Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

André Gemeinhardt, Matthew Paul McDonald, Katharina König, Michael Aigner, Andreas Mackensen, Vahid Sandoghdar

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

Intracellular Mass Density Increase Is Accompanying but Not Sufficient for Stiffening and Growth Arrest of Yeast Cells

Shada Abuhattum, Kyoohyun Kim, Titus M. Franzmann, Anne Esslinger, Daniel Midtvedt, Raimund Schluessler, Stephanie Mollmert, Hui-Shun Kuan, Simon Alberti, et al.

Many organisms, including yeast cells, bacteria, nematodes, and tardigrades, endure harsh environmental conditions, such as nutrient scarcity, or lack of water and energy for a remarkably long time. The rescue programs that these organisms launch upon encountering these adverse conditions include reprogramming their metabolism in order to enter a quiescent or dormant state in a controlled fashion. Reprogramming coincides with changes in the macromolecular architecture and changes in the physical and mechanical properties of the cells. However, the cellular mechanisms underlying the physical-mechanical changes remain enigmatic. Here, we induce metabolic arrest of yeast cells by lowering their intracellular pH. We then determine the differences in the intracellular mass density and stiffness of active and metabolically arrested cells using optical diffraction tomography (ODT) and atomic force microscopy (AFM). We show that an increased intracellular mass density is associated with an increase in stiffness when the growth of yeast is arrested. However, increasing the intracellular mass density alone is not sufficient for maintenance of the growth-arrested state in yeast cells. Our data suggest that the cytoplasm of metabolically arrested yeast displays characteristics of a solid. Our findings constitute a bridge between the mechanical behavior of the cytoplasm and the physical and chemical mechanisms of metabolically arrested cells with the ultimate aim of understanding dormant organisms.

Pili mediated intercellular forces shape heterogeneous bacterial microcolonies prior to multicellular differentiation

Wolfram Poenisch, Kelly B. Eckenrode, Khaled Alzurqa, Hadi Nasrollahi, Christoph Weber, Vasily Zaburdaev, Nicolas Biais

Microcolonies are aggregates of a few dozen to a few thousand cells exhibited by many bacteria. The formation of microcolonies is a crucial step towards the formation of more mature bacterial communities known as biofilms, but also marks a significant change in bacterial physiology. Within a microcolony, bacteria forgo a single cell lifestyle for a communal lifestyle hallmarked by high cell density and physical interactions between cells potentially altering their behaviour. It is thus crucial to understand how initially identical single cells start to behave differently while assembling in these tight communities. Here we show that cells in the microcolonies formed by the human pathogen Neisseria gonorrhoeae (Ng) present differential motility behaviors within an hour upon colony formation. Observation of merging microcolonies and tracking of single cells within microcolonies reveal a heterogeneous motility behavior: cells close to the surface of the microcolony exhibit a much higher motility compared to cells towards the center. Numerical simulations of a biophysical model for the microcolonies at the single cell level suggest that the emergence of differential behavior within a multicellular microcolony of otherwise identical cells is of mechanical origin. It could suggest a route toward further bacterial differentiation and ultimately mature biofilms.

Exactly solvable dynamics of forced polymer loops

Wenwen Huang, Yen Ting Lin, Daniela Froemberg, Jaeoh Shin, Frank Juelicher, Vasily Zaburdaev

Here, we show that a problem of forced polymer loops can be mapped to an asymmetric simple exclusion process with reflecting boundary conditions. The dynamics of the particle system can be solved exactly using the Bethe ansatz. We thus can fully describe the relaxation dynamics of forced polymer loops. In the steady state, the conformation of the loop can be approximated by a combination of Fermi-Dirac and Brownian bridge statistics, while the exact solution is found by using the fermion integer partition theory. With the theoretical framework presented here we establish a link between the physics of polymers and statistics of many-particle systems opening new paths of exploration in both research fields. Our result can be applied to the dynamics of the biopolymers which form closed loops. One such example is the active pulling of chromosomal loops during meiosis in yeast cells which helps to align chromosomes for recombination in the viscous environment of the cell nucleus.

Correction-free force calibration for magnetic tweezers experiments

Eugen Ostrofet, Flavia Stal Papini, David Dulin

Magnetic tweezers are a powerful technique to perform high-throughput and high-resolution force spectroscopy experiments at the single-molecule level. The camera-based detection of magnetic tweezers enables the observation of hundreds of magnetic beads in parallel, and therefore the characterization of the mechanochemical behavior of hundreds of nucleic acids and enzymes. However, magnetic tweezers experiments require an accurate force calibration to extract quantitative data, which is limited to low forces if the deleterious effect of the finite camera open shutter time (tau(sh)) is not corrected. Here, we provide a simple method to perform correction-free force calibration for high-throughput magnetic tweezers at low image acquisition frequency (f(ac)). By significantly reducing tau(sh) to at least 4-fold the characteristic times of the tethered magnetic bead, we accurately evaluated the variance of the magnetic bead position along the axis parallel to the magnetic field, estimating the force with a relative error of similar to 10% (standard deviation), being only limited by the bead-to-bead difference. We calibrated several magnets - magnetic beads configurations, covering a force range from similar to 50 fN to similar to 60 pN. In addition, for the presented configurations, we provide a table with the mathematical expressions that describe the force as a function of the magnets position.

Controlled generation of intrinsic near-infrared color centers in 4H-SiC via proton irradiation and annealing

M. Ruehl, C. Ott, Stephan Götzinger, M. Krieger, H.B. Weber

We report on the generation and annihilation of color centers in 4H silicon carbide (SiC) by proton irradiation and subsequent annealing. Using low-temperature photoluminescence (PL), we study the transformation of PL spectra for different proton doses and annealing temperatures. Among well reported defect signatures, we observe omnipresent but not yet identified PL signatures consisting of three sharp and temperature stable lines (denoted TS1,2,3) at 768.8 nm, 812.0 nm, and 813.3 nm. These lines show a strong correlation throughout all measurement parameters, suggesting that they belong to the same microscopic defect. Further, a clear dependence of the TS1,2,3 line intensities on the initial implantation dose is observed after annealing, indicating that the underlying defect is related to implantation induced intrinsic defects. The overall data suggest a sequential defect transformation: proton irradiation initially generates isolated silicon vacancies which are transformed into antisite vacancy complexes which are, in turn, transformed into presumably intrinsic-related defects, showing up as TS1,2,3 PL lines. We present recipes for the controlled generation of these color centers. Published by AIP Publishing.

High-Speed Microscopy of Diffusion in Pore-Spanning Lipid Membranes

Susann Spindler, Jeremias Sibold, Reza Gholami Mahmoodabadi, Claudia Steinem, Vahid Sandoghdar

Pore-spanning membranes (PSMs) provide a highly attractive model system for investigating fundamental processes in lipid bilayers. We measure and compare lipid diffusion in the supported and suspended regions of PSMs prepared on a microfabricated porous substrate. Although some properties of the suspended regions in PSMs have been characterized using fluorescence studies, it has not been possible to examine the mobility of membrane components on the supported membrane parts. Here, we resolve this issue by employing interferometric scattering microscopy (iSCAT). We study the location-dependent diffusion of DOPE 1,2-dioleoylsn-glycero-3-phosphoethanolamine) lipids (DOPE) labeled with gold nanoparticles in (l,2-dioleoyl-sn-glycero-3-phosphocholine) (DOPC) bilayers prepared on holey silicon nitride substrates that were either (i) oxygen-plasma-treated or (ii) functionalized with gold and 6-mercapto-l-hexanol. For both substrate treatments, diffusion in regions suspended on pores with diameters of 5 mu m is found to be free. In the case of functionalization with gold and 6-mercapto-l-hexanol, similar diffusion coefficients are obtained for both the suspended and the supported regions, whereas for oxygen-plasma-treated surfaces, diffusion is almost 4 times slower in the supported parts of the membranes. We attribute this reduced diffusion on the supported parts in the case of oxygen-plasma-treated surfaces to larger membrane-substrate interactions, which lead to a higher membrane tension in the freestanding membrane parts. Furthermore, we find clear indications for a decrease of the diffusion constant in the freestanding regions away from the pore center. We provide a detailed characterization of the diffusion behavior in these membrane systems and discuss future directions.

Genetic noise mechanism for power-law switching in bacterial flagellar motors

M. I. Krivonosov, Vasily Zaburdaev, S. V. Denisov, M. V. Ivanchenko

Switching of the direction of flagella rotations is the key control mechanism governing the chemotactic activity of E. coli and many other bacteria. Power-law distributions of switching times are most peculiar because their emergence cannot be deduced from simple thermodynamic arguments. Recently, it was suggested that by adding finite-time correlations into Gaussian fluctuations regulating the energy height of the barrier between the two rotation states, it is possible to generate switching statistics with an intermediate power-law asymptotics. By using a simple model of a regulatory pathway, we demonstrate that the required amount of correlated 'noise' can be produced by finite number fluctuations of reacting protein molecules, a condition common to the intracellular chemistry. The corresponding power-law exponent appears as a tunable characteristic controlled by parameters of the regulatory pathway network such as the equilibrium number of molecules, sensitivities, and the characteristic relaxation time.

Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria

David Dulin, David L. V. Bauer, Anssi M. Malinen, Jacob J. W. Bakermans, Martin Kaller, Zakia Morichaud, Ivan Petushkov, Martin Depken, Konstantin Brodolin, et al.

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription.

Manipulation of Quenching in Nanoantenna–Emitter Systems Enabled by External Detuned Cavities: A Path to Enhance Strong-Coupling

Burak Gürlek, Vahid Sandoghdar, Diego-Martin Cano

We show that a broadband Fabry Perot microcavity can assist an emitter coupled to an off-resonant plasmonic nanoantenna to inhibit the nonradiative channels that affect the quenching of fluorescence. We identify the interference mechanism that creates the necessary enhanced couplings and bandwidth narrowing of the hybrid resonance and show that it can assist entering into the strong coupling regime. Our results provide new possibilities for improving the efficiency of solid-state emitters and accessing diverse realms of photophysics with hybrid structures that can be fabricated using existing technologies.

Visualizing single-cell secretion dynamics with single protein sensitivity

Matthew Paul McDonald, André Gemeinhardt, Katharina König, Marek Piliarik, Stefanie Schaffer, Simon Völkl, Andreas Mackensen, Vahid Sandoghdar

Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa-1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity.