Publications by authors named "Matthias D Koch"

9 Publications

  • Page 1 of 1

Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili.

Proc Natl Acad Sci U S A 2021 02;118(8)

Department of Molecular Biology, Princeton University, Princeton, NJ 08540;

Type IV pili (TFP) function through cycles of extension and retraction. The coordination of these cycles remains mysterious due to a lack of quantitative measurements of multiple features of TFP dynamics. Here, we fluorescently label TFP in the pathogen and track full extension and retraction cycles of individual filaments. Polymerization and depolymerization dynamics are stochastic; TFP are made at random times and extend, pause, and retract for random lengths of time. TFP can also pause for extended periods between two extension or two retraction events in both wild-type cells and a slowly retracting PilT mutant. We developed a biophysical model based on the stochastic binding of two dedicated extension and retraction motors to the same pilus machine that predicts the observed features of the data with no free parameters. We show that only a model in which both motors stochastically bind and unbind to the pilus machine independent of the piliation state of the machine quantitatively explains the experimentally observed pilus production rate. In experimental support of this model, we show that the abundance of the retraction motor dictates the pilus production rate and that PilT is bound to pilus machines even in their unpiliated state. Together, the strong quantitative agreement of our model with a variety of experiments suggests that the entire repetitive cycle of pilus extension and retraction is coordinated by the competition of stochastic motor binding to the pilus machine, and that the retraction motor is the major throttle for pilus production.
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http://dx.doi.org/10.1073/pnas.2014926118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923367PMC
February 2021

Pseudomonas aeruginosa detachment from surfaces via a self-made small molecule.

J Biol Chem 2021 Jan-Jun;296:100279. Epub 2021 Jan 12.

Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA. Electronic address:

Pseudomonas aeruginosa is a significant threat in both healthcare and industrial biofouling. Surface attachment of P. aeruginosa is particularly problematic as surface association induces virulence and is necessary for the ensuing process of biofilm formation, which hampers antibiotic treatments. Previous efforts have searched for dispersal agents of mature biofilm collectives, but there are no known factors that specifically disperse individual surface-attached P. aeruginosa. In this study, we develop a quantitative single-cell surface-dispersal assay and use it to show that P. aeruginosa itself produces factors that can stimulate its dispersal. Through bioactivity-guided fractionation, mass spectrometry, and nuclear magnetic resonance, we elucidated the structure of one such factor, 2-methyl-4-hydroxyquinoline (MHQ). MHQ is an alkyl quinolone with a previously unknown activity and is synthesized by the PqsABC enzymes. Pure MHQ is sufficient to disperse P. aeruginosa, but the dispersal activity of natural P. aeruginosa conditioned media requires additional factors. Whereas other alkyl quinolones have been shown to act as antibiotics or membrane depolarizers, MHQ lacks these activities and known antibiotics do not induce dispersal. In contrast, we show that MHQ inhibits the activity of Type IV Pili (TFP) and that TFP targeting can explain its dispersal activity. Our work thus identifies single-cell surface dispersal as a new activity of P. aeruginosa-produced small molecules, characterizes MHQ as a promising dispersal agent, and establishes TFP inhibition as a viable mechanism for P. aeruginosa dispersal.
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http://dx.doi.org/10.1016/j.jbc.2021.100279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949062PMC
January 2021

Substrate-rigidity dependent migration of an idealized twitching bacterium.

Soft Matter 2019 Aug 23;15(30):6224-6236. Epub 2019 Jul 23.

Theoretical Soft Matter and Biophysics, Institute of Complex Systems and Institute for Advanced Simulation, Forschungszentrum Juelich, D-52425 Juelich, Germany.

Mechanical properties of the extracellular matrix are important determinants of cellular migration in diverse processes, such as immune response, wound healing, and cancer metastasis. Moreover, recent studies indicate that even bacterial surface colonization can depend on the mechanics of the substrate. Here, we focus on physical mechanisms that can give rise to substrate-rigidity dependent migration. We study a "twitcher", a cell driven by extension-retraction cycles, to idealize bacteria and perhaps eukaryotic cells that employ a slip-stick mode of motion. The twitcher is asymmetric and always pulls itself forward at its front. Analytical calculations show that the migration speed of a twitcher depends non-linearly on substrate rigidity. For soft substrates, deformations do not lead to build-up of significant force and the migration speed is therefore determined by stochastic adhesion unbinding. For rigid substrates, forced adhesion rupture determines the migration speed. Depending on the force-sensitivity of front and rear adhesions, forced bond rupture implies an increase or a decrease of the migration speed. A requirement for the occurrence of rigidity-dependent stick-slip migration is a "sticky" substrate, with binding rates being an order of magnitude larger than unbinding rates in absence of force. Computer simulations show that small stall forces of the driving machinery lead to a reduced movement on high rigidities, regardless of force-sensitivities of bonds. The simulations also confirm the occurrence of rigidity-dependent migration speed in a generic model for slip-stick migration of cells on a sticky substrate.
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http://dx.doi.org/10.1039/c9sm00541bDOI Listing
August 2019

Microfluidic-based transcriptomics reveal force-independent bacterial rheosensing.

Nat Microbiol 2019 08 13;4(8):1274-1281. Epub 2019 May 13.

Department of Molecular Biology, Princeton University, Princeton, NJ, USA.

Multiple cell types sense fluid flow as an environmental cue. Flow can exert shear force (or stress) on cells, and the prevailing model is that biological flow sensing involves the measurement of shear force. Here, we provide evidence for force-independent flow sensing in the bacterium Pseudomonas aeruginosa. A microfluidic-based transcriptomic approach enabled us to discover an operon of P. aeruginosa that is rapidly and robustly upregulated in response to flow. Using a single-cell reporter of this operon, which we name the flow-regulated operon (fro), we establish that P. aeruginosa dynamically tunes gene expression to flow intensity through a process we call rheosensing (as rheo- is Greek for flow). We further show that rheosensing occurs in multicellular biofilms, involves signalling through the alternative sigma factor FroR, and does not require known surface sensors. To directly test whether rheosensing measures force, we independently altered the two parameters that contribute to shear stress: shear rate and solution viscosity. Surprisingly, we discovered that rheosensing is sensitive to shear rate but not viscosity, indicating that rheosensing is a kinematic (force-independent) form of mechanosensing. Thus, our findings challenge the dominant belief that biological mechanosensing requires the measurement of forces.
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http://dx.doi.org/10.1038/s41564-019-0455-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656604PMC
August 2019

Dynamics of a Protein Chain Motor Driving Helical Bacteria under Stress.

Biophys J 2018 04;114(8):1955-1969

Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany. Electronic address:

The wall-less, helical bacterial genus Spiroplasma has a unique propulsion system; it is not driven by propeller-like flagella but by a membrane-bound, cytoplasmic, linear motor that consists of a contractile chain of identical proteins spanning the entire cell length. By a coordinated spread of conformational changes of the proteins, kinks propagate in pairs along the cell body. However, the mechanisms for the initiation or delay of kinks and their coordinated spread remain unclear. Here, we show how we manipulate the initiation of kinks, their propagation velocities, and the time between two kinks for a single cell trapped in an optical line potential. By interferometric three-dimensional shape tracking, we measured the cells' deformations in response to various external stress situations. We observed a significant dependency of force generation on the cells' local ligand concentrations (likely ATP) and ligand hydrolysis, which we altered in different ways. We developed a mechanistic, mathematical model based on Kramer's rates, describing the subsequent cooperative and conformational switching of the chain's proteins. The model reproduces our experimental observations and can explain deformation characteristics even when the motor is driven to its extreme. Nature has invented a set of minimalistic mechanical driving concepts. To understand or even rebuild them, it is essential to reveal the molecular mechanisms of such protein chain motors, which need only two components-coupled proteins and ligands-to function.
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http://dx.doi.org/10.1016/j.bpj.2018.02.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937200PMC
April 2018

Label-free Imaging and Bending Analysis of Microtubules by ROCS Microscopy and Optical Trapping.

Biophys J 2018 01;114(1):168-177

Laboratory for Bio- and Nano-Photonics, Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany. Electronic address:

Mechanical manipulation of single cytoskeleton filaments and their monitoring over long times is difficult because of fluorescence bleaching or phototoxic protein degradation. The integration of label-free microscopy techniques, capable of imaging freely diffusing, weak scatterers such as microtubules (MTs) in real-time, and independent of their orientation, with optical trapping and tracking systems, would allow many new applications. Here, we show that rotating-coherent-scattering microscopy (ROCS) in dark-field mode can also provide strong contrast for structures far from the coverslip such as arrangements of isolated MTs and networks. We could acquire thousands of images over up to 30 min without loss in image contrast or visible photodamage. We further demonstrate the combination of ROCS imaging with fast and nanometer-precise 3D interferometric back-focal-plane tracking of multiple beads in time-shared optical traps using acoustooptic deflectors to specifically construct and microrheologically probe small microtubule networks with well-defined geometries. Thereby, we explore the frequency-dependent elastic response of single microtubule filaments between 0.5 Hz and 5 kHz, which allows for investigating their viscoelastic response up to the fourth-order bending mode. Our spectral analysis reveals constant filament stiffness at low frequencies and frequency-dependent stiffening following a power law ∼ω with a length-dependent exponent p(L). We find further evidence for the dependence of the MT persistence length on the contour length L, which is still controversially debated. We could also demonstrate slower stiffening at high frequencies for longer filaments, which we believe is determined by the molecular architecture of the MT. Our results shed new light on the nanomechanics of this essential, multifunctional cytoskeletal element and pose new questions about the adaptability of the cytoskeleton.
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http://dx.doi.org/10.1016/j.bpj.2017.10.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773756PMC
January 2018

Force generation by groups of migrating bacteria.

Proc Natl Acad Sci U S A 2017 07 27;114(28):7266-7271. Epub 2017 Jun 27.

Lewis-Sigler Institute for Integrative Genomics, Princeton University, NJ 08544;

From colony formation in bacteria to wound healing and embryonic development in multicellular organisms, groups of living cells must often move collectively. Although considerable study has probed the biophysical mechanisms of how eukaryotic cells generate forces during migration, little such study has been devoted to bacteria, in particular with regard to the question of how bacteria generate and coordinate forces during collective motion. This question is addressed here using traction force microscopy. We study two distinct motility mechanisms of , namely, twitching and gliding. For twitching, powered by type-IV pilus retraction, we find that individual cells exert local traction in small hotspots with forces on the order of 50 pN. Twitching bacterial groups also produce traction hotspots, but with forces around 100 pN that fluctuate rapidly on timescales of <1.5 min. Gliding, the second motility mechanism, is driven by lateral transport of substrate adhesions. When cells are isolated, gliding produces low average traction on the order of 1 Pa. However, traction is amplified approximately fivefold in groups. Advancing protrusions of gliding cells push, on average, in the direction of motion. Together, these results show that the forces generated during twitching and gliding have complementary characters, and both forces have higher values when cells are in groups.
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http://dx.doi.org/10.1073/pnas.1621469114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514709PMC
July 2017

Single microtubules and small networks become significantly stiffer on short time-scales upon mechanical stimulation.

Sci Rep 2017 06 26;7(1):4229. Epub 2017 Jun 26.

Laboratory for Bio- and Nano-Photonics, Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 102, 79110, Freiburg, Germany.

The transfer of mechanical signals through cells is a complex phenomenon. To uncover a new mechanotransduction pathway, we study the frequency-dependent transport of mechanical stimuli by single microtubules and small networks in a bottom-up approach using optically trapped beads as anchor points. We interconnected microtubules to linear and triangular geometries to perform micro-rheology by defined oscillations of the beads relative to each other. We found a substantial stiffening of single filaments above a characteristic transition frequency of 1-30 Hz depending on the filament's molecular composition. Below this frequency, filament elasticity only depends on its contour and persistence length. Interestingly, this elastic behavior is transferable to small networks, where we found the surprising effect that linear two filament connections act as transistor-like, angle dependent momentum filters, whereas triangular networks act as stabilizing elements. These observations implicate that cells can tune mechanical signals by temporal and spatial filtering stronger and more flexibly than expected.
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http://dx.doi.org/10.1038/s41598-017-04415-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484680PMC
June 2017

Introduction to Optical Tweezers.

Methods Mol Biol 2017 ;1486:3-24

Joseph Henry Laboratory of Physics and Lewis-Sigler Institute for Integrative Genomics, Department of Physics, Princeton University, 244 Carl C. Icahn Laboratory, Washington Road, Princeton, NJ, 08544, USA.

Thirty years after their invention by Arthur Ashkin and colleagues at Bell Labs in 1986 [1], optical tweezers (or traps) have become a versatile tool to address numerous biological problems. Put simply, an optical trap is a highly focused laser beam that is capable of holding and applying forces to micron-sized dielectric objects. However, their development over the last few decades has converted these tools from boutique instruments into highly versatile instruments of molecular biophysics. This introductory chapter intends to give a brief overview of the field, highlight some important scientific achievements, and demonstrate why optical traps have become a powerful tool in the biological sciences. We introduce a typical optical setup, describe the basic theoretical concepts of how trapping forces arise, and present the quantitative position and force measurement techniques that are most widely used today.
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http://dx.doi.org/10.1007/978-1-4939-6421-5_1DOI Listing
January 2018
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