Mechanical forces guide the biophysics of immune cells

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Colin-York H1, Barbieri L1, and Fritzsche M1,2 
1 MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, OX3 9DS Oxford, UK
2 Kennedy Institute for Rheumatology, Roosevelt Drive, University of Oxford, Oxford OX3 7LF Oxford, UK
Marco Fritzsche completed his initial training with a MSc in theoretical physics and two BSc in physics and mathematics at the University of Saarland, Germany. He then undertook his PhD with Professor Guillaume Charras at the London Centre for Nanotechnology at University College London in the UK and with Professor Ned Wingreen at Princeton University, USA. Marco continued his Postdoctoral training with Professor Christian Eggeling at the Weatherall Institute of Molecular Medicine at the University of Oxford in close collaboration with Professor Eric Betzig at the Howard Hughes Medical Institute, Janelia Farm, USA. Dr Marco Fritzschenow now leads the Biophysical Immunology ( group within the Human Immunology Unit at the Weatherall Institute for Molecular Medicine and a satellite laboratory at the Kennedy Institute for Rheumatology at the University of Oxford.
Biomedical research must aim to speed up the development and applications of cutting edge physical science insights, methods and techniques. JPK Instruments, manufacturer of nanoanalytic instrumentation for research in life sciences and soft matter enables the NanoWizard® AFM system being combined with a toolbox of fluorescence microscopy techniques in the Biophysical Immunology group at the University of Oxford.
Corresponding author
Medical sciences increasingly recognise the importance of biophysical research for human health.1, 2 Consequently to-date, most groups at the fore-front of their disciplines apply biophysical tools to their research on a daily basis – knowing that these technologies will transform the landscape of medical research in the years to come. Yet, very few research environments offer a platform for physical scientists, engineers, and life scientists to develop and apply together their own custom techniques and instrumentation to target key challenges in health and the life sciences - although it is apparent that such multidisciplinary research will lead to improved understanding of disease, standard of life, and long-term economic wealth.
The Biophysical Immunology (BPI) group led by the author, Dr Marco Fritzsche, is located within the MRC Human Immunology Unit at the Weatherall Institute of Molecular Medicine and the Kennedy Institute for Rheumatology at the University of Oxford, UK. Having expertise across multiple disciplines from theoretical-physics to immunology, the BPI group (@FritzscheLab and is determined to carve out a niche and help in closing this gap in knowledge in biomedical sciences. 
The BPI laboratory develops and employs custom-built new state-of-the-art technology at the interface of biophysics and immunology such as the new opto-mechanical platform combining JPK’s NanoWizard® AFM system and a toolbox of fluorescence microscopy techniques (Fig. 1). This technology aligns with the main research interests of the BPI laboratory that are focused on the understanding of the biophysics of the immune system in health and disease. The laboratory is especially interested in the biophysical functioning of a vital cellular skeleton – the main determinant of the biophysics of immune cells.
Figure 1. Photo of the Imaging-based opto-mechanical platform during acquisition and force probing in live HeLa cells. Liliana Barbieri,left, is operating the AFM-FRAP system comprised of the NanoWizard AFM head (blue) on a Leica DMI8 (white) microscope under supervision of Dr Huw Colin-York. Three control screens display the information from the Leica DMI8 imaging system (upper left screen), the NanoWizard AFM force probing control software (upper right screen), and the FRAP control module (lower right screen). Further details are described in the main text. 
The ability of immune cells to perform their function often involves biophysical changes e.g. due to motion, so called mechanical forces.3, 4, 5 These mechanical forces are produced and maintained by the cytoskeleton,6 the cellular skeleton, which are active structures composed of polymers that continuously undergo turnover of their proteic components.7 This holds notably for the cortical actin cytoskeleton of immune cells that comprises polydisperse actin filaments undergoing continuous turnover with constant growth of the filaments at their barbed ends and shrinkage at their pointed ends.
On the one hand, the filaments are crosslinked over finite periods of time and redistributed by the action of molecular motors, such as myosin-II. On the other hand, two filament subpopulations compose the cortex in eukaryotic cells. T lymphocytes have 10-fold differing turnover rates of the actin monomers and arise from distinct nucleation pathways (Fig. 2):7 (1) polymerization of long filamentous actin (F-actin) driven by formin proteins, which associate with the fast-growing barbed end of actin; and (2) branching of short F-actin driven by the Arp2/3 complex, which binds to pre-existing F-actin and nucleates new filaments (Fig. 3). The latter population has been shown to account for 80% of the total F-actin in different cell types, such as cervical HeLa, Melanoma and T cells.7 Filament crosslinking by myosin-II as well as the fact that the Arp2/3 complex nucleates new filaments at a distinct angle of 70° from its mother filament has direct implications on the actin cytoskeletal ultra-structure (such as the cortex mesh-size), and thus the mechanobiology of immune cells.
Figure 2. Quantification of the actin filament turnover in the equatorial actin cortex and ventral stress fibers in live HeLa cells. Logarithmic representation of FRAP recovery curves measured in the cortical actin and ventral stress fibers revealed two types of actin filaments in live HeLa cells nucleated by the Arp2/3 complex and formins. Cortical actin (blue and red lines) and stress fibers (magenta and green lines) display quantitative changes in the turnover dynamics of the Arp2/3- and formin-nucleated in response to 20nN force compared to control conditions.
Figure 3. Quantification of the actin filament lengths in the actin cortex of live HeLa cells. The cortical actin filament length-distribution shows changes including in the average actin filament lengths in response to 20nN force (blue line) compared to control conditions (red line). Note, dots indicate individual measurements and lines corresponding fits. Further information can be found in (7), and details to the Figure are described in the main text.
To quantify the turnover dynamics of the actin-assembly-pathways, the BPI laboratory previously developed an analysis pipe-line based on fluorescence recovery after photo-bleaching (FRAP).8 This broadly-applicable approach allows the determination of how many reaction processes participate in the turnover of any given protein of interest, for characterising their apparent association and dissociation rates, and for determining their relative importance in the turnover of the overall protein population. 
For example, by analysing FRAP recovery curves and by measuring the individual recovery rates of the F-actin subpopulations, one can gain detailed insight into Arp2/3- and formin-nucleated filament turnover within the dense interwoven actin networks of immune cells. This analysis and process identification strategy enables the determination of these changes, thereby providing valuable quantitative data for systems biology approaches measured in physiologically relevant conditions.
Historically, efforts to understand immune mechanobiology employed methodologies to independently study either actin-assembly-pathways or cellular mechanics. However, detailed understanding of how interactions among cytoskeletal proteins and their translation into dynamic architectures regulate cell mechanics remains largely elusive. Specifically, it is known that cell rheology (time-dependent mechanical properties) vastly differs at different time- and length-scales, and yet no consistent bottom-up framework exists that explains cell rheological behaviour.
For example, on short time-scales (tens of milli-seconds) and large length-scales (micro-meters) cells show poroelastic properties/behaviour, and at long time-scales (~minutes) they exhibit a power law behaviour in response to application of external forces. While a network of reconstituted cross-linked actin can be tuned to show similar mechanical features, their characteristic mechanical properties (such as elasticity and viscosity) drastically deviate from cells, and also the transition between their mechanical regimes occurs at time- and length-scales that differ by several orders of magnitude.
It is assumed that cells actively adapt their dynamic cytoskeletal structures to mechanical conditions by remodelling actin filaments and reassembly of associated crosslinkers. The BPI laboratory has uncovered how these nanoscale changes in the biophysical properties of immune cells result into mechanical forces that guide the physiology of immune cells.7
To this end, recent experiments provide convincing evidence that the life-time of the two major actin-assembly factors in immune cells, the Arp2/3 complex and formins, govern the characteristic turnover time and length of actin filaments that in turn control the length- and time-scale dependent mechanical behaviour of the cell. To this end, mechanical probing of living immune cells depends on the compliance and the length-scale of the biological material, the frequency (time scale) at which mechanics are characterised as well as the environmental and experimental conditions.
Cell mechanical properties such as cell stiffness and elasticity (the ability to resist mechanical forces), in turn, are utterly different depending on the speed of the applied forces, including poroelastic and viscoelastic stress responses, as well as protein-turnover-dependent responses. Similarly, probing of active mechanical force production is challenging due to the complexity of mechanosensitivity, mechanotransduction and mechanical feedback mechanisms of cell mechanics. Consequently, quantifying cell mechanics in immune cells including both mechanical properties and mechanical force production is challenging, primarily due to the physics of mechanical force measurements themselves. Since cells do not emit mechanical signals that could be detected and analysed in a contact-less manner, such quantification demands direct engagement of the force probing technology with the cell.
Measuring mechanical force production of immune cells is challenging but the methodology of traction force microscopy (TFM) is perhaps the most successful force probing technology.9 In TFM experiments, cells interact with a thin (20–30 μm) elastic hydrogel by adhering to a protein functionalised surface. Within the hydrogel, immobilised fluorescent beads serve as fiducial markers, and imaging of the bead positions over time in two or three dimensions (2D/3D) during the application of cellular tractions allows the 2D, 3D elastic displacement of the gel to be quantified. Combining the displacement measurements with knowledge about the mechanical properties of the hydrogel allows the forces applied by the cell to be recovered.
The greatest shortcoming of classical TFM is its limited sensitivity due to the finite density at which the displacement field can be sampled within the gel, which must be high enough to reflect the complexity of the traction field that is applied by the cell.10 To overcome these challenges, the BPI laboratory recently improved the spatial resolution and accuracy of TFM using optical super-resolution stimulated emission depletion (STED) microscopy.
The increased spatial resolution of STED-TFM (STFM) allows a greater than five-fold higher sampling of the forces generated by the cell than conventional TFM, leading to more accurate quantification of cellular tractions. (S)TFM has been successful in quantifying cellular force production but precludes the characterisation of mechanical properties. For example, the mechanical stiffness of cells in the form of the elastic modulus is determined by physically indenting the cell surface by a given force using e.g. atomic force microscopy (AFM), which is not possible in TFM experiments.
To overcome these limitations, the BPI laboratory combined a toolbox of fluorescence microscopy techniques with the NanoWizard® AFM.
Previously, no system had been available within the Oxford microscope facilities serving biological research for directly measuring or applying forces on cells. The new JPK AFM setup is fully integrated into the image facility at the HIU, offering the possibility for less-experienced users to address questions about force involvement in their biomedical research. This opto-mechanical platform allows simultaneous FRAP and AFM to directly quantify molecular responses to external forces at multiple length- and time-scales. To fully exploit the advances of measuring and applying forces, it is important to simultaneously observe the molecular processes that underlie the measured mechanical behaviour. In particular, the dynamic behaviour and turnover of the cellular cytoskeleton is essential for transferring forces. The opto-mechanical platform enables the BPI group now to simultaneously measure and correlate nanoscale actin turnover and cell mechanics with the combination of AFM and FRAP (AFM-FRAP; Fig. 4). 
Figure 4. Imaging-based opto-mechanical platform. (a) Schematic of the opto-mechanical platform combining the JPK NanoWizard AFM and the FRAP system, which allows imaging and quantification of actin associated turnover processes by the FRAP module and under application of precise mechanical forces by the AFM. The AFM-FRAP platform with super-resolution STED enables high-resolution imaging during force probing and turnover quantifications. Detailed information for the FRAP analysis can be found in.8
In light of the increasing importance of mechanobiology in immune cell physiology, we envisage AFM-FRAP to become a major player for quantifying mechanical forces in living cells. Also, JPK are interested in combining their microscopes with other technologies such as fluorescence microscopy. 
The BPI laboratory finally aims to integrate AFM with other technologies in embedded systems including super-resolution microscopy modes. This project therefore provides a unique opportunity for the industrial partners and the researchers to benefit from each other and elucidate how mechanical forces guide the biophysics of immune cells.
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