European Journal of Cell Biology 93 (2014) 380–387

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Mini review

Podosomes revealed by advanced bioimaging: What did we learn? Marjolein B.M. Meddens 1 , Koen van den Dries 2 , Alessandra Cambi ∗ Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein Zuid 26-28, 6525 GA Nijmegen, The Netherlands

a r t i c l e

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Article history: Received 23 June 2014 Received in revised form 4 September 2014 Accepted 28 September 2014 Keywords: Podosomes Super-resolution microscopy Cytoskeleton Actin fibers Cell migration

a b s t r a c t Podosomes are micrometer-sized, circular adhesions formed by cells such as osteoclasts, macrophages, dendritic cells, and endothelial cells. Because of their small size and the lack of methods to visualize individual proteins and protein complexes, podosomes have long been considered a simple two-module structure with a protrusive actin core and a surrounding adhesive ring composed of integrins and cytoskeletal adaptor proteins such as vinculin and talin. In the past decade, the applications of fluorescence based techniques that circumvent the diffraction limit of conventional light microscopy took a major leap forward. Podosomes have been imaged by a variety of these super-resolution methods, and in this concise review we discuss how these super-resolution data have increased our understanding of the podosome ultra-structure and function. © 2014 Elsevier GmbH. All rights reserved.

Introduction Podosomes are small (1 ␮m), circular adhesions formed by cells such as osteoclasts, macrophages, dendritic cells (DCs), and under specific conditions by endothelial cells (Linder et al., 2011). In addition, megakaryocytes were also recently shown to form podosomes (Schachtner et al., 2013). They classically consist of a dense actin core (≈0.5 ␮m radius and ≈0.5 ␮m high) surrounded by a ring of integrins and various cytoskeletal adaptor proteins such as talin and vinculin. Podosomes are often organized into large clusters that are associated with an actin network. Although no direct evidence has been presented yet, the abundant presence of Arp2/3 (Linder et al., 2000) and fimbrin (Babb et al., 1997; Evans et al., 2003) suggests that the core actin is organized in highly branched and cross-linked fibers, whereas the localization of myosin IIA in the area surrounding the core (Labernadie et al., 2010; Van den Dries et al., 2013a) suggests an antiparallel arrangement of actin filaments in the network. This organization nicely correlates with the proposed stabilizing, contractile activity of the actin network and the proposed protrusive function of the core. The latter, however, has been widely debated since podosomes, compared to invadopodia, were thought to have a limited protrusive capacity (Linder,

∗ Corresponding author. Tel.: +31 24 3617600; fax: +31 24 3540339. E-mail address: [email protected] (A. Cambi). 1 Current address: Department of Physics and Astronomy and Department of Pathology, University of New Mexico, Albuquerque, NM, United States. 2 Current address: Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, New Haven, CT, United States. http://dx.doi.org/10.1016/j.ejcb.2014.09.002 0171-9335/© 2014 Elsevier GmbH. All rights reserved.

2007). However, recent evidence indicates that this might only be true for podosomes formed by cells adhering to stiff substrates. Studies from our laboratory and others demonstrated that on compliant substrates, podosomes develop into elongated structures that can protrude for several micrometers into the substrate, while mostly maintaining the typical core-ring organization characteristic for podosomes (Gawden-Bone et al., 2010; Baranov et al., 2014). Podosomes are highly dynamic with a lifetime of 2–12 min and continuous turnover of actin (half-life = 30–60 s) within the core. Moreover, they undergo actomyosin dependent stiffness oscillations (Labernadie et al., 2010) that most likely correlate with actin density oscillations (Van den Dries et al., 2013a). Evidently, the structural complexity of podosomes and podosome clusters and their highly dynamic nature provide a continuous challenge to the ever increasing platform of advanced microscopy techniques. In this short review, we will describe and discuss the recent efforts made by our groups and others to generate knowledge about the nanoscale organization of individual podosomes exploiting super-resolution fluorescence microscopy. In addition, we will highlight the structural and dynamic properties of podosomes that motivate the need to image these adhesion structures at high spatiotemporal resolution. Complex multimolecular adhesion structures require advanced bioimaging Proteins and protein complexes such as podosomes are too small to be directly observed within a fixed or living cell. Therefore, many kinds of fluorescent dyes and proteins have been developed that

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can be tagged or targeted to proteins to visualize them in situ, forming the basis for the field of fluorescence microscopy. In the last 30 years, much effort has been devoted to adapt existing fluorescence microscopy techniques or to develop new techniques that have greatly increased our understanding of complex multimolecular assemblies in the cell including adhesion structures such as podosomes. Conventional fluorescence-based methods have revealed many structural components of podosomes, but it has become increasingly clear that the organization of adhesions is regulated at the single molecule level, which urges the need for techniques that are able to study localization and interactions of molecules at higher resolution. Conventional microscopy techniques lack the spatial resolution to visualize single molecules, therefore, much effort has been devoted to the development of techniques that truly push the spatial and temporal resolution limits of light microscopy. Up until only two decades ago, information on localization in situ of multiple proteins was mainly provided by immunofluorescence stainings with antibodies or other labeling reagents. Using correlated fluorescence microscopy with internal reflection microscopy (IRM) to visualize cellular structures that are in close contact with the substrate, many focal adhesion (FA) components, such as vinculin, talin and paxillin, were identified (Burridge and Connell, 1983; Geiger, 1979; Turner et al., 1990) that later have been found in podosomes as well (Tarone et al., 1985; Zambonin Zallone et al., 1989; Duong et al., 1998). In fact, two- and threecolor fluorescence microscopy allowed the identification of novel podosome components and their classification as either core or ring constituents, however insight into the nanoscale structural organization of these adhesion complexes was still lacking. For many years electron based techniques such as scanning and transmission electron microscopy (SEM and TEM, respectively) were the only available imaging methods to study the ultrastructure of FAs and podosomes in fixed cells (Marchisio et al., 1984; Trotter, 1981). By SEM, podosome cores and the associated network were first identified in spreading macrophages (Trotter, 1981). Later, in a series of papers, the group of Marchisio used both SEM and TEM to characterize podosomes in RSV-transformed BHK cells (Gavazzi et al., 1989; Nitsch et al., 1989; Tarone et al., 1985). Initially, using TEM, they showed that podosomes are short cylindrical protrusions that contact the substratum at the tip (Tarone et al., 1985). Next, they further characterized these structures by SEM and TEM, labeling for different podosome components such as vinculin, gelsolin, phosphotyrosine-containing proteins and Src kinase (Gavazzi et al., 1989). Interestingly, this early publication already demonstrates that vinculin is associated with the ends of actin filaments within the network (Gavazzi et al., 1989). Later, TEM studies in transformed chondrocytes by Marchisio indicated the presence of an invagination within the podosome core (Nitsch et al., 1989), something that has later also been directly observed in RSVtransformed fibroblasts and transformed BHK cells (Nermut et al., 1991; Ochoa et al., 2000) but could not be observed in osteoclasts (Akisaka et al., 2008). Interestingly, a recent TEM-based study by Gawden-Bone et al. (2010) using dendritic cells reported about a plasma membrane invagination in the ring of podosomes protruding into a gelatin matrix. Podosomal invaginations might engulf degraded matrix or, in the case of dendritic cells and macrophages, foreign material but their exact frequency of occurrence, function and nanoarchitecture remain to be investigated. Lastly, SEM has proven to be invaluable to study the organization of the actin network since this network cannot be directly observed using conventional fluorescence microscopy techniques. In previous studies, the body of podosome bearing cells was removed while leaving the ventral plasma membrane with podosomes and actin network still intact (Trotter, 1981). This feature was used in osteoclasts to show that podosomes are highly interconnected by numerous actin

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filaments, collectively called the “actin network” (Luxenburg et al., 2007; Akisaka et al., 2008). By myosin S1 fragment labeling, it was shown that the barbed ends of the actin filaments in the network are oriented toward the core (Akisaka et al., 2008). More recently, SEM was exploited to show that Kindlin-3 (Schmidt et al., 2011) and actin polymerization (Luxenburg et al., 2012) are essential for the formation of a proper actin network. In summary, electron microscopy studies have greatly contributed to the general knowledge on podosome organization. However, specific labeling of multiple individual components is limited with SEM and TEM, and both techniques are highly invasive, requiring complex fixation and dehydration procedures, thus being not suited to address the mutual organization of several podosome components simultaneously. Enhancements in spatial resolution have rapidly progressed in the past decade with the advent of super-resolution light microscopy techniques that allowed unprecedented nanoscale (10–90 nm) imaging of many cellular structures (Schermelleh et al., 2010). Several nanoscopy techniques have been developed to circumvent the diffraction limit of light such as stimulated emission depletion (STED) (Hell and Wichmann, 1994), near-field scanning optical microscopy (NSOM) (Van Zanten et al., 2010) and the family of single fluorophore localization microscopy which includes photoactivated localization microscopy (PALM) (Betzig et al., 2006; Hess et al., 2006), stochastic optical reconstruction microscopy (STORM) (Rust et al., 2006) and ground state depletion microcopy (GSDIM) (Fölling et al., 2008). Although less powerful than the approaches described so far, structured illumination microscopy (SIM) also belongs to the family of super-resolution techniques providing a 2-fold increase in lateral and axial resolution with respect to diffraction limited microscopy (Gustafsson, 2000). Some of these methods, discussed in several recent reviews (Schermelleh et al., 2010; Lippincott-Schwartz, 2011; Lidke and Lidke, 2012; Heintzmann and Ficz, 2013) and schematically summarized in Table 1, have been successfully applied to study podosomes (Fig. 1). PALM and STORM exploit photoactivatable (PA) and photoswitchable (PS) fluorescent proteins and dyes. By sequentially activating a selected subset of PA or PS fluorophores during image acquisition, subsequently localizing these fluorophores and mapping their localizations to reconstruct a single image, PALM and STORM can theoretically achieve a spatial resolution of 10–20 nm (Table 1). These type of approaches have greatly enhanced our understanding of the nanoarchitecture of adhesions as shown by Shroff and colleagues in 2007, who used two color PALM to determine the spatial relationship between various focal adhesion components revealing substantial segregation of these molecules with respect to each other within individual adhesions (Shroff et al., 2007). More recently, single particle tracking PALM (sptPALM) has been exploited to investigate the dynamics of different fibronectinbinding integrins within individual focal adhesion (Rossier et al., 2012). This elegant study demonstrated for the first time that the powerful combination of super-resolution microscopy with single molecule tracking revealed how the life cycle of focal adhesions is controlled by the fast remodeling of an integrin mosaic with which intracellular scaffold proteins such as talin interact (Rossier et al., 2012). Below we will highlight the recent studies that used advanced fluorescence microscopy to gain novel insights into the nanoscale organization of podosomes.

Application of super-resolution microscopy to study podosomes Recently, we used direct Stochastic Optical Reconstruction Microscopy (dSTORM) to shed light on the nano-architecture

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Table 1 Super-resolution methods applied to study podosomes. Method

Brief description

Limitations

Advantages

What has it revealed?

Electron microscopy (Bozzola and Russell, 1999)

An electron beam is used to illuminate a sample and transmitted (TEM) or scattered (SEM) electrons or other signals generated upon the interaction of the beam with the sample can be captured to produce an image

Harsh fixation Extensive sample preparation Limited protein specificity

Widely available Very high spatial resolution due to very small wavelength of electrons Sections can be made vertically to obtain very high axial resolution

Single fluorophore localization microscopy (Betzig et al., 2006; Hess et al., 2009; Rust et al., 2006) 3D localization microscopy (Huang et al., 2008; Juette et al., 2008; Shtengel et al., 2009; York et al., 2011) SIM (Gustafsson, 2000; Gustafsson et al., 2008; Kner et al., 2009)

Localization of individual molecules with high precision to build a high resolution image. Variants include PALM, STORM and GSDIM. Determine z-position of fluorophore by PSF modeling or interference based methods

Slow Limited axial resolution

High lateral resolution (10–50 nm)

Actin filament arrangement in core and network (Gavazzi et al., 1989; Nitsch et al., 1989; Tarone et al., 1985) Structure of podosomes in 3D environments (Carman et al., 2007; Cougoule et al., 2012; Gawden-Bone et al., 2010; Van Goethem et al., 2013) Nano-islets of vinculin, talin and integrins in the podosome ring (Van den Dries et al., 2013b)

Slow

High lateral and axial resolution (10–50 nm)

Height differences among podosomes (this review)

Specific microscope setup required

Also increased z-resolution

Confirm actin network and vinculin localization (this review)

Computationally intensive Artifacts might be difficult to predict and interpret

Conventional fluorophores Conventional microscopes Fast, live cell imaging possible

Talin and vinculin ring is composed of short strands surrounding the core (Cox et al., 2012; Walde et al., 2014)

3B (Cox et al., 2012)

Moire fringes caused by gridded illumination allow imaging of up to two times higher spatial frequencies, effectively doubling resolution Determine most likely positions of fluorophores from conventional image series using Bayesian statistics

of podosomes (Fig. 1). We showed that the standard model of podosomes as structures with a well-defined core and ring is in fact too simplified (Van den Dries et al., 2013b). The ring only appears as a ring in conventional and confocal microscopy due to diffraction limitations, whereas super-resolution microscopy now reveals a heterogeneous architecture of the ring. Our dSTORM observations in fact indicate that the adhesive apparatus of podosomes rather consists of homogeneously distributed islets of talin-bound integrins (Van den Dries et al., 2013b). Furthermore, using dual color dSTORM, we were able to reveal that vinculin molecules specifically localize in close vicinity to the core or colocalize with the actin filaments radiating from each podosome core (Van den Dries et al., 2013b). Although a difference in distribution between talin and vinculin has be shown using conventional microscopy combined with automated image analysis (Meddens et al., 2013), observing the distribution of these proteins in small islets requires super-resolution methods. The distribution of the integrins and cytoskeletal adaptor proteins within these small islets implies a more dynamic arrangement of these molecules than was suggested previously with the

core-ring model. Although the link between the integrins islets and the podosome core still needs to be elucidated, the small islets can be expected to readily adapt to small changes in the environment and coordinate the spatial arrangement of the podosomes within the cluster. Importantly, our model also implies that each ring component has its own specific function within podosomes. In this respect, it is interesting to note that, for FAs, the function of specific adaptor proteins such as vinculin and talin are well characterized but only poorly defined for podosomes. Therefore in our view, super-resolution imaging provided evidence that key podosome components such as integrins, vinculin and talin are not arranged in a ‘circular’ geometry around the core (as the term ‘ring’ implies), meaning that the heterogeneous spatial arrangements of these components with respect to actin core and the radiating filaments cannot be oversimplified by collectively calling this area a ‘ring’, especially when podosomes are not isolated features but members of large super-structures like podosome belts or clusters. Altogether, our studies directly emphasize the need for a better functional characterization of these proteins and a better

Fig. 1. Podosomes imaged by different fluorescence microscopy approaches. Immature human DCs adhering to a glass coverslips were fixed, permeabilized and labeled for actin by phalloidin (red signal in left and right images, orange signal in middle image) and for vinculin by specific monoclonal antibody (green signal in left and right images, blue signal in middle image) prior to imaging by fluorescence-based microscopy. Note the strong yellow signal in the confocal microscopy image that indicates highly overlapping (colocalizing) actin and vinculin fluorescence intensities, whereas super-resolution imaging is capable of resolving the signals providing a much sharper view of these podosome components. The scale bar refers to all three images.

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characterization of the link between the podosome core and the integrin islets and, as such, our work highlights how superresolution imaging can spark new questions about the function and organization of sub-cellular structures. Walde et al. (2014) confirmed and further characterized the spatial organization of vinculin by imaging podosomes by Bayesian analysis of blinking and bleaching (3B analysis) and SIM. This study proposes that the vinculin ring is composed by straight strands of vinculin arranged preferentially at angles between 116◦ and 135◦ , conferring a polygonal shape to the podosome ring and possibly representing points for podosome growth (Walde et al., 2014). Although the exact relevance of this polygonal shape for podosome dynamics and function remains to be fully determined, this study puts forward the existence of geometric and perhaps regular spatial arrangements of podosome constituents that might be involved in force sensing and re-distribution. The same group previously pioneered 3B analysis for imaging of podosomes both in fixed and living cells (Cox et al., 2012). The 3B analysis method allows superresolution data to be extracted from conventional, wide field image series of cells labeled with standard fluorescent proteins. Using Bayesian statistics the whole image series is modeled as number of fluorophores being on and off with certain predefined bleaching and blinking parameters. The most likely positions of each fluorophore can be determined with up to 50 nm accuracy and a temporal resolution of 4 s. This approach allowed, for the first time, the investigation of nanoscale dynamics of podosome formation and dissolution, revealing that in some cases during podosome formation the talin ring is not formed instantaneously, but rather closes around or seals the podosomes. During dissolution a similar process was observed, where talin retracted from one point on the ring in an unwinding fashion (Cox et al., 2012). A major advantage of the 3B analysis method is that conventional microscopes and fluorophores can be used, however, the analysis of the time series is highly computational intensive. Nevertheless, also because the analysis method is available as ImageJ plugin the method is accessible for many researchers without other super-resolution equipment (Rosten et al., 2013). These results clearly demonstrate the power of super-resolution microscopy over conventional and confocal microscopy in elucidating the nanoscale organization of podosomes and challenging old assumptions about how podosome constituents are spatially arranged.

Podosomes in the third dimension Nanoscale 3D imaging of podosome architecture Although the various super-resolution approaches have given valuable insights into the nanoscale organization of several podosome components (Cox et al., 2012; Van den Dries et al., 2013b), 3D information was lacking. Using a combination of TEM, immunogold labeling and computational methods Gawden-Bone et al. (2010) have mapped for the first time the localization of actin, paxillin, gelsolin and phosphotyrosine in podosomes on 2D substrates and in podosomes protruding in collagen impregnated porous membranes, demonstrating that the core-ring distribution of components is largely maintained. However, this TEM approach prevents the direct imaging of specific components, and the sparse gold labeling density greatly limits the spatial resolution of the final 3D distribution map of the investigated core and ring components. Recently, several methods have been described that substantially increase the axial resolution of light microscopy (Lidke and Lidke, 2012). Various approaches to obtain this 3D information have been used, including the use of a PSF that is asymmetrical in the

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z-dimension (Huang et al., 2008; York et al., 2011) splitting different z-planes onto different regions of a detector or by the use of interferometry (Shtengel et al., 2009). The latter has been used in combination with PALM (iPALM) to resolve the 3D nanoscale structure of FAs, revealing their composite multilaminar architecture at an unprecedented lateral and axial resolution (Kanchanawong et al., 2010). This study elegantly demonstrated that FAs consist of three distinct layers, a bottom integrin signaling layer containing FAK and paxillin, an intermediate mechanosensing layer containing talin and vinculin and an upper actin regulatory layer containing zyxin, VASP and ␣-actinin. Furthermore, by comparing the height of N and C terminally tagged talin, they revealed its polarized localization with its N-terminus being localized closer to the glass then its C-terminus, with an axial resolution of 10–15 nm (Kanchanawong et al., 2010). We have recently exploited 3D STORM based on dual focal plane imaging combined with phase retrieved pupil functions (Liu et al., 2013) to image podosome actin at the ventral site of DCs (Fig. 2). Although we overall confirmed the observations obtained by 2D STORM (Van den Dries et al., 2013b), with the 3D STORM approach we could directly observe the variation in height among podosomes (Fig. 2), which so far has not been possible using conventional microscopy, due to its inferior axial resolution. It would be very interesting to further exploit this approach to see whether a hierarchical organization of adhesion complex proteins, as reported for FAs, exists in podosomes as well. Furthermore, a better understanding of the 3D organization of other podosome components, such as the other adaptor proteins paxillin and zyxin, or the localization of microtubules, formins, matrix-metalloproteases and signaling small GTPases would greatly increase our understanding of the structure–function relationship of these proteins within podosomes. Podosomes in a 3D world The next obvious step would be to image podosomes of DCs within living 3D tissues; however imaging in thick biological samples or tissues is still a major challenge in fluorescence microscopy. This is also the main reason why it remains difficult to fully assess the relevance and existence of podosomes directly in real threedimensional tissues. In fact, a few laboratories have only recently addressed this issue, and despite some controversy, FAs have been observed in a (semi) physiological 3D environment and found to be quite similar to the 2D situation (Kubow and Horwitz, 2011; Kim et al., 2012). Similar efforts have been made for podosomes, which have been described in osteoclasts when cultured on bone (Luxenburg et al., 2007). Moreover, the osteoclast sealing zone, which is a podosome based cellular apparatus (Lakkakorpi and Väänänen, 1996), is well characterized in vivo (Georgess et al., 2014). Also other cell types have been described to form podosomes on substrates other than glass and in 3D conditions in various cell types, mostly maintaining the same composition observed in 2D (Cougoule et al., 2012). Van Goethem et al. (2013) showed that macrophages migrating through a collagen I 3D matrix model formed F-actin rich protrusions that contained typical podosome markers like gelsolin, vinculin, talin, paxillin and integrins. Similar podosome-like structures were shown to be formed by DCs seeded on gelatin drained filters (Baranov et al., 2014; Gawden-Bone et al., 2010). Carman et al. (2007) demonstrated that lymphocytes form podosomes that are used by these cells to probe the surface of, and ultimately form trans-cellular pores through the endothelial cell layer. Furthermore podosomes were shown to be formed in the endothelium of native arterial vessel exposed to TGF␤ (Rottiers et al., 2009). Most studies mentioned above used conventional fluorescence microscopy combined with TEM of vertical thin slices to show

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Fig. 2. Podosomes by 3D STORM. Immature DCs were seeded on glass coverslips for 3 h, fixed and stained with Alexa Fluor647-conjugated phalloidin to visualize actin. Shown is the 3D-STORM reconstruction of a part of a representative podosome cluster with podosome cores and associated network. The height information is displayed in a color coding, with blue representing fluorophores localized close to the glass substrate and red indicating fluorophore localized further away from glass substrate, and higher into the cell. The top image shows a 2D projection of color coded localizations. The bottom image shows a 3D projection of the same area with the white open arrow indicating a high podosome, and the black closed arrowhead indicating a low podosome. Scale bar represents 1 ␮m.

the protrusions, and immunogold labeling has been used to identify their components; however, how podosomes are organized at the nanoscale within these various 3D environments has not been revealed yet. There are several reasons as to why imaging in 3D tissue environment is challenging. The penetration of visible light into tissues is poor due to scattering and tissues are optically heterogeneous and therefore cause aberrations of the wavefront leading to poor image quality. During the last decade, multiphoton excitation has been applied to image immune responses and cellular adhesion in 3D tissues and live animals (Germain et al., 2012; Jayo and Parsons, 2012). Multiphoton microscopy exploits the fact that a fluorophore can be excited by multiple photons if their combined energy is the same as their excitation wavelength. Since the energy of a photon is inversely proportional to its wavelength, much higher wavelengths can be used than in single photon excitation. Higher wavelength light is scattered much less in biological tissues and therefore the penetration depth is much larger allowing imaging in thick biological samples. Another advancement in deep tissue imaging is the application of adaptive optics (Girkin et al., 2009). In adaptive optics the excitation wavefront is shaped in order to correct for aberrations caused by inhomogeneity of the refractive index of the tissue and is a method that was originally designed for uses in astronomy (Beckers, 1993). Adaptive optics has recently been applied to in vivo imaging of mouse bone marrow vasculature and mouse brain (Wang et al., 2014a, 2014b), demonstrating its potential for in vivo imaging applications. Hopefully, the combination of multiphoton imaging and adaptive optics could yield the resolution necessary to provide unambiguous proof of the in vivo existence of podosomes in various cell types directly in tissues. This will be of key importance to establish the podosome structure and unravel the function of podosomes and podosome clusters in real tissues in vivo.

Capturing podosome dynamics: Current knowledge and future developments The well-known ‘resolution trade-off’ in the field of light microscopy says that high spatial resolution is accompanied with a low temporal resolution and vice versa. While live-cell confocal microscopy is well suited to study the dynamics of proteins on the second timescale, no information is provided about the spatial localization of the studied molecules. On the other hand, STORM microscopy has a spatial resolution of 10–20 nm but since the acquisition of a single image of fixed specimens takes minutes, sometimes hours, temporal information is lost. Despite a limited palette of live-cell imaging approaches in the early years of fluorescence microscopy, microinjection of fluorescently labeled proteins combined with techniques such as fluorescence recovery after photobleaching (FRAP) revealed key features of FA assembly and disassembly (Burridge and Feramisco, 1980; Geiger et al., 1984). Yet, it was the discovery and adaptation of the green fluorescent protein (GFP) in the early 1990s that greatly expanded the possibilities of visualizing the dynamics of cellular adhesions and probing the molecular dynamics and interactions of their components in situ (Chalfie et al., 1994; Heim et al., 1994). GFP-tagged proteins were used to study the assembly, disassembly and organization of FAs (Balaban et al., 2001; Rottner et al., 2001; Zamir et al., 2000), and FRAP studies were performed to study the dynamics of individual FA components (Edlund et al., 2001; Fraley et al., 2005; Lele et al., 2006). At podosomes, the expression of GFP-tagged podosome components revealed the highly dynamic behavior of these structures (Evans et al., 2003), and FRAP was used to show that continuous actin turnover occurs within the podosome core (Destaing et al., 2003). We studied the diffusion of the cytoskeletal adaptor proteins vinculin, talin, paxillin and zyxin with FRAP microscopy and found that these proteins are highly

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dynamic within podosome rings (Van den Dries et al., 2013a). Although the turnover of podosomes themselves is in the order of minutes, the exchange rates of their components are in the order of seconds and maybe even milliseconds. Interestingly, we found that the exchange rates of the cytoskeletal adaptor proteins are strikingly similar to values found for FAs suggesting that the interactions among these proteins are similar in both structures (Van den Dries et al., 2013a). Therefore, while FAs and podosomes are structurally very different at the micron scale, our FRAP data suggests that the nanoarchitecture of the link between the integrins and the cytoskeleton in both structures might be more similar than previously anticipated. Whether and how differently shaped micron size structures are assembled from similar nanoscale building blocks certainly deserves further investigation. Several components such as WASP, Tks5 and FBP17 have been demonstrated to be exclusively present in podosomes (Linder et al., 1999; Abram et al., 2003; Tsuboi et al., 2009), something that might account for their different micron scale appearance with respect to focal adhesions. Recently developed microscopy techniques like the ones discussed in this review will be instrumental to address these questions. Part of the progress in studying protein dynamics on cells has been made in using complex software algorithms that extract information about molecular dynamics from images taken on conventional microscopy setups (Wiseman et al., 2004). With image correlation spectroscopy (ICS) based techniques such as raster image correlation spectroscopy (RICS) and cross correlation RICS (ccRICS) we are now able to study protein dynamics and protein–protein interactions at the molecular level within adhesions (Digman and Gratton, 2009). For example, exploiting the RICS microscopy technique, it has been shown that paxillin is recruited to FAs as monomers but leaves FAs as large aggregates (Digman et al., 2008). Pioneering work from the Wiseman and Horwitz laboratories has demonstrated the application of ICS and ICS-based methods, including spatiotemporal ICS (STICS) to study the dynamics of both integrins and signaling molecules involved in cell adhesion and migration. They used ICS to determine the dynamics, density and interactions of integrin molecules in living cells, revealing differences between nascent and mature adhesions (Wiseman et al., 2004). Subsequently, STICS, which maps the transport properties of fluorescently tagged proteins using correlation analysis of fluorescence intensity fluctuations in image series in space and time, has been shown to detect flow and directionally biased diffusion of proteins within subcellular regions (Kolin and Wiseman, 2007). In particular, STICS has been applied to characterize the integrin-actin linkage efficiency in protrusions of migrating cells (Brown et al., 2006) and to investigate fluxing behavior of different integrin–ligand pairs at adhesions formed by migrating fibroblasts (Chen et al., 2012). It would be very interesting to image podosomes by STICS to determine whether transport of podosome components also occurs and whether this contributes to regulate podosome mediated adhesion, protrusion and substrate sensing. Although ICS approaches could potentially reveal podosome dynamics at very high temporal resolution, the spatial resolution is limited by the microscope used. The ongoing improvements in instrumentation and analysis algorithms have substantially speeded up super-resolution imaging approaches (Huang et al., 2013). A very elegant example of the insights fast super-resolution methods can yield in cytoskeletal dynamics is a recent study by Lippincott-Schwartz and colleagues in which they use live cell imaging by SIM to show a contractile adhesion system consisting of actin arcs and repeating myosin II filaments (Burnette et al., 2014). Because of the high spatial (both axial and lateral) and temporal resolution of SIM, this imaging approach could become a very useful method to investigate the spatiotemporal dynamics of podosomes by simultaneously monitoring constituents involved in their adhesive, protrusive and matrix degrading activities.

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Conclusions The pioneering application of super-resolution light microscopy to the study of podosomes has revealed novel insight into the spatial organization of these complex structures. Podosome rings were shown to be composed of nano-islets of integrins and talin with vinculin populating these islets at sites of actin fibers and at the core rim, showing a carpet-like architecture around the core, thus challenging the model of a well-defined core–ring structure. Despite this novel information, many aspects of podosome nanoscale organization remain to be determined including the exact localization of membrane curvature sensing proteins such as the known podosome constituents FBP17 and IRSp53, the nature of the molecular linker between protruding core and radiating actin filaments or the spatial regulation of matrix metalloprotease release. Possibly, this should be imaged in living cells, in 3D and in multi-color modes. To achieve this level of detail, efforts should be directed toward the optimization of microscopy setups to simultaneously provide high temporal and spatial resolution. The development of fast twoand three-dimensional STORM microscopy, selective plane illumination microscopy and Bayesian localization microscopy open up new possibilities for high resolution 3D live imaging of complex cellular structures in the near future.

Acknowledgements We are indebted to A.B. Houtsmuller, J. Slotman from the Erasmus MC Rotterdam and L. Paardekoper for the SIM image of podosomes and Keith A. Lidke, Sheng Liu and Samantha L. Schwartz from the University of New Mexico for the 3D STORM image of podosomes. Work in the Cambi laboratory is supported by EU grant NANO-VISTA (FP7-2011-7-ICT-288263) and a HFSP research grant (RGP0027/2012). AC is the recipient of a Meervoud grant (836.09.002) from The Netherlands Organization for Scientific Research (NWO).

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