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Peroxisome Motility Measurement and Quantification Assay
过氧化物酶体能动性测量和定量分析   

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Abstract

Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation with mitochondria and the endoplasmic reticulum (ER) is essential for the β-oxidation of fatty acids and the synthesis of myelin lipids and polyunsaturated fatty acids. A key assay to assess peroxisome motility in mammalian cells is the expression of a fluorescent fusion protein with a peroxisomal targeting signal (e.g., GFP-PTS1), which targets the peroxisomal matrix and allows live-cell imaging of peroxisomes. Here, we first present a protocol for the transfection of cultured mammalian cells with the peroxisomal marker EGFP-SKL to observe peroxisomes in living cells. This approach has revealed different motile behaviour of peroxisomes and novel insight into peroxisomal membrane dynamics (Rapp et al., 1996; Wiemer et al., 1997; Schrader et al., 2000). We then present a protocol which combines the live-cell approach with peroxisome motility measurements and quantification of peroxisome dynamics in mammalian cells. More recently, we used this approach to demonstrate that peroxisome motility and displacement is increased when a molecular tether, which associates peroxisomes with the ER, is lost (Costello et al., 2017b). Silencing of the peroxisomal acyl-CoA binding domain protein ACBD5, which interacts with ER-localised VAPB, increased peroxisome movement in skin fibroblasts, indicating that membrane contact sites can modulate organelle distribution and motility. The protocols described can be adapted to other cell types and organelles to measure and quantify organelle movement under different experimental conditions.

Keywords: Peroxisome motility(过氧化物酶体动力学), Live-cell imaging(活细胞成像), Organelle cooperation(细胞器协作), Membrane contact(膜接触), GFP-PTS1(GFP-PTS1), ACBD5(ACBD5), ACBD4(ACBD4)

Background

An important feature of eukaryotic cells is the presence of membrane-bound compartments (organelles), which create distinct optimised environments to promote various metabolic reactions required to sustain life. For the entire cell to function as a unit, coordination and cooperation between specialized organelles must take place. This requires a dynamic spatial organization, which allows the movement of organelles to areas of greatest metabolic need, the positioning of organelles in those areas, and the interaction with other compartments, which permits metabolic cooperation and communication amongst organelles. This is often mediated through interorganellar membrane contacts, whereby two organelles come into close apposition (Prinz, 2014; Eisenberg-Bord et al., 2016).

Peroxisomes are multifunctional organelles that play pivotal cooperative roles in the metabolism of cellular lipids and reactive oxygen species (ROS) and are thus essential for human health and development (Islinger and Schrader, 2011; Wanders et al., 2015; Waterham et al., 2016). Peroxisomes interact with many organelles involved in cellular lipid metabolism such as the endoplasmic reticulum (ER), mitochondria, lipid droplets or lysosomes (Schrader et al., 2013 and 2015). We revealed that the peroxisomal tail-anchored membrane proteins ACBD5 and ACBD4 directly interact with tail-anchored VAPB at the ER (Costello et al., 2017a; 2017b and 2017c). This interaction links both organelles together and allows transfer of lipids between them (Costello et al., 2017b; Hua et al., 2017). Whereas in plants and yeast, peroxisomes move along the actin cytoskeleton by interacting with myosin motors (Jedd and Chua, 2002; Fagarasanu et al., 2009), in mammalian cells (and filamentous fungi), peroxisomes use microtubules and dynein/kinesin motors to distribute uniformly within the cell and to reach new neighbourhoods (Schrader et al., 1996 and 2003; Guimaraes et al., 2015; Lin et al., 2016). We also found that ACBD5-VAPB interaction, which tethers peroxisomes to the ER, influences peroxisome motility (Costello et al., 2017b). Using the peroxisome motility measurement and quantification assay, we showed that loss of ACBD5, which resulted in reduced peroxisome-ER association, also increased peroxisome movement (Costello et al., 2017b). Our data indicate that organelle contact sites can modulate peroxisome (organelle) distribution and motility.

Peroxisomes are highly versatile organelles, which respond to environmental stimuli with changes in their number, size, and enzyme composition (Islinger et al., 2010). Certain stress conditions, in particular in plants, can lead to changes in the motile behaviour of peroxisomes and altered distribution (Rodríguez-Serrano et al., 2009). There is currently great interest in the measurement of peroxisome (organelle) motility and its quantification in order to understand the fundamental principles of organelle distribution, its regulation and role in organelle interaction and metabolic cooperation. Understanding these mechanisms is not just important for comprehending fundamental physiological processes but also for understanding pathogenic processes in disease etiology (Ferdinandusse et al., 2017; Yagita et al., 2017).

Materials and Reagents

  1. 10 cm culture dishes (Greiner Bio One International, catalog number: 664160 )
  2. Microporation tips
  3. 15 ml centrifuge tubes (Greiner Bio One International, catalog number: 188271 )
  4. 1.5 ml microcentrifuge tubes (Greiner Bio One International, catalog number: 616201 )
  5. 3.5 cm round glass bottom dishes (Cellview, Greiner Bio One International, catalog number: 627861 )
  6. Serological pipettes, 10 ml (Greiner Bio One International, catalog number: 607180 )
  7. Mammalian cell line of interest, here: human skin fibroblasts (see Note 1)
  8. Peroxisome marker (fluorescent reporter with a C-terminal peroxisomal targeting signal, e.g., EGFP-SKL plasmid [Schrader et al., 2000])
  9. Optional: siRNA for silencing of candidate genes/proteins in mammalian cells, here: ACBD5 (Ambion, catalog number: s40666 ); control scrambled siRNA (GE Healthcare Dharmacon, catalog number: D-001206-14-05 ) (50-100 µM stock) (in sterile, RNase/DNase-free water or buffer supplied by manufacturer)
  10. 70% (v/v) ethanol
  11. TrypLETM Express solution (1x) (Thermo Fisher Scientific, GibcoTM, catalog number: 12604013 ) (store at 4 °C)
  12. Immersion oil (Olympus, catalog number: IMMOIL-F30CC )
  13. Dulbecco’s modified Eagle medium (DMEM) high glucose (4.5 g/L) ( Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 ) for complete growth medium for cell culture of human skin fibroblasts
  14. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
  15. Penicillin/streptomycin solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  16. Sodium chloride (NaCl)
  17. Potassium chloride (KCl)
  18. Sodium phosphate dibasic (Na2HPO4)
  19. Potassium phosphate dibasic (K2HPO4)
  20. Complete growth medium for cell culture of human skin fibroblasts (see Recipes)
  21. Phosphate-buffered saline (1x PBS) (see Recipes)

Equipment

  1. Pipetting aid (Greiner Bio One International, model: Sapphire MAXIPETTE, catalog number: 847070 )
  2. Class II biological safety cabinet/tissue culture hood (Faster, model: SafeFAST Top 209-D, catalog number: F00000050000 ) (see Note 2)
  3. Humidified CO2 incubator (95% air, 5% CO2, 37 °C) (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 240i , catalog number: 510263330)
  4. Inverted light microscope (phase contrast) (ZEISS, model: Primo VertTM )
  5. 37 °C water bath (Grant Instruments, model: JBA12 )
  6. TC20TM Automated Cell Counter (Bio-Rad Laboratories, catalog number: 145-0101 )
  7. Vacuum aspiration system (Fisher Scientific, catalog number: 11636620)
    Manufacturer: INTEGRA Biosciences, catalog number: 158320 .
  8. Table top centrifuge equipped with a swing-out rotor for 15-ml conical tubes (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM BiofugeTM StratosTM , catalog number: 75005282)
  9. Microcentrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM PicoTM 17 , catalog number: 75002491)
  10. Microporator Neon® Transfection System (Thermo Fisher Scientific, InvitrogenTM, catalog number: MPK5000S )
  11. Spinning disk microscope with controlled temperature-CO2 chamber and objective warmer
    Note: An Olympus IX81 microscope (Olympus, model: IX81 ) equipped with a Yokogawa CSUX1 spinning disk (Yokogawa Electric, model: CSU-X1 ) head, CoolSNAP HQ2 CCD camera (Photometrics, model: CoolSNAP HQ2 ) and a UPlanSApo 60x/1.35 oil objective was used. Image acquisition was performed using a 488 nm solid state laser at 20% of max. intensity.

Software

  1. VisiView software (Visitron Systems, Germany)
  2. Fiji (ImageJ)
  3. Custom Python data analysis pipeline

Procedure

  1. Cell culture and transfection
    1. Perform all cell culture based work in a class II biological safety cabinet/tissue culture hood and disinfect the work surface and materials (e.g., pipettes) with 70% (v/v) ethanol.
    2. Grow mammalian cells of choice (here, human skin fibroblasts) in complete growth medium (see Recipes) (10 cm Ø cell culture dishes or cell culture flasks) in a humidified CO2 incubator (95% air, 5% CO2, 37 °C).
    3. For maintenance of cells, refresh the cell culture medium every 2-3 days and split the cells before they reach 100% confluency using standard cell culture procedures (see Note 3).
    4. One or two days prior to transfection split the cells so that they reach 70-80% confluency at the day of transfection (10 cm Ø cell culture dishes or cell culture flasks).
    5. Prior to transfection prepare 3.5 cm glass bottom dishes with complete growth medium (without antibiotics), and pre-incubate in the CO2 incubator at 37 °C.
    6. In addition, place the Neon® Microporation device in the biological safety cabinet. Fill a Neon tube with 3 ml of electrolyte buffer E and insert the tube into the pipette station. Set the transfection parameters (here, 1,700 V, 20 msec pulse width, 1 pulse) on the device (see Notes 4 and 5).
    7. For transfection, wash the cells (from 10 cm Ø cell culture dishes or cell culture flasks) once with 1x PBS (see Recipes) and incubate for 2-5 min with 1 ml of TrypLE Express at 37 °C.
    8. Resuspend detached cells in 9 ml of complete growth medium (without antibiotics), and determine the cell number using a TC20TM Automated Cell Counter (see Note 6).
    9. For each transfection reaction use 1-2 x 105 cells (number of cells used with a 10 µl microporation tip) (see Note 7). Transfer the total number of cells for all transfections to a 15 ml tube and centrifuge for 3 min at RT in a table top centrifuge (500 x g).
    10. Resuspend the cell pellet in 1 ml of 1x PBS and transfer cells to a 1.5 ml microcentrifuge tube.
    11. Centrifuge cells in a microcentrifuge for 3 min at 500 x g and resuspend the pellet in buffer R. The amount of buffer R will depend on the number of transfections and the microporation tip used (e.g., 6 transfections with a 10 µl microporation tip–6 x 10 µl = 60 µl).
    12. For each transfection, pre-mix 1-2 µg of plasmid DNA (here, EGFP-SKL) and 50-100 nM of siRNA (optional) in a microcentrifuge tube and add 10 µl of cells resuspended in buffer R. To facilitate pipetting, mix enough reagents for at least 2 transfections in each microcentrifuge tube (see Note 8).
    13. Mount a 10 µl Neon tip onto the Neon pipette.
    14. Immerse the tip into the cell-DNA-siRNA mixture and slowly aspirate 10 µl of the sample. Avoid generating air bubbles in the tip.
    15. Insert the pipette into the E buffer–containing tube in the pipette station, and press start on the touch screen.
    16. After delivery of the electric pulse, quickly remove the pipette from the pipette station and immediately transfer the cells from the tip to the 3.5 cm dishes containing the pre-warmed growth medium (without antibiotics) (see Note 9).
    17. Gently move the dish horizontal and vertical to evenly distribute the cells (see Note 10).
    18. Incubate the cells in a humidified CO2 incubator (95% air, 5% CO2, 37 °C) for 48-72 h to allow efficient silencing (see Note 11).
    19. Discard the Neon tip in an appropriate biological hazardous waste container and repeat steps A12 to A18 for the remaining cell-DNA-siRNA mixtures (e.g., control siRNA and other plasmids of interest) (see Note 12).

  2. Live-cell imaging
    1. Prior to image acquisition set up a controlled-temperature and CO2 chamber at the microscope stage, as well as an objective warmer. In the absence of a CO2 regulator change cells to a CO2–independent medium (e.g., HEPES buffered).
    2. Set up a glass bottom dish in the controlled-temperature and CO2 chamber, switch on the mercury lamp and scan the sample for transfected cells (i.e., green GFP signal).
    3. Image collection is performed using VisiView software. For imaging, 250 stacks of 9 planes (0.5 µm thickness, 100 msec exposure) were taken from each cell in a continuous stream. All conditions and laser intensities were kept between experiments.
    4. Collected images are converted into a Maximum intensity projection file using VisiView. This option compresses the images from each stack into a single one and binds them in a continuous time frame (Videos 1 and 2).

      Video 1. Peroxisome movement in control fibroblasts. Video 1 shows movement of peroxisomes in human skin fibroblasts (control) transfected with the peroxisome marker EGFP-SKL (fluorescent reporter with a C-terminal peroxisomal targeting signal). Note the spherical, punctate labelling of peroxisomes and some long-range, directed movements, which are likely microtubule-dependent. Bar = 20 µm. Video 1 (2 min observation, 10 x original speed) is taken from Costello et al. (2017b).

      Video 2. Peroxisome movement in fibroblasts after loss of ACBD5. Video 2 shows increased movement of peroxisomes in human skin fibroblasts silenced for ACBD5. Peroxisomes were labelled as described (Video 1). Note that loss of ACBD5, which tethers peroxisomes to the ER, results in increased peroxisomal movement. Not all of the movements may depend on microtubules. Bar = 20 µm. Video 2 (2 min observation, 10 x original speed) is taken from Costello et al. (2017b).

  3. Peroxisome motility measurements The following workflow is implemented as a Python module, which uses Numpy, Scipy, and Scikit-Image libraries.
    1. Each frame is filtered using Laplace of Gaussian scale-space filtering (Lindeberg, 1998).
    2. The threshold for each filtered image is determined using Median Absolute Deviation as a robust estimator of foreground locations (Hampel, 1974).
    3. Peroxisome positions are calculated as maxima in the filtered image above threshold.
    4. Positions between frames are tracked using a global optimization subroutine using a modified version of the Jonker-Volgenant algorithm (Jonker and Volgenant, 1987).
    5. Tracking results are manually verified for accuracy.
    6. Peroxisome trajectories are displayed as trajectory and density plots, and converted into speed distributions which are subsequently plotted as cumulative distribution functions (CDFs) (Figure 1).
    7. Pseudocode for the tracking is:
      # Detection
      allpositions = []
      for t in range(len(frames)):
              filtered = scale_space_filter(frames[t])
              threshold = median(filtered) + 3*median_absolute_deviation(filtered)
              positions = peak_locations(filtered, mask=filtered>threshold)
              allpositions.append(positions)

      # Tracking
      allidentities = [ range(len(allpositions[0])) ]
      max_identity = max(allidentites[0])
      for t in range(1, len(allpositions)+1):
              cost = distance_matrix(allpositions[t-1], allpositions[t])
              identities, final_cost = lapjv(cost)
              identities[final_cost > maximum_distance] = -1
              max_identity = max(allidentites[-1], max_identity)
              num_new = count(identities == -1)
              identities[identities == -1] = range(max_identity+1, max_identity+num_new+1)
              allidentities.append(identities)

      # Create trajectories
      for t in range(len(allidentities)):
              for i in range(len(allidentities[t])):

      trajectories[allidentities[t][i]].append(allpositions[t][i])

Data analysis

  1. Trajectory plots (Figures 1A-1C)
    1. Randomly select a fixed number of peroxisome trajectories for each condition being analysed–we used 100 trajectories.
    2. Smooth trajectories using Kalman filtering and an Expectation-Maximization algorithm (https://github.com/pykalman/pykalman: PyKalman: Kalman Filter, Smoother, and EM Algorithm for Python).
    3. Plot the initial 20 time frames of each trajectory, starting at the center (x = 0, y = 0), i.e., subtract the initial position from each trajectory before plotting (see Costello et al., 2017b). 
  2. Density plots (Figures 1D-1F)
    1. Select all significant trajectories–in our case we chose trajectories with 20 or more time frames.
    2. Smooth the trajectories using Kalman filtering and an Expectation-Maximization algorithm.
    3. Perform pooling and binning of the x and y coordinates of the trajectories; we used the interval -3.3 µm in x and y directions and 50 bins along each dimension.
    4. Plot the log-scaled 2D histogram of these points to accentuate the ‘wings’ of the distribution; we recommend a high contrast colormap, e.g., the ‘jet’ color-map available in Matplotlib (and MATLAB).
  3. ECDF plots (Figure 1G)
    1. Select all significant trajectories–in our case we chose trajectories with 20 or more time frames (see Costello et al., 2017b).
    2. Smooth the trajectories using Kalman filtering and an Expectation-Maximization algorithm.
    3. Calculate the instantaneous trajectory speed profiles by calculating the distance moved between each time point in the trajectory.
    4. Pool the speeds and convert them into an empirical cumulative distribution function (ECDF) by sorting the speeds.
    5. By pooling speeds for all datasets for a given condition, a single ECDF is generated for each (in our case we had a minimum of 38,175 trajectories from 24 videos per condition).
    6. The ECDF is generated using the following pseudocode:
      xvals = sort(speeds)
      yvals = range(N)/N
      plot(xvals, yvals)


      Figure 1. Analysis of peroxisome movement. A-C. For trajectory plots, 100 peroxisome trajectories were retrieved for each condition and the first 20 time-frames were plotted starting at a center. D-F. For density plots, the x and y coordinates of all trajectories ≥ 20 time-frames were pooled and binned in the interval -3,3 µm in x and y directions, using 50 bins. The log-scaled 2D histogram of these points was plotted using ‘jet’ colormap. G. For ECDF plots, instantaneous trajectory speed profiles were estimated by calculating distance moved between each time-point in the trajectory. These speeds were pooled and converted to an Empirical Cumulative Distribution Function (ECDF). By pooling speeds for all datasets for a given condition, a single ECDF was generated for each (min. 38,175 trajectories from 24 videos/condition). All three approaches for analysis of peroxisome motility revealed an increase in peroxisome movement under conditions where ACDB5, which is involved in the tethering of peroxisomes to the ER, is lost. Figure 1 is taken from Costello et al. (2017b).

Notes

  1. Other mammalian cell lines (e.g., COS-7 or HepG2 cells) can also be used. Those do not necessarily require microporation for transfection of plasmids or siRNA.
  2. Follow the biosafety and GMO guidelines of your institution.
  3. We usually pre-warm 1x PBS, TrypLETM Express solution, and complete growth medium to 37 °C. Medium is removed from the cell culture dish with a Pasteur pipette by vacuum aspiration. Cells are washed once with 3-4 ml of 1x PBS before TryplExpress solution is added (approx. 1 ml/10 cm dish) (gently tilt to cover the surface and incubate for 2-5 min at 37 °C). Upon detachment, cells are harvested in complete growth medium (10 ml/10 cm dish), carefully resuspended by pipetting the cell suspension 2-3 times up and down and transferred to a 15 ml conical tube. Cells are pelleted by centrifugation in a table top centrifuge (500 x g, 3 min at RT) and resuspended in 10 ml of complete growth medium. For maintenance, approx. 5 x 105 cells are transferred to a new 10 cm cell culture dish containing 10 ml of complete growth medium and incubated in a humidified CO2 incubator (95% air, 5% CO2, 37 °C).
  4. A detailed instruction manual of how to use the Neon® device can be found on the supplier’s website (http://tools.thermofisher.com/content/sfs/manuals/neon_device_man.pdf).
  5. Transfection conditions for other cell types need to be optimized. Further information is provided on the supplier’s website (https://www.thermofisher.com/be/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html).
  6. Cell number can also be determined manually, e.g., with a Neubauer chamber.
  7. It is advisable to collect a higher number of cells than required for each transfection (e.g., double the amount) to account for possible microporation failures due to air bubbles in the electroporation tip (visible electric discharge), and to facilitate Neon tip pipetting.
  8. If transfecting with more than one plasmid, pre-mix 1-2 µg of each plasmid.
  9. In the case of an electric spark, discard the tip with the cells and repeat the microporation using the backup cells.
  10. Do not swirl to avoid accumulation of the cells in the centre of the dish.
  11. The silencing efficiency should be tested and optimised prior to motility analysis by setting up similar dishes, and perform cell lysis and Western blotting. Efficient silencing can also be confirmed by immunofluorescence.
  12. The Neon pipette tips and tubes can be regenerated and reused to minimise costs of microporation (Brees and Fransen, 2014).

Recipes

  1. Complete growth medium for cell culture of human skin fibroblasts
    Dulbecco’s modified Eagle medium (DMEM) high glucose (4.5 g/L), supplemented with:
    10% fetal bovine serum (FBS)
    100 U/ml of penicillin and 100 μg/ml of streptomycin
    Store at 4 °C
  2. Phosphate-buffered saline (1x PBS)
    140 mM NaCl
    2.5 mM KCl
    6.5 mM Na2HPO4
    1.5 mM K2HPO4, pH 7.35

Acknowledgments

We would like to thank J. Costello and T. Schrader for helpful comments and suggestions. This protocol was adapted from Costello et al. (2017b) J Cell Biol 216(2): 331-342. DOI: 10.1083/jcb.201607055. This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/K006231/1 and BB/N01541X/1 to M. Schrader). J. Metz and M. Schrader are supported by a Wellcome Trust Institutional Strategic Support Award (WT097835MF and WT105618MA). M. Schrader is supported by Marie Curie Initial Training Network action PerFuMe (316723).

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  28. Yagita, Y., Shinohara, K., Abe, Y., Nakagawa, K., Al-Owain, M., Alkuraya, F. S. and Fujiki, Y. (2017). Deficiency of a retinal dystrophy protein, acyl-CoA binding domain-containing 5 (ACBD5), impairs peroxisomal β-oxidation of very-long-chain fatty acids. J Biol Chem 292(2): 691-705.

简介

细胞器运动,分布和相互作用有助于真核细胞的组织。过氧化物酶体是有助于细胞脂质代谢和ROS稳态的多功能细胞器。它们在哺乳动物细胞中均匀分布,并通过驱动蛋白和动力蛋白电动机沿微管移动。他们与线粒体和内质网(ER)的代谢合作对脂肪酸的β-氧化和髓鞘脂质和多不饱和脂肪酸的合成至关重要。评估哺乳动物细胞中过氧化物酶体运动性的关键测定是具有过氧化物酶体靶向信号(例如GFP-PTS1)的荧光融合蛋白的表达,其靶向过氧化物酶体基质并允许过氧化物酶体的活细胞成像。在这里,我们首先提出了用过氧化物酶体标记物EGFP-SKL转染培养的哺乳动物细胞的方案,以观察活细胞中的过氧化物酶体。这种方法揭示了过氧化物酶体的不同运动行为和对过氧化物酶体膜动力学的新见解(Rapp等,1996; Wiemer等,1997; Schrader等,2000)。然后,我们提出一种方案,其将活细胞方法与过氧化物酶体运动性测量结合并在哺乳动物细胞中定量过氧化物酶体动力学。最近,我们使用这种方法来证明当将过氧化物酶体与ER结合的分子系链丢失时,过氧化物酶体运动和位移增加(Costello等,2017b)。与ER定位的VAPB相互作用的过氧化物酶体酰基辅酶A结合结构域蛋白ACBD5的沉默增加了皮肤成纤维细胞中的过氧化物酶体运动,表明膜接触位点可以调节细胞器分布和运动。所描述的方案可以适用于其他细胞类型和细胞器,以在不同的实验条件下测量和定量细胞器移动。
【背景】真核细胞的一个重要特征是膜结合的隔室(细胞器)的存在,其产生不同的优化环境以促进维持生命所需的各种代谢反应。要使整个细胞发挥作用,必须发挥专业细胞器之间的协调与配合。这需要一个动态的空间组织,其允许细胞器移动到代谢需求最大的区域,细胞器在这些区域中的定位,以及与其他区间的相互作用,这允许细胞器之间的代谢协调和通信。这通常通过间质膜接触来介导,由此两个细胞器紧密结合(Prinz,2014; Eisenberg-Bord et al。,2016)。
  过氧化物酶体是多功能细胞器,其在细胞脂质和活性氧(ROS)的代谢中起着关键的协同作用,因此对人类健康和发育至关重要(Islinger和Schrader,2011; Wanders等,2015; Waterham et al。 2016)。过氧化物酶体与参与细胞脂质代谢的许多细胞器相互作用,如内质网(ER),线粒体,脂滴或溶酶体(Schrader等,2013和2015)。我们发现过氧化物酶体尾锚定蛋白ACBD5和ACBD4在ER处与尾锚VAPB直接相互作用(Costello等,2017a; 2017b和2017c)。这种相互作用将两个细胞器连接在一起,并允许它们之间转移脂质(Costello等,2017b; Hua等,2017)。而在植物和酵母中,过氧化物酶体通过与肌球蛋白电动机(Jedd和Chua,2002; Fagarasanu等人,2009)在哺乳动物细胞(和丝状真菌)中相互作用而沿着肌动蛋白细胞骨架移动,过氧化物酶体使用微管和动力蛋白/驱动蛋白发动机在细胞内均匀分布并到达新的社区(Schrader et al。,1996 and 2003; Guimaraes et al。,2015; Lin et al。,2016)。我们还发现ACBD5-VAPB相互作用(其将过氧化物酶体系到ER)影响过氧化物酶体的运动(Costello等,2017b)。使用过氧化物酶体动力学测量和定量测定,我们显示导致过氧化物酶体 - ER结合减少的ACBD5的丧失也增加了过氧化物酶体运动(Costello等,2017b)。我们的数据表明细胞器接触点可以调节过氧化物酶体(细胞器)分布和运动。
  过氧化物酶体是高度通用的细胞器,其响应于环境刺激的数量,大小和酶组成的变化(Islinger等,2010)。某些胁迫条件,特别是在植物中,可导致过氧化物酶体的运动行为发生变化并改变分布(Rodríguez-Serrano et al。,2009)。目前对过氧化物酶体(细胞器)运动性及其量化的测量有很大的兴趣,以了解细胞器分布的基本原理,其在细胞器相互作用和代谢合成中的作用。了解这些机制不仅对于理解基本的生理过程也是对于了解疾病病因学中的致病过程非常重要(Ferdinandusse等,2017; Yagita等,2017)。

关键字:过氧化物酶体动力学, 活细胞成像, 细胞器协作, 膜接触, GFP-PTS1, ACBD5, ACBD4

材料和试剂

  1. 10厘米培养皿(Greiner Bio One International,目录号:664160)
  2. 微量提示
  3. 15ml离心管(Greiner Bio One International,目录号:188271)
  4. 1.5ml微量离心管(Greiner Bio One International,目录号:616201)
  5. 3.5厘米圆形玻璃底盘(Cellview,Greiner Bio One International,目录号:627861)
  6. 血清移液管,10 ml(Greiner Bio One International,目录号:607180)
  7. 感兴趣的哺乳动物细胞系,这里:人皮肤成纤维细胞(见注1)
  8. 过氧化物酶体标志物(具有C末端过氧化物酶体靶向信号的荧光报告物,例如EGFP-SKL质粒[Schrader等人,2000])
  9. 任选的:用于在哺乳动物细胞中沉默候选基因/蛋白质的siRNA,这里:ACBD5(Ambion,目录号:s40666);控制加扰siRNA(GE Healthcare Dharmacon,目录号:D-001206-14-05)(50-100μM储备液)(在无菌的RNase /无DNA酶水或由制造商提供的缓冲液中)
  10. 70%(v / v)乙醇
  11. TrypLE TM Express溶液(1x)(Thermo Fisher Scientific,Gibco TM,目录号:12604013)(在4℃下储存)
  12. 浸油(Olympus,目录号:IMMOIL-F30CC)
  13. 用于人皮肤成纤维细胞细胞培养的完全生长培养基的Dulbecco改良的Eagle培养基(DMEM)高葡萄糖(4.5g / L)(Thermo Fisher Scientific,Gibco TM,目录号:11965092)
  14. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10082147)
  15. 青霉素/链霉素溶液(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  16. 氯化钠(NaCl)
  17. 氯化钾(KCl)
  18. 磷酸氢二钠(Na 2 HPO 4)
  19. 磷酸二氢钾(K 2/2 HPO 4)
  20. 用于人皮肤成纤维细胞的细胞培养的完整生长培养基(参见食谱)
  21. 磷酸盐缓冲盐水(1x PBS)(参见食谱)

设备

  1. 移液助剂(Greiner Bio One International,型号:Sapphire MAXIPETTE,目录号:847070)
  2. II类生物安全柜/组织培养罩(更快,型号:SafeFAST Top 209-D,目录号:F00000050000)(见注2)
  3. 加湿的CO 2培养箱(95%空气,5%CO 2,37℃)(Thermo Fisher Scientific,Thermo Scientific,superson TM,型号: Heracell TM 240i,目录号:510263330)
  4. 倒置光学显微镜(相位对比度)(ZEISS,型号:Primo Vert TM )
  5. 37°C水浴(Grant仪器,型号:JBA12)
  6. TC20 TM 自动细胞计数器(Bio-Rad Laboratories,目录号:145-0101)
  7. 真空吸气系统(Fisher Scientific,目录号:11636620)
    制造商:INTEGRA Biosciences,目录号:158320。
  8. 配备有用于15ml锥形管的摆动转子的台式离心机(Thermo Fisher Scientific,Thermo Scientific& TM),型号为Heraeus< Biofuge< sup> Stratos TM ,目录号:75005282)
  9. 微量离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Pico TM,目录号:75002491)
  10. Microporator Neon ®转染系统(Thermo Fisher Scientific,Invitrogen TM,目录号:MPK5000S)
  11. 旋转磁盘显微镜带有受控温度的CO 2室和客观温度计
    注意:配备有横河电机CSUX1旋转盘(Yokogawa Electric,型号:CSU-X1)的Olympus IX81显微镜(Olympus,型号:IX81),CoolSNAP HQ2 CCD相机(Photometrics,型号:CoolSNAP HQ2)和UPlanSApo使用60x / 1.35石油物镜。使用最大20%的488nm固体激光器进行图像采集。强度。

软件

  1. VisiView软件(Visitron Systems,德国)
  2. 斐济(ImageJ)
  3. 自定义Python数据分析管道

程序

  1. 细胞培养和转染
    1. 在II类生物安全柜/组织培养罩中进行所有基于细胞培养的工作,并用70%(v / v)乙醇对工作表面和材料(例如,移液管)进行消毒。 >
    2. 在加湿的CO 2培养箱(95%空气,95%空气中)培养完整生长培养基(参见食谱)(10cmØ细胞培养皿或细胞培养瓶)中选择的哺乳动物细胞(这里是人皮肤成纤维细胞) 5%CO 2,37℃)
    3. 为了维持细胞,每2-3天刷新细胞培养基,并使用标准细胞培养程序在细胞达到100%汇合之前分裂细胞(见注3)。
    4. 在转染前一天或两天,分裂细胞,使其在转染当天达到70-80%汇合(10cm×细胞培养皿或细胞培养瓶)。
    5. 在转染前,准备3.5cm玻璃底层培养皿(不含抗生素),并在37℃的CO 2培养箱中预孵育。
    6. 另外,将Neon ® Microporation设备放在生物安全柜中。用3ml电解液缓冲液E填充氖管,并将管插入移液器。在设备上设置转染参数(这里为1,700 V,20 msec脉冲宽度,1脉冲)(见注4和5)。
    7. 对于转染,用1x PBS洗涤细胞(10厘米Ø细胞培养皿或细胞培养瓶)一次(参见食谱),并在37℃下用1ml TrypLE Express孵育2-5分钟。
    8. 将分离的细胞重悬在9ml完全生长培养基(无抗生素)中,并使用TC20 自动细胞计数器(参见附注6)确定细胞数目。
    9. 对于每个转染反应,使用1-2×10 5个细胞(10μl微孔尖端使用的细胞数)(参见附注7)。将所有转染的细胞总数转移到15ml管中,并在室温离心机(500×g)中离心3分钟。
    10. 将细胞沉淀重悬于1ml 1x PBS中,并将细胞转移到1.5ml微量离心管中
    11. 将微量离心机中的细胞以500×g离心3分钟,并将沉淀重新悬浮在缓冲液R中。缓冲液R的量将取决于所用转染次数和使用的微孔末端(例如< em,6,用10μl微孔尖端转染6×10μl=60μl)
    12. 对于每次转染,在微量离心管中预先混合1-2μg质粒DNA(这里是EGFP-SKL)和50-100nM siRNA(任选的),并将10μl细胞重新悬浮于缓冲液R中。为了便于移液,在每个微量离心管中混合足够的试剂至少2次转染(见附注8)
    13. 将10μl氖尖装入氖气移液管。
    14. 将细胞浸入细胞DNA-siRNA混合物中并缓慢吸出10μl样品。避免在尖端产生气泡。
    15. 将移液器插入移液器站中的E缓冲液管中,然后按触摸屏上的开始。
    16. 电脉冲输送后,迅速从移液管中取出移液器,并立即将细胞从尖端转移到含有预热的生长培养基(不含抗生素)的3.5厘米培养皿中(见附注9)。
    17. 轻轻地将餐具水平和垂直移动,以均匀分布细胞(见附注10)。
    18. 将细胞在加湿的CO 2培养箱(95%空气,5%CO 2,37℃)中孵育48-72小时以允许有效的沉默(参见附注11 )
    19. 将适当的生物危险废物容器中的氖尖丢弃,并对剩余的细胞-DNA-siRNA混合物(例如,对照siRNA和感兴趣的其他质粒)重复步骤A12至A18(参见附注12) 。

  2. 活细胞成像
    1. 在图像采集之前,在显微镜阶段设置受控温度和CO 2室,以及客观的加热器。在不存在CO 2调节子的情况下,将细胞切换到CO 2 - 非依赖性介质(例如HEPES缓冲)。
    2. 在受控温度和CO 2 2室内设置玻璃底盘,打开汞灯,扫描样品进行转染细胞(即绿色GFP信号) 。
    3. 使用VisiView软件进行图像采集。对于成像,从连续流中的每个细胞取出250个堆叠的9个平面(0.5μm厚度,100msec曝光)。所有条件和激光强度保持在实验之间。
    4. 收集的图像使用VisiView转换为最大强度投影文件。此选项将每个堆叠中的图像压缩为单个图像,并以连续的时间范围(视频1和2)进行绑定。

      Video 1. Peroxisome movement in control fibroblasts. Video 1 shows movement of peroxisomes in human skin fibroblasts (control) transfected with the peroxisome marker EGFP-SKL (fluorescent reporter with a C-terminal peroxisomal targeting signal). Note the spherical, punctate labelling of peroxisomes and some long-range, directed movements, which are likely microtubule-dependent. Bar = 20 µm. Video 1 (2 min observation, 10 x original speed) is taken from Costello et al. (2017b).

      To play the video, you need to install a newer version of Adobe Flash Player.

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      Video 2. Peroxisome movement in fibroblasts after loss of ACBD5. Video 2 shows increased movement of peroxisomes in human skin fibroblasts silenced for ACBD5. Peroxisomes were labelled as described (Video 1). Note that loss of ACBD5, which tethers peroxisomes to the ER, results in increased peroxisomal movement. Not all of the movements may depend on microtubules. Bar = 20 µm. Video 2 (2 min observation, 10 x original speed) is taken from Costello et al. (2017b).

      To play the video, you need to install a newer version of Adobe Flash Player.

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  3. 过氧化物酶体动力学测定 以下工作流被实现为一个Python模块,它使用Numpy,Scipy和Scikit-Image库。
    1. 每个帧使用拉普拉斯高斯尺度空间滤波进行滤波(Lindeberg,1998)
    2. 使用中间绝对偏差作为前景位置的鲁棒估计器来确定每个滤波图像的阈值(Hampel,1974)。
    3. 过氧化物酶体位置被计算为超过阈值的过滤图像中的最大值
    4. 使用使用Jonker-Volgenant算法的修改版本的全局优化子程序来跟踪帧之间的位置(Jonker和Volgenant,1987)。
    5. 跟踪结果是手动验证的准确性。
    6. 过氧化物酶体轨迹显示为轨迹和密度图,并转化为速度分布,随后将其绘制为累积分布函数(CDF)(图1)。
    7. 跟踪的伪代码是:
      #检测
      allpositions = []
      for t in range(len(frames)):
      &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; filtered = scale_space_filter(frames [t])
      &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; threshold = median(filtered)+ 3 * median_absolute_deviation(filters) > &nbsp; &NBSP; &NBSP; &NBSP; position = peak_locations(filtered,mask = filtered&gt; threshold)
      &nbsp; &NBSP; &NBSP; &NBSP; allpositions.append(位置)

      #追踪
      allidentities = [range(len(allpositions [0]))]
      max_identity = max(allidentites [0])
      for t in range(1,len(allpositions)+1):
      &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; cost = distance_matrix(allpositions [t-1],allpositions [t])
      &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;身份,final_cost = lapjv(cost)
      &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;身份[final_cost&gt; maximum_distance] = -1
      &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; max_identity = max(allidentites [-1],max_identity)
      &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; num_new = count(identityities == -1)
      &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; identities [identityities == -1] = range(max_identity + 1,max_identity + num_new + 1 )
      &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; allidentities.append(identity)

      #创建轨迹
      for t in range(len(allidentities)):
      &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; for in in range(len(allidentities [t])): />
      轨迹[allidentities [t] [i]]。append(allpositions [t] [i])

数据分析

  1. 轨迹图(图1A-1C)
    1. 对每个被分析的条件随机选择一个固定数量的过氧化物酶体轨迹 - 我们使用了100个轨迹。
    2. 使用卡尔曼滤波和期望最大化算法的平滑轨迹( https://github.com/ pykalman / pykalman :PyKalman:用于Python的卡尔曼滤波器,平滑器和EM算法)
    3. 绘制每个轨迹的初始20个时间帧,从中心(x = 0,y = 0),即开始,在绘制前从每个轨迹中减去初始位置(参见Costello et al。 al 。,2017b)。&nbsp;
  2. 密度图(图1D-1F)
    1. 选择所有重要的轨迹 - 在我们的例子中,我们选择了具有20个或更多时间帧的轨迹。
    2. 使用卡尔曼滤波和期望最大化算法平滑轨迹。
    3. 执行轨迹的x和y坐标的汇总和合并;我们在x和y方向上使用了-3.3μm的间隔,并且沿着每个维度使用了50个箱。
    4. 绘制这些点的对数尺度的2D直方图以强调分布的“翅膀”;我们建议使用MatRlotlib(和MATLAB)中可用的“jet”颜色映射的高对比度色彩映射,例如。
  3. ECDF图(图1G)
    1. 选择所有重要的轨迹 - 在我们的例子中,我们选择了具有20个或更多时间帧的轨迹(参见Costello等人,,2017b)。
    2. 使用卡尔曼滤波和期望最大化算法平滑轨迹。
    3. 通过计算轨迹中每个时间点之间移动的距离来计算瞬时轨迹速度曲线。
    4. 通过对速度进行排序,将速度转换成经验累积分布函数(ECDF)。
    5. 通过在给定条件下汇总所有数据集的速度,为每个数据集生成单个ECDF(在我们的情况下,我们每个条件从24个视频中至少有38,175个轨迹)。
    6. ECDF使用以下伪代码生成:
      xvals = sort(speed)
      yvals = range(N)/ N
      plot(xvals,yvals)


      图1.过氧化物酶体运动分析 A-C。对于轨迹图,为每个条件检索100个过氧化物酶体轨迹,并且从中心开始绘制前20个时间框架。 D-F。对于密度图,将所有轨迹≥20个时间帧的x和y坐标合并,并在x和y方向的间隔-3,3μm中采样,使用50个分箱。使用'jet'色彩图绘制这些点的对数刻度的2D直方图。 G.对于ECDF图,通过计算在轨迹中的每个时间点之间移动的距离来估计瞬时轨迹速度分布。将这些速度合并并转换为经验累积分布函数(ECDF)。通过汇总给定条件下所有数据集的速度,每个生成一个ECDF(从24个视频/条件中最少38,175个轨迹)。用于分析过氧化物酶体运动的所有三种方法显示,在涉及过氧化物酶体连接到ER的ACDB5被丢失的条件下,过氧化物酶体运动的增加。图1取自Costello等人。 (2017b)。

笔记

  1. 也可以使用其它哺乳动物细胞系(例如COS-7或HepG2细胞)。那些不一定需要用于转染质粒或siRNA的微孔。
  2. 遵循贵机构的生物安全和转基因生物指导方针。
  3. 我们通常预热1x PBS,TrypLE TM Express溶液,并将成长培养基完成至37°C。通过真空抽吸用巴斯德吸管从培养皿中取出培养基。在加入TryplExpress溶液(约1ml / 10cm皿)(轻轻倾斜以覆盖表面并在37℃下孵育2-5分钟)之前,用3-4ml 1x PBS洗涤细胞一次。分离后,将细胞在完全生长培养基(10ml / 10cm皿)中收获,通过将细胞悬液移液2-3次上下移液小心地重新悬浮并转移到15ml锥形管中。通过在台式离心机(500×g,在室温下3分钟)离心将细胞沉淀并重新悬浮于10ml完全生长培养基中。对于维护,约将5×10 5个细胞转移到含有10ml完全生长培养基的新的10cm细胞培养皿中,并在加湿的CO 2培养箱(95%空气,5 %CO 2,37℃)。
  4. 有关如何使用Neon ®设备的详细说明手册可以在供应商的网站上找到( http://tools.thermofisher.com/content/sfs/manuals/neon_device_man.pdf )。
  5. 需要优化其他细胞类型的转染条件。更多信息请参见供应商网站( https://www.thermofisher.com/be/en/home/life-science/cell - 文化/转染/转染---选择杂志/霓虹灯转染系统/ neon-protocols-cell-line-data.html )。
  6. 也可以使用Neubauer腔室手动确定细胞数目,例如。
  7. 建议收集比每次转染所需数量更多的细胞(例如,,双倍的量)以考虑由于电穿孔尖端中的气泡(可见放电)引起的可能的微孔破坏,并促进霓虹笔尖移液。
  8. 如果转染多于一个质粒,则预先混合1-2μg每个质粒。
  9. 在电火花的情况下,用细胞丢弃尖端,并使用备用电池重复微孔。
  10. 不要旋转,以避免细胞在盘中心积聚。
  11. 在运动分析之前,应通过设置类似的菜肴来测试和优化沉默效率,并进行细胞裂解和Western印迹。免疫荧光也可以确认有效的沉默
  12. 氖管吸头和管可以再生和重新使用,以最大程度地降低微孔成本(Brees and Fransen,2014)。

食谱

  1. 用于人皮肤成纤维细胞的细胞培养的完全生长培养基
    Dulbecco改良的Eagle培养基(DMEM)高葡萄糖(4.5g / L),补充:
    10%胎牛血清(FBS)
    100 U / ml青霉素和100μg/ ml链霉素 储存于4°C
  2. 磷酸缓冲盐水(1x PBS)
    140 mM NaCl
    2.5 mM KCl
    6.5mM Na 2 HPO 4
    1.5mM K 2 HPO 4,pH 7.35

致谢

我们要感谢J. Costello和T. Schrader的有用的意见和建议。该协议由Costello等人改编。 (2017b)J Cell Biol 216(2):331-342。 DOI:10.1083 / jcb.201607055。这项工作得到了生物技术和生物科学研究理事会(BB / K006231 / 1和BB / N01541X / 1至M. Schrader)的资助。 J. Metz和M. Schrader由Wellcome Trust机构战略支持奖(WT097835MF和WT105618MA)支持。 M. Schrader由Marie Curie初步培训网络Action PerFuMe(316723)支持。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Metz, J., Castro, I. G. and Schrader, M. (2017). Peroxisome Motility Measurement and Quantification Assay. Bio-protocol 7(17): e2536. DOI: 10.21769/BioProtoc.2536.
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