搜索

Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay
无细胞荧光高尔基体内逆向囊泡转运分析   

编审
Jia Li
引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
The Journal of Clinical Investigation
Jan 2017

Abstract

Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was tagged with either CFP or YFP. Given that GalT is recycled to the cisterna where it is located at steady state, GalT-containing vesicles target GalT-containing cisternal membranes. Golgi membranes were therefore isolated from GalT-CFP expressing cells, while vesicles were prepared from GalT-YFP expressing ones. Incubating CFP-labelled Golgi with YFP-labelled vesicles in the presence of cytosol and an energy regeneration mixture at 37 °C produced a significant increase in CFP-YFP co-localization upon fluorescent imaging of the mixture compared to incubation on ice. The assay was validated to require energy, proteins and physiologically important trafficking components such as Rab GTPases and the conserved oligomeric Golgi tethering complex. This assay is useful for the investigation of both physiological and pathological changes that affect the Golgi trafficking machinery, in particular, vesicle tethering.

Keywords: Golgi apparatus (高尔基体), Fluorescent imaging (荧光成像), Galactosyltransferase (半乳糖转移酶), Vesicle trafficking (囊泡转运), Vesicle tethering (囊泡栓系), Conserved oligomeric Golgi complex (保守寡聚高尔基体复合物)

Background

The molecular mechanisms of intracellular vesicle targeting are important to decipher to understand processes as diverse as glycosylation homeostasis, neurotransmitter release, regulation of signaling receptors and nutrient uptake (Ungar and Hughson, 2003; Fisher and Ungar, 2016). The Golgi apparatus is an excellent test case, as it maintains a network of target compartments, called cisternae, that require the specific delivery of different vesicles (Cottam and Ungar, 2012). The Golgi can also be isolated in a functional form retaining its ability for vesicle transport (Balch et al., 1984). Fluorescent labelling of vesicles and target cisternae offers a direct readout of vesicle targeting by measuring the co-localization of the two membrane fractions following a cell-free incubation. This type of measurement has some caveats. The size of vesicles is below the resolution limit of conventional microscopy, and there are only single fluorophores in the majority of the vesicles (C. Baumann and D. Ungar, University of York, unpublished data). This means that very high quality optics and sensitive detection has to be combined with automated exposure control during microscopy to avoid photobleaching, and sophisticated image processing to obtain images that are free of noise.

The assay was set up to investigate the molecular requirements of vesicle tethering at the trans-Golgi (Cottam et al., 2014). Accordingly, it was found to be dependent on functional Rab GTPases (Rabs), as the protein Rab-GDI, which extracts Rabs from membranes (Soldati et al., 1993), was found to inhibit the signal (Cottam et al., 2014). Moreover, the assay was sensitive to various defects of the conserved oligomeric Golgi (COG) tethering complex. Cytosol is an essential component of the assay mixture for obtaining activity, and when used from cells harboring patient-derived COG mutations (Wu et al., 2004; Luebbehusen et al., 2010), it inhibited the assay signal (Cottam et al., 2014). Moreover, the assay was able to differentiate the contributions to retrograde trafficking of two different COG mutants (Cog1- and Cog2-null mutations, Cottam et al., 2014), which have essentially identical cellular phenotypes in CHO cells (Kingsley et al., 1986).

Materials and Reagents

  1. Microscope slides and coverslips (purchased from Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: MNJ-400-030Y and MNJ-100-030J )
    Note: The quality of the slides and coverslips is critical, the mentioned product does work, if others are to be used it is advised to test these.
  2. Silica beads (5 micron silica beads) (Bangs Laboratories, catalog number: SS06N )
  3. 0.5 ml and 1.5 ml microfuge tubes, 0.2 ml PCR tubes
  4. T175 flasks (each 175 cm2) (Corning BV Life Sciences, Amsterdam, the Netherlands)
  5. 10 ml serological pipette
  6. PD-10 desalting column (GE Healthcare, Buckinghamshire, UK)
  7. Wild type HEK293 cells
  8. HEK293 cells stably expressing CFP/YFP-GalT
  9. Water
    Note: Molecular biology grade water was used for all procedures, including the washing of microscope slides and coverslips.
  10. Detergent decon90 (Decon Laboratories Limited, Sussex, UK)
  11. Potassium chloride (KCl)
  12. Geneticin (GibcoTM)
  13. Dulbecco’s Modified Eagle Medium (DMEM, high glucose, pyruvate, no glutamine) (Thermo Fisher Scientific, GibcoTM, catalog number: 21969 )
  14. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 12657029 )
  15. GlutaMAX-I (an L-alanyl-L-glutamine dipeptide substitute for L-glutamine) (Thermo Fisher Scientific, GibcoTM, catalog number: A12860-01 )
  16. Penicillin-streptomycin (10,000 U/ml; 100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  17. Ham’s F-12 Nutrient Mix (Sigma-Aldrich, catalog number: N4888 )
  18. Liquid nitrogen
  19. Tris
  20. Optiprep density gradient media (Axis-Shield PoC) (Cosmo Bio, catalog number: AXS-1114542 )
  21. α-HA antibody (monoclonal anti-HA.11) (BioLegend, catalog number: 901513 )
  22. Dithiothreitol (DTT)
  23. Golgi membranes, vesicles, cytosol (see preparation of working aliquots under Recipes)
  24. Sucrose (Fisher Scientific, catalog number: S/8600/60 )
  25. HEPES
  26. Creatine phosphokinase
  27. GTP
  28. ATP
  29. Creatine phosphate
  30. Potassium hydroxide (KOH)
  31. Magnesium acetate (Mg(OAc)2)
  32. Magnesium chloride (MgCl2)
  33. Trypsin, 2.5% (10x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
  34. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  35. Buffers (see Recipes)
    1. Assay sucrose
    2. ATP/GTP mixture (10x)
    3. Cytosol buffer
    4. HM buffer
    5. KHM buffer
    6. Reaction buffer (10x)
    7. Trypsin-PBS buffer

Note: All reagents and buffers should be stored in convenient sized aliquots at -80 °C. When running low on critical aliquots (membranes, cytosol, ATP/GTP mixture), prepare a new set and test it against the old ones to ensure reproducibility.

Equipment

  1. Microwave oven
  2. Water bath
  3. Sonicating water bath (Grant Instruments, model number: XUBA3 )
  4. Incubator
  5. 1 ml Dounce homogenizer (DWK Life Sciences, Wheaton, catalog number: 357538 )
  6. Centrifuge
  7. Ultracentrifuge
  8. SW41 rotor (Beckman Coulter, model: SW 41 Ti ) and 13.2 ml thinwall ultra-clearTM tubes (Beckman Coulter, catalog number: 344059 )
  9. Sugar refractometer (range 0-50%) (Bellingham and Stanley Ltd, UK)
  10. TLA 100.3 rotor (Beckman Coulter, model: TLA-100.3 ) and 3.5 ml thickwall polycarbonate tubes (Beckman Coulter, catalog number: 349622 )
  11. TLS-55 rotor (Beckman Coulter, model: TLS-55 ) and 2.2 ml thinwall ultra-clearTM tubes (Beckman Coulter, catalog number: 347356 )
  12. Standard mammalian cell culture apparatus
  13. Evolve 512 EMCCD (electron multiplying charged coupled device) Camera (Photometrics, model: Evolve® 512 )
  14. Zeiss Axiovert 200M fully motorized inverted microscope (Carl Zeiss, model: Axiovert 200M )
  15. X-Cite 120Q excitation light source (Excelitas Technologies, model: X-Cite 120Q )
  16. CFP filter (Chroma Technology, catalog number: 49001 )
  17. YFP filter (Chroma Technology, catalog number: 49003 )
  18. Objective lens (Zeiss Plan-Apochromat 63x/1.40 Oil DIC, Carl Zeiss Ltd, Cambridge, UK)
  19. Black card to exclude room light from samples during imaging

Software

  1. ZEN 2009 software (www.zeiss.com)
  2. PM Capture Pro software (http://www.photometrics.com)
  3. AutoHotkey (www.autohotkey.com) (AutoHotkey Foundation LLC)
  4. ImageJ (https://imagej.nih.gov/ij/)

Procedure

  1. Experimental preparation
    1. Water
      Note: Molecular biology grade water was used for all procedures, including the washing of microscope slides and coverslips.
    2. Microscope slides and coverslips
      1. To fully submerge the slides and coverslips in their racks, sufficient water was warmed to ~40 °C in a microwave, then the detergent decon90 (Decon Laboratories Limited, Sussex, UK) was added to 3% (v/v). In their racks, slides and coverslips were dipped up and down 10 times in the detergent solution to help dislodge surface contaminants, then left to soak for ~7 h.
      2. After dipping up and down a further 5 times, the solution was discarded and the glassware rinsed once with water as follows: sufficient water was added to the container to fully submerge glassware which was then dipped up and down 10 times. Fresh 3% (v/v) decon90 solution was made up, and the glassware left to soak in this for at least 12 h. Glassware was then thoroughly rinsed at least 5 times in water, then placed in an oven at 60 °C until dry. Once dry, slides and coverslips were stored in covered boxes to prevent dust contamination.
    3. Silica beads
      1. A suspension containing 5 micron silica beads was prepared for addition to assay mixtures prior to mounting. The beads acted as a spacer between the slide and the coverslip. A small amount of bead powder (~5 μl dry volume) was deposited in a 1.5 ml microfuge tube to which 500 μl of 150 mM KCl, 10 mM HEPES, pH 7.2 was added.
      2. The beads were distributed in this buffer by vigorous vortexing followed by 30 sec agitation in a sonicating water bath (power 35 W, intensity 44 kHz)–this was repeated once more to make the beads become mono-dispersed. A bead density was required such that 1 μl of this suspension added to a 25 μl assay mixture would provide ~1-2 beads per field of view when 3 μl of the mixture was mounted and viewed under the 63x objective. Therefore, when a new stock of beads was made, the bead density was tested by mixing 1 μl of the stock with 25 μl water and mounting 3 μl of this suspension as described below and viewing under a microscope. If the number of beads was too low, the bead stock was briefly spun down and enough buffer removed to increase bead density when resuspended. More buffer could be added to the bead stock for the opposite effect.
    4. Mammalian cell culture
      Wild type and HEK293 cell clones stably expressing GalT-XFP constructs (Cottam et al., 2014) were used. These were generated by stable transfection of a pCR3.1 vector (Invitrogen) based plasmid containing human β-1,4-galactosyltransferase 1 with a CFP or a YFP tag, and selection with 800 µg/ml Geneticin (GibcoTM). Single cell-derived clones were selected for use if the expressed GalT-XFP was found localized in the Golgi only, rather than partly distributed in cytosolic puncta. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM GlutaMAX-I, and 100 U of penicillin and 100 μg of streptomycin per ml. Most other cell lines were cultured in the same media, although CHO lines needed Ham’s F12 media supplemented with 5% FBS and the same antibiotics composition as for HEK cell culture. All cell lines were grown in a humidified incubator at 37 °C and 5% CO2.
    5. Golgi membranes
      1. The following method is adapted from Balch et al. (1984). Typically, two T175 flasks (each 175 cm2) of stably expressing GalT-CFP HEK cells (Cottam et al., 2014) were grown to a confluent monolayer (~7-10 x 107 cells total), harvested, and washed twice in 0.25 M sucrose, 10 mM HEPES, pH 7.4. The cell pellet (~500-600 μl) was resuspended in four times the pellet volume using the same 0.25 M sucrose buffer. The suspension was treated with 25 strokes in a 1 ml tight fitting Dounce homogenizer. The homogenate was flash frozen in liquid nitrogen and then stored at -80 °C for later subcellular fractionation.
      2. Golgi membranes were isolated from homogenates by floatation on a discontinuous sucrose gradient by centrifugation. Frozen homogenates were rapidly thawed at 37 °C and then maintained on ice. The sucrose concentration of the homogenate was adjusted to 1.4 M by adding ice-cold 2.3 M sucrose, 10 mM HEPES, pH 7.4 at a volume of 1.28-fold to the homogenate volume, and mixed thoroughly by pipetting with a 10 ml serological pipette.
      3. The mixture was loaded into an SW41 tube to form the bottom layer and then overlaid with 4.94 ml of 1.2 M sucrose, 10 mM HEPES, pH 7.4. These two layers were then overlaid with enough 0.8 M sucrose, 10 mM HEPES, pH 7.4 to fill the tube to its maximum capacity of 12 ml (between 2.5 ml and 3 ml depending on the volume of the bottom layer). The gradient was then centrifuged for 80 min at 39,000 rpm, 4 °C in an SW41 rotor. In a cold room, the turbid band at the 1.2/0.8 M sucrose interface was then drawn off in a minimum volume (~700-800 μl) by manual pipetting from the top of the gradient downwards. This fraction was mixed well, aliquoted into 20 μl volumes in PCR tubes, and flash frozen in liquid nitrogen. The sucrose concentration of the final sample was measured using a 0-50% sugar refractometer (Bellingham and Stanley Ltd, UK). The sucrose concentration was always very close to and slightly above 1 M.
      4. In the assay, 1 μl from a stock of Golgi isolated in the above fashion gave ~32 particles per field of view by EMCCD imaging.
    6. Vesicles
      1. The following method is adapted from Love et al. (1998). Typically, four T175 flasks of stably expressing GalT-YFP HEK cells were grown to a confluent monolayer (~14-20 x 107 cells total), harvested, and washed once in PBS and then in 0.2 M sucrose, 10 mM Tris, pH 7.2. The pellet (~1 ml) was resuspended in four times the pellet volume using the same 0.2 M sucrose buffer, flash frozen in liquid nitrogen and then stored at -80 °C until needed.
      2. Vesicle membranes were isolated by floatation on a discontinuous gradient of Optiprep density gradient media by centrifugation. Frozen cell suspensions were gradually thawed in a water bath at 21 °C. This caused permeabilization of cells by membrane fracture.
      3. The cells were centrifuged for 5 min at 1,000 x g, 4 °C, which left vesicles in the supernatant. This supernatant was centrifuged once more for 5 min at 1,000 x g, 4 °C then twice more for 20 min at 20,000 x g, 4 °C to remove all debris and large cellular membranes. Vesicles in the supernatant were then pelleted by ultracentrifugation at 55,000 rpm for 45 min at 4 °C in a TLA 100.3 rotor. The vesicle pellet (typically ~30 μl) was resuspended in KHM buffer (see Recipes) to a volume of 320 μl. This 320 μl was mixed with 480 μl of 50% Optiprep in HM buffer (see Recipes). The mixture was transferred to a TLS-55 tube and overlayed with 800 μl of 25% Optiprep in KHM buffer and then with 400 μl of 10% Optiprep in KHM buffer.
      4. Vesicles were floated to the 10%/25% interface by ultracentrifugation at 55,000 rpm for 3 h 10 min at 4 °C in a TLS-55 rotor. The turbid band at the interface was harvested by manual pipetting in a minimum volume of typically 300-400 μl and mixed well. 10-15 μl aliquots were made into PCR tubes which were flash frozen in liquid nitrogen. In the assay, 1 μl from a stock of vesicles isolated in the above fashion gave ~150-250 particles per field of view by EMCCD imaging.
      5. A larger, more concentrated batch of vesicles was produced for studies requiring cytosol dependence by using a scaled-up version of the above method. ~3.5 x 109 GalT-YFP HEK cells were harvested. The Optiprep gradient was increased 6.25 times to 12.5 ml total volume in an SW41 tube, then ultracentrifuged at 41,000 rpm for 8 h at 4 °C. 250 μl fractions were manually drawn off the gradient by pipetting. Western blot analysis of the fractions (using an α-HA antibody against an HA tag in the GalT-YFP construct for detection) showed six consecutive fractions around the 10%/25% interface region which had the highest signal (indicating vesicles). These fractions were pooled (1.5 ml total), aliquoted and flash frozen as above. 1 μl from this concentrated stock of vesicles in the assay gave ~1,000-1,500 particles per field of view by EMCCD imaging.
    7. Cytosol
      1. The method for cytosol preparation was adapted from (Balch et al., 1984). Four T175 flasks of cells were grown to full confluency (~14-20 x 107 HEK293 cells in total). Cells were trypsinized using trypsin-PBS buffer, then washed 4 times with PBS before washing twice in ice-cold 0.25 M sucrose, 10 mM HEPES, pH 7.4. The pellet was resuspended in 1.5 ml of the same buffer with fresh DTT added to 1 mM from a 1 M aqueous stock.
      2. The suspension was treated with 30 strokes in a 1 ml tight fitting Dounce homogenizer. The homogenate was transferred to a TLA 100.3 ultracentrifuge tube and centrifuged at 60,000 rpm for 45 min at 4 °C. The supernatant was removed and centrifuged again in the same way to remove all cell debris. Meanwhile, a PD-10 desalting column was equilibrated with 100 mM KCl, 1 mM DTT, 10 mM HEPES, pH 7.2. The cleared cytosol supernatant was added to the column. The desalted cytosol was eluted with the same equilibration buffer. Most of the protein was contained within the first 2.5 ml of eluate as determined by Bradford protein assay. Protein concentration was typically 7.5-8.5 mg/ml. The eluates were mixed well then aliquoted and flash frozen in liquid nitrogen.

  2. Set-up, incubation and mounting
    1. Set-up
      Note: All samples, except for water, must be kept on ice that is mixed with cold water (to allow for good heat exchange) prior to use in the assay.
      1. Set the water bath to 37 °C. Prepare dilutions of all other reagents before defrosting membranes, cytosol and ATP/GTP mixture.
        Note: Defrost membrane aliquots and cytosol quickly in your hand and then put on wet ice.
      2. Aliquots of vesicles, Golgi, cytosol and ATP must be briefly spun down (~3 sec) in a microfuge straight after defrosting.
        Note: Do not vortex or flick membranes, cytosol or ATP/GTP, rather mix these by setting pipette volume to ~10% less than the aliquot volume and pipet up and down 7 times before any volume is removed. Buffers, in contrast, should be mixed by vortexing after defrosting.
      3. Dilute the vesicle stock to ensure about 70-150 vesicles per image.
        Notes:
        1. This should be tested for new vesicle preps, but is generally in the range of a 10-30x dilution.
        2. Assay mixtures should be made up in 0.5 ml microfuge tubes. Mix assay components in the order: water, reaction buffer and other buffers, cytosol, Golgi, vesicles, and ATP/GTP mixture.
    2. Incubation
      1. A typical assay will contain: 3.8 µl Golgi (see Note 1), 4 µl diluted vesicle stock, cytosol equivalent to 57 mg total protein, cytosol buffer (see Recipes) such that the sum of this and the cytosol volume is 15.5 µl, 5 µl 10x concentrated reaction buffer (see Recipes), 2.75 µl assay sucrose (see Recipes), 5 µl 10x concentrated ATP/GTP mixture (see Recipes) in a 50 µl total volume. The assay mixture is gently pipetted up and down 10 times before placing 25 µl into a separate tube. Half of the assay mixture is left on wet ice while the other is transferred to the 37 °C water bath.
      2. Samples are incubated at 37 °C for 40 min in the dark, then transferred back onto wet ice. Assay mixtures are mounted immediately after incubation.
    3. Mounting
      Prior to mounting, the silica bead suspension was briefly vortexed, and then 1 µl of this added to the assay mixture. The assay mixture was pipetted up and down 20 times with a pipet set to 21 µl, then quickly 3 µl was applied as two separate drops onto one half of a slide and immediately covered with a clean square coverslip. The edges were quickly sealed with clear nail varnish to prevent sample evaporation and fix in place. The 37 °C and ice incubated halves of the same assay were mounted in pairs onto the same slide. Slides were stored protected from light in a 4 °C fridge until imaging. Figure 1 shows how sample mounting is performed in the overall context of how the assay is executed.


      Figure 1. Execution of the assay. GalT-YFP vesicles and GalT-CFP Golgi are mixed together in a total reaction volume of 50 μl containing the desired reaction conditions. The mixture is equally divided into two tubes. One is incubated at 0 °C for 40 min as an internal control, and the other is incubated at 37 °C for the same period. After incubation, 5 micron silica beads are mixed with each sample, then 3 μl of the mixture is immediately delivered in two roughly equally-sized spots onto a microscope slide. The sample is covered with a 22 x 22 mm coverslip, sealed around the edges with clear nail varnish with the silica beads acting as spacers. The slide is kept in the dark at 4 °C until imaging by epifluorescence microscopy at room temperature.

  3. Imaging
    1. Equipment set-up and control
      1. Images were collected by bright-field microscopy using an Evolve 512 EMCCD camera, attached to a ZEISS Axiovert 200M fully motorized inverted microscope. Illumination was provided by an X-Cite 120Q excitation light source. The adjustable iris of the X-Cite was set to level 3 of 4, where level 4 is fully open for maximum output. The objective lens was a ZEISS Plan-Apochromat 63x/1.40 Oil DIC. The microscope was controlled by ZEN 2009 software (ZEISS) and the camera was controlled by PM Capture Pro software (Photometrics) running on the same computer system as the ZEISS software. CFP and YFP filters from Chroma Technology Corporation were used. Although confocality is not used, it is essential to use a fully motorized stage, filter wheel and shutters, hence the use of this microscope. Extraneous room light on the sample during imaging was minimized by using low-light conditions and by also shielding the sample with matt black card as follows. On a circular disc of the card (~7 cm in diameter), a circular hole was cut in the center that was slightly smaller than the diameter of the outer casing of the retractable front lens on the 63x objective. The front lens was gently pushed through the hole so that the disc formed a wide ‘collar’ to block light from below, then a sheet of card with a central rectangular hole was placed over the objective to cover gaps. Subsequently the universal mounting frame (i.e., the part which clips into the motorized stage) was placed above the rectangular card to hold the slide. On the microscope stage, a box lid made of the same card of dimensions approximately 15 x 10 x 2 cm (L x W x H), was placed over the sample (Figure 2).


        Figure 2. Shielding the mounted sample during imaging. Although low-light room conditions are used during imaging, extraneous light on the sample is also minimized by shielding. Matt black card is utilized as follows: 1. a wide collar is fitted around the objective lens to block light from below; 2. a sheet with a rectangular hole is placed over the objective to cover gaps; and 3. a shallow box lid is placed on top to block light from above. The slide is placed in the universal mounting frame on top of the rectangular sheet in image 2.

      2. The open source software AutoHotkey was used to write scripts to automate control of the ZEN 2009 and PM Capture Pro software. The scripts (programmed to be launched by pressing the keyboard F-keys or buttons of a joypad i.e., as ‘hotkeys’) moved the cursor to activate commands in both the microscope and camera software windows with appropriate time delays. The scripts were programmed so that the operations of changing filters, opening/closing the lamp shutter, and camera image acquisition occurred in sequence to obtain 1 image each for vesicles (YFP channel) and Golgi (CFP channel). This ensured all images were captured under the same conditions, and without unnecessary delay to avoid bleaching of the fluorophores.
    2. Sample handling and image acquisition
      1. The sample should be removed from the fridge a couple minutes before imaging to stop condensation build-up. Once on the microscope, the room must be switched to low-light conditions and the sample must be shielded from external light by covering it with matt black card from top and bottom as described above. Focusing is tricky due to the lack of contrast of the membranes under bright-field illumination. Initial focusing should aim for the 5 µm glass beads using the halogen lamp on low intensity.
      2. Then, for finer focus using the YFP fluorescence of the vesicles, the high gain preview mode of the EMCCD camera must be used (see Note 2). They are too faint to be seen via the eyepiece and the camera needs to ‘see’ the precise focal plane of these sub-micron particles. It is easy to overfocus, thereby squashing the sample, in which case it has to be re-mounted. Once the focal plane with the vesicles is found, care should be taken so that all images are collected from the particles sitting on the coverslip rather than the ones on the slide. A quick refocusing away from the coverslip will verify this by showing the slide-attached particles; this check should be performed regularly. Moving the distance of a few fields of view in the x-y plane usually keeps the focus close enough to require only little adjustments to regain focus.
      3. For each assay condition, 12-16 images per incubation temperature need to be collected. Particles bleach quickly, so care should be taken that the focusing time overall does not exceed 7 sec before image acquisition is started. Camera settings may need to be adjusted depending on the age of the excitation lamp. A set of ideal settings are in Note 2. A very short exposure time with binning and a high gain is used for finding an appropriate area and focusing. Conversely, a long exposure time without binning and low gain is used to acquire images of vesicles and Golgi with higher resolution and lower noise. Scripts need to be written (using for example the AutoHotkey program for PC) to automate the sequence of operations performed by the microscope and camera to acquire images for the YFP and CFP channels (described above). These scripts should be programmed to launch upon pressing single ‘hotkeys’ such as the F-keys, or even the buttons of a joypad can be assigned. This allows single click initiation of the three presets (see Note 2) that allow preview mode for focusing, followed by YFP and CFP image acquisition.
        Note: We found that programming further keys for saving the YFP (vesicles) and CFP (Golgi) channel images with a V_ or G_ prefix followed by a time-stamp in the file name made data acquisition and subsequent processing much more user-friendly. This also ensured that the correct vesicle and Golgi images from the same field of view were always paired up together afterwards.

Data analysis

  1. ImageJ was used to process and analyze all images. For Golgi, images were converted to 8 bit, then background noise reduced using the ‘subtract background’ command (rolling ball radius 2.0 pixels, sliding paraboloid). The threshold was set to ‘Triangle dark’, and the lower threshold value was multiplied by a factor of 1.1 to ensure that particles were selected above background noise. A binary image was made of the selection, and any selected particles containing holes were filled in. A summary of the number of particles was generated. The Look Up Table for the binary image was changed to ‘red’ and saved. The same process was conducted for vesicle images except that after the ‘Triangle dark’ threshold, the lower threshold value was multiplied by a factor of 1.4. The Look Up Table for the binary image was changed to ‘green’ and saved. The multiplying factor, for both Golgi and vesicles images, can be increased in case images are very noisy, or even decreased if noise is low. However, the same factor should be used for all images collected on the same day. Both the Golgi (red) and vesicle (green) binary images were opened, converted to RGB color images, and then merged into a composite image where overlapping red and green particles were additive to give yellow pixels. Using the ‘connection thresholding’ plugin (obtained from http://imagejdocu.tudor.lu), the thresholds in the dialogue box for this plugin were adjusted so that Golgi were selected in blue, overlapping (colocalized) pixels in red, and non-colocalizing vesicle particles were excluded. The ‘Hyst’ (Hysteresis) command in the dialogue box was selected. This caused any Golgi particle containing one or more colocalized pixels to be filled in as a single colocalization event, and output as a binary image. A threshold of ‘255, 255’ was applied and the number of such colocalization events was counted. These image processing operations are summarized in Figure 3. The above operations carried out on Golgi and vesicle images were compiled into ImageJ macros (Note 3) which allowed multiple images to be processed as a batch. Following the processing, all images should be looked at to remove false positive colocalization events caused by the 5 µm beads, which occasionally give fluorescent signals in both channels, but can easily be distinguished from real events due to the shape of the putative particle.


    Figure 3. Image processing workflow (Cottam, 2012). A. Raw images of Golgi and vesicles are treated with background subtraction. This also corrects slight uneven illumination of the sample generating the images in (B). B. An intensity threshold is applied to images to select particles above the average background noise. C. The selection is converted to a binary image and re-colored to magenta and green for Golgi and vesicles respectively. D. The binary images are overlaid to reveal colocalized areas as white pixels. E. A hysteresis operation is applied to the overlaid image which completely fills any particle containing white pixels to become a single colocalization event. The number of colocalization events is expressed as a percentage of the total number of particles (colocalization events/(Golgi + vesicles)) to give the measure of assay activity. In our previous work, we have used the calculation (Golgi + vesicles + colocalization events) for total particle numbers, which is not correct but should be considered when comparing data to our published results. Given the generally low number of colocalization events (Figure 4), the distortion by the incorrect particle total does not change the biological conclusions when comparing results for different assay conditions. Scale bar = 10 μm.

  2. In Excel, the number of colocalized particles was expressed as a percentage of the total number of particles ((colocalized/(Golgi + vesicles)); see explanation in the legend of Figure 3). This gave the percentage of activity in the sample. Activity for the 37 °C incubation within each assay condition was then normalized to the activity in the corresponding ice control, giving the normalized activity of each condition (i.e., the fold increase compared to the ice control) (see Figure 4). Background activity does vary from day to day. However, we found that it was not practical to perform assays with more than six separate conditions on one day. Therefore a further normalization step was introduced, in which each condition’s normalized activity was further normalized to a wild type (or other suitable) control sample measured on the day. Activities normalized in this manner to the same control on different measurement days could be robustly compared and averaged.


    Figure 4. Typical data sheet of the image processing results. Particle numbers as well as the numbers of co-localizing particles are shown for each image.

Notes

  1. When following the preparation protocol above, the concentration of the prep allows the amount of Golgi membranes in the assay to be very close to 3.8 µl giving about 70-150 particles per image. If the concentration of the Golgi preparation is very different, care needs to be taken to balance the amount of Golgi added with the sucrose buffer such that the osmolarity of the final assay mixture is not too far off the 310 mOsm of DMEM.
  2. Camera preset parameters in the PM Capture Pro software:
    1. Preset 1–preview mode (high gain, high noise) for finding the right area and focusing quickly:
      2 x 2 binning, analogue-to-digital gain 3, exposure 120 msec, electron multiplication gain = 220
    2. Preset 2–YFP capture (low gain, low noise):
      1 x 1 binning, analogue-to-digital gain 3, exposure 4.5 sec, electron multiplication gain = 22
    3. Preset 3–CFP capture (low gain, low noise):
      1 x 1 binning, analogue-to-digital gain 3, exposure 2.6 sec, electron multiplication gain = 8
  3. Macros for ImageJ
    1. YFP vesicle processing:

      for (i=1; i<=nImages; i++) {
      selectImage(i);
      run("8-bit");
      run("Subtract Background...", "rolling=2 sliding");
      setAutoThreshold("Triangle dark");
      getThreshold(lower, upper);
      setThreshold(lower*1.4, 255);
      run("Make Binary", "thresholded remaining black");
      run("Invert");
      run("Fill Holes");
      setThreshold(255, 255);
      run("Analyze Particles...", "size=0-infinity circularity=0.00-1.00 show=Nothing clear include summarize");
      run("Green");
      }
      run("Images to Stack", "name=YFP_stack stack title=[] use");
      saveAs("Tiff")

    2. CFP Golgi processing:

      for (i=1; i<=nImages; i++) {
      selectImage(i);
      run("8-bit");
      run("Subtract Background...", "rolling=2 sliding");
      setAutoThreshold("Triangle dark");
      getThreshold(lower, upper);
      setThreshold(lower*1.1, 255);
      run("Make Binary", "thresholded remaining black");
      run("Invert");
      run("Fill Holes");
      setThreshold(255, 255);
      run("Analyze Particles...", "size=0-infinity circularity=0.00-1.00 show=Nothing clear include summarize");
      run("Red");
      }
      run("Images to Stack", "name=CFP_stack stack title=[] use");
      saveAs("Tiff")

    3. To generate a merged RGB stack:

      for (i=1; i<=nImages; i++) {
      selectImage(i);
      run("RGB Color");
      }
      run("Merge Channels...", "red=CFP_stack.tif green=YFP_stack.tif blue=*None* gray=*None* create");
      saveAs("Tiff")

Recipes

  1. Assay sucrose
    1.2 M sucrose
    10 mM HEPES, pH 7.4
  2. ATP/GTP mixture (10x)
    Creatine phosphokinase 1,500 U/ml
    10 mM GTP
    5 mM ATP
    200 mM creatine phosphate
    7.5 mM KOH to neutralise the ATP
    20 mM HEPES, pH 7.4
  3. Cytosol buffer
    100 mM KCl
    1 mM DTT
    10 mM HEPES, pH 7.2
  4. HM buffer
    10 mM HEPES, pH 7.2
    2.5 mM Mg(OAc)2
  5. KHM buffer
    150 mM KCl
    2.5 mM Mg(OAc)2
    10 mM HEPES, pH 7.2
  6. Reaction buffer (10x)
    250 mM HEPES, pH 7.4
    20 mM MgCl2
  7. Trypsin-PBS buffer
    0.25% (v/v) trypsin in PBS

Acknowledgments

We are grateful to the Imaging Facility at the University of York, Department of Biology’s Bioscience Technology Facility. This work was supported by a BBSRC PhD studentship supporting NPC awarded to DU and Marie Curie grant (201098) to DU. The protocol was published in (Cottam et al., 2014). The authors declare that there’s no conflicts of interest.

References

  1. Balch, W. E., Dunphy, W. G., Braell, W. A. and Rothman, J. E. (1984). Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell 39(2 Pt 1): 405-416.
  2. Cottam, N. P. (2012). A cell-free vesicle tethering assay. PhD thesis, University of York.
  3. Cottam, N. P. and Ungar, D. (2012). Retrograde vesicle transport in the Golgi. Protoplasma 249(4): 943-955.
  4. Cottam, N. P., Wilson, K. M., Ng, B. G., Korner, C., Freeze, H. H. and Ungar, D. (2014). Dissecting functions of the conserved oligomeric Golgi tethering complex using a cell-free assay. Traffic 15(1): 12-21.
  5. Fisher, P., and Ungar, D. (2016). Bridging the gap between glycosylation and vesicle traffic. Front Cell Dev Biol 4: 15.
  6. Kingsley, D. M., Kozarsky, K. F., Segal, M. and Krieger, M. (1986). Three types of low density lipoprotein receptor-deficient mutant have pleiotropic defects in the synthesis of N-linked, O-linked, and lipid-linked carbohydrate chains. J Cell Biol 102(5): 1576-1585.
  7. Love, H. D., Lin, C. C., Short, C. S. and Ostermann, J. (1998). Isolation of functional Golgi-derived vesicles with a possible role in retrograde transport. J Cell Biol 140(3): 541-551.
  8. Luebbehusen, J., Thiel, C., Rind, N., Ungar, D., Prinsen, B. H., de Koning, T. J., van Hasselt, P. M. and Koerner, C. (2010). Fatal outcome due to deficiency of subunit 6 of the conserved oligomeric Golgi complex leading to a new type of congenital disorders of glycosylation. Hum Mol Genet 19(18): 3623-3633.
  9. Soldati, T., Riederer, M. A. and Pfeffer, S. R. (1993). Rab GDI: a solubilizing and recycling factor for rab9 protein. Mol Biol Cell 4(4): 425-434.
  10. Ungar, D., and Hughson, F. M. (2003). SNARE protein structure and function. Annu Rev Cell Dev Biol 19: 493-517.
  11. Wu, X., Steet, R. A., Bohorov, O., Bakker, J., Newell, J., Krieger, M., Spaapen, L., Kornfeld, S. and Freeze, H. H. (2004). Mutation of the COG complex subunit gene COG7 causes a lethal congenital disorder. Nat Med 10(5): 518-523.

简介

高尔基体内的逆行囊泡运输被用来运送和分类高尔基酶到适当的池内位置。建立了一个检测方法来研究无细胞系统中囊泡靶向的分子细节。生成了稳定的细胞系,其中反式 - 高尔基酶半乳糖基转移酶(GalT)用CFP或YFP标记。考虑到GalT被循环到稳定状态的小池中,含有GalT的囊泡将靶向含有GalT的池内膜。因此从表达GalT-CFP的细胞分离高尔基体膜,而从GalT-YFP表达细胞制备囊泡。在胞质溶胶和能量再生混合物的存在下,在37℃孵育CFP标记的高尔基体和YFP标记的囊泡,与在冰上孵育相比,在混合物的荧光成像后CFP-YFP共定位显着增加。该测定被验证需要能量,蛋白质和生理学重要的运输组分,如Rab GTP酶和保守寡聚体高尔基体系复合物。该测定法可用于调查影响高尔基体运输机器的生理和病理变化,特别是囊泡束缚。

【背景】细胞内囊泡靶向的分子机制对于解释糖基化稳态,神经递质释放,信号受体的调节和营养摄取等方面的解释是重要的(Ungar和Hughson,2003; Fisher和Ungar,2016)。高尔基体是一个很好的测试案例,因为它维持着一个被称为cisternae的目标室的网络,这个网络需要不同囊泡的特定输送(Cottam和Ungar,2012)。高尔基体也可以以保持其囊泡运输能力的功能性形式被分离(Balch等人,1984)。囊泡和靶标池的荧光标记通过在无细胞温育之后测量两个膜片段的共定位来提供囊泡靶向的直接读数。这种类型的测量有一些警告。囊泡的大小低于常规显微镜的分辨率极限,并且在大多数囊泡中仅有单一荧光团(C.Baumann和D.Ungar,University of York,未公开的数据)。这意味着,高质量的光学元件和灵敏的检测必须与显微镜过程中的自动曝光控制相结合,以避免光漂白和复杂的图像处理,以获得无噪声的图像。

为了研究反式-Golgi(Cottam等人,2014)中的囊泡束缚的分子需求,设立了该测定法。因此,发现它依赖于功能性Rab GTP酶(Rabs),因为发现从膜中提取Rabs的蛋白质Rab-GDI(Soldati等人,1993)被发现能抑制信号(Cottam et al。,2014)。此外,该测定对保守寡聚高尔基(COG)系链复合物的各种缺陷敏感。胞质溶胶是用于获得活性的测定混合物的重要组分,并且当从含有患者衍生的COG突变的细胞中使用时(Wu等人,2004; Luebbehusen等人 ,2010),它抑制了测定信号(Cottam et al。,2014)。此外,该测定能够区分在CHO中具有基本上相同的细胞表型的对两种不同COG突变体(Cog1-和Cog2-无效突变,Cottam等人,2014)逆行运输的贡献细胞(Kingsley等人,1986)。

关键字:高尔基体, 荧光成像, 半乳糖转移酶, 囊泡转运, 囊泡栓系, 保守寡聚高尔基体复合物

材料和试剂

  1. 显微镜载玻片和盖玻片(购自Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:MNJ-400-030Y和MNJ-100-030J)
    注:幻灯片和盖玻片的质量是至关重要的,上述产品确实工作,如果其他人被使用,建议测试这些。
  2. 二氧化硅珠(5微米硅珠)(Bangs Laboratories,目录号:SS06N)
  3. 0.5 ml和1.5 ml微量离心管,0.2 ml PCR管
  4. T175烧瓶(每个175cm2)(Corning BV Life Sciences,荷兰阿姆斯特丹)
  5. 10毫升血清移液器
  6. PD-10脱盐柱(GE Healthcare,英国白金汉郡)
  7. 野生型HEK293细胞
  8. 稳定表达CFP / YFP-GalT的HEK293细胞

  9. 注:分子生物学级别的水用于所有程序,包括显微镜载玻片和盖玻片的洗涤。
  10. 洗涤剂decon90(Decon Laboratories Limited,Sussex,UK)
  11. 氯化钾(KCl)
  12. 遗传霉素(Gibco TM)
  13. Dulbecco改良的Eagle培养基(DMEM,高葡萄糖,丙酮酸,无谷氨酰胺)(Thermo Fisher Scientific,Gibco TM,目录号:21969)
  14. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,产品目录号:12657029)
  15. GlutaMAX-I(用于L-谷氨酰胺的L-丙氨酰-L-谷氨酰胺二肽替代物)(Thermo Fisher Scientific,Gibco TM,目录号:A12860-01)
  16. 青霉素 - 链霉素(10,000U / ml; 100x)(Thermo Fisher Scientific,Gibco,产品目录号:15140122)
  17. Ham's F-12营养素混合物(Sigma-Aldrich,目录号:N4888)
  18. 液氮
  19. Tris
  20. Optiprep密度梯度介质(Axis-Shield PoC)(Cosmo Bio,目录号:AXS-1114542)
  21. α-HA抗体(单克隆抗HA.11)(BioLegend,目录号:901513)
  22. 二硫苏糖醇(DTT)
  23. 高尔基膜,囊泡,胞质溶胶(参见配方下工作等分试样的制备)
  24. 蔗糖(Fisher Scientific,目录号:S / 8600/60)
  25. HEPES
  26. 肌酸磷酸激酶
  27. GTP
  28. ATP
  29. 磷酸肌酸
  30. 氢氧化钾(KOH)
  31. 醋酸镁(Mg(OAc)2)
  32. 氯化镁(MgCl 2)
  33. 胰蛋白酶,2.5%(10x)(Thermo Fisher Scientific,Gibco TM,目录号:15090046)
  34. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  35. 缓冲区(见食谱)
    1. 分析蔗糖
    2. ATP / GTP混合物(10x)
    3. Cytosol缓冲液
    4. HM缓冲区
    5. KHM缓冲区
    6. 反应缓冲液(10x)
    7. 胰蛋白酶-PBS缓冲液

注意:所有的试剂和缓冲液都应该储存在-80°C的方便大小的等分试样中。当在关键的等分试样(膜,胞质溶胶,ATP / GTP混合物)上运行时,准备一套新的装置并对照旧装置进行测试,以确保重现性。

设备

  1. 微波炉
  2. 水浴
  3. 超声波水浴(格兰特仪器,型号:XUBA3)
  4. 孵化器
  5. 1毫升Dounce匀浆器(DWK Life Sciences,Wheaton,目录号:357538)
  6. 离心机
  7. 超速离心机
  8. SW41转子(型号:SW 41 Ti)和13.2 ml薄壁超清晰TM < (Beckman Coulter,目录号:344059)
  9. 糖折射计(范围0-50%)(英国贝灵汉和斯坦利有限公司)
  10. TLA 100.3转子(Beckman Coulter,型号:TLA-100.3)和3.5ml厚壁聚碳酸酯管(Beckman Coulter,目录号码:349622)
  11. TLS-55转子(型号:TLS-55)和2.2毫升薄壁超清晰 TM管(Beckman Coulter,目录号:347356)
  12. 标准的哺乳动物细胞培养仪器
  13. 进化512 EMCCD(电子倍增电荷耦合器件)相机(Photometrics,型号:Evolve 512)
  14. 蔡司Axiovert 200M全电动倒置显微镜(卡尔蔡司,型号:Axiovert 200M)
  15. X-Cite 120Q激发光源(Excelitas Technologies,型号:X-Cite 120Q)
  16. CFP过滤器(Chroma Technology,目录号:49001)
  17. YFP过滤器(Chroma Technology,目录号:49003)
  18. 物镜(Zeiss Plan-Apochromat 63x / 1.40 Oil DIC,Carl Zeiss Ltd,剑桥,英国)
  19. 在成像过程中使用黑卡排除样品中的房间光线

软件

  1. ZEN 2009软件( www.zeiss.com
  2. PM Capture Pro软件( http://www.photometrics.com
  3. AutoHotkey( www.autohotkey.com )(AutoHotkey Foundation LLC)
  4. ImageJ( https://imagej.nih.gov/ij/

程序

  1. 实验准备

    1. 注:分子生物学级别的水用于所有程序,包括显微镜载玻片和盖玻片的洗涤。
    2. 显微镜幻灯片和盖玻片
      1. 为了将载玻片和盖玻片完全浸没在其架子中,将足够的水在微波中加热至约40℃,然后将洗涤剂decon90(Decon Laboratories Limited,Sussex,UK)加入到3% v)中。在他们的衣架上,载玻片和盖玻片在洗涤剂溶液中上下倾斜10次以帮助清除表面污染物,然后浸泡约7小时。
      2. 再浸5次,将溶液弃去,用水冲洗玻璃器皿一次,如下:将足够的水加入到容器中以完全浸没玻璃器皿,然后上下浸渍10次。将新鲜的3%(v / v)decon90溶液制成,并将玻璃器皿浸泡在其中至少12小时。然后将玻璃器皿在水中至少彻底冲洗5次,然后放入60℃的烘箱中直至干燥。一旦干燥,幻灯片和盖玻片存放在有盖的箱子,以防止灰尘污染。
    3. 硅胶珠
      1. 在安装之前,制备含有5微米二氧化硅珠的悬浮液以添加到测定混合物中。珠子作为幻灯片和盖玻片之间的间隔。将少量珠粒粉末(~5μl干体积)沉积在1.5毫升微量离心管中,向其中加入500μl150mM KCl,10mM HEPES,pH7.2。
      2. 通过剧烈涡旋将珠分散在该缓冲液中,接着在超声水浴(功率35W,强度44kHz)中进行30秒的搅拌 - 重复一次以使珠变为单分散。珠密度需要使得当将3μl的混合物安装并在63×物镜下观察时,将各1μl的该悬浮液加入到25μL的测定混合物中将提供每个视野~1-2个珠子。因此,当制备新的珠子储备物时,通过将1μl的储备物与25μL水混合并且如下所述安装3μL该悬浮液并在显微镜下观察来测试珠子密度。如果珠的数量太低,珠悬浮液短暂旋转下来,并且重新悬浮时除去足够的缓冲液以增加珠密度。
        更多的缓冲液可以被添加到珠子股票相反的效果
    4. 哺乳动物细胞培养
      使用稳定表达GalT的-XFP构建体的野生型和HEK293细胞克隆(科塔姆等人,2014)。这些是通过用CFP或YFP标记稳定转染含有人β-1,4-半乳糖基转移酶1的PCR3如果发现表达的GalT-XFP只在高尔基体中定位,而不是部分分布在胞质泪点中,选择单细胞来源的克隆使用。将细胞在补充有10%胎牛血清(FBS),2mM GlutaMAX-I和100U青霉素和100μg链霉素/ ml的Dulbecco's Modified Eagle Medium(DMEM)中培养。尽管CHO细胞系需要添加5%FBS的Ham's F12培养基和与HEK细胞培养物相同的抗生素组合物,但大多数其他细胞系在相同培养基中培养。所有的细胞系在37℃和5%CO 2下在潮湿的培养箱中生长。
    5. 高尔基膜
      1. 以下方法改编自Balch等人(1984)。通常,将两个稳定表达GalT-CFP HEK细胞(Cottam等人,2014)的T175烧瓶(每个175cm 2)培养至融合单层(〜7总共约10×10 7个细胞),收获,并在0.25M蔗糖,10mM HEPES,pH 7.4中洗涤两次。使用相同的0.25M蔗糖缓冲液将细胞沉淀物(〜500-600μl)重悬于沉淀物体积的四倍。在1ml紧密装配的Dounce匀浆器中用25次冲击处理悬浮液。将匀浆在液氮中快速冷冻,然后储存在-80℃下进行亚细胞分级分离。
      2. 高尔基体膜通过在不连续的蔗糖梯度上通过离心漂浮从匀浆分离。冷冻的匀浆物在37℃快速解冻,然后保持在冰上。将匀浆体积为1.28倍的冰冷的2.3M蔗糖,10mM HEPES,pH7.4的匀浆物的蔗糖浓度调节至1.4M,通过用10ml血清移液管移液充分混合。
      3. 将该混合物装入SW41管形成底层,然后用4.94ml 1.2M蔗糖,10mM HEPES,pH7.4覆盖。然后用足够的0.8M蔗糖,10mM HEPES(pH7.4)覆盖这两层以将管填充至其最大容量为12ml(取决于底层的体积在2.5ml和3ml之间)。然后将该梯度在SW41转子中以39,000rpm,4℃离心80分钟。在冷室中,通过从梯度的顶部向下手动移液,将1.2 / 0.8M蔗糖界面处的浑浊带以最小体积(〜700-800μl)抽出。将该部分充分混合,在PCR管中等分成20μl体积,并在液氮中快速冷冻。使用0-50%糖折射计(Bellingham and Stanley Ltd,UK)测量最终样品的蔗糖浓度。蔗糖浓度总是非常接近并略高于1 M.
      4. 在该测定中,以上述方式分离的1微升高尔基体通过EMCCD成像给每个视野〜32个粒子。
    6. 囊泡
      1. 以下方法改编自Love等人(1998)。通常,将稳定表达GalT-YFP HEK细胞的四个T175烧瓶生长至汇合单层(总共〜14-20×10 7个细胞),收获,并在PBS中洗涤一次,然后在0.2M蔗糖,10mM Tris,pH7.2。使用相同的0.2M蔗糖缓冲液将沉淀(〜1ml)重悬浮于沉淀体积的四倍中,在液氮中快速冷冻,然后储存于-80℃直至需要。
      2. 在Optiprep密度梯度介质的不连续梯度上通过离心浮选分离囊泡膜。冷冻的细胞悬浮液在21℃的水浴中逐渐解冻。这导致细胞通过膜破裂的透化。
      3. 将细胞在1,000℃×g,4℃下离心5分钟,使囊泡留在上清液中。将该上清液在1000×g,4℃下再次离心5分钟,然后在20000×g,4℃下再次离心2分钟,以除去所有的碎片和大的细胞膜。然后通过TLA 100.3转子在4℃以55,000rpm超速离心45分钟沉淀上清液中的囊泡。将囊泡沉淀物(通常约30μl)重悬于KHM缓冲液(参见食谱)至320μl体积。将320μl与480μl50%Optiprep在HM缓冲液中混合(见食谱)。将混合物转移至TLS-55管中并用800μl25%Optiprep在KHM缓冲液中覆盖,然后用400μl10%Optiprep在KHM缓冲液中覆盖。
      4. 通过在TLS-55转子中于4℃以55,000rpm超速离心3小时10分钟使囊泡漂浮至10%/ 25%界面。在界面处的混浊带通过手动吸取以通常300-400μl的最小体积收集并充分混合。将10-15μl等分试样制成在液氮中快速冷冻的PCR管。在该测定中,从以上述方式分离的囊泡储液中取1μl通过EMCCD成像给每个视场约150-250个颗粒。
      5. 通过使用上述方法的放大版本,为需要胞质溶胶依赖性的研究产生了更大,更浓缩的囊泡批次。收获约3.5×10 9个GalT-YFP HEK细胞。在SW41管中Optiprep梯度增加了6.25倍,达到12.5ml总体积,然后在4℃以41,000rpm超速离心8小时。通过移液手动从梯度中抽取250μl级分。 Western blot分析(使用针对GalT-YFP构建体中的HA标签的α-HA抗体进行检测)显示具有最高信号(指示囊泡)的10%/ 25%界面区域附近的六个连续级分。汇集这些级分(总共1.5ml),等分并如上所述快速冷冻。在该测定中从该浓缩的囊泡储液中取出1μl通过EMCCD成像在每个视场中给出约1,000-1,500个颗粒。
    7. 胞液
      1. 胞质溶胶制备方法改编自(Balch等,1984)。将四个T175细胞瓶培养至完全融合(总共约14-20×10 7个HEK293细胞)。使用胰蛋白酶-PBS缓冲液将细胞胰蛋白酶消化,然后用PBS洗涤4次,然后在冰冷的0.25M蔗糖,10mM HEPES,pH7.4中洗涤两次。将沉淀重悬于1.5ml相同的缓冲液中,从1M水溶液中加入新鲜的DTT至1mM。
      2. 在1ml紧密配合的Dounce匀浆器中用30次冲击处理悬浮液。将匀浆物转移到TLA100.3超速离心管中,并在4℃以60,000rpm离心45分钟。取出上清液并以相同方式再次离心以除去所有细胞碎片。同时,PD-10脱盐柱用100mM KCl,1mM DTT,10mM HEPES,pH7.2平衡。将清除的胞质溶胶上清液加入柱中。用相同的平衡缓冲液洗脱脱盐的胞质溶胶。通过Bradford蛋白质测定确定,大部分蛋白质包含在第一个2.5ml洗脱液中。蛋白质浓度通常为7.5-8.5mg / ml。将洗脱液充分混合,然后等分并在液氮中快速冷冻。

  2. 设置,孵化和安装
    1. 设置
      注意:除了水以外,所有样品必须放置在与冷水混合的冰上(以使热交换良好),然后才能用于分析。
      1. 将水浴温度调至37°C。在解冻膜,细胞溶质和ATP / GTP混合物之前,准备所有其他试剂的稀释液。
        注意:在您的手中快速除霜膜等分试样和胞质溶胶,然后放在湿冰上。
      2. 囊泡,高尔基体,胞质溶胶和ATP的等分试样必须在除霜后直接在微型离心机中短暂离心(〜3秒)。
        注意:不要涡旋或轻弹膜,胞质溶胶或ATP / GTP,而是将移液器的体积设定为比等分量少10%,并在移除任何体积之前上下移液7次。相反,缓冲液应该在解冻后涡旋混合。
      3. 稀释囊泡储备以确保每个图像约70-150个囊泡。
        注意:
        1. 这应该测试新的囊泡准备,但一般在10-30倍稀释范围。
        2. 测定混合物应该在0.5ml微量离心管中组成。按以下顺序混合测定组分:水,反应缓冲液和其他缓冲液,细胞溶胶,高尔基体,囊泡和ATP / GTP混合物。
    2. 孵化
      1. 典型的试验将含有:3.8微升高尔基体(见注1),4微升稀释囊泡股票,胞质溶胶中相当于57毫克总蛋白,胞质溶胶缓冲液(见配方),使得此总和和胞质溶胶体积为15.5微升,5微升10X浓缩的反应缓冲液(见配方),2.75微升测定蔗糖(见配方),5μl的10×浓缩的ATP / GTP混合物(见配方)在50μl总体积。在将25μl置于单独的试管中之前,将试验混合物轻轻地上下移液10次。将一半的测定混合物放在湿冰上,另一半转移到37℃的水浴中。
      2. 样品在黑暗中37℃孵育40分钟,然后转移回湿冰上。孵化后立即安装检测混合物。
    3. 安装
      在安装之前,将硅珠悬浮液短暂涡旋,然后将1μl加入到测定混合物中。使用移液管设置为21μl将分析混合物上下移液20次,然后快速地将3μl作为两个单独的滴剂施加到载玻片的一半上并且立即用干净的方形盖玻片覆盖。用清晰的指甲油迅速密封边缘以防止样品蒸发并固定到位。同一试验的37℃和冰孵育的一半成对地安装在同一张载玻片上。幻灯片存放在4°C的冰箱中避光保存,直到成像。图1显示了样品安装是如何在整个执行化验的情况下执行的。


      图1.测定的执行将GalT-YFP囊泡和GalT-CFP高尔基体以50μl的总反应体积混合在一起,其中含有所需的反应条件。混合物被等分成两个管子。一个在0℃孵育40分钟作为内部对照,另一个在37℃孵育相同的时间。温育后,将5微米的二氧化硅珠与每个样品混合,然后立即将3μl的混合物以两个大致相同大小的斑点递送到显微镜载玻片上。样品用22×22毫米的盖玻片覆盖,在边缘密封,用二氧化硅珠子作为间隔物的清漆指甲油。幻灯片保持在4°C的黑暗,直到在室温下通过epifluorescence显微镜成像。

  3. 成像
    1. 设备设置和控制
      1. 使用Evolve 512 EMCCD照相机通过明场显微镜采集图像,该照相机连接到ZEISS Axiovert 200M全电动倒置显微镜。照明由X-Cite 120Q激发光源提供。 X-Cite的可调光圈设置为4的3级,其中4级完全打开以获得最大输出。物镜是蔡司Plan-Apochromat 63x / 1.40油DIC。显微镜由ZEN 2009软件(ZEISS)控制,相机由与ZEISS软件相同的计算机系统运行的PM Capture Pro软件(Photometrics)控制。使用来自Chroma Technology Corporation的CFP和YFP滤光片。尽管没有使用共焦,但使用全电动舞台,滤光轮和百叶窗是必要的,因此使用这种显微镜。在成像过程中样品上的异常室内光线通过使用弱光条件而被最小化,并且还通过如下所述用亚光黑色卡片来遮蔽样品。在卡片的圆盘上(直径约7厘米),在中心切割一个圆孔,该圆孔略小于63x物镜上可伸缩前透镜外壳的直径。将前透镜轻轻地推入孔内,使圆盘形成一个宽的“领子”,从下面挡住光线,然后将一张带有中央矩形孔的卡片放在物镜上以覆盖间隙。随后,将通用安装框架(即,夹在机动平台中的部件)放置在矩形卡的上方以保持滑动件。在显微镜台上,将尺寸约为15×10×2厘米(L×W×H)的相同卡片的盒盖置于样品上(图2)。


        图2.在成像过程中屏蔽已安装的样品虽然在成像过程中使用了低照度的房间条件,但通过屏蔽,样品上的外来光线也被最小化。亚光黑卡的使用方法如下:1.在物镜周围安装一个宽领,以遮挡下方的光线; 2.将具有矩形孔的片放置在物镜上以覆盖间隙; 3.将浅箱盖置于顶部以阻挡来自上方的光线。图片2中的幻灯片放置在矩形片的顶部的通用安装框架中。

      2. 使用开源软件AutoHotkey编写脚本来自动控制ZEN 2009和PM Capture Pro软件。脚本(编程为通过按下键盘F键或游戏手柄的按钮(如<热键>)启动的脚本)移动光标,在适当的时间在显微镜和相机软件窗口中激活命令延迟。对脚本进行编程,使得改变滤波器,打开/关闭灯快门和摄像机图像采集的操作顺序发生,以获得囊泡(YFP通道)和高尔基体(CFP通道)各1个图像。这确保了所有的图像在相同的条件下被捕获,没有不必要的延迟,以避免荧光团的漂白。
    2. 样品处理和图像采集
      1. 在成像之前几分钟,样品应该从冰箱中取出,以防止结露。一旦在显微镜上,房间必须切换到低光照条件,样品必须从上面和底部用上面和下面的无光泽黑色卡片遮盖外部光线。由于在明场照明下膜的对比度不足,所以聚焦是棘手的。初始聚焦应该针对使用低强度卤素灯的5μm玻璃珠。
      2. 然后,为了使用囊泡的YFP荧光进行更精细的对焦,必须使用EMCCD相机的高增益预览模式(请参见注释2)。它们太微弱,不能通过目镜看到,相机需要“看到”这些亚微米颗粒的精确焦平面。过度聚焦很容易,因此挤压样品,在这种情况下必须重新安装。一旦发现有囊泡的焦平面,应该小心,以便从坐在盖玻片上的粒子而不是在载玻片上的所有图像收集。从盖玻片快速重新聚焦将通过显示滑动附着的颗粒来验证这一点;这个检查应定期进行。在x-y平面上移动几个视野的距离通常会使焦点足够靠近,只需稍作调整即可重新获得焦点。
      3. 对于每个测定条件,需要收集每个温育温度12-16个图像。颗粒很快就会漂白,所以在拍摄图像之前,请注意整体对焦时间不要超过7秒。相机设置可能需要根据激发灯的使用年限进行调整。一组理想的设置在注2中。使用分档和高增益的非常短的曝光时间来找到合适的区域和聚焦。相反,长时间的曝光时间没有装箱和低增益被用来获取更高分辨率和更低噪声的囊泡和高尔基体的图像。需要编写脚本(例如使用用于PC的AutoHotkey程序)以使显微镜和相机执行的操作序列自动化以获取YFP和CFP通道的图像(如上所述)。这些脚本应该被编程为在按下单个“热键”(例如F键)时启动,或者甚至可以分配游戏手柄的按钮。这允许三个预设(参见注2)的单击启动,允许预览模式进行对焦,然后是YFP和CFP图像采集。
        注意:我们发现,用V_或G_前缀保存YFP(囊泡)和CFP(高尔基)通道图像,然后在文件名中加上一个时间标记进行编程的其他键,使得数据采集和后续处理的用户更多-友善。这也确保了来自同一视野的正确的囊泡和高尔基图像总是在一起后再配对。

数据分析

  1. ImageJ被用来处理和分析所有图像。对于高尔基,图像被转换为8位,然后使用“减去背景”命令(滚动球半径2.0像素,滑动抛物面)减少背景噪声。阈值设置为“三角暗”,并将下阈值乘以系数1.1以确保选择高于背景噪声的粒子。选择二值图像,并填充任何包含孔的选定粒子。生成粒子数的总结。二进制图像的查找表已被更改为“红色”并保存。对泡囊图像进行相同的处理,除了在“三角暗”阈值之后,下阈值乘以因子1.4。二进制映像的查找表已更改为“绿色”并保存。高尔基和囊泡的倍增因子如果图像非常嘈杂,图像可能会增加,如果噪点较低,图像可能会增加。但是,同一个因素应该用于同一天收集的所有图像。打开高尔基(红色)和囊泡(绿色)二值图像,转换成RGB彩色图像,然后合并成复合图像,其中重叠的红色和绿色粒子相加以产生黄色像素。使用“连接阈值”插件(从 http://imagejdocu.tudor.lu 获得),阈值这个插件的对话框进行了调整,使高尔基选择蓝色,重叠(colocalized)的像素在红色,非colocalizing囊泡颗粒被排除在外。在对话框中选择“Hyst”(迟滞)命令。这导致包含一个或多个共定位像素的任何高尔基体粒子被填充为单个共定位事件,并且输出为二值图像。应用“255,255”的阈值并计数这样的共定位事件的数量。图3总结了这些图像处理操作。对高尔基体和囊泡图像进行的上述操作被编译成ImageJ宏(注3),允许将多个图像作为一批进行处理。在处理之后,应该查看所有图像以去除由5μm珠子引起的假阳性共定位事件,偶尔在两个通道中给出荧光信号,但是由于推定的粒子的形状,可以容易地将其与真实事件区分开。 br />

    图3.图像处理工作流程(Cottam,2012)。 A.高尔基体和囊泡的原始图像用背景扣除处理。这也校正了(B)中产生图像的样本的轻微不均匀照明。 B.将强度阈值应用于图像以选择高于平均背景噪声的粒子。 C.选择被转换成二进制图像,并分别重新着色为洋红色和绿色的高尔基体和囊泡。 D.二进制图像被覆盖以将共定位区域显示为白色像素。 E.将滞后操作应用于重叠的图像,其完全填充任何包含白色像素的粒子以成为单个共定位事件。共定位事件的数量表示为粒子总数(共定位事件/(高尔基体+囊泡))的百分比,以给出测定活性的量度。在我们以前的工作中,我们使用了计算(高尔基+囊泡+共定位事件)的总粒子数量,这是不正确的,但应该考虑到数据与我们公布的结果进行比较时。考虑到共定位事件的数量通常较低(图4),当比较不同测定条件的结果时,由不正确的颗粒总量引起的失真不会改变生物学结果。比例尺= 10微米。

  2. 在Excel中,共定位颗粒的数量表示为颗粒总数的百分比((colocalized /(Golgi + vesicles));参见图3的说明中的解释)。这给了样本中活动的百分比。然后将每个测定条件下37℃孵育的活性标准化为相应的冰对照的活性,给出每个条件的标准化活性(即,与冰对照相比倍数增加)(见图4)。背景活动每天都有所不同。但是,我们发现在一天内进行超过六个独立条件的测定是不实际的。因此引入了进一步的标准化步骤,其中将每个条件的标准化活性进一步标准化为当天测量的野生型(或其他合适的)对照样品。按照这种方式在不同的测量日对相同的控制进行标准化的活动可以进行稳健的比较和平均。


    图4.图像处理结果的典型数据表每个图像都显示了粒子数量以及共定位粒子的数量。

笔记

  1. 当遵循上述制备方案时,制备物的浓度允许测定中高尔基体膜的量非常接近3.8μl,每个图像约70-150个颗粒。如果高尔基制剂的浓度差别很大,则需要注意平衡加入蔗糖缓冲液的高尔基体的量,使得最终测定混合物的渗透压不会离310mOsm的DMEM差不多太远。 >
  2. PM Capture Pro软件中的摄像机预设参数:
    1. 预设1预览模式(高增益,高噪音),以找到正确的区域和快速对焦:
      2 x 2分档,模拟数字增益3,曝光120毫秒,电子倍增增益= 220
    2. 预置2-YFP捕捉(低增益,低噪音):
      1×1分档,模拟 - 数字增益3,曝光4.5秒,电子倍增= 22
    3. 预设3-CFP捕捉(低增益,低噪音):
      1×1组合,模拟数字增益3,曝光2.6秒,电子倍增增益= 8
  3. ImageJ的宏
    1. YFP小泡处理:

      for(i = 1; i <= nImages; i ++){
      selectImage(i);
      run(“8-bit”);
      run(“Subtract Background ...”,“rolling = 2 sliding”);
      setAutoThreshold(“Triangle dark”);
      getThreshold(lower,upper);
      setThreshold(lower * 1.4,255);
      run(“Make Binary”,“thresholded remaining black”);
      run(“Invert”);
      run(“Fill Holes”);
      setThreshold(255,255);
      run(“Analyze Particles ...”,“size = 0-infinity circularity = 0.00-1.00 show = Nothing clear include summarize”); /> run(“Green”);
      }
      run(“Images to Stack”,“name = YFP_stack stack title = [] use”);
      saveAs(“Tiff”)

    2. CFP高尔基处理:

      for(i = 1; i <= nImages; i ++){
      selectImage(i);
      run(“8-bit”);
      run(“Subtract Background ...”,“rolling = 2 sliding”);
      setAutoThreshold(“Triangle dark”);
      getThreshold(lower,upper);
      setThreshold(lower * 1.1,255);
      run(“Make Binary”,“thresholded remaining black”);
      run(“Invert”);
      run(“Fill Holes”);
      setThreshold(255,255);
      run(“Analyze Particles ...”,“size = 0-infinity circularity = 0.00-1.00 show = Nothing clear include summarize”); /> run(“Red”);
      }
      run(“Images to Stack”,“name = CFP_stack stack title = [] use”);
      saveAs(“Tiff”)

    3. 要生成合并的RGB堆栈:

      for(i = 1; i <= nImages; i ++){
      selectImage(i);
      run(“RGB Color”);
      }
      run(“合并通道...”,“red = CFP_stack.tif green = YFP_stack.tif blue = * None * gray = * None * create”); / span>
      saveAs(“Tiff”)

食谱

  1. 分析蔗糖
    1.2 M蔗糖
    10 mM HEPES,pH 7.4
  2. ATP / GTP混合物(10x)
    肌酸磷酸激酶1,500 U / ml
    10 mM GTP
    5 mM ATP
    200毫克磷酸肌酸
    7.5 mM KOH中和ATP
    20 mM HEPES,pH 7.4
  3. Cytosol缓冲液
    100 mM KCl
    1 mM DTT
    10 mM HEPES,pH 7.2
  4. HM缓冲区
    10 mM HEPES,pH 7.2
    2.5mM Mg(OAc)2•/ 2
  5. KHM缓冲区
    150 mM KCl
    2.5mM Mg(OAc)2•/ 2 10 mM HEPES,pH 7.2
  6. 反应缓冲液(10x)
    250 mM HEPES,pH 7.4
    20 mM MgCl 2 2/2
  7. 胰蛋白酶-PBS缓冲液
    PBS中0.25%(v / v)胰蛋白酶

致谢

我们非常感谢约克大学生物科学技术研究所的成像设施。这项工作得到了杜博士和居里夫人(201098)支持的BBSRC博士生支持。该协议发表于(Cottam et al。,2014)。作者声明不存在利益冲突。

参考

  1. Balch,W.E.,Dunphy,W.G.,Braell,W.A。和Rothman,J.E。(1984)。 通过偶联并入N-乙酰葡糖胺测量高尔基连续区室之间蛋白质运输的重建。 Cell 39(2 Pt 1):405-416。
  2. Cottam,N.P。(2012)。无细胞囊泡束缚测定。博士论文,约克大学。
  3. Cottam,N.P。和Ungar,D。(2012)。 高尔基逆行囊泡转运原生质 249( 4):943-955。
  4. Cottam,N.P.,Wilson,K.M.,Ng,B.G.,Korner,C.,Freeze,H.H。和Ungar,D。(2014)。 使用无细胞试验解剖保守寡聚体高尔基体系复合物的功能 <交通 15(1):12-21。
  5. Fisher,P.和Ungar,D。(2016)。 缩小糖基化和囊泡通量之间的差距 Front Cell Dev Biol < / em> 4:15.
  6. Kingsley,D.M。,Kozarsky,K.F。,Segal,M。和Krieger,M。(1986)。 三种低密度脂蛋白受体缺陷型突变体在N-连接的合成中具有多效性缺陷, O-连接的和脂质连接的碳水化合物链。 J Cell Biol 102(5):1576-1585。
  7. Love,H.D.,Lin,C.C.,Short,C.S。和Ostermann,J。(1998)。 功能性高尔基体囊泡的分离,可能在逆行运输中发挥作用。 > J细胞生物学 140(3):541-551。
  8. Luebbehusen,J.,Thiel,C.,Rind,N.,Ungar,D.,Prinsen,B.H.,de Koning,T.J.,van Hasselt,P.M。和Koerner,C。(2010)。 由于保守寡聚体高尔基复合体的亚基6缺陷导致一种新型的先天性致命的结果糖尿病的障碍。Hum Mol Genet 19(18):3623-3633。
  9. Soldati,T.,Riederer,M.A。和Pfeffer,S.R。(1993)。 Rab GDI:rab9蛋白的增溶和回收因子 Mol Biol细胞 4(4):425-434。
  10. Ungar,D.和Hughson,F.M。(2003)。 SNARE蛋白的结构和功能。
  11. Wu,X.,Steet,R.A。,Bohorov,O.,Bakker,J.,Newell,J.,Krieger,M.,Spaapen,L.,Kornfeld,S.and Freeze,H.H。(2004)。 COG复合体亚基基因突变COG7导致致命的先天性疾病。 Nat Med 10(5):518-523。
  • 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: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cottam, N. P. and Ungar, D. (2017). Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay. Bio-protocol 7(22): e2616. DOI: 10.21769/BioProtoc.2616.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。