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Time-lapse Observation of Chromosomes, Cytoskeletons and Cell Organelles during Male Meiotic Divisions in Drosophila
果蝇雄性减数分裂期间染色体、细胞骨架和细胞器的延时观察   

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Abstract

In this protocol, we provide an experimental procedure that perform time-lapse observation of intra-cellular structures such as chromosomes, cytoskeletons and cell organelles during meiotic cell divisions in Drosophila males. As primary spermatocyte is the largest dividing diploid cell in Drosophila, which is equivalent in size to mammalian cultured cells, one can observe dynamics of cellular components during division of the model cells more precisely. Using this protocol, we have showed that a microtubule-associated protein plays an essential role in microtubule dynamics and initiation of cleavage furrowing through interaction between microtubules and actomyosin filaments. We have also reported that nuclear membrane components are required for a formation and/or maintenance of the spindle envelope essential for cytokinesis in the Drosophila cells.

Keywords: Drosophila(果蝇), Male meiosis(雄性减数分裂), Time-lapse observation(延时观察), GFP-tagged protein(GFP标签蛋白), Chromosome dynamics(染色体动力学), Microtubules(微管), Cytokinesis(胞质分裂), Mitochondria(线粒体)

Background

In Drosophila, good cultured cell lines that proliferate well in a standard culture condition are also available. However, their cell size, particularly cytoplasmic volume, is much smaller than that of mammalian cells. This compromises the examination of cellular components during cell division. Spermatocytes, on the other hand, achieve distinct cell growth before initiation of first meiotic division. The primary spermatocytes are the largest diploid cells among proliferative cells to appear in Drosophila development. Thus, one can easily perform detailed observation of cellular structures in dividing cells using optical microscopes. In Drosophila melanogaster, well-advanced and sophisticated genetic techniques are available (Ashburner et al., 2004). Meiotic defects in chromosome segregation and in cytokinesis appear in cellular organization of spermatids just after completion of 2nd meiotic division. By observation of such early spermatids, one can easily find out even subtle meiotic abnormalities (Giansanti et al., 2012; Inoue et al., 2012). Furthermore, if a loss of microtubule integrity or dynamics would have occurred in normal cultured cells, their cell cycle progression should be arrested before metaphase. Therefore, it is hard to examine how microtubules would influence later processes of cell divisions in the somatic cells. Spermatocytes, on the other hand, are less sensitive to microtubule abnormalities at microtubule assembly checkpoint before metaphase. One can, therefore, examine a role of microtubule-related genes in cytokinesis without arresting cell cycle. We and other groups have established systems to facilitate dynamics of chromosomes or microtubules by expression of proteins fused with GFP fluorescence tag (Clarkson and Saint, 1999; Inoue et al., 2004; Rebollo and Gonzalez, 2004; Kitazawa et al., 2012).

Previous protocols can trap the male meiotic cells in a narrow space sandwiched between a coverslip and a slide glass, ensured by a small cushion materials and observe chromosome segregation under an upright microscope (Savoian et al., 2000; Inoue et al., 2004; Savoian, 2015). These protocols allowed us to collect clear images of microtubules. However, a preparation that makes the cells flattened often prevents initiation and/or progression of cytokinesis. In addition, it was difficult to add drugs or inhibitors to the living cells while time-lapse observation.

Therefore, we have established a new method that allows us to observe a whole meiosis I from prophase I to end of cytokinesis in an open chamber under an inverted microscope. We can add drugs in the cell culture in any timing of the imaging. We also improved the protocol so that we can perform a simultaneous observation of chromosomes and other cellular components such as microtubules, actin filaments, endoplasmic reticulum, Golgi apparatuses or mitochondria during male meiosis I. It can be achieved by a simultaneous expression of proteins fused with different fluorescent tags showing spectrally separable colors. As most aspects of division process seen in Drosophila meiotic cells are shared among higher eukaryotes, this protocol should be useful for studying cell division processes of other organisms as well as Drosophila somatic cell mitosis.

Materials and Reagents

  1. Cover slips (22 x 22 mm, No. 1, thickness 0.12-0.17 mm) (Matsunami Glass, catalog number: C022221 )
    Note: It was argued that thorough cleaning steps of cover slips are required for maintenance of cell viability and for a success prolonged observation of living cells (Savoian, 2015). However, if these cover slips are used, any pretreatment is not basically necessary except a wipe with 70% ethanol just before using.
  2. Plastic cover slip folder (76 x 26 mm)
    Note: The folders were customized. It was 76 x 32 mm in length and width and 1.7 mm in thickness. There was a concavity (25 mm square) where the cover slip fell in the surface and a circular hole of 15 mm diameter in the center of the concavity) (see Figure 1).


    Figure 1. A plastic cover slip holder. The holder should be set on the microscope stage. There was a concavity where the cover slip (22 x 22 mm) fell in the surface and a circular hole with 15 mm diameter in the center of the concavity.

  3. 10 x 10 mm open frame that had adhesives on the bottom side (Gene Frame 25 μm) (Thermo Fisher Scientific Co., Waltham, USA)
  4. Plastic Petri dish (90 mm diameter) (AsOne, catalog number: 1-7484-01 )
  5. Kim-wipe (KCWW, Kimberly-Clark)
  6. bam-Gal4::vp16 (abbreviated as bam-Gal4) can be used as a Gal4 driver for testis-specific ectopic expression of fluorescence proteins (Kitazawa et al., 2012)
  7. bam-Gal4::vp16 UAS-dir2 was used as a Gal4 driver for testis-specific depletion (Kitazawa et al., 2014)
  8. P{His2AvT:Avic\GFP-S65T} (abbreviated as Histone2Av-GFP) can be used for expression of Histone 2Av fused with a GFP tag to visualize chromatin in living meiotic cells (Bloomington Drosophila Stock Center, catalog number: BL5941 )
  9. P{Ubi-mRFP-βTub85D} (abbreviated as RFP-βTubulin) can be used for ubiquitous expression of β-tubulin fused with a mRFP tag to visualize microtubules in living meiotic cells (Kitazawa et al., 2014)
  10. P{sqh-EYFP-Golgi} can be used for ubiquitous expression of Golgi components fused with a YFP tag to visualize Golgi apparatus in living meiotic cells (Bloomington Drosophila Stock Center, catalog number: BL7193 )
  11. P{sqh-EYFP-ER} can be used for ubiquitous expression of ER components fused with a YFP tag to visualize endoplasmic reticulum in living meiotic cells (Bloomington Drosophila Stock Center, catalog number: BL7195 )
  12. P{sqh-EYFP-Mito} can be used for ubiquitous expression of mitochondrial target sequences fused with a YFP tag to visualize mitochondria in living meiotic cells (Bloomington Drosophila Stock Center, catalog number: BL7194 )
  13. P{UASp-GFP-Orbit}, P{UASp-mRFP-Orbit}, and P{UASp-Venus-Orbit} can be used for visualization of Orbit protein, an essential microtubule-associated protein. UAS-stocks that can induce Orbit proteins fused with three different fluorescent tags are available (Miyauchi et al., 2013)
  14. P{UASp-mRFP-Actin5C} (Bloomington Drosophila Stock Center, catalog number: BL24777 ), P{UAS-GFP-Actin5C} (Bloomington Drosophila Stock Center, catalog number: BL9257 ) and P{UAS-CFP-Actin5C} (Miyauchi et al., 2013) can be used for ectopic expression of F-actin components fused with different tags to visualize F-actin in living meiotic cells
  15. P{UAS-GFP-anillin} (Bloomington Drosophila Stock Center, catalog number: BL51348 ) and P{Ub-mRFP-anillin} (Bloomington Drosophila Stock Center, catalog number: BL52220 ) can be used for visualization of a contractile ring in a living meiotic cell (gifts from J.A. Brill, now available from Bloomington Drosophila Stock Center)
  16. P{w[+mC]=sqh-GFP.RLC}3 can be used for ubiquitous expression of myosin light chain components fused with GFP tag to visualize MLC in living meiotic cells (a gift from R. Karess)
  17. P{PTT-GA}Pdi[G00198], a protein trap stock expressing GFP-Protein disulfide isomerase for visualization of intracellular membranous structures (a gift from L. Cooley, now available from Bloomington Drosophila Stock Center as catalog number: BL6839 ). A protein stock expressing GFP-LamC for visualization of nuclear lamina (a gift from L. Wallrath)
  18. P{UAS-PLCg-PH-GFP} for visualization of plasma membrane in male meiotic cells (a gift from J. A. Brill)
  19. P{UAS-mRFP-Nup107.K} 7.1 for visualization of nuclear pore complex in nuclear envelope of male meiotic cells (a gift from V. Doyle, now available from Bloomington Drosophila Stock Center as catalog number: BL35516 )
  20. P{UAS-GFP-Pav} and P{UAS-GFP-Polo} were used for visualization of a microtubule motor and an important cell division regulator in a living meiotic cell, respectively (gifts from D. Glover)
  21. For RNAi experiments in male meiotic cells, UAS-RNAi stocks for ectopic expression of dsRNA for each protein were obtained from VDRC stock center and Bloomington stock center. P{UAS-GFP RNAi} (Bloomington Drosophila Stock Center, catalog number: BL9330 ) can be used as a negative control of RNAi experiments
  22. Colchicine (50 μg/ml in BRB80 buffer) (≥ 95% colchicine) (Sigma-Aldrich, catalog number: C9754 )
    Note: To examine requirement of microtubules for cellular dynamics, colchicine that is an inhibitor of microtubule polymerization was used. The BRB80 buffer containing colchicine was prepared before the dissection every time. The testes were dissected in the buffer containing colchicine and then, meiotic cells were spread under mineral oil.
  23. Cytochalasin D (10 μg/ml in BRB80 buffer) (≥ 98% cytochalasin D) (Sigma-Aldrich, catalog number: C8273 )
    Note: To examine requirement of F-actin for cellular dynamics, cytochalasin D that is an inhibitor of actin polymerization was used. The BRB80 buffer containing cytochalasin D was prepared before the dissection every time. The male flies were dissected to collect the testes in the buffer containing cytochalasin D and then, meiotic cells were spread under mineral oil.
  24. Fetal calf serum (Thermo Fisher Scientific, GibcoTM, catalog number: 451456 or 10437 )
    Note: The fetal calf serum can be kept at 4 °C for a month.
  25. Mineral oil (Trinity Biotech, catalog number: 400-5-1000 )
    Note: The mineral oil was replaced to a fresh one every time-lapse recording.
  26. Brefeldin A (Cell Signaling, catalog number: 9972 ) or Exo1 (Sigma-Aldrich, catalog number: E8280 )
    Note: To examine whether membrane trafficking mediated by COPI is required for cellular dynamics, each compound was used to inhibit αCOPI. They were directly added to the culture medium. The BRB80 buffer containing Brefeldin A or Exo1 was prepared before the dissection every time. The testis can be incubated in the culture medium for up to 14 h before isolation of spermatocytes.
  27. PIPES (Dojindo Mecular Technologies, catalog number: 340-08255 )
  28. Magnesium chloride hexahydrate (MgCl2·6H2O) (Nacalai Tesque, catalog number: 20908-65 )
  29. Ethylene Glycol Bis (EGTA) (Nacalai Tesque, catalog number: 08907-42 )
  30. Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 162-17942 )
  31. Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
  32. Sodium phosphate dibasic (Na2HPO4)
  33. Potassium dihydrogen phosphate (KH2PO4)
  34. M3 medium (Sigma-Aldrich, catalog number: S8398 )
  35. BRB80 buffer (pH 6.8) (see Recipes)
  36. Insect M3 medium (see Recipes)
  37. Phosphate-buffered saline (PBS) (see Recipes)

Equipment

  1. Super fine forceps (Fine Science Tools, model: Dumont #5 )
  2. Dissection needles sharpened tungsten wire with 0.5 mm diameter
  3. Inverted fluorescence microscope (Olympus, model: IX81 ) outfitted with excitation, emission filter wheels (Olympus, Tokyo, Japan)
  4. Objectives; UPLFLN40XPH (NA=0.75), UPLSAPO60XO (NA=1.4), UPLSAPO100XO (NA=1.4) (Olympus, Tokyo, Japan)
  5. Hg lump (Olympus, catalog number: USH-1030L )
  6. Cooled CCD camera (Hamamatsu Photonics, model: C10600-10B )
  7. Autoclave

Software

  1. Metamorph software version 7.6 (Molecular Devices, Sunnyvale, USA)

Procedure

  1. To label cellular components of interest with fluorescence, one can induce fluorescence-tagged proteins, which consists of each target component by Drosophila ectopic gene expression system, Gal4/UAS (Ashburner et al., 2004; White-Cooper, 2012). Alternatively, one may use strains expressing such fluorescence proteins continuously. In former cases, F1 progenies obtained by crossing bam-Gal4::vp16 with an UAS stock in which a cDNA encoding the protein with a fluorescence tag is placed under a control of the UAS sequences can be used for the time-lapse observation (see the schematic diagram for genetic cross in Figure 2). In testes cells in the F1 progeny males, expression of the fluorescence protein is specifically induced. The F1 progenies are reared at 28 °C (not beyond this temperature) in order to induce effective expression. For depletion experiments, another UAS stock carrying a transgene so as to induce dsRNA for each target protein is used instead of the UAS stock described above. After selection of male flies for the dissection, any preparation should be performed at an ambient room temperature below 25 °C.
    Note: One should adjust the room temperature around the microscope stage to 25 °C at least 1 h before observation.


    Figure 2. Genetic cross to generate F1 progenies producing fluorescence-tagged protein to label intracellular structures or F1 progenies expressing dsRNA to deplete a target protein. For labeling intra-cellular structures, induced expression of cDNA for a fusion protein X, which was consisting of the structures, with GFP fluorescence tag is carried out by Gal4/UAS system. For a depletion experiment, dsRNA representing a portion of target mRNA was induced instead of a fluorescence protein X.

  2. A pair of testis was carefully collected from pharate adults or newly eclosed adult flies (0-1 day old) described above at a room temperature (Figure 3). To collect adult testes, a movie uploaded to the internet web site would be useful as a guide (https://www.youtube.com/watch?v=-ej8nF1YsRg). The fly was placed ventral side up in BRB80 buffer on a plastic Petri dish under a dissecting microscope. Using a pair of the forceps, their abdomens were clamped by forceps and gently pulled the external genitalia in opposite direction by another one. After carefully removed associated accessory tissues away from a pair of testes, a pair of coiled testes were isolated as shown in Figure 3C. In a case of colchicine or cytochalasin D treatment, this dissection step was carried out in a BRB80 buffer containing 50 μg/ml colchicine and 10 μg/ml cytochalasin D, respectively. For a longer treatment, the spermatocytes isolated from testes can be incubated in M3 culture medium containing 10% fetal calf serum and 50% male cell extracts with a drug until 14 h (Kitazawa et al., 2012).
    Note: It is important to select young flies as much as possible because testes in aged flies contained less numbers of living spermatocytes instead of a large amount of sperm bundles. To collect young flies, all adult flies were discarded from fly culture tubes and newly eclosed flies were collected 12 h to 24 h after the discard. Remove a pair of testis gently and quickly using forceps. It is recommended that one complete the testis dissection (steps 1 to 4) within three minutes.


    Figure 3. A reproductive tract including a pair of testis collected from a normal male adult. A. A ventral view of a wild-type adult male. An arrow indicates external genitalium. Scale bar = 1 mm. B. A reproductive tract collected from a normal pharate adult male. The male reproductive tract is consisting of a pair of testis, seminal vesicle (sv) and accessory gland (ag). Ejaculatory duct (ed) and ejaculatory bulb (eb) are also attached. Scale bar = 1 mm. C. A pair of testis isolated from an adult male after removal of associated tissues such as seminal vesicles and accessory glands away. Note that flies carrying a w mutation have colorless testes, while wild type testes display pale yellow as shown in this photo. Scale bar = 100 μm. D. A phase construct micrograph of a half pair of testis. There would be at least one or more meiotic cysts consisted of 16 primary spermatocytes undergoing meiosis I at one-third the away from the apical tip of the testis (arrow). The dissection should be carried out to tear the sheath open at this point. Apical tip (asterisk). Scale bar = 100 μm.

  3. One or two testes were transferred and laid out under mineral oil (Trinity Biotech, Bray, Ireland) filled in open chambers on a clean glass cover slip (Figure 4). To prevent a drop of the oil to spread over the cover slip, it was surrounded by a 10 x 10 mm open frame that had adhesives on the bottom side (Gene Frame 25 μm, Thermo Fisher Scientific Co., Waltham, USA). Extra liquid associated around the testis should be sucked up using a string of Kim-wipe carefully in order to avoid an unexpected slip down of the testis cells due to the liquid remaining beneath the cells during a time-lapse recording (Figure 3D).
    Note: It is recommended to collect intact and a health-looking testis complex in which ejaculatory pump is still actively contracting, if possible (Figure 3B). The testes punctured in the process of dissection should not be selected.


    Figure 4. Schematic diagram of procedure to make cysts and cells within testis to spill into the mineral oil. 1. A half of testis pair illustrated here in a darker gray is located straight in a drop of mineral oil. One or two pairs of testis can be dissected in the drop of the oil. 2. Using a pair of tungsten needles, a sheath covering a testis is torn open from the apical tip (asterisks) until one-third of the testis. 3. By gently moving the testis sheath outward using needles, cysts consisting of 16 primary spermatocytes can be spread into the oil. The cells and cysts spread out are indicated in a light gray. On the other hand, each cyst should be kept intact as much as possible. The testis sheath is removed from the oil drop. 4. Using a pair of tungsten needles attached at the tip of holders in each hand, the sheath covering a testis is torn at a position at one third from the apical tip (arrow in Figure 3D) so as to allow a whole cyst consisting of 16 primary spermatocytes to release into the oil outside of the testis, while keeping intact. The oil should cover every testis cell at any time to prevent desiccation. Under these conditions, the cells within intact cysts remained viable for several hours (Kitazawa et al., 2012; Savoian, 2015).

    Note: A few germline stem cells are located at the apical tip of testis (Ueishi et al., 2009). And 16 cell cysts undergoing 1st meiotic division are localized around one third from the apical tip (Figures 3C, 3D and Figure 4).


    Figure 5. A phase contrast micrograph of a living 16-cell cyst of spermatocytes at early stage of meiosis I under mineral oil. Primary spermatocytes that have initiated meiotic division I are indicated by arrows. These cells should be selected for the time-lapse recording. (Inset) A premeiotic cell from a primary spermatocyte cyst at earlier stage. Note that it has a larger nucleolus (arrowhead). An intact 16-cell cyst is encircled by a yellow line. Bar = 10 μm.

  4. The cover slips set on the plastic cover slip holder are placed on an inverted fluorescence microscope (Olympus, Tokyo, Japan), outfitted with excitation, emission filter wheels (Olympus, Tokyo, Japan). The fluorescence signals are collected using a 40x dry objective lens or a 100x oil immersion lens.
  5. As Drosophila spermatocytes are extremely sensitive to light (Rebollo and Gonzalez, 2004), one of most crucial points for the time-lapse imaging is to restrict the dose of irradiation to the living cell specimens as much as possible. Thus, after setting the holder on the microscope stage, one should look for prophase I cysts in which mature spermatocytes would initiate meiotic division I soon after using phase contrast optics under a transmission light through ND25 filter. In the prophase I cells, round-shaped multi-layers of nuclear envelope are formed, while oval-shaped elongated envelopes are observed at stages after prometaphase I. Primary spermatocytes within an intact cyst rather than cells stayed alone should be selected for the imaging as much as possible.
  6. A recording of time-lapse images started from timing when a fluorescence of GFP-Tubulin initiated to accumulate at spindle poles located opposite to each other (t = 0 min). Alternatively, one should start image collection from timing when nucleoli became almost disappeared under a phase contrast (t = 0 min) (Figure 6).
    Note: It is advisable to initiate time-lapse imaging within ten minutes from the initial step of the dissection.


    Figure 6. Phase-contrast observation and fluorescence observation of testis cells including primary spermatocytes expressing GFP-βTubulin just before or at onset of meiosis I. Primary spermatocytes at prophase I or cells that would initiate meiotic division I soon after should be found by a phase contrast observation under a transmission light in order to avoid a prolonged irradiation of excitation light from a Hg lamp. The spermatocytes before and at prophase I show round-shaped cell morphology, while cells elongate to shape oval morphology as meiosis progress after prometaphase I. Premeiotic spermatocytes at S5 stage (small arrows) contain single larger nucleoli. As meiotic cell cycle progress, the nucleoli become smaller and disintegrated at S6 stage (arrows). The spermatocytes at prophase I, which contained tiny almost invisible nucleoli (arrowheads, A), also show that intensity of a fluorescence of GFP-Tubulin at the spindle poles separated apart toward opposite direction becomes vigorous (B). Asterisk (*) indicates bundled tails of elongated spermatids. Image collection can start from timing when a nucleolus has disappeared under a phase contrast (t = 0 min). In the case that a fluorescence of GFP-Tubulin is simultaneously collected, the recording should be initiated at timing when the GFP fluorescence becomes stronger at the poles (t = 0 min). Scale bar = 10 μm.

  7. At each 30-sec time interval, fluorescence-tagged proteins in the interested cells are excited by irradiation light from mercury lamp. Specimens are illuminated with UV filtered and shuttered light using the appropriate filter wheel combinations through a GFP/RFP filter cube. Near-simultaneous GFP and/or RFP fluorescence images are captured with a CCD camera (Hamamatsu Photonics, Shizuoka, Japan). For instance, a sequential collection of fluorescence images (10 msec exposure) and phase contrast images (300 msec exposure using ND12 filter) can be carried out for time-lapse observation to examine dynamics of cellular components in the primary spermatocytes. Image acquisition is controlled through the Metamorph software version 7.6 (Molecular Devices, Sunnyvale, CA, USA).
    Note: One can adjust the focus and the microscope stage so as to make target cellular components clearly visible while looking at the computer screen.
  8. For a drug treatment, we carried out short term in vitro culture of primary spermatocytes (Rebollo and Gonzalez, 2004; Kitazawa et al., 2012).
    1. A testis complex attached with accessory gland, ejaculatory duct and pomp was collected from adult males. A living testis complex in which ejaculatory pomp was actively contracting should be selected and transferred into the M3 culture medium.
    2. For a longer incubation with special drugs such as Brefeldin A or Exo1, these inhibitors for intracellular vesicle transport can be directly added to the modified culture medium consisting of the M3 medium without bicarbonates containing 10% fetal calf serum and 50% male cell extracts as prepared in according with (Kitazawa et al., 2012).
    3. The testis was incubated in the culture medium for 14 h before isolation of spermatocytes at room temperature.

Note: Using this protocol, one can carry out continuous observation of primary spermatocytes undergoing proper chromosome segregation and cytokinesis for at least an hour without any distinct abnormalities (Kitazawa et al., 2012 and 2014; Hayashi et al., 2016). A time-lapse imaging can continue to the end of meiosis II through meiosis I without medium changes, although a prolonged incubation of the cells without media replacement may result in cellular toxicity due to extra accumulation of metabolic wastes.

Data analysis

  1. This protocol usually allows us to perform a time-lapse observation of chromosomes, microtubules, F-actin, Golgi stacks, ER-based structures and mitochondria in primary spermatocytes undergoing meiosis I with good reproducibility. Spermatocytes carrying a transgene(s) (reagents 7, 8, 15, 16) to induce expression of fluorescence-tagged proteins consisting the intracellular structures can be used for the time-lapse experiments. Alternatively, spermatocytes are prepared from males generated by a cross between bam-Gal4 stock (reagent 5) and the UAS stocks (reagents 12-14, 17-19) to induce fluorescence proteins by Gal4/UAS system.
  2. One can also perform a simultaneous recording of multiple fluorescence having different wavelengths emitting from, for example GFP-Histone 2Av and RFP-βTubulin proteins. At prophase I, chromatins that have been homogeneously distributed before become assembled to form chromosomes at timing when accumulation of RFP-βtubulin becomes distinct at spindle poles (t = 0 min). Four foci of GFP-Histone 2Av corresponding to bivalents between two major autosomes, X-Y chromosomes and tiny 4th chromosomes should be formed at prometaphase I (t = 15 min). Bivalents of smaller 4th chromosomes are not usually observed because of overlapping with major chromosomes.
  3. At prometaphase I, the condensed bivalent chromosomes appeare within a nucleus in which nuclear membrane seems to be intact (t = 5 min). Astral microtubules emanating from spindle poles are constructed at prometaphase I. Developing asters have moved around nuclear membrane as to reach at opposite poles. Then, all chromosome complements appears to congress into a single chromosome mass at the center of the bipolar spindle structure until metaphase I (t = 55 min). Each chromosome performs poleward movement with an average velocity of 11.2 ± 1.2 µm/min until bipolar kinetochore attachment (Savoian et al., 2000). State of microtubule assembly is surveyed at the spindle checkpoint, although the checkpoint at male meiosis is less strict than that in somatic cells (Rebollo and Gonzalez, 2000). Anaphase I takes around 8 min and that the chromosomes moves poleward at 1.9 ± 0.1 µm/min after dyad disjoining (Savoian et al., 2000). At this stage, the nuclear membrane around spindle poles has already disintegrated and the spindle microtubules are free to elongate into the inside of the nuclear space. At onset of anaphase I, multi-layers of nuclear membrane surrounding the nuclear space separate spindle microtubules including thick kinetochore microtubules form astral microtubules. Two populations of central spindle microtubules appear after disjunction of bivalents (t = 60 to 70 min). A peripheral set of the microtubules become more dynamic as if they look for the cytoplasm towards the cell equator (Inoue et al., 2004). Another set of the microtubule bundles corresponding spindle microtubules is localized interiorly at the middle of the cell. The peripheral microtubules from opposite poles meet at equator and formed bubble-like structures protruding outwards (t = 60 min). The interior and most of the peripheral central spindles are then released from each pole and they form independent bundles at the equator. Furrow ingression is then observed soon after the peripheral microtubules from both poles contact the cell cortex. Chromosomes are de-condensing as the furrowing progresses (t = ~80 min).
  4. According to the procedure and stocks described above (reagents 9-11, 16-18), dynamics of Golgi stacks, endoplasmic reticulum-based structures, nuclear envelopes, plasma membranes and mitochondria in male meiosis can be also reproducibly examined (Inoue et al., 2012; Kitazawa et al., 2012; Hayashi et al., 2016). Distribution of F-actin and other cytoskeletons including contractile rings at cleavage furrow sites can also be visualized using several markers generated by stocks described above (reagents 13-15). A cellular localization of regulatory proteins for microtubules dynamics in cell division such as Orbit, Pavarotti and Polo can be examined in living spermatocytes using stocks (reagents 12 and 19). Excessive overexpression of a microtubule-associated protein, Orbit that stabilizes microtubules result in generation of abnormal spindle structures at a certain frequency (more than 10%). The details of the dynamics have been described elsewhere (Inoue et al., 2012; Kitazawa et al., 2014)
  5. In RNAi experiments by ectopic expression of dsRNA for target genes specifically in spermatocytes, one can examine whether dynamics of chromosomes, cytoskeletons and other cell organelles would be affected. Before the time-lapse recording, one should test whether UAS-RNAi stocks could perturb chromosome segregation, cytokinesis or mitochondrial partition in the presence of bam-Gal4 driver. It can be investigated by observation of post-meiotic spermatocytes under phase contrast microscope (see supplement table in Kitazawa et al., 2014). On the basis of overlapped phenotypes observed in multiple RNAi experiments using different UAS-RNAi stocks for the same genes, one should argue a role of the gene in male meiotic division.
  6. In a drug treatment, primary spermatocytes are pre-treated in a dissection buffer containing the drug. Alternatively, the time-lapse recording is carried out in the presence of the drug. One should consider the abnormalities observed exclusively in cells treated with the drug as cell phenotypes by the drug treatment. The cell phenotypes should be observed in a manner dependent on drug concentration. One should confirm that they appear at a higher frequency and that the phenotypes are enhanced as its concentration increases.

Notes

As a result of this protocol, dynamics of both chromosomes and microtubules as described above can be observed at good reproducibility. One should stop the time-lapse recording and discard the cells, if the cells quit cell division or abnormal microtubule structures are detected, such as multi-polar spindles that appear due to improper physiological condition. Homozygotes for P{His2Av-GFP} as well as those for P{His2Av-mRFP} generated abnormal spermatocytes which meiotic progression was arrested in the middle at a low frequency (less than 10%).

Recipes

Note: The ingredients of buffers or media and catalog number of each reagent described above are as follows. For preparation of all buffers, media and reagents, ultrapure water prepared by a water purification system such as Sartorius arium® should be used. Buffers described below can be kept at room temperature unless otherwise noted. Any materials used in this protocol were subject to MTAs.

  1. BRB80 buffer (pH 6.8)
    80 mM PIPES
    1 mM MgCl2
    1 mM EGTA
  2. Insect M3 medium
    Insect M3 medium containing 10% fetal calf serum and 50% male cell extracts (Kitazawa et al., 2012) was prepared just before each time-lapse recording experiment under sterile working conditions
  3. Phosphate-buffered saline (PBS) (pH 7.4)
    137 mM NaCl
    2.68 mM KCl
    10.14 mM Na2HPO4
    1.76 mM KH2PO4
    To make 1 L of PBS, 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4 were combined and dissolved in H2O to a total volume of 1 L. The pH was adjusted to 7.4. The buffer was sterilized by autoclaving. The buffer can be stored at room temperature

Acknowledgments

We are grateful to M. S. Savoian (Massey University, New Zealand) for sharing information about experimental procedures. We acknowledge V. Doyle (Institut Jacques Monod, Paris, France), L. Wallrani (University of Iowa, Iowa City, USA), D. Glover (University of Cambridge, Cambridge, UK), L. Cooley (Yale University, USA) and J. A. Brill (Toronto University, Toronto, Canada). We also thank Vienna Drosophila RNAi Center, Bloomington Stock Center and Drosophila Genetic Resource Center for providing fly stocks.
No competing or financial interests that may impact the design and implementation of their protocol. This work was partially supported by Japan Society for the Promotion of Science [grant number 26440188 to Y.H.I.]. This protocol was adapted or modified from our previous studies (Inoue et al., 2004; Kitazawa et al., 2012; Kitazawa et al., 2014; Hayashi et al., 2016).

References

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  2. Clarkson, M. and Saint, R. (1999). A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behavior. DNA Cell Biol 18(6): 457-462.
  3. Giansanti, M. G., Sechi, S., Frappaolo, A., Belloni, G. and Piergentili, R. (2012). Cytokinesis in Drosophila male meiosis. Spermatogenesis 2(3): 185-196.
  4. Hayashi, D., Tanabe, K., Katsube, H. and Inoue, Y. H. (2016). B-type nuclear lamin and the nuclear pore complex Nup107-160 influences maintenance of the spindle envelope required for cytokinesis in Drosophila male meiosis. Biol Open 5(8): 1011-1021.
  5. Inoue, Y. H., Miyauchi, C., Ogata, T. and Kitazawa, D. (2012). Dynamics of cellular components in meiotic and premeiotic divisions in Drosophila males. In: Swan, A. (Ed.). InTech. Open Access Publisher 67-86.
  6. Inoue, Y. H., Savoian, M. S., Suzuki, T., Mathe, E., Yamamoto, M. T. and Glover, D. M. (2004). Mutations in orbit/mast reveal that the central spindle is comprised of two microtubule populations, those that initiate cleavage and those that propagate furrow ingression. J Cell Biol 166(1): 49-60.
  7. Kitazawa, D., Matsuo, T., Kaizuka, K., Miyauchi, C., Hayashi, D. and Inoue, Y. H. (2014). Orbit/CLASP is required for myosin accumulation at the cleavage furrow in Drosophila male meiosis. PLoS one 9: e93669.
  8. Kitazawa, D., Yamaguchi, M., Mori, H. and Inoue, Y. H. (2012). COPI-mediated membrane trafficking is required for cytokinesis in Drosophila male meiotic divisions. J Cell Sci 125(Pt 15): 3649-3660.
  9. Miyauchi, C., Kitazawa, D., Ando, I., Hayashi, D. and Inoue, Y. H. (2013). Orbit/CLASPis required for germline cyst formation through its developmental control of fusomes and ring canals in Drosophila males. PLoS one 8(3): e58220.
  10. Rebollo, E. and Gonzalez, C. (2004). Time-lapse imaging of male meiosis by phase-contrast and fluorescence microscopy. Methods Mol Biol 247: 77-87.
  11. Savoian, M. S. (2015). Using photobleaching to measure spindle microtubule dynamics in primary cultures of dividing Drosophila meiotic spermatocytes. J Biomol Tech 26(2): 66-73.
  12. Savoian, M. S., Goldberg, M. L. and Rieder, C. L. (2000). The rate of poleward chromosome motion is attenuated in Drosophila zw10 and rod mutants. Nat Cell Biol 2(12): 948-952.
  13. Ueishi, S., Shimizu, H. and Inoue, Y. H. (2009). Male germline stem cell division and spermatocyte growth required insulin signaling in Drosophila. Cell Struct Funct 34: 61-69.
  14. White-Cooper, H. (2012). Tissue, cell type and stage-specific ectopic gene expression and RNAi induction in Drosophila testis. Spermatogenesis 2(1):11-12.

简介

在这个协议中,我们提供了一个实验程序,在果蝇男性的减数分裂细胞分裂期间,进行细胞内结构如染色体,细胞骨架和细胞器的延时观察。由于主要精母细胞是果蝇中最大的分裂二倍体细胞,其大小与哺乳动物培养细胞相当,可以更准确地观察模型细胞分裂期间细胞组分的动力学。使用该方案,我们已经表明,微管相关蛋白在微管动力学中起重要作用,并通过微管和肌动球蛋白丝之间的相互作用开始裂解沟槽。我们还报道说,需要核膜组分来形成和/或维持在果蝇细胞中细胞分裂所必需的主轴包络。

在果蝇中,也可以在标准培养条件下良好培养的良好培养细胞系。然而,它们的单元尺寸,特别是细胞质体积,比哺乳动物细胞的小得多。这在细胞分裂过程中损害了细胞成分的检查。精母细胞,在另一方面,实现第一次减数分裂开始之前不同的细胞生长。主要精母细胞是出现在果蝇发育中的增殖细胞中最大的二倍体细胞。因此,可以使用光学显微镜容易地细分观察分裂细胞中的细胞结构。在果蝇黑腹果蝇中,提供了先进和复杂的遗传技术(Ashburner等人,2004)。染色体分离和细胞分裂中的减数分裂缺陷出现在完成2< 减数分裂后精子细胞的细胞组织中。通过观察这种早期精子细胞,人们可以很容易地发现甚至微小的减数分裂异常(2012); 2012年; Inoue等人,2012)。此外,如果在正常培养细胞中发生微管完整性或动力学损失,则其细胞周期进程应在中期前停止。因此,很难检查微管如何影响体细胞细胞分裂的后期过程。另一方面,精母细胞对中后期微管组装检查点的微管异常敏感性较低。因此,可以检测微管相关基因在细胞分裂中的作用,而不阻止细胞周期。我们和其他团体已建立的系统通过用GFP荧光标签(Clarkson和圣,1999融合蛋白质的表达,以促进染色体或微管的动力学;井上等人,2004; Rebollo的。和Gonzalez,2004; Kitazawa等人,2012)。
 以前的方案可以将夹带在盖玻片和载玻片之间的窄空间中的雄性减数分裂细胞捕获,通过小的缓冲材料确保并在直立显微镜下观察染色体分离(Savoian等人, 2000; Inoue等人,2004; Savoian,2015)。这些协议允许我们收集微管的清晰图像。然而,制备使经常扁平细胞阻止起始和或胞质分裂的进展。此外,这是很难的药物或抑制剂增加而延时观察活细胞。
因此,我们建立了一种新的方法,允许我们在倒置显微镜下,在开放室中从前期I观察到完整的减数分裂I到细胞分裂结束。我们可以在成像的任何时间在细胞培养物中添加药物。我们还改进了协议,使我们可以执行染色体和其它细胞组分如雄性减数分裂期间I.微管,微丝,内质网,高尔基体装置或线粒体它可以通过具有稠合蛋白的同时表达来实现的同时观察不同的荧光标签显示光谱可分离的颜色。如在果蝇减数分裂细胞高等真核生物之间共享,该协议应该是为研究其它生物体的细胞分裂过程,以及有用的果蝇体细胞可见分裂过程的大多数方面有丝分裂。

关键字:果蝇, 雄性减数分裂, 延时观察, GFP标签蛋白, 染色体动力学, 微管, 胞质分裂, 线粒体

材料和试剂

  1. 盖板(22×22mm,No.1,厚度0.12-0.17mm)(Matsunami Glass,目录号:C022221)
    注意:有人认为,为了维持细胞活力和成功长期观察活细胞,需要彻底清洁盖玻片的清洁步骤(Savoian,2015)。然而,如果使用这些盖玻片,除了在使用前使用70%乙醇的擦拭物,基本上不需要任何预处理。
  2. 塑料盖滑动夹(76 x 26毫米)
    注意:文件夹是自定义的。长76宽32mm,厚1.7mm。有一个凹面(25毫米正方形),其中盖板滑落在表面上,在凹面中心有一个直径为15毫米的圆形孔)(见图1)。


    图1.塑料盖滑盖。支架应置于显微镜平台上。有一个凹面,其中凹面(22×22mm)落在表面上,在凹面中心有一个直径为15mm的圆形孔。

  3. 10×10毫米开口框架,底部有粘合剂(Gene Frame 25μm)(Thermo Fisher Scientific Co.,Waltham,USA)
  4. 塑料培养皿(直径90mm)(AsOne,目录号:1-7484-01)
  5. Kim-wipe(KCWW,Kimberly-Clark)
  6. 可以将bam-Gal4 :: vp16 (缩写为 bam-Gal4 )用作用于睾丸特异性异位表达荧光蛋白的Gal4驱动子(Kitazawa等人,,2012)
  7. UAS-dir2 被用作用于睾丸特异性消耗的Gal4驱动程序(Kitazawa等人,2014) br />
  8. P {His2Av T:Avic \ GFP-S65T }(简写为Histone2Av-GFP)可用于表达与GFP标签融合的组蛋白2Av,以在活的减数分裂细胞中观察染色质(Bloomington Drosophila Stock Center,目录号:BL5941)
  9. P(Ubi-mRFP-βTub85D)(简写为RFP-β微管蛋白)可用于与mRFP标签融合的β-微管蛋白的普遍表达,以显现活的减数分裂细胞中的微管(Kitazawa et al。 al ,2014)
  10. 可以将P {sqh-EYFP-Golgi} 用于与YFP标签融合的高尔基成分的普遍存在的表达,以在活的减数分裂细胞中显现高尔基体(Bloomington Drosophila Stock Center,目录号:BL7193) />
  11. 可以将P {sqh-EYFP-ER} 用于与YFP标签融合的ER成分的普遍表达,以显现活的减数分裂细胞中的内质网(Bloomington Drosophila Stock Center,目录号:BL7195) />
  12. 可以将P {sqh-EYFP-Mito} 用于与YFP标签融合的线粒体靶序列的普遍表达,以观察活的减数分裂细胞中的线粒体(Bloomington Drosophila Stock Center,目录号:BL7194) />
  13. P P {UASp-Venus-Orbit} 可用于轨道蛋白的可视化,必需的微管相关蛋白。可以诱导与三种不同荧光标记融合的轨道蛋白的UAS-库存(Miyauchi等人,2013)
  14. (UASp-mRFP-Actin5C)(Bloomington Drosophila Stock Center,目录号:BL24777),P(UAS-GFP-Actin5C)(Bloomington Drosophila Stock Center,目录号:BL9257)和 P(UAS-CFP-Actin5C)(Miyauchi等人,2013)可用于与不同标签融合的F-肌动蛋白成分的异位表达在活体减数分裂细胞中观察F-肌动蛋白
  15. (UAS-GFP-anillin)(Bloomington Drosophila Stock Center,目录号:BL51348)和P(Ub-mRFP-anillin)(Bloomington Drosophila Stock Center,目录号:BL52220)可用于生活在减数分裂细胞中的收缩环的可视化(来自布鲁明顿果蝇库中心的JA Brill的赠品)
  16. p {w [+ mC] = sqh-GFP.RLC} 3可用于与GFP标签融合的肌球蛋白轻链组分的普遍表达,以在活体减数分裂细胞中显现MLC(来自R. Karess)
  17. P(PTT-GA)Pdi [G00198],一种蛋白质捕获物,表达GFP-蛋白质二硫键异构酶,用于细胞内膜结构的可视化(来自L.Cooley的礼物,现在可从布卢明顿果蝇库存中心获得目录号:BL6839)。表达GFP-LamC的蛋白质库,用于可视化核层(来自L. Wallrath的礼物)
  18. 用于可视化雄性减数分裂细胞(来自J.A.Billill的礼物)的P {UAS-PLCg-PH-GFP}
  19. 用于可视化的男性减数分裂细胞核膜中的核孔复合物的可见性(来自V. Doyle的礼物,现在可从Bloomington获得)的可视化的 果蝇库中心目录号:BL35516)
  20. 使用P(UAS-GFP-Pav)和 P(UAS-GFP-Polo)可见微生物运动者和活的减数分裂细胞中重要的细胞分裂调节剂,分别(D.Glover的礼物)
  21. 对于雄性减数分裂细胞的RNAi实验,从VDRC股票中心和Bloomington股票中心获得了每种蛋白质的dsRNA异位表达的UAS-RNAi库。可以使用P(UAS-GFP RNAi)(Bloomington Drosophila Stock Center,目录号:BL9330)作为RNAi实验的阴性对照
  22. 秋水仙碱(BRB80缓冲液中为50μg/ml)(≥95%秋水仙碱)(Sigma-Aldrich,目录号:C9754)
    注意:为了检查微管细胞动力学的需要,使用作为微管聚合抑制剂的秋水仙素。含有秋水仙素的BRB80缓冲液在每次解剖前制备。在含有秋水仙素的缓冲液中解剖睾丸,然后将减数分裂细胞铺在矿物油上。
  23. 细胞松弛素D(BRB80缓冲液中10μg/ml)(≥98%细胞松弛素D)(Sigma-Aldrich,目录号:C8273)
    注意:为了检查细胞动力学中F-肌动蛋白的需要,使用作为肌动蛋白聚合抑制剂的细胞松弛素D。含有细胞松弛素D的BRB80缓冲液在每次解剖前制备。解剖雄性苍蝇以在含有细胞松弛素D的缓冲液中收集睾丸,然后将减数分裂细胞铺在矿物油上。
  24. 胎牛血清(Thermo Fisher Scientific,Gibco TM,目录号:451456或10437)
    注意:胎牛血清可以在4℃保存一个月。
  25. 矿物油(Trinity Biotech,目录号:400-5-1000)
    注意:每次延时记录,矿物油被更换为新鲜的。
  26. Brefeldin A(Cell Signaling,目录号:9972)或Exo1(Sigma-Aldrich,目录号:E8280)
    注意:为了检查细胞动力学是否需要由COPI介导的膜转运,使用每种化合物抑制αCOPI。将它们直接加入到培养基中。每次清除前制备含有Brefeldin A或Exo1的BRB80缓冲液。睾丸可以在分离精母细胞之前在培养基中培养长达14小时。
  27. PIPES(Dojindo Mecular Technologies,目录号:340-08255)
  28. 氯化镁六水合物(MgCl 2·6H 2 O)(Nacalai Tesque,目录号:20908-65)
  29. 乙二醇双(EGTA)(Nacalai Tesque,目录号:08907-42)
  30. 氯化钾(KCl)(Wako Pure Chemical Industries,目录号:162-17942)
  31. 氯化钠(NaCl)(Wako Pure Chemical Industries,目录号:191-01665)
  32. 磷酸氢二钠(Na 2 HPO 4)
  33. 磷酸二氢钾(KH 2 PO 4)<>>
  34. M3培养基(Sigma-Aldrich,目录号:S8398)
  35. BRB80缓冲液(pH 6.8)(参见食谱)
  36. 昆虫M3培养基(见食谱)
  37. 磷酸盐缓冲盐水(PBS)(见食谱)

设备

  1. 超细镊子(精细科学工具,型号:Dumont#5)
  2. 解剖针锋利切割直径为0.5mm的钨丝
  3. 配备激发发射滤光轮(Olympus,Tokyo,Japan)的倒置荧光显微镜(Olympus,型号:IX81)
  4. 目标; UPLFLN40XPH(NA = 0.75),UPLSAPO60XO(NA = 1.4),UPLSAPO100XO(NA = 1.4)(Olympus,东京,日本)
  5. 汞块(Olympus,目录号:USH-1030L)
  6. 冷却CCD相机(滨松光子,型号:C10600-10B)
  7. 高压灭菌器

软件

  1. Metamorph软件版本7.6(Molecular Devices,Sunnyvale,USA)

程序

  1. 为了用荧光标记感兴趣的细胞组分,可以诱导荧光标记的蛋白质,其由果蝇异位基因表达系统的每个靶组分组成,Gal4/UAS(Ashburner等人) >,2004; White-Cooper,2012)。或者,可以连续使用表达这种荧光蛋白的菌株。在前一种情况下,通过将具有荧光标签的蛋白质编码的cDNA的UAS原种杂交获得的F1后代置于UAS序列的控制下,可以用于延时观察(参见图2中遗传交叉的示意图)。在F1后代男性的睾丸细胞中,特异性诱导荧光蛋白的表达。 F1后代在28℃(不超过该温度)饲养,以诱导有效表达。对于耗尽实验,使用携带转基因以诱导每种靶蛋白的dsRNA的另一UAS原料代替上述UAS原料。在选择用于解剖的雄性蝇后,任何制备应在低于25℃的环境室温下进行。
    注意:观察前应将显微镜台周围的室温调整至少25°C。


    图2.遗传交叉产生产生荧光标记蛋白的F1后代以标记细胞内结构或表达dsRNA以消耗靶蛋白的F1后代。为了标记细胞内结构,诱导用于融合的cDNA的表达由GFP结构组成的蛋白质X,由GFP荧光标记进行,由Gal4/UAS系统进行。对于耗尽实验,诱导代表靶mRNA的一部分的dsRNA代替荧光蛋白X.

  2. 从室温下的上述成年人或新近成年的苍蝇(0-1日龄)仔细收集睾丸(图3)。要收集成人睾丸,上传到互联网网站的电影将作为指南( https://www.youtube.com/watch?v=-ej8nF1YsRg )。在解剖显微镜下将苍白腹侧面放置在塑料培养皿上的BRB80缓冲液中。使用一对镊子,他们的腹部被镊子夹紧,并将外生殖器轻轻拉向另一个相反的方向。在仔细地将相关附属组织远离一对睾丸后,如图3C所示分离出一对线圈睾丸。在秋水仙碱或细胞松弛素D处理的情况下,该解剖步骤分别在含有50μg/ml秋水仙碱和10μg/ml细胞松弛素D的BRB80缓冲液中进行。对于较长时间的治疗,从睾丸分离的精母细胞可以在含有10%胎牛血清和50%雄性细胞提取物的M3培养基中用药物孵育至14小时(Kitazawa等人,2012) 。
    注意:重要的是尽可能选择年轻的苍蝇,因为老年苍蝇中的睾丸含有较少数量的活精母细胞而不是大量的精子束。收集年轻苍蝇,将所有成年苍蝇从飞行培养管中弃去,丢弃后12小时至24小时收集新近放养的苍蝇。使用镊子轻轻快速地取出一副睾丸。建议您在三分钟内完成睾丸解剖(步骤1至4)。


    图3.包括从正常男性成年人收集的一对睾丸的生殖道。A.野生型成年男性的腹侧视图。一个箭头表示外生殖器。比例尺= 1 mm。 B.从正常的成年男性收集的生殖道。男性生殖道由一对睾丸,精囊(sv)和附属腺(ag)组成。还附有射精管(ed)和射精球(eb)。比例尺= 1 mm。 C.从成年男性中分离出的一对睾丸,除去相关组织如精囊和附属腺体。注意,携带w突变的苍蝇具有无色睾丸,而野生型睾丸显示为淡黄色,如该照片所示。刻度棒=100μm。 D.一对睾丸的相构造显微照片。至少有一个或多个减数分裂囊肿由16个初级精母细胞组成,经过减数分裂I,距离睾丸顶端三分之一(箭头)。此时应进行解剖以撕开鞘。顶端(星号)。比例尺= 100μm。

  3. 将一个或两个睾丸转移和铺设在矿物油(Trinity Biotech,Bray,爱尔兰)上,将其装在干净的玻璃盖板上的开放室中(图4)。为了防止油滴在盖板上滑落,它被一个10 x 10 mm的开放框架包围,底部有粘合剂(Gene Frame 25μm,Thermo Fisher Scientific Co.,Waltham,USA)。应使用一串Kim-wipe仔细吸取额外的液体,以避免由于在延时记录期间残留在细胞下面的液体而导致睾丸细胞意外滑落(图3D)。 br /> 注意:如果可能,建议收集完整和健康的睾丸复合体,其中射精泵仍然积极收缩(图3B)。不应选择在解剖过程中穿刺的睾丸。


    图4.使睾丸内的囊肿和细胞溅入矿物油中的程序示意图。 1.这里以较深的灰色示出的一半睾丸对直接位于一滴矿物油。一滴或两只睾丸可以在油滴中解剖。 2.使用一对钨针,覆盖睾丸的鞘从顶端(星号)撕开直到睾丸的三分之一。 3.用针头向外轻轻移动睾丸鞘,由16个初级精母细胞组成的囊肿可以扩散到油中。展开的细胞和囊肿以浅灰色表示。另一方面,每个囊肿应尽可能保持完好。从油滴中取出睾丸鞘。 4.使用一只连接在每只手的支架尖端的钨针,将覆盖睾丸的鞘撕开在顶端三分之一(图3D中的箭头)的位置,以便允许由16个初级精母细胞释放到睾丸外的油中,同时保持完整。油应随时覆盖每个睾丸细胞,以防止干涸。在这些条件下,完整囊肿内的细胞保持存活数小时(Kitazawa等人,2012; Savoian,2015)。

    注意:少数种系干细胞位于睾丸顶端(Ueishi et al。,2009)。经历第一次减数分裂的16个细胞囊肿位于顶端的三分之一左右(图3C,3D和图4)。


    图5.矿物油在减数分裂I早期的精母细胞活体16细胞囊肿的相差显微照片。已经开始减数分裂I的初级精母细胞由箭头指示。应选择这些细胞进行延时记录。 (Inset)早期阶段主要精母细胞囊肿的premeiotic细胞。注意它具有较大的核仁(箭头)。完整的16细胞囊肿被黄线包围。 Bar = 10μm
  4. 放置在塑料盖滑盖上的盖板放置在倒置荧光显微镜(Olympus,Tokyo,Japan)上,配备激发发射滤光轮(Olympus,Tokyo,Japan)。使用40x干物镜或100x油浸透镜收集荧光信号。
  5. 由于果蝇精子细胞对光线极度敏感(Rebollo和Gonzalez,2004),延时成像的最关键点之一是尽可能限制照射到活细胞标本的剂量。因此,在显微镜平台上设置支架后,应该寻找前期I囊肿,其中成熟的精母细胞将在通过ND25过滤器的透射光下使用相差光学器件后不久开始减数分裂。在前期I细胞中,形成圆形多层核包膜,而在前一期的阶段观察到椭圆形细长的信封I.应当选择完整囊肿而不是单独停留的原代精母细胞进行成像尽可能的
  6. 时间推移图像的记录从GFP-微管蛋白的荧光开始积累在彼此相对的心轴极点(t = 0分钟)时的定时开始。或者,应该在相位对比(t = 0分钟)内核仁几乎消失的时间开始图像采集(图6)。
    注意:建议您从最初的解剖步骤后十分钟内启动延时成像。


    图6.在减数分裂前或开始时表达GFP-βTubulin的初级精母细胞的睾丸细胞的相差观察和荧光观察I。前期I的初级精母细胞或将启动减数分裂的细胞我很快应在透射光下通过相位对比观察发现,以避免长时间照射来自Hg灯的激发光。前期和前期的精母细胞表现出圆形细胞形态,而细胞延伸形成椭圆形形态,因为在前一期后减数分裂进展.5阶段(小箭头)的premeiotic精母细胞含有单个较大的核仁。随着减数分裂细胞周期的进展,核仁在S6期(箭头)变小并分解。包含微小的几乎看不见的核仁(箭头,A)的前期I的精母细胞也显示出在相对方向分离的主轴极上的GFP-微管蛋白的荧光强度变得剧烈(B)。星号(*)表示细长的精子细胞的捆扎尾巴。当相位对照(t = 0分钟)内的核仁消失时,图像采集可以从时间开始。在同时收集GFP-微管蛋白的荧光的情况下,记录应当在荧光在极点(t = 0分钟)变强时的时刻开始。比例尺= 10μm
  7. 在每30秒的时间间隔,感兴趣的细胞中的荧光标记的蛋白质被来自汞灯的照射光激发。使用适当的滤光轮组合通过GFP/RFP过滤器立方体,用UV过滤和快门光照亮样品。用CCD相机(Hamamatsu Photonics,Shizuoka,Japan)捕获近同时的GFP和/或RFP荧光图像。例如,可以进行荧光图像(10毫秒曝光)和相位图像(使用ND12滤光片的300毫秒曝光)的连续收集用于时间推移观察以检查初级精母细胞中细胞组分的动力学。图像采集通过Metamorph软件版本7.6(Molecular Devices,Sunnyvale,CA,USA)进行控制。
    注意:可以调整焦点和显微镜平台,以便在查看计算机屏幕时使目标细胞组分清晰可见。
  8. 对于药物治疗,我们进行了初级精母细胞的短期体外培养(Rebollo和Gonzalez,2004; Kitazawa等人,2012)。
    1. 从成年男性收集附属于附属腺体,射精管和肛门的睾丸复合物。射精活动正在收缩的活睾丸复合体应选择并转移到M3培养基中。
    2. 对于与特异性药物如布雷菲德菌素A或Exo1进行更长时间的孵育,可将这些细胞内小泡运输抑制剂直接加入到由M3培养基组成的修饰培养基中,所述培养基不含有10%胎牛血清和50%雄性细胞提取物的碳酸氢盐根据(Kitazawa等人,2012),
    3. 将睾丸在培养基中孵育14小时,然后在室温下分离精母细胞。

注意:使用该方案,可以连续观察经历适当的染色体分离和细胞分裂的原代精母细胞至少一个小时而没有任何明显的异常(Kitazawa等,2012和2014; Hayashi等,2016) 。延迟成像可以通过减数分裂I继续减数分裂II的终止,而无中等变化,尽管无介质置换的细胞的延长孵育可能由于代谢废物的额外积累而导致细胞毒性。

数据分析

  1. 该协议通常允许我们对具有良好重现性的减数分裂I的原代精母细胞中的染色体,微管,F-肌动蛋白,高尔基体,ER-结构和线粒体进行延时观察。携带转基因的精母细胞(试剂7,8,15,16)可诱导由细胞内结构组成的荧光标记蛋白的表达,用于延时实验。或者,由通过bam-Gal4原液(试剂5)和UAS原液(试剂12-14,17-19)之间的杂交产生的雄性制备精母细胞,以通过Gal4/UAS系统诱导荧光蛋白。
  2. 也可以同时记录从例如GFP-组蛋白2Av和RFP-β微管蛋白发射的具有不同波长的多个荧光。在前期I期间,在聚合RFP-β微管蛋白在主轴极(t = 0分钟)处变得不均匀时,在组装前均匀分布的染色质组装形成染色体。对应于两个主要常染色体,X-Y染色体和微小的第4染色体之间的二价体的GFP-组蛋白2Av的四个灶点应在前一期I(t = 15分钟)形成。由于与主要染色体重叠,通常不会观察到较小的第4染色体的二价体。
  3. 在前一期I,浓缩的二价染色体位于细胞核内,其中核膜似乎是完整的(t = 5分钟)。在前一期构建了从主轴极发出的星体微管。开发的微粒已经在核膜周围移动,达到相反的极点。然后,所有染色体互补物似乎在双极纺锤体结构的中心进入单个染色体质量,直到中期I(t = 55分钟)。每个染色体进行平均速度为11.2±1.2μm/min的极向运动,直到双相类动物连接(Savoian等人,2000)。虽然检测点在男性减数分裂方面比体细胞检测点不那么严格(Rebollo和Gonzalez,2000),但在主轴检查点进行微管装配状态调查。后期我花了约8分钟,染色体在染色体分离后以1.9±0.1μm/min的速度向前移动(Savoian等人,2000)。在这个阶段,主轴极周围的核膜已经分解,主轴微管自由伸长到核空间的内部。在后期I的开始,围绕核空间的多层核膜将包含粗细胞微管的纺锤体微管分开形成星形微管。两个中心主轴微管群体出现在二价体分离后(t = 60〜70分钟)。微管的外围组件变得更加动态,就像它们向细胞赤道寻找细胞质一样(Inoue等人,2004)。对应的微管束的另一组微管束位于细胞中部。来自相反极的周边微管在赤道处相遇,并形成向外突出的气泡状结构(t = 60分钟)。然后,内部和大部分外围中心心轴从每个极点释放,并在赤道处形成独立的束。然后在两极的外周微管接触细胞皮层之后立即观察到沟槽侵入。随着沟槽的进行,染色体脱水(t =〜80 min)。
  4. 根据上述程序和试剂(试剂9-11,16-18),还可以重复检查雄性减数分裂中高尔基体积,内质网结构,核包膜,质膜和线粒体的动力学(Inoue <等人,2012; Kitazawa等人,2012; Hayashi等人,2016)。也可以使用由上述库存产生的几种标记(试剂13-15)来显现F-肌动蛋白和其它包括收缩环在分裂沟部位的细胞骨架的分布。在细胞分裂中的微管动力学的调节蛋白的细胞定位可以在使用库存(试剂12和19)的活细胞细胞中检查,如Orbit,Pavarotti和Polo。微管相关蛋白过度表达的过度表达使稳定微管的轨道导致在特定频率(超过10%)产生异常纺锤体结构。动态的细节已经在其他地方描述(Inoue等人,2012; Kitazawa等人,2014)
  5. 在RNAi实验中,通过特异性在精子细胞中靶基因的dsRNA的异位表达,可以检查染色体,细胞骨架和其他细胞器的动力学是否会受到影响。在延期记录之前,应该测试UAS-RNAi库存是否可以扰乱染色体分离,细胞分裂或线粒体分布,这种分布在bam-Gal4驱动程序的存在下。可以通过在相差显微镜下观察减数分裂后的精母细胞进行调查(参见Kitazawa等人,2014年的补充表)。基于在使用不同UAS-RNAi种群的相同基因的多个RNAi实验中观察到的重叠表型的基础上,应该认为该基因在男性减数分裂中的作用。
  6. 在药物治疗中,将初级精母细胞在含有药物的夹层缓冲液中进行预处理。或者,延迟记录在药物存在下进行。应该考虑通过药物治疗在药物治疗的细胞中唯一观察到的异常作为细胞表型。应以取决于药物浓度的方式观察细胞表型。应该确认它们出现频率更高,并且随着浓度的增加,表型增强。

笔记

作为该方案的结果,可以以良好的再现性观察到如上所述的染色体和微管的动力学。如果检测到细胞退出细胞分裂或异常微管结构,则应停止延时记录并丢弃细胞,例如由于不适当的生理条件而出现的多极轴。 P(His2Av-GFP)以及P {His2Av-mRFP} 的纯合子产生异常精母细胞,减数分裂进展在中期以较低的频率被阻止超过10%)。

食谱

注意:上述每种试剂的缓冲液或培养基的成分和目录号如下。为了制备所有缓冲液,培养基和试剂,应使用通过诸如Sartorius arium等水净化系统制备的超纯水 。除非另有说明,缓冲液可以保持在室温。本协议中使用的任何材料均经过MTA。

  1. BRB80缓冲液(pH6.8)
    80 mM PIPES
    1mM MgCl 2
    1 mM EGTA
  2. 昆虫M3培养基
    在无菌工作条件下的每次延时记录实验之前准备含有10%胎牛血清和50%雄性细胞提取物(Kitazawa等人,2012)的昆虫M3培养基
  3. 磷酸盐缓冲盐水(PBS)(pH 7.4)
    137 mM NaCl
    2.68 mM KCl
    10.14mM Na 2 HPO 4

    1.76mM KH PO 4
    为了制成1L PBS,8g NaCl,0.2g KCl,1.44g Na 2 HPO 4和0.24g KH 2 N/> PO 4结合并溶解在H 2 O中,总体积为1L。将pH调节至7.4。缓冲液通过高压灭菌消毒。缓冲液可以在室温下储存

致谢

我们感谢萨维亚先生(马西大学,新西兰),分享关于实验程序的信息。我们承认V. Doyle(法国巴黎的Jacques Monod),L. Wallrani(美国爱荷华州爱荷华大学),D. Glover(剑桥大学,英国剑桥),L. Cooley(耶鲁大学,美国) )和JA Brill(多伦多大学,加拿大多伦多)。我们还感谢维也纳果蝇RNAi中心,布卢明顿股票中心和果蝇遗传资源中心提供飞股。
没有可能影响其协议设计和实施的竞争或经济利益。这项工作得到日本科学促进会的部分支持[授予Y.H.I.的授权号26440188]。本协议经过我们以前的研究(Inoue等人,2004; Kitazawa等人,2012; Kitazawa等人)的改编或修改。 ,2014; Hayashi等人,2016)。

参考文献

  1. Ashburner,M.,Golic,KG和Hawley,RS(2004)。  果蝇:实验室手册。 2 nd 冷泉港实验室出版社。
  2. Clarkson,M。和Saint,R。(1999)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/10390154"target ="_ blank" > His2AvDGFP融合基因补充致死性His2AvD突变体等位基因,并提供用于果蝇染色体行为的体内标记物。细胞生物学 18(6):457-462。
  3. Giansanti,MG,Sechi,S.,Frappaolo,A.,Belloni,G.和Piergentili,R。(2012)。  果蝇中的细胞因子男性减数分裂。精子发生 2(3):185-196。 br />
  4. Hayashi,D.,Tanabe,K.,Katsube,H.and Inoue,YH(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pupmed/27402967"target ="_ blank"> B型核层和核孔复合体Nup107-160影响维生素在果蝇中的细胞分裂所需的包膜。男性减数分裂。 > Biol Open 5(8):1011-1021。
  5. Inoue,YH,Miyauchi,C.,Ogata,T.和Kitazawa,D。(2012)。< a class ="ke-insertfile"href ="http://www.intechopen.com/books/meiosis-分子机制和细胞遗传学多样性/动态变化 - 雄性减数分裂 - 果蝇中的细胞成分"目标="_ blank">果蝇中减数分裂和premeiotic分裂中细胞成分的动力学男性。在:Swan,A.(Ed。)。 InTech。 Open Access Publisher 67-86。
  6. Inoue,YH,Savoian,MS,Suzuki,T.,Mathe,E.,Yamamoto,MT和Glover,DM(2004)。< a class ="ke-insertfile"href ="http://www.ncbi在轨道/桅杆上的突变显示中心主轴由两个微管群体组成,即引发裂隙的那些群体,以及传播犁沟入侵的群体。 (J Cell Biol)166(1):49-60。
  7. Kitazawa,D.,Matsuo,T.,Kaizuka,K.,Miyauchi,C.,Hayashi,D。和Inoue,YH(2014)。< a class ="ke-insertfile"href ="http:对于果蝇中的分裂沟,男性减数分裂所需的肌球蛋白积聚是必需的,所以需要使用轨道/CLASP来进行轨迹/CLASP。 PLoS one 9:e93669。
  8. Kitazawa,D.,Yamaguchi,M.,Mori,H。和Inoue,YH(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pailmed/22553212"target ="_ blank">在果蝇中的细胞分裂是COPD介导的膜运输所必需的男性减数分裂。细胞科学125(Pt 15 ):3649-3660。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tanabe, K., Okazaki, R., Kaizuka, K. and Inoue, Y. H. (2017). Time-lapse Observation of Chromosomes, Cytoskeletons and Cell Organelles during Male Meiotic Divisions in Drosophila. Bio-protocol 7(8): e2225. DOI: 10.21769/BioProtoc.2225.
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