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Jul 2017

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A Quantitative Heterokaryon Assay to Measure the Nucleocytoplasmic Shuttling of Proteins
定量异核体分析测定蛋白质核质穿梭   

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Abstract

Many proteins appear exclusively nuclear at steady-state but in fact shuttle continuously back and forth between the nucleus and the cytoplasm. For example, nuclear RNA-binding proteins (RBPs) often accompany mRNAs to the cytoplasm, where they can regulate subcellular localization, translation and/or decay of their cargos before shuttling back to the nucleus. Nucleocytoplasmic shuttling must be tightly regulated, as mislocalization of several RBPs with prion-like domains such as FUS and TDP-43 causes the cytoplasmic accumulation of solid pathological aggregates that have been implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Traditionally, interspecies heterokaryon assays have been used to determine whether a nuclear protein of interest shuttles; those assays are based on the fusion between donor and recipient cells from two different species (e.g., mouse and human), which can be distinguished based on different chromatin staining patterns, and detecting the appearance of the protein in the recipient nucleus. However, identification of heterokaryons requires experience and is prone to error, which makes it difficult to obtain high-quality data for quantitative studies. Moreover, transient overexpression of fluorescently tagged RBPs in donor cells often leads to their aberrant subcellular localization. Here, we present a quantitative assay where stable donor cell lines expressing near-physiological levels of eGFP-tagged RBPs are fused to recipient cells expressing the membrane marker CAAX-mCherry, allowing to readily identify and image a large number of high-confidence heterokaryons. Our assay can be used to measure the shuttling activity of any nuclear protein of interest in different cell types, under different cellular conditions or between mutant proteins.

Keywords: RNA-binding protein (RNA结合蛋白), Nucleocytoplasmic shuttling (核质穿梭), Heterokaryon assay (异核体分析), Quantification of shuttling capacities (穿梭能力定量), Nuclear proteins (核蛋白)

Background

To understand the various functions of a protein, it is important to find out where it localizes within cells. Standard microscopic and biochemical methods only reveal the presence of a protein when its steady-state concentration is above the detection threshold. They do not rule out the possibility that it plays additional, important roles where it localizes only transiently (Gama-Carvalho and Carmo-Fonseca, 2001). For example, many RBPs perform functions in different cellular compartments where they accompany their bound mRNAs (often going undetected) and connect multiple steps in eukaryotic gene expression (Müller-McNicoll and Neugebauer, 2013). SR proteins (SRSF1 to SRSF12) are a family of RBPs that regulate transcription, pre-mRNA splicing, 3’end processing and mRNP packaging in the nucleus and appear exclusively nuclear at steady state (Howard and Sanford, 2015; Jeong, 2017). However, most family members shuttle continuously (but to different extents) between the nucleus and the cytoplasm, performing additional functions in mRNA export and translation (Caceres et al., 1998; Sapra et al., 2009; Maslon et al., 2014; Müller-McNicoll et al., 2016; Botti et al., 2017). Changes in RBP shuttling have been described in viral infections, early development, cellular differentiation and neurodegenerative diseases such as ALS and FTD, where pathological accumulation of prion-like RBPs such as FUS and TDP-43 in the cytoplasm forms solid neurotoxic aggregates (Ederle and Dormann, 2017; Liu et al., 2017). Thus, it is very important to know whether an RBP normally shuttles between the nucleus and the cytoplasm and if so, under which circumstances and how it is controlled.

With the tools currently available, it has been difficult to study the cytoplasmic functions of nuclear RBPs and to compare their shuttling abilities. An ingenious method was developed almost thirty years ago–the interspecies heterokaryon assay–in which donor and recipient cells from two different species (e.g., mouse and human) are fused and a protein present only in the donor nuclei gradually appears in the recipient nuclei if it shuttles (Borer et al., 1989). However, this assay provides only qualitative information. The fusion events are identified based on phase-contrast images and donor and recipient nuclei are identified based on distinct chromatin features, which makes the assay laborious, subjective and produces only small numbers of high-confidence heterokaryons. Moreover, fluorescently tagged RBPs are often expressed from transiently transfected plasmids, which results in very different RBP levels in different cells, ranging from barely detectable to non-physiologically high expression that may lead to partially aberrant cytoplasmic or subnuclear localization of RBPs [(Maharana et al., 2018) our unpublished observations]. Altogether, these limitations preclude any comparative analyses.

Here, we present a detailed experimental protocol to perform quantitative shuttling assays in cultured mammalian cells (Figure 1). Our assay is an improvement of the classical heterokaryon assay, with two novelties and a standardized imaging pipeline to perform quantitative measurements. The first novelty is the use of recipient cell lines expressing a fluorescently tagged membrane marker (CAAX-mCherry), which greatly facilitates the identification of heterokaryons containing both a donor and a recipient nucleus and thus allows the rapid and easy identification of a large number of high-confidence heterokaryons. The second novelty is the use of stable clonal donor cell lines, where a fluorescently (eGFP) tagged RBP of interest is expressed from a bacterial artificial chromosome (BAC), which has been integrated into the genome (Botti et al., 2017; Poser et al., 2008). Subsequent clonal selection of cells ensures equal and near-physiological levels of tagged RBPs in every donor cell to facilitate image acquisition, analysis and comparisons.

Our assay has been successfully applied to compare the shuttling activities of different SR proteins in the same cell line, between different cell lines and between differentiation states. Moreover, it allowed us to study the requirements for the shuttling of individual SR proteins using mutated proteins and knockdowns of nuclear export factors (Botti et al., 2017). We have used various cell lines as either donor or recipient cells: these comprise mouse (P19 and NIH3T3) and human (HeLa) cells. Although other cell lines remain to be tested, we are confident that any adherent cell line in which fluorescent RBPs can be expressed at physiological levels is suitable for our assay. This should include primary cells obtained from transgenic animals expressing a fluorescently tagged RBP of interest. We have successfully used P19 cells differentiated into neural cells as donors in our assays (Botti et al., 2017), and it should be possible to study and quantify shuttling of RBPs in other cellular models of differentiation, for example in mouse embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), or to compare shuttling of RBPs in distinct cellular differentiation fates (Hammarskjold and Rekosh, 2017). Moreover, our assay should allow to quantify changes in shuttling during viral infections and cellular stress, or to assess the impact of disease mutations in RBPs. In principle, our assay could even be adapted to visualize shuttling of long-noncoding RNAs (lncRNAs), for example through the insertion of binding sites for fluorescent MS2 binding protein (MS2-BP) or by inserting an aptamer sequence that binds a fluorescent dye (Ouellet, 2016).

Materials and Reagents

  1. 10 μl filter tips long (SARSTEDT, catalog number: 70.1116.210 )
  2. 20 μl filter tips (SARSTEDT, catalog number: 70.760.213 )
  3. 300 μl filter tips (SARSTEDT, catalog number: 70.765.210 )
  4. 1,000 μl filter tips (SARSTEDT, catalog number: 70.762.211 )
  5. 10-cm cell culture dishes (VWR, Thermo Fisher Scientific, catalog number: 734-2043 )
  6. Cloning discs, size 5 mm (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z374458-100EA )
  7. Disposable Glass Pasteur Pipettes 150 mm (VWR, catalog number: 612-1701 )
  8. Serological pipettes, 2 ml (VWR, Corning, catalog number: 734-1690 )
  9. Serological pipettes, 5 ml (VWR, Corning, catalog number: 734-1737 )
  10. 15-ml Centrifuge tubes (Corning, catalog number: 430791 )
  11. 2-ml microcentrifuge tubes (SARSTEDT, catalog number: 72.691 )
  12. 1,000 µl pipette tips ART® 1000E Barrier Tips (Thermo Fisher Scientific, catalog number: 2079E )
  13. 12-well plates for cell culture (VWR, Thermo Fisher Scientific, catalog number: 734-2156 )
  14. Precision coverslips, 18 mm, borosilicate glass 0.17 ± 0.005 mm (Carl Roth, catalog number: LH23.1 )
  15. Microscope slides (VWR, catalog number: 631-1550 )
  16. Bacterial artificial chromosome (BAC) containing the gene encoding an eGFP-tagged RBP of interest (Botti et al., 2017 and Poser et al., 2008; see Notes 1-3)
  17. Plasmid for expression of fluorescent plasma membrane marker of a different color (e.g., CAAX-mCherry, plasmid TH0477, Stewart et al., 2011; Botti et al., 2017; see Note 4)
  18. DMEM, high glucose, GlutaMAXTM Supplement, pyruvate (Thermo Fisher Scientific, catalog number: 31966047 )
  19. Fetal Bovine Serum (Thermo Fisher Scientific, catalog number: 10270106 )
  20. Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, catalog number: 15140122 )
  21. Puromycin 10 mg/ml (Thermo Fisher Scientific, GibcoTM, catalog number: A1113803 )
  22. Geneticin® Selective Antibiotic (G418 Sulfate) (50 mg/ml) (Thermo Fisher Scientific, catalog number: 10131035 )
  23. Trypsin 0.05% EDTA (Thermo Fisher Scientific, catalog number: 25300054 )
  24. Dulbecco's Phosphate Buffered Saline (Sigma-Aldrich, catalog number: D8537 )
  25. Gelatin solution bioreagent 2% in H2O (Sigma-Aldrich, catalog number: G1393 )
  26. Cycloheximide solution 100 mg/ml in DMSO (Sigma-Aldrich, catalog number: C4859 )
  27. Polyethylene Glycol (PEG) 1500 in 75 mM HEPES (Roche Diagnostics, catalog number: 10783641001 )
  28. Phosphate buffered saline 10x (Sigma-Aldrich, catalog number: P5493 )
  29. Pierce 16% Formaldehyde (w/v), Methanol-free (Thermo Fisher Scientific, catalog number: 28908 )
  30. 30. Water, Molecular Biology Reagent (Sigma-Aldrich, catalog number: W4502 )
  31. ProLong® Diamond Antifade Mountant (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36970 )
  32. TWEEN® 20 (Sigma-Aldrich, catalog number: P7949 )
  33. Tris (Carl Roth, catalog number: 4855.2 )
  34. Hoechst 34580 (Sigma-Aldrich, catalog number: 63493 )
  35. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  36. Sodium Chloride (NaCl), BioXtra (Sigma-Aldrich, catalog number: S7653 )
  37. Hydrochloric acid 36.5-38.0% (HCl), for molecular biology (Sigma-Aldrich, catalog number: H1758 )
  38. DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (see Recipes)
  39. PBS containing 0.1% gelatin (see Recipes)
  40. 10x TBS (see Recipes)
  41. TBST (see Recipes)
  42. 4% Formaldehyde in 1x PBS (see Recipes)
  43. Hoechst 34580 stock solution (1 mg/ml) (see Recipes)
  44. TBST containing 0.25 μg/ml Hoechst 34580 (see Recipes)

Equipment

  1. Hemocytometer
  2. Timer
  3. Fume hood
  4. Cell culture hood (Thermo Fisher Scientific, model: HerasafeTM KSP , type KSP 12)
  5. Fine tip curved tweezers
  6. P10 pipettor (VWR, catalog number: 613-5259 )
  7. P20 pipettor (VWR, catalog number: 613-5260 )
  8. P200 pipettor (VWR, catalog number: 613-5263 )
  9. P1000 pipettor (VWR, catalog number: 613-5265 )
  10. Mini vacuum pump, KNF (A. Hartenstein, catalog number: AP86 )
  11. Fluid aspiration system (VACCUBRAND, model: VHCpro )
  12. CO2 incubator (Thermo Fisher Scientific, model: HeracellTM 150i )
  13. Vortex mixer (VWR, catalog number: 444-1372 )
  14. Pipette controller (accu-jet® pro, Brand, catalog number: 26300 )
  15. -20 °C freezer
  16. Refrigerator
  17. Inverted microscope (Motic, model: AE31 )
  18. Confocal laser-scanning microscope (ZEISS, model: LSM 780 )

Software

  1. Fiji (ImageJ Version 2.0.0-rc-43/1.51h or more recent)
  2. Microsoft Excel (version 14.6.7 or more recent)

Procedure

See Figure 1 for an overview of the quantitative heterokaryon shuttling assay.


Figure 1. Overview of the quantitative heterokaryon shuttling assay. Donor cells expressing a GFP-tagged nuclear RBP are seeded together with recipient cells expressing CAAX-mCherry tethered to their cell membranes. Cells are fused with PEG1500 in the presence of cycloheximide to block new protein synthesis, i.e., to avoid appearance of the RBP in the common cytoplasm not due to shuttling. After 3 h, cells are fixed and imaged by confocal microscopy. Heterokaryons suitable for analysis can be readily identified as they contain only two nuclei, at least one of which is green, and both are surrounded by a red membrane. Nuclei are imaged by confocal microscopy and GFP fluorescence is quantified in both the donor and the recipient nucleus. Given that shuttling is allowed to occur in the presence of a protein synthesis inhibitor (cycloheximide), any fluorescence in the recipient nucleus is due to shuttling of the RBP.


  1. Generate clonal transgenic donor and recipient cell lines
    1. Generate a donor cell line expressing an eGFP-tagged RBP of interest from a BAC randomly integrated into the genome as described in Botti et al. (2017) and Poser et al. (2008) (see Notes 1-3).
    2. Generate a recipient cell line expressing a plasma membrane-localized marker of a different color (e.g., CAAX-mCherry) from a plasmid randomly integrated into the genome as described in Botti et al. (2017) (see Note 4).
    3. Using fluorescence activated cell sorting (FACS) or cloning discs, isolate clones with different fluorescence levels for the donor cell line and at least one clone with high fluorescence for the recipient cell line (see Note 5).
    4. If a specific antibody against the RBP of interest is available, estimate by Western blotting the expression levels of the eGFP-tagged RBP in different clones and select a clone with near-endogenous levels.

  2. Prepare individual cultures of donor and recipient cells
    Passage donor and recipient cells onto separate 10-cm culture dishes to obtain nearly confluent cultures 1-3 days later.

  3. Mix and seed donor and recipient cells (Timing: 30 min) (see Note 6)
    1. For each combination of donor and recipient cells, place 4 sterile 18-mm coverslips into different wells of a 12-well plate. If using cell lines that normally require a pre-coated surface to increase their adherence, pre-coat the coverslips accordingly. For example, P19 cells require coating with PBS containing 0.1% gelatin, and we normally pre-coat the coverslips for 24 h at 37 °C when using this cell line.
    2. Estimate the confluence (e.g., 50%, 75%, 100%) of both donor and recipient cells.
    3. Aspirate medium and gently wash with 5 ml of pre-warmed sterile PBS. Aspirate the PBS thoroughly and add 1.5 ml of 0.05% Trypsin. Incubate for 3-5 min at RT or 37 °C (optimal conditions must be determined for each cell line).
    4. Help the cells detach from the dishes by tilting the plate in every direction.
    5. Add 3 ml of appropriate medium (e.g., DMEM) containing 10% FCS to inhibit trypsin activity.
      Critical Step: Disrupt the cell clumps by pipetting up and down 20 times in the cell culture dish using the fastest speed setting on your pipet controller. Make sure you obtain single cell suspensions of both donor and recipient cells by visual inspection under the microscope.
    6. Transfer each single cell suspension to a 15-ml tube.
    7. Calculate the concentration of both donor and recipient cell suspensions using a standard method (e.g., using a hemocytometer). Alternatively, use approximate values according to your cell line, culture dish (i.e., surface area) and confluence. For example, a 10-cm dish with HeLa cells grown at confluence contains approximately 8.8 x 106 cells, and following Steps C3 to C6, the corresponding cell suspension (in a volume of 4.5 ml) would contain approximately 2.0 x 106 cells/ml. 
    8. In a 2-ml tube, prepare 2-ml of a 1:1 donor:recipient mixed cell suspension containing approximately 1 x 106 to 2 x 106 cells from each cell suspension. For example, if the donor cell culture was approximately 50% confluent (~4.4 x 106 cells per dish) and the recipient cell culture was approximately 100% confluent (~8.8 x 106 cells per dish), mix 1.333 ml of donor cell suspension (~9.8 x 105 cells/ml) with 0.667 ml of recipient cell suspension (~2.0 x 106 cells/ml) for a total of approximately 2.6 x 106 cells. 
    9. Vortex the mixed cell suspension for 10 sec at medium speed.
    10. Transfer 1 ml of the mixed cell suspension to a 2-ml tube containing 1 ml of medium (2-fold dilution). Vortex for 10 sec at medium speed. This and the subsequent dilutions should help to obtain the optimal density for cell fusion and imaging (see Procedure I). 
    11. Similarly, perform two more 2-fold serial dilutions (corresponding to 4-fold and 8-fold dilutions). The 8-fold dilution should contain approximately 2.5 x 105 to 5.0 x 105 cells.
    12. Transfer 1 ml of each mixed cell suspension (undiluted, 2-fold, 4-fold and 8-fold dilutions) onto a coverslip in a 12-well plate prepared at Step C1. Make sure the coverslips are not floating at the surface of the medium. If some of them do, help them sink at the bottom using a sterile tip. If the coverslips were pre-coated (e.g., with PBS containing 0.1% gelatin), aspirate the coating solution from each well just before adding the mixed cell suspension.

  4. Attachment of cells to coverslips (Timing: overnight [8-16 h])
    Incubate at 37 °C, 5% CO2 for 8-16 h.
    Critical Step: Incubate long enough to allow the cells to attach to the coverslips but briefly enough to avoid cell division. Recently divided cells readily fuse together, often resulting in heterokaryons containing more than two nuclei, which should not be used for shuttling quantification (see Procedure I and J). For example, mouse P19 cells divide every 16 h in the exponential growth phase, and we incubate for a maximum of 8 h at this step when using this cell line (see Note 6).

  5. Inhibition of protein synthesis (Timing: 2.5 h) (see Note 7)
    Critical Step: Some cell lines might not be tightly attached to the coverslips at this stage. To prevent the loss of cells, be very careful when performing each medium change or wash. This advice should be followed until the cells are fixed (Step H3). Always add medium or buffer on the sidewall of the well, not directly onto the coverslip.
    1. Aspirate medium and gently add 1 ml of medium containing 50 μg/ml cycloheximide (CHX). Incubate for 2 h at 37 °C with 5% CO2.
    2. Aspirate medium and gently add 1 ml of medium containing 100 μg/ml CHX. Incubate for 30 min at 37 °C with 5% CO2.

  6. Cell fusion (Timing: 30 min) (see Note 8)
    1. Aspirate medium and gently add 1 ml of pre-warmed PBS containing 100 μg/ml CHX.
    2. Start the timer for 2 min and 10 sec. Then, one coverslip at a time, aspirate PBS/CHX and gently add 0.25 ml of pre-warmed PEG 1500.
      Critical Step: Proceed swiftly (approx. 10 sec) from coverslip to coverslip. Each coverslip should be incubated for 2 min (± a few seconds). Hold the fluid aspiration device with one hand and the pipette controller with the other. For aspiration, use the same glass Pasteur pipette for all samples. To distribute PEG 1500, use the same 2-ml pipette for all samples. We do not recommend processing more than 12 coverslips within one batch.
    3. Without removing the PEG 1500, gently add 3 ml of PBS to each coverslip. Maintain a pace of about 10 sec per coverslip.
    4. Aspirate PBS and perform two more washes with 3 ml of PBS each.
    5. Aspirate PBS and gently add 1 ml of medium containing 100 μg/ml CHX.
    6. Optional: Briefly visually inspect the overall fusion efficiency under the microscope. Many cells containing several nuclei can be observed at this stage.

  7. Shuttling (Timing: 3 h)
    Incubate for 3 h at 37 °C with 5% CO2 (see Note 9).

  8. Cell fixation and preparation of microscope slides (Timing: 1.5 h, overnight)
    1. Aspirate medium containing 100 μg/ml CHX and add 1 ml of ice-cold PBS containing 100 μg/ml CHX.
    2. Perform a second wash with 1 ml of ice-cold PBS containing 100 μg/ml CHX.
    3. One coverslip at a time, aspirate PBS containing 100 μg/ml CHX and gently add 0.5 ml of ice-cold PBS containing 4% formaldehyde. Protect from light and incubate at RT for 20 min.
      Caution: Formaldehyde is toxic and should be handled in a fume hood.
    4. Wash twice each with 1 ml of PBS.
    5. Remove PBS and add 0.5 ml of TBST containing 0.25 μg/ml Hoechst 34580. Protect from light and incubate at RT for 30 min.
      Caution: Hoechst 34580 is potentially carcinogenic, so precautions should be taken for handling and disposal.
    6. Remove TBST containing 0.25 μg/ml Hoechst 34580 and wash twice each with 1 ml of PBS. Leave the coverslips in the PBS from the second wash.
    7. Using fine curved tweezers, retrieve each coverslip, remove as much PBS as possible by touching a napkin with the edge of the coverslip, and transfer (facing up) onto a sheet of paper. Label each coverslip by writing on the sheet of paper next to it.
    8. Protect from light and allow to dry completely (at least 15 min).
    9. One coverslip at a time, add 13.5 μl of ProLong® Diamond Antifade mounting reagent to a microscope slide, pick up the coverslip using tweezers and invert it onto the drop of mounting reagent.
      Critical: Avoid pipetting air bubbles onto the slide.
    10. Protect from light and incubate at RT for at least 16 h on a level surface.
      Pause Point: Slides may be stored up to 24 h at RT and then for several weeks at 4 °C until imaging.

  9. Imaging (Timing: 4-6 h per sample for 15 heterokaryons)
    Note: We recommend that the imaging settings (excitation/emission wavelengths, laser power, gain, offset, etc.) be optimized for each donor cell line (e.g., expressing an eGFP-tagged protein of interest) and recipient cell line (e.g., expressing CAAX-mCherry) grown separately on coverslips and processed in parallel with the samples. Make sure that the combination of fluorophores and settings do not generate any cross-talk.
    1. For each sample (i.e., for each pair of donor-recipient cell lines), choose the coverslip containing the optimal cell density by visual inspection of Hoechst fluorescence. Cell density should be high enough so that pairs of nuclei often appear in close proximity to each other but clearly separated from surrounding nuclei (see Notes 10-12).
    2. Move the objective toward the periphery of the coverslip until cell density is reduced and/or cells appear damaged, then move the objective back into the region suitable for analysis (see Note 13).
    3. Perform a systematic search for the desired cell fusion (donor-recipient) events using Hoechst fluorescence. Nuclei in fused cells are very close to each other and often display complementary shapes, although it is not always the case (see examples in Figures 2 and 3). When using cells from two different species (e.g., mouse and human), finding heterokaryons can sometimes be facilitated by the presence of distinct heterochromatin patterns specific to each species (Borer et al., 1989). Note that heterokaryons suitable for analysis (i.e., clearly containing only one donor and one recipient nuclei) constitute rare events (< 0.01% of the cells on the examined coverslip).
    4. Before imaging, confirm that both nuclei are surrounded by a common, red membrane in the mCherry channel (see Note 14).
    5. Using the confocal microscope acquisition settings, find the optimal focus (middle of the cells on the Z-axis), define the area to be imaged and take a picture in all three channels (mCherry, eGFP and Hoechst, see Figures 2 and 3). Use the same frame size and resolution for all pictures in a given analysis; according to our experience, imaging an area of 67.4 x 67.4 μm with a resolution of 1,024 x 1,024 pixels generates high-quality images with a reasonable scanning time and almost always allows us to include at least one nucleus from an unfused recipient cell (or at least part of it) for background subtraction.
    6. Acquire z-stacks of eGFP and Hoechst fluorescence with the same number of slices for all pictures in a given analysis. In our experience, 10 to 12 slices with an interval of 0.31 μm allow covering almost the entire nucleus in the Z-axis. To minimize scanning time and bleaching of eGFP fluorescence, we recommend imaging first all slices in the eGFP channel and then all slices in the Hoechst channel. We do not deem it necessary to acquire z-stacks of CAAX-mCherry as it significantly increases scanning time; however, make sure to keep the picture generated in Step I5) as evidence that the two nuclei originate from a fused donor-recipient cell.
    7. Resume the systematic search for desired donor-recipient cell fusion events and repeat Steps I4 to I6. We recommend imaging a minimum of 15 pairs of nuclei per sample for proper statistical analysis.


      Figure 2. Examples of clonal donor and recipient cell lines that can be used in quantitative heterokaryon assays. Upper left panel, mouse P19 cells expressing GFP-tagged SRSF5, an RBP with nucleocytoplasmic shuttling activity (Botti et al., 2017), are used as donor cells. SRSF5-GFP is expressed at near endogenous levels from a BAC randomly integrated into the genome, resulting in physiological localization of the protein throughout the nucleoplasm with enrichment in nuclear speckles. Note the highly similar GFP fluorescence levels in all cells due to the use of a clonal cell line. An inset shows the characteristic Hoechst staining pattern for mouse P19 cells. Upper right panel, human HeLa cells expressing CAAX-mCherry in their cell membranes are used as recipient cells. CAAX-mCherry is expressed at uniform levels in all recipient cells due to the selection of a high-expressing clone following random integration of plasmid TH0477 into the genome. An inset shows the characteristic Hoechst staining pattern for HeLa cells. Lower panel, donor and recipient cell lines shown in upper panels were used in the quantitative heterokaryon assay. The image shows a heterokaryon, i.e., a cell containing one nucleus from a donor P19 cell and one nucleus from a recipient HeLa cell, both surrounded by a common membrane marked with CAAX-mCherry. An inset shows the Hoechst staining patterns of both nuclei of the heterokaryon. The orange asterisk indicates an unfused HeLa cell and the yellow asterisk, an unfused P19 cell. Note the conspicuous presence of GFP signal in the recipient HeLa nucleus and its absence from the unfused HeLa nucleus (see also Figure 3). Also note the absence of CAAX-mCherry signal in membranes surrounding unfused P19 nuclei. Scale bars = 10 μm.


      Figure 3. Cell type-specific shuttling activity can be studied using quantitative heterokaryon assays. Left and middle panels, mouse P19 cells expressing SRSF5-GFP were used as donor cells before (left) and after (middle) 9 days of differentiation into neural cells. Right panel, a mouse NIH3T3 clonal cell line expressing SRSF5-GFP was used as donor cell line. SRSF5-GFP shuttles efficiently in pluripotent (P19) cells but poorly in differentiated (P19 and NIH3T3) cells. See Botti et al. (2017) for quantification and comparison of the shuttling activity of SRSF5 in these different cell types. Note the complete absence of GFP signal from unfused HeLa nuclei (indicated by orange asterisks). Scale bars: left panel = 5 µm; middle panel = 5 µm; right panel = 10 μm.

  10. Image analysis and quantification of fluorescence (Timing: 2-3 h for 15 images)
    1. Open Fiji (ImageJ Version 2.0.0-rc-43/1.51h or more recent).
    2. Open the Region Of Interest (ROI) Manager by selecting “Analyze → Tools → ROI Manager…”.
    3. Open image (select “View stack with: Hyperstack”, “Color mode: Composite” and “Autoscale”.
    4. Select “Image → Stacks → Z Project…”, “Start slice: 1”, “Stop slice: 10” (or 12, include all slices), “Projection type: Max Intensity”.
    5. Select “Image → Color → Split Channels”.
    6. Click on the picture generated in the Hoechst channel and select “Image → Lookup Tables → Grays”.
    7. Select “Image → Adjust → Brightness/Contrast” and make both the donor and recipient nuclei as visible as possible while still being able to distinguish them from each other.
    8. If the donor and recipient nuclei are in very close proximity to each other, draw a line between them using the Pencil Tool.
    9. Adjust the brightness and contrast to see the entire donor and recipient nuclei as clearly as possible.
    10. Zoom on the donor/recipient nuclei as much as possible so that they are entirely visible in the window. Using Freehand selections, draw the border of the donor cell.
    11. In the ROI Manager window, click “Add [t]”. This will generate an ROI with a specific number.
    12. In the list, click on the number corresponding to the ROI and select “Rename…” and then “Rename As: Image 1 donor”.
    13. Draw the border of the recipient nucleus, add to the ROI Manager and rename as “Image1 recipient”.
    14. Zoom out as necessary and zoom in on the nucleus of an unfused recipient cell.
    15. Draw the border of the nucleus from the unfused recipient cell, add to the ROI Manager and rename as “Image 1 background”.
    16. Click on the original (.czi) picture and select “Image → Stacks → Z Project…”, “Start slice: 1”, “Stop slice: 10” (or 12, include all slices), and “Projection type: Sum Slices” (see Note 15).
    17. Select “Image → Color → Split Channels.
    18. Click on the image of the eGFP fluorescence and select “Image → Type → 16-bit”.
    19. In the ROI Manager window, select the ROI corresponding to the donor nucleus and click on “Measure”. A Results window will open. Make sure the following data have been generated: Area, Mean, Min, Max and Slice.
    20. In the ROI Manager window, select the ROI corresponding to the recipient nucleus and click on “Measure”.
    21. Repeat for the background nucleus.
    22. Close all images and repeat Steps J3 to J21 with image 2.
    23. To save the ROIs for documentation, select in the ROI Manager “More >>… Save”.
    24. Save the results in a Microsoft Excel file by clicking on the Results window and selecting “File → Save As…”.
    25. Open the Excel File. For each image, subtract the mean pixel intensity (Mean) of the unfused recipient nucleus from that of both the donor and recipient nuclei. This yields the background subtracted mean pixel intensity for each nucleus.
    26. For both the donor and recipient nuclei, multiply the background subtracted mean pixel intensity by the area to obtain the total pixel intensity.
    27. Divide the total pixel intensity of the recipient nucleus by that of the donor nucleus and multiply by 100 to obtain the value of the Shuttling capacity (%).

Data analysis

  1. To compare the shuttling of a protein in two different conditions or the shuttling of two different proteins, we recommend imaging a minimum of 15 heterokaryons per condition or protein from at least two independent experiments (e.g., 7-8 heterokaryons per coverslip from two independent experiments). 
  2. The mean shuttling activity of a protein in a given condition should be calculated from at least 15 heterokaryon images. 
  3. We apply Student's t-tests and paired Wilcoxon rank-sum tests to test whether the shuttling activity is significantly different between the different conditions or proteins. For this, all quantified and background subtracted shuttling capacity values are assembled into columns with the protein name as header into a .txt file. Plots and tests are performed in R.
    Example:
    read.table("/path/Quantification_HKA.txt", head=TRUE, sep="\t") -> experiment_HKA
    summary(experiment_HKA)
    boxplot(experiment_HKA, las = 2, ylab = "Shuttling capacity HKA)", boxfill=c("lightgrey"))
    wilcox.test(experiment_HKA $Control, experiment_HKA $Mutant, paired=TRUE)

Notes

  1. Protein tagging in bacterial artificial chromosomes (BACs) and subsequent generation of transgenic cell lines has been described in detail in (Poser et al., 2008).
  2. The readout of shuttling assays is the re-distribution of a protein from a donor to a recipient nucleus. In the original assay (Borer et al., 1989), the emergence of proteins of interest in recipient nuclei (from a different species) was qualitatively assessed by indirect immunofluorescence using species-specific monoclonal antibodies; however, for some proteins, specific antibodies are difficult–if not impossible–to generate. To circumvent this, cells have been transiently transfected with epitope-tagged cDNAs encoding proteins of interest prior to fusion with recipient cells (Pinol-Roma and Dreyfuss, 1991; Caceres et al., 1998; Lin et al., 2005). However, transient expression from plasmids often leads to overexpression, which in turn can affect the functions and subcellular distribution of RBPs (Maharana et al., 2018; our unpublished observations). Moreover, RBP levels will vary substantially from cell to cell, precluding the identification of comparable heterokaryons. To overcome these limitations, we strongly recommend using clonal cell lines stably expressing fluorescently tagged proteins from bacterial artificial chromosomes (BACs) that have been integrated into the genome in single copy (Poser et al., 2008; Botti et al., 2017). The large size of BACs ensures the presence of all gene regulatory elements (e.g., promoters, enhancers, intronic sequences and untranslated regions), which preserves endogenous ratios of splice isoforms and results in expression levels similar to those of the endogenous gene (Figure 2, Änkö et al., 2012; Müller-McNicoll et al., 2016; Botti et al., 2017). Alternatively, fluorescent tags can be introduced into RBPs expressed from their endogenous loci using genome editing by CRISPR/Cas9 (Van Nostrand et al., 2017).
  3. We also recommend using control cell lines in each experiment. We have used cells expressing eGFP-tagged PRPF8 (a spliceosomal component) and the nuclear export factor (NXF1) as negative and positive controls for shuttling, respectively (Botti et al., 2017). Other RBPs for which shuttling has been confirmed or ruled out can be used. However, one should bear in mind that shuttling of a given RBP might vary depending on the cell type and/or differentiation state (Figure 3, Botti et al., 2017; Hammarskjold et al., 2017).
  4. To readily identify true heterokaryons, our assay requires the use of a membrane marker that is expressed only in recipient cells. We have successfully used clonal recipient cell lines expressing CAAX-mCherry from a plasmid randomly integrated into their genomes (TH0477, Stewart et al., 2011; Botti et al., 2017). CAAX is an isoprenylated peptide which is tethered to cell membranes (Wright and Philips, 2006). However, other membrane markers might be equally suitable. Stable transgenic cell lines using plasmids can be generated in essentially the same way as when using BACs. See Note 1.
  5. Our assay is based on quantitative cell imaging, which requires a standardized imaging protocol with identical settings (laser power, gain, digital offset, etc.) for acquisition of all images. To achieve this, we strongly recommend the use of clonal lines for both donor and recipient cell lines, preferentially selected for endogenous expression level of the studied RBP. Clonal cell lines can be obtained either by FACS sorting, which requires access to a specialized core facility or the help of a qualified technician, or without any specialized equipment or expertise using cloning discs.
  6. The most critical part of the protocol: To produce reliable results, only heterokaryons containing one donor and one recipient nucleus should be quantified. To increase the frequency of examinable heterokaryons–and thereby reduce microscopy time/increase statistical power–experimental conditions should be optimized for different combinations of donor-recipient cell lines in the following three ways.
      First, use an appropriate ratio of donor to recipient cells. In principle, a 1:1 ratio should be used (and is a good starting point) to increase the probability that heterokaryons contain one donor and one recipient nucleus. However, different cell types adhere more or less tightly to glass coverslips, and given that several medium changes/washes must be carried out prior to cell fusion, this ratio might have to be optimized according to the combination of cells used. In addition, some cell lines have a faster growth rate than others, and a compromise should be made between strong attachment and low number of cell division, as recently divided cells tend to fuse together and reduce the frequency of examinable heterokaryons. For example, when we use rapidly dividing mouse P19 cells as donors and HeLa cells as recipients, we allow a shorter (6 to 8 h) time for adhesion than when using mouse NIH3T3 cells with the same recipients (12-16 h) to avoid P19 cell division, but we use a P19:HeLa cell ratio of 2:1 or 3:1 as P19 cells adhere less quickly and strongly than HeLa and NIH3T3 cells to the coverslips and many P19 cells are lost during the washes. When using cell types that require coating (e.g., with gelatin, laminin, etc.), we recommend pre-incubating the coverslips for a sufficient time, preferably several hours at 37 °C, before seeding the cells.
      Second, it is crucial to obtain a single-cell suspension of both donor and recipient cells prior to mixing and seeding. This might require pipetting up and down harshly to break the cell clumps. Do not hesitate to sacrifice viability of some cells to obtain true single-cell suspensions.
      Third, use an optimal cell density. To obtain heterokaryons, cells should adhere to the coverslip close enough to each other so that their membranes can fuse upon chemical treatment (see below); however, cell suspensions should be diluted enough to avoid multiple fusion events. To find the best compromise between single and multiple fusions, we recommend performing serial 2-fold dilutions of a mixed (donor-recipient) cell suspension prior to seeding and determining by microscopy the optimal dilution to search for suitable heterokaryons. Although the use of recipient cell lines expressing a fluorescent membrane marker greatly facilitates the identification of heterokaryons appropriate for analysis, finding clear fusion events between a single donor and a single recipient cell can remain challenging, and it is worth investing some time to determine the optimal dilutions with which to proceed to the search.
  7. Nuclear proteins are synthesized in the cytoplasm. To determine whether a nuclear protein shuttles to the cytoplasm and back to the nucleus, it is therefore essential to block protein synthesis before and during the shuttling assay. In this way, any newly emerging fluorescent protein in a recipient nucleus should originate from the donor nucleus, not from the pool of newly synthesized proteins in their common cytoplasm. A confirmed non-shuttling nuclear RBP (e.g., splicing factor PRPF8; Sapra et al., 2009) should always be used as a negative control; the absence of shuttling confirms efficient protein synthesis inhibition. We have used two different inhibitors of protein synthesis (cycloheximide and puromycin) yielding essentially similar results (Botti et al., 2017).
  8. Polyethylene glycol (PEG) is water soluble, so it can be easily removed by washing; however, a prolonged incubation can be toxic for most cell lines (our observations). It is important to use the same incubation time for all samples in a comparative shuttling analysis.
  9. Following cell fusion, sufficient time should be allowed in order to detect and measure the shuttling of an RBP of interest. Several factors can affect the shuttling efficiency of a given protein. We have shown that different members of the SR protein family shuttle at different rates, which correlated with the length of their phosphorylatable RS domain (Botti et al., 2017). Shuttling rates can differ greatly depending on the biological functions of the RBP. For example, whereas some nuclear proteins such as nucleolin appear to slowly “leak” from to the cytoplasm, requiring about 24 h to be detected in a recipient nucleus, shuttling of nucleocytoplasmic transport factors can be detected within minutes (Gama-Carvalho et al., 2001) and reach equilibrium (i.e., 50% in both donor and recipient nuclei) within 3 h (Caceres et al., 1998; Sapra et al., 2009; Botti et al., 2017). When comparing the shuttling efficiencies of different RBPs, it is crucial to fix the cells before they reach equilibrium. Moreover, in order to minimize variation, the exact same incubation time should be allowed for shuttling in each replicate of the analysis.
      Another factor that can affect shuttling efficiency is the combination of donor and recipient nuclei. For example, if a given protein is exported from the donor nucleus but not from the recipient nucleus, the protein might get trapped and accumulate in the recipient nucleus, and the shuttling capacity of the protein might appear greater than it is in reality, sometimes reaching over 50% (unpublished observation). In such a case, it might be necessary to reduce the shuttling time to be able to quantify differences (e.g., between mutants and WT proteins).
      Finally, the cell cycle stage of donor and recipient nuclei might, in some cases, affect shuttling capacity of a protein under study. The nuclear volume and the number of NPCs almost double during interphase in dividing cells (Maeshima et al., 2011). A larger nucleus should, in theory, allow a faster import and a slower export of shuttling proteins due to its larger surface area being in contact with the cytoplasm and its smaller surface area being in contact with the nucleoplasm. We have observed that larger recipient nuclei tend to harbor higher total fluorescence levels (unpublished observation); however, such an effect does not significantly affect the results when a sufficient number of heterokaryons are imaged. Nevertheless, the shuttling of some proteins might vary throughout the cell cycle, and in some cases it might be useful to synchronize the cells (e.g., in G1) before fusion.
  10. If none of the coverslips shows an optimal density, adjust dilutions in subsequent experiments.
  11. Some cell fusion events might seem to happen only between cells of the same cell line (donor or recipient cell line). One possible reason is the faster growth rate of either donor or recipient cells. If one cell line grows too quickly, try to reduce the attachment time at Step D1.
  12. In some cases, although a donor:recipient cell ratio of 1:1 has been used at Step C8, one cell type (either donor or recipient) appears to predominate on the slides. One possible reason is that different cell types attach more or less firmly to coverslips, and either donor or recipient cells might be lost at a higher rate during medium changes and washes.
  13. Cells at the periphery of the coverslip are sometimes not suitable for imaging/analysis: according to the Hoechst fluorescence, their nuclei might appear smaller, brighter and irregularly shaped, and their specific chromatin features might be less apparent. The fluorescence levels of such cells tend to be lower in both the eGFP and mCherry channels. These are regions containing presumably damaged cells.
  14. Acquiring Z-stacks, which are necessary for proper quantification, is time consuming. The use of recipient cells with a fluorescent marker in their membranes (e.g., CAAX-mCherry) allows to readily distinguish true heterokaryons from donor/recipient cells that are merely in close proximity but not fused. If a quick look under the microscope suggests that it is a true heterokaryon, proceed to imaging. Otherwise, resume the search for suitable heterokaryons.
  15. Even if care has been taken to avoid pixel saturation during imaging, the sum of slices will make some of the pixels appear saturated. This should affect only a minority of the pixels in the region of interest, and in our experience it doesn’t significantly impact the results. However, if one suspects that too many pixels appear saturated in the sum of slices and that this might affect the analysis, fluorescence should be quantified in separate slices and then summed.

Recipes

  1. DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin
    1. Add 50 ml of heat-inactivated FBS to 500 ml of DMEM
    2. Add 5 ml of Penicillin-Streptomycin (10,000 U/ml)
    3. Mix well and store at 4 °C (keep sterile)
  2. PBS containing 0.1% gelatin
    1. Add 25 ml of 2% gelatin to 500 ml of sterile 1x PBS
    2. Mix well and store at 4 °C (keep sterile)
  3. 10x TBS (1 L)
    1. Dissolve 24 g of Trizma® base and 88 g of NaCl in 900 ml of dH2O
    2. Adjust pH to 7.6 with HCl
    3. Adjust volume to 1 L and verify pH
    4. Store at RT
  4. TBST (1 L)
    1. Add 100 ml of 10x TBS to 900 ml of dH2O
    2. Add 0.5 ml of Tween® 20
    3. Mix well and store at RT
  5. 4% Formaldehyde in 1x PBS (40 ml, for 80 coverslips)
    1. In a 50-ml tube, add 4 ml of 10x PBS to 26 ml of H2O (Sigma)
    2. In a fume hood, add 10 ml of 16% formaldehyde
    3. Mix by vortexing for 10-15 sec
    4. Store at -20 °C in 4-ml aliquots (each enough for 8 coverslips)
  6. Hoechst 34580 stock solution, 1 mg/ml (5 ml)
    1. Dissolve the entire contents of the vial (5 mg) in 5 ml of H2O (Sigma)
    2. Store in aliquots at -20 °C and protect from light
  7. Hoechst 34580, 0.25 μg/ml in TBST (4 ml, for 8 cover slips)
    1. In a 15-ml tube, add 1 μl of 1 mg/ml Hoechst 34580 to 4 ml of H2O (Sigma)
    2. Mix by vortexing for 10-15 sec
    3. Prepare fresh, keep on ice and protect from light

Acknowledgments

This work was supported by funding from the German Research Foundation (DFG) to MMM (CEF-MC and SFB902). We are grateful to M. C. Steiner for developing the first version of the assay and K. M. Neugebauer for inspiration and guidance. We thank H. Schewe (FCAM) for support on confocal microscopes. This protocol was adapted from procedures published in Botti et al. (2017).

Competing interests

The authors declare no competing financial interests.

References

  1. Änkö, M. L., Muller-McNicoll, M., Brandl, H., Curk, T., Gorup, C., Henry, I., Ule, J. and Neugebauer, K. M. (2012). The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes. Genome Biol 13(3): R17.
  2. Borer, R. A., Lehner, C. F., Eppenberger, H. M. and Nigg, E. A. (1989). Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56(3): 379-390.
  3. Botti, V., McNicoll, F., Steiner, M. C., Richter, F. M., Solovyeva, A., Wegener, M., Schwich, O. D., Poser, I., Zarnack, K., Wittig, I., Neugebauer, K. M. and Müller-McNicoll, M. (2017). Cellular differentiation state modulates the mRNA export activity of SR proteins. J Cell Biol 216(7): 1993-2009.
  4. Caceres, J. F., Screaton, G. R. and Krainer, A. R. (1998). A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm. Genes Dev 12(1): 55-66.
  5. Ederle, H. and Dormann, D. (2017). TDP-43 and FUS en route from the nucleus to the cytoplasm. FEBS Lett 591(11): 1489-1507.
  6. Gama-Carvalho, M. and Carmo-Fonseca, M. (2001). The rules and roles of nucleocytoplasmic shuttling proteins. FEBS Lett 498(2-3): 157-163.
  7. Gama-Carvalho, M., Carvalho, M. P., Kehlenbach, A., Valcarcel, J. and Carmo-Fonseca, M. (2001). Nucleocytoplasmic shuttling of heterodimeric splicing factor U2AF. J Biol Chem 276(16): 13104-13112.
  8. Hammarskjold, M. L. and Rekosh, D. (2017). SR proteins: To shuttle or not to shuttle, that is the question. J Cell Biol 216(7): 1875-1877.
  9. Howard, J. M. and Sanford, J. R. (2015). The RNAissance family: SR proteins as multifaceted regulators of gene expression. Wiley Interdiscip Rev RNA 6(1): 93-110.
  10. Jeong, S. (2017). SR Proteins: Binders, regulators, and connectors of RNA. Mol Cells 40(1): 1-9.
  11. Lin, S., Xiao, R., Sun, P., Xu, X. and Fu, X. D. (2005). Dephosphorylation-dependent sorting of SR splicing factors during mRNP maturation. Mol Cell 20(3): 413-425.
  12. Liu, E. Y., Cali, C. P. and Lee, E. B. (2017). RNA metabolism in neurodegenerative disease. Dis Model Mech 10(5): 509-518.
  13. Maeshima, K., Iino, H., Hihara, S. and Imamoto, N. (2011). Nuclear size, nuclear pore number and cell cycle. Nucleus 2(2): 113-118.
  14. Maharana, S., Wang, J., Papadopoulos, D. K., Richter, D., Pozniakovsky, A., Poser, I., Bickle, M., Rizk, S., Guillen-Boixet, J., Franzmann, T., Jahnel, M., Marrone, L., Chang, Y. T., Sterneckert, J., Tomancak, P., Hyman, A. A. and Alberti, S. (2018). RNA buffers the phase separation behavior of prion-like RNA binding proteins. Science 360(6391): 918-921.
  15. Maslon, M. M., Heras, S. R., Bellora, N., Eyras, E. and Caceres, J. F. (2014). The translational landscape of the splicing factor SRSF1 and its role in mitosis. Elife: e02028.
  16. Müller-McNicoll, M. and Neugebauer, K. M. (2013). How cells get the message: dynamic assembly and function of mRNA-protein complexes. Nat Rev Genet 14(4): 275-287.
  17. Müller-McNicoll, M., Botti, V., de Jesus Domingues, A. M., Brandl, H., Schwich, O. D., Steiner, M. C., Curk, T., Poser, I., Zarnack, K., and Neugebauer, K. M. (2016). SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export. Genes Dev 30: 553-566.
  18. Ouellet, J. (2016). RNA Fluorescence with light-up aptamers. Front Chem 4: 29.
  19. Pinol-Roma, S. and Dreyfuss, G. (1991). Transcription-dependent and transcription-independent nuclear transport of hnRNP proteins. Science 253(5017): 312-314.
  20. Poser, I., Sarov, M., Hutchins, J. R., Heriche, J. K., Toyoda, Y., Pozniakovsky, A., Weigl, D., Nitzsche, A., Hegemann, B., Bird, A. W., Pelletier, L., Kittler, R., Hua, S., Naumann, R., Augsburg, M., Sykora, M. M., Hofemeister, H., Zhang, Y., Nasmyth, K., White, K. P., Dietzel, S., Mechtler, K., Durbin, R., Stewart, A. F., Peters, J. M., Buchholz, F. and Hyman, A. A. (2008). BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat Methods 5(5): 409-415.
  21. Sapra, A. K., Änkö, M. L., Grishina, I., Lorenz, M., Pabis, M., Poser, I., Rollins, J., Weiland, E. M. and Neugebauer, K. M. (2009). SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo. Mol Cell 34(2): 179-190.
  22. Stewart, M. P., Helenius, J., Toyoda, Y., Ramanathan, S. P., Muller, D. J. and Hyman, A. A. (2011). Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding. Nature 469(7329): 226-230.
  23. Van Nostrand, E. L., Gelboin-Burkhart, C., Wang, R., Pratt, G. A., Blue, S. M. and Yeo, G. W. (2017). CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins. Methods 118-119: 50-59.
  24. Wright, L. P. and Philips, M. R. (2006). Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras. J Lipid Res 47(5): 883-891.

简介

许多蛋白质在稳态下仅出现核,但事实上在细胞核和细胞质之间连续地来回穿梭。例如,核RNA结合蛋白(RBP)通常伴随mRNA到达细胞质,在那里它们可以在穿梭回到细胞核之前调节其货物的亚细胞定位,翻译和/或腐烂。必须严格调节核质穿梭,因为几种RBP与朊病毒样结构域如FUS和TDP-43的错误定位导致固体病理性聚集体的细胞质积累,这些聚集体与肌萎缩侧索硬化症(ALS)和额颞叶痴呆等神经退行性疾病有关。 (FTD)。传统上,种间异核体分析已被用于确定感兴趣的核蛋白是否穿梭;这些分析是基于来自两个不同物种(例如,小鼠和人类)的供体和受体细胞之间的融合,可以根据不同的染色质染色模式区分,并检测蛋白质的外观。受体核。然而,异核体的鉴定需要经验并且容易出错,这使得难以获得用于定量研究的高质量数据。此外,荧光标记的RBP在供体细胞中的瞬时过表达通常导致其异常的亚细胞定位。在这里,我们提出定量测定,其中表达接近生理水平的eGFP标记的RBP的稳定供体细胞系与表达膜标记物CAAX-mCherry的受体细胞融合,允许容易地鉴定和成像大量高可信度异核体。我们的测定法可用于测量任何感兴趣的核蛋白在不同细胞类型,不同细胞条件下或突变蛋白之间的穿梭活性。

【背景】要了解蛋白质的各种功能,重要的是找出它在细胞内定位的位置。标准的微观和生物化学方法仅在其稳态浓度高于检测阈值时才揭示蛋白质的存在。他们不排除它在短暂地定位的情况下扮演其他重要角色的可能性(Gama-Carvalho和Carmo-Fonseca,2001)。例如,许多RBP在不同的细胞区室中发挥作用,它们伴随着它们的结合mRNA(通常未检测到)并连接真核基因表达的多个步骤(Müller-McNicoll和Neugebauer,2013)。 SR蛋白(SRSF1至SRSF12)是一系列RBP,其调节细胞核中的转录,前mRNA剪接,3'末端加工和mRNP包装,并且在稳态下仅显示核(Howard和Sanford,2015; Jeong,2017)。然而,大多数家庭成员在细胞核和细胞质之间连续(但程度不同)穿梭,在mRNA输出和翻译中发挥其他功能(Caceres et al。,1998; Sapra et al。 ,2009; Maslon et al。,2014;Müller-McNicoll et al。,2016; Botti et al。,2017 )。 RBP穿梭的变化已经在病毒感染,早期发育,细胞分化和神经退行性疾病如ALS和FTD中被描述,其中细胞质中朊病毒样RBP如FUS和TDP-43的病理积累形成固体神经毒性聚集体(Ederle和Dormann,2017; Liu et al。,2017)。因此,了解RBP是否通常在细胞核和细胞质之间穿梭是非常重要的,如果是,在何种情况下以及如何控制它。

利用目前可用的工具,很难研究核RBP的细胞质功能并比较它们的穿梭能力。近三十年前开发了一种巧妙的方法 - 种间异核体分析 - 来自两个不同物种(例如,小鼠和人类)的供体和受体细胞融合,蛋白质仅存在于供体细胞核中如果它穿梭,它会逐渐出现在受体细胞核中(Borer et al。,1989)。然而,该测定仅提供定性信息。基于相位对比图像识别融合事件,并且基于不同的染色质特征鉴定供体和受体核,这使得测定费力,主观并且仅产生少量高可信度异核体。此外,荧光标记的RBP通常由瞬时转染的质粒表达,这导致不同细胞中RBP水平差异很大,从几乎不可检测到非生理学高表达,可能导致RBP的部分异常细胞质或亚核定位[(Maharana et al。,2018)我们未发表的观察结果]。总而言之,这些限制排除了任何比较分析。

在这里,我们提出了一个详细的实验方案,在培养的哺乳动物细胞中进行定量穿梭测定(图1)。我们的分析是对经典异核体分析的改进,具有两个新颖性和标准化的成像管道以进行定量测量。第一个新颖性是使用表达荧光标记的膜标记物(CAAX-mCherry)的受体细胞系,这极大地促进了包含供体和受体细胞核的异核体的鉴定,因此允许快速且容易地鉴定大量的高可信度的异核体。第二个新颖性是使用稳定的克隆供体细胞系,其中感兴趣的荧光(eGFP)标记的RBP由细菌人工染色体(BAC)表达,其已经整合到基因组中(Botti 等人,2017; Poser et al。,2008)。随后的细胞克隆选择确保了每个供体细胞中标记的RBP的相等和接近生理水平,以促进图像采集,分析和比较。

我们的测定已成功应用于比较不同SR蛋白在同一细胞系,不同细胞系之间和分化状态之间的穿梭活性。此外,它使我们能够研究使用突变蛋白和核输出因子的敲除来穿梭个体SR蛋白的要求(Botti 等。,2017)。我们使用各种细胞系作为供体或受体细胞:这些细胞包括小鼠(P19和NIH3T3)和人(HeLa)细胞。虽然其他细胞系仍有待测试,但我们相信,荧光RBP可在生理水平表达的任何贴壁细胞系都适合我们的测定。这应该包括从表达荧光标记的目标RBP的转基因动物获得的原代细胞。我们在我们的检测中成功地使用了分化为神经细胞的P19细胞作为供体(Botti et al。,2017),并且应该可以研究和量化其他细胞分化模型中RBP的穿梭,例如,在小鼠胚胎干细胞(ESCs)或诱导多能干细胞(iPSC)中,或比较不同细胞分化命运中RBP的穿梭(Hammarskjold和Rekosh,2017)。此外,我们的分析应该可以量化病毒感染和细胞应激期间穿梭的变化,或评估疾病暴露在RBP中的影响。原则上,我们的测定甚至可以用于显示长非编码RNA(lncRNA)的穿梭,例如通过插入荧光MS2结合蛋白(MS2-BP)的结合位点或插入结合荧光染料的适体序列(Ouellet,2016)。

关键字:RNA结合蛋白, 核质穿梭, 异核体分析, 穿梭能力定量, 核蛋白

材料和试剂

  1. 10μl过滤器吸头长(SARSTEDT,目录号:70.1116.210)
  2. 20μl过滤嘴(SARSTEDT,目录号:70.760.213)
  3. 300μl过滤嘴(SARSTEDT,目录号:70.765.210)
  4. 1,000μl过滤嘴(SARSTEDT,目录号:70.762.211)
  5. 10-cm细胞培养皿(VWR,Thermo Fisher Scientific,目录号:734-2043)
  6. 克隆盘,尺寸5 mm(Sigma-Aldrich,SP Scienceware - Bel-Art Products - H-B Instrument,目录号:Z374458-100EA)
  7. 一次性玻璃巴斯德吸管150毫米(VWR,目录号:612-1701)
  8. 血清移液器,2 ml(VWR,Corning,目录号:734-1690)
  9. 血清移液器,5 ml(VWR,Corning,目录号:734-1737)
  10. 15毫升离心管(Corning,目录号:430791)
  11. 2毫升微量离心管(SARSTEDT,目录号:72.691)
  12. 1,000μl移液器吸头ART ® 1000E阻隔吸头(Thermo Fisher Scientific,目录号:2079E)
  13. 用于细胞培养的12孔板(VWR,Thermo Fisher Scientific,目录号:734-2156)
  14. 精密盖玻片,18 mm,硼硅酸盐玻璃0.17±0.005 mm(Carl Roth,目录号:LH23.1)
  15. 显微镜载玻片(VWR,目录号:631-1550)
  16. 含有编码eGFP标记的目的RBP的基因的细菌人工染色体(BAC)(Botti et al。, 2017和Poser et al。,2008;见注释1- 3)
  17. 用于表达不同颜色的荧光质膜标记物的质粒(例如,CAAX-mCherry,质粒TH0477,Stewart 等人,2011; Botti 等。 ,2017;见注4)
  18. DMEM,高葡萄糖,GlutaMAX TM 补充剂,丙酮酸盐(Thermo Fisher Scientific,目录号:31966047)
  19. 胎牛血清(Thermo Fisher Scientific,目录号:10270106)
  20. 青霉素 - 链霉素(10,000 U / ml)(Thermo Fisher Scientific,目录号:15140122)
  21. 嘌呤霉素10 mg / ml(Thermo Fisher Scientific,Gibco TM ,目录号:A1113803)
  22. Geneticin ®选择性抗生素(G418硫酸盐)(50 mg / ml)(Thermo Fisher Scientific,目录号:10131035)
  23. 胰蛋白酶0.05%EDTA(Thermo Fisher Scientific,目录号:25300054)
  24. Dulbecco的磷酸盐缓冲盐水(Sigma-Aldrich,目录号:D8537)
  25. H 2 O中2%明胶溶液生物试剂(Sigma-Aldrich,目录号:G1393)
  26. 环己酰亚胺溶液100 mg / ml,在DMSO中(Sigma-Aldrich,目录号:C4859)
  27. 聚乙二醇(PEG)1500在75 mM HEPES中(Roche Diagnostics,目录号:10783641001)
  28. 磷酸盐缓冲盐水10x(Sigma-Aldrich,目录号:P5493)
  29. Pierce 16%甲醛(w / v),不含甲醇(Thermo Fisher Scientific,目录号:28908)
  30. 30.水,分子生物学试剂(Sigma-Aldrich,目录号:W4502)
  31. ProLong ®金刚石抗褪色剂(Thermo Fisher Scientific,Invitrogen TM ,目录号:P36970)
  32. TWEEN ® 20(Sigma-Aldrich,目录号:P7949)
  33. Tris(Carl Roth,目录号:4855.2)
  34. Hoechst 34580(Sigma-Aldrich,目录号:63493)
  35. Trizma ® base(Sigma-Aldrich,目录号:T1503)
  36. 氯化钠(NaCl),BioXtra(Sigma-Aldrich,目录号:S7653)
  37. 盐酸36.5-38.0%(HCl),用于分子生物学(Sigma-Aldrich,目录号:H1758)
  38. DMEM含有10%FBS,100 U / ml青霉素和100μg/ ml链霉素(见食谱)
  39. 含0.1%明胶的PBS(见食谱)
  40. 10倍TBS(见食谱)
  41. TBST(见食谱)
  42. 1x PBS中含4%甲醛(参见食谱)
  43. Hoechst 34580原液(1 mg / ml)(见食谱)
  44. 含有0.25μg/ ml Hoechst 34580的TBST(见食谱)

设备

  1. 血球
  2. 计时器
  3. 通风柜
  4. 细胞培养罩(Thermo Fisher Scientific,型号:Herasafe TM KSP,型号KSP 12)
  5. 细尖弯曲镊子
  6. P10移液器(VWR,目录号:613-5259)
  7. P20移液器(VWR,目录号:613-5260)
  8. P200移液器(VWR,目录号:613-5263)
  9. P1000移液器(VWR,目录号:613-5265)
  10. 微型真空泵,KNF(A. Hartenstein,目录号:AP86)
  11. 液体抽吸系统(VACCUBRAND,型号:VHCpro)
  12. CO 2 培养箱(Thermo Fisher Scientific,型号:Heracell TM 150i)
  13. 涡旋混合器(VWR,目录号:444-1372)
  14. 移液器控制器(accu-jet ® pro,品牌,目录号:26300)
  15. -20°C冰柜
  16. 冰箱
  17. 倒置显微镜(Motic,型号:AE31)
  18. 共聚焦激光扫描显微镜(蔡司,型号:LSM 780)

软件

  1. 斐济(ImageJ版本2.0.0-rc-43 / 1.51h或更近)
  2. Microsoft Excel(最新版本14.6.7或更高版本)

程序

有关定量异核运动穿梭试验的概述,请参见图1。


图1.定量异核运动穿梭试验概述。将表达GFP标记的核RBP的供体细胞与表达CAAX-mCherry的受体细胞一起接种到其细胞膜上。在环己酰亚胺存在下将细胞与PEG1500融合以阻断新的蛋白质合成,以避免RBP在普通细胞质中出现,而不是由于穿梭。 3小时后,固定细胞并通过共聚焦显微镜成像。适合于分析的异核体可以容易地鉴定,因为它们仅含有两个核,其中至少一个是绿色的,并且两者都被红色膜包围。通过共聚焦显微镜对细胞核进行成像,并在供体和受体细胞核中定量GFP荧光。鉴于允许在蛋白质合成抑制剂(环己酰亚胺)存在下进行穿梭,受体核中的任何荧光都是由于RBP的穿梭。


  1. 产生克隆转基因供体和受体细胞系
    1. 如Botti 等(2017)和Poser 等所述,从随机整合到基因组中的BAC产生表达eGFP标记的目标RBP的供体细胞系。 (2008)(见注1-3)。
    2. 如Botti 等所述,从随机整合到基因组中的质粒中产生表达不同颜色的质膜定位标记(例如,CAAX-mCherry)的受体细胞系。 em>(2017)(见注4)。
    3. 使用荧光激活细胞分选(FACS)或克隆盘,分离供体细胞系具有不同荧光水平的克隆和至少一个受体细胞系具有高荧光的克隆(参见注释5)。
    4. 如果可获得针对目标RBP的特异性抗体,则通过蛋白质印迹法估计不同克隆中eGFP标记的RBP的表达水平,并选择具有接近内源水平的克隆。

  2. 准备供体和受体细胞的个体培养物
    将供体和受体细胞传代到单独的10-cm培养皿上,1-3天后获得几乎融合的培养物。

  3. 混合并播种供体和受体细胞(时间:30分钟)(见注6)
    1. 对于供体和受体细胞的每种组合,将4个无菌18-mm盖玻片放入12孔板的不同孔中。如果使用通常需要预涂表面的细胞系来增加其粘附性,则相应地预涂盖玻片。例如,P19细胞需要用含有0.1%明胶的PBS涂覆,并且当使用该细胞系时,我们通常在37℃下将盖玻片预涂覆24小时。
    2. 估计供体和受体细胞的汇合(例如,50%,75%,100%)。
    3. 吸出培养基并用5ml预热的无菌PBS轻轻洗涤。彻底吸出PBS并加入1.5ml 0.05%胰蛋白酶。在室温或37°C孵育3-5分钟(必须确定每个细胞系的最佳条件)。
    4. 通过向每个方向倾斜板帮助细胞从餐具上脱离。
    5. 加入3ml含有10%FCS的适当培养基(例如,DMEM)以抑制胰蛋白酶活性。
      关键步骤:使用移液器控制器上的最快速度设置,通过在细胞培养皿中上下移液20次来破坏细胞团块。确保通过在显微镜下目视检查获得供体和受体细胞的单细胞悬浮液。
    6. 将每个单细胞悬浮液转移到15ml管中。
    7. 使用标准方法(例如,使用血细胞计数器)计算供体和受体细胞悬浮液的浓度。或者,根据您的细胞系,培养皿(即,表面积)和汇合使用近似值。例如,具有在汇合处生长的HeLa细胞的10-cm培养皿含有约8.8×10 6个细胞,并且在步骤C3至C6之后,相应的细胞悬浮液(体积为4.5ml)将包含大约2.0×10 6 细胞/ ml。&nbsp;
    8. 在2毫升管中,制备2毫升1:1供体:受体混合细胞悬液,每个细胞悬液含有大约1×10 6个至2×10 6个细胞。细胞悬液。例如,如果供体细胞培养物约为50%汇合(每个培养皿约4.4×10×10 6个细胞),则受体细胞培养物约100%汇合(~8.8×10 6 < / sup>每个培养皿中的细胞),将1.333ml供体细胞悬浮液(~9.8×10 5 细胞/ ml)与0.667ml受体细胞悬浮液混合(~2.0×10 6 细胞/ ml)总共约2.6×10 6个细胞。&nbsp;
    9. 以中等速度将混合细胞悬浮液涡旋10秒。
    10. 将1ml混合细胞悬浮液转移到含有1ml培养基(2倍稀释)的2ml管中。以中速涡旋10秒。这种和随后的稀释应有助于获得细胞融合和成像的最佳密度(参见程序I)。&nbsp;
    11. 类似地,再进行两次2倍连续稀释(相当于4倍和8倍稀释)。 8倍稀释应含有约2.5×10 5个/5μl至5.0×10 5个细胞。
    12. 将1ml每种混合细胞悬浮液(未稀释的,2倍,4倍和8倍稀释)转移到步骤C1中制备的12孔板中的盖玻片上。确保盖玻片不浮在介质表面。如果他们中的一些人这样做,帮助他们使用无菌尖端在底部下沉。如果盖玻片预先涂覆(例如,含有0.1%明胶的PBS),则在添加混合细胞悬浮液之前从每个孔中吸取涂层溶液。

  4. 将细胞附着到盖玻片上(时间:过夜[8-16小时])
    在37°C,5%CO 2 孵育8-16小时。
    关键步骤:孵育足够长的时间以使细胞附着在盖玻片上但短暂地足以避免细胞分裂。最近分裂的细胞容易融合在一起,通常导致含有两个以上核的异核体,这不应用于穿梭定量(参见方法I和J)。例如,小鼠P19细胞在指数生长期每16小时分裂一次,当使用该细胞系时,我们在此步骤孵育最多8小时(见注6)。

  5. 抑制蛋白质合成(时间:2.5小时)(见注7)
    关键步骤:在此阶段,某些细胞系可能没有紧密附着在盖玻片上。为了防止细胞损失,在进行每次更换介质或清洗时要非常小心。应该遵循该建议直到细胞被固定(步骤H3)。始终在孔的侧壁添加培养基或缓冲液,而不是直接添加到盖玻片上。
    1. 吸出培养基并轻轻加入1ml含有50μg/ ml放线菌酮(CHX)的培养基。在37℃下用5%CO 2 孵育2小时。
    2. 吸出培养基并轻轻加入1ml含有100μg/ ml CHX的培养基。在37℃下用5%CO 2 孵育30分钟。

  6. 细胞融合(时间:30分钟)(见注8)
    1. 吸出培养基并轻轻加入1ml含有100μg/ ml CHX的预热PBS。
    2. 启动计时器2分钟和10秒。然后,一次一个盖玻片,吸出PBS / CHX并轻轻加入0.25毫升预热的PEG 1500.
      关键步骤:从盖玻片到盖玻片迅速(约10秒)进行。每个盖玻片应孵育2分钟(±几秒)。用一只手握住液体抽吸装置,用另一只手握住移液器控制器。对于抽吸,对所有样品使用相同的玻璃巴斯德吸管。要分发PEG 1500,请对所有样品使用相同的2 ml移液器。我们不建议在一批中处理超过12个盖玻片。
    3. 在不移除PEG 1500的情况下,向每个盖玻片中轻轻添加3ml PBS。每个盖玻片保持约10秒的速度。
    4. 吸出PBS并再用3ml PBS洗涤两次。
    5. 吸出PBS并轻轻加入1ml含有100μg/ ml CHX的培养基。
    6. 可选:在显微镜下简要地目视检查整体融合效率。在这个阶段可以观察到许多含有多个细胞核的细胞。

  7. 穿梭(时间:3小时)
    在37°C孵育3小时,5%CO 2 (见注9)。

  8. 细胞固定和显微镜载玻片的制备(定时:1.5小时,过夜)
    1. 吸出含有100μg/ ml CHX的培养基,加入1 ml含有100μg/ ml CHX的冰冷PBS。
    2. 用1ml含有100μg/ ml CHX的冰冷PBS进行第二次洗涤。
    3. 每次一个盖玻片,吸出含有100μg/ ml CHX的PBS,并轻轻加入0.5ml含有4%甲醛的冰冷PBS。避光,在室温下孵育20分钟。
      注意:甲醛有毒,应在通风橱中处理。
    4. 用1ml PBS洗涤两次。
    5. 取出PBS并加入0.5 ml含0.25μg/ ml Hoechst 34580的TBST。避光保存,在室温下孵育30分钟。
      注意:Hoechst 34580具有潜在的致癌性,因此应采取预防措施进行处理和处置。
    6. 去除含有0.25μg/ ml Hoechst 34580的TBST,并用1ml PBS洗涤两次。将盖玻片从第二次洗涤中留在PBS中。
    7. 使用精细弯曲的镊子,取出每个盖玻片,通过用盖玻片边缘接触餐巾尽可能多地去除PBS,然后转移(面朝上)到一张纸上。通过在旁边的纸张上书写来标记每个盖玻片。
    8. 避光,让其完全干燥(至少15分钟)。
    9. 每次一个盖玻片,将13.5μlProLong® Diamond Antifade安装试剂添加到显微镜载玻片上,用镊子取出盖玻片并将其倒置在滴安装试剂上。
      严重:避免将气泡吸移到载玻片上。
    10. 避光,在室温下在水平表面上孵育至少16小时。
      暂停点:幻灯片可以在室温下存放24小时,然后在4°C下存放数周,直到成像。

  9. 成像(时间:每个样本4-6小时,15个异核体)
    注意:我们建议针对每个供体细胞系(例如,表达感兴趣的eGFP标记的蛋白质)和受体细胞系优化成像设置(激发/发射波长,激光功率,增益,偏移等)。 (例如,表达CAAX-mCherry)在盖玻片上单独生长并与样品平行处理。确保荧光团和设置的组合不会产生任何串扰。
    1. 对于每个样品(即,对于每对供体 - 受体细胞系),通过视觉检查Hoechst荧光选择含有最佳细胞密度的盖玻片。细胞密度应该足够高,以便成对的细胞核经常彼此非常接近,但与周围的细胞核明显分开(见注10-12)。
    2. 将物镜移向盖玻片的外围,直到细胞密度降低和/或细胞出现损坏,然后将物镜移回适合分析的区域(见注13)。
    3. 使用Hoechst荧光对所需的细胞融合(供体 - 受体)事件进行系统搜索。融合细胞中的细胞核彼此非常接近并且通常显示出互补的形状,但情况并非总是如此(参见图2和3中的实例)。当使用来自两个不同物种(例如,小鼠和人类)的细胞时,有时可以通过存在对每个物种特异的不同异染色质模式来促进发现异核体(Borer et al。,1989)。注意,适于分析的异核体(即,明显仅含有一个供体和一个受体核)构成罕见事件(在检查的盖玻片上<0.01%的细胞)。
    4. 在成像之前,确认两个细胞核都被mCherry通道中的一个共同的红色膜包围(见注14)。
    5. 使用共聚焦显微镜采集设置,找到最佳焦点(Z轴上的细胞中间),定义要成像的区域并在所有三个通道中拍照(mCherry,eGFP和Hoechst,见图2和图3) 。对给定分析中的所有图片使用相同的帧大小和分辨率;根据我们的经验,成像面积为67.4 x67.4μm,分辨率为1,024 x 1,024像素,可生成具有合理扫描时间的高质量图像,并且几乎总是允许我们从未融合的受体细胞中包含至少一个细胞核(或在至少部分)用于背景减法。
    6. 在给定分析中,对于所有图片,使用相同数量的切片获取eGFP和Hoechst荧光的z-堆叠。根据我们的经验,间隔为0.31μm的10到12个切片允许覆盖Z轴上的几乎整个核。为了最大限度地减少扫描时间和eGFP荧光的漂白,我们建议首先对eGFP通道中的所有切片进行成像,然后对Hoechst通道中的所有切片进行成像。我们认为没有必要获得CAAX-mCherry的z-stack,因为它显着增加了扫描时间;但是,请确保将步骤I5中生成的图片作为两个细胞核来源于融合供体 - 受体细胞的证据。
    7. 恢复对所需供体 - 受体细胞融合事件的系统搜索,并重复步骤I4至I6。我们建议每个样本至少成像15对核,以进行适当的统计分析。


      图2.可用于定量异核体分析的克隆供体和受体细胞系的实例。 左上图,表达GFP标记的SRSF5的小鼠P19细胞,RBP与细胞质穿梭活性(Botti et al。,2017)用作供体细胞。 SRSF5-GFP在来自随机整合到基因组中的BAC的近内源水平表达,导致蛋白质在整个核质中的生理定位,同时富集核斑点。注意由于使用克隆细胞系,所有细胞中的GFP荧光水平高度相似。插图显示了小鼠P19细胞的特征Hoechst染色模式。 右上图,在其细胞膜中表达CAAX-mCherry的人HeLa细胞用作受体细胞。由于在将质粒TH0477随机整合到基因组中后选择高表达克隆,CAAX-mCherry在所有受体细胞中以均一水平表达。插图显示了HeLa细胞的特征Hoechst染色模式。 下图,上图中显示的供体和受体细胞系用于定量异核体测定。该图显示了异核体,即,一个细胞含有来自供体P19细胞的一个细胞核和一个来自受体HeLa细胞的细胞核,两者都被标有CAAX-mCherry的共同膜包围。插图显示了异核体的两个核的Hoechst染色模式。橙色星号表示未融合的HeLa细胞和黄色星号,未融合的P19细胞。注意GFP信号在受体HeLa细胞核中的显着存在以及其与未融合的HeLa细胞核的缺失(也参见图3)。还要注意在未融合的P19核周围的膜中没有CAAX-mCherry信号。比例尺=10μm。


      图3.使用定量异核体分析可以研究细胞类型特异性穿梭活动。 左侧和中间小组,表达SRSF5-GFP的小鼠P19细胞用作供体细胞之前(左)和后(中)9天分化成神经细胞。 右图,表达SRSF5-GFP的小鼠NIH3T3克隆细胞系用作供体细胞系。 SRSF5-GFP在多能(P19)细胞中有效穿梭,但在分化的(P19和NIH3T3)细胞中很差。参见Botti 等人(2017),以量化和比较SRSF5在这些不同细胞类型中的穿梭活性。注意未融合的HeLa细胞核完全没有GFP信号(用橙色星号表示)。比例尺:左图=5μm;中图=5μm;右图=10μm。

  10. 荧光图像分析和定量(定时:15个图像2-3小时)
    1. 打开斐济(ImageJ版本2.0.0-rc-43 / 1.51h或更近)。
    2. 选择“分析→工具→ROI管理器...”,打开感兴趣区域(ROI)管理器。
    3. 打开图像(选择“查看堆栈:Hyperstack”,“颜色模式:复合”和“自动缩放”)。
    4. 选择“图像→堆栈→Z项目...”,“开始切片:1”,“停止切片:10”(或12,包括所有切片),“投影类型:最大强度”。
    5. 选择“图像→彩色→分割通道”。
    6. 单击Hoechst通道中生成的图片,然后选择“图像→查找表→灰色”。
    7. 选择“图像→调整→亮度/对比度”并使供体和受体核尽可能可见,同时仍能够将它们彼此区分开。
    8. 如果供体和受体核彼此非常接近,则使用铅笔工具在它们之间画一条线。
    9. 调整亮度和对比度,尽可能清楚地看到整个供体和受体细胞核。
    10. 尽可能放大供体/受体核,使它们在窗口中完全可见。使用手绘选择,绘制供体细胞的边界。
    11. 在ROI Manager窗口中,单击“Add [t]”。这将生成具有特定数字的ROI。
    12. 在列表中,单击与ROI对应的数字,然后选择“重命名...”,然后选择“重命名为:图像1捐赠者”。
    13. 绘制收件人核心的边框,添加到ROI管理器并重命名为“Image1收件人”。
    14. 根据需要缩小并放大未融合的受体细胞的细胞核。
    15. 从未融合的收件人单元格中绘制核心的边界,添加到ROI管理器并重命名为“图像1背景”。
    16. 单击原始(.czi)图片并选择“图像→堆栈→Z项目...”,“开始切片:1”,“停止切片:10”(或12,包括所有切片)和“投影类型:总和切片” “(见注15)。
    17. 选择“图像→彩色→分割通道”。
    18. 单击eGFP荧光图像,然后选择“图像→类型→16位”。
    19. 在ROI Manager窗口中,选择与供体细胞核相对应的ROI,然后单击“测量”。 “结果”窗口将打开。确保生成以下数据:面积,平均值,最小值,最大值和切片。
    20. 在ROI Manager窗口中,选择与收件人核相对应的ROI,然后单击“Measure”。
    21. 重复背景核。
    22. 关闭所有图像并使用图像2重复步骤J3至J21。
    23. 要保存文档的投资回报率,请在投资回报率管理器中选择“更多&gt;&gt; ...保存”。
    24. 通过单击“结果”窗口并选择“文件→另存为...”将结果保存在Microsoft Excel文件中。
    25. 打开Excel文件。对于每个图像,从供体核和受体核中减去未融合的受体核的平均像素强度(平均值)。这产生每个核的背景减去的平均像素强度。
    26. 对于供体核和受体核,将背景减去的平均像素强度乘以该面积以获得总像素强度。
    27. 将受体核的总像素强度除以供体核的总像素强度并乘以100以获得穿梭能力的值(%)。

数据分析

  1. 为了比较两种不同条件下蛋白质的穿梭或两种不同蛋白质的穿梭,我们建议至少在两次独立实验中每种条件或蛋白质至少成像15个异核体(例如,7-8来自两个独立实验的每个盖玻片的异核体。)&nbsp;
  2. 在给定条件下蛋白质的平均穿梭活动应该从至少15个异核体图像计算出来。&nbsp;
  3. 我们应用学生的 t - 测试和配对的Wilcoxon秩和检验来测试不同条件或蛋白质之间的穿梭活动是否显着不同。为此,将所有量化的和减去背景的穿梭能力值组合成列,将蛋白质名称作为标题放入.txt文件中。绘图和测试在R中进行。
    示例:
    read.table(“/ path / Quantification_HKA.txt”,head = TRUE,sep =“\ t”) - &gt; experiment_HKA
    摘要(experiment_HKA)
    boxplot(experiment_HKA,las = 2,ylab =“穿梭容量HKA”),boxfill = c(“lightgrey”))
    wilcox.test(experiment_HKA $ Control,experiment_HKA $ Mutant,配对= TRUE)

笔记

  1. 细菌人工染色体(BAC)中的蛋白质标记和随后的转基因细胞系的产生已在(Poser 等人,,2008)中详细描述。
  2. 穿梭测定的读数是蛋白质从供体到受体细胞核的重新分布。在最初的测定中(Borer et al。,1989),使用物种特异性单克隆抗体通过间接免疫荧光定性评估受体核(来自不同物种)中感兴趣的蛋白质的出现。然而,对于某些蛋白质,特异性抗体很难 - 如果不是不可能的话 - 产生。为了避免这种情况,在与受体细胞融合之前,用编码感兴趣的蛋白质的表位标记的cDNA瞬时转染细胞(Pinol-Roma和Dreyfuss,1991; Caceres et al。,1998; Lin 等人,2005)。然而,来自质粒的瞬时表达经常导致过表达,这反过来会影响RBP的功能和亚细胞分布(Maharana et al。,2018;我们未发表的观察结果)。此外,RBP水平将在不同细胞之间发生显着变化,从而排除了可比较的异核体的鉴定。为了克服这些限制,我们强烈建议使用稳定表达来自细菌人工染色体(BAC)的荧光标记蛋白的克隆细胞系,这些蛋白已经以单拷贝整合到基因组中(Poser et al。,2008; Botti) e t al。,2017)。大尺寸的BAC确保所有基因调控元件(例如,启动子,增强子,内含子序列和非翻译区域)的存在,这保留了剪接同种型的内源比率,并导致表达水平与内源基因(图2,Änkö et al。,2012;Müller-McNicoll et al。,2016; Botti et al。,2017 )。或者,可以使用CRISPR / Cas9的基因组编辑将荧光标签引入由其内源基因座表达的RBP中(Van Nostrand 等人,,2017)。
  3. 我们还建议在每个实验中使用对照细胞系。我们已经使用表达eGFP标记的PRPF8(剪接体组分)和核输出因子(NXF1)的细胞分别作为穿梭的阴性和阳性对照(Botti 等人,,2017)。可以使用已经确认或排除穿梭的其他RBP。然而,应该记住,给定RBP的穿梭可能根据细胞类型和/或分化状态而变化(图3,Botti 等。,2017; Hammarskjold et al。 ,2017)。
  4. 为了容易地鉴定真正的异核体,我们的测定法需要使用仅在受体细胞中表达的膜标记物。我们成功地使用克隆受体细胞系来表达随机整合到其基因组中的质粒的CAAX-mCherry(TH0477,Stewart et al。,2011; Botti et al。,2017 )。 CAAX是异戊二烯化肽,其与细胞膜结合(Wright和Philips,2006)。然而,其他膜标记可能同样适合。使用质粒的稳定转基因细胞系可以基本上与使用BAC时相同的方式产生。见注1。
  5. 我们的测定基于定量细胞成像,其需要具有相同设置(激光功率,增益,数字偏移,等)的标准化成像协议以获取所有图像。为了实现这一点,我们强烈建议对供体和受体细胞系使用克隆系,优先选择研究的RBP的内源表达水平。克隆细胞系可以通过FACS分选获得,其需要访问专门的核心设施或合格技术人员的帮助,或者没有使用克隆盘的任何专门设备或专业知识。
  6. 该方案最关键的部分:为了产生可靠的结果,只应量化含有一个供体和一个受体细胞核的异核体。为了增加可检查的异核体的频率 - 从而减少显微镜检查时间/增加统计功效 - 应该以下列三种方式针对供体 - 受体细胞系的不同组合优化实验条件。
    &NBSP;首先,使用适当比例的供体和受体细胞。原则上,应使用1:1的比例(并且是一个良好的起点)以增加异核体含有一个供体和一个受体核的可能性。然而,不同的细胞类型或多或少地紧密粘附于玻璃盖玻片,并且鉴于在细胞融合之前必须进行若干培养基更换/洗涤,该比例可能必须根据所用细胞的组合进行优化。此外,一些细胞系具有比其他细胞系更快的生长速率,并且应该在强附着和低数量的细胞分裂之间进行折衷,因为最近分裂的细胞倾向于融合在一起并且降低可检查的异核体的频率。例如,当我们使用快速分裂的小鼠P19细胞作为供体和HeLa细胞作为受体时,我们允许比使用具有相同接受者的小鼠NIH3T3细胞(12-16小时)更短(6至8小时)的粘附时间以避免P19细胞分裂,但我们使用P19:HeLa细胞比例为2:1或3:1,因为P19细胞比HeLa和NIH3T3细胞更快和更强地粘附到盖玻片上,并且在洗涤期间许多P19细胞丢失。当使用需要涂层的细胞类型(例如,明胶,层粘连蛋白,等)时,我们建议将盖玻片预孵育足够的时间,最好是37小时。 °C,在播种细胞之前。
    &NBSP;其次,在混合和接种之前获得供体和受体细胞的单细胞悬浮液是至关重要的。这可能需要严厉地上下移液以打破细胞团块。不要犹豫牺牲一些细胞的活力来获得真正的单细胞悬浮液。
    &NBSP;第三,使用最佳细胞密度。为了获得异核体,细胞应粘附在彼此足够接近的盖玻片上,以便它们的膜可以在化学处理时融合(见下文);但是,应将细胞悬液稀释到足以避免多次融合事件。为了找到单次和多次融合之间的最佳折衷,我们建议在接种前进行连续2倍稀释的混合(供体 - 受体)细胞悬液,并通过显微镜确定最佳稀释度以寻找合适的异核体。尽管使用表达荧光膜标记的受体细胞系极大地促进了适合于分析的异核体的鉴定,但是在单个供体和单个受体细胞之间发现明显的融合事件仍然具有挑战性,并且值得投入一些时间来确定最佳用于进行搜索的稀释液。
  7. 核蛋白质在细胞质中合成。为了确定核蛋白是否穿梭于细胞质并返回细胞核,因此在穿梭测定之前和期间阻断蛋白质合成是必要的。以这种方式,受体核中任何新出现的荧光蛋白应该来自供体细胞核,而不是来自其共同细胞质中新合成蛋白质的库。确认的非穿梭核RBP(例如,剪接因子PRPF8; Sapra 等,2009)应始终用作阴性对照;没有穿梭确认了有效的蛋白质合成抑制。我们使用了两种不同的蛋白质合成抑制剂(放线菌酮和嘌呤霉素),产生了基本相似的结果(Botti et al。,2017)。
  8. 聚乙二醇(PEG)是水溶性的,因此可以通过洗涤容易地除去;然而,延长孵育对大多数细胞系可能是有毒的(我们的观察结果)。在比较穿梭分析中对所有样品使用相同的孵育时间是很重要的。
  9. 细胞融合后,应该允许足够的时间来检测和测量感兴趣的RBP的穿梭。有几个因素会影响给定蛋白质的穿梭效率。我们已经证明SR蛋白家族的不同成员以不同的速率穿梭,这与其可磷酸化的RS结构域的长度相关(Botti 等,,2017)。根据RBP的生物学功能,穿梭率可能有很大差异。例如,虽然一些核蛋白如核仁蛋白似乎慢慢“泄漏”到细胞质,需要在受体细胞核中检测到约24小时,但可以在几分钟内检测到核质转运因子的穿梭(Gama-Carvalho et al。,2001)并在3小时内达到平衡(即,50%在供体和受体细胞核中)(Caceres et al。,1998; Sapra et al。,2009; Botti et al。,2017)。在比较不同RBP的穿梭效率时,在细胞达到平衡之前固定细胞至关重要。此外,为了使变化最小化,应该允许在每次重复分析中穿梭完全相同的孵育时间。
    &NBSP;影响穿梭效率的另一个因素是供体和受体核的组合。例如,如果给定的蛋白质从供体细胞核输出但不从受体细胞核输出,则蛋白质可能被捕获并在受体细胞核中积聚,蛋白质的穿梭能力可能看起来比实际上更大,有时达到超过50%(未发表的观察)。在这种情况下,可能需要减少穿梭时间,以便能够量化差异(例如,突变体和WT蛋白之间)。
    &NBSP;最后,供体和受体细胞核的细胞周期阶段在某些情况下可能影响研究中蛋白质的穿梭能力。在分裂细胞的间期期间,核体积和NPC的数量几乎翻倍(Maeshima et al。,2011)。理论上,较大的核应该允许更快的进口和较慢的穿梭蛋白输出,因为其较大的表面积与细胞质接触并且其较小的表面区域与核质接触。我们观察到较大的受体核往往具有较高的总荧光水平(未发表的观察结果);然而,当对足够数量的异核进行成像时,这种效果不会显着影响结果。然而,一些蛋白质的穿梭可能在整个细胞周期中变化,并且在一些情况下,在融合之前同步细胞(例如,在G1中)可能是有用的。
  10. 如果没有盖玻片显示最佳密度,则在随后的实验中调整稀释度。
  11. 一些细胞融合事件似乎只发生在相同细胞系(供体细胞系或受体细胞系)的细胞之间。一个可能的原因是供体或受体细胞的生长速度更快。如果一个细胞系生长太快,请尝试在步骤D1减少附着时间。
  12. 在一些情况下,尽管在步骤C8中使用了供体:受体细胞比例为1:1,但是一种细胞类型(供体或受体)似乎在载玻片上占优势。一个可能的原因是不同的细胞类型或多或少地牢固地附着在盖玻片上,并且在培养基更换和洗涤期间供体细胞或受体细胞可能以更高的速率丢失。
  13. 盖玻片周边的细胞有时不适合成像/分析:根据Hoechst荧光,它们的细胞核可能看起来更小,更亮和不规则,并且它们的特定染色质特征可能不太明显。这些细胞的荧光水平在eGFP和mCherry通道中倾向于较低。这些区域包含可能受损的细胞。
  14. 获取正确量化所必需的Z-堆栈是耗时的。在其膜中使用具有荧光标记的受体细胞(例如,,CAAX-mCherry)允许容易地区分真正的异核体与仅仅非常接近但未融合的供体/受体细胞。如果在显微镜下快速观察表明它是真正的异核体,那么继续进行成像。否则,继续搜索合适的异核体。
  15. 即使在成像期间已经注意避免像素饱和,切片的总和将使一些像素看起来饱和。这应仅影响感兴趣区域中的少数像素,并且根据我们的经验,它不会显着影响结果。但是,如果有人怀疑太多像素在切片总和中出现饱和并且这可能会影响分析,那么荧光应该在单独的切片中量化,然后求和。

食谱

  1. DMEM含有10%FBS,100U / ml青霉素和100μg/ ml链霉素
    1. 将50ml热灭活的FBS加入500ml DMEM中
    2. 加入5毫升青霉素 - 链霉素(10,000 U / ml)
    3. 充分混合并储存在4°C(保持无菌)
  2. 含有0.1%明胶的PBS
    1. 将25毫升2%明胶加入500毫升无菌1x PBS中
    2. 充分混合并储存在4°C(保持无菌)
  3. 10x TBS(1升)
    1. 将24克Trizma ®碱和88克NaCl溶于900毫升dH 2 O中
    2. 用HCl调节pH至7.6
    3. 将体积调节至1 L并验证pH值
    4. 在RT存储
  4. TBST(1升)
    1. 将100ml 10x TBS加入900ml dH 2 O中
    2. 加入0.5毫升吐温® 20
    3. 混合均匀并在室温下储存
  5. 1x PBS中4%甲醛(40 ml,80粒盖玻片)
    1. 在50毫升管中,加入4毫升10倍PBS至26毫升H 2 O(Sigma
    2. 在通风橱中,加入10毫升16%的甲醛
    3. 通过涡旋混合10-15秒
    4. 以-20毫升的等分试样储存于-20°C(每个等于8个盖玻片)
  6. Hoechst 34580原液,1 mg / ml(5 ml)
    1. 将小瓶(5 mg)的全部内容物溶解在5 ml H 2 O(Sigma)中
    2. 以-20℃的等分试样储存,避光
  7. Hoechst34580,0.25μg/ ml TBST(4 ml,8次盖玻片)
    1. 在15毫升管中,加入1微升1毫克/毫升Hoechst 34580至4毫升H 2 O(Sigma)
    2. 通过涡旋混合10-15秒
    3. 准备新鲜,保持在冰上并避光

致谢

这项工作得到了德国研究基金会(DFG)向MMM(CEF-MC和SFB902)的资助。我们感谢M. C. Steiner开发第一个测定版本和K. M. Neugebauer的灵感和指导。我们感谢H. Schewe(FCAM)对共聚焦显微镜的支持。该方案改编自Botti 等人发表的程序。 (2017)。

利益争夺

作者声明没有竞争性的经济利益。

参考

  1. Änkö,M。L.,Muller-McNicoll,M.,Brandl,H.,Curk,T.,Gorup,C.,Henry,I.,Ule,J。和Neugebauer,K.M。(2012)。 两种SR蛋白的RNA结合区域揭示了独特的功能并与不同的RNA类别结合。 Genome Biol 13(3):R17。
  2. Borer,R.A.,Lehner,C.F.,Eppenberger,H.M。和Nigg,E.A。(1989)。 主要的核仁蛋白在细胞核和细胞质之间穿梭。 细胞 56(3):379-390。
  3. Botti,V.,McNicoll,F.,Steiner,MC,Richter,FM,Solovyeva,A.,Wegener,M.,Schwich,OD,Poser,I.,Zarnack,K.,Wittig,I.,Neugebauer,KM和Müller-McNicoll,M。(2017年)。 细胞分化状态调节SR蛋白的mRNA输出活性。 J细胞Biol 216(7):1993-2009。
  4. Caceres,J.F.,Screaton,G.R。和Krainer,A.R。(1998)。 SR蛋白的特定子集在细胞核和细胞质之间连续穿梭。 Genes Dev 12(1):55-66。
  5. Ederle,H。和Dormann,D。(2017)。 TDP-43和FUS 途径从细胞核到细胞质。 FEBS Lett 591(11):1489-1507。
  6. Gama-Carvalho,M。和Carmo-Fonseca,M。(2001)。 核质穿梭蛋白的规则和作用。 FEBS Lett 498(2-3):157-163。
  7. Gama-Carvalho,M.,Carvalho,M。P.,Kehlenbach,A.,Valcarcel,J。和Carmo-Fonseca,M。(2001)。 异二聚体剪接因子U2AF的细胞质穿梭。 J Biol Chem 276(16):13104-13112。
  8. Hammarskjold,M。L.和Rekosh,D。(2017)。 SR蛋白质:要穿梭或不穿梭,这就是问题。 J Cell Biol 216(7):1875-1877。
  9. Howard,J。M.和Sanford,J。R.(2015)。 RNAissance系列:SR蛋白作为基因表达的多面调节因子。 Wiley Interdiscip Rev RNA 6(1):93-110。
  10. Jeong,S。(2017)。 SR蛋白质:RNA的结合剂,调节因子和连接体。 Mol细胞 40(1):1-9。
  11. Lin,S.,Xiao,R.,Sun,P.,Xu,X。和Fu,X.D。(2005)。 在mRNP成熟期间依赖于去磷酸化的SR剪接因子分选。 Mol Cell 20(3):413-425。
  12. Liu,E。Y.,Cali,C。P. and Lee,E。B.(2017)。 神经退行性疾病中的RNA代谢。 Dis Model Mech 10 (5):509-518。
  13. Maeshima,K.,Iino,H.,Hihara,S。和Imamoto,N。(2011)。 核大小,核孔数和细胞周期。 Nucleus 2(2):113-118。
  14. Maharana,S.,Wang,J.,Papadopoulos,DK,Richter,D.,Pozniakovsky,A.,Poser,I.,Bickle,M.,Rizk,S.,Guillen-Boixet,J.,Franzmann,T。 ,Jahnel,M.,Marrone,L.,Chang,YT,Sterneckert,J.,Tomancak,P.,Hyman,AA和Alberti,S。(2018)。 RNA缓冲朊病毒样RNA结合蛋白的相分离行为。 科学 360(6391):918-921。
  15. Maslon,M.M.,Heras,S.R.,Bellora,N.,Eyras,E。和Caceres,J.F。(2014)。 剪接因子SRSF1的转化前景及其在有丝分裂中的作用。 Elife :e02028。
  16. Müller-McNicoll,M。和Neugebauer,K.M。(2013)。 细胞如何得到信息:mRNA-蛋白质复合物的动态组装和功能。 Nat Rev Genet 14(4):275-287。
  17. Müller-McNicoll,M.,Botti,V.,de Jesus Domingues,AM,Brandl,H.,Schwich,OD,Steiner,MC,Curk,T.,Poser,I.,Zarnack,K。和Neugebauer,KM (2016)。 SR蛋白是NXF1适配器,可将替代RNA加工与mRNA输出联系起来。 Genes Dev 30:553-566。
  18. Ouellet,J。(2016年)。 带荧光适体的RNA荧光。 Front Chem 4:29。
  19. Pinol-Roma,S。和Dreyfuss,G。(1991)。 hnRNP蛋白的转录依赖性和转录非依赖性核转运。 Science 253(5017):312-314。
  20. Poser,I.,Sarov,M.,Hutchins,JR,Heriche,JK,Toyoda,Y.,Pozniakovsky,A.,Weigl,D.,Nitzsche,A.,Hegemann,B.,Bird,AW,Pelletier,L 。,Kittler,R.,Hua,S.,Naumann,R.,Augsburg,M.,Sykora,MM,Hofemeister,H.,Zhang,Y.,Nasmyth,K.,White,KP,Dietzel,S。, Mechtler,K.,Durbin,R.,Stewart,AF,Peters,JM,Buchholz,F。和Hyman,AA(2008)。 BAC TransgeneOmics:探索哺乳动物蛋白质功能的高通量方法。 Nat Methods 5(5):409-415。
  21. Sapra,A.K.,Änkö,M。L.,Grishina,I.,Lorenz,M.,Pabis,M.,Poser,I.,Rollins,J.,Weiland,E。M. and Neugebauer,K.M。(2009)。 SR蛋白家族成员在体内新生和成熟mRNPs的形成中表现出不同的活动。 Mol Cell 34(2):179-190。
  22. Stewart,M.P.,Helenius,J.,Toyoda,Y.,Ramanathan,S.P.,Muller,D.J。和Hyman,A.A。(2011)。 静水压力和肌动球蛋白皮质驱动有丝分裂细胞四舍五入。 自然 469(7329):226-230。
  23. Van Nostrand,E.L.,Gelboin-Burkhart,C.,Wang,R.,Pratt,G.A.,Blue,S.M。和Yeo,G.W。(2017)。 CRISPR / Cas9介导的整合使TAG-eCLIP成为内源性标记的RNA结合蛋白。 方法 118-119:50-59。
  24. Wright,L。P.和Philips,M。R.(2006)。 专题评论系列:脂质翻译后修饰。 Ras的CAAX修饰和膜靶向。 J Lipid Res 47(5):883-891。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. McNicoll, F. and Müller-McNicoll, M. (2018). A Quantitative Heterokaryon Assay to Measure the Nucleocytoplasmic Shuttling of Proteins. Bio-protocol 8(17): e2472. DOI: 10.21769/BioProtoc.2472.
  2. Botti, V., McNicoll, F., Steiner, M. C., Richter, F. M., Solovyeva, A., Wegener, M., Schwich, O. D., Poser, I., Zarnack, K., Wittig, I., Neugebauer, K. M. and Müller-McNicoll, M. (2017). Cellular differentiation state modulates the mRNA export activity of SR proteins. J Cell Biol 216(7): 1993-2009.
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