Name: Using LEXY and LINuS Optogenetics Tools and Automated Image Analysis to Quantify Nucleocytoplasmic Transport Dynamics in Live Cells (Video) | JoVE Journal
Uploaded: 2025-07-22T13:20:00.000+00:00
Duration: 8 min 46 s
Description: Learn automated analysis of nucleocytoplasmic transport dynamics using LEXY and LINuS optogenetic tools in live cells. This protocol enhances efficiency in tracking cellular transport mechanisms for cell biology research.

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5 minutes

We acknowledge the imaging facility MRI, member of the national infrastructure France-BioImaging (https://ror.org/01y7vt929), supported by the French National Research Agency (ANR-24-INBS-0005 FBI BIOGEN), and especially Marie-Pierre Blanchard, Julio Mateos-Langerak and Baptiste Monterroso for their help with microscope settings and FIJI analyses. We are grateful to all the Boumendil lab members for their advice and fruitful discussions, in particular Parisa Nobari for her help at the initiation of the project. We thank Joey Dufourd for proofreading of the manuscript.
Work in the CB group is supported by the Centre National de la Recherche Scientifique (CNRS), the Institut National du Cancer (French National Cancer Institute) / Canceropole Grand Sud-Ouest (Grant Emergence 2023 E07), the Agence Nationale pour la recherche (ANR-23-TERC-0015-01 and ANR-21-CE12-0039), the Institut National du Cancer (Grant INCa PLBIO23-207-2023-180) and the University of Montpellier (program I-SITE, France 2030, project NAChO, project ChOICe).

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<li>Lataster, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+cycle+control+by+optogenetically+regulated+cell+cycle+inhibitor+protein+p21%5BTitle%5D)+AND+%22Biology%22%5BJournal%5D)">Cell cycle control by optogenetically regulated cell cycle inhibitor protein p21.</a> Biology. 12 (9), 1194(2023).</li>
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<li>Schmidt, U., Weigert, M., Broaddus, C., Myers, G. Cell detection with star-convex polygons. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSchmidt%2C+U+%22Medical+Image+Computing+and+Computer+Assisted+Intervention+-+MICCAI+2018%22">Medical Image Computing and Computer Assisted Intervention - MICCAI 2018</a>. Frangi, A. F., Schnabel, J. A., Davatzikos, A. F., Alberola-López, C., Fichtinger, G. , Springer International Publishing. Cham. 265-273 (2018).</li>
<li>Yoo, T. Y., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((O-GlcNAc+modification+of+nuclear+pore+complexes+accelerates+bidirectional+transport%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">O-GlcNAc modification of nuclear pore complexes accelerates bidirectional transport.</a> J Cell Biol. 220 (7), e202010141(2021).</li>
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<li>Lukinavičius, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SiR-Hoechst+is+a+far-red+DNA+stain+for+live-cell+nanoscopy%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy.</a> Nat Commun. 6, 8497(2015).</li>
<li>Kudo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leptomycin+B+inactivates+CRM1%2Fexportin+1+by+covalent+modification+at+a+cysteine+residue+in+the+central+conserved+region%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region.</a> Proc Natl Acad Sci U S A. 96 (16), 9112-9117 (1999).</li>
<li>Soderholm, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Importazole%2C+a+small+molecule+inhibitor+of+the+transport+receptor+importin-%CE%B2%5BTitle%5D)+AND+%22ACS+Chem+Biol%22%5BJournal%5D)">Importazole, a small molecule inhibitor of the transport receptor importin-β.</a> ACS Chem Biol. 6 (7), 700-708 (2011).</li>
<li>Henderson, B. R., Eleftheriou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparison+of+the+activity%2C+sequence+specificity%2C+and+CRM1-dependence+of+different+nuclear+export+signals%5BTitle%5D)+AND+%22Exp+Cell+Res%22%5BJournal%5D)">A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals.</a> Exp Cell Res. 256 (1), 213-224 (2000).</li>
<li>Lu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Types+of+nuclear+localization+signals+and+mechanisms+of+protein+import+into+the+nucleus%5BTitle%5D)+AND+%22Cell+Commun+Signaling%22%5BJournal%5D)">Types of nuclear localization signals and mechanisms of protein import into the nucleus.</a> Cell Commun Signaling. 19 (1), 60(2021).</li>
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The authors have no conflicts of interest to disclose.

As mentioned in the protocol, the creation of stable cell lines greatly improves the quality of the analyses. First, having 100% of the cells expressing the probes increases the number of nuclei tracked and analyzed, which strengthens the robustness of the analyses. Second, it reduces cell death induced by the overexpression of the probes and the toxicity of the transfection reagents used. And finally, it reduces the strong heterogeneity of fluorescent signals amongst the cells. This parameter is of importance since a saturated signal will not allow proper detection of the change of intensity after illumination. The same conclusion is true for weak signals, which may bleach or not allow for the quantification of differences. Therefore, we recommend first testing the probes by transfection to ensure that they work properly, setting up the acquisition parameters, and then rapidly creating stable cell lines. We created lentivirus vectors for both LEXY and LINuS probes, which are available to share upon request, as well as the NIH/3T3 LEXY or LINuS stable lines created with these lentiviruses.
If the probes are transfected or if cells are sparse, the parameters suggested will likely decrease the number of tracks detected, and the analyses will be less robust. If this is the case, the users can be more permissive with the spots omitted in the tracking by decreasing the filter Number of spots in tracks (we suggest keeping at least 45 out of 50 spots). Caution: if this filter is decreased, the Excel file provided (Supplemental Table S1) should be manually adjusted: indeed, the values pasted from the CSV imported will not take into account the gaps. The user needs to find where the missing spots are with the TrackID number and correlate them with the nucleus number, which usually takes a few copy-paste rounds.
A critical parameter to guarantee the success of the experiment is to keep the cells in the dark. As the probes react to blue light, users must ensure the transport of the cells in the dark for at least 30 min before acquisition. A small closed box or using aluminum to cover the dish is sufficient. Similarly, it is necessary to be very careful to only use the mCherry light to search for the cells at the microscope, since illumination with blue light activates the cells. If an eventual illumination happens under the microscope, search for another distant field for the acquisition. Plotting the Z-axis Profile in step 3.6 will help to know if the cells were impacted by an illumination, if the curve is decreasing instead of being a plateau.
The imaging sequence is set up to consist of three phases. First, the stable phase, to assess for potential bleaching but also to ensure that LEXY&#39;s localization is steady without activation (1). Second, the activation phase, consisting of rounds of pulsatile blue light illumination, to induce the translocation of LEXY from the nucleus to the cytosol (2). Finally, the optional recovery phase, where the signal progressively goes back to its initial location in the nucleus (3).
The sequence described for acquisitions can be cell-type-specific19,20. For the first experiment, the user should test and modify the laser power and timings to see the system progressively translocate between the nucleus and cytosol. Of importance, a laser power too strong, as a pause too short, might prevent seeing a progressive translocation, but instead a brutal drop/enrichment (depending on the probe used) of the mCherry intensity after illumination.
If the sequence&#39;s optimization does not solve the problem, it is likely that the NES and/or NLS are not suited for the cell type used. Users can refer to the LINuS method article8 to engineer and modify the probes. Users can also consult this article for imaging LINuS with an epifluorescence microscope.
The nuclei segmentation is a critical step of the analysis. A robust and reliable nuclear marker should be used for live cell imaging of the probes. Since illumination with blue light activates the probe&#39;s translocation, and because the probes are monitored with mCherry fluorescent protein, this marker must be visible in the far-red spectrum or close. In our case, we use SiR-DNA that stains the cells during 1 to ~24 h of incubation, but using SPY-DNA 650 is also an option (see Table of&#160;Materials for references).
Finally, if the segmentation with StarDist combined with the filtered tracking of TrackMate proves its efficiency, it is important to note that the user should always visually check the results of the segmentation and the quantification. A dividing or dying cell might be included in the quantification, whereas it should not. To reduce the risks of analysis of such events, the provided Excel sheet attributes a color code to the relative nuclear intensity (blue = low values, red = high values). By inspecting the colors before plotting the values in GraphPad Prism, the user should be able to notice any unusual values. For example, LEXY&#39;s relative nuclear intensities should vary from high (red, strong amounts of the probe in the nuclei) during the stable phase to low (blue, reduced amounts of the probe in the nuclei) at the end of the illumination phase, before coming back to high values (red), at the recovery phase (as displayed in the Supplemental Table S2).
This protocol presents various advantages over previously proposed methods5,6. The semi-automatic pipeline facilitates the analysis of LEXY and LINuS nucleocytoplasmic transport, and provides more accuracy than previously proposed manual quantification5,6. Using our protocol will enable beginners in the field to perform efficient analyses, but also provide a new tool for experienced users, who will need only one open-access interface to perform semi-automated analyses of the nucleocytoplasmic transport.
This method can be used for further applications than the study of global nucleocytoplasmic transport rate in NIH3T3 cell lines. Indeed, with a few optimization steps, it can be adapted to study the probe&#39;s transport rate in various cell types or living animals8. Furthermore, by fusing a protein of interest to either LEXY or LINuS, it is also possible to study its transport rate or the downstream effects of its light-induced translocation in or out of the nucleus. For example, it is possible to sequester a protein in the cytoplasm with LEXY, or to force its localization into the nucleus with LINuS5,6,21,22,23. Finally, the analyses described can also be extended to more applications than LEXY and LINuS, to track any signal that translocates between subcellular compartments that can be segmented.

The importance of nucleocytoplasmic transport (NCT) to regulate key cellular pathways and maintain cellular homeostasis has been well studied over the last years1. In consequence, several tools were generated to study the NCT's dynamics2,3,4. Amongst these tools, LINuS5 and LEXY6 optogenetic probes, published respectively in 2014 and 2016, provide a powerful way to monitor NCT in live cells. These tools, designed to follow either the inducible export (LEXY) or import (LINuS) of a fluorescent reporter, are inducible by exposure to blue light and reversible.
In brief, these two small probes use the properties of the LOV2 domain from the protein phototropin-1 of the plant Avena sativa7. In the absence of blue light, the Jα helix from the LOV2 domain is photocaged and embedded into the protein core. As the Jα helix can respond to light stimulus, illumination by blue light induces a conformational change and the subsequent undocking of the Jα helix from the LOV2 domain. The authors of LEXY and LINuS used this light sensitivity to fuse either a Nuclear Localization Signal (NLS) or a Nuclear Export Signal (NES) to the Jα helix, for LINuS and LEXY, respectively (Figure 1). The resulting tool is a probe that translocates into the nucleus (LINuS) or the cytosol (LEXY) upon blue light illumination.
To track the probes with fluorescent imaging, a mCherry fluorescent reporter was also added to the constructs. Finally, a weak NES (for LINuS) or NLS (for LEXY) was added to the system to ensure that the probes were not in their final localization before blue light illumination, which would prevent observing LEXY or LINuS translocation between the nucleus and cytosol.
<img alt="Protein localization mechanism under blue light; diagram shows nucleus-cytoplasm interaction." class="xfigimg" src="/files/ftp_upload/68585/68585fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Representation of LEXY and LINuS probes. Both probes consist of a mCherry protein fused to the light-sensitive LOV2 domain. The difference between the two tools resides in the signaling sequence hidden by the Jα-helix: an NES for LEXY and an NLS for LINuS. Both probes also possess a weak signal sequence to control their initial localization: a weak NLS for LEXY and a weak NES for LINuS. The activation of the systems by blue light illumination therefore induces either their export for LEXY or their import for LINuS, allowing users to monitor nuclear export or import. Abbreviations: LEXY = light-inducible nuclear export system; LINuS = light-inducible nuclear localization signal; NLS = nuclear localization signal; NES = nuclear export signal. <a href="https://www.jove.com/files/ftp_upload/68585/68585fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The localization of mCherry fluorescence reports the shuttle of LEXY and LINuS probes between nuclei and cytosol, and can therefore be followed in live cell imaging, allowing users to monitor NCT. In addition to being used as simple probes, LEXY and LINuS can also be engineered to control the localization and study proteins of interest, in vitro5,6,8,9 or in vivo10,11.
The quantification method proposed in LEXY and LINuS publications shows the relative nuclear intensity of the probes. The suggested analysis is done manually and relies on the measurement of a Region of Interest (ROI) in the nucleus and the cytosol. Although this method gives a good representation of the probe's signal over time, it is very long and tedious, especially when experiments and conditions are repeated. Of importance, this method also lacks accuracy since only a small portion of the designed compartment is represented.
Here we propose a new protocol for LEXY and LINuS fluorescent signal quantification. We implemented automated techniques, which facilitate the analyses and provide greater accuracy. This will ease the process for new users of these tools and help to increase throughput. Our quantification protocol uses only one interface, the open-source TrackMate12 plugin of FIJI. Notably, to improve the reliability of the quantifications, we used a live-imaging compatible far-red DNA marker to perform whole nucleus segmentation with StarDist13, a trained convolutional neural network, combined with tracking and measuring of the probe's fluorescent signal with TrackMate12.
The data obtained can then easily be exported into the provided and ready-to-use spreadsheet (Supplemental Table S1) to obtain the relative nuclear intensity of each identified nucleus. This protocol also explains how to pool the obtained values in a graph by fitting the time course curve obtained to a monoexponential model, as in Yoo and Mitchison14 and how to calculate the transport rate for the different conditions tested. With this protocol and the provided spreadsheet for analysis, users will be able to easily quantify live-cell imaging data, even with limited bioinformatics skills.

<table><tbody><tr><td>Collagen</td><td>Sigma-Aldrich</td><td>08-115</td><td></td></tr><tr><td>Confocal microscope</td><td>LEICA</td><td>SP8</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>31966021</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>FIJI software</td><td>FIJI</td><td></td><td>ImageJ 2.16.0/1,54p; Java1.8.0_322</td></tr><tr><td>Fluorodishes</td><td>Dutscher</td><td>FD3510-100</td><td>Alternative: Attofluor chamber (Invitrogen #A7816)</td></tr><tr><td>GraphPad Prism 10.4.1</td><td>GraphPad Software, Inc.</td><td></td><td></td></tr><tr><td>Importazole</td><td>Sigma-Aldrich</td><td>SML0341</td><td></td></tr><tr><td>Leptomycin B</td><td>Sigma-Aldrich</td><td>L2913</td><td></td></tr><tr><td>LEXY plasmid</td><td>Addgene</td><td>72655</td><td>pDN122</td></tr><tr><td>LINuS Plasmid</td><td>Addgene</td><td>61347</td><td>pDN77</td></tr><tr><td>Pencillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Phenol-red free DMEM</td><td>Gibco</td><td>21063-029</td><td></td></tr><tr><td>SiR-DNA</td><td>Spirochrome</td><td>SC007</td><td>Alternative: SPY-DNA650 (Spirochrome #SC501)</td></tr><tr><td>Stardist 2D plugin</td><td>StarDist</td><td></td><td>StarDist_0.3.0</td></tr><tr><td>TrackMate plugin</td><td>TrackMate</td><td></td><td>TrackMate-7.14.0</td></tr></tbody></table>

using lexy and linus optogenetics tools and automated image analysis to quantify nucleocytoplasmic transport dynamics in live cells

Using LEXY and LINuS Optogenetics Tools and Automated Image Analysis to Quantify Nucleocytoplasmic Transport Dynamics in Live Cells

Nucleocytoplasmic transport (NCT) is essential for maintaining cellular homeostasis, and its disruption is involved in various diseases, including neurodegenerative disorders and amyotrophic lateral sclerosis. This underscores the need to develop tools to monitor and quantify NCT. Amongst these tools, the fast and reversible optogenetics probes, LEXY (light-inducible nuclear export system) and LINuS (light-inducible nuclear localization signal), allow the measurement of NCT dynamics in live cells.
The original publications describe manual segmentation and quantification of the fluorescent probe signal in the nucleus and cytosol upon transfection of LEXY and LINuS constructs in live-cell imaging. However, both transfection and manual segmentation limit the number of cells that can be analyzed and are subject to imprecision due to potential user-dependent errors. While the high speed and reversibility provided by optogenetics should, in principle, allow for high sensitivity in detecting changes in NCT dynamics, it depends on the acquisition parameters and analysis of a sufficient number of cells.
We have therefore established lentiviral vectors expressing LEXY and LINuS to create stable cell lines, tested live imaging markers and control conditions, and implemented a semi-automated image analysis pipeline that allows for the analysis of hundreds of cells. This analysis method uses the open-access software FIJI, is accessible to beginners in bioinformatics, and does not require advanced computer setups. Here we provide a step-by-step protocol to set up LEXY as an example of these optogenetic tools to monitor nuclear export, from preparation of the samples to live-cell imaging acquisition and automated analysis, while demonstrating how to adapt the protocol for other conditions, controls, or models in any lab. All plasmids and cell lines used in this protocol will be made available to the scientific community, therefore further increasing the accessibility of the method.

The above-mentioned protocol should allow the users to visualize the probe's localization and translocation during the acquisition in live cell imaging. An example of the expected images acquired to visualize nuclear export with LEXY is shown in Figure 4. A rapid decrease of the signal from the nucleus and an enrichment in the cytosol should be observed after a couple of rounds of blue light illumination. A progressive recovery of the probe's signal can be visible after stopping the illumination cycles. To control the system, Leptomycin B, an export inhibitor, attests that a change in export can be visualized with the analyses, since it prevents LEXY from being exported upon blue light illumination (Figure 4).
<img alt="Fluorescence microscopy series; mCherry-tagged proteins; methanol vs. Leptomycin B treatments." class="xfigimg" src="/files/ftp_upload/68585/68585fig4.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Visualization of LEXY's signal over time. Capture of representative time points of the stable, illumination, and recovery phases, acquired with live cell imaging. The methanol condition represents the control condition. Upon pulsatile blue light illumination, in red, the mCherry fluorescent signal, reporter of LEXY's location, is translocated from the nucleus to the cytosol. After stopping the illumination, the signal is recovered in the nucleus. Treatment with the export inhibitor Leptomycin B at 1 nM or 0.5 nM shows the absence or decrease of LEXY's export when the export is blocked. Scale bar = 30 µm. <a href="https://www.jove.com/files/ftp_upload/68585/68585fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Regarding the analyses, while values of the relative nuclear intensity curve during the stable phase should be around 1, after blue light activation, they should display a slow decrease until reaching a plateau at the end of the illumination phase (Figure 5A). If the decrease is too fast or too slow, acquisition parameters can be optimized (see discussion). Leptomycin B treatment at sufficient concentrations can also confirm that the analyses and technique are sensitive enough to show that nuclear export is completely blocked, with relative nuclear intensity values stable at around 1 for the whole experiment. Intermediate Leptomycin B concentrations, however, should display a mild nuclear export (Figure 5A). These visual observations should be confirmed quantitatively with the obtention of the export rate (Figure 5B).
<img alt="Graph of Leptomycin B impact on nuclear intensity; violin plot details export rates in cellular assay." class="xfigimg" src="/files/ftp_upload/68585/68585fig5.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Expected relative nuclear intensity of LEXY and transport rate. (A) The graph represents the relative nuclear intensity of LEXY's fluorescent signal over time, normalized to the mCherry mean intensity of the stable phase. The relative nuclear intensity of the methanol (control, red) condition decreases by half after illumination, in contrast to the cells treated with the export inhibitor Leptomycin B, at a concentration of 0.5 nM (light blue) or 1 nM (dark blue). Error bars: SEM. (B) Export rate of the control and Leptomycin B conditions, obtained by fitting the curve to a monoexponential model. The values obtained when the export is blocked by Leptomycin B are significantly lower than the control condition. N = 18 cells for Methanol (red), 19 cells for Leptomycin B 0.5 nM (light blue), and 15 cells for Leptomycin B 1 nM (dark blue). P values were calculated by a two-sided Welch's t test. ***: P &lt; 1×10−4, ****: P &lt; 1×10−5. <a href="https://www.jove.com/files/ftp_upload/68585/68585fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table S1: Spreadsheet with predefined calculations. This spreadsheet is provided to facilitate the analysis. The intensity values obtained with TrackMate can be directly copied and pasted into the spreadsheet to obtain the normalized relative nuclear intensity values. <a href="https://www.jove.com/files/ftp_upload/68585/JOVE-Supplemental_Table1.zip" target="_blank">Please click here to download this File.</a>
Supplemental Table S2: Example of the expected results for a control condition of LEXY's analysis. The spreadsheet represents an example of the obtained values for a control condition of LEXY's stable cell line. <a href="https://www.jove.com/files/ftp_upload/68585/JOVE-Supplemental_Table2.xlsx" target="_blank">Please click here to download this File.</a>

This protocol describes how to efficiently set up and perform automated analyses of live cell imaging data obtained from tracking the nucleocytoplasmic transport via optogenetic tools.

Nucleocytoplasmic transport (NCT) is essential for maintaining cellular homeostasis, and its disruption is involved in various diseases, including ...

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Research

JoVE Journal

Biology

Single molecule microscopy approch provided novel insights into nuclear transport.

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Using LEXY and LINuS Optogenetics Tools and Automated Image Analysis to Quantify Nucleocytoplasmic Transport Dynamics in Live Cells (Video) | JoVE Journal

Using LEXY and LINuS Optogenetics Tools and Automated Image Analysis to Quantify Nucleocytoplasmic Transport Dynamics in Live Cells

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