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May 2019

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Live Cell Imaging of Male Meiosis in Arabidopsis by a Landmark-based System
地标系统在拟南芥雄性减数分裂活体细胞成像中的应用   

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Abstract

Live cell imaging has tremendously promoted our understanding of cellular and subcellular processes such as cell division. Here, we present a step-by-step protocol for a robust and easy-to-use live cell imaging approach to study male meiosis in the plant Arabidopsis thaliana as recently established. Our method relies on the concomitant analysis of two reporter genes that highlight chromosome configurations and microtubule dynamics. In combination, these reporter genes allowed the discrimination of five cellular parameters: cell shape, microtubule array, nucleus position, nucleolus position, and chromatin condensation. These parameters can adopt different states, e.g., the nucleus position can be central or lateral. Analyzing how tightly these states are associated gives rise to landmark stages that in turn allow a quantitative and qualitative dissection of meiotic progression. We envision that such an approach can also provide valuable criteria for the analysis of cell differentiation processes outside of meiosis.

Keywords: Cell division (细胞分裂), Cell differentiation (细胞分化), Meiosis (减数分裂), Chromosome (染色体), Microtubules (微管), Cytoskeleton (细胞骨架), Arabidopsis (拟南芥)

Background

Meiosis is a special cell division cycle that serves two major purposes. First, the DNA content of the meiotic mother cell is reduced by half that leads, in the case of a diploid organism, to haploid meiotic products. This reduction is necessary for sexually reproducing organisms so that, after the fusion of two gametes during fertilization, the original genome size is restored. Second, meiosis promotes genetic diversity through the exchange of DNA segments between the parental chromosomes (homologous chromosomes or shortly homologs), named meiotic recombination, and by the generation of new, yet complete chromosome sets in which randomly either the homolog of the mother or the father is present for each chromosome (in case of a diploid organism). Thus, understanding meiosis is interesting for different fields of research ranging from cell biology and reproductive biology via genetics to evolutionary biology (Wijnker and Schnittger, 2013; Mercier et al., 2015; Melamed-Bessudo et al., 2016; Lambing and Heckmann, 2018; Pelé et al., 2018; Wang and Copenhaver, 2018).

Plants are a powerful model system to study the various aspects of meiosis. In addition, research in plant meiosis is also promoted by an applied interest since a low level of recombination often restricts breeding programs.

Research on plant meiosis has largely relied on the analysis of fixed cells, e.g., for chromosome spreads and immunolocalization studies, both for Arabidopsis (Armstrong et al., 2009; Martinez-Garcia and Pradillo, 2017; Parra-Nunez et al., 2020; Sims et al., 2020) as well as for other plant species (Chelysheva et al., 2013; Sepsi et al., 2018; Darrier et al., 2020; Stack et al., 2020). These techniques have been and continue to be important tools as they offer a great spatial resolution to address the structure of meiotic chromosomes and the localization of meiotic regulators for instance demonstrated by an immuno-cytological analysis of the 3D configuration of meiotic chromosomes (Hurel et al., 2018). However, these studies provide little to no information about the underlying dynamics of meiosis. In addition, temporal aspects of meiosis have to be indirectly deduced by the frequency of observed stages, a procedure that is inherently error-prone and can easily misguide the researcher e.g., when two or more populations of meiocytes exist in the same sample undergoing an altered course of meiosis that can be mistaken as one population with cells at different stages (Prusicki et al., 2019; Sofroni et al., unpublished). Moreover, short-lived phases, for instance nuclear envelope breakdown, are difficult to catch, and there has been for instance a long discussion in the field whether the nuclear envelop is reformed in Arabidopsis after the first meiotic division.

Live cell imaging of meiosis can complement the analyses of fixed material and build together with these techniques a powerful approach to reach molecular mechanistic insights into this important cell division program. Live cell-imaging of plant meiosis has been previously initiated in maize (Yu et al., 1997; Sheehan and Pawlowski, 2009; Nannas et al., 2016). However, only short phases of meiosis in maize could be recorded with a genetic reporter for microtubules and a chemical stain that highlights DNA. In addition, a recent protocol for live cell imaging of meiosis by light-sheet microscopy has been published (Valuchova et al., 2020). While this set-up is very powerful to follow meiosis in entire flower buds, it still does not reach the subcellular resolution as obtained by confocal laser scanning microscopy (CLSM).

Here, we describe in detail a method to follow meiosis in Arabidopsis anthers based on a recently established procedure by CLSM (Prusicki et al., 2019). Importantly, this method allows keeping the samples alive up to several days allowing the analysis of meiosis in its entirety. Furthermore, the use of Arabidopsis allowed the generation of plants containing fluorescent reporters for different meiotic regulators, foremost the meiosis-specific kleisin subunit of the cohesin complex called RECOMBINATION 8 (REC8), ASYNAPTIC 1 (ASY1) and ASY3, two components of the chromosome axis, and ZYP1, a component of the central region of the synaptonemal complex (Prusicki et al., 2019; Yang et al., 2019 and 2020). This set up has recently been used to study the control of cohesin in mutants in which REC8 is prematurely cleaved (Cromer et al., 2019).

A major challenge is the quantitative analysis of the obtained movies. As typical for biological processes, meiosis is a continuous and gradual succession of events. To dissect these movies, we focused on five cellular parameters as visualized by the KINGBIRD reporter system (REC8 labeled with GFP in combination with microtubules highlighted in red; Prusicki et al., 2019). These five cellular parameters are: cell shape, microtubule array, nucleus position, nucleolus position and chromatin state. Each of these parameters can adapt different states, see Table 1 (Table 1 Excel file), this publication and Figure 3 in Prusicki et al., 2019.

Looking then at the association of these different parameter states revealed that they are not randomly associated but often tightly linked. This gives rise to a biological landmark system where one landmark is a prominent cellular configuration with distinct parameter states. In turn, this landmark system can be used as a map to qualitatively (appearance of the same or new landmarks) and quantitatively (duration of these landmarks) dissect meiotic progression in mutants or different environmental conditions. The use of other meiotic reporters can then be used to refine and/or complement this landmark system. The principle of this analysis can be easily translated to other cellular differentiation processes, of course including other cellular parameters, which need to be identified.

Materials and Reagents

  1. Black marker
  2. Squared Petri dishes 100 x 100 x 20 mm (Sardstedt, catalog number: 82.9923.422 )
  3. Round Petri dishes 35 x 10 mm (Sarstedt, catalog number: 82.1135.500 )
  4. Round Petri dishes 60 x 15 mm (Sarstedt, catalog number: 82.1194.500 )
  5. Safeseal tube 1.5 ml (Sarstedt, catalog number: 72.706 )
  6. Parafilm M (Neolab, Bemis, 3-1011, PM-996)
  7. Sterilizing filter Millex-GV 0.22 μm (Merck, catalog number: SLGV033RS )
  8. Needle 30 G ½” 0.3 x 13 mm (BD Microlance, model: 304000 )
  9. Seeds of Arabidopsis thaliana plants containing meiotic fluorescent reporter constructs as a hallmark of meiosis such as the KINGBIRD line presented in Prusicki et al., 2019 as well as reporters for the meiotic chromosome axis and the synaptonemal complex (Yang et al., 2019 and 2020)
  10. NaClO in H2O, solution 13% (Applichem GmbH, ITW Reagents, catalog number: 213322.0715 )
  11. HCl 37% (VWR Chemicals, catalog number: 20255.29 0)
  12. Agarose (Sigma, catalog number: A9539 )
  13. Agar, powdered food grade (Applichem GmbH, ITW Reagents, catalog number: A00917,5000 )
  14. MS Basal Salt Mixture (Duchefa Biochemie, catalog number: M0221.0050 )
  15. Myo-Inositol (Duchefa Biochemie, catalog number: I0609.0100 )
  16. Nicotinic acid (Duchefa, catalog number: N0611 )
  17. Pyridoxin hydrochloride (Duchefa, catalog number: P0612 )
  18. Thiamine hydrochloride (Duchefa, catalog number: T0614 )
  19. Glycine (Sigma, catalog number: G-7126 )
  20. Sterile dH2O
  21. Acetocarmine
  22. Isopropanol
  23. Sucrose
  24. KOH
  25. Murashike and Skoog medium (MS medium) for plant germination (see Recipes)
  26. Arabidopsis Apex Culture Medium (ACM) for imaging (see Recipes)

Equipment

  1. Pipettes 10/100/1000 (e.g., Eppendorf Research plus pipette)
  2. Growth chambers MobyLux GroBanks (CLF Plant Climatics, model: BrightBoy )
  3. Vacuum chamber
  4. Glass bicker
  5. Clean bench
  6. Fine tweezers, thickness: 0.02/0.01 mm (Dumont, Dumoxel, catalog numbers: 0203-4-PO and 0203-5-PO )
  7. Dissection microscopes (e.g., Zeiss, models: Stemi2000 and Stemi508)
  8. Laser Scanning Microscope (e.g., Zeiss, model: LSM880 ), equipped with a water dipping objective (e.g., Zeiss, model: W Plan-Apochromat 40x/1.0 DIC objective )

Software

  1. ZEN 2.3 SP1 FP1 black (Zeiss) (or the related imaging software of your microscope system)
  2. MetaMorph Version 7.8.0.0 (Molecular devices)
  3. Fiji version 1.52b (ImageJ, https://imagej.net/Fiji) (Schindelin et al., 2012)
  4. Excel (Microsoft Office)
  5. Python programming language (Version 3.6, Python Software Foundation)

Procedure

  1. Preparation of plant material
    1. Surface-sterilize seeds of Arabidopsis thaliana, carrying a fluorescent reporter expressed at meiotic stages (an example is available in Prusicki et al., 2019). One efficient method to surface-sterilize seeds is by chloride gas: pour the seeds in 1.5 ml tubes, and mark the tubes with a black marker (other colors will faint due to the sterilization process). Leave the lid of the tubes open, and place the tubes in a racket inside a vacuum chamber. In the same chamber place a glass bicker containing 25 ml of a 7.5% bleach solution (eluted with tap water). Add to the bicker 1 ml of 37% HCl, quickly close the chamber and apply vacuum. The whole procedure should be carried out under a chemical hood. Be careful not to breathe the chloride gas! Leave the seeds in the chamber for 3-4 h. After the treatment, the seeds are ready to be sawn. If instead you are planning to store the sterilized seeds in the tube for some time, let the tubes open under a clean bench for a minimum of 30 min to ensure the gas has completely evaporated.
    2. Saw the seeds on Murashike and Skoog (½ MS, Recipe 1) squared Petri dishes enriched with selecting agent according with the resistance of the inserted construct. e.g., add 25 mg/L Hygromycin B to select for PROREC8:REC8:GFP as done in Prusicki et al. (2019). Alternatively, the selection can be performed by genotyping or based on fluorescence detection, if the protein of interest is expressed in seedlings.
    3. Store the plates for stratification in the dark for 3 days at 4 °C and then transfer them to long-day conditions (16 h day/8 h night regime at 22 °C/18 °C) for germination.
    4. 10 days after germination transfer the seedlings on soil and grow them at long-day conditions.
    5. Plants will be ready to image in 3 to 4 weeks, depending on growth conditions and genotypes.

  2. Preparation of imaging samples
    1. Prepare Apex Culture Medium (ACM) with agarose to 0.8%, 1% and 2% concentrations and 1,000x vitamin stock as indicated in the session (Recipe 2).
    2. Sterilize the ACM by autoclaving.
    3. When the autoclaved (or reheated) ACM with 0.8% agarose is hand warm, add the vitamin mix to a 1x working concentration (e.g., to 50 ml of ACM add 50 μl of a 1,000x vitamin stock solution), and pour it into small Petri dishes (35 x 15 mm), seal with parafilm and store at 4 °C.
    4. Pour the ACM with 1% agarose in medium-sized Petri dishes (60 x 15 mm), seal with parafilm and store at 4 °C.
    5. Aliquot the ACM with 2% agarose in sterile 1.5 ml Eppendorf tubes and store them at 4 °C.

  3. Sample mounting
    1. Cut an inflorescence and anchor it on a Petri dish with ACM with 1% agarose (Video 1).

      Video 1. Removal of large flower buds and selection of the flower bud for image acquisition, Steps C1 and C2

    2. Remove all the open and the elongated flowers at the base of the pedicel but the flower bud with a roundish shape and the size between 0.4-0.6 mm containing early meiocytes (Video 1). The characteristics of flower buds with meiocytes in an early stage can vary depending on the growth conditions and the genotype, e.g., mutants in which meiosis is delayed usually takes places in flower buds of larger sizes. It is therefore recommended to analyze several flower buds with acetocarmine or similar staining to estimate in which flower buds meiotic stages can be found. In general, flower buds at developmental stage 9 (Smyth et al., 1990) contain male meiocytes undergoing meiosis.
    3. Remove the upper sepal of the selected flower bud, being careful to not touch the inner organs of the flower, in particular the anthers (Videos 2 and 3).

      Video 2. Removal of sepal to give access to two anthers in the selected flower bud, Steps C3 and C4

      Video 3. Close up of a flower bud ready to image, after Steps C3 and C4

    4. Remove the remaining flower buds (Videos 2 and 3). The next smallest/youngest flower buds can be left attached as a backup flower in the case the main flower bud gets damaged during the removal of the sepal.
    5. Lift the inflorescence from the preparation medium and cleanly cut the stem to a length of circa (ca.) 0.5 cm (with forceps or better using a needle) (Video 4). This will facilitate the uptake of nutrients from the medium and will keep the sample in good condition for a long time.

      Video 4. Positioning of the selected and processed flower bud for imaging, steps C5 and C6

    6. Transfer the sample onto the small Petri dish with ACM with 0.8% agarose, anchor it in the middle of the plate and cover the flower bud with a drop of ACM with 2% agarose (Video 4 and Figures 1A and 1B).
    7. It is possible to follow more than one flower bud in one microscopy session. In this case, prepare a new sample as previously explained (starting from Procedure C1 of the Bio-protocol), and locate all flower buds as close as possible in the center of the Petri dish (Video 4).


      Figure 1. Sample mounting and positioning at the microscope. A. The sample is placed in the middle of a Petri dish and covered by a drop of 2% agarose. B. Close up of the sample from panel A. C Overview and D zoom onto the microscope stage with the sample positioned under the water immersion objective.

  4. Image acquisition
    1. It is important to have stable acquisition conditions, e.g., room temperature, humidity, etc. Therefore, it is advisable to turn on the confocal microscope in advance and position the sample in the room with a few ml of water covering the mounted samples.
    2. After approximately one hour you can start the image acquisition.
    3. Position the sample on the microscope stage, submerge the water-dipping objective and fill up the Petri dish to the top with autoclaved water (Figures 1C and 1D). It is advisable to thoroughly clean the objective with isopropanol before the acquisition to reduce the risk of bacteria growth during image acquisition.
    4. Using the visual function (or related software tools), identify the position of each flower bud in the sample and save it using the multi-position function on ZEN black software.
    5. The set-up for image acquisition needs to be adapted to the purpose of the experiment and to the characteristics of the used fluorophores. To follow a complete meiotic division in a time-lapse experiment, and to identify the meiotic landmarks using the KINGBIRD line as presented in Prusicki et al. (2019), the following set-up can be used:
      1. Use the Argon laser at λ 488 nm to excite GFP and the DPSS 561-10 laser (λ 561) to excite TagRFP.
      2. Use two tracks in sequential line mode for signal detection, and the Beam splitter MBS 488/561. In the first track, use the GaAsP detector to record the GFP signal filtered for λ between 498 and 550 nm. In the second track, use the GaAsP detector to observe the TagRFP signal filtered for λ between 578 and 649 nm. A third channel to detect the autofluorescence of chloroplast can be added to the second track and filtered for λ between 680 and 750 nm. Additionally, the Transmitted Light detection can be added to one of the two tracks.
      3. Set the Pinhole at 1 Airy Unity for the TagRFP detection, use bidirectional scan function and set the pixel dwell to a value around 2 μs.
      4. Parameters for image acquisition such as laser intensity, gain and offset have to be adapted to the individual imaging conditions. Among others, they depend on the laser status, as well as on the reporter line used, and thus the level of protein expression. In general, a compromise between sample viability and high-resolution imaging has to be found to obtain the best image quality while maintaining the sample in good conditions. In our case, we set the intensity of the Argon laser between 1% and 4.5%, while the DPSS 561-10 laser was set between 0.3% and 1%. The detector gain to collect GFP and TaqRFP signals was set between 700 and 850, and was set between 650 and 750 for the detection of chloroplast autofluorescence. In all the cases, the offset parameter was 0.
      5. Perform averaging on 2 lines in case of the KINGBIRD line; for other reporters, different setting may be needed.
      6. Acquire time lapse of multiple positions with 10 min interval time. At each position, perform a Z-stack of 6 to 7 planes with a step size to a maximum of 50 μm per time point. To follow the entire meiotic division, images should be acquired for ca. 30 h (Prusicki et al., 2019).
    6. The use of the Autofocus function at each position and each time point is suggested, especially for long acquisition times.
    7. In case the sample is still going through meiosis but the data acquisition needs to be shortly interrupted (e.g., in case of readjustment of the focal plane or in case of reaching the last acquisition cycle) it is advisable to fit the time of interruption to match the interval time, so to start the new time-lapse acquisition at the end of the 10 min interval.

  5. Image processing
    1. Save each position of the acquired time-lapse as a separate file, for instance in the case of the ZEN black software: processing → copy→ subset.
    2. Open the image in Fiji and save it in a separate folder as image sequence (save as → image sequence → format TIFF → digits must be enough to cover all time points e.g., 110 time points need 3 digits).
    3. Open the image sequence using MetaMorph (App → review multiple image sequence file → open sequence → follow the instructions), and select for each time point the z-stack layer, which shows the cells of interest. Finally, load the selected z-stacks for each wavelength and save them as tiff. files. This step helps to correct for the drift of the sample in the z-direction, and together with the autofocus function activated during image acquisition (see Procedure D6) allows following the same cells for the complete duration of the time lapse.
    4. Open the new files in Fiji, combine the different channels, adjust their brightness and contrast and generate an RGB version of the file.
    5. If an x-y drift is detectable, it can be corrected by using the plug-in Stackreg from Fiji (Plugins → Stackreg → Rigid body) (Thévenaz et al., 1998).
    6. An example of a time-lapse series after image processing is given in Video 5.

      Video 5. Example of the outcome of a time lapse experiment after image processing as explained in Steps E1-E6

Data analysis

Landmark identification using the KINGBIRD line

  1. Data set annotation
    1. Give an ID number to each visible cell within one anther. The ID number could be impressed on each frame of the RGB.tiff file using the Text tool of Fiji (Figure 2).


      Figure 2. Data analysis. A. One frame of an acquired time-lapse movie depicting an anther of an Arabidopsis plant carrying the KINGBIRD reporter (microtubules are in magenta, REC8-chromatin is in green and chloroplasts are blue). Each meiocyte is given a number, which will be its ID on the spreadsheet in B. B. Example of a spreadsheet used for analysis as described in the paragraph Data-analysis A2. The numbering of the stage refers to the numbers in Table 1. Each stage is color-coded to facilitate the data organization.

    2. Prepare a table using Microsoft Excel that includes 7 columns: frame number, time in min, cell shape, microtubule array, nucleus position, nucleolus position and chromatin state (Figure 2). The number of rows will be equal to the number of frames acquired for a single cell. The first imaged frame will be counted as Time 0 (Figure 2).
    3. Fill in each column with a number that corresponds to the state of the analyzed cellular parameter as annotated in Table 1 and Figure 2 (for a further reference see Prusicki et al., 2019). For instance, if at time 0 the cell presents a round shape, the number 4 must be filled in in the third column. Proceed with the annotation of each cellular parameter at each time point. If the cell is not visible at one time point, or the parameter state cannot be recognized, fill in the table an n for non-visible.
    4. Annotate all the visible cells of one another on different Excel sheets (one sheet per cell). This file constitutes the data set that will be analyzed as explained in the following session.

      Table 1. Cell features of meiocytes as obtained with the KINGBIRD reporter system. The table provides microscopy images that illustrate each described cell feature (adapted from Figures 3 and 4 in Prusicki et al., 2019. Creative Commons Attribution License). The original table file could be accessed here (Table 1 Excel file).


  2. Data set analysis
    A Python script to perform analysis of the annotated data set created in part A. is available at https://gitlab.com/wurssb/arabidopsis-thaliana---landmark-analysis. This script contains the procedure to extract landmarks from the data and takes the following steps:
    1. Prepare the data for analysis by filtering out invalid or duplicate entries. Each entry, consisting of the observed state for each cellular parameter should have an identifier for the sample and the specific cell within the sample.
    2. If the data is not sampled at regular intervals, resample to a common interval. However, take care when resampling, as going from short to large intervals might remove short-lived states from the data set while the reverse could introduce (large) errors from interpolating the states.
    3. A cellular state is defined by the ensemble of all parameter states. For example, cell 11 from Figure 2 at time point 10 has the cellular state: 1-1-2-2-2. Cellular states diverging for up to two different parameter states were defined as neighboring states (or neighbors). Hence, the cellular states 1-1-2-2-1, 1-1-2-3-2 and 1-1-2-3-3 but not 1-1-3-3-3 are neighboring states to 1-1-2-2-2.
    4. Partially observed states can be either discarded, or interpolated from neighboring states. Again, take care when interpolating, for example, by setting a maximum time interval that can be safely interpolated depending on the observed processes.
    5. Calculate how much time is spent in each cellular state.
    6. For each observed cellular state, list the neighboring states.
    7. For each cellular state, calculate the neighboring score using the time spent in the specific state (tstate) and the time spent its neighbors (tneighbors):



    8. Sort the cellular states by their score. Landmarks can now be assigned with a suitable cut-off value (an example in Prusicki et al., 2019). An example of the final result of the neighboring analysis is given in Table 2 (Table 2 Excel file), where all the cellular states observed are listed together with their counts (number of cases they have been observed), their percentage of appearance in relation to the other cellular states and finally their neighboring score. The source of the data listed in Table 2 are the results published in Prusicki et al., 2019.
    9. To assess the robustness of the analysis, bootstrap the analysis by resampling the data with replacement and re-running the whole analysis multiple times.
      Bootstrapping can be done on the level of single observations, or by resampling groups of observations of specific cells or anthers to uncover different sources of possible bias in the analysis.

      Table 2. Example of an outcome of the data analysis. The original table file could be accessed here (Table 2 Excel file).

Recipes

  1. Murashike and Skoog medium (MS medium) for plant germination
    Ingredient
    Concentration
    For 1 L
    MS basal medium
    0.5x
    2.2 g/L
    Sucrose
    1%
    10 g/L
    Agar
    1%
    10 g
    dH2O
    ---
    up to 1 L of volume
    KOH
    ---
    ---
    1. Dissolve MS basal and sucrose in 800 ml of dH2O
    2. Adjust the pH to 5.8 using KOH and bring to the final volume of 1 L
    3. Add the agar to the solution
    4. Sterilize by autoclaving and store at room temperature until usage
  2. Arabidopsis Apex Culture Medium (ACM) for imaging

    Ingredient
    Concentration
    Quantity
    MS-Medium base
    MS basal medium
    0.5x
    2.2 g/L

    Sucrose
    1%
    10 g/L

    Agarose
    0.8%-1%-2%
    8 g-10 g-20 g

    dH2O
    ---
    up to 1 L of volume

    KOH
    ---
    ---
    Vitamins (1,000x)
    Myo-Inositol
    10%
    5 g/50 ml

    Nicotinic acid
    0.1%
    0.05 g/50 ml

    Pyridoxin hydrochloride
    0.1%
    0.05 g/50 ml

    Thiamine hydrochloride
    1%
    0.5 g/50 ml

    Glycine
    0.2%
    0.1 g/50 ml

    dH2O
    ---
    50 ml
    1. Prepare the MS base as described in the previous recipe, being careful to add agarose to the three different concentrations (0.8%, 1% and 2%) instead of agar and store it at room temperature
    2. Dissolve the vitamins in dH2O to obtain the 1,000x stock solution
    3. Sterilize the vitamin stock using a 0.22 μm filter, aliquot them and store at -20 °C

Acknowledgments

This work was supported by the European Union Marie-Curie “COMREC” network FP7 ITN-606956 to M.A.P. and A.S. In addition, core funding of the University of Hamburg to A.S. is gratefully acknowledged. The here-presented protocol was presented in Prusicki et al. (2019) and further described in Prusicki et al. (2020).

Competing interests

Arp Schnittger on behalf of all authors confirms that none of the authors has competing financial interests in this work.

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  20. Sofroni K., Takatsuka H., Yang, C., Dissmeyer N., Komaki, S., Hamamura, Y., Böttger, L., Umeda M. and Schnittger A. CDKD-dependent activation of CKDA;1 controls microtubule dynamics and cytokinesis during meiosis. (Unpublished).
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简介

[摘要 ] 活细胞成像极大地促进了我们对细胞分裂等亚细胞过程的了解。在此,我们提出了一种循序渐进的方案,以一种可靠且易于使用的活细胞成像方法来研究雄性减数分裂。植物拟南芥。由于最近建立了我们的方法依赖于伴随分析中的两个报告基因,突出染色体构和微管动态组合,这些记者基因准许入住歧视五细胞参数:细胞形状,微管阵列,核POSITI 在,核仁位置,染色质凝聚,这些参数可以采用不同的国家,例如,该核位置可以是中央或外侧。分析如何紧密这些国家有关联产生了里程碑阶段,反过来允许定量和定性的解剖减数分裂过程我们设想,这种方法还可以为细胞分化分析提供有价值的标准 减数分裂之外的增强过程。

[背景 ] 减数分裂是一种特殊的细胞分裂周期,有两个主要目的:首先,减数分裂母细胞的DNA含量降低一半,在二倍体生物体中会导致单倍体减数分裂产物。有性繁殖的生物,以便在受精过程中融合了两个配子后,恢复了原始的基因组大小。其次,减数分裂通过亲本染色体(同源染色体或短同源)之间的DNA片段交换(称为减数分裂重组)促进了遗传多样性。通过生成新的但完整的染色体集,其中每个染色体随机存在母亲或父亲的同系物(如果是二倍体生物体)。因此,减数分裂对于不同领域的研究很有趣,从细胞生物学和生殖生物学,从遗传学到进化生物学(Wijn ker 和Schnittger ,2013; Mercier 等,2015;Melamed- Bessudo 等人,2016; Lambing 和Heckmann ,2018; Pelé 等人,2018; Wang和Copenhaver ,2018)。

植物是研究减数分裂各个方面的强大模型系统。此外,由于低水平的重组常常限制了育种程序,因此对植物减数分裂的研究也受到了应用兴趣的推动。

植物减数分裂的研究在很大程度上依赖于固定细胞的分析,例如拟南芥的染色体扩散和免疫定位研究(Armstrong 等,2009; Martinez-Garcia和Pra dillo ,2017; Parra-Nunez 等,2020; Sims 等,2020)以及其他植物物种(Cherysheva 等,2013; Sepsi 等,2018; Darrier 等,2020 ; Stack 等,2020 )。并继续是重要的工具,因为它们为解决减数分裂染色体的结构和减数分裂调节子的定位提供了巨大的空间分辨率,例如通过对减数分裂染色体的3D构型进行免疫细胞学分析证明的(Hurel 等,2018)然而,这些研究几乎没有提供关于减数分裂的基本动力的信息。此外,减数分裂的时间方面必须通过观察阶段的频率间接推论,这是一种固有的容易出错且容易误导的过程。Ë研究员例如,当两个或多个群体性母细胞存在于同一样品发生改变,当然减数分裂可被误认为是一个群体随着细胞在不同阶段(Prusicki 等,2019 ; Sofroni 。等人,未发表)。此外,寿命短的阶段,例如核被膜破裂很困难,而且,例如,在第一次减数分裂分裂后,是否在拟南芥中改造核被膜在该领域进行了长时间的讨论。

减数分裂的活细胞成像可以补充固定材料的分析,并与这些技术一起建立一种强大的方法,以获取对该重要细胞分裂程序的分子力学见解。玉米减数分裂的活细胞成像先前已在玉米中启动(Yu et al。 。,1997; 小号Heehan 而罗斯基,2009; Nannas 。等..,2016)。然而,只有短阶段减数分裂玉米可以记录具有遗传记者为微管和化学染色,突出DNA此外,最近协议活细胞成像减数分裂通过光片显微镜已经出版(Valuchova 等人。,虽然这组2020年)。- 最多是非常强大的跟随减数分裂在整个花芽,但仍然没有达到亚细胞分辨率所获得通过共聚焦激光扫描显微镜(CLSM)。

在此,我们详细介绍了一种基于CLSM 最近建立的程序追踪拟南芥花药减数分裂的方法(P rusicki et al。,2019)。重要的是,该方法可以将样品保存多达几天,从而可以分析减数分裂在全部内容。此外,利用拟南芥允许的代含有荧光记者针对不同的减数分裂调节植物的,最重要的减数分裂特定Kleisin 亚基的黏合复称重组8(REC8),ASYNAPTIC 1(ASY1)和ASY3,两染色体轴的组成部分和ZYP1是突触复合体中心区域的组成部分(Prusicki 等,2019; Yang 等,2019和2020)。最近该研究已用于研究黏附素的控制突变体,其中在REC8过早切割(克罗默等人。,2019)。

一个典型的挑战是对获得的电影进行定量分析。减数分裂是连续不断的事件,这是生物学过程中典型的减数分裂过程。为了解剖这些电影,我们着重研究了KINGBIRD报告系统(REC8标记为GFP结合以红色突出显示的微管; Prusicki 等人,2019)。这五个细胞参数是:细胞形状,微管阵列,核位置,核仁位置和染色质状态,这些参数中的每一个都可以适应不同的状态,请参见表1 (表 1 Excel文件),此出版物以及Prusicki 等人,2019年的图3 。

大号Ooking然后在该协会的这些不同的参数国透露,他们不是随机相关,但通常紧密联系在一起的。这引起了生物标体系,其中一个地标是一个突出的细胞形态具有鲜明的参数状态。反过来,这一里程碑式系统可以用作定性(相同或新地标的出现)和定量(这些地标的持续时间)剖析突变体或不同环境条件下减数分裂进程的映射图,然后可以使用其他减数分裂报告子来完善和/或这种分析的原理可以轻松地转化为其他细胞分化过程,当然还包括需要识别的其他细胞参数。

关键字:细胞分裂, 细胞分化, 减数分裂, 染色体, 微管, 细胞骨架, 拟南芥

材料和试剂


 


黑色标记
平方培养皿100 x 100 x 20 mm(Sardstedt ,目录号:82.9923.422)
圆形培养皿35 x 10毫米(Sarstedt ,目录号:82.1135.500)
圆形培养皿60 x 15毫米(Sarstedt ,目录号:82.1194.500)
Safeseal 管1.5毫升(萨尔斯塔特,目录号:72.706)
Parafilm M(Neolab,Bemis,3-1011,PM-996)
过滤消毒的Millex -GV 0.22 Myuemu (Merck公司,目录号:SLGV033RS)
针30 G½英寸0.3 x 13毫米(BD Microlance ,型号:304000)
种子的拟南芥植株的减数分裂的荧光报告构建作为一个标志减数分裂如必胜鸟线呈现在Prusicki 等人,2019以及记者对减数分裂染色体轴和联会复合体(杨等人,2019年和2020年)
NaClO 在H 2 O中的溶液13%(Applichem GmbH,ITW试剂,目录号:213322.0715)
HCl 37%(VWR Chemicals,目录号:20255.290)
琼脂糖(Sigma,目录号:A9539 )。
食品级琼脂粉(Applichem GmbH,ITW试剂,目录号:A00917,5000)
MS基础盐混合物(Duchefa Biochemie ,目录号:M0221.0050)
肌肌醇(DUCHEFA Biochemie ,目录号:I0609.0100)
烟酸(Duchefa ,目录号:N0611)
盐酸吡ido 醇(Duchefa ,目录号:P0612)
盐酸硫胺素(Duchefa,目录号:T0614)
甘氨酸(Sigma,目录号:G-7126)
无菌dH 2 O
乙酰胭脂红
异丙醇
蔗糖
酸值
Murashi ke 和Skoog培养基(MS培养基),用于植物发芽(请参见食谱)
用于成像的拟南芥Apex培养基(ACM)(请参阅食谱)
 


配套设备


 


移液器10/100/1000(例如Eppendorf Research加上移液器)
生长室MobyLux GroBanks (CLF Plant Climatics ,型号:BrightBoy )。
真空室
玻璃瓶
无尘工作台
细镊子,厚度:0.02 / 0.01毫米(Dumont,Dumoxel ,目录号:0203-4-PO和0203-5-PO)
显微镜解剖(例如,蔡司,型号小号:Stemi2000和Stemi508)
配有水浸物镜的激光扫描显微镜(例如,蔡司,型号:LSM880)(例如,蔡司,型号:W Plan-Apochromat 40 x /1.0 DIC物镜)
 


软体类


 


ZEN 2.3 SP1 FP1黑色(Zeiss)(或显微镜系统的相关成像软件)
MetaMorph 版本7.8.0.0(分子设备)
斐济1.52b版本(Image J,https: //imagej.net/Fiji )(Schindelin 等人,2012年)
Excel(Microsoft Office)
Python编程语言(版本3.6,Python软件基金会)
 


程序


 


制备植物材料
拟南芥种子的表面灭菌种子,带有在减数分裂阶段表达的荧光报告分子(Prusicki 等,2019中有一个例子)。一种有效的种子表面灭菌方法是用氯气:将种子倒入1.5 ml管中,并用黑色记号笔标记试管(由于灭菌过程,其他颜色会变暗)。打开试管盖,将试管放入真空室内的球拍中。 25 ml的7.5%漂白剂溶液(用自来水洗脱)。向搅拌器中加入1 ml的37%HCl,快速关闭反应室并施加真空,整个过程应在化学通风橱中进行。吸入氯气!将种子留在室中3-4小时。处理后即可准备锯切种子。如果您打算将消毒后的种子在试管中存放一段时间,则让试管打开超净工作台下一个最低中30分钟Ensur 气体已完全蒸发。
看到的种子在Murashike 和Skoog(½ MS,配方1)的平方的Petri 培养皿富含选择代理根据用电阻的插入的构建体例如。,添加25的Mg / 大号潮霉素乙以选择用于PRO REC8 :REC8 :GFP 为已完成(Prusicki et al。,2019)。不同的是,如果目的蛋白在幼苗中表达,则可以通过基因分型或基于荧光检测进行选择。
将平板在黑暗中于4°C下存放3天,然后将其转移至长日条件下(在22 °C / 18 °C下为16h 白天/ 8h夜间模式)进行发芽。
发芽后10天,将幼苗转移到土壤上,并在长日照条件下生长。
根据生长条件和基因型,植物将在3-4周内准备成像。
 


成像样品的制备
如会话中所示,准备含0.8%,1%和2%浓度的琼脂糖的Apex培养基(ACM)和1,000倍的维生素储备(配方2)。
通过高压灭菌器对ACM进行灭菌。
蒸压当(或者再热)ACM 0.8 Pasento琼脂糖手暖,添加复合维生素的1X工作浓度(例如,向50ml ACM添加50 Myueru 的1,000倍维生素贮存液),倒成小培养皿(35 x 15毫米),用封口膜密封并储存在4°C.
用倾1%琼脂糖的培养基中的ACM - 尺寸的Petri 在4℃下培养皿(60×15毫米), -密封用封口膜和存储
将ACM与2%琼脂糖分装在无菌的1.5 ml Eppendorf管中,并将其储存在4°C下。
 


样品安装
用ACM和1%琼脂糖将花序切开并固定在陪替氏培养皿上(视频1)。




 






视频1. 步骤C1和C2 ,移除大花蕾并选择花蕾进行图像采集


 


去除花梗根部的所有开放和细长花,但花蕾呈圆形,大小在0.4-0.6 mm之间,包含早期的肌细胞(视频1)。早期具有肌细胞的花蕾的特征可以根据生长条件和基因型而变化,例如,减数分裂被延迟的突变体通常发生在较大尺寸的花蕾中。因此,建议分析具有乙酰胭脂红或类似染色的数个花蕾,以估计哪些花蕾减数分裂通常,处于发育阶段9的花蕾(Smyth 等人,1990)包含经历减数分裂的雄性减数分裂细胞。
移开所选流动芽的上萼片,注意不要触碰花的内部器官,尤其是花药(Vi deo s 2和3)。
 






视频2.移除萼片以进入选定花蕾中的两个花药,步骤C3和C4


 






视频3.在步骤C3和C4 之后关闭准备成像的花蕾


 


移走剩余的花蕾(视频第2和3节)。如果在移除萼片时主花蕾受到损坏,则可以保留下一个最小/最小的花蕾作为备用花。
花序从电梯制备介质,干净切茎的长度协卡(Ca的)0.5厘米(用钳子或更好用针)(视频4)。这将有利于吸收的养分中,并会不断样本中的好条件一较长时间。
 






4.定位视频的选择和处理的花蕾成像,小号TEPS C5和C6


 


将样品转移到装有0.8%琼脂糖的ACM 的小培养皿中,将其锚定在板的中间,并用一滴含2%琼脂糖的ACM覆盖花蕾(视频4和图1A和1B)。
它可追多一个花蕾在一个显微镜会话。在这种情况下,准备一个新的样品如先前解释的(起始神父嗡程序C1 中牛逼他生物协议),并找到所有的花蕾尽可能接近在培养皿的中心(视频4)。




1图,样品的安装和定位在显微镜。A. 样品被放置在中间的皮氏培养皿中并通过一滴2 Pasento琼脂糖,覆盖B. 收上来的样品从面板一。Ç 概述和d 放大将样品放在显微镜镜台上,并将样品置于水浸物镜下方。


 


图像采集
重要的是要有稳定的采集条件,例如室温,湿度等。因此,建议事先打开共聚焦显微镜,然后将样品放在室内,并用几毫升水覆盖已安装的样品。
大约一小时后,您可以开始图像采集。
将样品放在显微镜载物台上,浸入水的物镜中,并用高压灭菌的水将培养皿充满至顶部(图1C和1D)。建议在采集前用异丙醇彻底清洁物镜以降低风险图像获取过程中细菌的生长。
使用视觉功能(或相关软件工具),确定样品中每个花蕾的位置,并使用ZEN black软件上的多位置功能将其保存。
该套装- 。最多的图像采集需要适应的宗旨实验并在2002的荧光团遵循一个完整的减数分裂在时间的特点- 流逝实验,并确定减数分裂的地标使用必胜鸟行中介绍Prusicki 。等人(2019),下面的一组- 最多可使用:
使用λ488 nm的氩激光激发GFP,使用DPSS 561-10激光(λ561)激发TagRFP 。
在顺序线模式下使用两个轨道进行信号检测,然后使用分束器MBS 488/561。在第一个轨道中,使用GaAsP 检测器记录在498和550 nm之间的λ滤波的GFP信号。在第二个轨道中,使用用GaAsP 检测器观察在578和649 nm之间被λ滤除的TagRFP 信号,可以在第二条光道中添加检测叶绿体自发荧光的第三通道,并过滤680至750 nm之间的λ。添加到两个轨道之一。
将Taghole 设置为1 Airy Unity上的Pinhole以进行TagRFP 检测,使用双向扫描功能并将像素驻留时间设置为2μs 左右的值。
用于图像采集的参数(例如激光强度,增益和偏移)必须适合于各个成像条件,其中尤其取决于激光状态,所使用的报告分子以及蛋白质表达水平。一般情况下,样品的生存能力和高分辨率成像之间的妥协必须找到以获得最佳的图像质量,同时维护样品在良好的条件。在我们的例子中,我们设置1 Pasento和4.5 Pasento之间的氩激光的强度,而将DPSS 561-10激光设置在0.3 %至1%之间,将收集GFP和TaqRFP 信号的检测器增益设置在700至850之间,并将其设置在650至750之间以检测叶绿体自发荧光。 offset参数为0。
对于KINGBIRD线,在2条线上执行平均;对于其他报告器,可能需要不同的设置。
以10分钟的间隔时间获取多个位置的时间流逝。在每个位置上,执行6到7个平面的Z堆栈,步长为每个时间点最大50μm。获得性的Ca. 30 H(Prusicki 等人,2019)。
建议在每个位置和每个时间点都使用自动对焦功能,尤其是对于较长的采集时间。
如果样品仍在进行减数分裂,但需要短暂中断数据采集(例如,在重新调整焦平面或到达最后一个采集周期的情况下),建议将中断时间调整为匹配间隔时间,因此要开始新的时间- 在10分钟间隔结束时获取圈数。
 


影像处理
将获取的时间的每个位置- 间隔保存为一个单独的文件,例如在ZEN black软件的情况下:处理 复制 子集。
在斐济打开图像并将其作为图像序列保存在单独的文件夹中(另存为 图像序列 格式TIFF 数字必须足以覆盖所有时间点,例如110个时间点需要3位数字)。
使用MetaMorph 打开图像序列(应用程序 查看多个图像序列文件 打开序列 按照说明进行操作),并为每个时间点选择显示感兴趣的单元格的z-stack层,最后,加载选定的z-stacks 。对于每个波长,并将其保存为TIFF文件此步骤有助于纠正漂移样品中的TH éž - 方向,并连同激活在图像采集期间(见自动对焦功能过程D6 )允许按照相同的细胞来时间间隔的完整持续时间。
新的文件打开在斐济,结合不同的渠道,调整自己的亮度和对比度,并产生ň RGB版本的文件。
答:如果ñ XY漂移检测的,它可以修正通过使用插件Stackreg 从斐济(插件 Stackreg  刚体)(Thevenaz 等,1998)。
视频5中给出了图像处理后的延时序列示例。
 






V IDEO 5. 实施例的时间流逝实验结果后图像处理如所解释的在小号TEPS E1-E6


 


资料分析


 


使用KINGBIRD 线进行地标识别


数据集注释
给一个花药中的每个可见单元格分配一个ID号。可以使用斐济文本工具(图2)将ID号压在RGB.tiff文件的每一帧上。
 






2图,数据分析。A. 一帧的收购定时短片描绘花药的拟南芥植物携带必胜鸟记者(微管在品红,REC8染色质是绿色和叶绿体是蓝色的)。每个性母细胞被赋予数A,这将是它的ID在电子表格中乙。B. 示例的电子表格用于分析所描述的段落中的数据分析A2。编号的阶段是指号码表1.每个阶段的颜色编码以方便数据组织。


 


使用Microsoft Excel准备一个包含7列的表格:帧数,以分钟为单位的时间,细胞形状,微管阵列,核位置,核仁位置和染色质状态(图2)。行数将等于获取的帧数对于单个单元格,第一个成像的帧将被计为时间0(图2)。
在每列中填写与表1和图2中注释的分析的细胞参数状态相对应的数字(f 或其他参考文献,请参阅Prusicki 等,2019),例如,如果在时间0时出现该单元格圆形,则必须在第三栏中填写数字4。在每个时间点都对每个单元格参数进行注释。如果在某个时间点看不到单元格,或者无法识别参数状态,请填写表n为不可见。
所有可见的注释细胞的另一种不同的Excel工作表小号(一张纸上每个细胞),该文件构成数据集进行分析作为解释下一届会议。
../../../../../Downloads/Creative%20commons%20grey1个


 




说明:logonew                                                                  


 


表1 。细胞特征性母细胞作为获得具有必胜鸟报告系统。该表提供了举例说明每个所描述的小区特征显微镜图像(甲Dapted 从图小号3和4 Prusicki 等人,2019 。知识共享署名许可)。该可以在此处访问原始表文件(表1 Excel文件)。






../../../../../Downloads/Creative%20commons%20grey1个


 




说明:logonew                


数据集分析
https://gitlab.com/wurssb/arabidopsis-thaliana--landmark-analysis上提供了一个Python脚本,用于对A部分中创建的带注释的数据集进行分析。该脚本包含从数据中提取地标的过程。并采取以下步骤:


通过过滤掉无效或重复的条目来准备要分析的数据。每个条目(由每个细胞参数的观察状态组成)应具有样品的标识符和样品中的特定细胞。
如果没有按固定的时间间隔对数据进行采样,请重新采样到一个公共间隔,但是在重新采样时要小心,因为从短间隔到大间隔可能会删除数据集中的短暂状态,而相反的间隔可能会导致(大)错误插值状态。
蜂窝状态由所有参数状态的集合来定义,例如,图2中在时间点10的单元11具有蜂窝状态:1-1-2-2-2。蜂窝状态发散最多两个不同的参数状态。被定义为相邻状态(或相邻状态)。在那里,蜂窝状态1-1-2-2-1、1-1-2-3-2和1-1-2-3-3而不是1-1- 3-3-3是1-1-2-2-2的相邻状态。
可以丢弃部分观察到的状态,也可以从相邻状态进行内插,同样,在进行内插时要格外小心,例如,根据观察到的过程设置可以安全内插的最大时间间隔。
计算在每种细胞状态下花费了多少时间。
对于每个观察到的蜂窝状态,请列出相邻状态。
对于每个蜂窝状态,使用在特定状态下花费的时间(t state )和其邻居所花费的时间(t neighbors )计算邻居分数:
 






 


现在可以为地标分配适当的分界值(Prusicki 等人的示例,2019)。表2给出了邻近分析最终结果的示例(表2 Ë Xcel公司文件),WH ERE的所有细胞状态观察到的一起列出了它们的计数(许多情况下,他们已经观察到的),他们的百分比的外观相对于其他蜂窝美和菲娜LLY它们相邻的得分。来源的列于表2的数据中所发表结果Prusicki 等人。,2019。
要评估分析的鲁棒性,请通过替换数据重新采样并多次重新运行整个分析来引导分析。
可以在单个观测值的水平上进行引导,也可以通过对特定细胞或花药的观测值进行重采样来发现分析中可能存在偏差的不同来源。






2表。例如结果的数据分析,在原表文件仍可被访问这里(表2 Excel文件)。






菜谱


 


Murashike 和Skoog培养基(MS培养基),用于植物发芽
1升成分浓度                           


MS基础培养基0.5x 2.2克/升                           


蔗糖1%10克/升                           


琼脂1%10克                           


DH 2 Ø --- 补足至1升容积的                           


KOH --- ---                           


将MS基础和蔗糖溶解在800 ml的dH 2 O中
用KOH调节pH值至5.8,使最终体积达到1 L
将琼脂加到溶液中
通过高压灭菌,并在室温下储存直至使用
拟南芥Apex培养基(ACM)用于成像
 


成分


专心


数量


MS- 中碱


MS基础培养基


0.5倍


2.2克/ 升


 


蔗糖


1%


10克/ 升


 


琼脂糖


0.8%-1%-2%


8克至10克至20克


 


DH 2 Ø


---


高达1 L的体积


 


酸值


---


---


维生素(1,000倍)


肌醇


10%


5克/ 50毫升


 


烟酸


0.1%


0.05克/ 50毫升


 


盐酸吡ido醇


0.1%


0.05克/ 50毫升


 


盐酸硫胺素


1%


0.5克/ 50毫升


 


甘氨酸


0.2%


0.1克/ 50毫升


 


DH 2 Ø


---


50毫升


按照之前的配方中所述准备MS碱,小心地将琼脂糖添加到三种不同浓度(0.8%,1%和2%)而不是琼脂中,并在室温下保存
将维生素溶解在dH 2 O中以获得1,000倍的原液
维生素股票灭菌器采用0.22 Myuemu 过滤,分装他们并储存在-20℃
 


致谢


 


这项工作得到了MAP和AS的欧盟Marie-Curie“ COMREC”网络FP7 ITN-606956的支持。此外,还非常感谢汉堡大学对AS的核心资助。本文介绍的协议在Prusicki 等人中提出。。(2019),并且进一步描述于Prusicki 等人(2020)。


 


竞争利益


 


Arp Schnittger代表所有作者确认,没有任何作者在这项工作中有相互竞争的财务利益。


 


参考文献


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Prusicki, M. A., Keizer, E. M., van Rosmalen, R. P., Fleck, C. and Schnittger, A. (2020). Live Cell Imaging of Male Meiosis in Arabidopsis by a Landmark-based System. Bio-protocol 10(9): e3611. DOI: 10.21769/BioProtoc.3611.
  2. Prusicki, M. A., Keizer, E. M., van Rosmalen, R. P., Komaki, S., Seifert, F., Muller, K., Wijnker, E., Fleck, C. and Schnittger, A. (2019). Live cell imaging of meiosis in Arabidopsis thaliana. Elife 8: 42834 .
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