参见作者原研究论文

本实验方案简略版
Mar 2020
Advertisement

本文章节


 

Quantitation of Secretory Granule Size in Drosophila Larval Salivary Glands
果蝇幼虫唾液腺分泌颗粒大小的定量研究   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Maturation of secretory granules is a crucial process that ensures the bioactivity of cargo proteins undergoing regulated secretion. In Drosophila melanogaster, the larval salivary glands produce secretory granules that are up to four-fold larger in cross-sectional area after maturation. Therefore, we developed a live imaging microscopy approach to quantitate the size of secretory granules with a view to identifying genes involved in their maturation. Here, we describe the procedures of larval salivary gland dissection and sample preparation for live imaging with a fluorescence confocal microscope. Furthermore, we describe the workflow for measuring the size of secretory granules by cross-sectional surface area and statistical analysis. Our live imaging microscopy method provides a reliable read-out for the status of secretory granule maturation in Drosophila larval salivary glands.

Keywords: Drosophila (果蝇), Genetics (遗传学), Organelle biogenesis (细胞器生源论), Live microscopy (实时显微镜), Image analysis (图象分析)

Background

Regulated secretion is a process during which biologically active molecules such as hormones, digestive enzymes, and mucus are secreted from specialized secretory cells in a coordinated manner. Hence, regulated secretion is critical for maintaining physiological homeostasis in animals. Examples include the release of insulin after a meal, the release of mucin in response to pathogenic microorganisms, and the release of sweat during elevated body temperature. These biologically active molecules are produced by endocrine or exocrine cells and stored in long-lasting secretory organelles termed secretory granules.


Biogenesis of secretory granules begins at the trans-Golgi network, where cargoes of secretory granules aggregate and bud off as immature secretory granules (Tooze, 1991 and 1998; Borgonovo et al., 2006). Immature secretory granules then undergo a maturation process to become fully functional and competent for secretion. The maturation process includes homotypic fusion of immature secretory granules, removal of unwanted materials, and processing of cargoes (Tooze, 1991; Arvan and Castle, 1998). Failure of secretory granules to mature can lead to reduced bioactivity of their cargoes. For example, most hormones enter immature secretory granules as inactive prohormones. During maturation, the lumen of secretory granules is acidified, and prohormone convertases cleave prohormones into biologically active hormones (Moore et al., 2002). Therefore, failed granule maturation can have physiological consequences, leading to reduced activity or inefficient secretion of cargo proteins.


Although secretory granule maturation is a critical step in regulated secretion, assays for secretory granule maturation are not simple. Transmission electron microscopy is one of the standard methods to assay secretory granule maturation. Secretory granules are electron dense in electron micrographs (Nitsch and Rinne, 1981; Tatsuoka and Reese, 1989). Reduced secretory granule maturation is often correlated with a reduction in electron density or in secretory granule number (Edwards et al., 2009; Cao et al., 2013; Du et al., 2016; Emperador-Melero et al., 2018; Hummer et al., 2017; Rao et al., 2020). If antibodies are available, a decrease in hormone secretion or an increased ratio of prohormone to hormone can be measured in blood plasma or cell culture media by western blotting and densitometry or ELISA following stimulation (Cao et al., 2013; Du et al., 2016; Hummer et al., 2017). Immunofluorescence using a combination of anti-prohormone antibodies and secretory granule markers can also reveal defects in hormone processing, as indicated by an increase in intensity of the prohormone signal (Bogan et al., 2012; Cao et al., 2013).


The Drosophila larval salivary gland is a powerful genetic model for studying secretory granule biogenesis (Biyasheva et al., 2001; Burgess et al., 2011 and 2012; Torres et al., 2014; Csizmadia et al., 2018; Ma et al., 2020; Neuman et al., 2020). The larval salivary glands start producing glue protein-containing secretory granules 24 h after entry into the third instar larval stage. Immature secretory granules then mature over the next 18 h and are secreted all at once in response to a pulse of the hormone ecdysone (Biyasheva et al., 2001; Burgess et al., 2011). The secreted glue proteins adhere pupal cases onto a solid surface during metamorphosis. Size differences between immature and mature secretory granules can be 2- to 4-fold in cross-sectional surface area (5 μm2 vs. 10-25 μm2) (Ma et al., 2020). Thus, the Drosophila larval salivary gland is an excellent system to identify genes required for secretory granule maturation.


The Andres laboratory previously generated transgenic lines expressing one of the glue proteins (Sgs3) tagged with GFP or DsRed under the control of its endogenous promoter (Biyasheva et al., 2001; Costantino et al., 2008). Using these lines, secretory granules can be visualized by confocal microscopy. Our laboratory has used these transgenic lines in combination with a salivary gland-specific Gal4 driver and UAS-controlled RNAi transgenic lines to identify genes needed for secretory granule maturation. Here, we provide a detailed protocol for visualizing secretory granules via live imaging with a spinning-disc confocal microscope. Acquired data are analyzed with the imaging software Volocity 6.3 to quantitate secretory granule size. Secretory granule size distribution is then used as a readout for secretory granule maturation in the Drosophila larval salivary gland.

Materials and Reagents

  1. Microscope slides 75 × 25 × 1 mm (VWR, catalog number: 16004-368)

  2. Glass coverslips 18 × 18 mm No. 1.5 (VWR, catalog number: 48366-205)

  3. Self-adhesive reinforcement labels (Avery, catalog number: 32203)

  4. Syringe 5 ml (BD, catalog number: 309603)

  5. Needle 18 G or lower (BD, catalog number: 305195)

  6. Dissection needle (Fisher Scientific, catalog number: 13820024)

  7. Petri dishes 100 × 15 mm (VWR, catalog number: 25384-088)

  8. Petri dishes 35 × 10 mm (Corning, catalog number: 351008)

  9. P{w+, Sgs3-GFP} (Bloomington Drosophila Stock Center, catalog number: 5884, 5885) or P{w+, Sgs3-DsRed} (Andres lab, University of Nevada, Las Vegas, USA) 3rd instar larva

  10. High-vacuum M grease 100 g (Apiezon, Sigma, catalog number: Z273589)

  11. Pipette tips (P200)

  12. SYLGARDTM 184 Silicone Elastomer Kit 0.5 kg (Dow Chemical, Sigma, catalog number: 4019862)

  13. KCl (Sigma, catalog number: P3911-500G)

  14. NaCl (Bio Basic, catalog number: CA99501-558)

  15. CaCl2·2H2O (Sigma, catalog number: C-5080-500G)

  16. Tris-HCl (VWR, catalog number: 0234-1KG)

  17. Drosophila Ringer’s solution (see Recipe 1)

  18. Silicone dissection plate (see Recipe 2)

Equipment

  1. Dumont #5 fine forceps (Fine Science Tools, catalog number: 11251-20)

  2. Stereomicroscope (Leica Microsystems, model: Leica MZ6)

  3. Spinning-disc confocal coupled with an Olympus IX81 microscope (Quorum Technologies, Puslinch, Ontario, Canada)

Software

  1. Volocity 6.3 (Quorum Technologies, Puslinch, ON, Canada)

  2. Adobe Creative Cloud Photoshop (Adobe, San Jose, CA, USA)

  3. Microsoft Office 365 Excel (Microsoft, Redmond, WA, USA)

Procedure

  1. Dissection and sample preparation

    1. Pick 3rd instar wandering larvae with a dissection needle and transfer to a 35-mm Petri dish with Drosophila Ringer’s solution. Use fine forceps to hold the larvae and swirl in Drosophila Ringer’s solution to wash off any food. 3rd instar wandering larvae are those moving away from the food source; they can be easily located on the side of vials or bottles housing flies.

      Note: It is best to pick larvae that are still moving and have food left in the gut. If molasses is included in the Drosophila food recipe, food in the gut can easily be identified by the dark brown color in the middle of the larvae. Otherwise, consumer food coloring can also be added to the Drosophila food to help identify larvae in the right stage. Larvae that have stopped moving or have no food left in the gut are ready to pupariate; therefore, their secretory granules would be secreted and would not be useful in determining secretory granule size.

    2. Transfer individual clean larva with fine forceps to a droplet of Drosophila Ringer’s solution (50 μl) on a silicone dissection plate.

    3. Dissect the larva under a stereomicroscope with fine forceps. Immobilize the larva by gently holding it down with forceps using the non-dominant hand. Pinch the mouth hook of the larva with the dominant hand and pull to remove the salivary glands.

    4. Clean unwanted tissues away from the salivary glands using forceps (Figure 1A) and mount the samples as demonstrated in the illustration (Figure 1B and 1C).



      Figure 1. Mounting of Drosophila Larval Salivary Glands for Live Microscopy. A. DIC image of a larval salivary gland (translucent) with accompanying fat body (opaque) taken with 10× objective. B. Mounting of larval salivary glands on microscope slide with self-adhesive reinforcement label. C. Mounting of larval salivary glands on microscope slide with vacuum grease.


    5. Mounting the samples with self-adhesive reinforcement labels is fast and consistent (Figure 1B, step 6). However, to prevent anoxia, samples should be mounted with vacuum grease if a longer imaging time (>30 min) is desired (Figure 1C, step 7).

    6. Place one self-adhesive reinforcement label on the microscope slide. Ensure that the label completely adheres to the slide. Pipette 8 µl Drosophila Ringer’s solution to the middle of the label. Transfer salivary glands to the solution (3-5 pairs of salivary glands per label). Place cover slip on and seal the slide with quick-drying nail polish. Samples are good for up to 30 min (following 10 min dissection time).

    7. Dispense vacuum grease into a 5-ml syringe. Use a needle with a small gauge number. Dispense vacuum grease by drawing a square on the microscope slide using the syringe. Leave a small gap and do not complete the square. Pipette 30 µl Drosophila Ringer’s solution into the middle of the square. Transfer salivary glands to the solution (up to 8 pairs). Place cover slip on and gently push down with fine forceps until the vacuum grease seals the sample. Seal the slide with quick-drying nail polish. Samples are good for up to 50 min (following 10 min dissection time).


  2. Imaging

    1. Transfer slide to the stage of spinning-disc confocal microscope.

    2. Open Volocity 6.3 in acquisition mode. Image samples with the 60× objective. Use corresponding setting to acquire images in the green or red channel for Sgs3-GFP or Sgs3-DsRed, respectively.

    3. Capture 20 μm in Z with 0.3 µm per slice. Image at least three representative cells for each pair of glands (Figure 2).



      Figure 2. Secretory Granules of Drosophila Larval Salivary Glands. A representative spinning-disc confocal image of one Z-slice of a Drosophila larval salivary gland cell expressing the secretory granule marker Sgs3-DsRed.


    4. Image at least three independent glands for each genotype.

Data analysis

  1. Quantitation and Statistical Analysis

    1. Launch Volocity 6.3 in analysis mode. Open the files saved from acquisition. Create a point spread function (PSF) file for deconvolution (Actions > Create New > Calculated PSF…). Enter values corresponding to the acquisition setup (Figure 3A).



      Figure 3. Deconvolution in Volocity 6.3. A. Settings for creating a new point spread function file to perform deconvolution on data from the red channel in Volocity 6.3. Values may need to be adjusted for a different setup. Here, the objective magnification is increased by 1.5-fold (60× to 90×) because the detector used in our system further magnifies the image by 1.5-fold. B. Deconvolved data will be listed as a new channel in Volocity 6.3.


    2. Run deconvolution on acquired data (Tools > Iterative Restoration…). Set the confidence limit to 99% and the iteration limit to 30. A new channel (deconvolved data) will be generated in the right panel (Figure 3B).

    3. Split the Z-stacks into individual images (Tools > Split Volumes, select Organize the images into folders; Figure 4A). A new folder containing two subfolders will be generated in the left panel (Figure 4B). Perform analysis on deconvolved images in the second subfolder.



      Figure 4. Split Volumes. A. Split volume function to organize Z-slices from raw data and deconvolved data into different folders. B. Folders of images created to the left panel of Volocity 6.3 after split volume function.


    4. Select the first image (z = 1) and go to the Measurements tab. Drag Find Objects and Separate Touching Objects tasks to the measurement protocol panel at the top of the screen (Figure 5). Make sure the two tasks are under the same protocol, generating only one population of measurements.

    5. Click the gear icon beside the Find Objects function. For the Threshold, using drop down menu, select Automatic. In the Offset threshold by field, enter -10%. In the Minimum object size field, enter 4 μm2. Press OK. Under the Separate Touching Objects function, enter 12 μm2 in the Object size guide field (Figure 5). This setting works well for secretory granules in wild-type salivary glands. The values need to be adapted for genotypes with very large or very small secretory granules.



      Figure 5. Functions to Measure Secretory Granules. A screen shot displaying a protocol containing Find Objects and Separate Touching Objects in Volocity 6.3 (top left). Settings for the Find Objects function by thresholding (bottom right).


    6. Click Measure under the function generated from steps 4 and 5. Check Intensity and Volume measurements and press OK. Identified objects will be outlined in the color chosen in the protocol created from steps 4 and 5 (Figure 6A). A list of objects should be populated in the bottom right panel (Figure 6B). The function can be saved (Measurements > Save Protocol…) and later restored (Measurements > Restore Protocol…).



      Figure 6. Measuring Secretory Granule Size. A. Secretory granules (red) outlined in purple using the protocol described in steps 4-5. B. Output of secretory granule cross-sectional area.


    7. Go to the list of objects. Double click on the Area (μm2) tab until the objects are sorted by size in descending order. Select all the data excluding the those that are less than 4 μm2. Copy the data and paste into Excel 365 for statistical analysis.

    8. Repeat Steps 4-7 for z = 10 and z = 20 to account for variability in secretory granule cross-sectional area throughout the cell. If the measurement protocol was saved, steps 4-5 can be avoided by the Restore Protocol… function described in step 6.

    9. Repeat Steps 4-8 for three representative cells from the same salivary gland.

    10. Repeat Steps 4-9 for three independent salivary glands for each genotype.

    11. Pool all the data for each genotype in Excel 365.

    12. Box plots (Insert > Insert Statistic Chart > Box and whisker plot) can be used to visualize the distribution of secretory granule size by cross-sectional surface area (Figure 7).



      Figure 7. Box Plot Showing Secretory Granule Size Distribution. Distribution of secretory granule sizes by cross-sectional area in wild type salivary glands. Each line of the box from bottom to top represents the 25th, 50th (median), and 75th percentile of the population. X denotes the mean of the sample. Bottom and top whiskers represent minimum and maximum values determined by 1.5× the interquartile range. Dots outside of the whiskers are samples considered as statistical outliers. n = 1597 secretory granule cross-sections from 3 salivary gland cells (3 Z-slices from each cell for a total of 9 Z-slices).


    13. t-Test (Data > Data Analysis > t-Test: Two-Sample Assuming Unequal Variances) can be used to evaluate the statistical difference in secretory granule cross-sectional area between two genotypes.

Recipes

  1. Drosophila Ringer’s solution (pH 7.2)
    KCl                               182 mM

    NaCl                             46 mM

    CaCl2·2H2O                3 mM

    Tris-HCl                       10 mM

  2. Silicone dissection plate

    1. Mix the base and curing agent from the SYLGARD 184 kit (10:1, weight:weight) directly in a 100-mm Petri dish with a spatula. All procedures should be conducted in a fume hood.

    2. Allow to cure inside the fume hood for 48 h.

Acknowledgments

This protocol was adapted from our previous work (Ma et al., 2020). We acknowledge the following funding sources: SickKids Restracomp Studentship, Canadian Institutes of Health Research Strategic Training Fellowship #TGF-53877, University of Toronto Open Fellowship, and Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarships–Doctoral Program Studentship (to Cheng-I J. Ma); Canadian Institutes of Health Research Grants #PJT-162165 and #MOP-119483 and Canadian Institutes of Health Research Institute of Genetics #IG1-115714 (to Julie A. Brill).

Competing interests

There are no competing interests to declare.

References

  1. Arvan, P. and Castle, D. (1998). Sorting and storage during secretory granule biogenesis: looking backward and looking forward. Biochem J 332 (Pt 3): 593-610.
  2. Biyasheva, A., Do, T. V., Lu, Y., Vaskova, M. and Andres, A. J. (2001). Glue secretion in the Drosophila salivary gland: a model for steroid-regulated exocytosis. Dev Biol 231(1): 234-251.
  3. Bogan, J. S., Xu, Y. and Hao, M. (2012). Cholesterol accumulation increases insulin granule size and impairs membrane trafficking. Traffic 13(11): 1466-1480.
  4. Borgonovo, B., Ouwendijk, J. and Solimena, M. (2006). Biogenesis of secretory granules. Curr Opin Cell Biol 18(4): 365-370.
  5. Burgess, J., Jauregui, M., Tan, J., Rollins, J., Lallet, S., Leventis, P. A., Boulianne, G. L., Chang, H. C., Le Borgne, R., Kramer, H. and Brill, J. A. (2011). AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila. Mol Biol Cell 22(12): 2094-2105.
  6. Burgess, J., Del Bel, L. M., Ma, C. I., Barylko, B., Polevoy, G., Rollins, J., Albanesi, J. P., Kramer, H. and Brill, J. A. (2012). Type II phosphatidylinositol 4-kinase regulates trafficking of secretory granule proteins in Drosophila. Development 139(16): 3040-3050.
  7. Cao, M., Mao, Z., Kam, C., Xiao, N., Cao, X., Shen, C., Cheng, K. K., Xu, A., Lee, K. M., Jiang, L. and Xia, J. (2013). PICK1 and ICA69 control insulin granule trafficking and their deficiencies lead to impaired glucose tolerance. PLoS Biol 11(4): e1001541.
  8. Costantino, B. F., Bricker, D. K., Alexandre, K., Shen, K., Merriam, J. R., Antoniewski, C., Callender, J. L., Henrich, V. C., Presente, A. and Andres, A. J. (2008). A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila. PLoS Genet 4(6): e1000102.
  9. Csizmadia, T., Lorincz, P., Hegedus, K., Szeplaki, S., Low, P. and Juhasz, G. (2018). Molecular mechanisms of developmentally programmed crinophagy in Drosophila. J Cell Biol 217(1): 361-374.
  10. Du, W., Zhou, M., Zhao, W., Cheng, D., Wang, L., Lu, J., Song, E., Feng, W., Xue, Y., Xu, P. and Xu, T. (2016). HID-1 is required for homotypic fusion of immature secretory granules during maturation. Elife 5: e18134.
  11. Edwards, S. L., Charlie, N. K., Richmond, J. E., Hegermann, J., Eimer, S. and Miller, K. G. (2009). Impaired dense core vesicle maturation in Caenorhabditis elegans mutants lacking Rab2. J Cell Biol 186(6): 881-895.
  12. Emperador-Melero, J., Huson, V., van Weering, J., Bollmann, C., Fischer von Mollard, G., Toonen, R. F. and Verhage, M. (2018). Vti1a/b regulate synaptic vesicle and dense core vesicle secretion via protein sorting at the Golgi. Nat Commun 9(1): 3421.
  13. Hummer, B. H., de Leeuw, N. F., Burns, C., Chen, L., Joens, M. S., Hosford, B., Fitzpatrick, J. A. J. and Asensio, C. S. (2017). HID-1 controls formation of large dense core vesicles by influencing cargo sorting and trans-Golgi network acidification. Mol Biol Cell 28(26): 3870-3880.
  14. Ma, C. J., Yang, Y., Kim, T., Chen, C. H., Polevoy, G., Vissa, M., Burgess, J. and Brill, J. A. (2020). An early endosome-derived retrograde trafficking pathway promotes secretory granule maturation. J Cell Biol 219 (3): e201808017.
  15. Moore, H. H., Andresen, J. M., Eaton, B. A., Grabe, M., Haugwitz, M., Wu, M. M. and Machen, T. E. (2002). Biosynthesis and secretion of pituitary hormones: dynamics and regulation. Arch Physiol Biochem 110(1-2): 16-25.
  16. Neuman, S. D., Terry, E. L., Selegue, J. E., Cavanagh, A. T. and Bashirullah, A. (2020). Mistargeting of secretory cargo in retromer-deficient cells. Dis Model Mech 14(1): dmm046417.
  17. Nitsch, C. and Rinne, U. (1981). Large dense-core vesicle exocytosis and membrane recycling in the mossy fibre synapses of the rabbit hippocampus during epileptiform seizures. J Neurocytol 10(2): 201-209.
  18. Rao, A., McBride, E. L., Zhang, G., Xu, H., Cai, T., Notkins, A. L., Aronova, M. A. and Leapman, R. D. (2020). Determination of secretory granule maturation times in pancreatic islet β-cells by serial block-face electron microscopy. J Struct Biol 212(1): 107584.
  19. Tatsuoka, H. and Reese, T. S. (1989). New structural features of synapses in the anteroventral cochlear nucleus prepared by direct freezing and freeze-substitution. J Comp Neurol 290(3): 343-357.
  20. Tooze, S. A. (1991). Biogenesis of secretory granules. Implications arising from the immature secretory granule in the regulated pathway of secretion. FEBS Lett 285(2): 220-224.
  21. Tooze, S. A. (1998). Biogenesis of secretory granules in the trans-Golgi network of neuroendocrine and endocrine cells. Biochim Biophys Acta 1404(1-2): 231-244.
  22. Torres, I. L., Rosa-Ferreira, C. and Munro, S. (2014). The Arf family G protein Arl1 is required for secretory granule biogenesis in Drosophila. J Cell Sci 127(Pt 10): 2151-2160.

简介

[摘要]分泌颗粒的成熟是一个关键过程,可确保货物蛋白质受调节分泌的生物活性。在果蝇中,幼虫唾液腺产生的分泌颗粒在成熟后的横截面积最多增加四倍。因此,我们制定了活体成像显微镜方法孔定量泰特分泌颗粒的大小,以期查明荷兰国际集团参与其成熟的基因。在这里,我们描述了用荧光共聚焦显微镜对幼虫唾液腺进行解剖的程序和用于实时成像的样品制备方法。此外,我们描述了通过横截面表面积和统计分析来测量分泌颗粒大小的工作流程。我们的实时成像显微镜方法为果蝇幼虫唾液腺分泌颗粒成熟的状态提供了可靠的读数。

[背景]调节分泌是一个过程,在此过程中,诸如激素,消化酶和粘液等生物活性分子以协调的方式从专门的分泌细胞中分泌出来。因此,调节分泌对于维持动物体内的生理稳态至关重要。实例包括饭后释放胰岛素,响应病原微生物释放粘蛋白以及在体温升高时释放汗液。这些生物活性分子由内分泌或外分泌细胞产生,并储存在称为分泌颗粒的长效分泌细胞器中。

分泌颗粒的生物发生始于反高尔基网络,在那儿,分泌颗粒的货物聚集并萌芽为未成熟的分泌颗粒(Tooze,1991年和1998年; Borgonovo等人,2006年)。然后,未成熟的分泌性小颗粒会经历成熟过程,以完全发挥功能并具有分泌能力。成熟过程包括未成熟分泌颗粒的同型融合,去除不需要的物质以及加工货物(Tooze,1991; Arvan和Castle,1998)。分泌颗粒无法成熟会导致其货物的生物活性降低。例如,大多数激素以不活跃的激素形式进入未成熟的分泌颗粒。在成熟期间,分泌颗粒的内腔被酸化,并且激素原转化酶将激素原裂解为具有生物活性的激素(Moore等,2002)。因此,失效编颗粒成熟可具有生理后果,从而导致降低的活性或inefficie货物蛋白的核苷酸分泌。

尽管分泌颗粒成熟是调节分泌的关键步骤,但分泌颗粒成熟的测定并不简单。透射电子显微镜是测定分泌性颗粒成熟的标准方法之一。在电子显微照片中,分泌颗粒是电子致密的(Nitsch和Rinne,1981; Tatsuoka和Reese,1989)。分泌颗粒成熟的减少通常与电子密度或分泌颗粒数量的减少相关(Edwards等,2009; Cao等,2013; Du等,2016; Emperador-Melero等,2018; Hummer等人,2017; Rao等人,2020)。如果有抗体,可以在刺激后通过Western blotting和光密度法或ELISA测定血浆或细胞培养基中激素分泌的减少或激素原与激素的比率增加(Cao等人,2013; Du等人, 2016; Hummer等人,2017)。结合使用抗激素原抗体和分泌性颗粒标记的免疫荧光也可以揭示激素加工中的缺陷,如激素原信号强度的增强所表明的那样(Bogan等,2012; Cao等,2013)。

在果蝇幼虫唾液腺是一个功能强大的基因为研究分泌颗粒的生物合成模型(Biyasheva等,2001;伯吉斯等人,2011和2012;托雷斯等人。2014; Csizmadia等人。2018年,马等。 ,2020; Neuman et al 。,2020)。进入第三龄幼虫阶段后24小时,幼虫唾液腺开始产生含胶蛋白的分泌颗粒。未成熟的分泌颗粒然后成熟在接下来的18小时,并响应于激素蜕皮激素的脉冲被分泌一次全部(Biyasheva等人,2001;伯吉斯等人。,2011) 。在变态过程中,分泌的胶蛋白将cases盒粘附在固体表面上。未成熟和成熟的分泌颗粒之间大小的差异可以是在横截面表面积(5微米2至4倍2 VS 。10-25微米2 )(马等人。,2020) 。因此,果蝇幼虫唾液腺是鉴定分泌性颗粒成熟所需的基因的优良系统。

安德列斯实验室先前在其内源启动子的控制下产生了表达一种用GFP或DsRed标记的胶蛋白(Sgs3)的转基因亚麻(Biyasheva等,2001; Costantino等,2008)。使用这些品系,分泌颗粒可以通过共聚焦显微镜观察。我们的实验室已联合使用这些转基因株系与唾液腺-特定Gal4驱动子和UAS -控制RNAi的转基因株系,以确定所需要的分泌颗粒成熟的基因。在这里,我们提供了一个详细的协议,通过旋转盘共聚焦显微镜通过实时成像来可视化分泌颗粒。获取的数据进行分析与所述成像软件Volocity 6.3至QUANTI泰特分泌颗粒大小。分泌颗粒大小分布随后用作果蝇幼虫唾液腺中分泌颗粒成熟的读数。

关键字:果蝇, 遗传学, 细胞器生源论, 实时显微镜, 图象分析



材料和试剂


显微镜载玻片75 × 25 × 1 mm(VWR,目录号:16004-368 )
玻璃盖玻片18 × 18毫米1.5号(VWR,目录号:48366-205)
自粘增强标签(每个,目录号:32203)
注射器5 ml(BD,货号:309603)
18 G或更低的针(BD,目录号:305195)
解剖针(Fisher Scientific,目录号13820024)
培养皿100 × 15毫米(VWR,目录号:25384-088)
培养皿35 × 10毫米(Corning,目录号:351008)
p {w ^ + ,SGS3-GFP} (布卢明顿果蝇库存中心,目录号:5884,5885 )或p {w ^ + ,SGS3,红色荧光蛋白} (安德烈斯实验室,内华达大学拉斯维加斯分校,美国)3届龄幼虫
高真空M润滑脂100克(Apiezon ,Sigma,目录号:Z273589)
移液器吸头(P200)
SYLGARD TM 184硅胶弹性体套件0.5千克(Dow Chemical,Sigma ,目录号:4019862)
氯化钾(Sigma,目录号:P3911-500G)
氯化钠(Bio Basic,目录号:CA99501-558)
氯化钙2· 2H 2 O(Sigma,目录号:C-5080-500G)
Tris-HCl(VWR,目录号:0234-1KG)
果蝇林格氏液(见配方1)
小号ilicone夹层板(见配方2 )


设备


杜蒙#5细钳(Fine Science Tools,目录号:11251-20)
体视显微镜(Leica Microsystems,型号:Leica MZ6 )
旋转盘共聚焦与Olympus IX81显微镜(Quorum Technologies,Puslinch ,安大略省,加拿大)


软件


Volocity 6.3(Quorum Technologies,Puslinch ,ON,加拿大)
Adobe Creative Cloud Photoshop(Adobe,美国加利福尼亚州圣何塞)
Microsoft Office 365 Excel(Microsoft,美国华盛顿州雷德蒙德)


程序


解剖和样品制备
选择3次龄用解剖针和徘徊幼虫转移到35 -毫米培养皿果蝇林格的r溶液。用细镊子握住幼虫,并在果蝇林格氏液中旋转,以洗净任何食物。3 RD龄幼虫徘徊是那些从食品源移开; 吨哎可以容易地定位在小瓶或瓶子壳体苍蝇的一侧。
注:我t是最好挑选幼虫还在移动,并有留在肠道的食物。如果果蝇食品食谱中包括糖蜜,则可以通过幼虫中间的深棕色轻松识别出肠道中的食物。否则,也可以在果蝇食品中添加食用食用色素,以帮助在正确的阶段识别幼虫。停止运动或肠道内没有食物的幼虫准备进行化ari。因此,它们的分泌颗粒将被分泌出来,并且在确定分泌颗粒大小时将无用。


将单独的干净幼虫用细镊子转移到硅胶解剖板上的果蝇林格氏溶液(50μl )的液滴中。
在幼虫下用细镊子解剖幼虫。用不占优势的手用镊子轻轻按住幼虫,以固定幼虫。用惯用的手捏住幼虫的嘴钩,然后拉动以除去唾液腺。
使用镊子将多余的组织从唾液腺中清除(图1A),并如图所示安装样品(图1B和1 C )。




图1的安装果蝇大号arval小号alivary ģ土地用于大号香港专业教育学院中号icroscopy。A.用10倍物镜拍摄的幼虫唾液腺(半透明)和伴随的脂肪体(不透明)的DIC图像。B.将幼虫唾液腺安装在带有自粘增强标签的显微镜载玻片上。C.用真空油脂将幼虫唾液腺安装在显微镜载玻片上。


使用自粘增强标签安装样品快速且一致(图1B,第6步)。但是,为防止缺氧,如果需要更长的成像时间(> 30分钟),则应使用真空油脂安装样品(图1C,步骤7)。
P花边在显微镜载玻片一个自粘标签加固。确保该标签完全粘附在幻灯片。移取8 µl果蝇林格氏液到标签中间。将唾液腺转移到溶液中(每个标签3-5对唾液腺)。P花边上盖玻片并密封与快干滑动ING指甲油。样品最长可使用30分钟(解剖时间为10分钟)。
分配真空润滑脂成5 -毫升注射器。使用小口径的针。通过使用注射器在显微镜载玻片上画一个正方形来分配真空润滑脂。留一点缝隙,不要完成正方形。移液器30微升果蝇林格氏液中的广场中央。将唾液腺转移至溶液中(最多8对)。P花边上盖玻片并轻轻向下推用细镊子,直到真空润滑脂密封样品。密封采用快干幻灯片ING指甲油。样品最长可使用50分钟(解剖时间为10分钟以下)。


影像学
将幻灯片转移到旋转盘共聚焦显微镜的阶段。
在采集模式下打开Volocity 6.3。图像样本与所述60 ×物镜。使用对应设置获取图像中的用于SGS3-GFP或SGS3-DsRed的绿色或红色通道,分别。
以每片0.3 µm的量捕获Z中的20 µm 。为每对腺体成像至少三个代表性细胞(图2)。




图2.分泌ģ ranules的果蝇大号arval小号alivary ģ土地。果蝇幼虫唾液腺细胞的一个Z切片的代表性旋转盘共聚焦图像,表达分泌颗粒标记Sgs3-DsRed。


为每种基因型至少成像三个独立的腺体。


数据一nalysis


孔定量牛逼通货膨胀和统计分析
在分析模式下启动Volocity 6.3。打开通过采集保存的文件。创建用于解卷积的点扩展函数(PSF)文件(“操作”>“新建”>“计算出的PSF…” )。输入与采集设置相对应的值(图3A)。




图3. Volocity 6.3中的反卷积。A.用于在Volocity 6.3中创建新点扩散函数文件以对来自红色通道的数据执行反卷积的设置。可能需要针对其他设置调整值。在这里,由于我们系统中使用的检测器将图像进一步放大了1.5倍,因此物镜放大倍率提高了1.5倍(从60 ×到90 × )。B.反卷积的数据将在Volocity 6中被列为新通道。3 。


对获取的数据运行反卷积(“工具”>“迭代还原” )。设置的置信限至99%,并且所述迭代限制到30,一种新的信道(去卷积的数据)将在右侧面板(图3B)来生成。
将Z堆栈拆分为单个图像(工具>拆分卷,选择将图像组织到文件夹中;图4A)。包含两个子文件夹的新文件夹将在左侧面板中生成(图4B)。对第二个子文件夹中的反卷积图像执行分析。




图4 。拆分V olumes。A.分割卷功能可以将原始数据和解卷积后的数据的Z切片组织到不同的文件夹中。B.分割音量功能后,在Volocity 6.3左面板上创建的图像文件夹。


选择第一张图像(z = 1),然后转到“测量”选项卡。将“查找对象”和“分离触摸对象”任务拖到屏幕顶部的“测量协议”面板中(图5)。确保两个任务都在相同的协议下,仅生成一组测量值。
点击旁边的齿轮图标在找对象的功能。对于“阈值” ,使用下拉菜单,选择“自动” 。在“偏移阈值依据”字段中,输入-10%。在最小对象大小字段,输入4微米2 。按确定。下的单独的触摸物体的功能,输入12微米2的对象大小引导场(图5)。此设置适用于野生型唾液腺中的分泌性颗粒。该值需要调整为具有非常大或非常小的分泌颗粒的基因型。




图5.测量分泌颗粒的功能。屏幕快照显示了协议,该协议包含Volocity 6.3中的“查找对象”和“单独的触摸对象” (左上方)。通过阈值设置“查找对象”功能的设置(右下)。


点击测量从步骤4和5,检查所产生的作用下强度和V olume测量,然后按OK。所标识的对象将以在步骤4和5(图6A)创建的协议中选择的颜色进行概述。对象列表应填充在右下方的面板中(图6B)。可以保存该功能(Measurements>保存协议… ),然后再恢复该功能(Measurements> Restore Protocol ... )。




图6.测量分泌颗粒的大小。A.使用步骤4-5中所述的方案,紫色概述的分泌颗粒(红色)。B.分泌颗粒横截面积的输出。


转到对象列表。在双击区(微米2 )选项卡,直至对象按照大小降序排列。选择所有不包括所述数据的那些是小于4μm 2 。复制数据并粘贴到Excel 365中以进行统计分析。
对于z = 10和z = 20重复步骤4-7,以说明整个细胞中分泌颗粒横截面积的变化。如果保存了测量协议,则可以通过第6步中描述的Restore Protocol…功能来避免步骤4-5 。
对来自同一唾液腺的三个代表性细胞重复步骤4-8 。
对于每种基因型,对三个独立的唾液腺重复步骤4-9。
合并Excel 365中每个基因型的所有数据。
箱形图(插入>插入统计图>箱形图和晶须图)可用于通过横截面面积可视化分泌颗粒大小的分布(图7)。




图7.显示分泌颗粒大小分布的箱形图。野生型唾液腺中按横截面积的分泌颗粒大小分布。从底部到顶部的盒子的每一行代表的25个,50个(中位数),和75个百分点的人口。X表示样品的平均值。底部和顶部晶须代表由1.5 ×四分位间距确定的最小值和最大值。晶须之外的点是被视为统计异常值的样本。n = 3597个唾液腺细胞的分泌颗粒横截面(每个细胞3个Z切片,总共9个Z切片)。


吨-Test(数据>数据分析> t-检验:双样本异方差假设)可用于评估统计学差异在分泌颗粒横截面面积两种基因型之间。


菜谱


果蝇林格氏液(pH 7.2)
KCl            182 mM                           
氯化钠          46 mM                         

CaCl 2 · 2H 2 O 3 mM               

Tris-HCl        10毫米           

硅胶解剖板
混合基料和固化剂从所述(1:1,10 SYLGARD 184套件重量:重量)直接在100 -毫米P用刮刀ETRI菜。所有程序均应在通风橱中进行。
允许到通风柜内固化48小时。


致谢


该协议改编自我们先前的工作(Ma等,2020)。我们认可以下资金来源:SickKids Restracomp学生奖学金,加拿大卫生研究院战略培训奖学金#TGF-53877,多伦多大学开放奖学金和加拿大自然科学与工程研究理事会研究生奖学金–博士生奖学金(对马成一教授);加拿大卫生研究院拨款#PJT-162165和#MOP-119483以及加拿大卫生研究院遗传研究所#IG1-115714(授予Julie A. Brill)。


利益争夺


没有竞争利益可以宣布。


参考


Arvan ,P.和Castle,D.(1998)。分泌性颗粒生物发生过程中的分类和存储:回顾和展望。Biochem J 332(Pt 3):593-610。              
Biyasheva ,A.,Do,TV,Lu,Y.,Vaskova ,M。和Andres,AJ(2001)。果蝇唾液腺中的胶水分泌:类固醇调节胞吐作用的模型。Dev Biol 231(1):234-251。              
Bogan ,JS,Xu,Y.和Hao,M.(2012)。胆固醇积聚会增加胰岛素颗粒的大小并损害膜运输。交通13(11):1466-1480。
Borgonovo ,B.,Ouwendijk ,J。和Solimena ,M。(2006)。分泌颗粒的生物发生。Curr Opin Cell Biol 18(4):365-370。
Burgess,J.,Jauregui,M.,Tan,J.,Rollins,J.,Lallet ,S.,Leventis ,PA,Boulianne ,GL,Chang,HC,Le Borgne,R.,Kramer,H. and Brill, JA(2011)。AP-1和网格蛋白对于果蝇分泌性颗粒生物发生至关重要。分子生物学细胞22(12):2094-2105。
Burgess,J.,Del Bel,LM,Ma,CI,Barylko ,B.,Polevoy ,G.,Rollins,J.,Albanesi ,JP,Kramer,H.和Brill,JA(2012)。II型磷脂酰肌醇4-激酶调节果蝇中分泌性颗粒蛋白的运输。发展139(16):3040-3050。
曹明,毛泽东,甘长成,肖楠,曹新成,沉长成,成KK,徐爱玲,李明明,姜丽玲和夏, J.(2013)。PICK1和ICA69控制胰岛素颗粒的运输,其不足会导致葡萄糖耐量降低。PLoS Biol 11(4):e1001541。              
Costantino,BF,Bricker,DK,Alexandre,K.,Shen,K.,Merriam,JR,Antoniewski ,C.,Callender ,JL,Henrich,VC,Presente ,A.和Andres,AJ(2008)。一种新的蜕皮激素受体介导果蝇三分之一龄中龄期间类固醇调节的发育事件。PLoS Genet 4(6):e1000102。
Csizmadia ,T.,洛林奇,P.,Hegedus的,K.,Szeplaki ,S.,低,P。和Juhasz ,G。(2018)。果蝇发育程序吞噬的分子机制。J细胞生物学217(1):361-374。
杜威,周敏,赵威,程大成,王琳,卢健,宋恩恩,冯威,薛Y,徐平和徐婷(2016)。在成熟过程中,未成熟分泌颗粒的同型融合需要HID-1。Elif e 5:e18134 。
Edwards,SL,Charlie,NK,Richmond,JE,Hegermann ,J.,Eimer ,S。和Miller,KG(2009)。缺乏Rab2的秀丽隐杆线虫突变体的致密核心囊泡成熟受损。J Cell Biol 186(6):881-895。
Emperador-Melero ,J.,Huson ,V.,van Weering ,J.,Bollmann,C.,Fischer von Mollard ,G.,Toonen ,RF和Verhage ,M.(2018)。Vti1a / b通过高尔基体中的蛋白质分选来调节突触小泡和致密核心小泡的分泌。Nat Commun 9(1):3421。
B.Hummer,BH,NF de Leeuw,Burns,C.,Chen,L.,Joens ,MS,Hosford,B.,Fitzpatrick,JAJ和Asensio,CS(2017)。HID-1通过影响货物分选和反高尔基网络酸化来控制大型致密核心囊泡的形成。分子生物学细胞28(26):3870-3880。
Ma,CJ,Yang,Y.,Kim,T.,Chen,CH,Polevoy ,G.,Vissa,M.,Burgess,J. and Brill,JA(2020)。早期的内体来源的逆行运输途径促进分泌性颗粒成熟。J Cell Biol 219 (3):e201808017 。
Moore,HH,Andresen,JM,Eaton,BA,Grabe ,M.,H augwitz ,M.,Wu,MM和Machen,TE(2002)。垂体激素的生物合成和分泌:动力学和调节。Arch Physiol Biochem 110(1-2):16-25。              
Neuman,SD,Terry,EL,Selegue ,JE,Cavanagh,AT和Bashirullah ,A.(2020年)。在缺乏逆转录酶的细胞中分泌物的靶向错误。Dis Model Mec h 14(1):dmm046417。
Nitsch,C.和Rinne,U.(1981)。癫痫样癫痫发作过程中,兔子海马长满苔藓的纤维突触中有大的密集的核心囊泡胞吐作用和膜再循环。Ĵ Neurocytol 10(2):201-209。
Rao,A.,McBride,EL,Zhang,G.,Xu,H.,Cai,T.,Notkins ,AL,Aronova ,MA和Leapman ,RD(2020)。用串联块面电子显微镜测定胰岛β细胞分泌颗粒的成熟时间。结构生物学杂志212(1):107584。              
Tatsuoka ,H。和Reese,TS(1989)。直接冷冻和冷冻替代制备的前腹耳蜗核突触的新结构特征。J Comp Neurol 290(3):343-357。
Tooze ,SA(1991)。分泌颗粒的生物发生。未成熟的分泌颗粒在调节的分泌途径中产生的影响。FEBS Lett 285(2):220-224。              
Tooze ,SA(1998)。神经内分泌和内分泌细胞的反式高尔基网络中分泌颗粒的生物发生。Biochim Biophys Acta 1404(1-2):231-244。
伊利诺斯州的托雷斯(Torres),罗莎·费雷拉(Rosa-Ferreira)和南的蒙罗(Munro)(2014)果蝇分泌颗粒的生物发生需要Arf家族G蛋白Arl1 。J Cell Sci 127(Pt 10):2151-2160。              
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ma, C. J. and Brill, J. A. (2021). Quantitation of Secretory Granule Size in Drosophila Larval Salivary Glands. Bio-protocol 11(11): e4039. DOI: 10.21769/BioProtoc.4039.
  2. Ma, C. J., Yang, Y., Kim, T., Chen, C. H., Polevoy, G., Vissa, M., Burgess, J. and Brill, J. A. (2020). An early endosome-derived retrograde trafficking pathway promotes secretory granule maturation.J Cell Biol 219 (3): e201808017.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。