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Jun 2020
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Dual Color, Live Imaging of Vesicular Transport in Axons of Cultured Sensory Neurons
培养感觉神经元轴突囊泡运输的双色实时成像   

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Abstract

The function of neurons in afferent reception, integration, and generation of electrical activity relies on their strikingly polarized organization, characterized by distinct membrane domains. These domains have different compositions resulting from a combination of selective targeting and retention of membrane proteins. In neurons, most proteins are delivered from their site of synthesis in the soma to the axon via anterograde vesicular transport and undergo retrograde transport for redistribution and/or lysosomal degradation. A key question is whether proteins destined for the same domain are transported in separate vesicles for local assembly or whether these proteins are pre-assembled and co-transported in the same vesicles for delivery to their cognate domains. To assess the content of transport vesicles, one strategy relies on staining of sciatic nerves after ligation, which drives the accumulation of anterogradely and retrogradely transported vesicles on the proximal and distal side of the ligature, respectively. This approach may not permit confident assessment of the nature of the intracellular vesicles identified by staining, and analysis is limited to the availability of suitable antibodies. Here, we use dual color live imaging of proteins labeled with different fluorescent tags, visualizing anterograde and retrograde axonal transport of several proteins simultaneously. These proteins were expressed in rat dorsal root ganglion (DRG) neurons cultured alone or with Schwann cells under myelinating conditions to assess whether glial cells modify the patterns of axonal transport. Advantages of this protocol are the dynamic identification of transport vesicles and characterization of their content for various proteins that is not limited by available antibodies.

Keywords: Axonal transport (轴突输送), Vesicular transport (膜泡运输), Dual color live imaging (双色实时成像), Node of Ranvier (兰氏结), Neuronal polarity (神经元极性)

Background

Neurons are highly polarized cells. This polarity is critical for neuronal function, i.e., integrating pre-synaptic inputs on the somatodendritic compartment and initiating and propagating action potentials along axons. Myelinated axons are further subdivided into a series of sub-domains, notably including the nodes of Ranvier, the gaps located between adjacent myelin sheaths where action potentials regenerate during saltatory conduction. Nodes are highly enriched in voltage gated Na+ channels, their associated beta subunits, and several neuronal cell adhesion molecules (CAMs). Among the latter is neurofascin (NF) 186, which binds to cognate receptors on Schwann cells, the glial cells that myelinate axons in the peripheral nervous system. Sodium channels and CAMs at the node are tethered to and stabilized by interactions with ankyrin G (AnkG), which forms a specialized submembranous cytoskelon with βIV spectrin. Together, these components form a multimeric nodal complex that differs strikingly from that of other multimeric protein complexes present in other domains of myelinated axons (Salzer et al., 2008). These latter domains include the paranodes, which flank the node, and the juxtaparanodal and internodal domains, which lie underneath the compact myelin sheath.


A key question is whether the components of the node, and of other domains, are transported from the soma to the axon separately to be assembled locally or whether they are transported to their respective domains as pre-assembled complexes. Transport vesicles that shuttle transmembrane proteins from the cell body to the axon (i.e., anterogradely) would predominantly contain a single protein cargo in the former case, whereas they would contain a mixture of protein cargoes in the latter case. A related question is whether vesicles retrogradely transported from the axon to the soma also contain single or multiple cargoes.


One strategy to assess prospective co-expression of proteins in transport vesicles relies on immunofluorescence of proteins in sections of fixed ligated nerves (Cavalli et al., 2005). This method depends on the availability of suitable antibodies against the proteins of interest. In addition, the precise nature of the vesicles being visualized can be potentially ambiguous in the absence of active transport.


As an alternative strategy, we have dynamically imaged vesicles during active transport using multi-color live imaging, a powerful tool to characterize the transport of membrane proteins in neurons (Kaether et al., 2000). To examine whether components of the nodes are transported separately from each other, and from components of other domains, we first infected cultured rat dorsal root ganglia (DRG) neurons with a doxycycline-inducible lentiviral vector to drive simultaneous expression of various tagged proteins. These lentiviral-infected neurons were grown on poly-L-lysine (PLL)/laminin-coated glass bottom dishes either alone or with Schwann cells (SCs) under myelinating conditions. We then carried out pair-wise comparisons of these different cargoes in anterogradely and retrogradely transported vesicles by dual color live imaging and assessed whether vesicles contained single or multiple cargoes. Dynamic imaging allows for the unambiguous identification of vesicles undergoing active transport. Analyzing the transport of multiple proteins relies on distinct fluorescent tags of sufficient brightness to live image over extended time periods and does not require corresponding antibodies. Here, we provide a detailed protocol for dual-color live imaging of the transport of transmembrane proteins in cultured rat DRG neurons.


Materials and Reagents

  1. 35 mm glass bottom dishes (MatTek Corporation, catalog number: P35G-1.5-14-C)

  2. 100 mm tissue culture dishes (TPP, catalog number: 93100)

  3. Syringe filter unit, 0.45 μm (Millipore Sigma, catalog number: SLHV033RS)

  4. Timed pregnant Sprague Dawley rats (for E15-16 DRG dissection)

  5. 293FT cells (Thermo Fisher, catalog number: R7007)

  6. Modified pSLIK (Addgene #25737; Shin et al., 2006) (storage at -20°C)

  7. Modified pFUGW lentiviral vector (Addgene #14883; Lois et al., 2002) (storage at -20°C)

  8. pCMV-VSV-G (gift from J. Milbrandt, Washington University) (storage at -20°C)

  9. pCMV-Δ8.9 (gift from J. Milbrandt, Washington University) (storage at -20°C)

    Note: pSLIK was engineered to remove hygromycin resistance and IRES sequences to provide additional space to accommodate larger cDNAs for various cargoes (Zhang et al., 2012). pFUGW was modified to lack the GFP reporter and to add a unique cloning site (Dzhashiashivili et al., 2007).

  10. Gateway LR Clonase II Enzyme Mix (Thermo Fisher, catalog number: 11791020) (storage at -80°C)

  11. Poly-L-lysine (Sigma-Aldrich, catalog number: P5899) (storage at -80°C)

    Note: Used for 293FT cell culture.

  12. Poly-L-lysine (Sigma-Aldrich, catalog number: P1274) (storage at -80°C)

    Note: Used to coat glass bottom dishes.

  13. Natural mouse laminin (Invitrogen, catalog number: 23017-015) (storage at -80°C)

  14. Dulbecco’s phosphate-buffered saline (Lonza, catalog number: 17-512Q) (store at room temperature)

  15. LipoD293 DNA transfection reagent (Signagen, catalog number: SL100668) (storage at 4°C)

  16. 0.25% trypsin (Thermo Fisher, catalog number: 15050065) (storage at -20°C)

  17. Fetal bovine serum (Thermo Fisher, catalog number: 16000-044) (storage at -80°C)

  18. MEM (Thermo Fisher, catalog number: 11090-073) (storage at 4°C)

  19. MEM, minus phenol red (Thermo Fisher, catalog number: 51200-038) (storage at 4°C)

  20. Neurobasal medium (Thermo Fisher, catalog number: 21103-049) (storage at 4°C)

  21. Neurobasal medium, minus phenol red (Thermo Fisher, catalog number: 12348-017) (storage at 4°C)

  22. DMEM (Lonza, catalog number: 12-614F) (storage at 4°C)

  23. MEM non-essential amino acid solution (Thermo Fisher, catalog number: 11140050) (storage at 4°C)

  24. Sodium pyruvate (Thermo Fisher, catalog number: 11360070) (storage at 4°C)

  25. Glucose (Sigma-Aldrich, catalog number: G5146) (storage at 4°C)

  26. L-Glutamine (Thermo Fisher, catalog number: 25030-081) (storage at -80°C)

  27. B27 supplement (ThermoFisher, catalog number: 17504-054) (storage at -20°C)

  28. 2.5S nerve growth factor (AbD Serotec, catalog number: PMP04Z) (storage at -80°C)

  29. Doxycycline (Sigma-Aldrich, catalog number: D9891) (storage at -80°C)

  30. HEPES (Thermo Fisher, catalog number: 15630106) (storage at 4°C)

  31. Penicillin-streptomycin (Thermo Fisher, catalog number: 15140122) (storage at -80°C)

  32. Vitamin C (Sigma-Aldrich, catalog number: A0278) (storage at room temperature)

  33. Fluorodexyuridine (FdU) (Sigma-Aldrich, catalog number: F0503) (storage at room temperature)

  34. Uridine (Sigma-Aldrich, catalog number: U3003) (storage at room temperature)

  35. HEK293 cell medium (see Recipes)

  36. NB medium (see Recipes)

  37. NBF medium (see Recipes)

  38. C medium (see Recipes)

  39. CF medium (see Recipes)

  40. Live imaging medium (see Recipes)

Equipment

  1. CO2 incubator (Thermo Fisher, model: Heracell 240)

  2. Microscope and requirements:

    1. Inverted epifluorescence microscope with plan Apo Lambda 100×/1.45 NA objective (Nikon, model: Eclipse Ti-E) equipped with a motorized Epi-fl rotating filter turret.

    2. CCD cameras (Andor Technology, model: Clara; Nikon, model: DS-Qi2)

    3. Heating Insert P (PECON, catalog number: 130-800 207)

    4. Tempcontrol 37-2 digital (PECON, catalog number: 0503.000)

    5. GFP filter cube (Nikon, catalog number: 96362)

    6. 594-nm laser bandpass set filter cube (Chroma, catalog number: 49911)

Software

  1. NIS-Elements Advanced Research Software (Nikon, version: 4.30.01)

  2. ImageJ Manual Tracking (Fiji, https://imagej.net/Manual_Tracking)

  3. Excel (Microsoft, version: 2013)

  4. Prism Software (GraphPad, version: 8)

Procedure

  1. Generation of fluorescently tagged proteins

    1. Fuse either EGFP or mKate2 (far red) (see Notes 1, 4, 5, 6) to the C-terminus or other suitable sites of the protein of interest using unique restriction enzyme sites present in the expression vectors. Add restriction enzyme sites as needed via PCR or by first transferring to other vectors with multiple restriction sites prior to subcloning into pSLIK or FUGW. If suitable sites for tags are in between domains, add restriction enzyme sites, linker sequences, and tags by patch PCR (Squinto et al., 1990).

    2. Subclone the resultant fusion protein cDNA into a doxycycline inducible-lentiviral vector, e.g., modified pSLIK (Addgene #25737) using the Gateway LR Clonase II or the constitutively expressing modified pFUGW lentiviral vector (Addgene #14883) using the unique restriction sites present in this vector. Details of the constructs used for live imaging have been described previously (Dzhashiashivili et al., 2007; Zhang et al., 2012; Bekku and Salzer, 2020).


  2. Lentivirus production

    1. Coat 100-mm dishes with 5 ml poly-L-lysine (10 μg/ml; P5899, No.11 in the reagent list) in PBS for 20 min at room temperature; rinse 3 times with PBS at room temperature.

    2. Seed dishes with 6 × 106 293FT cells 24 h prior to transfection and allow cell density to reach ~90% confluence at the time of transfection.

    3. Add fresh HEK293 cell medium (5 ml) 30-60 min before transfection.

    4. Transfect 293FT cells with 5 μg pSLIK or FUGW lentiviral constructs together with helper plasmids Δ8.9 (6.25 μg) and VSVg (3.3 μg) using the LipoD293TM in vitro DNA transfection reagent.

    5. Replace the DNA/LipoD293 complex containing medium with 7 ml HEK293 cell medium 5 h after transfection.

    6. Collect media from the cultures 48 h after transfection and transfer to 50-ml centrifuge tubes. Centrifuge for 15 min at 1,700 × g at room temperature and filter the supernatants through a 0.45-μm filter unit. Filtered supernatants are aliquoted as 0.6-ml samples and stored at -80°C until use.


  3. Preparation of dissociated rat DRG neuron cultures and myelinating co-cultures for live imaging

    1. Coat the glass coverslip at the bottom of each MatTek dish with 0.5 mg/ml PLL (P1274, No.12 in the reagent list) in 300 μl PBS. Incubate at 37°C for 30 min, then wash 3 times with PBS.

    2. Next, coat the glass coverslips with 10 μg/ml laminin in 200 μl PBS. Incubate at 37°C for 30 min.

    3. Prepare dissociated E15-E16 rat DRG neuron cultures for growth as neuron-only cultures or myelinating cocultures as per previously published protocols (Taveggia and Bolino, 2018). Briefly, remove DRGs from rat embryos, incubate them with 1.5 ml trypsin at 37°C for 45 min, add 1 ml C medium, dissociate DRG neurons by repetitive pipetting to triturate ganglia, and centrifuge cells at 193 × g at room temperature for 5 min.

    4. Resuspend the pellet by gentle trituration in ~200 μl CF medium containing penicillin-streptomycin (PS) per DRG. Seed dissociated rat DRG neurons, corresponding to the equivalent of 1 ganglion (approx. 2.1 × 104 cells), onto the Matek PLL/laminin-coated coverslip and incubate for 24 h.

    5. Cultures are cycled on NBF and NB media every other day for a total of 12 days to deplete all non-neuronal cells, leaving neuron-only cultures.

    6. To establish myelinating co-cultures, add 3 × 105 post-natal rat SCs to neuron-only cultures. SCs are prepared from sciatic nerves, and individual aliquots can be stored in liquid N2 as previously described (Kim and Maurel, 2009). To initiate myelination, after 5 days, add vitamin C to the C media to a final concentration of 50 μg/ml; this concentration of vitamin C does not affect the pH of the medium. Myelination typically ensues several days after adding vitamin C.


  4. Dual viral infection and induction of fluorescently tagged proteins in neuron-only and myelinating cocultures

    1. Dilute the first viral supernatant in C medium containing PS. For neuron-only cultures, add 1:1 (virus: C medium) diluted mKate2-tagged pSLIK virus to one-day-old DRG neuron cultures. For myelinating co-cultures, add 1:1 (virus: C medium) diluted EGFP-tagged pSLIK virus to one-day-old neuron cultures. Incubate cultures with viral supernatants for 24 h.

    2. The next day, dilute the second viral supernatant in C medium containing PS. For neuron-only cultures, change the viral medium to freshly diluted EGFP-tagged pSLIK virus (1:1 dilution). For co-cultures, replace the viral medium with new diluted mKate2-tagged FUGW virus (dilution 1:15 of virus: C medium) onto the neuron culture (see Note 9). Incubate for 24 h.

    3. Remove the viral medium, add NBF, and culture the infected neurons for an additional 1-2 days until expression is induced. If longer incubation times are desirable to allow further neuron growth, change the medium to NB for an additional 2 days.

    4. Add 2 µg/ml doxycycline (final concentration) to either NB or NBF in neuron-only cultures depending on the medium cycle on the day doxycycline is added, or into C plus vitamin C in co-cultures to induce protein expression 24-48 h before live imaging (see Note 8).

      Note: The schematic time courses of the experiment for neuron only cultures and myelinating cultures (procedures C and D) are shown in Figure 1.



      Figure 1. Time course of the dual-color live imaging


  5. Live imaging

    1. Change medium to live imaging media immediately prior to live imaging.

    2. Place cultures growing on a MatTek dish onto a 37°C heated microscope stage (Heat Insert P) connected to a temperature control unit (Tempcontrol 37-2).

    3. Determine the direction of axonal transport for each vesicle relative to the position of the neuronal soma before or at the onset of live imaging.

    4. Capture images at 2 s intervals for ≥2 min with CCD cameras and the 100× objective (see Notes 2, 3, 8) using the perfect focus setting of the NIS-Elements Advanced Research Software (Nikon) to correct focus drift. Process images using real time deconvolution of the NIS-Elements Advanced Research Software. EGFP-tagged proteins were captured with a GFP filter cube (96362, Nikon); those for mKate2 were captured with a 594-nm laser bandpass set filter (49911, Chroma) cube. See the representative still images of dual color live imaging in Figure 2.

    5. Take images of more than 20 neurons for analysis. Loss of focus due to microscope stage drift may require refocusing and may also reduce image numbers available for generating kymographs.



      Figure 2. Representative still images of vesicular co-transport and independent transport. Upper panels: Vesicles containing NF186-mKate2 and NrCAM-EGFP are anterogradely co-transported in the axon as evidenced by the yellow color from the overlap of mKate2-labeled NF186 and EGFP-labeled NrCAM in the same vesicles; an example of co-transported vesicles is highlighted by the yellow arrow. Lower panels: Vesicles containing NF186-mKate2 and Navβ1-EGFP are transported separately in the retrograde direction along the axon; examples of vesicles containing Navβ1-EGFP only are highlighted by the white arrows.

Data analysis

  1. Build kymographs by live imaging 15-µm axon segments for 2 min. One option for building kymographs is to use the NIS-Elements Advanced Research Software, which displays pixel intensity changes along a defined linear section over time (Figure 3). ImageJ or other microscopy analysis software is another option for building kymographs. Extract trajectories of individual vesicles from the kymographs and manually count vesicle numbers in each direction. Analyze ≥ 10 neurons for each combination of transfected proteins and compare the rates of co-transport for each protein. For statistical analysis, analyze ≥ 100 vesicles (≥ 10 neurons); sample size may vary depending on the experimental design. Sample size can be calculated by software, e.g., G power.

  2. Confirm quantitative results using ImageJ Manual Tracking, particularly for some anterograde vesicles that can be of limited brightness.

  3. Measure the average velocities of vesicles using ImageJ Manual Tracking.

  4. Compare the cotransport rates by averaging the results from each imaged axon.

  5. Assess statistical significance with either the two-tailed Welch’s t-test or the two-tailed Student’s t-test for unpaired data using the Excel or GraphPad Prism software.



    Figure 3. Representative kymograph of co-transported vesicles. This kymograph was generated from live imaging of neurons expressing NF186-EGFP and NF186-mKate2 by the NIS-Elements Advanced Research Software. The trajectory of a vesicle is highlighted by blue circles, which are numbered in temporal sequence. In the kymograph, two retrogradely co-transported vesicles are detectable. Additional representative live images and movies, kymographs, and data analysis are provided in J Cell Biol (Bekku and Salzer, 2020).

Notes

  1. This protocol describes dual color imaging but can be adapted for three or more fluorophores, depending on the microscope’s ability to distinguish the different fluorophores used as tags.

  2. A limitation of live imaging by single-laser epifluorescence microscopy is the time lag that occurs during switching between filters to monitor the different fluorophores. During this lag, if the vesicles are actively being transported, the vesicle will continue to move and exhibit an artefactual spatial separation of different fluorophores demarcating the same vesicle.

  3. Spinning disk microscopy with two cameras is an excellent alternative to simultaneously live image multi-colored vesicles with no time lag as there is no filter switching. In our study, we also used spinning disk microscopy to confirm the results. In terms of image quality, the epifluorescence microscope takes clear images of individual vesicles.

  4. In our studies, proteins were tagged with either EGFP or mKate2 at their C-termini, and their trafficking was analyzed by pairwise comparisons. Unlike mKate2, all other red-fluorophore tags that were tried, i.e. mRFP, mCherry, and DsRed, induced some degree of protein aggregation that impacted trafficking, which is in agreement with Katayama et al. (2008).

  5. It is important to confirm that protein trafficking is not affected by the addition of the fluorescent protein tags. For example, dual expression of a protein tagged with EGFP and the same protein tagged with mKate2 showed that these proteins exhibited complete overlap during intracellular trafficking in neurons (Bekku and Salzer, 2020).

  6. It is also important to show that the addition of the fluorescent tag does not interfere with targeting of the tagged protein to its normal plasma membrane destination (e.g., to the node, synapse, etc.). For a description of how to design fusion proteins see Snapp (2005).

  7. To express two proteins simultaneously in neurons, tagged proteins were subcloned into the inducible viral construct pSLIK, and their expression was simultaneously induced by doxycycline.

  8. For longer live imaging (> 5 min), it may be necessary to refocus on the region of interest as the microscope stage may drift out of focus despite installation of autofocus software.

  9. For myelinating cultures, which require prolonged periods of culture, co-expression of two constructs was best achieved by driving the expression of one construct in the FUGW vector and pSLIK for the other. Co-infection of neurons with two pSLIK constructs adversely affected expression in older cultures, potentially by either impairing the survival of dually infected neurons or dual expression.

Recipes

  1. HEK293 cell medium

    DMEM

    10% FBS

    2 mM L-glutamine

    2 mM NEAA

    1 mM sodium pyruvate

    1× penicillin-streptomycin

  2. NB medium

    Neurobasal medium (prepared with or without phenol red)

    0.4% glucose

    2% B27 supplement

    2 mM L-glutamine

    50 ng/ml 2.5S NGF

  3. NBF medium

    NB medium

    FUDR (final concentration: 10 μM FdU + 10 μM Uridin)

  4. C medium

    MEM (prepared with or withour phenol red)

    0.4% glucose

    10% FBS

    2 mM L-glutamine

    50 ng/ml 2.5S NGF

  5. CF medium

    C medium

    FUDR (final concentration: 10 μM FdU + 10 μM Uridine)

  6. Live imaging medium

    Phenol red-free NB (for neuron-only culture) or C (for co-culture) media

    10 mM HEPES

Acknowledgments

We thank Erik Snapp for advice on the use of fluorescent protein tags for live imaging and Michael Cammer for supporting the spinning disk microscopy live imaging. This work was supported by an NIH grant (Grant Number NS043474) to JLS. The protocol is based on work published in J Cell Biology (Bekku and Salzer, 2020; doi: 10.1083/jcb.201906071).

Competing interests

The authors declare no competing interests.

Ethics

All animal experiments were performed in compliance with the relevant policies and institutional guidelines issued by the New York University School of Medicine Institutional Animal Care and Use Committee.

References

  1. Bekku, Y. and Salzer, J. L. (2020). Independent anterograde transport and retrograde cotransport of domain components of myelinated axons. J Cell Biol 219(6).
  2. Cavalli, V., Kujala, P., Klumperman, J. and Goldstein, L. S. (2005). Sunday Driver links axonal transport to damage signaling. J Cell Biol 168(5): 775-787.
  3. Dzhashiashvili, Y., Zhang, Y., Galinska, J., Lam, I., Grumet, M. and Salzer, J. L. (2007). Nodes of Ranvier and axon initial segments are ankyrin G-dependent domains that assemble by distinct mechanisms. J Cell Biol 177(5): 857-870.
  4. Kaether, C., Skehel, P. and Dotti, C. G. (2000). Axonal membrane proteins are transported in distinct carriers: a two-color video microscopy study in cultured hippocampal neurons. Mol Biol Cell 11(4): 1213-1224.
  5. Katayama, H., Yamamoto, A., Mizushima, N., Yoshimori, T and Miyawaki, A. (2008). GFP-like proteins stability accumulate in lysosome. Cell Struc Funct 33 (1): 1-12.
  6. Kim, H. A. and Maurel, P. (2009). Primary Schwann Cell Cultures. In: Doering, L. (Ed). Protocols for Neural Cell Culture. Springer Protocols Handbooks. Humana Press.
  7. Lois, C., Hong, E. J., Pease, S., Brown, E. J. and Baltimore, D. (2002). Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295(5556): 868-872.
  8. Salzer, J. L., Brophy, P. J. and Peles, E. (2008). Molecular domains of myelinated axons in the peripheral nervous system. Glia 56(14): 1532-1540.
  9. Shin, K. J., Wall, E. A., Zavzavadjian, J. R., Santat, L. A., Liu, J., Hwang, J. I., Rebres, R., Roach, T., Seaman, W., Simon, M. I. and Fraser, I. D. (2006). A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proc Natl Acad Sci U S A 103(37): 13759-13764.
  10. Snapp, E. (2005). Design and use of fluorescent fusion proteins in cell biology. Curr Protoc Cell Biol 21.4.1-21.4.13.
  11. Squinto, S. P., Aldrich, T. H., Lindsay, R. M., Morrissey, D. M., Panayotatos, N., Bianco,. S. M., Furth, M. E. and Yancopoulos, G. D. (1990). Identification of functional receptors for ciliary neurotrophic factor on neuronal cell lines and primary neurons. Neuron 5(6):757-766.
  12. Taveggia, C. and Bolino, A. (2018). DRG Neuron/Schwann Cells Myelinating Cocultures. In: Woodhoo, A. (Ed). Myelin. Methods in Molecular Biology, vol 1791. Humana Press, New York, NY.
  13. Zhang, Y., Bekku, Y., Dzhashiashvili, Y., Armenti, S., Meng, X., Sasaki, Y., Milbrandt, J. and Salzer, J. L. (2012). Assembly and maintenance of nodes of ranvier rely on distinct sources of proteins and targeting mechanisms. Neuron 73(1):92-107.

简介

[摘要]在传入接收,集成神经元的功能,和生成的电活动的依赖于他们的惊人地极化组织,其特征在于由不同的膜结构域。由于选择性靶向和膜蛋白保留的组合,这些结构域具有不同的组成。在神经元中,大多数蛋白质通过顺行囊泡运输从它们在胞体中的合成位点传递到轴突,并进行逆行运输以进行重新分配和/或溶酶体降解。一个关键问题关键在于运往同一结构域的蛋白质是否在单独的囊泡中运输以进行局部组装,或者这些蛋白质是否在相同的囊泡中预先组装并共同运输以输送到其同源结构域。为了评估运输囊泡的含量,一种策略依赖于结扎后坐骨神经的染色,这会分别在结扎的近端和远端推动顺行和逆行运输的囊泡的积累。这种方法可能无法对通过染色鉴定的细胞内囊泡的性质进行可靠的评估,并且分析仅限于合适抗体的可用性。在这里,我们使用的蛋白质双色实时成像标签用不同的荧光标记编,可视化顺行和逆行同时几种蛋白质的轴突运输。这些蛋白质在大鼠背根神经节 (DRG) 神经元中表达,在髓鞘形成条件下单独培养或与雪旺细胞一起培养,以评估神经胶质细胞是否改变了轴突运输的模式。该协议的优点是运输囊泡的动态识别及其对各种蛋白质的含量表征,不受可用抗体的限制。

[背景]神经元是高度极化的细胞。该极性对神经元functi临界上,即,在积分突触前输入体树突隔室以及发起和沿轴突传播动作电位。髓轴突进一步细分为一系列子域,特别是包括郎飞的节点,位于相邻的髓鞘其中动作电位跳跃式传导期间再生之间的间隙。节点高度富含电压门控 Na +通道、它们相关的 β 亚基和几种神经元细胞粘附分子 (CAM) 。在后者中是neurofascin (NF)186 ,其结合于雪旺氏细胞的同源受体小号,神经胶质细胞š在周围神经系统,髓鞘的轴突。节点处的钠离子通道和 CAM 通过与锚蛋白G ( AnkG )的相互作用而稳定下来,锚蛋白G ( AnkG )与β IV血影蛋白形成特殊的亚膜细胞骨架。这些成分一起形成了多聚体节点复合物,该复合物与存在于有髓轴突其他域中的其他多聚体蛋白质复合物显着不同(Salzer等,2008)。这些后面的域包括位于节点两侧的节段,以及位于致密髓鞘下方的近节段和节间域。

一个关键问题是节点和其他域的组件是否从体细胞单独运输到轴突以在本地组装,或者它们是否作为预组装复合物运输到各自的域。运输囊泡梭跨膜蛋白从细胞体的轴突(即。,顺行)将主要含有在前者的情况下的单一蛋白货物,而它们将包含在后者的情况下蛋白的货物的混合物。一个相关的问题是从轴突逆行运输到体细胞的囊泡是否也包含单个或多个货物。

一种评估蛋白质在转运囊泡中的预期共表达的策略依赖于固定结扎神经切片中蛋白质的免疫荧光(Cavalli等,2005)。该方法取决于针对感兴趣蛋白质的合适抗体的可用性。此外,在没有主动运输的情况下,被可视化的囊泡的精确性质可能不明确。

作为一种替代策略,我们动态地主动运输期间使用的多色实时成像成像囊泡,一个强大的工具来表征所述膜蛋白的神经元中传输(Kaether等人,2000)。为了检查节点的组件是否彼此分开运输,以及从其他域的组件运输,我们首先用强力霉素诱导的慢病毒载体感染培养的大鼠背根神经节 (DRG) 神经元,以驱动各种标记蛋白的同时表达。这些慢病毒-感染的神经元生长在聚-L-赖氨酸(PLL)/层粘连蛋白包被的玻璃底培养皿单独或与雪旺氏细胞(SC小号)髓鞘的条件下。然后,我们通过双色实时成像对顺行和逆行运输的囊泡中的这些不同货物进行了成对比较,并评估了囊泡是否包含单个或多个货物。动态成像允许明确识别正在进行主动运输的囊泡。分析多种蛋白质的运输依赖于足够亮度的不同荧光标签,以在较长时间内实时成像,并且不需要相应的抗体。在这里,我们提供一种用于双重的详细协议-跨膜蛋白在培养的大鼠DRG神经元的传输的颜色实时成像。

关键字:轴突输送, 膜泡运输, 双色实时成像, 兰氏结, 神经元极性



材料和试剂


1. 35毫米克小姑娘底菜ES (马蒂克公司,目录号:P35G-1.5-14-C)     

2. 100毫米吨问题培养皿ES (TPP,目录号:93100)     

3.注射器过滤器单元,0.45       μ m (Millipore Sigma,目录号:SLHV033RS)


4.定时怀孕 Sprague Dawley 大鼠(用于 E15-16 DRG 解剖)     

5. 293FT 细胞(Thermo Fisher,目录号:R7007)     

6.改良的pSLIK (Addgene #25737;Shin等人,2006)(储存在 -20 °C)     

7.改良的pFUGW慢病毒载体 ( Addgene #14883; Lois et al ., 2002)(储存在 -20 °C)     

8.的pCMV -VSV-G(礼物从J. Milbrandt ,华盛顿大学)(存储于-20 ℃)     

9.的pCMV- Δ 8.9(礼物从J. Milbrandt ,华盛顿大学)(存储于-20 ℃)     

注意:pSLIK被设计为去除潮霉素抗性和 IRES 序列,以提供额外的空间来容纳各种货物的更大的 cDNA(Zhang 等人,2012)。pFUGW被修改为缺少 GFP 报告基因并添加了一个独特的克隆位点(Dzhashiashivili等,2007)。


10. Gateway LR Clonase II Enzyme Mix(Thermo Fisher,目录号:11791020)(储存在- 80 °C ) 

11.聚-L-赖氨酸(Sigma-Aldrich,目录号:P5899)(- 80 °C储存) 

注:ü sed的用于293FT细胞培养。


12.聚-L-赖氨酸(Sigma-Aldrich,目录号:P1274)(- 80 °C储存) 

注:ü用sed到外套玻璃底菜。


13.天然米乌斯升aminin(Invitrogen公司,目录号:23017-015) (在-80存储℃下) 

14. Dulbecco 磷酸盐缓冲盐水(Lonza,目录号:17-512Q)(室温储存) 

15. LipoD293 DNA转染试剂(Signagen ,目录号:SL100668 )(4 ℃保存) 

16. 0.25% 胰蛋白酶(Thermo Fisher,目录号:15050065)(-20 °C储存) 

17.胎牛血清(Thermo Fisher,目录号:16000-044)(- 80 °C储存) 

18. MEM(Thermo Fisher,目录号:11090-073)(在 4 °C 下储存) 

19. MEM,米INUS p苯酚的制备- [R ED(热费舍尔,目录号:51200-038) (在4存储℃下) 

20. Neurobasal 培养基(Thermo Fisher,目录号:21103-049)(在 4 °C 下储存) 

21. Neurobasal培养基,米INUS p苯酚的制备- [R ED(热费舍尔,目录号:12348-017) (在4存储℃下) 

22. DMEM(Lonza,目录号:12-614F)(在 4 °C 下储存) 

23. MEM Ñ导通Ë ssential一个蓑一个CID小号olution(热费舍尔,目录号:11140050) (在4存储℃下) 

24.钠p yruvate(热费舍尔,目录号:11360070) (在4存储℃下) 

25.葡萄糖(Sigma-Aldrich,目录号:G5146)(在4 °C下储存) 

26. L-谷氨酰胺(Thermo Fisher,目录号:25030-081)(储存在-80 °C ) 

27. B27补充剂(ThermoFisher ,目录号:17504-054)(- 20 °C储存) 

28. 2.5S Ñ ERVE克rowth ˚F演员(ABD Serotec公司,目录号: PMP04Z) (存储于-80 ℃下) 

29.强力霉素(Sigma-Aldrich,目录号:D9891)(储存在-80 °C ) 

30. HEPES(Thermo Fisher,目录号:15630106)(在 4 °C 下储存) 

31.青霉素小号treptomycin(热费舍尔,目录号:15140122) (在-80存储℃下) 

32. V itaminÇ (Sigma-Aldrich公司,目录号:A0278)(在室温下储存) 

33.氟脱氧尿苷(FdU )(Sigma-Aldrich,目录号:F0503)(室温储存) 

34.尿苷(Sigma-Aldrich,目录号:U3003)(室温储存) 

35. HEK293细胞培养基(见ř ecipes) 

36. NB培养基(参见ř ecipes) 

37. Ñ乙˚F介质(见ř ecipes) 

38. Ç介质(见ř ecipes) 

39. Ç F培养基(见ř ecipes) 

40.大号香港专业教育学院IMAG荷兰国际集团介质(见ř ecipes) 



设备


CO 2培养箱(Thermo Fisher,型号:Heracell 240 )
显微镜及要求:
倒置落射荧光显微镜,带有平面 Apo Lambda 100 × /1.45 NA 物镜(尼康,型号:Eclipse Ti -E ),配备电动 Epi- fl旋转过滤器转盘。
CCD 相机(安道尔科技,型号:Clara;尼康,型号:DS-Qi2)
加热插件 P(PECON,目录号:130-800 207)
温度控制37-2 数字(PECON,目录号:0503.000)
GFP过滤器立方体(尼康,目录号:96362)
594 - nm 激光带通设置滤波器立方体(色度,目录号:49911)


软件


NIS-Elements 高级研究软件(尼康,版本:4.30.01 )
我mageJ 手动跟踪(斐济,https: //imagej.net/Manual_Tracking )
E excel(微软,版本:2013)
Prism软件(g ^ raphPad,版本:8)


程序


荧光标记蛋白的产生
保险丝或者EGFP或mKate2(远红外)(小号EE注1,4,5,6 )在C末端或使用独特的限制性酶位点感兴趣的蛋白质的其他合适的位点存在于表达载体中。根据需要通过 PCR 添加限制性酶切位点,或者在亚克隆到pSLIK或 FUGW之前首先转移到具有多个限制性位点的其他载体。如果标签的合适位点在域之间,则通过补丁 PCR添加限制性离子酶位点、接头序列和标签(Squinto等,1990)。
亚克隆第Ë所得融合蛋白的cDNA为doxycycli NE诱导型慢病毒载体,例如,改性pSLIK (Addgene公司使用Gateway LR#25737)克隆酶II或组成型表达改性pFUGW慢病毒载体(Addgene公司#14883使用独特的限制性位点存在的)在这个向量中。用于实时成像的构建体的细节之前已经描述过(Dzhashishivili等,2007;Zhang等,2012;Bekku和Salzer ,2020)。


Lentivir我们生产
外套100 - mm培养皿用5 ml聚-L-赖氨酸(10 μ克/毫升; P5899,号11中第ë试剂列表)的PBS在室温下20分钟; 室温下用 PBS 冲洗 3 次。
用6个菜肴× 10 6 293FT细胞前24小时转染,并允许细胞密度达到〜90%confluenc Ë在转染时。
添加新鲜HEK293细胞培养基(5ml)中30 -在转染前60分钟。
使用 LipoD293 TM体外DNA 转染试剂,用 5 μg pSLIK或 FUGW 慢病毒构建体以及辅助质粒Δ 8.9 (6.25 μg ) 和VSVg (3.3 μg )转染 293FT 细胞。
取代的用7ml DNA / LipoD293复杂含有培养基HEK293细胞转染之后的培养基5小时。
从培养物中48小时收集培养基的转染和转移后,以50 -毫升离心管中。离心机在1700 15分钟×在室温克和过滤的通过0.45上清- μ微米的过滤器单元。过滤的上清液等分为 0.6 - ml 样品并储存在 -80°C直至使用。


P赔偿分离的大鼠DRG神经元培养物的髓鞘和共培养物用于LIV ë成像
Ç燕麦在各底部的玻璃盖玻片马蒂克培养皿(P1274,用号0.5mg / ml的PLL 12在试剂列表)在300 μ升PBS。37 °C孵育30 分钟,然后用 PBS 洗涤 3 次。
接着,涂覆的玻璃盖玻片上,用10 μ克/ ml的层粘连蛋白在200 μ升PBS。在 37 °C 下孵育30 分钟。
根据先前发布的协议 ( Taveggia和Bolino , 2018),准备分离的 E15-E16 大鼠 DRG 神经元培养物作为仅神经元培养物或髓鞘共培养物生长。简而言之,从大鼠胚胎中取出 DRG,用 1.5 ml 胰蛋白酶在 37 °C下孵育45 分钟,加入 1 ml C 培养基,通过重复移液分离神经节,并在室温下以 193 × g离心细胞5分钟
重悬的在〜200通过温和研磨粒料μ升CF介质含有p enicillin-小号treptomycin每DRG(PS)。种子解离的大鼠 DRG 神经元,相当于 1 个神经节(约 2.1 × 10 4 个细胞),放在Matek PLL/层粘连蛋白涂层盖玻片上并孵育 24 小时。
每隔一天在 NBF 和 NB 培养基上循环培养共 12 天,以耗尽所有非神经元细胞,只留下神经元培养物。
要建立髓鞘共培养,将 3 × 10 5出生后大鼠 SCs 添加到仅神经元培养物中。SCs 是从坐骨神经制备的,可以将单独的等分试样储存在液体 N 2 中,如前所述(Kim 和Maurel ,2009)。为了启动髓鞘形成,5 天后,将维生素 C 添加到 C 培养基中,最终浓度为 50 μg /ml;这种维生素 C 浓度不会影响培养基的 pH 值。髓鞘形成通常在添加维生素 C 几天后发生。


在仅神经元和髓鞘共培养物中双重病毒感染和荧光标记蛋白的诱导
在含有PS 的C 培养基中稀释第一个病毒上清液。仅适用于神经元的文化,加入1:1(病毒:C为介质)稀释mKate2标记pSLIK病毒一个-天-老DRG神经元的文化。对于髓鞘共培养,加入1:1(病毒:C为介质)稀释EGFP标记pSLIK病毒一个-天-老神经元的文化。用病毒上清液孵育培养物 24 小时。
Ť他第二天,稀释用C介质中的第二病毒上清含有PS。对于仅神经元培养,将病毒培养基更改为新鲜稀释的 EGFP -标记的pSLIK病毒(1:1 稀释)。对于共培养,更换的新的稀释mKate2病毒介质-标记FUGW病毒:在(稀释病毒的1:15 C培养基)的神经元培养物(小号EE注9 )。孵育 24 小时。
除去的病毒培养基,加入NBF,并培养所述另外的1感染的神经元- 2天直到表达被诱导。如果需要更长的孵育时间以允许神经元进一步生长,请将培养基改为 NB再延长2 天。
根据添加强力霉素当天的培养基循环,将 2 µg/ml 强力霉素(最终浓度)添加到仅神经元培养物中的 NB 或 NBF 中,或加入共培养物中的 C 加维生素 C 以诱导蛋白质表达 24 - 48 小时之前实时成像(S ^ EE注8 )。
注:概略时间过程š实验对神经元培养物仅和髓鞘培养物(程序C和d)是图1中所示。




图1.时间过程的双-彩色实时成像


现场IMAG ING
1.在实时成像之前立即将介质更改为实时成像介质。     

2. P花边培养成长荷兰国际集团上的马蒂克菜到37 ℃下加热显微镜载物台(热插入P)连接到温度控制单元(Tempcontrol 37-2) 。     

3.在实时成像之前或开始时,确定每个囊泡相对于神经元体的位置的轴突运输方向。     

4.使用 CCD 相机和 100 ×物镜(见注释 2、3、8 )使用 NIS-Elements 高级研究软件(尼康)的完美对焦设置以 2 秒的间隔捕获图像,持续时间≥2分钟,以校正焦点漂移。使用NIS-Elements 高级研究软件的实时解卷积处理图像。EGFP -用 GFP 过滤器立方体 (96362, Nikon) 捕获标记的蛋白质;mKate2 的那些是用 594 - nm 激光带通设置过滤器(49911,色度)立方体捕获的。请参阅图 2 中双色实时成像的代表性静止图像。     

5.拍摄 20 多个神经元的图像进行分析。焦点的损失由于显微镜载物台漂移可能需要重新聚焦和可能也减少可用于生成kymographs图像号。     





图 2.囊泡协同运输和独立运输的代表性静止图像。上部面板小号:含有囊泡NF186-mKate2和NRCAM -EGFP是顺行共同输运轴突如eviden土木工程署通过从mKate2标记NF186和EGFP标记的重叠黄色NRCAM在相同的囊泡; 黄色箭头突出显示了共同运输的囊泡s 的一个例子。下部面板小号:含有囊泡NF186-mKate2和的Na v β 1 -EGFP在沿轴突逆行方向分开运输; 囊泡的实例含有荷兰国际集团的Na v β 1-EGFP仅由白色箭头突出显示。


数据分析


1.通过实时成像 15 - μm 轴突段 2 分钟构建 kymographs 。构建 kymographs 的一种选择是使用 NIS-Elements 高级研究软件,该软件显示像素强度随时间沿定义的线性部分的变化(图 3)。ImageJ或其他显微镜分析软件是构建 kymographs的另一种选择。的提取物轨迹个体从囊泡的kymographs并手动在每个方向上计数囊泡号码。甲nalyz Ë ≥ 10个神经元转染蛋白的每个组合和比较所述的速率协同转运每种蛋白质。对于统计分析,分析≥ 100 个囊泡(≥ 10 个神经元);样本大小可以根据变化的实验设计。样本大小可以通过软件计算,例如,G power。     

2.确认定量结果使用ImageJ手动跟踪,特别是对于一些顺行囊泡其可以是有限的亮度。     

3.使用 ImageJ 手动跟踪测量囊泡的平均速度。     

4.比较了通过平均协同转运率的从每个被拍轴突结果。     

5.评估与任双尾韦尔奇统计学意义牛逼-测试或双尾学生牛逼-使用非成对数据检验的Excel或的GraphPad Prism软件。     





图 3.共同转运囊泡的代表性 kymograph 。这ķ ymograph从表达NF186-EGFP和NF186-mKate2由神经元的实时成像产生的NIS-元素的高级研究软件。囊泡的轨迹由蓝色圆圈突出显示,按时间顺序编号。在 kymograph 中,可以检测到两个逆行共运输的囊泡。另外的代表性实时图像和电影,kymographs ,在设置和数据分析细胞与生物学(Bekku餐厅和萨尔泽,2020)。


笔记


1.该协议描述了双色成像,但可适用于三个或更多荧光团,具体取决于显微镜区分用作标签的不同荧光团的能力。     

2.由单一活体成像的限制-激光荧光显微镜是滤波器之间切换以监视不同的荧光团时产生的时间延迟。在此滞后期间,如果囊泡被主动运输,囊泡将继续移动并表现出划分同一囊泡的不同荧光团的人工空间分离。     

3.带有两个摄像头的旋转圆盘显微镜是同时实时成像多色囊泡的绝佳替代方案,没有时滞,因为没有过滤器切换。在我们的研究中,我们还使用了转盘显微镜来确认结果。来讲图像质量,落射荧光显微镜需要清晰图像小号个别囊泡。     

4.在我们的研究中,蛋白质在其 C 端用 EGFP 或 mKate2 标记,并通过成对比较分析它们的运输。不像mKate2,都进行了尝试,其他红色荧光标记,即mRFP的,mCherry的,和红色荧光蛋白,引起一定程度的蛋白质聚集是受影响贩运的是与片山协议等。, 2008.     

5.确认添加荧光蛋白标签不会影响蛋白质运输是很重要的。例如,用 EGFP 标记的蛋白质和用 mKate2 标记的相同蛋白质的双重表达表明,这些蛋白质在神经元的细胞内运输过程中表现出完全重叠(Bekku和Salzer ,2020)。     

6.同样重要的是,以显示其在加入荧光标记的不与所述标记蛋白的靶向到其正常质膜目的地(干扰例如,到节点,突触,等等)。有关如何设计融合蛋白的说明,请参见(Snapp, 2005)。     

7.若要在神经元中同时表达两种蛋白,标记的蛋白亚克隆到诱导型病毒构建pSLIK ,并且它们的表达瓦特作为同时通过多西环素诱导的。     

8.对于更长的实时成像(> 5 分钟),可能需要重新聚焦感兴趣的区域,因为尽管安装了自动对焦软件,显微镜载物台仍可能会偏离焦点。     

9.对于需要长时间培养的髓鞘形成培养物,通过驱动一个构建体在 FUGW 载体中的表达和pSLIK驱动另一个构建体的表达,可以最好地实现两种构建体的共表达。神经元与两个pSLIK构建体的共同感染对旧培养物中的表达产生不利影响,可能通过损害双重感染神经元的存活或双重表达。     



食谱


HEK293细胞培养基
d MEM


10% 胎牛血清


2 mM L-谷氨酰胺


2 毫米 NEAA


1 mM 丙酮酸钠


1 × p enicillin-小号treptomycin


NB培养基
Neurobasal 培养基(用或不用酚红制备)


0.4%克葡萄糖


2% B27 补充剂


2 mM L-谷氨酰胺


50 纳克/毫升 2.5S NGF


NBF培养基
NB培养基


FUDR(最终浓度:10 μ M FdU + 10 μ M Uridin )


中号
MEM(用或不用酚红制备)


0.4%克葡萄糖


10% 胎牛血清


2 mM L-谷氨酰胺


50 纳克/毫升 2.5S NGF


CF培养基
中号


FUDR(最终浓度:10 μ M FdU + 10 μ M尿苷)


实时成像介质
酚红-无 NB(仅用于神经元培养)或 C(用于共培养)培养基


10 毫米 HEPES


致谢


我们感谢 Erik Snapp 关于使用荧光蛋白标签进行实时成像的建议,感谢 Michael Cammer支持旋转盘显微镜实时成像。这项工作得到了JLS的NIH 赠款(赠款编号 NS043474)的支持。该协议基于J Cell Biology上发表的工作(Bekku和Salzer ,2020 年;doi :10.1083/jcb.201906071 )。


利益争夺


作者声明没有竞争利益。


伦理


所有动物实验均按照纽约大学医学院动物护理和使用委员会发布的相关政策和机构指南进行。


参考


Bekku , Y. 和Salzer , JL (2020)。有髓轴突域成分的独立顺行运输和逆行协同运输。细胞生物学杂志219(6)。
Cavalli, V.、Kujala , P.、Klumperman , J. 和 Goldstein, LS (2005)。Sunday Driver 将轴突运输与损伤信号联系起来。J Cell Biol 168(5): 775-787。
Dzhashiashvili , Y.、Zhang, Y.、Galinska , J.、Lam, I.、Grumet , M. 和Salzer , JL (2007)。Ranvier 节点和轴突初始段是通过不同机制组装的锚蛋白 G 依赖性域。J Cell Biol 177(5): 857-870。
Kaether , C., Skehel , P. 和 Dotti, CG (2000)。轴突膜蛋白在不同的载体中运输:在培养的海马神经元中进行的双色视频显微镜研究。分子生物学细胞11(4): 1213-1224。
Katayama, H.、Yamamoto, A.、Mizushima, N.、Yoshimori , T 和Miyawaki , A. (2008)。GFP 样蛋白稳定性在溶酶体中积累。细胞STRUC功能该33(1):1-12。
Kim, HA 和Maurel , P. (2009)。原代施万细胞培养物。在:Doering ,L.(E d)。神经细胞培养方案。Springer 协议手册。人类出版社。
Lois, C., Hong, EJ, Pease, S., Brown, EJ 和 Baltimore, D. (2002)。慢病毒载体传递的转基因的种系传递和组织特异性表达。科学295(5556):868-872。
Salzer , JL, Brophy, PJ 和Peles , E. (2008)。周围神经系统中髓鞘轴突的分子结构域。神经胶质56(14):1532-1540。
Shin, KJ, Wall, EA, Zavzavadjian , JR, Santat , LA, Liu, J., Hwang, JI, Rebres , R., Roach, T., Seaman, W., Simon, MI 和 Fraser, ID (2006) . 用于基于 microRNA 的条件性 RNA 干扰和协调转基因表达的单一慢病毒载体平台。Proc Natl Acad Sci USA 103(37): 13759-13764。
Snapp, E. (2005)。荧光融合蛋白在细胞生物学中的设计和使用。CURR Protoc细胞生物学21.4.1-21.4.13 。
斯奎托,S 。P 。,奥尔德里奇,T 。H . ,林赛,R 。米。,莫里西,D 。米。,Panayotatos ,N 。,比安科,。小号。米。,弗斯,M 。乙。和扬科普洛斯,G 。d 。(1990) 。神经元细胞系和原代神经元上睫状神经营养因子的功能受体的鉴定。神经元5 (6) :757-766。
Taveggia, C. 和 Bolino, A. (2018)。DRG 神经元/施万细胞髓鞘共培养。在:伍德胡,A.(E d)。髓磷脂。分子生物学方法,第 1791 卷。Humana 出版社,纽约,纽约。
Zhang, Y., Bekku, Y., Dzhashiashvili, Y., Armenti, S., Meng, X., Sasaki, Y., Milbrandt, J. 和 Salzer, JL (2012)。ranvier 节点的组装和维护依赖于不同的蛋白质来源和靶向机制。神经元73(1):92-107。
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引用:Bekku, Y. and Salzer, J. L. (2021). Dual Color, Live Imaging of Vesicular Transport in Axons of Cultured Sensory Neurons. Bio-protocol 11(12): e4067. DOI: 10.21769/BioProtoc.4067.
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