Mechanical forces play a pivotal role in cellular adhesions, where a vast array of proteins interacts with the cell cytoskeleton, affecting focal adhesions and adherens junctions, and regulating cell behavior and fate. To directly observe such events, we developed an experimental assay that combines several advanced single molecule techniques. Here, ultrafast force-clamp spectroscopy is employed to directly probe the force-dependence of molecular interactions between a single actin filament and a binding protein with sub-ms time resolution. Stabilization of the microscope through local gradient localization enables the resolution of protein conformational changes and binding position with sub-nm accuracy. An experimental arrangement, termed oriented dumbbell, allows us to determine the actin filament orientation and, thus, asymmetries in the force response of the interacting proteins. We applied our methodology to the interaction between α-catenin and F-actin, revealing that α-catenin switches between a slip and an asymmetric cooperative catch-bond with F-actin. This mechanism may underlie fluid-to-solid phase transitions that occur at the membrane-cytoskeleton interface.
Highly Inclined and Laminated Optical sheet (HILO) microscopy is an optical technique that employs a highly inclined laser beam to illuminate the sample with a thin sheet of light that can be scanned through the sample volume1 . HILO is an efficient illumination technique when applied to fluorescence imaging of thick samples owing to the confined illumination volume that allows high contrast imaging while retaining deep scanning capability in a wide-field configuration. The restricted illumination volume is crucial to limit background fluorescence originating from portions of the sample far from the focal plane, especially in applications such as single molecule localization and super-resolution imaging2-4. Despite its widespread use, current literature lacks comprehensive reports of the actual advantages of HILO in these kinds of microscopies. Here, we thoroughly characterize the propagation of a highly inclined beam through fluorescently labeled samples and implement appropriate beam shaping for optimal application to single molecule and super-resolution imaging. We demonstrate that, by reducing the beam size along the refracted axis only, the excitation volume is consequently reduced while maintaining a field of view suitable for single cell imaging. We quantify the enhancement in signal-tobackground ratio with respect to the standard HILO technique and apply our illumination method to dSTORM superresolution imaging of the actin and vimentin cytoskeleton. We define the conditions to achieve localization precisions comparable to state-of-the-art reports, obtain a significant improvement in the image contrast, and enhanced plane selectivity within the sample volume due to the further confinement of the inclined beam.
Mechanical signals occurring at the interface between cell membrane and extracellular matrix and at intercellular junctions trigger biochemical signals that are fundamental for cell growth, development and regulation. Adaptor proteins, which link the cell membrane to the actin cytoskeleton, seem to partake in this process of mechanotransduction. In particular, catenins play a key role in intercellular junctions, where they act as a bridge between the cell membrane and actin. Studies suggest that α-catenin contains a domain that normally masks vinculin binding sites, which can become accessible after a conformational change induced by an external force. Here we demonstrate a single-molecule technique for investigating actin-protein interactions at different forces (up to ~17 pN) with adequate temporal resolution (sub-ms). This system is based on the ultrafast force-clamp spectroscopy technique that has been recently developed by our group and is adapted to study and measure force-dependent kinetics of the catenin-actin interaction, as well as the amplitude of the expected conformational changes such as force-induced protein unfolding.
We assembled an ultra-fast infrared optical trapping system to detect mechanical events that occur less than a millisecond
after a ligand binds to its filamentous substrate, such as myosin undergoing its 5 – 10 nm working stroke after actin binding.
The instrument is based on the concept of Capitanio et al.1, in which a polymer bead-actin-bead dumbbell is held in two
force-clamped optical traps. A force applied by the traps causes the filament to move at a constant velocity as
hydrodynamic drag balances the applied load. When the ligand binds, the filament motion stops within 100 μs as the total
force from the optical traps is transferred to the attachment. Subsequent translations signal active motions, such as the
magnitude and timing of the motor’s working stroke. In our instrument, the beads defining the dumbbell are held in
independent force clamps utilizing a field-programmable gate array (FPGA) to update the trap beam positions at 250 kHz.
We found that in our setup, acousto-optical deflectors (AODs) steering the beams were unsuitable for this purpose due to
a slightly non-linear response in the beam intensity and deflection angle vs. the AOD ultra-sound wavelength, likely caused
by low-amplitude standing acoustic waves in the deflectors. These aberrations caused instability in the force feedback
loops leading to artefactual ~20 nm jumps in position. This type of AOD non-linearity has been reported to be absent in
electro-optical deflectors (EODs)2. We demonstrate that replacement of the AODs with EODs improves the performance
of our instrument. Combining the superior beam-steering capability of the EODs, force acquisition via back-plane
interferometry, and the dual high-speed FPGA-based feedback loops, we smoothly and precisely apply constant loads to
study the dynamics of interactions between biological molecules such as actin and myosin.
Here we present development of optical techniques for the study of single processive myosin motors based on the combination of high-speed optical tweezers force spectroscopy and single molecule fluorescence imaging. Ultrafast force-clamp spectroscopy1 is applied to study the dependence of single chemo-mechanical steps of processive myosin motors on the applied load. On the other hand, single molecule localization through FIONA (Fluorescence Imaging with One Nanometer Accuracy)2, 3 is applied to in vitro motility assay to measure parameters such as the runlength, velocity and step size of single myosin V motors, labeled with Quantum Dots, under unloaded conditions.
Recently, tremendous improvements have been achieved in the precision of localization of single fluorescent molecules, allowing localization and tracking of biomolecules at the nm level. Since the behaviour of proteins and biological molecules is tightly influenced by the cell’s environment, a growing number of microscopy techniques are moving from in vitro to live cell experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). To satisfy these requirements we developed an automated routine that allow 3D tracking of single fluorescent molecules in living cells with nanometer accuracy, by exploiting the properties of the point-spread-function of out-of-focus Quantum Dots bound to the protein of interest.
Since the behaviour of proteins and biological molecules is tightly related to cell’s environment, more and more microscopy techniques are moving from in vitro to in living cells experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution. Since protein dynamics inside a cell involve all three dimensions, we developed an automated routine for 3D tracking of single fluorescent molecules inside living cells with nanometer accuracy, by exploiting the properties of the point-spread-function of out-of-focus Quantum Dots bound to the protein of interest.
The lac operon is a well-known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). We recently developed an ultrafast force-clamp laser trap technique capable of probing molecular interactions with sub-ms temporal resolution, under controlled pN-range forces. With this technique, we tested the interaction of LacI with different DNA constructs. Based on position along the DNA sequence, the observed interactions can be interpreted as specific binding to operator sequences and transient interactions with nonspecific sequences.
The lac operon is a well known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). LacI and other DNA-binding proteins bind their specific target sequences with rates higher than allowed by 3D diffusion alone. Generally accepted models predict a combination of free 3D diffusion and 1D sliding along non-specific DNA. We recently developed an ultrafast force-clamp laser trap technique capable of probing molecular interactions with sub-ms temporal resolution, under controlled pN-range forces. With this technique, we tested the interaction of LacI with two different DNA constructs: a construct with two copies of the O1 operator separated by 300 bp and a construct containing the native E.coli operator sequences. Our measurements show at least two classes of LacI-DNA interactions: long (in the tens of s range) and short (tens of ms). Based on position along the DNA sequence, the observed interactions can be interpreted as specific binding to operator sequences (long events) and transient interactions with nonspecific sequences (short events). Moreover, we observe continuous sliding of the protein along DNA, passively driven by the force applied with the optical tweezers.
Since the behaviour of proteins and biological molecules is tightly related to the cell’s environment, more and more microscopy techniques are moving from in vitro to in living cells experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). We developed an apparatus that combines different microscopy techniques to satisfy all the technical requirements for 3D tracking of single fluorescent molecules inside living cells with nanometer accuracy. To account for the optical sectioning of thick samples we built up a HILO (Highly Inclined and Laminated Optical sheet) microscopy system through which we can excite the sample in a widefield (WF) configuration by a thin sheet of light that can follow the molecule up and down along the z axis spanning the entire thickness of the cell with a SNR much higher than traditional WF microscopy. Since protein dynamics inside a cell involve all three dimensions, we included a method to measure the x, y, and z coordinates with nanometer accuracy, exploiting the properties of the point-spread-function of out-of-focus quantum dots bound to the protein of interest. Finally, a feedback system stabilizes the microscope from thermal drifts, assuring accurate localization during the entire duration of the experiment.
In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.
The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes,
critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The
mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety
of methods in biology. A large interest in these processes stems from the faster-than-diffusion association rates,
explained in current models by a combination of 3D and 1D diffusion. Here, we describe the combination of optical
tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the
opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in
other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence.
Optical tweezers allow recording mechanical data from single biological molecules such as molecular motors, DNA
processing enzymes, nucleic acids. Such data consist of time series that are dominated by thermal noise and such noisy
recordings require proper analysis to correctly extract kinetic and mechanical information. Several different analysis
approaches have been established in the past years. Here, we propose an analysis method for optical trapping recordings
of non-processive motor proteins. The method does not assume any particular interaction kinetics, allows detection of
sub-millisecond interactions and quantification of the number of false and lost events. Precise alignment of interaction
events and ensemble averaging allow the investigation of the stepping dynamics of non-processive motors with a
temporal resolution of few tens of microseconds and a spatial resolution of few angstroms. Our analysis is applied to the
study of the motor protein myosin from fast skeletal muscle. Thanks to the high spatio-temporal resolution, we can
distinguish three mechanical pathways in the acto-myosin interaction, with several orders of magnitude different
kinetics, which contribute in a load-dependent manner to the myosin working stroke.
Forces play a fundamental role in a wide array of biological processes, regulating enzymatic activity, kinetics of
molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the
investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular
complexes. We developed an ultrafast force-clamp spectroscopy technique that uses a dual trap configuration to apply
constant loads to a single intermittently interacting biological polymer and a binding protein. Our system displays a delay
of only ∼10 μs between formation of the molecular bond and application of the force and is capable of detecting
interactions as short as 100 μs. The force-clamp configuration in which our assay operates allows direct measurements of
load-dependence of lifetimes of single molecular bonds. Moreover, conformational changes of single proteins and
molecular motors can be recorded with sub-nanometer accuracy and few tens of microseconds of temporal resolution.
We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA
interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and
active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our
technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to
now, opening new perspectives for the investigation of the effects of forces on biological processes.
We describe a microscopy technique that, combining wide-field single molecule microscopy, bifocal imaging and Highly
Inclined and Laminated Optical sheet (HILO) microscopy, allows a 3D tracking with nanometer accuracy of single
fluorescent molecules in vitro and in living cells.
We recently developed an ultrafast force-clamp laser trap capable to probe, under controlled force, bimolecular
interactions with unprecedented temporal resolution. Here we present the technique in the framework of protein-DNA
interactions, specifically on Lactose repressor protein (LacI). The high temporal resolution of the method reveals the
kinetics of both short- and long-lived interactions of LacI along the DNA template (from ∼100 μs to tens of seconds), as
well the dependence on force of such interaction kinetics. The two kinetically well-distinct populations of interactions
observed clearly represent specific interactions with the operator sequences and a fast scanning of LacI along non-cognate
DNA. These results demonstrate the effectiveness of the method to study the sequence-dependent affinity of
DNA-binding proteins along the DNA and the effects of force on a wide range of interaction durations, including μs time
scales not accessible to other single-molecule methods. This improvement in time resolution provides also important
means of investigation on the long-puzzled mechanism of target search on DNA and possible protein conformational
changes occurring upon target recognition.
Forces play a fundamental role in a wide array of biological processes, regulating enzymatic activity, kinetics of
molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the
investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular
complexes. We developed an ultrafast force-clamp spectroscopy technique that uses a dual trap configuration to apply
constant loads to a single intermittently interacting biological polymer and a binding protein. Our system displays a delay
of only ∼10 μs between formation of the molecular bond and application of the force and is capable of detecting
interactions as short as 100 μs. The force-clamp configuration in which our assay operates allows direct measurements of
load-dependence of lifetimes of single molecular bonds. Moreover, conformational changes of single proteins and
molecular motors can be recorded with sub-nanometer accuracy and few tens of microseconds of temporal resolution.
We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA
interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and
active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our
technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to
now, opening new perspectives for the investigation of the effects of forces on biological processes.
Here we report the effect of DNA tension on lac repressor 1D-diffusion through a combination of single-molecule
localization and optical trapping. The diffusion coefficient shows a parabolic dependence on DNA tension.
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