Name: mass-sensitive particle tracking to characterize membrane-associated macromolecule dynamics
Uploaded: 2022-02-18T15:00:00
Duration: 13 min 30 s
Description: Watch this Scientific Journal Video about Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics at JoVE.com

We sincerely appreciate the support from Philipp Kukura, Gavin Young, and the Refeyn software team and acknowledge their assistance by sharing parts of the image analysis code. We thank the Cryo-EM MPIB Core Facility for providing access to the commercial Refeyn mass photometer. F.S. gratefully acknowledges the support and funding granted by J&#252;rgen Plitzko and Wolfgang Baumeister. T.H. and P.S. received funding through the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID 201269156 - SFB 1032 (A09). N.H. was supported by a DFG return grant HU 2462/3-1. P.S. acknowledges support through the research network MaxSynBio via the joint funding initiative of the German Federal Ministry of Education and Research (BMBF) and the Max Planck Society.

<ol>
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The authors have no conflicts of interest.

The presented protocol extends mass photometry21, a technique that analyzes the mass of single biomolecules adsorbing on glass, to an even more versatile tool capable of simultaneously measuring the mass and diffusion of unlabeled membrane-interacting biomolecules. This analysis extension is achieved through the implementation of a modified background removal strategy adapted to the lateral motion of molecules24,25. In general, the background removal is of utmost importance for iSCAT-based approaches, since the strong scattering of the glass surface roughness represents the main analysis impediment, and the accurate determination of each pixel&#39;s local background is essential for quantification of particle mass and location. Besides the image-analysis adapted to particle motion, subsequent particle detection, trajectory linking, and data analysis complete the novel expansion of MP into mass-sensitive particle tracking (MSPT).
In general, thoroughly cleaned glass cover slides and a clean working environment are critical requirements for the successful performance of MSPT experiments. Due to the absence of macromolecule labeling, the acquired signal is inherently nonselective. Clean samples, as well as proper sample handling, are hence crucial to ensure that observations cannot be misinterpreted. In particular, when molecules of low molecular weight are examined, control measurements of protein-free membranes are endorsed to assess background contributions (Supplementary Figure 1). Besides the inclusion of control measurements, it is thus recommended to follow the preparation steps displayed in Figure 2 for each flow chamber. When combined, these safety measures will ensure that the detected signal originates from the biomolecule of interest and not, for example, a contaminated flow chamber, buffer, or membrane.
Besides the precautions regarding the experimental design, care needs to also be taken during MSPT image processing. During video processing, the value for three parameters should be chosen carefully to ensure correct results: i) the length of the median window for background removal, ii) the threshold for particle detection, and iii) the maximum search radius during the linking assignment. A larger median window (i) generally facilitates the separation of diffusing particles from the superimposed quasi-constant background. However, for too large window sizes sample drift will eventually become noticeable and diminish the accuracy of the background estimation. Optimal settings heavily depend on sample properties and measurement conditions. Nevertheless, a value of 1,001 can be used as a robust starting point. The threshold parameter (ii) must be tuned depending on the lowest molecular mass expected in the sample. A value below 0.0005 is not recommended for measurements taken with the mass photometer used in this study. To speed up analysis times, higher values can be chosen if a sample with high molecular weight is expected. The search radius in trajectory linking (iii) specifies the maximum radial distance in pixels in which the particle&#39;s shifted location will be searched for in consecutive frames. It should be adapted to the fastest particle in the sample, and, if favored, an adaptive search range (see documentation of trackpy) could be used instead to reduce computation time. Especially during the initial phase of a project, re-analyzing the movies with varying parameters is recommended to validate the obtained results.
In light of the single-molecule nature of MSPT, it should be avoided to measure at high membrane particle densities as those can interfere with accurate contrast and mass determination. It has been shown that densities below one particle per squared micrometer are favorable for MSPT measurements24. An additional consideration is the expected diffusion coefficients in the sample. Although applicable to a broad range of diffusion coefficients, MSPT has a lower limit of accessible diffusion coefficients. Local confinement to a region of few pixels during a significant portion of the median window period merges the particle with the static background. For the imaging conditions used in this protocol, measurement of diffusion coefficients below 0.01 &#181;m2/s is not recommended. At this diffusion speed, for example, the mean squared displacement of a particle during the median window half-size is about 4 pixels and hence of similar size as the extent of the PSF. As a consequence, the static background estimate is likely to contain signal contributions from the particle itself, which results in an apparently reduced contrast of the particle until it eventually approaches the noise level. However, macromolecule diffusion coefficients ranging between 0.05 and 10 &#181;m2/s can clearly be resolved.
To further extend the range of MSPT applications, one can envision an advancement of the median-based background algorithm through the elimination of pixels that are temporarily occupied with a particle, or by sample drift correction enabling larger median window sizes. Both approaches would alleviate the problems regarding measurements at high particle densities and slow diffusion. Improvements in terms of lower mass sensitivity are on the horizon with a new generation of mass photometers, which may provide access to biomolecules smaller than 50 kDa. Therefore, future MSPT experiments will be able to study single-molecule dynamics and membrane-related interactions for an even wider range of membrane mimicries such as cushioned bilayers and macromolecular systems.

Once merely perceived as a barrier against the wide range of ambient physical conditions, biological membranes are nowadays considered functional entities and catalytic platforms1,2. Based on their ability to localize, amplify, and direct signals in response to membrane-associated macromolecule reactions, lipid interfaces constitute a crucial element for a wide variety of cellular processes such as membrane trafficking and signaling cascades3,4,5. Serving as a nucleation site for the assembly of stable complexes, membrane attachment often relies on a dynamic equilibrium between membrane-associated and cytosolic forms of macromolecules and is hence of transient nature6,7.
Despite their great importance in biology, it has so far been challenging to develop methods that can provide access to the compositional, spatial, and temporal heterogeneities of membrane-associated macromolecule reactions in real-time7,8. To resolve the underlying molecular processes, two experimental aspects are decisive: sufficient time resolution and single-particle sensitivity. Therefore, ensemble average techniques such as fluorescence recovery after photobleaching (FRAP) but also the much more sensitive fluorescence correlation spectroscopy (FCS) do have limitations, since they largely uncouple spatial information from temporal information9. An important step toward the characterization of individual molecule dynamics has thus been the advent of single-particle tracking (SPT) in combination with highly sensitive microscopy. In particular, two SPT approaches have proven to be effective in this regard. Firstly, the utilization of fluorophores as labels and the corresponding fluorescence detection systems paved the way for nanometer precision and millisecond time resolution10,11,12. Secondly, scattering-based detection using gold nanoparticles improved both localization precision and time resolution to the sub-nanometer and microsecond range, respectively13,14,15,16. Despite the many advantages of both approaches and their significant contributions regarding the mechanistic understanding of membrane-associated systems17,18, both techniques have so far been limited:&#160;they require labeling of the molecules of interest, which potentially perturbs their native behavior and are&#160;insensitive to the molecular composition of membrane-associated particles19,20.
Both these limitations have recently been overcome by the introduction of a novel interferometric scattering (iSCAT)-based approach termed mass photometry (MP)21,22,23. This technique allows the determination of in-solution mass distributions of biomolecules according to their iSCAT contrast when landing on a glass interface. However, for the detection and characterization of mobile molecules diffusing on lipid membranes, a more sophisticated image analysis approach had to be developed. This has meanwhile been successfully implemented and allows to detect, track, and determine the molecular mass of single unlabeled biomolecules diffusing on a lipid interface24,25. Referred to as dynamic mass photometry or mass-sensitive particle tracking (MSPT), this technique now enables the assessment of complex macromolecule interactions by directly recording changes in the molecular mass of the tracked entities and hence opens up new possibilities for the mechanistic analysis of membrane-associated molecular dynamics.
Here, a detailed protocol for sample preparation, imaging, and the data analysis pipeline required for MSPT is presented. In particular, sample requirements and potential problems that may occur during measurement and analysis are discussed. Furthermore, the unparalleled potential to analyze membrane-interacting macromolecule systems is showcased through various representative results.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>annexin V</td>
			<td>Sigma Aldrich</td>
			<td>#SRP8026</td>
			<td>examplary membrane-interacting protein</td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay</td>
			<td>Bio-Rad Laboratories Inc.</td>
			<td>#5000006</td>
			<td>bradford assay kit to determine protein stock concentrations</td>
		</tr>
		<tr>
			<td>biotin labeled bovine albumin</td>
			<td>Sigma Aldrich</td>
			<td>#A8549</td>
			<td>examplary protein that can be used as standard protein for MSPT</td>
		</tr>
		<tr>
			<td>cholera toxin subunit B</td>
			<td>Sigma Aldrich</td>
			<td>#SAE0069</td>
			<td>examplary membrane-interacting protein</td>
		</tr>
		<tr>
			<td>cover glasses, #1.5, 24 x 24 mm</td>
			<td>Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>#0102062</td>
			<td></td>
		</tr>
		<tr>
			<td>cover glasses, #1.5, 24 x 60 mm</td>
			<td>Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>#0102242</td>
			<td></td>
		</tr>
		<tr>
			<td>dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td>Avanti Polar Lipids</td>
			<td>#850375</td>
			<td>lipid - in the form of extruded small unilamellar vesicles required for supported lipid bilayer formation</td>
		</tr>
		<tr>
			<td>dioleoyl-sn-glycero-3-phosphoethanolamine-N-cap biotinyl (18:1 Biotinyl Cap PE</td>
			<td>Avanti Polar Lipids</td>
			<td>#870273</td>
			<td>lipid - in the form of extruded small unilamellar vesicles required for supported lipid bilayer formation</td>
		</tr>
		<tr>
			<td>dioleoyl-sn-glycero-3-phosphoglycerol (DOPG)</td>
			<td>Avanti Polar Lipids</td>
			<td>#840475</td>
			<td>lipid - in the form of extruded small unilamellar vesicles required for supported lipid bilayer formation</td>
		</tr>
		<tr>
			<td>dioleoyl-sn-glycero-3-phospho-L-serine (DOPS)</td>
			<td>Avanti Polar Lipids</td>
			<td>#840035</td>
			<td>lipid - in the form of extruded small unilamellar vesicles required for supported lipid bilayer formation</td>
		</tr>
		<tr>
			<td>double-sided tape</td>
			<td>tesa</td>
			<td>#57912-00000-02</td>
			<td>needed for the assembly of glass sample chambers</td>
		</tr>
		<tr>
			<td>Extruder</td>
			<td>Avanti Polar Lipids</td>
			<td>#610023</td>
			<td>Lipid extruder to enable monodisperse vesicle distributions</td>
		</tr>
		<tr>
			<td>EZ-Link Maleimide-PEG2-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>#A39261</td>
			<td>maileimide-fused biotin that can be used to biotinylate standard proteins for MSPT</td>
		</tr>
		<tr>
			<td>Fibronectin (Biotinylated)</td>
			<td>Cytoskeleton Inc.</td>
			<td>#FNR03-A</td>
			<td>examplary protein that can be used as standard protein for MSPT</td>
		</tr>
		<tr>
			<td>Gel Filtration HMW Calibration Kit</td>
			<td>Cytiva</td>
			<td>#28403842</td>
			<td>standard proteins, e.g. aldolase that can be biotinylated and used as molecular weight standards for MSPT</td>
		</tr>
		<tr>
			<td>GM1 Ganglioside (Brain, Ovine-Sodium Salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>#860065</td>
			<td>lipid - in the form of extruded small unilamellar vesicles required for supported lipid bilayer formation</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Biotin</td>
			<td>Thermo Fisher Scientific, Waltham, USA)</td>
			<td>#31820</td>
			<td>examplary protein to highlight the existence of different protein states</td>
		</tr>
		<tr>
			<td>Isopropanol, 99.5%, for spectroscopy</td>
			<td>Thermo Fisher Scientific</td>
			<td>#10003643</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Autofluorescence Immersion Oil</td>
			<td>Olympus K.K.</td>
			<td>#IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr>
			<td>pET21a-Streptavidin-Alive</td>
			<td>Addgene</td>
			<td>#20860</td>
			<td>required to express and purify divalent streptavidin in combination with each other</td>
		</tr>
		<tr>
			<td>pET21a-Streptavidin-Dead</td>
			<td>Addgene</td>
			<td>#20859</td>
			<td>required to express and purify divalent streptavidin in combination with each other</td>
		</tr>
		<tr>
			<td>Pierce Alkaline Phosphatase, biotinylated</td>
			<td>Thermo Fisher Scientific</td>
			<td>#29339</td>
			<td>examplary protein that can be used as standard protein for MSPT</td>
		</tr>
		<tr>
			<td>Pierce Protein A, Biotinylated</td>
			<td>Thermo Fisher Scientific</td>
			<td>#29989</td>
			<td>examplary protein that can be used as standard protein for MSPT</td>
		</tr>
		<tr>
			<td>Refeyn Acquire</td>
			<td>Refeyn Ltd.</td>
			<td></td>
			<td>control software for Refeyn OneMP</td>
		</tr>
		<tr>
			<td>Refeyn One</td>
			<td>Refeyn Ltd.</td>
			<td>-</td>
			<td>mass photometer</td>
		</tr>
		<tr>
			<td>sterile syringe filters 0.45 &#181;m cellulose acetate membrane</td>
			<td>VWR International</td>
			<td>#514-0063</td>
			<td>needed to filter particles from the buffer of interest</td>
		</tr>
		<tr>
			<td>tetravalent streptavidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>#SNN1001</td>
			<td>tetravalent streptavidin to enable the presence of several biotin binding sites</td>
		</tr>
		<tr>
			<td>Whatman Nuclepore Hydrophilic Membrane, 0.05 &#181;m Pore Size, 25 mm Circle</td>
			<td>Cytiva</td>
			<td>#110603</td>
			<td>a pore size of 50 nm is recommended for supported lipid bilayer formation in the context of MSPT</td>
		</tr>
		<tr>
			<td>Zepto model 2 plasma cleaner</td>
			<td>Diener electronic GmbH</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

mass-sensitive particle tracking to characterize membrane-associated macromolecule dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

Watch this Scientific Journal Video about Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics at JoVE.com

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...

Research

JoVE Journal

Biochemistry

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Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method ...