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Force measuring optical tweezers system for long time measurements of P pili stability
Umeå University, Faculty of Science and Technology, Department of Physics.
Umeå University, Faculty of Science and Technology, Department of Physics.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Uhlin)
Umeå University, Faculty of Science and Technology, Department of Physics.
2006 (English)In: Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV / [ed] Farkas, DL, Nicolau, DV, Leif, RC, 2006, Vol. 6088, 608810- p.Conference paper (Refereed)
Abstract [en]

A force-measuring optical tweezers instrumentation and long time measurements of the elongation and retraction of bacterial fimbriae from Uropathogenic E. coli (UPEC) under strain are presented. The instrumentation is presented in some detail. Special emphasis is given to measures taken to reduce the influence of noise and drifts in the system and from the surrounding, which makes long term force measurements possible. Individual P pili from UPEC bacteria were used as a biological model system for repetitive unfolding and refolding cycles of bacterial fimbriae under equilibrium conditions. P pili have evolved into a three-dimensional helix-like structure, the PapA rod, that can be successively and significantly elongated and/or unfolded when exposed to external forces. The instrumentation is used for characterization of the force-vs.-elongation response of the PapA rod of individual P pili, with emphasis on the long time stability of the forced unfolding and refolding of the helical structure of the PapA rod. The results show that the PapA rod is capable of withstanding extensive strain, leading to a complete unfolding of the helical structure, repetitive times during the life cycle of a bacterium without any noticeable alteration of the mechanical properties of the P pili. This function is believed to be importance for UPEC bacteria in vivo since it provides a close contact to a host cell (which is an initial step of invasion) despite urine cleaning attempts.

Place, publisher, year, edition, pages
2006. Vol. 6088, 608810- p.
, Proceedings of the society of photo-optical instrumentation engineers (SPIE), ISSN 0277-786X ; 6088
Keyword [en]
force measuring optical tweezers, bacterial fimbriae, E. coli, P pili, PapA, unfolding and refolding, macromolecules
National Category
Other Physics Topics
URN: urn:nbn:se:umu:diva-2746DOI: 10.1117/12.642266ISI: 000237107800032ScopusID: 2-s2.0-33646201092ISBN: 0-8194-6130-XISBN: 978-081946130-8OAI: diva2:141005
Conference on Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV. San Jose, CA. JAN 23-25, 2006. SPIE.
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2014-11-18Bibliographically approved
In thesis
1. Construction of force measuring optical tweezers instrumentation and investigations of biophysical properties of bacterial adhesion organelles
Open this publication in new window or tab >>Construction of force measuring optical tweezers instrumentation and investigations of biophysical properties of bacterial adhesion organelles
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Optical tweezers are a technique in which microscopic-sized particles, including living cells and bacteria, can be non-intrusively trapped with high accuracy solely using focused light. The technique has therefore become a powerful tool in the field of biophysics. Optical tweezers thereby provide outstanding manipulation possibilities of cells as well as semi-transparent materials, both non-invasively and non-destructively, in biological systems. In addition, optical tweezers can measure minute forces (< 10-12 N), probe molecular interactions and their energy landscapes, and apply both static and dynamic forces in biological systems in a controlled manner. The assessment of intermolecular forces with force measuring optical tweezers, and thereby the biomechanical structure of biological objects, has therefore considerably facilitated our understanding of interactions and structures of biological systems.

Adhesive bacterial organelles, so called pili, mediate adhesion to host cells and are therefore crucial for the initial bacterial-cell contact. Thus, they serve as an important virulence factor. The investigation of pili, both their biogenesis and their expected in vivo properties, brings information that can be of importance for the design of new drugs to prevent bacterial infections, which is crucial in the era of increased bacterial resistance towards antibiotics.

In this thesis, an experimental setup of a force measuring optical tweezers system and the results of a number of biomechanical investigations of adhesive bacterial organelles are presented. Force measuring optical tweezers have been used to characterize three different types of adhesive organelles under various conditions, P, type 1, and S pili, which all are expressed by uropathogenic Escherichia coli. A quantitative biophysical force-extension model, built upon the structure and force response, has been developed. It is found, that this model describes the biomechanical properties for all three pili in an excellent way. Various parameters in their energy landscape, e.g., bond lengths and transition barrier heights, are assessed and the difference in behavior is compared. The work has resulted in a method that in a swift way allows us to probe different types of pili with high force and high spatial resolution, which has provided an enhanced understanding of the biomechanical function of these pili.

Abstract [sv]

Optisk pincett är en teknik i vilken mikrometerstora objekt, inkluderande levande celler och bakterier, beröringsfritt kan fångas och förflyttas med hög noggrannhet enbart med hjälp av ljus. Den optiska pincetten har därmed blivit ett kraftfullt verktyg inom biofysiken, som möjliggör enastående precisions-manipulering av celler och semi-transparenta objekt. Dessutom kan denna manipulation göras intracellulärt, dvs. utan att fysiskt öppna eller penetrera cellernas membran. Den optiska pincetten kan även mäta mycket små krafter och interaktioner (< 10-12 N) samt applicera både statiska och dynamiska krafter i biologiska system med utmärkt precision. Optisk pincett är därför en utmärkt teknik för mätning av intermolekylära krafter och för bestämning av biomekaniska strukturer och dess funktioner.

Vissa typer av bakterier har specifika vidhäftningsorganeller som kallas för pili. Dessa förmedlar vidhäftningen till värdceller och är därför viktiga vid bakteriens första kontakt. En djupare förståelse av pilis uppbyggnad och biomekanik kan därmed ge information, som kan vara vital i framtagandet av nya mediciner som förhindrar bakteriella infektioner. Detta är av stor vikt i skenet av den ökande antibiotikaresistensen i vårt samhälle.

I denna avhandling presenteras konstruktionen av en experimentell uppställning av kraftmätande optiskt pincett tillsammans med resultat från biomekaniska undersökningar av vidhäftande bakteriella organeller. Kraftmätande optisk pincett har använts för att karakterisera tre olika typer av pili, P, typ 1, och S pili, vilka kan uttryckas av uropatogena Escherichia coli. En kvantitativ biofysikalisk modell som beskriver deras förlängningsegenskaper under pålagd kraft har konstruerats. Modellen bygger på pilis strukturella uppbyggnad samt på dess respons som uppmäts med den kraftmätande optiska pincetten. Modellen beskriver de biomekaniska egenskaperna väl för alla tre pili. Dessutom kan ett antal specifika bindnings- och subenhetsparametrar bestämmas, t.ex. interaktionsenergier och bindningslängder. Skillnaden mellan dessa parametrar hos de tre pilis samt deras olika kraftrespons har jämförts. Detta arbete har dels resulterat i en förbättrad förståelse av pilis biomekaniska funktion och dels i en metod som, med hög noggrannhet, tillåter oss att bestämma ett antal biomekaniska egenskaper hos olika organeller på ett effektivt sätt.

Place, publisher, year, edition, pages
Umeå: Fysik, 2007. 79 p.
optical tweezers, biological physics, unfolding, Escherichia coli, force measurements, energy landscape, dynamic force spectroscopy, manipulation, polymers, pili
National Category
Physical Sciences
urn:nbn:se:umu:diva-1425 (URN)978-91-7264-435-9 (ISBN)
Public defence
2007-11-30, N450, Naturvetarhuset, Umeå Universitet Campus, Umeå, 13:00 (English)
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2009-08-19Bibliographically approved

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