Real-time assembly of an artificial virus elucidated at the single-particle level

AbstractWhile the structure of a variety of viruses has been resolved at atomistic detail, their assembly pathways remain largely elusive. Key unresolved issues in assembly are the nature of the critical nucleus starting particle growth, the subsequent self-assembly reaction and the manner in which the viral genome is compacted. These issues are difficult to address in bulk approaches and are effectively only accessible by tracking the dynamics of assembly of individual particles in real time, as we show here. With a combination of single-molecule techniques we study the assembly into rod-shaped virus-like particles (VLPs) of artificial capsid polypeptides, de-novo designed previously. Using fluorescence optical tweezers we establish that oligomers that have pre-assembled in solution bind to our DNA template. If the oligomer is smaller than a pentamer, it performs one-dimensional diffusion along the DNA, but pentamers and larger oligomers are essentially immobile and nucleate VLP growth. Next, using real-time multiplexed acoustic force spectroscopy, we show that DNA is compacted in regular steps during VLP growth. These steps, of ∼30 nm of DNA contour length, fit with a DNA packaging mechanism based on helical wrapping of the DNA around the central protein core of the VLP. By revealing how real-time, single particle tracking of VLP assembly lays bare nucleation and growth principles, our work opens the doors to a new fundamental understanding of the complex assembly pathways of natural virus particles.

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Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells

Molecules ◽

10.3390/molecules24152826 ◽

2019 ◽

Vol 24 (15) ◽

pp. 2826 ◽

Cited By ~ 4

Author(s):

Shangguo Hou ◽

Courtney Johnson ◽

Kevin Welsher

Keyword(s):

Fluorescence Spectroscopy ◽

Real Time ◽

Temporal Resolution ◽

Single Molecule ◽

Particle Tracking ◽

Single Particle ◽

Single Particle Tracking ◽

Single Molecule Fluorescence ◽

Single Molecule Fluorescence Spectroscopy ◽

Active Feedback

Single molecule fluorescence spectroscopy has been largely implemented using methods which require tethering of molecules to a substrate in order to make high temporal resolution measurements. However, the act of tethering a molecule requires that the molecule be removed from its environment. This is especially perturbative when measuring biomolecules such as enzymes, which may rely on the non-equilibrium and crowded cellular environment for normal function. A method which may be able to un-tether single molecule fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resolution over large axial ranges. This review gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single molecule tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed.

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Revealing in real-time a multistep assembly mechanism for SV40 virus-like particles

Science Advances ◽

10.1126/sciadv.aaz1639 ◽

2020 ◽

Vol 6 (16) ◽

pp. eaaz1639 ◽

Cited By ~ 3

Author(s):

Mariska G. M. van Rosmalen ◽

Douwe Kamsma ◽

Andreas S. Biebricher ◽

Chenglei Li ◽

Adam Zlotnick ◽

...

Keyword(s):

Real Time ◽

Optical Tweezers ◽

Self Assembly ◽

Persistence Length ◽

Simian Virus 40 ◽

Simian Virus ◽

Contour Length ◽

Sv40 Virus ◽

Virus Like Particles ◽

Assembly Mechanism

Many viruses use their genome as template for self-assembly into an infectious particle. However, this reaction remains elusive because of the transient nature of intermediate structures. To elucidate this process, optical tweezers and acoustic force spectroscopy are used to follow viral assembly in real time. Using Simian virus 40 (SV40) virus-like particles as model system, we reveal a multistep assembly mechanism. Initially, binding of VP1 pentamers to DNA leads to a significantly decreased persistence length. Moreover, the pentamers seem able to stabilize DNA loops. Next, formation of interpentamer interactions results in intermediate structures with reduced contour length. These structures stabilize into objects that permanently decrease the contour length to a degree consistent with DNA compaction in wild-type SV40. These data indicate that a multistep mechanism leads to fully assembled cross-linked SV40 particles. SV40 is studied as drug delivery system. Our insights can help optimize packaging of therapeutic agents in these particles.

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Imaging the behavior of molecules in biological systems: breaking the 3D speed barrier with 3D multi-resolution microscopy

Faraday Discussions ◽

10.1039/c5fd00090d ◽

2015 ◽

Vol 184 ◽

pp. 359-379 ◽

Cited By ~ 7

Author(s):

Kevin Welsher ◽

Haw Yang

Keyword(s):

Real Time ◽

Single Molecule ◽

Particle Tracking ◽

Single Particle ◽

Pid Control ◽

Single Particle Tracking ◽

Three Dimensions ◽

Temporal Precision ◽

Optical Sensitivity ◽

Position Sensitive

The overwhelming effort in the development of new microscopy methods has been focused on increasing the spatial and temporal resolution in all three dimensions to enable the measurement of the molecular scale phenomena at the heart of biological processes. However, there exists a significant speed barrier to existing 3D imaging methods, which is associated with the overhead required to image large volumes. This overhead can be overcome to provide nearly unlimited temporal precision by simply focusing on a single molecule or particle via real-time 3D single-particle tracking and the newly developed 3D Multi-resolution Microscopy (3D-MM). Here, we investigate the optical and mechanical limits of real-time 3D single-particle tracking in the context of other methods. In particular, we investigate the use of an optical cantilever for position sensitive detection, finding that this method yields system magnifications of over 3000×. We also investigate the ideal PID control parameters and their effect on the power spectrum of simulated trajectories. Taken together, these data suggest that the speed limit in real-time 3D single particle-tracking is a result of slow piezoelectric stage response as opposed to optical sensitivity or PID control.

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Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2006900117 ◽

2020 ◽

Vol 117 (35) ◽

pp. 21328-21335

Author(s):

Zhijie Chen ◽

Alan Shaw ◽

Hugh Wilson ◽

Maxime Woringer ◽

Xavier Darzacq ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Single Molecule ◽

Particle Tracking ◽

Single Particle ◽

Theoretical Models ◽

Single Particle Tracking ◽

Correlation Spectroscopy ◽

Single Molecule Level ◽

Diffusion Phenomena ◽

Diffusion Enhancement

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments usingp-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish thatpNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

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Studying the Interaction of Receptor Tyrosine Kinases and Adaptor Proteins at the Single-Molecule Level with Single-Particle Tracking

Biophysical Journal ◽

10.1016/j.bpj.2019.11.691 ◽

2020 ◽

Vol 118 (3) ◽

pp. 97a

Author(s):

Tim Niklas Baldering ◽

Johanna Rahm ◽

Sebastian Malkusch ◽

Marina S. Dietz ◽

Mike Heilemann

Keyword(s):

Single Molecule ◽

Particle Tracking ◽

Single Particle ◽

Tyrosine Kinases ◽

Receptor Tyrosine Kinases ◽

Adaptor Proteins ◽

Single Particle Tracking ◽

Single Molecule Level

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Adaptive Precision Real-Time 3D Single Particle Tracking Microscopy

Biophysical Journal ◽

10.1016/j.bpj.2017.11.929 ◽

2018 ◽

Vol 114 (3) ◽

pp. 166a

Author(s):

Shangguo Hou ◽

Kevin Welsher

Keyword(s):

Real Time ◽

Particle Tracking ◽

Single Particle ◽

Single Particle Tracking

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Automated single particle detection and tracking for large microscopy datasets

Royal Society Open Science ◽

10.1098/rsos.160225 ◽

2016 ◽

Vol 3 (5) ◽

pp. 160225 ◽

Cited By ~ 11

Author(s):

Rhodri S. Wilson ◽

Lei Yang ◽

Alison Dun ◽

Annya M. Smyth ◽

Rory R. Duncan ◽

...

Keyword(s):

Single Molecule ◽

Single Particle ◽

Image Data ◽

Ground Truth ◽

Detection Algorithm ◽

Large Datasets ◽

Single Particle Tracking ◽

Synthetic Image ◽

Particle Detection ◽

Very Large Datasets

Recent advances in optical microscopy have enabled the acquisition of very large datasets from living cells with unprecedented spatial and temporal resolutions. Our ability to process these datasets now plays an essential role in order to understand many biological processes. In this paper, we present an automated particle detection algorithm capable of operating in low signal-to-noise fluorescence microscopy environments and handling large datasets. When combined with our particle linking framework, it can provide hitherto intractable quantitative measurements describing the dynamics of large cohorts of cellular components from organelles to single molecules. We begin with validating the performance of our method on synthetic image data, and then extend the validation to include experiment images with ground truth. Finally, we apply the algorithm to two single-particle-tracking photo-activated localization microscopy biological datasets, acquired from living primary cells with very high temporal rates. Our analysis of the dynamics of very large cohorts of 10 000 s of membrane-associated protein molecules show that they behave as if caged in nanodomains. We show that the robustness and efficiency of our method provides a tool for the examination of single-molecule behaviour with unprecedented spatial detail and high acquisition rates.

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