scholarly journals Single-Molecule Tracking of Chromatin-Associated Proteins in the C. elegans Gonad

2021 ◽  
Author(s):  
Lexy von Diezmann ◽  
Ofer Rog
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Reference Channel ◽  
Free Diffusion ◽  
Single Molecule Tracking ◽  
C Elegans ◽  
Nuclear Signaling ◽  
Meiotic Chromosomes ◽  
Associated Proteins ◽  
Localization Precision

Biomolecules are distributed within cells by molecular-scale diffusion and binding events that are invisible in standard fluorescence microscopy. These molecular search kinetics are key to understanding nuclear signaling and chromosome organization, and can be directly observed by single-molecule tracking microscopy. Here, we report a method to track individual proteins within intact C. elegans gonads and apply it to study the molecular dynamics of the axis, a proteinaceous backbone that organizes meiotic chromosomes. Using either fluorescent proteins or enzymatically ligated dyes, we obtain multi-second trajectories with a localization precision of 15-25 nm in nuclei actively undergoing meiosis. Correlation with a reference channel allows for accurate measurement of protein dynamics, compensating for movements of the nuclei and chromatin within the gonad. We find that axis proteins exhibit either static binding to chromatin or free diffusion in the nucleoplasm, and we separately quantify the motion parameters of these distinct populations. Freely diffusing axis proteins selectively explore chromatin-rich regions, suggesting they are circumventing the central phase-separated region of the nucleus. This work demonstrates that single-molecule microscopy can infer nanoscale-resolution dynamics within living tissue, expanding the possible applications of this technique.

scholarly journals TRPV channel OCR-2 is distributed along C. elegans chemosensory cilia by diffusion in a local interplay with intraflagellar transport

2020 ◽  
Author(s):  
Jaap van Krugten ◽  
Noémie Danné ◽  
Erwin J.G. Peterman
Keyword(s):  
Single Molecule ◽  
Transmembrane Proteins ◽  
Intraflagellar Transport ◽  
Single Molecule Tracking ◽  
C Elegans ◽  
Normal Diffusion ◽  
Cellular Signal ◽  
Underlying Mechanisms ◽  
And Function

AbstractSensing and reacting to the environment is essential for survival and procreation of most organisms. Caenorhabditis elegans senses soluble chemicals with transmembrane proteins (TPs) in the cilia of its chemosensory neurons. Development, maintenance and function of these cilia relies on intraflagellar transport (IFT), in which motor proteins transport cargo, including sensory TPs, back and forth along the ciliary axoneme. Here we use live fluorescence imaging to show that IFT machinery and the sensory TP OCR-2 reversibly redistribute along the cilium after exposure to repellant chemicals. To elucidate the underlying mechanisms, we performed single-molecule tracking experiments and found that OCR-2 distribution depends on an intricate interplay between IFT-driven transport, normal diffusion and subdiffusion that depends on the specific location in the cilium. These insights in the role of IFT on the dynamics of cellular signal transduction contribute to a deeper understanding of the regulation of sensory TPs and chemosensing.

scholarly journals TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shipeng Shao ◽  
Hongchen Zhang ◽  
Yong Zeng ◽  
Yongliang Li ◽  
Chaoying Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Time Course ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Protein Protein Interactions ◽  
Spatiotemporal Resolution ◽  
Dynamic Interactions ◽  
Single Molecule Tracking

AbstractProtein-protein interactions (PPIs) are critical for cellular activity regulation. Visualization of PPIs using bimolecular fluorescence complementation (BiFC) techniques helps to understand how PPIs implement their functions. However, current BiFC is based on fluorescent proteins and the brightness and photostability are suboptimal for single molecule tracking experiments, resulting in either low spatiotemporal resolution or incapability of tracking for extended time course. Here, we developed the TagBiFC technique based on split HaloTag, a self-labeling tag that could conjugate an organic dye molecule and thus offered better brightness and photostability than fluorescent proteins for PPI visualization inside living cells. Through screening and optimization, we demonstrated that the reconstituted HaloTag exhibited higher localization precision and longer tracking length than previous methods. Using TagBiFC, we reveal that the dynamic interactions of transcription factor dimers with chromatin DNA are distinct and closely related to their dimeric states, indicating a general regulatory mechanism for these kinds of transcription factors. In addition, we also demonstrated the advantageous applications of TagBiFC in single nucleosome imaging, light-burden imaging of single mRNA, low background imaging of cellular structures. We believe these superior properties of our TagBiFC system will have broad applications in the studies of single molecule imaging inside living cells.

scholarly journals Choosing the right label for single-molecule tracking in live bacteria: Side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes

2018 ◽  
Author(s):  
Nehir Banaz ◽  
Jarno Mäkelä ◽  
Stephan Uphoff
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Single Molecule Tracking ◽  
Advantages And Disadvantages ◽  
Fluorescent Ligands ◽  
The Right

AbstractVisualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins. An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and single-molecule localization and tracking results. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.

Time-Resolved, Confocal Single-Molecule Tracking of Individual Organic Dyes and Fluorescent Proteins in Three Dimensions

ACS Nano ◽  
2012 ◽  
Vol 6 (10) ◽  
pp. 8922-8932 ◽  
Author(s):  
Jason J. Han ◽  
Csaba Kiss ◽  
Andrew R. M. Bradbury ◽  
James H. Werner
Keyword(s):  
Single Molecule ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Three Dimensions ◽  
Time Resolved ◽  
Single Molecule Tracking

scholarly journals Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jérôme Goudeau ◽  
Catherine S Sharp ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D Leonetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

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