Chromatin recruitment of effector proteins involved in gene regulation depends on multivalent interaction with histone post-translational modifications (PTMs) and structural features of the chromatin fiber. Due to the complex interactions involved, it is currently not understood how effectors dynamically sample the chromatin landscape. Here, we dissect the dynamic chromatin interactions of a family of multivalent effectors, heterochromatin protein 1 (HP1) proteins, using single-molecule fluorescence imaging and computational modeling. We show that the three human HP1 isoforms are recruited and retained on chromatin by a dynamic exchange between histone PTM and DNA bound states. These interactions depend on local chromatin structure, the HP1 isoforms as well as on PTMs on HP1 itself. Of the HP1 isoforms, HP1α exhibits the longest residence times and fastest binding rates due to DNA interactions in addition to PTM binding. HP1α phosphorylation further increases chromatin retention through strengthening of multivalency while reducing DNA binding. As DNA binding in combination with specific PTM recognition is found in many chromatin effectors, we propose a general dynamic capture mechanism for effector recruitment. Multiple weak protein and DNA interactions result in a multivalent interaction network that targets effectors to a specific chromatin modification state, where their activity is required.
Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report the discovery of a virulence mechanism by which changes to the mechanical stiffness of the mycobacterial cell surface confer refraction to killing during infection. Long-term time-lapse atomic force microscopy was used to reveal a process of “mechanical morphotype switching” in mycobacteria exposed to host intracellular stress. A “soft” mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Both pharmacologic treatment, with bedaquiline, and a genetic mutant lacking uvrA modified the basal mechanical state of mycobacteria into a soft mechanical morphotype, enhancing survival in macrophages. Our study proposes microbial cell mechanical adaptation as a critical axis for surviving host-mediated stressors.
Abstract Mycobacteria grow by inserting new cell wall material in discrete zones at the cell poles. This pattern implies that polar growth zones must be assembled de novo at each division, but the mechanisms that control the initiation of new pole growth are unknown. Here, we combine time-lapse optical and atomic force microscopy to measure single-cell pole growth in mycobacteria with nanometer-scale precision. We show that single-cell growth is biphasic due to a lag phase of variable duration before the new pole transitions from slow to fast growth. This transition and cell division are independent events. The difference between the lag and interdivision times determines the degree of single-cell growth asymmetry, which is high in fast-growing species and low in slow-growing species. We propose a biphasic growth model that is distinct from previous unipolar and bipolar models and resembles “new end take off” (NETO) dynamics of polar growth in fission yeast.
Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) is a valuable tool in biophysics to investigate the ligand-receptor interactions, cell adhesion and cell mechanics. However, the force spectroscopy data analysis needs to be done carefully to extract the required quantitative parameters correctly. Especially the large number of molecules, commonly involved in complex networks formation; leads to very complicated force spectroscopy curves. One therefore, generally characterizes the total dissipated energy over a whole pulling cycle, as it is difficult to decompose the complex force curves into individual single molecule events. However, calculating the energy dissipation directly from the transformed force spectroscopy curves can lead to a significant over-estimation of the dissipated energy during a pulling experiment. The over-estimation of dissipated energy arises from the finite stiffness of the cantilever used for AFM based SMFS. Although this error can be significant, it is generally not compensated for. This can lead to significant misinterpretation of the energy dissipation (up to the order of 30%). In this paper, we show how in complex SMFS the excess dissipated energy caused by the stiffness of the cantilever can be identified and corrected using a high throughput algorithm. This algorithm is then applied to experimental results from molecular networks and cell-adhesion measurements to quantify the improvement in the estimation of the total energy dissipation.
The accuracy and repeatability of atomic force microscopy (AFM) imaging significantly depend on the accuracy of the piezoactuator.However, nonlinear properties of piezoactuators can distort the image, necessitating sensor-based closed-loop actuators to achieve high accuracy AFM imaging.The advent of high-speed AFM has made the requirements on the position sensors in such a system even more stringent, requiring higher bandwidths and lower sensor mass than traditional sensors can provide.In this paper, we demonstrate a way for high-speed, high-precision closed-loop AFM nanopositioning using a novel, miniaturized MEMS position sensor in conjunction with a simple PID controller.The sensor was developed to respond to the need for small, lightweight, high-bandwidth, long-range and subnm-resolution position measurements in high-speed AFM applications.We demonstrate the use of this sensor for closed-loop operation of conventional as well as high-speed AFM operation to provide distortion-free images.The presented implementation of this closed-loop approach allows for positioning precision down to 2.1 Å, reduces the integral nonlinearity to below 0.2%, and allows for accurate closed loop imaging at line rates up to 300 Hz.
Abstract The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b-7 interacts with the lipid bilayer prior to recruiting C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.
Imaging living cells by atomic force microscopy (AFM) promises not only high-resolution topographical data, but additionally, mechanical contrast, both of which are not obtainable with other microscopy techniques. Such imaging is however challenging, as cells need to be measured with low interaction forces to prevent either deformation or detachment from the surface. Off-resonance modes which periodically probe the surface have been shown to be advantageous, as they provide excellent force control combined with large amplitudes, which help reduce lateral force interactions. However, the low actuation frequency in traditional off-resonance techniques limits the imaging speed significantly. Using photothermal actuation, we probe the surface by directly actuating the cantilever. Due to the much smaller mass that needs to be actuated, the achievable measurement frequency is increased by two orders of magnitude. Additionally, photothermal off-resonance tapping (PORT) retains the precise force control of conventional off-resonance modes and is therefore well suited to gentle imaging. Here, we show how photothermal off-resonance tapping can be used to study live cells by AFM. As an example of imaging mammalian cells, the initial attachment, as well as long-term detachment, of human thrombocytes is presented. The membrane disrupting effect of the antimicrobial peptide CM-15 is shown on the cell wall of Escherichia coli. Finally, the dissolution of the cell wall of Bacillus subtilis by lysozyme is shown. Taken together, these evolutionarily disparate forms of life exemplify the usefulness of PORT for live cell imaging in a multitude of biological disciplines.
High-speed atomic force microscopy (HS-AFM) is a technique capable of revealing the dynamics of biomolecules and living organisms at the nanoscale with a remarkable temporal resolution. The phase delay in the feedback loop dictates the achievable speed of HS-AFM instruments that rely on fast nanopositioners operated predominantly in conjunction with piezoelectric actuators (PEAs). The high capacitance and high operating voltage of PEAs make them difficult to drive. The limited bandwidth of associated high-voltage piezo-amplifiers is one of the bottlenecks to higher scan speeds. In this study, we report a high-voltage, wideband voltage amplifier comprised of a separate amplification and novel voltage-follower power stage, requiring no global feedback. The reported amplifier can deliver a current over ±2 amps, offers a small-signal bandwidth of 1 MHz, and exhibits an exceptionally low phase lag, making it particularly well suited for the needs of next-generation HS-AFMs. We demonstrate its capabilities by reporting its achievable bandwidth under various PEA loads and showcasing its merit for HS-AFM by imaging tubulin protofilament dynamics at sub-second frame rates.
New developments in MEMS (microelectromechanical systems) fabrication allowed the development of new types of atomic force microscopy (AFM) sensor with integrated readout circuit and actuator built in on the cantilever. Such a fully instrumented cantilever allows a much more direct measurement and actuation of the cantilever motion and interaction with the sample. This technology is expected to not only allow for high speed imaging but also the miniaturization of AFMs. Based on the complexity of these integrated MEMS devices, a thorough understanding of their behavior and a specialized controls approach is needed to make the most use out of this new technology. In this paper we investigate the intrinsic properties of such MEMS cantilevers and develop a combined approach for sensing and control, optimized for high speed detection and actuation. Further developments based on the results presented in this paper will help to expand the use of atomic force microscopy to a broad range of everyday applications in industrial process control and clinical diagnostics.
