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Sep 2018

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Lipid Mixing Assay for Murine Myoblast Fusion and Other Slow Cell-cell Fusion Processes
小鼠成肌细胞融合及其他慢细胞融合过程的脂质混合实验   

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Abstract

Lipid mixing (redistribution of lipid probes between fusing membranes) has been widely used to study early stages of relatively fast viral and intracellular fusion processes that take seconds to minutes. Lipid mixing assays are especially important for identification of hemifusion intermediates operationally defined as lipid mixing without content mixing. Due to unsynchronized character and the slow rate of the differentiation processes that prime the cells for cell-cell fusion processes in myogenesis, osteoclastogenesis and placentogenesis, these fusions take days. Application of lipid mixing assays to detect early fusion intermediates in these very slow fusion processes must consider the continuous turnover of plasma membrane components and potential fusion-unrelated exchange of the lipid probes between the membranes. Here we describe the application of lipid mixing assay in our work on myoblast fusion stage in development and regeneration of skeletal muscle cells. Our approach utilizes conventional in vitro model of myogenic differentiation and fusion based on murine C2C12 cells. When we observe the appearance of first multinucleated cells, we lift the cells and label them with either fluorescent lipid DiI as a membrane probe or CellTrackerTM Green as a content probe. Redistribution of the probes between the cells is scored by fluorescence microscopy. Hemifused cells are identified as mononucleated cells labeled with both content- and membrane probes. The interpretation must be supported by a system of negative controls with fusion-incompetent cells to account for and minimize contributions of fusion-unrelated exchange of the lipid probes. This approach with minor modifications has been used for investigating fusion of primary murine myoblasts, osteoclast precursors and fusion mediated by a gamete fusogen HAP2, and likely can be adopted for other slow cell-cell fusion processes.

Keywords: Membrane fusion (膜融合), Hemifusion (半融合), Myoblast fusion (成肌细胞融合), Lipid mixing (脂质混合), Syncytium formation (合胞体形成), Myomaker (Myomaker), Myomerger (Myomerger)

Background

Fusion of membrane lipid bilayers in cell biological processes as diverse as fusion of intracellular membranes in exocytosis, and fusion of viral and cell membranes in enveloped virus infection, and myoblast fusion in skeletal muscle development apparently involves similar lipid rearrangements (Figure 1) (Brukman et al., 2019). First, a merger of the apposing, contacting monolayers of two fusing bilayers generates a hemifusion connection and allows redistribution of lipid probes between these monolayers. A subsequent merger of the distal monolayers generates a fusion pore and allows redistribution of aqueous probes between fusing membrane compartments.


Figure 1. Schematic representation of the lipid rearrangements during hemifusion and fusion pore formation (modified from Figure 1 in Brukman et al., 2019).

Hemifusion can either transition into a fusion pore or represent a dead end of the fusion reaction aborted before fusion pore formation. In the latter case, hemifusion connections can then dissociate yielding two distinct bilayers. In their turn, nascent fusion pores can either close or expand advancing fusion towards its completion with full unification of the membrane compartments. The rates of formation and dissociation of the key fusion intermediates, hemifusion and fusion pores, and the rates of the transition between these intermediates vary between different fusion processes and are determined by the activity of the proteins involved and lipid compositions of the fusing membranes.

In most of the experimental studies, pores large enough to pass content probes in the range from ~1 kDa to ~100 kDa are detected by fluorescence microscopy. Hemifusion, operationally defined as lipid mixing without content mixing, is detected using fluorescence microscopy or spectrofluorometry. Different modifications of lipid mixing assays have been developed in studies on fusion of protein-free lipid bilayers, and fusion mediated by viral fusogens and intracellular fusogens (Brukman et al., 2019). The successful application of the lipid mixing assay in all these systems has been facilitated by a relative rapidness of these fusion processes with characteristic times of lipid mixing varying from seconds to minutes.

Application of lipid mixing assays to developmental cell-cell fusion, the subject of this work, is critically important for clarification of the pathways of the membrane rearrangements but more challenging than for faster viral and intracellular fusion reactions. For instance, formation of multinucleated myotubes, one of the best characterized examples of cell-cell fusion processes (Sampath et al., 2018), is preceded by myogenic differentiation that prepares the cells for fusion and takes days. As a result, by the time fusion is evaluated, fluorescent lipid probes added before fusion to label plasma membranes are already partially internalized. Moreover, labeling of intracellular membrane compartments containing internalized probes may appear brighter than labeling of the plasma membrane because of a higher membrane content and contrast. An additional concern in developing lipid mixing assays for very slow fusion processes is related to the ability of lipid probes to be transferred from membrane to membrane by lipid-exchanging proteins, lipid micelles or extracellular vesicles (reviewed in Merklinger et al., 2016), i.e., by mechanisms that neither involve hemifusion or fusion nor depend on the fusion machinery.

Here we present a protocol used to assay lipid mixing in fusion between C2C12 cells, immortalized mouse myoblasts that proliferate in high-serum medium and differentiate and fuse in low-serum medium. We also discuss applications of the modified versions of this protocol to other slow cell-cell fusion processes.

Materials and Reagents

Materials

  1. Falcon® 15 ml Polystyrene Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap (Corning, catalog number: 352095 )
  2. Tissue culture dishes, 35 x 10 mm REF (Corning, catalog number: 353001 )
  3. Tissue culture dishes, 60 x 15 mm (Corning, catalog number: 353002 )

Cells
  1. C2C12 cells (ATCC, catalog number: CRL-1772TM)
  2. Myomaker-deficient C2C12 cells and Myomerger-deficient C2C12 cells were generated in (Millay et al., 2016; Quinn et al., 2017) and grown on collagen coated substrates

Reagents
  1. VybrantTM DiI Cell-Labeling Solution (Thermo Fisher Scientific, catalog number: V22885 )
  2. CellTrackerTM Green CMFDA (5-chloromethylfluorescein diacetate) (Thermo Fisher Scientific catalog number: C7025 )
  3. Orange CMRA CellTrackerTM (Thermo Fisher Scientific, catalog number: C34551 )
  4. Trypsin-EDTA 0.05% (Thermo Fisher, catalog number: 25300054 )
  5. Hoechst 33342 Trihydrochloride, Trihydrate- 10 mg/ml Solution in Water (Thermo Fisher Scientific, catalog number: H3570 )
  6. Formalin 10% Buffered in Phosphate (Electron microscopy Sciences, catalog number: 15740 )
  7. Collagen from calf skin (Sigma, catalog number: C8919-20 ml )
  8. Dimethyl sulfoxide (DMSO, Sigma, catalog number: D2650-100 ml )

Media
  1. The proliferation medium (PM): DMEM, high glucose, GlutaMAXTM Supplement (Thermo Fisher, catalog number: 10566-016 ) + 10% Fetal Bovine Serum, Penicillin/Streptomycin (Thermo Fisher, catalog number: 10378016 )
  2. The differentiation medium (DM): DMEM, high glucose, GlutaMAXTM Supplement (Thermo Fisher, catalog number: 10566-016 ) + 5% Horse Fetal Serum, Penicillin/Streptomycin
  3. Fetal Bovine Serum, FBS (GIBCO Life Technologies, catalog number: 10437-028 )
  4. Horse Serum, heat inactivated, New Zealand origin (Thermo Fisher, catalog number: 26050088 )
  5. PBS, Corning® Dulbecco’s Phosphate-Buffered Saline (Life Sciences, catalog number: 21-030-CV ), 1x with calcium and magnesium

Equipment

  1. Zeiss Axioscope microscope
  2. Camera (Manufacturer pco-tech inc.; pco.edge 3.1 sCMOS)
  3. F-LD 32/0.4 Zeiss objective lens
  4. Single fluorophore bandpass filter for green cell tracker: excitation 472 nm/30 nm, emission 520 nm/35 nm, dichroic 495 nm LP from Semrock
  5. Single fluorophore bandpass filter for DiI: excitation 545 nm/25 nm, emission 605 nm/70 nm, dichroic 570 LP from Zeiss

Software

  1. Micro-Manager software (Edelstein et al., 2014)
  2. The open-source platform, ImageJ (National Institute of Health, Rockville Pike, Bethesda, MD

Procedure

Figure 2 gives a schematic presentation of the timeline for preparing the cells for lipid mixing experiments.


Figure 2. Schematic presentation of the timeline for preparing and labeling the cells for lipid mixing experiments. The labeling of the cells is started 48 h post differentiation (i.e., 48 h after placing the cells into DM). Differently labeled cells are mixed 1 h later at 49 h post differentiation.


  1. Collagen-coating dishes
    Coat the dishes (60 x 15 mm) (two dishes for each condition) and 35 x 10 mm dishes (5 dishes for each condition) with collagen by covering the dishes with 3 ml or 1.5 ml of 1:10 solution of collagen in sterile water for 60 x 15 mm dish and 35 x 10 mm dish, respectively, and, after overnight incubation at room temperature and two washes (3 ml and 2 ml each for large and small dishes, respectively) with water, dry the dishes in the biosafety cabinet.

  2. Cells
    Grow C2C12 cells in the proliferation medium (PM) in the collagen-coated dishes to 75% confluency. Transfer the cells into the differentiation medium (DM) (t = 0). By 48 h post differentiation (t = 48 h), the cells spread to ~90% confluency and the first myotubes are observed. At this time, wash the cells in each of the two dishes with 2 ml PBS and place the cells in 2 ml fresh PBS.

  3. Cell labeling
    Label the cells in dish 1 with CellTrackerTM Green and cells in dish 2 either with Orange CMRA CellTracker or with DiI. To label with CellTrackerTM Green; add 4 μl of the CellTrackerTM Green stock solution (50 μg of the probe in 20 μl of DMSO) to 2 ml of PBS and incubate the cells in this medium for 15 min at 37 °C. Then place the cells into complete DM for 30 min at 37 °C. Use the same procedure for labeling with Orange CMRA CellTracker. To label cells with DiI, inject 4 μl of 1 mM DiI stock solution into 2 ml of PBS at 37 °C and incubate the cells for 45 min. Wash the cells in the dishes 1 and 2 three times with 2 ml of DM and two times with 2 ml of PBS.

  4. Co-plating differently labeled cells
    1. Gently lift the cells in dishes 1 and 2 with 1 ml of trypsin-EDTA 0.05% applied for 1 min at 37 °C. During the subsequent 2-3 min incubation already at the room temperature, check whether the cells started to round up and lift. When they do, remove EDTA-trypsin and lift the cells into 4 ml of complete DM at 37 °C. Collect the cells from dishes 1 and 2 into the same 15 ml tube and mix the cells by vortexing. Adjust the volume to 10 ml. Then plate the cells (2 ml of cell suspension) onto each of five collagen-treated small dishes (35 x 10 mm). Two hours later, remove debris and non-attached cells by changing the medium for the fresh DM.
    2. Use coplating of the cells labeled with different CellTrackers to analyze content mixing between the cells. In the absence of content mixing, use coplating of the cells labeled with CellTrackerTM Green and cells labeled with DiI to analyze lipid mixing.
    3. At t = 72 h, fix the cells with 10% Formalin in phosphate buffer for 10 min at room temperature. Stain the nuclei by replacing Formalin with PBS supplemented with Hoechst (10 mg/ml stock diluted 1,000-fold) applied for 30 min at room temperature. Replace Hoechst-supplemented PBS with PBS. Take the images on a fluorescence microscope using appropriate excitation and emission filters.

Data analysis

  1. We analyze the images in ImageJ. First, using plates with only green and only red cells we verified that bleed-through between green and red channels is negligible at the used settings. In the images from the experiments with co-plated CellTrackerTM Green -labeled cells and Orange CMRA CellTracker -labeled cells, the cells that aborted fusion at the fusion pore opening stage would be detected as mononucleated cells co-labeled with both probes. So far, we have observed this phenotype only for fusions mediated by viral fusogens.
  2. In the images from the experiments with co-plated DiI-labeled cells and CellTrackerTM Green -labeled cells, the cells that aborted fusion at the hemifusion stage are seen and scored as mononucleated cells co-labeled with both probes. Figures 3, 4 and 5 show representative fluorescence microscopy images that we used to analyze the redistribution of lipid probe (DiI) and content probe (CellTrackerTM Green) in Myomerger−/− (Figure 3); Myomaker−/− (Figure 4) and WT (Figure 5) C2C12 cells.


    Figure 3. Representative fluorescence microscopy images of Myomerger-deficient C2C12 cells used to evaluate the efficiency of lipid mixing by analysis of redistribution of lipid probe (Dil) and content probe (CellTrackerTM Green). Images of the cells after co-incubation of Dil-labeled cells with CellTrackerTM green-labeled cells in the differentiation medium. Nuclei are stained by Hoechst. Arrows indicate examples of several distinct phenotypes counted or not counted as mononucleated cells co-labeled with Dil and CellTrackerTM Green. A and B mark colabeled cells with stronger (A) and weaker (B) levels of Dil fluorescence. Cells like the ones marked as A and B were counted as co-labeled mononucleated cells. Cells labeled with only CellTrackerTM Green (C), or only Dil (D), or green cells with just one red point (E) or cells with more than one nucleus (F) were not counted as co-labeled mononucleated cells. Scale bar = 50 μm.


    Figure 4. Representative fluorescence microscopy images of Myomaker-deficient C2C12 cells used to evaluate the efficiency of lipid mixing by analysis of redistribution of lipid probe (Dil) and content probe (CellTrackerTM Green). Images of the cells after co-incubation of Dil-labeled cells with CellTrackerTM Green-labeled cells in the differentiation medium. Nuclei are stained by Hoechst. Arrow indicates an example of mononucleated cell co-labeled with Dil and CellTrackerTM Green. Scale bar = 50 μm.


    Figure 5. Representative fluorescence microscopy images of wild type (WT) C2C12 cells used to evaluate the efficiency of lipid mixing by analysis of redistribution of lipid probe (Dil) and content probe (CellTrackerTM Green). Images of the cells after co-incubation of Dil-labeled cells with Cell TrackerTM Green-labeled cells in the differentiation medium. Nuclei are stained by Hoechst. Arrow indicates an example of mononucleated cell co-labeled with Dil and CellTrackerTM Green. Arrowhead marks an example of a co-labeled syncytium. Scale bar = 50 μm.

  3. To identify DiI labeled mononucleated CellTrackerTM Green labeled cells, we use the following procedure. First, we find the median of maximal DiI fluorescence levels Fmax in ~100 DiI-labeled cells that have no green fluorescence. We use this value to set the lower limit of fluorescence for detection of DiI redistribution using the following formula:

    Fmin=background+k(Fmax-background)

    We found that k = 0.1 works well to exclude cells that acquired DiI fluorescence by fusion-unrelated mechanisms. To count the co-labeled cells, we adjust image contrast by setting in Brightness/Contrast tool minimum to Fmin and select the maximum to reveal the faintest distinct puncta. After that, we count as co-labeled all green cells that have at least 3 distinct perinuclearly located DiI puncta. (1-2 bright red puncta that we occasionally find associated with cell-surface likely represent debris or extracellular vesicles and the corresponding cells are not scored as co-labeled.)
  4. To quantify the efficiency of hemifusion for different conditions we normalize the number of mononucleated double-labeled cells in the field of view by the total number of mononucleated cells in the field. For each condition, we score at least 10 randomly selected fields of view. The characteristic results for Myomerger-deficient cells, Myomaker-deficient cells and wild type (WT) cells are shown in Figure 6.


    Figure 6. Quantification of hemifusion by lipid mixing assay. Hemifusion was operationally defined as Dil redistribution in the absence of Cell TrackerTM Green and detected as formation of mononucleated cells labeled with both Dil and CellTrackerTM Green. Data presentation and statistical analyses: box-and-whisker plots show median (center line), 25th-75th percentiles (box) and minimum and maximum values (whiskers); statistical significance was evaluated with Mann-Whitney test.

  5. Syncytia are seen as bright double-labeled or single-labeled multinucleated (n ≥ 2) cells generated by fusion involving differently-labeled and similarly-labeled cells, respectively. Hemifusion efficiency, i.e., the probability that under given conditions the cells will hemifuse but will not proceed to fusion completion is quantified by normalizing the numbers of hemifusion events in the field of view (mononucleated double-labeled cells to the total number of nuclei in this field). Assuming that each complete fusion event proceeds through hemifusion intermediates, the efficiency of local membrane merger events yielding either hemi- or complete fusion can be quantified as (Nhf + Ncf)/Nt, where Nhf is the number of hemifusion events, Ncf, the number of complete fusion events (estimated as the number of nuclei in the cells with at least 2 nuclei) and Nt, the total number of the nuclei in this field.

Notes

Modifications of the assay for different cell-cell fusion processes

  1. Lipid mixing assay has been successfully adapted for fusion of primary murine myoblasts (Gamage et al., 2017; Leikina et al., 2013 and 2018), HAP2-mediated fusion of BHK cells (Valansi et al., 2017) and osteoclast precursors (Verma et al., 2018). The required modifications mostly reflected the differences in characteristic rates of the cell fusion processes necessitating adjustment of the relative timing of labeling stages. For instance, primary murine myoblasts that differentiate and fuse faster than C2C12 cells, were labeled in PM rather than in DM (Leikina et al., 2013). Then differently labeled cells were co-plated in DM to start myogenic differentiation and fusion.
  2. In Valansi et al., 2017 and Leikina et al., 2018, we used a modification of the lipid mixing assay, in which we co-plated cells labeled with both lipid and content probe with unlabeled cells, instead of co-plating cells labeled with membrane probe and cells labeled with content probe. In this experimental design, cell fusion stalled at the hemifusion stage produces cells labeled with membrane, but not content probe. Application of this assay requires control experiments with cells labeled with both membrane and content probes cultured in the absence of unlabeled cells to verify that cells labeled with only membrane probe are generated by interactions between labeled and unlabeled cells but not by content probe leakage.

Concerns and controls
  1. An important concern in developing lipid mixing assays for very slow fusion processes is related to the ability of lipid probes to be transferred from membrane to membrane by lipid-exchanging proteins, lipid micelles or extracellular vesicles (reviewed in Merklinger et al., 2016), i.e., by mechanisms that do not depend on the cell fusion machinery. DiI, lipid probe used in our assay, has been developed for long-term labeling and, after careful optimization, DiI and similar lipid probes have been used for tracking individual cells in tissues for several days and even weeks (reviewed in Progatzky et al., 2013). Still, application of DiI-based lipid mixing assay for slow fusion processes requires a set of controls to assure that redistribution of lipid probes is due to membrane merger rather than to the fusion-independent transfer of the probes. If, under some conditions, fusion-independent redistribution of lipid probes dominates fusion-dependent redistribution, the conditions should be altered to reduce fusion-unrelated components of the lipid probe transfer between the membranes. To optimize the experimental procedure, we use cells lacking functional fusion machinery (proliferating satellite cells, differentiating myomaker-deficient myoblasts in DM and differentiating myoblasts in the presence of hemifusion inhibitor lysophosphatidylcholine [Leikina et al., 2013]). The main factors contributing to the fusion-independent lipid mixing are the details of cell labeling protocol (probe concentration and incubation time), number of washes, cell viability (to minimize cell debris), and fusion efficiency of the cells (to increase the relative contribution of the membrane-merger-dependent lipid mixing). In addition, too high confluency of cell monolayer can lead to undetected overlaps between green cells and red cells in fluorescence microscopy images.
  2. Myomaker-deficient C2C12 cells and Myomerger-deficient C2C12 cells provide convenient controls for the lipid mixing assay. As seen in Figure 6, Myomerger-deficient cells demonstrate much higher levels of lipid mixing than Myomaker-deficient cells. Importantly, we show that lipid probe transfer for Myomerger-deficient cells depends on the contacts between differently labeled cells rather than on the transfer through the medium (for instance, by extracellular vesicles), by incubating unlabeled differentiating cells in the conditioned medium from DiI- labeled differentiating cells (Figure 7).


    Figure 7. Representative fluorescence microscopy image that illustrates cell-cell fusion independent acquisition of Dil by unlabeled cells incubated with conditioned medium from Dil-labeled cells. The conditioned medium from Myomerger-deficient cells committed to differentiation and labeled with Dil as described above was collected at t = 72 h post differentiation. In another dish, unlabeled differentiating Myomerger-deficient cells were placed into this conditioned medium at t = 24 h. At t = 72 h, these cells were fixed and analyzed. Scale bar = 50 μm.

    As expected, in these experiments we find only few cells that acquired lipid probe and no cells containing more than 3 distinct perinuclearly located DiI puncta. In this experimental design, finding significant numbers of DiI-labeled cells would suggest problems with DiI labeling.

  3. Identification of hemifusion phenotype in lipid mixing assay can be further validated by independent experimental approaches that do not utilize lipid mixing assays. These two approaches (short-term application of hypotonic shock or chlorpromazine) reveal hemifusion intermediates by converting them into easy-to-detect complete fusion (Melikyan et al., 1997; Chernomordik et al., 1998). Hypotonic osmotic shock generates membrane tension that breaks hemifusion structures, and chlorpromazine preferentially partitions to inner monolayers of plasma membranes and destabilizes hemifusion intermediate formed by these monolayers. Neither of the treatments induces complete fusion if applied to tightly bound rather than hemifused cells.

Acknowledgments

The research in L.V.C.’s laboratory was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, and by Grant Number 2013151 from the United States-Israel Binational Science Foundation (BSF). The protocol described here has been developed in Leikina et al., 2013 and 2018.

Competing interests

The authors declare no competing financial or non-financial interests.

References

  1. Brukman, N. G., Uygur, B., Podbilewicz, B. and Chernomordik, L. V. (2019). How cells fuse. J Cell Biol 218(5): 1436-1451. 
  2. Chernomordik, L. V., Frolov, V. A., Leikina, E., Bronk, P. and Zimmerberg, J. (1998). The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation. J Cell Biol 140(6): 1369-1382.
  3. Edelstein, A. D., Tsuchida, M. A., Amodaj, N., Pinkard, H., Vale, R. D. and Stuurman, N. (2014). Advanced methods of microscope control using muManager software. J Biol Methods 1(2).
  4. Gamage, D. G., Leikina, E., Quinn, M. E., Ratinov, A., Chernomordik, L. V. and Millay, D. P. (2017). Insights into the localization and function of myomaker during myoblast fusion. J Biol Chem 292(42): 17272-17289.
  5. Leikina, E., Gamage, D. G., Prasad, V., Goykhberg, J., Crowe, M., Diao, J., Kozlov, M. M., Chernomordik, L. V. and Millay, D. P. (2018). Myomaker and myomerger work independently to control distinct steps of membrane remodeling during myoblast fusion. Dev Cell 46(6): 767-780 e767.
  6. Leikina, E., Melikov, K., Sanyal, S., Verma, S. K., Eun, B., Gebert, C., Pfeifer, K., Lizunov, V. A., Kozlov, M. M. and Chernomordik, L. V. (2013). Extracellular annexins and dynamin are important for sequential steps in myoblast fusion. J Cell Biol 200(1): 109-123.
  7. Melikyan, G. B., Brener, S. A., Ok, D. C. and Cohen, F. S. (1997). Inner but not outer membrane leaflets control the transition from glycosylphosphatidylinositol-anchored influenza hemagglutinin-induced hemifusion to full fusion. J Cell Biol 136(5): 995-1005.
  8. Merklinger, E., Schloetel, J. G., Spitta, L., Thiele, C. and Lang, T. (2016). No evidence for spontaneous lipid transfer at ER-PM membrane contact sites. Journal of Membrane Biology 249(1-2): 41-56. 
  9. Millay, D. P., Gamage, D. G., Quinn, M. E., Min, Y. L., Mitani, Y., Bassel-Duby, R. and Olson, E. N. (2016). Structure-function analysis of myomaker domains required for myoblast fusion. Proc Natl Acad Sci U S A 113(8): 2116-2121. 
  10. Progatzky, F., Dallman, M. J. and Lo Celso, C. (2013). From seeing to believing: labelling strategies for in vivo cell-tracking experiments. Interface Focus 3(3): 20130001.
  11. Quinn, M. E., Goh, Q., Kurosaka, M., Gamage, D. G., Petrany, M. J., Prasad, V. and Millay, D. P. (2017). Myomerger induces fusion of non-fusogenic cells and is required for skeletal muscle development. Nat Commun 8: 15665. 
  12. Sampath, S. C., Sampath, S. C. and Millay, D. P. (2018). Myoblast fusion confusion: the resolution begins. Skelet Muscle 8(1): 3. 
  13. Valansi, C., Moi, D., Leikina, E., Matveev, E., Grana, M., Chernomordik, L. V., Romero, H., Aguilar, P. S. and Podbilewicz, B. (2017). Arabidopsis HAP2/GCS1 is a gamete fusion protein homologous to somatic and viral fusogens. J Cell Biol 216(3): 571-581.
  14. Verma, S. K., Leikina, E., Melikov, K., Gebert, C., Kram, V., Young, M. F., Uygur, B. and Chernomordik, L. V. (2018). Cell-surface phosphatidylserine regulates osteoclast precursor fusion. J Biol Chem 293(1): 254-270.

简介


[摘要 ] 脂质混合(脂质探针在融合膜之间的分布)已广泛用于研究相对快速的病毒和细胞内融合过程的各个阶段,这些过程耗时数秒至数分钟。脂质混合测定对于鉴定在操作上定义为没有内容混合的脂质混合的半融合中间体特别重要。由于不同步的特性以及分化过程的缓慢速度,这些分化过程使细胞在成肌,破骨细胞生成和胎盘生成中引发细胞间的融合过程,因此这些融合需要几天的时间。在这些非常缓慢的融合过程中应用脂质混合测定法检测早期融合中间体时,必须考虑质膜成分的连续转换以及脂质探针在膜之间的潜在融合无关交换。在这里,我们描述了脂质混合分析在骨骼肌细胞发育和再生中成肌融合阶段的工作中的应用。我们的方法利用了基于鼠C2C12细胞的成肌分化和融合的常规体外模型。当我们观察到第一个多核细胞的外观时,我们将细胞提起并用荧光脂质DiI 作为膜探针或CellTracker TM Green 作为含量探针标记它们。通过荧光显微镜对探针在细胞之间的重新分布进行评分。半融合细胞被鉴定为用内含物和膜探针标记的单核细胞。解释必须由具有融合能力不足细胞的阴性对照系统支持,以说明脂质探针的与融合无关的交换,并将其贡献降到最低。这种方法进行了较小的修改已用于调查原代鼠成肌细胞,破骨细胞前体的融合以及由配子融合原HAP2 介导的融合,并且很可能可以用于其他缓慢的细胞-细胞融合过程。

[Bac kground ] 膜脂质双层在细胞生物学过程中的融合多种多样,如胞吐过程中细胞内膜的融合,包膜病毒感染中病毒膜和细胞膜的融合以及骨骼肌发育中的成肌细胞融合显然涉及类似的脂质重排(图1)(Brukman 等人,2019)。首先,两个融合双层的对置,接触的单层的合并会产生半融合连接,并使脂质探针在这些单层之间进行重新分布。远端单层的后续合并会产生融合孔,并使水性探针在融合膜室之间重新分布。





图1. 在半融合和融合孔形成脂质重排的示意图(从改性˚F 在igure 1 Brukman 等人。,2019 )。



半融合可以转变成融合孔,或者代表在融合孔形成之前中止的融合反应的死角。在后一种情况下,半融合连接可以解离,产生两个不同的双层。反过来,新生的融合孔可以关闭或扩展前进的融合,直至完全融合膜室。关键融合中间体,半融合和融合孔的形成和解离速率,以及这些中间体之间的过渡速率在不同的融合过程之间变化,并且取决于所涉及的蛋白质活性和融合膜的脂质组成。

在大多数实验研究中,通过荧光显微镜检测到的大孔足以使含量探针通过〜1 kDa 至〜100 kDa 。使用荧光显微镜或荧光分光光度法检测半融合,在操作上定义为脂质混合而不进行内容混合。在关于无蛋白脂质双层的融合以及由病毒融合素和细胞内融合素介导的融合的研究中,已经开发了脂质混合测定的不同修饰(Brukman 等人,2019)。这些融合过程的相对快速性促进了脂质混合测定在所有这些系统中的成功应用,脂质混合的特征时间从数秒到数分钟不等。

脂质混合测定法在发育细胞-细胞融合中的应用是这项工作的主题,对于阐明膜重排的途径至关重要,但比更快的病毒和细胞内融合反应更具挑战性。例如,多核肌管的形成是细胞-细胞融合过程中最具特征的例子之一(Sampath et al 。,2018),其后是成肌分化,这为融合做好了准备,并且需要几天的时间。结果,通过评估融合的时间,在融合之前添加到标记质膜的荧光脂质探针已经被部分内化。而且,由于膜含量和对比度较高,因此包含内化探针的细胞内膜区室的标记可能比质膜的标记更亮。开发用于非常慢融合过程的脂质混合测定法的另一个问题是脂质探针通过脂质交换蛋白,脂质微团或细胞外囊泡从膜之间转移的能力(在Merklinger 等人,2016年综述),即,通过既不涉及半融合或融合也不依赖于融合机制的机制。

在这里,我们提出了一种协议,用于在C2C12细胞,永生化的小鼠成肌细胞(在高血清培养基中增殖并在低血清培养基中分化并融合)之间的融合中分析脂质混合。我们还将讨论该协议的修改版本在其他慢速细胞-细胞融合过程中的应用。

关键字:膜融合, 半融合, 成肌细胞融合, 脂质混合, 合胞体形成, Myomaker, Myomerger

材料和试剂


 


用料


鹘® 15米升聚苯乙烯离心管中,锥底,具有圆顶密封螺帽(康宁,目录号:352095)
组织培养皿,35 x 10 mm REF(Corning,目录号:353001)
组织培养皿,60 x 15毫米(Corning,目录号:353002)
 


细胞


C2C12电池(ATCC,目录号:CRL-1772 TM )
Millay 等人产生了Myomaker缺陷的C2C12细胞和Myomerger缺陷的C2C12细胞。(2016年); 奎因等。(2017)并在胶原蛋白涂层的基材上生长
 


试剂种类


Vybrant TM DiI 细胞标记溶液(Thermo Fisher Scientific,目录号:V22885)
CellTracker TM Green CMFDA(5-氯甲基荧光素二乙酸酯)(Thermo Fisher Scientific目录号:C7025)
橙色CMRA CellTracker TM (Thermo Fisher Scientific,目录号:C34551)
胰蛋白酶-EDTA 0.05%(Thermo Fisher,目录号:25300054)
Hoechst 33342三盐酸盐,三水合物-10 mg / ml水溶液(Thermo Fisher Scientific,目录号:H3570)
在磷酸盐中缓冲的10%福尔马林(电子显微镜科学,目录号:15740)
小牛皮肤的胶原蛋白(Sigma,目录号:C8919-20 ml)
二甲基亚砜(DMSO,Sigma,目录号:D2650-100 ml)
 


媒体


增殖培养基(PM):DMEM,高葡萄糖,GlutaMAX TM 补充剂(Thermo Fisher,目录号:10566-016)+ 10%胎牛血清,青霉素/链霉素(Thermo Fisher,目录号:10378016)
分化培养基(DM):DMEM,高葡萄糖,GlutaMAX TM 补充剂(Thermo Fisher,目录号:10566-016)+ 5%马胎血清,青霉素/链霉素
胎牛血清,FBS(GIBCO生命Ť echnologies,目录号:10437-028)
源自新西兰的热灭活马血清(Thermo Fisher,目录号:26050088)
PBS,康宁® 贝科的磷酸盐缓冲液(生命科学,目录号:21-030-CV),1个与钙和镁
 


设备


 


蔡司轴向显微镜
摄像头(厂商PCO -tech INC。 ,pco.edge 3.1 SCMOS )
F-LD 32 / 0.4蔡司物镜
用于绿色细胞跟踪仪的单个荧光团带通滤光片:Semrock 激发472 nm / 30 nm,发射520 nm / 35 nm,二向色性495 nm LP
用于DiI的单个荧光团带通滤光片:蔡司(Zeiss)激发545 nm / 25 nm,发射605 nm / 70 nm,二向色570 LP
 


软件


 


Micro-Manager软件(Edelstein 等,2014)
开源平台ImageJ(美国国立卫生研究院,罗克维尔·派克,贝塞斯达,医学博士
 


程序


 


图2给出了制备用于脂质混合实验的细胞的时间表的示意图。


 






图2.为脂质混合实验准备和标记细胞的时间表示意图。细胞的标记开始48 小时后的分化(即,48 将细胞置于成DM后小时)。分化后49 小时1小时后,将不同标记的细胞混合。


 


胶原蛋白涂层餐具
Ç 燕麦的菜(60×15 通过覆盖培养皿用3ml或1.5毫升1:10溶液毫米)(两个菜对于每种条件)和35×10mm的培养皿(5个菜对于每种条件)与胶原的胶原蛋白在将无菌水分别用于60 x 15毫米培养皿和35 x 10毫米培养皿,并在室温下过夜孵育并用水洗涤两次(分别用于大和小的培养皿,分别洗涤3毫升和2毫升),然后在生物安全柜。


 


细胞
使C2C12细胞在胶原蛋白涂层培养皿中的增殖培养基(PM)中生长至75%融合度。将细胞转移至分化培养基(DM)(t = 0)。分化后 48 小时(t = 48 小时),细胞扩散至约90%融合,并观察到第一个肌管。此时,用2 ml PBS清洗两个培养皿中的每个细胞,然后将细胞放入2 ml新鲜PBS中。


 


细胞标记
用CellTracker TM Green 标记培养皿1中的细胞,并用Orange CMRA CellTracker 或DiI 标记培养皿2中的细胞。用CellTracker TM 绿色标记;添加4 μ 升的的CellTracker TM (50绿原液μ 克在20探针的μ 升在此培养基中,在37 DMSO的)至2毫升的PBS孵育细胞15分钟° C.然后把细胞进入在37 ° C下30分钟完成DM处理。使用相同的步骤对Orange CMRA CellTracker 进行标记。为了标记细胞的DiI ,注射4 μ 升的1种mM的的DiI 储备溶液于2ml的PBS中,在37 ° C和孵育细胞45分钟。用2 ml DM洗涤皿1和2中的细胞三遍,并用2 ml PBS洗涤两次。


 


共镀不同标记的细胞
在37 ° C下用1 ml的胰蛋白酶-EDTA 0.05%轻轻孵育1和2皿中的细胞,持续1分钟。在随后的2-3分钟已经在室温下孵育的过程中,检查细胞是否开始聚集。电梯。完成后,取出EDTA-胰蛋白酶,将细胞在37 ° C的条件下移入4 ml完全DM中。将培养皿1和2的细胞收集到同一15 ml试管中,并涡旋混合。将体积调整为10毫升。然后将细胞(2 ml细胞悬液)铺在五个胶原蛋白处理过的小皿中(35 x 10 mm)。两个小时后,通过更换新鲜DM的培养基去除碎片和未附着的细胞。
使用标记有不同CellTracker 的细胞的复制来分析细胞之间的内容混合。在没有内容混合的情况下,对用CellTracker TM Green 标记的细胞和用DiI 标记的细胞进行交配以分析脂质混合。
在t = 72小时,在室温下用10%福尔马林在磷酸盐缓冲液中固定细胞10分钟。在室温下放置30 分钟,用补充了Hoechst的PBS(10 mg / ml稀释1,000 倍的PBS)代替福尔