Combined AFM and super-resolution localisation microscopy: Investigating the structure and dynamics of podosomes

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Abstract

Podosomes are mechanosensitive attachment/invasion structures that form on the matrix-adhesion interface of cells and protrude into the extracellular matrix to probe and remodel. Despite their central role in many cellular processes, their exact molecular structure and function remain only partially understood. We review recent progress in molecular scale imaging of podosome architecture, including our newly developed localisation microscopy technique termed HAWK which enables artefact-free live-cell super-resolution microscopy of podosome ring proteins, and report new results on combining fluorescence localisation microscopy (STORM/PALM) and atomic force microscopy (AFM) on one setup, where localisation microscopy provides the location and dynamics of fluorescently labelled podosome components, while the spatial variation of stiffness is mapped with AFM. For two-colour localisation microscopy we combine iFluor-647, which has previously been shown to eliminate the need to change buffer between imaging modes, with the photoswitchable protein mEOS3.2, which also enables live cell imaging.

Introduction

Podosomes are highly dynamic micron-sized conical integrin-based adhesion structures that form on the surface of certain cells, especially those of monocytic origin. They are mechanosensitive, i.e. they can sense the extracellular matrix (ECM) topography and rigidity, and besides adhesion they are capable of remodelling and digesting the ECM. They are pivotal in many cellular processes that require matrix remodelling, such as the spread of cancer cells and inflammatory responses of macrophages, bone resorption by osteoclasts, and remodeling of blood vessels and axons by endothelial cells. Podosomes are linked to diseases such as cancer metastasis, chronic inflammations, cardiomyopathy and age-related osteoporosis (Maridonneau-Parini (2014), Paterson and Courtneidge (2018)). Understanding how and why podosomes form, and the molecular mechanics behind how they function, will play a central role in the prevention and treatment of these diseases.

Podosomes have been studied extensively by conventional light microscopy (Marchisio et al. (1984), Pfaff and Jurdic (2001), Destaing et al. (2003)), electron microscopy (Luxenburg et al. (2007), Gawden-Bone et al. (2010), Schmidt et al. (2011), Joosten et al. (2018)) and atomic force microscopy (Labernadie et al., 2010, Labernadie et al. (2010, 2014). These studies have yielded a model of the podosome, consisting of an actin-rich core nucleated by the ARP2/3 complex, surrounded by a ring of adhesion-related proteins such as talin, vinculin, paxillin and zyxin, which together with integrins anchor the podosome onto the ECM and provide structural support during force generation. Podosomes generate forces to protrude into the ECM to probe the matrix topography and stiffness, but the mechanism behind this force generation is unclear. Podosomes are also capable of digesting and remodelling the ECM (Paterson and Courtneidge (2018)), but little is known about the molecular mechanism behind these processes.

Live cell imaging is essential in mechanobiological investigations which study function and mechanics, and it would be helpful to have many forms of dynamic information on a small spatial scale – ideally small enough to resolve single molecules. Atomic force microscopy (AFM) (Binnig et al. (1986)) and fluorescence microscopy are a powerful combination in providing different types of information that complement each other (Hinterdorfer and Dufrêne (2006), Müller and Dufrêne (2008)), and both are compatible with physiological buffers, allowing the observation of living biological specimens. Fluorescence microscopy allows the tagging of intracellular molecules and cellular components with high specificity, and their observation inside cells in a minimally invasive manner using non-destructive wavelengths of light in the visible spectrum. AFM, on the other hand, uses a sharp tip to measure the topography of the sample with sub-nanometer axial resolution, or other physical properties, such as adhesion or stiffness. It is also possible to functionalise AFM tips to recognise specific molecules and measure binding energies (Hinterdorfer and Dufrêne (2006), Miller et al. (2017)), or use AFM for manipulation of the sample in nanometer scale (Chacko et al. (2013b)).

In the past, combination of fluorescence microscopy and AFM was made difficult by the diffraction limit of light microscopy, which restricted the resolution in fluorescence microscopy to two orders of magnitude more than AFM. Recently developed super-resolution microscopy techniques are able to go beyond this limit, but the achievable resolution depends on the technique. Currently, structured illumination microscopy (SIM) is the most utilised super-resolution method for live cell imaging due to its speed. The combination of SIM and AFM has been demonstrated (Gomez-Varela et al., 2020),(and SIM has been applied to study podosomes (Cervero et al., 2018; Rafiq et al., 2019a; Rafiq et al., 2019; van den Dries et al., 2019). However, as SIM has only a maximum of 2-fold potential for resolution improvement over conventional fluorescence microscopy and a typical final resolution around 120–140 nm, its usefulness in molecular mechanobiology is very limited.

Fluorescence super-resolution techniques based on single molecule localisation microscopy (SMLM), such as stochastic optical reconstruction microscopy (STORM) (Rust et al. (2006)) and photoactivated localisation microscopy (PALM) (Betzig et al. (2006)), offer theoretically unlimited resolution which is in practise limited by the signal-to-noise ratio typically to few tens of nanometres, a similar scale to the typical lateral resolution of AFM when imaging soft biological samples (Miller et al. (2017), Hauser et al. (2017), Zhou et al. (2017), Chacko et al. (2013a)). Localisation microscopy methods have been applied to the study of podosomes with the aim of resolving the molecular architecture. While fixed cell SMLM imaging can shed light on the molecular architecture of podosomes (Bouissou et al. (2017), Joosten et al. (2018), Foxall et al. (2019)), many studies have also yielded conflicting results due to artefacts arising from sample preparation and image processing protocols (Cox et al. (2012), van den Dries et al. (2013), Meddens et al. (2014), Walde et al. (2014), Staszowska et al. (2017)).

Localisation microscopy techniques rely on the ability to switch a fluorophore between a bright and a dark state. In its most simple experimental form, direct STORM (dSTORM) (Heilemann et al. (2008), van de Linde et al. (2011)), the sample is illuminated with a high power laser while immersed in a switching buffer that makes the dye molecules blink, and a series of images is acquired where each frame contains only a few emitting fluorophores. While the images of the fluorophores are diffraction-limited, their centroid positions can be calculated with great accuracy, and the final image constructed by summing these locations together. However, to induce the blinking, a switching buffer containing enzymatic oxygen scavengers is normally required. Unfortunately the typical ingredients of this buffer stick to the AFM cantilevers, and make AFM image acquisition impossible. Most attempts to combine AFM and STORM report adding the switching buffer for STORM after AFM imaging (Odermatt et al. (2015), Bondia et al., 2017a, Bondia et al. (2017a, b), Monserrate et al. (2014)), which is time-consuming and cumbersome and can lead to sample movement and damage, seriously limiting the practical use of this technique. In this work, we perform STORM imaging with the fluorescent dye iFluor-647, which is especially suitable for imaging in a buffer without enzymatic oxygen scavenger (Hirvonen and Cox (2018)), allowing combined AFM and STORM imaging without any change of buffer.

The use of endogenous fluorescent proteins, such as the green fluorescent protein (GFP), offers an alternative to antibody labelling. With some fluorescent proteins – called photoswitchable fluorescent proteins – the on and off states can be controlled with specific wavelengths of light. Photoswitchable fluorescent proteins offer another way to perform localisation microscopy without special buffers, and while they also enable live cell imaging, the cost of better resolution in SMLM is increased acquisition time: typically thousands of images are required for the reconstruction of the final image, and the acquisition of this number of frames usually takes at least several minutes – too slow for live cell imaging. Acquisition speed can be increased by acquiring data with higher emitter density (i.e. more molecules/frame), but any overlap of molecules in the raw data can lead to artefacts in the reconstructed final image, such as artificial sharpening of sample structures and missing features. It can be very difficult to spot these artefacts, since there is no warning of failure by the reconstruction software, and quantification of resolution, for example by Fourier Ring Correlation (FRC) (Nieuwenhuizen et al. (2013)), indicates sharpened images have a better resolution.

Artificial sharpening has been shown to be a problem with most localisation reconstruction software where the overlapping molecules cannot be separated properly (Burgert et al. (2015), Sage et al. (2015), Fox-Roberts et al. (2017)), even methods designed for high density SMLM data. To address this problem, we have recently developed a new method, Haar Wavelet Kernel (HAWK) analysis (Marsh et al. (2018)), for separating overlapping molecules. HAWK is a preprocessing method that separates molecules by their blinking statistics, and has been shown to eliminate sharpening artefacts in high density localisation microscopy data. HAWK also works for extremely high density data, allowing reconstruction of a final image from just a few hundred frames, and bringing data acquisition time down to a few seconds – fast enough for many live cell imaging applications.

In this work we have labelled several podosome-related proteins with the photoconvertible fluorescent protein mEOS3.2 (Zhang et al. (2012)), which usually emits in green with 488 nm excitation but can convert to yellow-emitting conformation upon 405 nm illumination. We use mEOS3.2 and iFluor-647 labelling of podosome molecular components for combined two-colour localisation microscopy and AFM on a single microscope, where localisation microscopy provides resolution of <30 nm, while the spatial variation of stiffness is mapped with AFM. We also demonstrate typical artefacts created by super-resolution imaging, and how these can be reduced by HAWK imaging.

Section snippets

Sample preparation

mEOS3.2 sequence was amplified from a template (gift from Dylan Owen, King's College London) using PCR and cloned into an pLNT/Sffv-MCS vector via pCR Blunt vector (Invitrogen). cDNAs encoding the target sequences: residues 1975–2541 of human talin from a template plasmid we previously generated (Vijayakumar et al. (2015)), paxillin and lifeact (gifts from Maddy Parsons, King's College London), and ARPC3 (gift from Matthias Krause, King's College London), were amplified using PCR and then

Two-colour localisation microscopy

To test two-colour localisation microscopy in a buffer that is also compatible with AFM imaging, THP-1 cells expressing mEOS3.2 constructs were plated onto coverslip-bottom dishes and induced to form podosomes (Bombara and Ignotz (1992), Rafiq et al. (2017)), then fixed and immunolabelled with iFluor-647, and imaged in the MEA buffer. The 647 channel was acquired first, and the photoconverted form of mEOS3.2 was then imaged in the 561 channel.

Fig. 2 shows localisation microscopy images of

Discussion

In a quest to elucidate the mechanobiology behind podosome structure and function, a variety of different kinds of information is required. Advances in super-resolution microscopy methods have significantly improved the scale where we can make direct observations of the distribution of fluorescently labelled biomolecules inside cells from hundreds of nanometers to near molecular scale. However, there are drawbacks.

Firstly, all microscopy techniques are prone to artefacts. This is especially

Conclusion

In this work we demonstrate an easy and straightforward method for combined AFM and SMLM super-resolution imaging of podosomes. While the combination of these methods has previously required buffer change between the imaging modes, this problem is eliminated using iFluor-647 dye in a simple buffer without an enzymatic oxygen scavenger. The use of endogenous fluorescent proteins offers an alternative to antibody labelling, and enables live-cell imaging which can be difficult with dye labels. We

Conflicts of interest

The authors declare no competing interests.

Acknowledgements

Support from Human Frontier Science Program (grant number RGP0035/2016),Royal Society (research grantRG110451),MRC(Next Generation Optical Imaging grant, MR/K015664) andBBSRC (research grantBB/R021767/1) is gratefully acknowledged. SC acknowledges support from Royal Society University Research Fellowship, and GEJ from Leverhulme Trust Emeritus Fellowship (EM-2018-037).

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    1

    Current address: Centre for Microscopy, Characterisation and Analysis (CMCA), The University of Western Australia, 35 Stirling Highway, Perth WA 6009, Australia.

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