Biology subjects

Kukura, P.

Publications and source records attributed to Kukura, P..

5 recordsLinked to original sources

bioRxiv2018

Label-free visualization of type IV pili dynamics by interferometric scattering microscopy

Type IV pili (TFP) are slender objects that assemble by polymerization and secretion of protein subunits from bacterial cell surfaces. The mechanisms by which these surface structures of microscopic length and molecular diameter modulate the physical interaction of bacteria with their environment, however, remains poorly understood largely due to limitations in our ability to monitor and characterize the dynamics of individual TFP. Here, we demonstrate that interferometric scattering microscopy (iSCAT) enables label-free and dynamic visualization of TFP in intact cells of the opportunistic pathogen Pseudomonas aeruginosa. As a result, we can directly monitor extension, attachment and retraction events on millisecond timescale and nanometer length scale in three dimensions. These capabilities allow us to observe that P. aeruginosa is able to crawl against the direction of flow using short TFP filaments. Also, careful observation show that TFP retract rapidly after surface attachment, suggesting that P. aeruginosa senses contact of the pilus tip with the solid substrate. These results illustrate the power of iSCAT for the label-free visualization of small, dynamic microbial extracellular structures.

microbiology↗

bioRxiv2017

Complementary studies of lipid membrane dynamics using iSCAT and STED microscopy

Observation techniques with high spatial and temporal resolution, such as single-particle tracking (SPT) based on interferometric Scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye-tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work we have used lipid analogues tagged with a hybrid fluorescent tag - gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous Supported Lipid Bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50 [≤] t [≤] 100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye-tagged lipid analogues. These FCS measurements of hybrid fluorescent tag - gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2-3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially crosslinking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

biophysics↗

bioRxiv2017

Quantitative mass imaging of single molecules in solution

The cellular processes underpinning life are orchestrated by proteins and their interactions. Structural and dynamic heterogeneity, despite being key to protein and drug function, continues to pose a fundamental challenge to existing analytical and structural methodologies used to study these associations. Here, we use interferometric scattering microscopy to mass-image single biomolecules in solution with <2% mass error, up to 19-kDa resolution and 1-kDa precision. Thereby, we resolve oligomeric distributions at high dynamic range, detect small-molecule binding, and mass-image biomolecules composed not only of amino acids, but also heterogeneous species, such as lipo- and glycoproteins. These capabilities enable us to characterize the molecular mechanisms of processes as diverse as oligomeric selfassembly, glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry (iSCAMS) provides spatially resolved access to the dynamics of biomolecular interactions ranging from those involving small molecules to mesoscopic assemblies, one molecule at a time.

biophysics↗

bioRxiv2017

Revealing intricate acto-myosin dynamics at a membrane surface using interferometric scattering microscopy

The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood due to difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering (iSCAT) microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect, and image individual membrane attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as a function of ATP concentration, providing evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network. Statement of SignificanceHere, we show that interferometric scattering microscopy in combination with single particle tracking enables label-free, high contrast imaging of filament dynamics on surfaces, while distinguishing different species based on their mass. These results significantly broaden the available toolkit, and associated capabilities of researchers studying dynamics of biological machines at interfaces.

biophysics↗

bioRxiv2016

Revealing compartmentalised membrane diffusion in living cells with interferometric scattering microscopy

Single-particle tracking is a powerful tool for studying single molecule behaviour involving plasma membrane-associated events in cells. Here, we show that interferometric scattering microscopy (iSCAT) combined with gold nanoparticle labeling can be used to follow the motion of membrane proteins in the plasma membrane of live cultured mammalian cell lines and hippocampal neurons. The unique combination of microsecond temporal resolution and nanometer spatial precision reveals signatures of a compartmentalised plasma membrane in neurons.

biophysics↗