Combined optical trapping and nanometer-precision localization for the single-molecule study of DNA-binding proteins

Mónico, Carina; Belcastro, Gionata; Capitanio, Marco; Vanzi, Francesco

doi:10.1109/iwbp.2011.5954832

Cited by 8 publications

(3 citation statements)

References 8 publications

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“…We employed a custom-built microscopy setup with single-molecule imaging capabilities to perform quantitative PALM on E. coli TV001 cells in various conditions 29,58,59 . 405 nm and 532 nm diode lasers were employed to, www.nature.com/scientificreports/ respectively, photoactivate PAmCherry1 and excite the fluorescence of PAmCherry1 in its activated state.…”

Section: Imaging Setupmentioning

confidence: 99%

Highly inclined light sheet allows volumetric super-resolution imaging of efflux pumps distribution in bacterial biofilms

Vignolini,

Capitanio,

Caldini

et al. 2024

Sci Rep

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Bacterial biofilms are highly complex communities in which isogenic bacteria display different gene expression patterns and organize in a three-dimensional mesh gaining enhanced resistance to biocides. The molecular mechanisms behind such increased resistance remain mostly unknown, also because of the technical difficulties in biofilm investigation at the sub-cellular and molecular level. In this work we focus on the AcrAB-TolC protein complex, a multidrug efflux pump found in Enterobacteriaceae, whose overexpression is associated with most multiple drug resistance (MDR) phenotypes occurring in Gram-negative bacteria. We propose an optical method to quantify the expression level of the AcrAB-TolC pump within the biofilm volume at the sub-cellular level, with single-molecule sensitivity. Through a combination of super-resolution PALM with single objective light sheet and precision genome editing, we can directly quantify the spatial distribution of endogenous AcrAB-TolC pumps expressed in both planktonic bacteria and, importantly, within the bacterial biofilm volume. We observe a gradient of pump density within the biofilm volume and over the course of biofilm maturation. Notably, we propose an optical method that could be broadly employed to achieve volumetric super-resolution imaging of thick samples.

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Section: Imaging Setupmentioning

confidence: 99%

Highly inclined light sheet allows volumetric super-resolution imaging of efflux pumps distribution in bacterial biofilms

Vignolini,

Capitanio,

Caldini

et al. 2024

Sci Rep

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“…The sample (S) can be positioned through piezo translators (x-y and z) with nm accuracy and imaged on a CCD camera (CCD 200X). The apparatus is stabilized to less than 1 nm with both passive and active stabilization [10][11][12] . The bead where the myosin molecule lies is used as a landmark to determine the sample coordinates (CCD 2000X) and correct for thermal drifts and low-frequency noise by moving piezo translators (nmstabilization feedback in Fig.…”

Section: Operation Principlementioning

confidence: 99%

Force-clamp laser trapping of rapidly interacting molecules

Capitanio

Mónico²,

Vanzi

et al. 2013

SPIE Proceedings

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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.

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“…Single-molecule techniques have provided crucial contributions to the biophysical characterization of protein-DNA interactions and target search mechanisms 5,6 . These methods are usually based on single-molecule imaging of fluorescently labeled proteins, which are capable of reconstructing the protein trajectory along the DNA molecule with nanometer accuracy [7][8][9][10][11] . Nevertheless, the localization of single molecules by fluorescence imaging demonstrates an intrinsic compromise between localization accuracy and temporal resolution.…”

Section: Introductionmentioning

confidence: 99%

Mechanics of protein-DNA interaction studied with ultra-fast optical tweezers

Mónico¹,

Tempestini²,

Vanzi³

et al. 2014

Biophotonics: Photonic Solutions for Better Health Care IV

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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.

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Combined optical trapping and nanometer-precision localization for the single-molecule study of DNA-binding proteins

Cited by 8 publications

References 8 publications

Highly inclined light sheet allows volumetric super-resolution imaging of efflux pumps distribution in bacterial biofilms

Highly inclined light sheet allows volumetric super-resolution imaging of efflux pumps distribution in bacterial biofilms

Force-clamp laser trapping of rapidly interacting molecules

Mechanics of protein-DNA interaction studied with ultra-fast optical tweezers

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