with AFM
Soon after its invention, AFM became a valuable tool for imagingcells (Butt et al., 1990; Radmacher et al., 1992). However, AFMimaging of single cells requires their firm attachment to a surface,
which is not always a simple task. A straightforward approach isto exploit the ability of animal cells to spread and adhere to solidsupports (Radmacher et al., 1992). Coating the substrate withadhesion proteins might be used to enhance immobilization, and
this method has made it possible to observe, for example, actinfilament dynamics beneath the plasma membrane of glial cells(Henderson et al., 1992). In some cases, chemical fixation using
cross-linking agents such as glutaraldehyde might be requiredeither to prevent cell damage or detachment from the supportcaused by the scanning tip, or as a means to obtain high-
resolution images (Fig. 2A). Using these different protocols,various cell types have been investigated, includingmacrophages, CV-1 kidney cells, fibroblasts, Madin–Darby
canine kidney (MDCK) cells, platelets and cardiomyocytes(Fig. 2A; Dufrene, 2011; Jena and Horber, 2002).
In recent years, much progress has been made with regards tolive-cell imaging of various microbes (Fig. 2B; Dufrene, 2008b;
Dufrene, 2011). In order to gain reliable high-resolution imagesof microbial cells, sample preparation is of crucial importance.Unlike animal cells, microbes have a well-defined shape and
usually do not spread on surfaces under experimental conditions.As a result, the contact area between a cell and the support is verysmall, which often leads to cell detachment caused by thescanning tip. Therefore, several approaches have been developed
to ensure more stable cell attachment (Dufrene, 2008a). Forinstance, it is possible to attach the cells onto supports thathave been functionalized with either positively charged
macromolecules, like poly-L-lysine or polyethylenimine, orwith molecules containing hydrophobic groups. This methodhas been successfully applied to lactic acid and Gram-negative
bacteria, diatoms and fungi, and has yielded new insights intosurface structure and elasticity of these organisms. Cells can alsobe immobilized mechanically within gelatin-coated supports or
captured in the holes of porous polymer membranes. In the latterapproach, a concentrated cell suspension is driven through aporous membrane with a pore size similar to that of the cells to beinvestigated (Kasas and Ikai, 1995) (Fig. 2B). This procedure is
fairly simple and does not involve a macromolecular support,thus preventing the risk of contamination of the cell surface or theAFM tip.
Although AFM imaging offers key benefits over moreconventional microscopy techniques, newcomers should beaware that the current technology is still limited by a number
A
B
Ni2+-NTA
Fig. 1. Atomic force microscopy. (A) In the imaging mode, a very sharp tip
follows the contours of the cell surface with nanometer resolution. The lipid
bilayer of the plasma membrane is shown, with inserted proteins as yellow
objects. (B) In SMFS, the small interaction force between the AFM tip and
cell surface molecules is measured, while the distance between the tip and cell
is altered. The two examples show a tip labeled with a chemical group
(Ni2+-NTA, left) or a ligand (e.g. an antibody, right) to detect, determine the
location of and manipulate individual cell surface proteins, such as
mechanosensors (shown in magenta and green, respectively).
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of problems that hamper its widespread use in cell biology (Box
1). Nonetheless, AFM can be employed to address a number of
interesting cell biological questions, as we will discuss below.
Single-protein localization and manipulation
Unlike fluorescence microscopy, AFM topographic imaging lacks
biochemical specificity (Table 1). Thus, specific molecules, such
as a given receptor, cannot be unambiguously identified and
located on cell surfaces. This is an important limitation, because
the organization and interactions of the cell surface machinery is
an important parameter for controlling its functions. However, the
use of spatially resolved SMFS with AFM tips that are labeled with
specific chemical groups or ligands now enables the detection and
localization of single functional proteins on cell surfaces
(Hinterdorfer and Dufrene, 2006). The rationale behind this
approach is to use SMFS to record arrays of force curves across the
cell surface with a specifically labeled tip (Fig. 2C), estimate the
specific molecular recognition force value for each force curve and
then display it in terms of pixels, where the pixel brightness
reflects the magnitude of the recorded force (Fig. 2D). The cell
surface recognition map that can be obtained using this approach
enables researchers to understand the distribution of a given
receptor and to determine whether it is clustered or randomly
distributed across the cell surface (Fig. 2D). However, it should be
A B
C E D
Distance
Forc
e
100 nm 200
pN
1 µm 10 µm
Fig. 2. Atomic force microscopy in cell biology. (A,B) Imaging cells. AFM
images of gently fixed macrophages spread on glass (A) and of a single yeast
cell of Candida albicans trapped in a porous polymer membrane (B). Arrows
highlight a common artifact, namely the alteration of the cell surface by the
scanning tip. (C-E) Imaging and manipulating cell surface proteins: single-
molecule force spectroscopy with AFM tips attached to specific antibodies
makes it possible to detect single proteins on the cell surface (C), to map their
distribution (D) and to measure their mechanical response in relation to
function (E). The recognition map in (D) documents the detection of single
cell-adhesion protein (white pixels), and the notion that they are concentrated
into localized nanodomains. The force–distance curves in (E) document
multiple force peaks that reflect the force-induced unfolding of individual
proteins. Shown here are the results for the Candida albicans Als adhesion
proteins, whose unfolding and clustering are believed to have a role in
cell adhesion.
Box 1. Advantages and limitations of AFM incell biology
AFM shows distinct advantages over optical and electron
microscopy techniques for cell surface biology (Table 1), namely
(1) the ability to observe purified membranes and live cells at
nanometer resolution and under physiological conditions (e.g. in
buffer solution at room temperature and atmospheric pressure),
(2) the capability to track structural dynamics and remodeling of
the cell surface in response to environmental stimulants (e.g.
drugs), and (3) the possibility to determine the location of and
force-probe single constituents of the cell surface. Despite these
key benefits, there are still a number of limitations and
technological issues that must be solved before the full potential
of the technique to address cell biological questions can be
exploited (Table 1).
When observing cells using AFM topographic imaging, an
important source of problems is the resolution and interpretation of
the images, which directly depend on the imaging force, tip
geometry and physical properties of the sample (Dufrene, 2008a).
Large forces acting between tip and sample during imaging can
dramatically reduce the image resolution and cause molecular
damage or displacement (Fig. 2A,B). This is particularly true for
soft corrugated mammalian cells, where the tip generally deforms
the cell surface, pushes cellular material along the scanning
direction and easily becomes blind owing to contamination by
loosely bound macromolecules. Therefore, it is essential to control
the force that is applied during scanning (0.1-0.5 nN) by selecting
appropriate buffer conditions. In addition, unlike microbial cells,
which can be readily imaged in their native state (Fig. 2B), animal
cells generally require chemical fixation in order to be imaged by
AFM (Fig. 2A). The image resolution achieved with AFM on
mammalian cells is generally not better than 50-100 nm. One
approach to solve this problem is to use a nanopipette that does
not contact the surface directly, which gives resolutions from live
cells of ,20 nm (Novak et al., 2009).
Another limitation of AFM imaging compared with fluorescence
microscopy is its rather poor temporal resolution (typically
,1 minute per image), which is much slower than the time scale
at which dynamic processes usually occur in cell biology.
However, remarkable advances are being made in developing
high-speed AFM set-ups that can operate in the millisecond
timescale and thus offer new possibilities to explore cellular
dynamics (Shibata, 2010).
A crucial feature of SMFS is the functionalization of the AFM tips
with biomolecules. This can be achieved using essentially two
types of surface chemistries, which are based either on the strong
chemisorption of thiols onto gold surfaces or on the covalent
attachment of silanes or alcohols onto silicon oxide surfaces
(reviewed by Hinterdorfer and Dufrene, 2006). In addition, well-
defined procedures are available to accurately measure the
localization and forces of single proteins on live cells
(Hinterdorfer and Dufrene, 2006), but accurate data collection
and interpretation often remain technically challenging. In this
context, the main issues are those associated with the quality of
the tip and its possible alteration during the course of an
experiment. As with statistical validation, spatially resolved
SMFS should be performed on different locations of the cell
surface using different tips and the measurements should be
repeated using many cells from independent cultures. Finally, an
important drawback of AFM-based SMFS is that it probes cell
surface biomolecules, but is unable to access the interior of living
cells.
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noted that, as is the case for cell imaging, single-protein analysis by
SMFS is still a rather new method that suffers from a number of
drawbacks (Box 1).
SMFS cannot only be used for mapping single protein
molecules on a given cell surface, but can also be employed to
subject cell surface proteins to force in order to study their
mechanical properties (Fig. 2E). Early studies have demonstrated
that proteins can be subjected to controlled forces using this
approach, and have yielded details of protein unfolding pathways
(Rief et al., 1997). Since then, single-molecule AFM, combined
with molecular dynamics simulations and protein engineering,
has greatly enhanced our understanding of the mechanical
behavior of a great variety of proteins (Engel and Gaub, 2008;
Puchner and Gaub, 2009; Li and Cao, 2010; Marszalek and
Dufrene, 2012). Most of these single protein manipulation
experiments have been conducted in vitro, where the system
under study can be tightly controlled. However, this also means
that molecules are removed from the native cellular context that
controls their structural assembly and functional state (Dufrene
et al., 2011). In the following section, we will highlight some
recent breakthrough studies in which SMFS has been
successfully used to unravel the nanomechanics and clustering
of single proteins on the surface of living cells.
Measuring mechanics of single proteinsNanomechanics of membrane sensors
The biophysical mechanisms underlying cellular mechanosensing
are poorly understood at the molecular level. In cell wall integrity
(CWI) sensing and signaling in yeast, five plasma-membrane-
spanning proteins (Wsc1, Wsc2, Wsc3, Mid2 and Mtl1)
presumably detect mechanical forces acting on the cell wall
and membrane, and subsequently trigger an intracellular
signaling cascade, which ultimately results in the expression of
genes encoding either cell wall biosynthetic enzymes or cell wall
proteins (Levin, 2005; Rodicio and Heinisch, 2010). However,
the lack of an appropriate probing technique meant that, until
recently, it remained unclear how these membrane sensors
responded to mechanical force.
SMFS studies with genetically manipulated Wsc1 in live
Saccharomyces cerevisiae have recently unraveled the
mechanical behavior of single sensor molecules in relation to
their function (Dupres et al., 2009; Heinisch et al., 2010a). Central
to this discovery was the use of genetic manipulations to address a
conceptual problem: AFM is a surface-based technique, so how
can it probe protein sensors (such as Wsc1) that are embedded
within the cell wall? The yeast cell wall has a thickness of
,100 nm (Backhaus et al., 2010), whereas the maximal length of
the extracellular part of the Wsc1 sensor is 86 nm, meaning
it does not reach the outermost cell surface. Therefore, the
mechanosensors were artificially elongated to a length of 153 nm
by inserting a stiff serine/threonine-rich region from the Mid2
sensor. Furthermore, the addition of a histidine tag to the N-
terminal end of the protein allowed the proteins to be detected by a
AFM tip modified with Ni2+-nitrilotriacetate (Ni2+-NTA). Force–
extension curves obtained by pulling on the modified Wsc1
sensors revealed a fascinating behavior: the sensors display the
characteristics of a Hookean spring (i.e. their force curve contains
a linear region where force is directly proportional to extension)
Table 1. Comparison of high-resolution techniques for imaging single proteins in cells
Technique
AFMSuper-resolution fluorescence
microscopy (TIRF, PALM, STORM) Electron microscopy (TEM)Topographic imaging SMFS
Resolution <50 nma <20 nm <20–50 nm <1-10 nmLive cell Yes Yes Yes NoTime for sample
preparation10 minutes 1 dayb 1-3 hours 2-5 days
Time for imageacquisition
<5 minutes 25-50 minutes <5 minutes 5-10 minutes
Image processing 5 minutes A few hours Up to 24 hours 30 minutesSimultaneous detection
of different proteinsNo No Yes Yesc
Protein tracking No No Yes NoCost of equipment J150,000-250,000 J250,000-500,000 J<500,000Operational costs Low Moderate Moderate HighAdvantages Localization and force response of single proteins; protein
unfolding; dynamic processes; various environments(temperature, buffer, etc.)
Spatial-temporal resolution; onlybasic genetic manipulationsrequired; high sample throughput;study of live protein interactionsand their quantification
Imaging of the cellultrastructures at very highresolution
Disadvantages Only the cell surface is analyzed; only a single cell at atime; slow temporal resolution; various sources ofartifacts like cell or tip alteration
Frequently a bad signal-to-noiseratio; photobleaching; noinformation on physical propertiesof proteins; tendency offluorescent protein tags tomultimerize (i.e. interactionartifacts)
Fixation artifacts; no accessto dynamics; noinformation on physicalproperties of proteins
Only whole cell analyses are considered, i.e. purified proteins or membranes are not included. TIRF, total internal reflection fluorescence; PALM,photoactivated localization microscopy; STORM, stochastic optical reconstruction microscopy; TEM, transmission electron microscopy.
aIn mammalian cells (a 10-nm resolution can be routinely achieved on microbial cells).bIncluding tip preparation.cIndirect; e.g. gold beads of different sizes.
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(Dupres et al., 2009). These nanospring properties, which contrast
with the properties of most proteins (Rief et al., 1997; Marszalekand Dufrene, 2012), have subsequently been shown to be mediatedby the extracellular serine/threonine-rich region of the sensor
(Dupres et al., 2009). Notably, this technology has also provided atool for determining cell wall thickness in vivo in yeast, becausesensor molecules of different lengths can only be detected oncethey reach the surface (Dupres et al., 2010).
Accordingly, SMFS opens new avenues for investigating howproteins respond to forces in living cells and how mechanosensingevents proceed in vivo, thereby providing new insights into what has
been suggested to be one of the most ancient sensory mechanisms inevolution (Kee and Robinson, 2008). An exciting new developmentin this direction will be the combination of SMFS with fluorescencemicroscopy. Pulling on a sensor from the outside with SMFS should
lead to the recruitment of the corresponding intracellular signalingcomponents to the cytoplasmic tail of the sensor, which cansubsequently be observed by fluorescence microscopy. We also
anticipate that the single protein manipulations described hereshould allow researchers to address the mechanics of a variety ofother membrane sensors that would usually be hidden and not
accessible by AFM per se. For instance, mammalian cell sensors,such as integrins, are usually covered by a polysaccharide andprotein matrix, the glycocalyx, which prevents reliable SMFS
experiments. Elongation and tagging of such proteins shouldprovide a means to solve this issue.
Nanomechanics of cell adhesion proteins
During the past 15 years, SMFS has been widely used to measurethe molecular interactions of cell adhesion molecules, includingselectins, cadherins, integrins, oligosaccharides, proteoglycansand microbial adhesins (see Hinterdorfer and Dufrene, 2006;
Muller and Dufrene, 2011, and references therein). Because theseexperiments were conducted on purified molecules, it is of greatinterest to probe single cell-adhesion proteins directly in living
cells to further understand the interactions and functions ofadhesion molecules in vivo.
Adhesion of the pathogen Candida albicans is mediated by cell-surface glycoproteins referred to as the Als family. Microscopic
assays have revealed that Als-mediated adhesion involves aninitial adhesion step, followed by force-induced proteinconformational changes that result in the formation of strong
amyloid-like bonds that can ‘cement’ the adhering cells together(Dranginis et al., 2007; Lipke et al., 2012). Again, the moleculardetails of this mechanism remained essentially unknown. SMFS
was therefore used to study the adhesive and mechanical propertiesof Als5 in live cells (Alsteens et al., 2009). Pulling on single Als5proteins using a ligand-modified AFM tip has revealedcharacteristic force signatures with multiple well-defined peaks,
with each peak corresponding to the force-induced unfolding of anindividual tandem-repeat domain. AFM measurements alsorevealed that the unfolding probability increases with the number
of repeats and correlates with the level of cell–cell adhesion, whichconfirms that the modular domains have a role in many fungaladhesion molecules. Presumably, the force-induced unfolding of
Als proteins leads to extended conformations in which previouslymasked hydrophobic groups become exposed, thus favoringhydrophobic interactions between opposing cells. These single-
molecule measurements open up new avenues for understandingthe mechanical properties of adhesion molecules in a variety of celltypes. In particular, they should contribute to increasing our
understanding of how force-induced conformational changes in
proteins such as integrins modulate their binding strength (Vogel
and Sheetz, 2006; Brown and Discher, 2009).
Imaging protein clusteringUnderstanding how membrane proteins assemble to form
membrane micro- and nano-domains is another important
question in cell biology. A key example of such clustering is the
formation of adhesion domains (i.e. focal adhesion complexes)
composed of aggregated proteins that mediate the early stage of
adhesion (Vogel and Sheetz, 2006). The small size (,10-500 nm)
and dynamic nature of membrane domains make their direct
visualization in live cells very challenging. However, AFM is
emerging as a valuable tool to map the distribution of single-
proteins on cell surfaces, thereby unraveling the formation and
composition of such protein domains. Prominent examples include
the mapping of the distribution of prostaglandin receptors on live
CHO cells (Kim et al., 2006), the localization of vascular
endothelial cadherin-binding sites (Chtcheglova et al., 2007), and
the measurement of the organization and stiffness of neuron
membrane domains containing glycosylphosphatidylinositol
(GPI)-anchored proteins (Roduit et al., 2008). As discussed
below, functional protein clusters were recently found to form
and increase in size under stress, thereby activating cell signaling
and cell adhesion in yeasts.
Stress-induced clustering of membrane sensors
Fluorescence microscopy has shown that GFP–Wsc1 sensors
form membrane patches, but the structure–function relationships
of such clusters has long remained a mystery (Wilk et al., 2010).
To address this problem, spatially resolved SMFS was used to
map the distribution of single Wsc1 sensors on the surface of live
S. cerevisiae (Heinisch et al., 2010b). Molecular recognition
images were recorded on cells expressing His-tagged elongated
sensors using Ni2+-NTA tips. Many sensor molecules appeared
to form clusters of ,200 nm, which is in the range of the
large patches observed by fluorescence microscopy for
the microdomains containing the marker protein Can1 (MCC)
in S. cerevisiae (Malinsky et al., 2010). Both the surface density
of Wsc1 sensors and their tendency to cluster increases when
cells are stressed by heat or low osmolarity, which suggests that
clustering is a stress response that is intimately connected to
signaling.
An analysis of three different Wsc1 mutants has also indicated
that the Wsc1 cysteine-rich domain has a crucial function in
sensor clustering and signaling. Although mutant proteins display
surface density and spring behaviors that are similar to those of
wild-type Wsc1, they are more evenly distributed over the yeast
cell surface, instead of being clustered. In addition, Wsc1 sensor
clustering mediated by the cysteine-rich domain is dependent on
the formation of disulfide bridges (Dupres et al., 2011). As these
mutant proteins produce non-functional sensors that are unable to
activate the downstream signaling pathway, these findings
demonstrate the importance of the cysteine-rich domain in
sensor clustering and turnover (Heinisch et al., 2010b). The
studies also suggest that in yeast, like in higher eukaryotes, the
function of sensors is coupled to their localized enrichment in
membrane patches that are referred to as ‘sensosomes’. We are
confident that single-protein imaging by SMFS will be useful to
study similar clustering of sensor proteins in mammalian cells,
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especially following the further development of high-speedSMFS mapping.
Force-induced clustering of cell adhesion proteins
A fascinating phenomenon that is portrayed by cell adhesion
domains in mammalian cells is their ability to grow andstrengthen under force (Bershadsky et al., 2006). In Candida
albicans, macroscopic assays have suggested that adhesion-
triggered changes in the conformation of Als5 propagate aroundthe cell surface, thereby forming ordered adhesion domains(Lipke et al., 2012). This mechanism was recently experimentallyconfirmed by SMFS: mechanical force was shown to trigger the
formation of cell adhesion nanodomains in living yeast cells(Alsteens et al., 2010). Pulling on single adhesins was shown toinduce the formation of adhesion domains of 100-500 nm in size,
and these force-induced nano-domains propagate over the entirecell surface. Als5-mediated remodeling is independent of cellularmetabolic activity, because the protein shows the same behavior
in heat-killed cells, which are no longer metabolically active.Remarkably, a single-site mutation in the conserved amyloid-forming sequence of the protein has revealed that amyloid
interactions represent the driving force that underlies Als5clustering (Garcia et al., 2011). Hence, the strength of yeastcell–cell adhesion results from the force-induced amyloid-likeclustering of hundreds of proteins on the cell surface to form
arrays of ordered multimeric binding sites (Lipke et al., 2012).These results highlight the role that functional amyloids can havein cell adhesion, both in lateral clustering (in cis) of many
adhesion proteins to increase binding avidity, and in the potentialto form stable amyloid-type bonds between cells (in trans), amechanism that we expect to be detected in many more
organisms as more cell adhesion systems are examined(Romero et al., 2010).
Interestingly, the above mechanical restructuring is reminiscentof shear-induced unfolding and refolding of proteins during bloodclotting, where force induces the exposure of interaction sites in
the blood glycoprotein von Willebrand factor (VWF), which hasrecently been demonstrated by single-molecule laser tweezerexperiments (Kim et al., 2010). Shear-induced bond strengthening,
called ‘catch bonding’, is also crucial in arresting leukocyte rollingand extravasation (Alon and Dustin, 2007). Clearly, as the SMFStechnique further evolves and matures, it will contribute to the
discovery of similar remarkable phenomena in many other cellsurface proteins.
ConclusionsUnderstanding protein mechanics and protein clustering in vivo is acrucial challenge in cell biology, which, until now, has been
difficult to tackle owing to the lack of high-resolution probingtechniques. This Commentary has highlighted how the combinationof single-molecule AFM with genetic manipulations is a powerful
tool for probing functional receptors and sensors on cell surfaces, ina way that was not possible previously, and how this approachnicely complements other imaging techniques in cell biology
(Table 1). This integrated platform can be applied to measuring themechanical properties of single proteins, and can be used to imagetheir localization, thus revealing whether they are isolated or form
clusters.
Despite the key benefits of AFM, researchers should realizethat the technology is still under development and that there arestill a number of limitations that must be solved in order for the
technique to become more widespread in the cell biology
community (Box 1). Clearly, a detailed understanding of the
principles and limitations of the different AFM modalities is
essential before users start their first experiments. In SMFS,
protocols have been developed for attaching biomolecules to
AFM tips but they still require specific expertise that is usually
not found in cell biology laboratories. In the future, defining
simple standardized protocols for tip functionalization and data
interpretation, and automation of force spectroscopy analyses,
making them readily available to the biology community, will
contribute to make SMFS accessible for cell biologists.
Undoubtedly, the main limitation of both AFM imaging and
spatially resolved SMFS in cell biology is their rather slow
temporal resolution (Box 1). Hopefully, current efforts in
developing high-speed AFM techniques should soon provide
access to millisecond resolutions using live cells. Another
important direction for future research is to expand the range
of potential applications for single molecule analysis, by
combining AFM with advanced light microscopy techniques,
such as stimulated emission depletion microscopy (Hell, 2009).
Such combined imaging platforms would allow researchers to
simultaneously identify, track, observe and force probe the
individual constituents of the cell surface, thereby helping to
answer many unresolved problems in cell biology.
FundingWork in the Y.F.D. group was supported by the National Foundationfor Scientific Research (FNRS); the Foundation for Training inIndustrial and Agricultural Research (FRIA); the Universitecatholique de Louvain (Fonds Speciaux de Recherche); the FederalOffice for Scientific, Technical and Cultural Affairs (InteruniversityPoles of Attraction Programme); and the Research Department of theCommunaute francaise de Belgique (Concerted Research Action). Y.F. D. and D.A. are Senior Research Associate and PostdoctoralResearcher of the FRS-FNRS, respectively. Work in the P. N. L.laboratory was supported by National Institutes of Health [grantnumber SC1 GM083756]. Work on CWI sensors in the lab of J. J. H.is supported by a grant from the Deutsche Forschungsgemeinschaftin the frame of the SFB944. Deposited in PMC for release after 12months.
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