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{His2Av^{T: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, Gibco^TM, 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