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Oct 2019

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Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration
神经退行性细胞模型中核质转运的定量分析   

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Abstract

Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and which pathways are involved is of great importance. Here, we make use of CRISPR-Cas9 technology to design cell lines stably expressing fluorophore proteins shuttling between the nucleus and cytoplasm by karyopherins of choice. To validate this protocol, we measured an ALS-associated nucleocytoplasmic transport pathway in the presence of the disease-associated peptide poly-PR. This technique allows measuring a particular active nucleocytoplasmic transport pathway in intact cells in a neurodegenerative disease-associated context. Moreover, these experiments can be performed without the need for expensive equipment and have the potential to be upscaled for high-throughput screening purposes.

Keywords: Neurodegenerative disease (神经退行性疾病), Nucleocytoplasmic transport (核质运输), Amyotrophic lateral sclerosis (肌萎缩侧索硬化), C9orf72 (C9orf72), Poly-PR (Poly-PR), CRISPR-Cas9 (CRISPR-Cas9)

Background

Nucleocytoplasmic transport is crucial for cellular homeostasis and occurs across the nuclear membrane via large multi-protein complexes that form aqueous channels, called nucleopore complexes (NPCs) (Stoffler et al., 1999; Ryan and Wente, 2000). Although these channels allow small proteins to passively equilibrate across the nuclear membrane (passive transport), most proteins appear to be actively transported by karyopherins, namely importins or exportins (active transport). Cargo proteins that contain a nuclear localization signal (NLS) are targeted by importins. Various receptor-mediated import pathways have been identified, but the best characterized pathway involves importin-β1/importin-α (KPNB1/KPNAx) of which the cargos contain a classical NLS (cNLS), exemplified by the SV40 large T antigen NLS (Lange et al., 2007). Protein export from the nucleus into the cytoplasm is mediated by exportins. Several pathways of nuclear export have been identified. The most common type of nuclear export signal (NES) is a leucine-rich sequence motif recognized by exportin1 (XPO1), of which the NES from protein kinase inhibitor (PKI) is the most often used prototype (Ossareh-Nazari et al., 2001).

Altered subcellular distribution of proteins is a common characteristic among neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS) (Fahrenkrog and Harel, 2018). Increasing evidence indicates impaired nucleocytoplasmic transport as a possible mechanism explaining this aberrant protein localization in the degenerating neurons. For example, in the majority of ALS patients the RNA binding protein TDP-43 (Arai et al., 2006; Neumann et al., 2006) or, to a lesser extent, FUS (Kwiatkowski et al., 2009; Vance et al., 2009; Tyzack et al., 2019) becomes partially depleted from the nucleus and aggregates in the cytoplasm. Interestingly, the most common genetic cause associated with ALS, a G4C2-repeat expansion in the C9orf72 gene (Majounie et al., 2012), has been suggested to affect nucleocytoplasmic transport in various ways (Boeynaems et al., 2016; Yuva-Aydemir et al., 2018). One possible toxic pathway is the bidirectional translation of the repeat expansion by repeat-associated non-ATG (RAN) translation into dipeptide repeat proteins (DPRs), namely poly-GP, poly-GA, poly-GR, poly-PA and poly-PR (Ash et al., 2013; Zu et al., 2010; Mori et al., 2013a and 2013b). Here, we expressed the DPR poly-PR using a lentiviral vector transduced into our reporter cells, to subsequently measure nucleocytoplasmic transport.

Although there is growing evidence indicating that impaired nucleocytoplasmic transport is a component of neurodegenerative diseases, it is still under large debate whether nucleocytoplasmic transport deficits are a consequence of neurodegeneration or an instigator (Hutten and Dormann, 2019). As nucleocytoplasmic transport is a complex and stress-sensitive process, reliable assays with a minimal interference are essential to answer this question, as we did before (Vanneste et al., 2019). We developed several reporter cell lines stably expressing a shuttling-fluorophore allowing us to consistently measure nucleocytoplasmic import as a function of time and in intact cells without the need for transfection. Moreover, this method is cheap and can easily be up scaled for high throughput screening purposes. By fine-tuning the NLS and NES fused to the reporter, the subcellular localization of the reporter can be modified and different transport pathways of interest can be analyzed. While the experiments here are performed with Hela Kyoto cells, the protocol can easily be adapted for other cell types.

Materials and Reagents

  1. 6-well plate (Corning Life science, 3516)
  2. 96-well plate (TTP, catalog number: 92696 )
  3. T-25 culture flask (TTP, catalog number: 90026 )
  4. Cover Glass (VWR, catalog number: 631-0150 )
  5. Nunc CryoTube (Sigma, catalog number: V7884 )
  6. Hela Kyoto cells (Cellosaurus ID: CVCL_1922)
  7. HEK-293T cells (ATCC, catalog number: CRL-3216TM)
  8. pcDNATM 3.1 (+) plasmid (Thermo Fisher Scientific, catalog number: V790-20 )
  9. pGL4 plasmid (Addgene)
  10. Plasmid containing Cas9 was a kind gift from Jonathan L. Schmid-Burgk (Schmid-Burgk et al., 2016).
  11. Plasmid containing the gRNA was a kind gift from Jonathan L. Schmid-Burgk (Schmid-Burgk et al., 2016)
  12. GibcoTM Dulbecco’s Modified Eagle’s Medium (DMEM), high glucose (Thermo Fisher Scientific, catalog number: 11965092 )
  13. Fetal Bovine Serum (GE Healthcare, Hyclone, catalog number: SV30160.03 )
  14. GibcoTM Gentamicin (Thermo Fisher Scientific, catalog number: 15750045 )
  15. GibcoTM Puromycin Dihydrochloride (Thermo Fisher Scientific, catalog number: A1113802 )
  16. GibcoTM Trypsin-EDTA (0.05%), phenol red (Thermo Fisher Scientific, catalog number: 253000054 )
  17. LipofectamineTM 3000 Transfection Reagent (Thermo Fisher Scientific, catalog number: L3000015 )
  18. Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D2650-100ml )
  19. Importazole (Selleckchem, catalog number: S8446 )
  20. Leptomocyin B (Invivogen, catalog number: tlrl-lep )
  21. NucBlue Live ReadyProbesTM Reagent Hoechst 33342 (Thermo Fisher Scientific, catalog number: R37605 )
  22. GibcoTM Dulbecco’s Phosphate-Buffered Saline (DPBS), no calcium, no magnesium (Thermo Fisher Scientific, catalog number: 14190250 )
  23. Formaldehyde 16 % methanol free, Ultra Pure (Polysciences Inc., catalog number: 18814-20 )
  24. Hexadimethrine bromide (polybrene) (MedChem Express, catalog number: 2878-55-4 )
  25. X-tremeGENE9 DNA transfection reagent (Sigma-Aldrich/Roche, catalog number: 06 365 809 001 )
  26. ON-TARGETplus KPNB1 siRNA human (Horizon Discovery, catalog number: L-01523-00-0005 )
  27. ON-TARGETplus KPNA2 siRNA human (Horizon Discovery, catalog number: L-004702-00-0005 )
  28. Gene Pulser Electroporation Butter (Bio-Rad, catalog number:165-2677)
  29. siRNA buffer (Horizon Discovery, catalog number: B-00200-UB-100 )

Equipment

  1. Incubator
  2. Forceps
  3. Mr. Frosty freezing container (Thermo Fisher Scientific, catalog number: 5100-0001 )
  4. Scanning confocal microscope, e.g., SP8 confocal microscope (Leica, model: SP8 MDi8 )
  5. CellInsight CX5 high content screening platform (Thermo Fisher Scientific, catalog number: CX51110 )

Software

  1. ImageJ (Wayne Rasband (retired from), https://imagej.nih.gov/ij/download.html)
  2. CellProfiler (Broad Institute, https://cellprofiler.org/)
  3. Snapgene (GSL Biotech LLC, https://www.snapgene.com/)

Procedure

A summary of the protocol is shown in Figure 1. The first part of this protocol describes the generation of three different reporter cell lines (Figure 2), which allows us to investigate the import pathways involved in ALS pathology. The protocol can be easily adapted to investigate other transport pathways by simply changing the nuclear localization or nuclear export signal in the constructs. The second part describes how to measure and analyze nucleocytoplasmic transport.


Figure 1. Workflow summary


Figure 2. Generated reporter cell lines. Using CRISPR-Cas9 technology three different reporter cell lines were generated that stably express the indicated constructs. This allows us to investigate the import pathways that are involved in ALS: (1 + 3) KPNB1/KPNAx-mediated import pathway (pathway used by TDP43) and (2) the TPNO1-mediated pathway (pathway used by FUS). In cell line 1, the reporter mainly localizes into the cytoplasm. This allows, in combination with an XPO1-inhibitor, to induce a time-dependent transport from the cytoplasm into the nucleus and as such to measure KPNB1/KPNAx-mediated import over time. Cell lines 2 and 3 allow measuring a shift in the balance between import and export in a simple way.


  1. Generation of cell lines to study nucleocytoplasmic transport
    The reporter constructs, driven by a CMV promotor, were integrated in the AAVS1 safe harbor locus of Hela Kyoto cells using a CRISPR-Cas9 knock-in protocol. Three different CRISPR-Cas9 knock-in protocols were used.

    1. CRISPR-Cas9 knock-in protocols for creating nucleocytoplasmic transport cell lines
      Protocol 1 (Sakuma et al., 2016):
      Use classical PCR to insert the nucleocytoplasmic transport reporter cassette of interest into a pcDNA3.1 plasmid. When designing the insert, make sure the 5' and 3' ends contain two identical targeting sites and micro-homology sequences. The CRISPR-Cas9 targeting sites are needed to cut the insert out of the plasmid in the following step. The microhomology sequences are needed to specifically insert the cassette into the AAVS1 safe harbor site. Here, we cloned a cassette expressing the NLSSV40-mNeongreen2x-NESpki and fused it with P2A-PuromycinR (Figure 3A).
      Protocol 2 (Geisinger et al., 2016):
      A PuromycinR-P2A-NESpki2-mNeongreen2x-NLSFUS reporter was cloned into a pcDNA3.1 plasmid (Figure 3B) that serves as a template for a PCR reaction with 5′ phosphorothioate modified primers (fw primer: T*C*C*CCTCCACCCCACAGTGGGGCC ACTAACGGGCCAGATATACGCG; rv primer: T*C*C*CCTCCACCCCACAGTGGGGCCACTA CTGGTTCTTTCCGCCTCAGAA, with * denoting a phosphorothioate modified nucleotide). This PCR product is used as the donor for the CRISPR knock-in protocol. The PCR primers contain the 5′ end microhomology sequences to enable specific integration of the insert into the AAVS1 safe harbor site (similar as for protocol 1).
      Protocol 3 (Schmid-Burgk et al., 2016):
      NLScMYC-AcGFP2x-NESikb2 was cloned into a pGL4-plasmid that contains at the 5′ end the CRISPR-Cas9 targeting side, to linearize it for insertion into the AAVS1 safe harbor site using the CRISPaint knock-in protocol (Figure 3C).
      Notes:
      1. Pay attention to the final size of your reporter protein. If your goal is to study active transport, use a fusion protein of two or three times your fluorescent protein reporter (approximately 27 kDa). We used two copies of mNeonGreen to exceed the limit for passive transport (± 50 kDa).
      2. NESpki2 contains a K to P mutation (MSLNELALPLAGLDI) which weakens the original NESpki export signal. As such, the equilibrium between import and export is shifted towards import and the reporter is localized mainly inside the nucleus.
      3. Because the Schmid-Burgk protocol is not based on microhomology (Schmid-Burgk et al., 2016), in contrast to 1 and 2, this is the least efficient knock-in protocol. Moreover, the full donor plasmid is integrated in the Hela Kyoto AAVS1 locus in this protocol. The Sakuma protocol (Sakuma et al., 2016) has the highest knock-in efficiency, but is the most difficult one to clone.

    2. Stable cell line generation (for used plasmids see Figure 3)
      Cell culture medium used in all steps consists of Gibco DMEM medium supplemented with 10% fetal bovine serum (FBS) and 50 µg/ml gentamicin.
      1. Day 1: Seed 100,000 Hela Kyoto cells in a 6-well plate.
      2. Day 2:
        Protocol 1: Co-transfect these cells using Lipofectamine 3000 (Thermo Fisher Scientific). Add 500 ng of the respective pcDNA3.1-donor plasmid (Figure 3A), 1,000 ng of a plasmid expressing Cas9 plus a gRNA targeting the AAVS1 locus (GTCACCAATCCTGTCCCTAG) (Figure 3D) and 500 ng of a plasmid expressing a gRNA (GCCAGTACCCAAAAAGCGGG) that cleaves the reporter plasmid at two sites to excise the cassette (Figure 3E).
        Protocol 2: Co-transfect these cells using Lipofectamine 3000 (Thermo Fisher Scientific). Add 500 ng of the cassette PCR donor product (Figure 3B) and 1,000 ng of a plasmid expressing Cas9 plus a gRNA targeting the AAVS1 locus (GTCACCAATCCTGTCCCTAG) (Figure 3D).
        Protocol 3: Co-transfect these cells using Lipofectamine 3000 (Thermo Fisher Scientific). Add 500 ng of the respective pGL4-donor plasmid (Figure 3C), 1,000 ng of a plasmid expressing Cas9 plus a gRNA targeting the AAVS1 locus (GTCACCAATCCTGTCCCTAG) (Figure 3D) and 500 ng of a plasmid expressing a gRNA (GCCAGTACCCAAAAAGCGGG) that cleaves the reporter plasmid at one site to linearize the donor plasmid (Figure 3E).
        Tip: Include a negative control condition by omitting the donor plasmid expressing Cas9/gRNA in one well.
      3. Day 3: Change medium to limit toxicity caused by the transfection reagents.
      4. Day 4: Trypsinize transfected cells and select with puromycin (1 µg/ml).
      5. Analyze plates in the next days for discrete colonies that start to appear.
        Tip: To exclude falls positives: make sure to only continue when all cells are dead in the negative control (without donor plasmid). In addition, keep cells under selection with antibiotics in the following days.
      6. When the 6-well is completely confluent with this polyclonal cell mixture, monoclonal cells can be grown. Therefore, seed cells in a 96-well plate at a density of 0.5 cell/well. This can be achieved as follows:
        1. Take off medium and wash cells with 1 ml of 1x PBS.
        2. Remove PBS and add 500 µl of 0.05% trypsin per well. Return to the incubator for 5 min.
        3. Confirm that cells are detached. If not, place the cells again in the incubator for several minutes until they do.
        4. Add 1.5 ml medium per well. It is important to pipette up and down to mix and obtain single cells. Measure the obtained cell number.
        5. Make a 1/100 dilution (10 µl to 990 µl of medium) to facilitate the next step. Mix well.
        6. Add a volume that contains 50 cells to 10 ml of medium (10 ml is sufficient for one 96-well plate).
        7. Add 100 µl per well to obtain a concentration of 0.5 cells/well. As such, the majority of wells will have zero or one cells, with a small number containing two or more cells.
        Tip: Seed a higher density (e.g., 1,000 cells/well) in the left upper corner. As such, it will be easier to find the right focus on the microscope when you search for colonies.
      7. Scan the plate five days later for the formation of monoclonal colonies. Do not include wells that contain two (or more) separate colonies, as they most probably arose from more than one cell and will therefore not result in a monoclonal culture.
        Tip: Pay attention to the sides of the wells, as cells tend to attach there.
      8. When the 96-well is completely full, expand the monoclonal culture.
        Tip: Expand gradually by starting with a T-25 culture flask.
      9. Freeze cells at -80 °C before long-term storage in liquid nitrogen. This can be achieved as follows:
        1. Prepare freezing medium (for a total of 10 ml: 8.2 ml culturing medium + 800 µl DMSO + 1 ml FBS). Place the medium on ice, as DMSO is toxic to your cells at room temperature.
        2. Label Cryotubes and pre-chill them on ice.
        3. Take off medium and wash cells with 1x PBS.
        4. Remove PBS and add 0.05% trypsin. Return to the incubator for 5 min.
        5. Confirm that cells are detached. If not, return cells to the incubator for several minutes until they do.
        6. Add medium to inhibit the trypsin and bring everything into a 50 ml tube.
        7. Count cells.
        8. Spin off for 5 min at 500 rcf.
        9. Remove supernatant and resuspend pellet in pre-made freezing medium. Add 1 ml per 1.5 x 106-2 x 106 cells.
        10. Pipette 1 ml of the freezing medium in each pre-chilled cryotube.
        11. Place tubes at -80 °C with the help of a Mr. FrostyTM freezing container.
        12. For long-term storage, move your samples the next day to a liquid nitrogen container.


          Figure 3. Plasmids used to generate reporter cell lines. A. Donor plasmid containing the reporter NLSSV40-mNeongreen2x-NESpki used in protocol 1. B. Plasmid containing the reporter NESpki2-mNeongreen2x-NLSFUS, which served as a template for the PCR reaction performed in protocol 2. NESpki2* contains a K to P mutation. C. Donor plasmid containing the reporter NLScMYC-AcGFP2x-NESikb2 used in protocol 3. D. Plasmid expressing Cas9 plus a gRNA targeting the AAVS1 locus (Schmid-Burgk et al., 2016) used in protocols 1, 2 and 3. E. Plasmid expressing a gRNA to cleave the donor plasmid containing the reporter (Schmid-Burgk et al., 2016) used in protocols 1 and 3.

    3. Validation
      Note: It is important to validate your reporter. This can be done by:
      1)
      Treating your cells with available compounds e.g., leptomycin B to inhibit XPO1-mediated export (Kudo et al., 1999) or importazole to inhibit KPNB1-mediated import (Soderholm et al., 2011).
      2)
      Transfecting your cells with appropriate constructs e.g., M9M expression to inhibit TPNO1/TPNO2 (Cansizoglu et al., 2007).
      3)
      Knock down of targeted transport receptors e.g., siRNA against KPNB1 or KPNA2.

      Note: Cell culture medium used in all steps consists of Gibco DMEM medium supplemented with 10% FBS and 50 µg/ml gentamicin.
      1. Validate XPO1-mediated export with leptomycin B
        1. Day 1: Seed NLSSV40-mNeongreen2x-NESpki reporter cells at a density to obtain 60-70% confluency the next day.
        2. Day 2: Change half of the medium in the wells to obtain a final concentration of 50 nM of the XPO1-inhibitor leptomycin B (Kudo et al., 1999).
        3. Return cells to the incubator for 30 min.
        4. Fix cells with 4% PFA for 15 min.
        5. Wash cells three times with 1x PBS.
        6. Stain nuclei using NucBlue Live Cell Stain reagent (Invitrogen).
        7. Image cells to confirm increased nuclear localization of the reporter in the leptomycin B treated cells (Figure 4).


          Figure 4. Validation of XPO1-mediated export. Representative images of cells expressing the reporter NLSSV40-mNeongreen2x-NESpki. A. In steady state, the reporter localizes in the cytoplasm of cells. B. Addition of the XPO1-inhibitor leptomcyin B (LMB) results in nuclear localization of the reporter. Scale bars = 20 µm.

      2. Validate KPNB1/KPNAx-mediated import with importazole
        1. Day 1: Seed NLSSV40-mNeongreen2x-NESpki reporter cells at a density to obtain 60-70% confluency the next day.
        2. Day 2: Incubate the cells for 2 h with 50 µM of the KPNB1-inhibitor importazole (Soderholm et al., 2011).
        3. Change half of the medium in the wells to obtain a final concentration of 50 nM leptomycin B.
          Note: The reporter localizes steady state in the cytoplasm of the cells (due to XPO1-mediated export) and serves therefore, in combination with an XPO1-inhibitor (leptomycin B), as an import-reporter for KPNB1/KPNAx-mediated import.
        4. Return cells to the incubator and fix cells with 4% PFA after 30 min.
        5. Wash cells three times with 1x PBS.
        6. Stain nuclei using NucBlue Live Cell Stain reagent (Invitrogen).
        7. Image cells to confirm a decreased nuclear localization of the reporter in the importazole-treated cells (Figure 5).


          Figure 5. Validation of KPNB1/KPNAx-mediated import. Representative images of cells expressing the reporter NLSSV40-mNeongreen2x-NESpki. A. Addition of leptomycin B results in nuclear localization of the reporter, due to KPNB1/KPNAx-mediated import. B. Pre-incubation with importazole partially abolishes KPNB1/KPNAx-mediated import. Scale bars = 20 µm.

      3. Validate TPNO1/TPNO2-mediated import through M9M expression
        1. Day 1: Seed NESpki2-mNeongreen2x-NLSFUS reporter cells at a density to obtain 60-70% confluency the next day.
        2. Day 2: Transfect cells with a plasmid expressing the peptide M9M (Cansizoglu et al., 2007) using lipofectamine 3000 (Thermo Fisher Scientific).
          Note: Express M9M in fusion with a fluorescent protein (e.g., Red fluorescent protein) to identify transfected cells.
        3. Change medium 4 h after transfection to reduce toxicity.
        4. Incubate cells overnight.
        5. Day 3: Fix cells with 4% PFA for 15 min.
        6. Wash three times with 1x PBS.
        7. Stain nuclei using NucBlue Live Cell Stain reagent (Invitrogen).
        8. Image cells to confirm decreased nuclear localization of the reporter in the M9M-expressing cells (Figure 6).


          Figure 6. Validation of TPNO1/TPNO2-mediated import. Representative images of cells expressing the reporter NESpki2-mNeongreen2x-NLSFUS. A. In steady state, the reporter localizes inside the nucleus, caused by TPNO1/TPNO2-mediated import. Reporter cells were transfected with an mCherry-plasmid as negative control. B. Reporter cells transfected with an RFP-M9M-plasmid, which inhibits TPNO1/TPNO2-mediated import and results in cytoplasmic redistribution of the reporter. Scale bars = 20 µm.

      4. Validate KPNB1/KPNAx-mediated import through KPNB1/KPNA2-knock down
        1. Day 1: Seed NLScMYC-AcGFP2x-NESikb2 reporter cells at a density to obtain 60-70% confluency the next day.
        2. Day 2: Electroporate cells with siRNA against KPNB1 or KPNA2 (Horizon Discovery siRNA resuspension buffer, Bio-Rad: 230V, 950 µF, ∞Ω, 10,000 ms, #1 pulse).
        3. Incubate cells for 48 h.
        4. Fix cells with 4% PFA for 15 min.
        5. Wash three times with 1x PBS.
        6. Stain nuclei using NucBlue Live Cell Stain reagent (Invitrogen).
        7. Image cells to confirm decreased nuclear localization of the reporter (Figure 7).


          Figure 7. Validation of KPNB1/KPNAx-mediated import. Representative images of cells expressing the reporter NLScMYC-AcGFP2x-NESikb2. A. The reporter localizes steady state in the nucleus of the cells, due to KPNB1/KPNAx-mediated import. B. Expression of siRNA targeting KPNB1 or KPNA2 results in cytoplasmic localization of the reporter. Scale bars = 25 µm.

    4. Optimization of the NLSSV40-mNeongreen2x-NESpki reporter
      In steady state, the NLSSV40-mNeongreen2x-NESpki reporter localizes in the cytoplasm of the cells (caused by XPO1-mediated nuclear export) and serves therefore, in combination with an XPO1-inhibitor (leptomycin B; LMB), as an import-reporter for KPNB1/KPNAx-mediated import.
      Note: Because the nuclear intensity results from the balance between export and import, it is important to optimize LMB concentrations to maximize the sensitivity of the assay to measure import deficits. In addition, to minimalize variability it is recommended to work with a LMB concentration that induces a gradual import of the reporter and allows import measurements to occur over longer periods (30 min). Moreover, it is recommended to measure at least two different time points to assure that your measurement falls within the dynamic range of the nuclear import process and saturation is not yet reached.

      Note: Cell culture medium used in all steps consists of Gibco DMEM medium supplemented with 10% FBS and 50 µg/ml gentamicin.
      1. Day 1: Seed NLSSV40-mNeongreen2x-NESpki reporter cells in a 96-well plate at a density to obtain 70% confluency the next day.
        Note: Keep in mind that you will need three different time points and this in duplicate or triplicate per condition.
      2. Day 2: Replace old medium with 100 µl of medium containing 25 µM of the KPNB1-inhibitor importazole.
      3. Return cells to the incubator for 1.5 h.
        Note: The importazole concentration was chosen with the aim to induce average import inhibition, to not miss potential small import deficits induced by poly-PR.
      4. Meanwhile, prepare different dilutions of LMB (dissolved in ethanol) in pre-warmed medium at a final concentration of 0 nM, 15 nM, 30 nM, 45 nM, 60 nM, 75 nM and 90 nM. Vortex.
        Note: Keep in mind the dilution in the next steps. Therefore, prepare twice the final concentration, e.g., 120 nM for a final concentration of 60 nM.
      5. Treat the cells intended for the 30 min time point with the different concentrations of LMB, by adding 100 µl of medium containing LMB to the 100 µl of medium already in the wells.
      6. Return cells to the incubator for 15 min.
      7. Treat the cells intended for the 15 min time point with the different concentrations of LMB by adding 100 µl of the medium containing LMB.
      8. Return cells to the incubator for 15 min.
      9. Fix with 4% PFA for 15 min.
      10. Wash cells three times with 1x PBS.
      11. Stain nuclei using NucBlue Live Cell Stain reagent (Invitrogen).
      12. Cells can be analyzed automatically by the CellInsight CX5 high content screening platform (Thermo Fisher Scientific) to determine the LMB concentration that induces the largest difference between control cells and importazole-treated cells (Figure 8).
        Note: Use a multichannel pipette to reduce variability.


        Figure 8. Optimization of KPNB1/KPNAx-mediated import of the NLSSV40-mNeongreen2x-NESpki reporter measured over time. A-B. Import of importazole-treated cells relative to import of DMSO-treated cells for different concentrations of leptomycin B (LMB). A-15 min; B-30 min. A. 45 nM of LMB results in the strongest difference between control cells and cells-treated with importazole. B. Lower concentrations allow measuring import-inhibition, as import becomes saturated at this later time point for higher concentrations of LMB. C. Left graph: lower concentrations (e.g., 30 nM) of LMB result in less overall import and make it therefore harder to observe disturbed import after 15 min. Import defects can be observed at later time points (30 min). Middle graph: 45 nM of LMB revealed import deficits at both time points. Right graph: higher concentrations of LMB (e.g., 90 nM) can push import too hard to observe a minimal impediment of import. A-B. Dot represents the average of one experiment with 2,500-7,000 cells per experiment. Mean + S.D. C. Dot represent the average of one experiment with 2,500-7,000 cells per experiment.

  2. Measuring nucleocytoplasmic transport in the presence of mutant C9orf72-related peptides
    1. Production of lentiviral vectors expressing mutant C9orf72-related peptides
      DPR100-plasmids were a kind gift of Dr. Daisuke Ito (Department of Neurology, Keio University, Tokyo, Japan). Classical PCR was used to clone mCherry-DPR100 constructs into lentiviral vector plasmids (LentiCrisprV2_Puro from Addgene) (Figure 9). Production of lentiviral vectors was done using HEK-293T cells as previously described (Hart et al., 2017).
      Note: We strongly advise to use transduction instead of transfection to express desired disease-related constructs. Transfection is more stressful which can influence nucleocytoplasmic transport measurements.


      Figure 9. Lentiviral vector plasmids used to express mCherry or mCherry-PR100. A. Lentiviral vector containing mCherry. B. Lentiviral vector containing mCherry-PR100.

    2. Assay
      Note: Cell culture medium used in all steps consists of Gibco DMEM medium supplemented with 10% FBS and 50 µg/ml gentamicin.
      1. Passage cells and seed onto cover glasses
        1. Day 0: Place a single cover glass into each well of a sterile 24-well polystyrene tissue culture plate with the use of sterile forceps.
          Notes:
          1)
          Pre-coating coverslips with poly-L-lysine and poly-L-ornithine or other cell-surface treatments may be necessary to adhere other cell types to glass. Hela Kyoto cells adhere to glass without additional coating.
          2)
          For high throughput screens, cells can be seeded into 96-well plates or other formats.
          3)
          Keep in mind that you will need three different time points and this in duplicate or triplicate per condition.
        2. Seed at a density that will result in 70% confluency at the day you plan to perform the nucleocytoplasmic transport assay.
        3. Incubate cells overnight in a tissue culture incubator (37 °C with 5% CO2).
      2. Transduction of cells to express C9orf72-related peptides
        1. Day 1: Dilute polybrene in medium at a final concentration of 10 µg/ml. Add lentiviral particles to each well. Leave the viral particles overnight on the cells (about 18 h).
        2. Day 2: Remove the lentivirus-containing medium and replace with fresh DMEM medium.
        3. Incubate cell for an additional two days in a tissue culture incubator (37 °C with 5% CO2).
          Note: Addition of polybrene can significantly improve transduction efficiency. However, first confirm that polybrene is not toxic to your cells as it can induce cell death in certain cell types (e.g., iPSC-derived neurons).
      3. Measure nucleocytoplasmic transport
        1. Day 5: Prepare a desired volume of pre-warmed medium containing 45 nM LMB.
        2. Treat the cells intended for the 30 min time point with the medium containing LMB.
        3. Return cells to the incubator for 15 min.
        4. Treat the cells intended for the 15 min time point with the medium containing LMB.
        5. Return cells to the incubator for 15 min.
        6. Fix cells by adding 4% PFA (final concentration) to the medium for 15 min.
        7. Stain nuclei using NucBlue Live Cell Stain reagent (Invitrogen).
        8. Use a 20 µl pipette to place a drop of mounting medium on a slide, before placing the cover glass upside down on the slide using forceps. Remove excess mounting medium with a paper towel and seal the slides using nail polish.
        9. Image cells using a confocal microscope (Figure 10).
          Notes:
          1)
          Keep in mind to maintain the same setting between different conditions and to focus on the middle of the nuclei (nucleoli are clearly visible).
          2)
          For high-throughput screens, intensity can be automatically analyzed by using a high content analyzer (e.g., CellInsight CX5 high content screening platform from Thermo Fisher Scientific).


          Figure 10. Measuring KPNB1/KPNAx-mediated import over time in a neurodegenerative-associated context. Representative images of cells expressing the reporter NLSSV40-mNeongreen2x-NESpki. The reporter localizes steady state in the cytoplasm of the cells, as a consequence of XPO1-mediated nuclear export. Addition of an XPO1-inhibitor (leptomycin B; LMB) allows to measure KPNB1/KPNAx-mediated import over time. A. Reporter cells were transduced with a mCherry-lentiviral vector as control. B. Reporter cells transduced with a poly-PR100-mCherry-lentiviral vector. Poly-PR dipeptides have been linked to the neurodegenerative disease amyotrophic lateral sclerosis. Scale bar = 20 µm.

Data analysis

  1. Manual
    1. ImageJ
      Images taken on the confocal microscope can be manually analyzed with the free-online available software ImageJ.
      1. Import images in ImageJ.
        Note: It is recommended to analyze the images in 64-bit format.
      2. Select region of interest + Ctrl M (Figure 11).


        Figure 11. Manual analysis in ImageJ. Images are representative for NLSSV40-mNeongreen2x-NESpki reporter cells before addition of LMB and transfected with mCherry. Graph contains analyzed data of images from Figure 10. Mean ± S.D. are shown.

  2. Automated
    1. Cell profiler
      Cells imaged on the confocal microscope can be automatically analyzed with the free-online available software Cell profiler (Figure 12).


      Figure 12. Workflow for automatic analysis in Cell profiler. Images are representative for NLSsv40-mNeongreen2x-NESpki reporter cells before addition of LMB and transfected with mCherry. Graph contains analyzed data of images from Figure 10. Mean ± S.D. are shown.

    Statistical analysis
    1. We recommend to analyze nuclear-to-total (= cytoplasm + nucleus) ratios as nucleus-to-cytoplasm ratios may be susceptible to large variations. The difference between nuclear and cytoplasmic values can become very large, especially before addition of LMB (very low nuclear intensity) or at the end of the assay (very low cytoplasmic intensity). In addition, when the data have to be normalized to the initial intensity, a small difference in the nuclear and cytoplasmic intensity might create a large difference in the ratio.
    2. Ratios are intrinsically asymmetric, meaning that all decreases are expressed as ratios between zero and one, and all increases are expressed as ratios greater than 1.0. Therefore, we recommend analyzing the logarithm of the ratios. As such, no change will be zero (the logarithm of 1.0), increases will be positive and decreases will be negative.

Acknowledgments

We thank Liesbeth Mercelis and Bob Massant for technical assistance. This work was supported by the KU Leuven (C1 and Opening the Future Fund); Fund for Scientific Research Flanders (FWO-Vlaanderen GOB3318N – G098314N), the ALS Liga (Belgium). J.V. is doctoral fellow of FWO. P.V.D. holds a senior clinical investigator ship of FWO-Vlaanderen and is supported by Fund ‘Een hart voor ALS’ and Laevers Fund for ALS Research.

Competing interests

The authors declare they have no financial and non-financial competing interests.

References

  1. Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H., Mann, D., Tsuchiya, K., Yoshida, M., Hashizume, Y. and Oda, T. (2006). TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun 351(3): 602-611.
  2. Ash, P. E., Bieniek, K. F., Gendron, T. F., Caulfield, T., Lin, W. L., Dejesus-Hernandez, M., van Blitterswijk, M. M., Jansen-West, K., Paul, J. W., 3rd, Rademakers, R., Boylan, K. B., Dickson, D. W. and Petrucelli, L. (2013). Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77(4): 639-646.
  3. Boeynaems, S., Bogaert, E., Van Damme, P. and Van Den Bosch, L. (2016). Inside out: the role of nucleocytoplasmic transport in ALS and FTLD. Acta Neuropathol 132(2): 159-173. 
  4. Cansizoglu, A. E., Lee, B. J., Zhang, Z. C., Fontoura, B. M. and Chook, Y. M. (2007). Structure-based design of a pathway-specific nuclear import inhibitor. Nat Struct Mol Biol 14(5): 452-454.
  5. Fahrenkrog, B. and Harel, A. (2018). Perturbations in traffic: aberrant nucleocytoplasmic transport at the heart of neurodegeneration. Cells 7(12). DOI: 10.3390/cells7120232.
  6. Geisinger, J. M., Turan, S., Hernandez, S., Spector, L. P. and Calos, M. P. (2016). In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining. Nucleic Acids Res 44(8): e76.
  7. Hart, T., Tong, A. H. Y., Chan, K., Van Leeuwen, J., Seetharaman, A., Aregger, M., Chandrashekhar, M., Hustedt, N., Seth, S., Noonan, A., Habsid, A., Sizova, O., Nedyalkova, L., Climie, R., Tworzyanski, L., Lawson, K., Sartori, M. A., Alibeh, S., Tieu, D., Masud, S., Mero, P., Weiss, A., Brown, K. R., Usaj, M., Billmann, M., Rahman, M., Constanzo, M., Myers, C. L., Andrews, B. J., Boone, C., Durocher, D. and Moffat, J. (2017). Evaluation and design of genome-wide CRISPR/SpCas9 knockout screens. G3 (Bethesda) 7(8): 2719-2727. 
  8. Hutten, S. and Dormann, D. (2019). Nucleocytoplasmic transport defects in neurodegeneration - Cause or consequence? Semin Cell Dev Biol S1084-9521 (18). 301090-301093.
  9. Kudo, N., Matsumori, N., Taoka, H., Fujiwara, D., Schreiner, E. P., Wolff, B., Yoshida, M. and Horinouchi, S. (1999). Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc Natl Acad Sci U S A 96(16): 9112-9117.
  10. Kwiatkowski, T. J., Jr., Bosco, D. A., Leclerc, A. L., Tamrazian, E., Vanderburg, C. R., Russ, C., Davis, A., Gilchrist, J., Kasarskis, E. J., Munsat, T., Valdmanis, P., Rouleau, G. A., Hosler, B. A., Cortelli, P., de Jong, P. J., Yoshinaga, Y., Haines, J. L., Pericak-Vance, M. A., Yan, J., Ticozzi, N., Siddique, T., McKenna-Yasek, D., Sapp, P. C., Horvitz, H. R., Landers, J. E. and Brown, R. H., Jr. (2009). Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323(5918): 1205-1208.
  11. Lange, A., Mills, R. E., Lange, C. J., Stewart, M., Devine, S. E. and Corbett, A. H. (2007). Classical nuclear localization signals: definition, function, and interaction with importin α. J Biol Chem 282(8): 5101-5105. 
  12. Majounie, E., Renton, A. E., Mok, K., Dopper, E. G., Waite, A., Rollinson, S., Chio, A., Restagno, G., Nicolaou, N., Simon-Sanchez, J., van Swieten, J. C., Abramzon, Y., Johnson, J. O., Sendtner, M., Pamphlett, R., Orrell, R. W., Mead, S., Sidle, K. C., Houlden, H., Rohrer, J. D., Morrison, K. E., Pall, H., Talbot, K., Ansorge, O., Chromosome, A. L. S. F. T. D. C., French research network on, F. F. A., Consortium, I., Hernandez, D. G., Arepalli, S., Sabatelli, M., Mora, G., Corbo, M., Giannini, F., Calvo, A., Englund, E., Borghero, G., Floris, G. L., Remes, A. M., Laaksovirta, H., McCluskey, L., Trojanowski, J. Q., Van Deerlin, V. M., Schellenberg, G. D., Nalls, M. A., Drory, V. E., Lu, C. S., Yeh, T. H., Ishiura, H., Takahashi, Y., Tsuji, S., Le Ber, I., Brice, A., Drepper, C., Williams, N., Kirby, J., Shaw, P., Hardy, J., Tienari, P. J., Heutink, P., Morris, H. R., Pickering-Brown, S. and Traynor, B. J. (2012). Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurol 11(4): 323-330. 
  13. Mori, K., Arzberger, T., Grasser, F. A., Gijselinck, I., May, S., Rentzsch, K., Weng, S. M., Schludi, M. H., van der Zee, J., Cruts, M., Van Broeckhoven, C., Kremmer, E., Kretzschmar, H. A., Haass, C. and Edbauer, D. (2013a). Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins. Acta Neuropathol 126(6): 881-893.
  14. Mori, K., Weng, S. M., Arzberger, T., May, S., Rentzsch, K., Kremmer, E., Schmid, B., Kretzschmar, H. A., Cruts, M., Van Broeckhoven, C., Haass, C. and Edbauer, D. (2013b). The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 339(6125): 1335-1338.
  15. Neumann, M., Sampathu, D. M., Kwong, L. K., Truax, A. C., Micsenyi, M. C., Chou, T. T., Bruce, J., Schuck, T., Grossman, M., Clark, C. M., McCluskey, L. F., Miller, B. L., Masliah, E., Mackenzie, I. R., Feldman, H., Feiden, W., Kretzschmar, H. A., Trojanowski, J. Q. and Lee, V. M. (2006). Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314(5796): 130-133.
  16. Ossareh-Nazari, B., Gwizdek, C. and Dargemont, C. (2001). Protein export from the nucleus. Traffic 2(10): 684-689. 
  17. Ryan, K. J. and Wente, S. R. (2000). The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm. Curr Opin Cell Biol 12(3): 361-371.
  18. Sakuma, T., Nakade, S., Sakane, Y., Suzuki, K. T. and Yamamoto, T. (2016). MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat Protoc 11(1): 118-133.
  19. Schmid-Burgk, J. L., Höning, K., Ebert, T. S. and Hornung, V. (2016). CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism. Nat Commun 7: 12338.
  20. Soderholm, J. F., Bird, S. L., Kalab, P., Sampathkumar, Y., Hasegawa, K., Uehara-Bingen, M., Weis, K. and Heald, R. (2011). Importazole, a small molecule inhibitor of the transport receptor importin-β. ACS Chem Biol 6(7): 700-708. 
  21. Stoffler, D., Fahrenkrog, B. and Aebi, U. (1999). The nuclear pore complex: from molecular architecture to functional dynamics. Current Opinion Cell Biology 11(3): 391-401.
  22. Tyzack, G. E., Luisier, R., Taha, D. M., Neeves, J., Modic, M., Mitchell, J. S., Meyer, I., Greensmith, L., Newcombe, J., Ule, J., Luscombe, N. M. and Patani, R. (2019). Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis. Brain 142(9): 2572-2580. 
  23. Vance, C., Rogelj, B., Hortobagyi, T., De Vos, K. J., Nishimura, A. L., Sreedharan, J., Hu, X., Smith, B., Ruddy, D., Wright, P., Ganesalingam, J., Williams, K. L., Tripathi, V., Al-Saraj, S., Al-Chalabi, A., Leigh, P. N., Blair, I. P., Nicholson, G., de Belleroche, J., Gallo, J. M., Miller, C. C. and Shaw, C. E. (2009). Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323(5918): 1208-1211.
  24. Vanneste, J., Vercruysse, T., Boeynaems, S., Sicart, A., Van Damme, P., Daelemans, D. and Van Den Bosch, L. (2019). C9orf72-generated poly-GR and poly-PR do not directly interfere with nucleocytoplasmic transport. Sci Rep 9(1): 15728. 
  25. Yuva-Aydemir, Y., Almeida, S. and Gao, F. B. (2018). Insights into C9ORF72-Related ALS/FTD from Drosophila and iPSC Models. Trends Neurosci 41(7): 457-469. 
  26. Zu, T., Gibbens, B., Doty, N. S., Gomes-Pereira, M., Huguet, A., Stone, M. D., Margolis, J., Peterson, M., Markowski, T. W., Ingram, M. A., Nan, Z., Forster, C., Low, W. C., Schoser, B., Somia, N. V., Clark, H. B., Schmechel, S., Bitterman, P. B., Gourdon, G., Swanson, M. S., Moseley, M. and Ranum, L. P. (2011). Non-ATG-initiated translation directed by microsatellite expansions. Proc Natl Acad Sci U S A 108(1): 260-265.

简介

[摘要]核细胞质运输缺陷被认为在神经退行性疾病中发挥作用,包括肌萎缩性侧索硬化症(ALS)。鉴于这一过程的重要性和复杂性,了解这些畸变何时发生以及涉及哪些途径是非常重要的。在这里,我们利用CRISPR-Cas9技术来设计细胞系,稳定地表达荧光素蛋白在细胞核和细胞质之间穿梭的选择的卡里菲林。为了验证这个协议,我们测量了一个ALS相关的核细胞质运输途径,在疾病相关的多肽poly-PR的存在。这种技术允许测量在神经退行性疾病相关的背景下,在完整细胞中的一个特定的活性核细胞质运输途径。此外,这些实验可以在不需要昂贵的设备的情况下进行,并有可能升级为高通量筛选目的。

[背景]核细胞质运输对细胞稳态至关重要,并通过形成水通道的大型多蛋白复合物穿过核膜,称为核孔复合物(NPCs)(Stoffler et al., 1999; Ryan and Wente, 2000)。虽然这些通道允许小蛋白被动地平衡穿过核膜(被动运输),但大多数蛋白质似乎是由核素主动运输的,即进口蛋白或出口蛋白(主动运输)。含有核定位信号(NLS)的货物蛋白被导入素所定位。已经确定了各种受体介导的导入途径,但最有特色的途径涉及导入素-β1/导入素-α(KPNB1/KPNAx),其中货蛋白含有经典的NLS(cNLS),以SV40大T抗原NLS为例(Lange et al., 2007)......)。蛋白质从细胞核出口到细胞质是由出口素介导的。已经确定了几种核出口的途径。最常见的核出口信号(NES)是由exportin1(XPO1)识别的富含亮氨酸的序列基团,其中来自蛋白激酶抑制剂(PKI)的NES是最常用的原型(Ossareh-Nazari et al., 2001)。
蛋白质亚细胞分布的改变是神经退行性疾病中的一个共同特征,如阿尔茨海默病(AD)、帕金森病(PD)、亨廷顿病(HD)和肌萎缩侧索硬化症(ALS)(Fahrenkrog和Harel,2018)。越来越多的证据表明,核细胞质运输受损是解释这种退行性神经元中异常蛋白定位的可能机制。例如,在大多数ALS患者中,RNA结合蛋白TDP-43(Araiet al., 2006; Neumann et al., 2006)或,在较小的程度上,FUS(Kwiatkowskiet al., 2009; Vance et al., 2009; Tyzack et al., 2019)从细胞核中变得部分耗竭,并在细胞质中聚集。有趣的是,与ALS相关的最常见的遗传原因,C9orf72基因中的G4C2重复扩展(Majounie et al., 2012),已被认为以各种方式影响核细胞质运输(Boeynaems et al., 2016; Yuva-Aydemir et al., 2018)。一个可能的毒性途径是重复相关的非ATG(RAN)翻译成二肽重复蛋白(DPRs),即聚-GP、聚-GA、聚-GR、聚-PA和聚-PR,重复扩张的双向翻译(Ash et al., 2013; Zu et al., 2010; Mori et al., 2013a and 2013b)。在这里,我们表达DPR poly-PR使用慢病毒载体转导到我们的报告细胞,随后测量核细胞质运输。
虽然有越来越多的证据表明,核细胞质运输受损是神经退行性疾病的一个组成部分,但核细胞质运输缺陷是否是神经退行性疾病的结果,还是一种诱因,仍在大面积的争论中(Hutten和Dormann,2019)。由于核细胞质运输是一个复杂的和应激敏感的过程,可靠的检测与最小的干扰是必不可少的,以回答这个问题,就像我们以前做的那样(Vanneste et al., 2019)。我们开发了几个报告细胞系稳定地表达一个穿梭-荧光团,使我们能够一致地测量核细胞质导入作为时间的函数,并在完整的细胞中,而不需要转染。此外,这种方法是廉价的,可以很容易地扩大高通量筛选的目的。通过微调NLS和NES融合到记者,可以修改记者的亚细胞定位,并可以分析感兴趣的不同运输途径。虽然这里的实验是与Hela京都细胞进行,该协议可以很容易地适应其他细胞类型。

关键字:神经退行性疾病, 核质运输, 肌萎缩侧索硬化, C9orf72, Poly-PR, CRISPR-Cas9

材料和试剂


1. 6孔板(康宁生命科学,3516)
2. 96孔板(TTP,目录号:92696)
3. T-25培养瓶(TTP,目录号:90026)。
4. 盖板玻璃(VWR,目录号:631-0150)。
5. Nunc CryoTube(Sigma,目录号:V7884)。
6. 京都海拉细胞(Cellosaurus ID: CVCL_1922)
7. HEK-293T细胞(ATCC,目录号:CRL-3216TM)。
8. pcDNATM 3.1(+)质粒(赛默飞世尔科技公司,目录号:V790-20)
9. pGL4质粒(Addgene)
10. 含有Cas9的质粒是Jonathan L.Schmid-Burgk(Schmid-Burgk等人,2016年)的友好礼物。
11. 含有gRNA的质粒是Jonathan L.Schmid-Burgk(Schmid-Burgk等人,2016年)的一份厚礼。
12. GibcoTM Dulbecco's Modified Eagle's Medium (DMEM),高葡萄糖(赛默飞世尔科技,目录号:11965092)。
13. 胎牛血清(GE Healthcare,Hyclone,目录号:SV30160.03)。
14. GibcoTM庆大霉素(赛默飞世尔科技公司,目录号:15750045)
15. GibcoTM普罗霉素二盐酸盐(赛默飞世尔科技公司,目录号:A1113802)。
16. GibcoTM 胰蛋白酶-EDTA(0.05%),酚红(赛默飞世尔科技公司,目录号:253000054)。
17. LipofectamineTM 3000 转染试剂(Thermo Fisher Scientific,目录号:L3000015)。
18. 二甲基亚砜(DMSO)(Sigma,目录号:D2650-100ml)。
19. 因莫他唑(Selleckchem,目录号:S8446)
20. 钩端螺旋体素B(Invivogen,目录号:tlrl-lep)。
21. NucBlue Live ReadyProbesTM 试剂 Hoechst 33342(赛默飞世尔科技公司,目录号:R37605)。
22. GibcoTM Dulbecco's Phosphate-Buffered Saline (DPBS),不含钙,不含镁(赛默飞世尔科技,目录号:14190250)。
23. 甲醛16%,不含甲醇,超纯(Polysciences Inc.,目录号:18814-20)。
24. 溴化六氯菊酯(聚溴)(MedChem Express,目录号:2878-55-4)。
25. X-tremeGENE9 DNA转染试剂(Sigma-Aldrich/Roche,目录号:06 365 809 001)。
26. ON-TARGETplus KPNB1 siRNA 人类 (Horizon Discovery, 目录号: L-01523-00-0005)
27. ON-TARGETplus KPNA2 siRNA 人类(Horizon Discovery,目录号:L-004702-00-0005)。
28. 基因脉冲器电穿孔黄油(Bio-Rad,目录号:165-2677)。
29. siRNA缓冲液(Horizon Discovery,目录号:B-00200-UB-100)。


装备


1. 孵化器
2. 镊子
3. Frosty先生冷冻容器(赛默飞世尔科技公司,目录号:5100-0001)。
4. 扫描共聚焦显微镜,例如,SP8共聚焦显微镜(徕卡,型号:SP8 MDi8)。
5. CellInsight CX5高含量筛选平台(赛默飞世尔科技,目录号:CX51110)。


軟件


1. ImageJ(Wayne Rasband(已退休),https://imagej.nih.gov/ij/download.html)
2. CellProfiler (Broad Institute, https://cellprofiler.org/)
3. Snapgene (GSL Biotech LLC, https://www.snapgene.com/)


流程


该协议的摘要如图1所示。该协议的第一部分描述了三种不同的报告细胞系( 图2)的生成,这使我们能够调查ALS病理中涉及的导入途径。该协议可以很容易地适应调查其他运输途径,通过简单地改变核定位或核出口信号在构建。第二部分介绍了如何测量和分析核细胞质运输。




图1.工作流程总结工作流程概要


图2.生成的报告细胞系。生成的报告细胞系。使用CRISPR-Cas9技术,生成了三种不同的报告细胞系,稳定地表达指定的构建体。这使我们能够研究参与ALS的导入途径。1+3)KPNB1/KPNAx介导的导入途径(TDP43使用的途径)和(2)TPNO1介导的途径(FUS使用的途径)。在细胞系1中,记者主要定位到细胞质中。这允许,结合XPO1抑制剂,诱导时间依赖性运输从细胞质到细胞核,并以此来测量KPNB1/KPNAx介导的进口随着时间的推移。细胞系2和3允许以一种简单的方式测量进口和出口之间的平衡变化。


A. 研究核细胞质运输的细胞系的生成。
由CMV启动子驱动的报告构建体,使用CRISPR-Cas9基因敲入协议整合到Hela京都细胞的AAVS1安全港位点中。使用三种不同的CRISPR-Cas9敲入协议。


1. CRISPR-Cas9基因敲入协议创建核细胞质运输细胞系。
第1号议定书(佐久间et al., 2016):
使用经典的PCR将感兴趣的核细胞质运输报告盒插入pcDNA3.1质粒中。设计插入时,确保5'和3'端包含两个相同的靶向位点和微同源序列。CRISPR-Cas9靶向位点是需要的,以便在下面的步骤中从质粒中切出插入物。微组学序列是需要特异性地将插入盒插入AAVS1安全港位点。在这里,我们克隆了一个表达NLSSV40-mNeongreen2x-NESpki的盒,并将其与P2A-PuromycinR融合(图3A)。
议定书2(盖辛格et al., 2016):
将PuromycinR-P2A-NESpki2-mNeongreen2x-NLSFUS报告子克隆到pcDNA3.1质粒(图3B),作为模板,用5′硫代磷酸酯修饰的引物(fw引物:T*C*C*CCTCCACCCCACAGTGGGGCC ACTAACGGGCCAGATATACGCG;rv引物:T*C*C*CCTCCACCCCACAGTGGGGCCACTA CTGGTTCTTTCCGCCTCAGAA,用*表示硫代磷酸酯修饰的核苷酸)进行PCR反应。该PCR产物被用作CRISPR敲入协议的供体。该PCR引物包含5′端微同源序列,以使插入物特异性整合到AAVS1安全港位点(类似于协议1)。
第3号议定书(Schmid-Burgket al., 2016):
NLScMYC-AcGFP2x-NESikb2被克隆到pGL4-质粒中,该质粒在5′端含有CRISPR-Cas9的靶向侧,将其线性化,以便使用CRISPaint敲入协议插入AAVS1安全港位点(图3C)。
注:
a. 注意你的报告蛋白的最终大小。如果你的目标是研究主动运输,使用的融合蛋白的两个或三个倍你的荧光蛋白记者(约27 kDa)。我们使用两个副本的mNeonGreen超过被动运输的限制(±50 kDa)。
b. NESpki2含有K到P的突变(MSLNELALPLAGLDI),削弱了原有的NESpki导出信号。因此,导入和导出之间的平衡向导入转移,报告器主要定位在细胞核内。
c. 因为Schmid-Burgk协议不是基于微组学(Schmid-Burgk et al., 2016),与1和2相比,这是效率最低的敲入协议。此外,完整的供体质粒集成在Hela京都AAVS1位点在这个协议。Sakuma协议(Sakumaet al., 2016)具有最高的基因敲入效率,但却是最难克隆的协议。


2. 稳定的细胞系生成(使用的质粒见图3)。
所有步骤中使用的细胞培养基由补充有10%胎牛血清(FBS)和50微克/毫升庆大霉素的Gibco DMEM培养基组成。
a. 第1天:在6孔板中播种10万个Hela京都细胞。
b. 第2天:
协议1:使用Lipofectamine 3000(赛默飞世尔科技)共同转染这些细胞。加入500纳克各自的pcDNA3.1-供体质粒( 图3A),1000纳克表达Cas9的质粒加上针对AAVS1位点的gRNA(GTCACCAATCCTGTCCCTAG)(图3D)和500纳克表达gRNA(GCCAGTACCCAAAAAGCGGG)的质粒,该质粒在两个位点上切割报告质粒,以切除盒( 图3E)。
协议2:使用Lipofectamine 3000(赛默飞世尔科技)共同转染这些细胞。加入500纳克的盒式PCR供体产物( 图3B)和1000纳克表达Cas9的质粒加上针对AAVS1位点(GTCACCAATCCTGTCCCTAG)的gRNA( 图3D)。
协议3:使用Lipofectamine 3000(赛默飞世尔科技)共同转染这些细胞。添加500纳克各自的pGL4-供体质粒( 图3C),1000纳克表达Cas9的质粒加上针对AAVS1位点的gRNA(GTCACCAATCCTGTCCCTAG)( 图3D)和500纳克表达gRNA(GCCAGTACCCAAAAAGCGGG)的质粒,在一个位点切割报告质粒,以线性化供体质粒( 图3E)。
提示:包括一个阴性对照条件,通过省略供体质粒表达Cas9/gRNA在一个孔中。
c. 第三天:更换培养基,限制转染试剂引起的毒性。
d. 第4天:胰蛋白酶法转染的细胞,并用嘌呤霉素(1微克/毫升)进行选择。
e. 在接下来的日子里,分析板的离散菌落,开始出现。
提示:为了排除瀑布阳性:确保只有当所有的细胞都死在阴性对照(没有供体质粒)继续。此外,在接下来的日子里,用抗生素保持细胞的选择。
f. 当6孔与该多克隆细胞混合物完全汇合时,可种植单克隆细胞。因此,在96孔板中以0.5个细胞/孔的密度播种细胞。这可以通过以下方式实现。
i. 取下培养基,用1毫升的1x PBS洗细胞。
ii. 移去PBS,每孔加入500微升0.05%的胰蛋白酶。返回孵化器中5分钟。
iii. 确认细胞分离。如果没有,将细胞再次在孵化器中几分钟,直到他们做。
iv. 每孔加入1.5毫升介质。重要的是吸管上下混合,并获得单细胞。测量获得的细胞数。
v. 进行1/100稀释(10微升到990微升的培养基),以方便下一步的操作。搅拌均匀。
vi. 加入含有50个细胞的体积到10毫升培养基(10毫升是足够一个96孔板)。
vii. 每孔加入100微升,以获得0.5个细胞/孔的浓度。因此,大多数孔将有零或一个细胞,少量含有两个或多个细胞。
提示:在左上角播种较高的密度(如 ,1000个细胞/孔)。因此,它将更容易找到正确的焦点在显微镜上,当你搜索殖民地。
g. 扫描板五天后的单克隆菌落的形成。不要包括包含两个(或更多)独立的菌落的井,因为他们很可能从一个以上的细胞产生,因此不会导致单克隆培养。
小贴士注意井的两侧,因为细胞往往会附着在那里。
h. 当96孔完全充满,扩大单克隆培养。
小贴士:从T-25培养瓶开始逐步扩大。
i. 在液氮中长期储存之前,将细胞冻结在-80℃。可采用以下方法:
i. 准备冷冻培养基(共10毫升:8.2毫升培养基+800微升DMSO+1毫升FBS)。将培养基放在冰上,因为DMSO在室温下对细胞是有毒的。
ii. 给冷冻管贴上标签,并在冰上预冷。
iii. 取下培养基,用1x PBS清洗细胞。
iv. 取出PBS,加入0.05%胰蛋白酶。返回到孵化器中5分钟。
v. 确认细胞分离。如果没有,返回细胞到孵化器几分钟,直到他们做。
vi. 加入培养基以抑制胰蛋白酶,并将所有东西带入50毫升管中。
vii. 计数细胞。
viii. 在500 rcf下旋转5分钟。
ix. 取出上清液,并在预先制作的冷冻培养基中重悬颗粒。每1.5×106-2×106细胞加入1毫升。
x. 移液器1毫升的冷冻介质在每个预冷的冷冻管。
xi. 放置管在-80℃,借助Mr.FrostyTM冷冻容器。
xii. 对于长期储存,第二天将样品移到液氮容器中。




图3.用于生成报告细胞系的质粒。用于生成报告细胞系的质粒。A.供体质粒含有协议1中使用的记者NLSSV40-mNeongreen2x-NESpki。B.含有报告者NESpki2-mNeongreen2x-NLSFUS的质粒,它作为协议2中进行的PCR反应的模板。NESpki2*含有K到P的突变。C.供体质粒含有协议3中使用的报告NLScMYC-AcGFP2x-NESikb2。D.协议1、2和3中使用的表达Cas9的质粒加上靶向AAVS1位点的gRNA(Schmid-Burgk等人,2016)。E.在协议1和3中使用的表达gRNA的质粒来裂解含有记者的供体质粒(Schmid-Burgk等人,2016)。
3. 验证
注意:验证您的记者很重要。可以通过以下方式进行验证:
(1) 用现有的化合物处理你的细胞,例如 ,瘦肉精B抑制XPO1介导的导出(Kudo等人,1999)或importazole抑制KPNB1介导的导入(Soderholm等人,, 2011)1999)。
(2) 转染你的细胞与适当的构建体,如 ,M9M的表达,以抑制TPNO1/TPNO2(Cansizoglu等人,2007)。
(3) 敲除靶向运输受体,例如,针对KPNB1或KPNA2的siRNA。


注:在所有步骤中使用的细胞培养基由Gibco DMEM培养基补充10%FBS和50微克/毫升庆大霉素。
a. 用瘦肉精B验证XPO1介导的出口。
i. 第1天:种子NLSSV40-mNeongreen2x-NESpki报告细胞的密度,以获得60-70%汇合的第二天。
ii. 第2天:改变井中一半的培养基,以获得最终浓度为50 nM的XPO1抑制剂瘦肉精B(工藤 et al., 1999)。
iii. 返回细胞到孵化器30分钟。
iv. 用4%PFA固定细胞15分钟。
v. 用1x PBS洗涤细胞三次。
vi. 染色核使用NucBlue活细胞染色试剂(Invitrogen)。
vii. 图像细胞,以确认增加的核定位的记者在瘦肉精B处理的细胞( 图4)。




图4.验证XPO1介导的出口。XPO1介导的出口的验证。表达记者NLSSV40-mNeongreen2x-NESpki的细胞的代表图像。A.在稳定状态下,记者定位在细胞的细胞质中。B.添加XPO1抑制剂leptomcyin B (LMB)导致记者的核定位。比例尺=20 µm。


b. 用importazole验证KPNB1/KPNAx介导的导入。
i. 第1天:种子NLSSV40-mNeongreen2x-NESpki报告细胞的密度,以获得60-70%汇合的第二天。
ii. 第2天:用50μM的KPNB1抑制剂importazole(Soderholm et al., 2011)。
iii. 改变一半的培养基在井中,以获得50 nM瘦肉精B的最终浓度。
注:记者在细胞质中定位稳定状态(由于XPO1介导的出口),因此,与XPO1抑制剂(瘦肉精B)结合使用,作为KPNB1/KPNAx介导的进口-记者。
iv. 将细胞返回到培养箱中,30分钟后用4%PFA固定细胞。
v. 用1x PBS洗涤细胞三次。
vi. 染色核使用NucBlue活细胞染色试剂(Invitrogen)。
vii. 图像细胞,以确认在importazole处理的细胞( 图5)的记者的核定位减少。




图5.验证KPNB1/KPNAx介导的导入。KPNB1/KPNAx介导的导入的验证。表达记者NLSSV40-mNeongreen2x-NESpki的细胞的代表图像。A.加入瘦肉精B的结果在核定位的记者,由于KPNB1 / KPNNAx介导的进口。B. 用importazole预孵育部分取消KPNB1/KPNAx介导的导入。比例尺=20 µm。


c. 通过M9M表达验证TPNO1/TPNO2-介导的导入。
i. 第1天:种子NESpki2-mNeongreen2x-NLSFUS报告细胞的密度,以获得60-70%汇合的第二天。
ii. 第2天:用表达多肽M9M(Cansizogluet al., 2007)使用lipofectamine 3000(赛默飞世尔科技)的质粒转染细胞。
注:表达M9M与荧光蛋白(如 ,红色荧光蛋白)融合,以识别转染细胞。
iii. 转染后4小时更换培养基以降低毒性。
iv. 孵育细胞过夜。
v. 第3天:用4%PFA固定细胞15分钟。
vi. 用1x PBS洗三遍。
vii. 染色核使用NucBlue活细胞染色试剂(Invitrogen)。
viii. 图像细胞,以确认在M9M表达的细胞( 图6)的记者的核定位减少。




图6.验证TPNO1/TPNO2-介导的导入。验证TPNO1/TPNO2-介导的导入。表达记者NESpki2-mNeongreen2x-NLSFUS的细胞的代表图像。A.在稳定状态下,记者定位在细胞核内,由TPNO1/TPNO2-介导的导入引起。记者细胞转染了mCherry-plasmid作为阴性对照。B. 用RFP-M9M-质粒转染的报告者细胞,该质粒抑制TPNO1/TPNO2-介导的导入,导致报告者的细胞质重新分布。比例尺=20 µm。


d. 验证KPNB1/KPNAx通过KPNB1/KPNA2-knock down介导的导入。
i. 第1天:种子NLScMYC-AcGFP2x-NESikb2报告细胞的密度,以获得60-70%汇合的第二天。
ii. 第2天:用针对KPNB1或KPNA2的siRNA对细胞进行电穿孔(Horizon Discovery siRNA重悬缓冲液,Bio-Rad公司:230V,950 µF,∞Ω,10 000 ms,#1脉冲)。
iii. 孵育细胞48小时。
iv. 用4%PFA固定细胞15分钟。
v. 用1x PBS洗三遍。
vi. 染色核使用NucBlue活细胞染色试剂(Invitrogen)。
vii. 图像细胞,以确认减少核定位的记者( 图7)。






图7.验证KPNB1/KPNAx介导的导入。KPNB1/KPNAx介导的导入的验证。表达记者NLScMYC-AcGFP2x-NESikb2的细胞的代表图像。A.记者定位稳态在细胞核中,由于KPNB1/KPNAx介导的导入。B. 靶向KPNB1或KPNA2的siRNA表达导致记者的细胞质定位。比例尺=25 µm。


4. NLSSV40-mNeongreen2x-NESpki报告器的优化设计。
在稳定状态下,NLSSV40-mNeongreen2x-NESpki记者定位在细胞的细胞质中(由XPO1介导的核输出引起),因此,与XPO1抑制剂(瘦肉精B;LMB)结合使用,作为KPNB1/KPNAx介导的导入记者。
注:由于核强度的结果从出口和进口之间的平衡,重要的是要优化LMB浓度,以最大限度地提高测定的灵敏度,以测量进口赤字。此外,为了最大限度地减少变异性,建议与LMB浓度,诱导记者的逐步进口,并允许进口测量发生在较长的时间(30分钟)。此外,建议至少测量两个不同的时间点,以确保您的测量属于核进口过程的动态范围内,尚未达到饱和。


注:在所有步骤中使用的细胞培养基由Gibco DMEM培养基补充10%FBS和50微克/毫升庆大霉素。
a. 第1天:种子NLSSV40-mNeongreen2x-NESpki报告细胞在96孔板的密度,以获得70%汇合的第二天。
注意:请记住,你需要三个不同的时间点,这在每个条件下一式两份或三份。
b. 第2天:用100微升含有25μM的KPNB1抑制剂importazole的培养基替换旧培养基。
c. 将细胞返回到培养箱中1.5小时。
注:importazole浓度的选择,目的是诱导平均进口抑制,以不错过潜在的小进口赤字诱导的多PR。
d. 同时,制备不同稀释的LMB(溶解在乙醇中)在预热的培养基中的终浓度为0 nM,15 nM,30 nM,45 nM,60 nM,75 nM和90 nM。涡流。
注意:请记住在接下来的步骤中的稀释。因此,准备两倍的最终浓度,例如,120 nM的最终浓度为60 nM。
e. 处理打算30分钟的时间点与不同浓度的LMB的细胞,通过添加100微升的含有LMB的培养基到100微升的培养基已经在井中。
f. 返回细胞到孵化器15分钟。
g. 通过加入100微升的含有LMB的培养基,用不同浓度的LMB处理打算15分钟时间点的细胞。
h. 返回细胞到孵化器15分钟。
i. 用4%PFA固定15分钟。
j. 用1x PBS洗涤细胞三次。
k. 染色核使用NucBlue活细胞染色试剂(Invitrogen)。
l. 细胞可以通过CellInsight CX5高含量筛选平台(赛默飞世尔科技公司)自动分析,以确定诱导对照细胞和importazole处理细胞之间的最大差异的LMB浓度(图8)。
注意:使用多通道移液器以减少变异性。




图8.优化KPNB1/KPNAx介导的NLSSV40-mNeongreen2x-NESpki记者的导入,随着时间的推移测量。优化KPNB1/KPNAx介导的NLSSV40-mNeongreen2x-NESpki报告的导入随时间的测量。A-B。进口importazole处理的细胞相对于进口DMSO处理的细胞不同浓度的瘦肉精B(LMB)。A-15分钟;B-30分钟。A.45 nM的LMB导致对照细胞和用importazole处理的细胞之间的最强差异。B.较低的浓度允许测量导入抑制,因为导入变得饱和在这个较高浓度的LMB较晚的时间点。C.左图:较低浓度(例如 ,30 nM)的LMB导致较少的整体导入,因此更难观察到15分钟后受干扰的导入。进口缺陷可以在以后的时间点(30分钟)观察到。中图。45 nM的LMB在两个时间点都显示出导入缺陷。右图:更高浓度的LMB(例如 ,90 nM)可以推动进口太难观察到进口的最小障碍。A-B。点代表一个实验的平均值,每个实验有2500-7000个细胞。平均值+S.D.C.点代表一个实验的平均值,每个实验2500-7000个细胞。


B. M 在存在突变的C9orf72相关肽的情况下,确保核细胞质的运输。
1. 表达突变的C9orf72相关肽的慢病毒载体的制作。
DPR100-质粒是伊藤大介博士(日本东京庆应义塾大学神经学系)的友好礼物。经典的PCR 被用来克隆mCherry-DPR100构建体到慢病毒载体质粒(LentiCrisprV2_Puro从Addgene)(图9)。使用HEK-293T细胞生产慢病毒载体,如前所述(Hart et al., 2017)。
注意:我们强烈建议使用转导而不是转染来表达所需的疾病相关的结构。转染是更多的压力,这可能会影响核细胞质运输测量。




图9.用于表达mCherry或mCherry-PR100的慢病毒载体质粒。用于表达mCherry或mCherry-PR100的慢病毒载体质粒。A.含有mCherry的慢病毒载体。B.含有mCherry-PR100的慢病毒载体。


2. 化验
注:在所有步骤中使用的细胞培养基由Gibco DMEM培养基补充10%FBS和50微克/毫升庆大霉素。
a. 通过细胞和种子到盖板玻璃上
i. 第0天:使用无菌镊子将单个盖玻放入无菌24孔聚苯乙烯组织培养板的每个孔中。
注:
(1) 预涂盖玻片与聚L-赖氨酸和聚L-鸟氨酸或其他细胞表面处理可能是必要的,以坚持其他类型的细胞到玻璃。Hela Kyoto细胞无需额外的涂层就能粘附在玻璃上。
(2) 对于高通量筛选,细胞可以被播种到96孔板或其他格式。
(3) 请记住,你将需要三个不同的时间点,这在每个条件下一式两份或三份。
ii. 种子的密度,将导致在70%的汇合,在你计划执行核细胞质运输测定的日子。
iii. 在组织培养箱中孵育细胞过夜(37℃,5%的二氧化碳)。
b. 转导细胞表达C9orf72相关的多肽
i. 第1天,在培养基中稀释聚丙烯,最终浓度为10微克/毫升。在培养基中稀释聚丙烯,最终浓度为10微克/毫升。将慢病毒颗粒添加到每个孔中。让病毒颗粒在细胞上过夜(约18小时)。
ii. 第2天:取出含有慢病毒的培养基,换上新鲜的DMEM培养基。
iii. 孵育细胞在组织培养箱(37℃,5%的CO 2)额外的两天。
注:添加聚丁烯可以显著提高转导效率。然而,首先确认polybrene对你的细胞没有毒性,因为它可以诱导某些细胞类型(例如,iPSC衍生的神经元)的细胞死亡。
c. 测量核细胞质运输
i. 第5天:准备一个所需体积的预热培养基,含有45 nM LMB。
ii. 用含有LMB的培养基处理打算30分钟时间点的细胞。
iii. 返回细胞到孵化器15分钟。
iv. 用含有LMB的培养基处理打算15分钟时间点的细胞。
v. 返回细胞到孵化器15分钟。
vi. 通过添加4%PFA(最终浓度)到培养基中固定细胞15分钟。
vii. 染色核使用NucBlue活细胞染色试剂(Invitrogen)。
viii. 使用20微升移液器将一滴安装介质放在载玻片上,然后用镊子将盖玻片倒置在载玻片上。用纸巾去除多余的装片介质,并使用指甲油密封载玻片。
ix. 使用共聚焦显微镜( 图10)成像细胞。
注:
(1) 切记在不同条件下要保持相同的设置,并将重点放在核的中间(核仁清晰可见)。 
(2) 对于高通量筛选,强度可以通过使用高含量分析仪(如赛默飞世尔科技的CellInsight CX5高含量筛选平台)自动分析。




图10.测量KPNB1/KPNAx介导的导入随着时间的推移在神经退行性相关背景下。测量KPNB1/KPNAx介导的进口随着时间的推移在神经退行性相关的背景下。表达记者NLSsv40-mNeongreen2x-NESpki的细胞的代表图像 。记者定位在细胞质中的稳定状态,作为XPO1介导的核出口的结果。添加XPO1抑制剂(瘦肉精B; LMB)允许测量KPNB1/KPNAx介导的进口随着时间的推移。A.Reporter细胞用mCherry-慢病毒载体转导作为对照。B.用poly-PR100-mCherry-慢病毒载体转导的报告者细胞。Poly-PR二肽与神经退行性疾病肌萎缩侧索硬化症有关。比例尺=20 µm。


数据分析


A. 指南
1. 图片J
在共聚焦显微镜上拍摄的图像可以手动分析与免费在线可用的软件ImageJ。
a. 在ImageJ中导入图片。
注:建议分析64位格式的图像。
b. 选择感兴趣的区域+Ctrl M(图11)。




图11.在ImageJ中手动分析。图像是代表性的NLSsv40-mNeongreen2x-NESpki报告细胞添加LMB和转染mCherry之前。图中包含图10的图像分析数据。平均值±S.D.显示。


B. 自动
1. 细胞剖析仪
共聚焦显微镜上成像的细胞可以自动分析与免费在线可用的软件细胞剖析器( 图12)。




图12.Cell profiler自动分析的工作流程在Cell profiler自动分析的工作流程。图像是代表NLSsv40-mNeongreen2x-NESpki报告细胞添加LMB和转染mCherry之前。图包含从图10的图像的分析数据。平均值±S.D.显示。


统计分析
1. 我们建议分析核与总(=细胞质+核)的比值,因为核与细胞质的比值可能容易出现较大的变化。核与细胞质值之间的差异可能会变得非常大,特别是在添加LMB(核强度非常低)之前或在检测结束时(细胞质强度非常低)。此外,当数据必须归一化为初始强度时,核强度和细胞质强度的微小差异可能会造成比值的巨大差异。
2. 比值本质上是不对称的,也就是说,所有的减少都表示为0和1之间的比值,所有的增加都表示为大于1.0的比值。因此,我们建议分析比率的对数。因此,没有变化将是零(1.0的对数),增加将是正数,减少将是负数。


鸣谢


我们感谢Liesbeth Mercelis和Bob Massant的技术援助。这项工作得到了鲁汶大学(C1和开放未来基金)的支持,弗兰德斯科学研究基金(FWO-Vlaanderen GOB3318N - G098314N),ALS Liga(比利时)。J.V.是FWO的博士研究员。P.V.D.是FWO-Vlaanderen的高级临床研究员,并得到Een hart voor ALS基金和Laevers ALS研究基金的支持。


竞争性利益


提交人声明他们没有财务和非财务竞争利益。


参考文献


1. Arai,T.,Hasegawa,M.,Akiyama,H.,Ikeda,K.,Nonaka,T.,Mori,H.,Mann,D.,Tsuchiya,K.,Yoshida,M.,Hashizume,Y.和Oda,T.(2006)。TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Biochem Biophys Res Commun 351(3): 602-611.
2. Ash, P. E., Bieniek, K. F., Gendron, T. F., Caulfield, T., Lin, W. L., Dejesus-Hernandez, M., van Blitterswijk, M. M., Jansen-West, K., Paul, J. W., 3rd, Rademakers, R., Boylan, K. B., Dickson, D. W. and Petrucelli, L. (2013).Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS.Neuron 77(4):639-646。
3. Boeynaems,S.,Bogaert,E.,Van Damme,P.和Van Den Bosch,L.(2016)。Inside out: the role of nucleocytoplasmic transport in ALS and FTLD.Acta Neuropathol 132(2):159-173。
4. Cansizoglu, A. E., Lee, B. J., Zhang, Z. C., Fontoura, B. M. and Chook, Y. M. (2007).Structure-based design of a pathway-specific nuclear import inhibitor.Nat Struct Mol Biol 14(5): 452-454.
5. Fahrenkrog, B. and Harel, A. (2018).Perturbations in traffic: aberrant nucleocytoplasmic transport at the heart of neurodegeneration.Cells 7(12). DOI:10.3390/cells7120232。
6. Geisinger, J. M., Turan, S., Hernandez, S., Spector, L. P. and Calos, M. P.(2016).In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining.Nucleic Acids Res 44(8):e76.
7. Hart, T., Tong, A. H. Y., Chan, K., Van Leeuwen, J., Seetharaman, A., Aregger, M., Chandrashekhar, M., Hustedt, N., Seth, S., Noonan, A., Habsid, A., Sizova, O., Nedyalkova, L., Climie, R., Tworzyanski, L., Lawson, K., Sartori, M. A., Alibeh, S., Tieu, D., Masud, S., Mero, P., Weiss, A., Brown, K. R., Usaj,Sartori,M. A.,Alibeh,S.,Tieu,D.,Masud,S.,Mero,P.,Weiss,A.,Brown,K. R.,Usaj,M.,Billmann,M.,Rahman,M.,Constanzo,M.,Myers,C. L.,Andrews,B. J.,Boone,C.,Durocher,D.和Moffat,J.(2017)。Evaluation and design of genome-wide CRISPR/SpCas9 knockout screens.G3(Bethesda)7(8):2719-2727。
8. Hutten, S. and Dormann, D. (2019).Nucleocytoplasmic transport defects in neurodegeneration - Cause or consequence?Semin Cell Dev Biol S1084-9521(18).301090-301093.
9. Kudo, N.、Matsumori, N.、Taoka, H.、Fujiwara, D.、Schreiner, E. P.、Wolff, B.、Yoshida, M.和Horinouchi, S.(1999年)。Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region.Proc Natl Acad Sci U S A 96(16): 9112-9117。
10. Kwiatkowski, T. J., Jr., Bosco, D. A., Leclerc, A. L., Tamrazian, E., Vanderburg, C. R., Russ, C., Davis, A., Gilchrist, J., Kasarskis, E. J., Munsat, T., Valdmanis, P., Rouleau, G. A., Hosler, B. A.。Cortelli, P., de Jong, P.J., Yoshinaga, Y., Haines, J.L., Pericak-Vance, M.A., Yan, J., Ticozzi, N., Siddique, T., McKenna-Yasek, D., Sapp, P.C., Horvitz, H.R., Landers, J.E. and Brown, R.H., Jr. (2009).(2009).16号染色体上FUS/TLS基因的突变导致家族性肌萎缩性侧索硬化症。科学》323(5918): 1205-1208。
11. Lange, A., Mills, R. E., Lange, C. J., Stewart, M., Devine, S. E. and Corbett, A. H. (2007)。经典的核定位信号:定义、功能和与导入素α的相互作用。J Biol Chem 282(8): 5101-5105.
12. Majounie, E., Renton, A. E., Mok, K., Dopper, E. G., Waite, A., Rollinson, S., Chio, A., Restagno, G., Nicolaou, N., Simon-Sanchez, J., van Swieten, J. C., Abramzon, Y., Johnson, J. O., Sendtner, M., Pamphlett, R., Orrell, R. W.,Mead,S.,Sidle,K.C.,Houlden,H.,Rohrer,J.D.,Morrison,K.E.,Pall,H.,Talbot,K.,Ansorge,O.,Chromosome,A.L.S.F.T.D.C.,French research network on,F.A.,Consortium,I.,Hernandez,D.G.,Arepalli,S.,Sabatelli,M。Mora,G.,Corbo,M.,Giannini,F.,Calvo,A.,Englund,E.,Borghero,G.,Floris,G.L.,Remes,A.M.,Laaksovirta,H.,McCluskey,L.,Trojanowski,J.Q.,Van Deerlin,V.M.,Schellenberg,G.D.,Nalls,M.A.,Drory,V.E.,Lu,C.S.。Yeh,T. H.,Ishiura,H.,Takahashi,Y.,Tsuji,S.,Le Ber,I.,Brice,A.,Drepper,C.,Williams,N.,Kirby,J.,Shaw,P.,Hardy,J.,Tienari,P. J.,Heutink,P.,Morris,H. R.,Pickering-Brown,S.和Traynor,B. J.(2012)。C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study.Lancet Neurol 11(4):323-330。
13. Mori, K., Arzberger, T., Grasser, F. A., Gijselinck, I., May, S., Rentzsch, K., Weng, S. M., Schludi, M. H., van der Zee, J., Cruts, M., Van Broeckhoven, C., Kremmer, E., Kretzschmar, H. A., Haass, C.和Edbauer, D. (2013a)。Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins.Acta Neuropathol 126(6):881-893。
14. Mori, K., Weng, S. M., Arzberger, T., May, S., Rentzsch, K., Kremmer, E., Schmid, B., Kretzschmar, H. A., Cruts, M., Van Broeckhoven, C., Haass, C. and Edbauer, D. (2013b).C9orf72 GGGGCC重复在FTLD/ALS中被翻译成聚集性二肽重复蛋白。Science 339(6125): 1335-1338.
15. Neumann,M.,Sampathu,D.M.,Kwong,L.K.,Truax,A.C.,Micsenyi,M.C.,Chou,T.T.,Bruce,J.,Schuck,T.,Grossman,M.,Clark,C.M.。McCluskey, L. F., Miller, B. L., Masliah, E., Mackenzie, I. R., Feldman, H., Feiden, W., Kretzschmar, H. A., Trojanowski, J. Q. and Lee, V. M. (2006).Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis。科学314(5796): 130-133。
16. Ossareh-Nazari, B.、Gwizdek, C.和Dargemont, C.(2001年)。蛋白质从细胞核中输出。Traffic 2(10): 684-689。
17. Ryan, K. J. and Wente, S. R. (2000)。核孔复合体:连接细胞核和细胞质的蛋白质机器。Curr Opin Cell Biol 12(3): 361-371.
18. Sakuma,T.,Nakade,S.,Sakane,Y.,Suzuki,K.T. and Yamamoto,T.(2016).MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems.Nat Protoc 11(1):118-133.
19. Schmid-Burgk, J. L., Höning, K., Ebert, T. S. and Hornung, V. (2016).CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism.Nat Commun 7: 12338.
20. Soderholm, J. F., Bird, S. L., Kalab, P., Sampathkumar, Y., Hasegawa, K., Uehara-Bingen, M., Weis, K. and Heald, R. (2011).Importazole, a small molecule inhibitor of the transport receptor importin-β。ACS Chem Biol 6(7):700-708。
21. Stoffler, D., Fahrenkrog, B. and Aebi, U. (1999)。核孔复合体:从分子结构到功能动力学。Current Opinion Cell Biology 11(3):391-401。
22. Tyzack, G. E., Luisier, R., Taha, D. M., Neeves, J., Modic, M., Mitchell, J. S., Meyer, I., Greensmith, L., Newcombe, J., Ule, J., Luscombe, N. M. and Patani, R. (2019).Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis.Brain 142(9):2572-2580.
23. Vance, C., Rogelj, B., Hortobagyi, T., De Vos, K. J., Nishimura, A. L., Sreedharan, J., Hu, X., Smith, B., Ruddy, D., Wright, P., Ganesalingam, J., Williams, K. L.,Tripathi, V., Al-Saraj, S., Al-Chalabi, A., Leigh, P. N., Blair, I. P., Nicholson, G., de Belleroche, J., Gallo, J. M., Miller, C. C.和Shaw, C. E. (2009).FUS的突变,一种RNA处理蛋白,导致家族性肌萎缩性侧索硬化症6型。科学》323(5918): 1208-1211。
24. Vanneste,J.,Vercruysse,T.,Boeynaems,S.,Sicart,A.,Van Damme,P.,Daelemans,D.和Van Den Bosch,L.(2019)。C9orf72-generated poly-GR and poly-PR do not directly interfere with nucleocytoplasmic transport.Sci Rep 9(1): 15728.
25. Yuva-Aydemir,Y.,Almeida,S. and Gao,F. B.(2018).Insights into C9ORF72-Related ALS/FTD from Drosophila and iPSC Models.Trends Neurosci 41(7):457-469.
26. Zu,T.,Gibbens,B.,Doty,N. S.,Gomes-Pereira,M.,Huguet,A.,Stone,M. D.,Margolis,J.,Peterson,M.,Markowski,T. W.,Ingram,M. A.,Nan,Z.,Forster,C.。Low, W. C., Schoser, B., Somia, N. V., Clark, H. B., Schmechel, S., Bitterman, P. B., Gourdon, G., Swanson, M. S., Moseley, M. and Ranum, L. P. (2011).Non-ATG-initiated translation directed by microsatellite expansions.Proc Natl Acad Sci U S A 108(1):260-265。

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引用:Vanneste, J., Vercruysse, T., Van Damme, P., Van Den Bosch, L. and Daelemans, D. (2020). Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration. Bio-protocol 10(12): e3659. DOI: 10.21769/BioProtoc.3659.
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