搜索

Immunogold Localization of Molecular Constituents Associated with Basal Bodies, Flagella, and Extracellular Matrices in Male Gametes of Land Plants
与陆生植物雄配子中的基体、鞭毛和细胞外基质相关的分子成分的免疫金定位   

Karen Sue Renzaglia

Karen Sue Renzaglia

renzaglia@siu.edu
Affiliation:
Department of Plant Biology, Southern Illinois University, Carbondale, IL, USA
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Male gametes (spermatozoids) are the only motile cells produced during the life cycle of land plants. While absent from flowering and most cone-bearing plants, motile cells are found in less derived taxa, including bryophytes (mosses, liverworts and hornworts), pteridophytes (lycophytes and ferns) and some seed plants (Ginkgo and cycads). During development, these cells undergo profound changes that involve the production of a locomotory apparatus, unique microtubule (MT) arrays, and a series of special cell walls that are produced in sequence and are synchronized with cellular differentiation. Immunogold labeling in the transmission electron microscope (TEM) provides information on the exact location and potential function of macromolecules involved with this developmental process. Specifically, it is possible to localize epitopes to proteins that are associated with the cellular inclusions involved in MT production and function. Spermatogenesis in these plants is also ideal for examining the differential expression of carbohydrates and glycoproteins that comprise the extracellular matrixes associated with the dramatic architectural changes in gamete shape and locomotory apparatus development. Here we provide methodologies using monoclonal antibodies (MAbs) and immunogold labeling in the TEM to localize macromolecules that are integral to spermatozoid development.

Keywords: Arabinogalactan proteins(阿拉伯半乳聚糖蛋白), Carbohydrates(碳水化合物), Centrin(中心体蛋白), Extracellular matrix(细胞外基质), Flagella(鞭毛), Gamma tubulin(γ微管蛋白), Immunogold labeling(免疫金标记), Microtubule organizing centers(微管组织中心), Transmission electron microscopy(透射电子显微镜检查)

Background

Motile gametes of land plants are strikingly diverse with numbers of flagella ranging from two to greater than 40,000 (Renzaglia and Garbary, 2001). Following a series of synchronized mitotic divisions within antheridia, nascent sperm cells (spermatids) undergo a sequence of developmental changes within the confines of a dynamic and growing cell wall. A complex locomotory apparatus is produced and flagella elongate around the cell as the organelles are repositioned and shaped. Synchronized development yields hundreds of cells in a single stage of maturation and in different planes of section within a single antheridium.

This profound cellular differentiation involves the development of unique MT arrays, the spline and flagella, that emanate from discrete microtubule organizing centers (MTOCs), the only centriole-containing centrosomes in land plants. Because of the exclusive occurrence of basal bodies, flagella and associated complexes in developing male gametes, studies of spermatogenesis have revealed important information on the structure, composition, and developmental changes in MT arrays as they relate to the cell cycle, MTOCs and cellular differentiation in plants (Joshi et al., 1992; Lui et al., 1993; Vaughn and Renzaglia, 1993; Hoffman et al., 1994; Hoffman and Vaughn, 1995; Vaughn and Harper, 1998; Klink and Wolniak, 2003; Vaughn and Renzaglia, 2006; Vaughn and Bowling, 2008; Vaughn, 2013). A dynamic and flexible extracellular matrix is necessary for spermatogenesis to take place (Garbary and Renzaglia, 2001; Lopez and Renzaglia, 2014); thus spermatogenesis in plants provides an opportunity to examine cell wall changes during development.

The purpose of this review is to describe the methodologies used in localizing proteins, carbohydrates and glycoproteins during the development of motile gametes in land plants. One of the most powerful tools in these studies involves antibodies that recognize epitopes to macromolecules using immunogold labeling techniques at the TEM level (Vaughn, 2013). Here we provide images and a brief discussion of results using immunogold labeling to examine the molecular constituents involved in sperm cell development in plants. Two procedures for these investigations are provided that use the same Materials and Reagents, and Equipment: Procedure A describes the protocol for microtubule-related proteins and procedure B for localizing cell wall constituents.

Procedure A: Immunogold labeling has led to important advances in understanding the role of the proteins centrin and tubulin in plants (Figures 1A-1E). Centrin is a ~20 kDa Ca-binding protein first discovered in motile green algae (Satisbury, 1995), where it is localized to the stellate pattern in the transition zone of the flagella and a dense band of fibers (distal fibers) that connect the nucleus to the basal bodies. In spermatogenous cells, centrin localizes to specific, seemingly diverse structures (Figures 1A-1D). The plates of the multilayered structure (MLS) of the locomotory apparatus, but not the microtubules (MTs), are strongly labeled with antibodies that recognize centrin (Vaughn and Renzaglia, 1993; Vaughn and Harper, 1998) (Figures 1A and 1B). The transition zone that occurs in the flagella of most plants with motile cells also strongly labels with antibodies to centrin, indicating homology with a similar zone in the basal body apparatus in green algae (Vaughn and Renzaglia, 1993; Hoffman et al., 1994; Vaughn and Harper, 1998; Klink and Wolniak, 2003; Vaughn and Renzaglia, 2006) (Figures 1C and 1D). In tracheophytes, an electron opaque pericentriolar type material, called the amorphous zone (AZ), runs along the top of the spline (MT band), connects the basal bodies of adjacent flagella and labels with centrin antibodies (Figure 1B) (Hoffman et al., 1994; Hoffman and Vaughn, 1995). The AZ thus serves as that same sort of basal body connector as found in the green algae.

These localizations are indicative of two possible functions for centrin, as an MTOC protein, and as a contractile protein. In the MLS, centrin appears to be involved in MT nucleation and organization of the spline MT array. In the AZ and transition zone, it is more likely that the centrin is involved in contractile functions. The AZ might also be involved in nucleation/organization of MTs as it lies at the base of the basal body and is close to the spline MT array.

Gamma tubulin was the last of the tubulin proteins to be discovered (Oakley et al., 1990) and occurs in a much lower quantity than alpha and beta tubulin. In mammalian cells, gamma tubulin is restricted to the ends of MTs, where it forms a template for the MTs to form (Joshi et al., 1992). In contrast, gamma tubulin in plants occurs along MTs and not just at their termini (Liu et al., 1993; Liu et al., 1994; Hoffman et al., 1994; Vaughn and Harper, 1998). These sites are in fact new nucleating sites as plant MTs form more of a ‘fir tree’ or highly branched pattern than that is noted in mammalian cells (Murata et al., 2005; Murata and Hasebe, 2007).

The blepharoplast occurs in the last two spermatogenous cell divisions in pteridophytes and serves as the spindle pole body in these divisions as well as the template for basal body production (Hepler, 1976; Hoffman and Vaughn, 1995). In an attempt to determine its ability to nucleate MTs, Vaughn and Bowling (2008) treated Ceratopteris antheridia with the potent microtubule-disrupter oryzalin (ChemService Inc., West Chester, PA) leading to the loss of all microtubules except those in stabilized MT arrays such as in flagella. In these oryzalin-treated cells, the blepharoplast was clearly recognizable but free of MTs and covered with pits that were the size and structure of the MT templates (tubulin ring complexes) recognized in mammalian cells.


Figure 1. Immunogold labeling of basal bodies and flagella of land plants. A. Centrin localization in the lamella strip (ls) that subtends the band of microtubules (mt) and basal bodies (b) of Phaeoceros carolinianus, a hornwort. B. In the seed plant, Ginkgo biloba, centrin epitopes are localized in the lamellar strip (ls), amorphous zone (az) where the basal bodies insert and stellate pattern (sp) of the transition zone. C. Longitudinal section of the long stellate pattern in Ceratopteris basal bodies that label with anti-centrin. A faint outline of the stellate pattern is visible at the arrow. D. Cross section at the basal body of Ceratopteris showing centrin localizations in the stellate pattern and amorphous zone around the basal body. E. In Ceratopteris spermatogenous cells, gamma tubulin (arrows) localizes around the periphery of the blepharoplast following oryzalin treatment. Bars = 0.1 µm.

Gamma tubulin antibodies label the periphery of the blepharoplast in oryzalin treated cells (Figure 1E). When the oryzalin is washed from the antheridia, MTs are quickly reformed along this pitted surface, further indicating the ability of the blepharoplast to serve as an MTOC.

In mammalian cells, the centrioles are surrounded by an electron opaque material where spindle MTs emanate. To identify the components of this pericentriolar material, monoclonal antibodies (MAbs) were raised to mitotic cells and MAbs that recognize the centriolar material could be used not only for mammalian cells but also for other materials, including spermatogenous cells. For example, MPM-2 recognizes a phosphorylated-protein epitope (Davis et al., 1983; Vandre et al., 1984) in spermatogenous cells. In cells without blepharoplasts, this MAb recognizes the surface of the nuclear envelope immediately before mitosis (Hoffman et al., 1994; Klink and Wolniak, 2003). These are the sites where MTs appear to be produced prior to mitosis in all plant cells. In cells with a blepharoplast, these antibodies strongly label the interior of this structure, not the edges (Hoffman et al., 1994; Vaughn and Bowling, 2008). Interestingly, as the blepharoplast begins to reorganize, the reactivity of the antibody is lost and centrin labeling increases in the pericentriolar material. Thus, as different MT arrays are formed, changes occur in proteins of the MTOC.

Procedure B: Immunogold localizations of the sequential matrices that are made during spermatogenesis has revealed differential labeling of carbohydrate-specific MAbs during development and across phylogeny. Callose is a prominent wall constituent in spermatogenesis of ferns, especially in the thickened wall of rounded spermatids in the early stages of ontogeny. In this stage, the locomotory apparatus originates and consists of a multilayered structure (MLS) and basal bodies (Figure 2A). Pectin is absent in this thickened wall in ferns (Lopez and Renzaglia, 2017). In contrast, mosses have a comparable wall that is deposited as spermatids become round, but it is devoid of callose and contains scattered aggregates of esterified pectin as localized with the JIM7 MAb (Figure 2B). By far the most abundant polysaccharide in this thickened wall layer in mosses is hemicellulose that localizes with both LM15 and LM25 MAbs (Figures 2C and 2D) (Lopez-Swalls, 2016).

In addition to carbohydrates, the walls involved in plant spermatogenesis contain abundant but diverse arabinogalactan proteins (AGPs) (Figures 2E-2G). AGPs recognized by the LM2 MAb replace the hemicelluloses around moss spermatids (Figure 2E). As the spermatid matures and begins to develop flagella and assume a coiled configuration, a flexible extraprotoplasmic matrix forms between the plasmalemma and thick callosic wall in ferns and between the plasmalemma and hemicellulosic-pectinaceous wall of mosses (Figures 2F and 2G). The matrix does not label with monoclonal antibodies raised against standard cell wall polysaccharide epitopes such as pectins, cellulose, and hemicelluloses. Rather, MAbs that recognize sugar residues of AGPs abundantly label the matrix as well as the plasmalemma of elongating flagella in fern and moss spermatids (Figures 1F and 1G) (Lopez and Renzaglia, 2014). These results coupled with light and fluorescence microscopy and inhibitor experiments with Yariv (a reagent that binds and precipitates AGPs) suggest that AGPs are involved in growth and positioning of flagella. The implication of AGPs as calcium modulators through binding and release of Ca2+ (Lamport and Várnai, 2013) is a potential mechanism for the regulation of cellular development in plants, and spermatogenesis is an ideal system in which to further pursue this hypothesis.


Figure 2. Immunogold labeling of spermatogenous cell walls in land plants. A. Callose localization in the unevenly-thickened wall layer that surrounds spermatids of Ceratopteris richardii during the formation of the locomotory apparatus which includes an anterior mitochondrion (am), a multilayered structure (mls) and basal bodies (b). B. A thickened wall layer comparable to that in Ceratopteris spermatids, is deposited by young spermatids in the moss, Physcomitrella patens. This wall labels intensely with the JIM7 MAb that binds to esterified pectin epitopes. C-D. Immunogold labeling of hemicelluloses in the thickened wall layer of young spermatids in the moss Aulacomnium palustre. C. This wall layer contains abundant xyloglucan epitopes recognized by the LM15 MAb. D. Similarly, galactoxyloglucan epitopes (LM25 MAb) are a rich component of these thickened walls. (Note: Spermatids in C. were post-fixed in osmium tetroxide (OsO4) that reveals the loose fibrillar consistency of the wall compared to the spermatids in D. that were not post-fixed in OsO4.) E-G. Immunogold labeling of arabinogalactan proteins (AGPs) in spermatid walls. E. AGP epitopes recognized by the LM2 MAb replace the hemicelluloses in the wall around spermatids in P. patens. F. JIM13, a monoclonal antibody to the epitope structure (β)-D-GlcpA1-(1,3)-α-D-GalpA-(1,2)-L-Rha of AGPs, is expressed in the extracellular matrix (*) around flagella during development in C. richardii. The microtubule band (mt), basal body (b) and a hub extension (h) are visible inside the developing spermatid. G. Cross sections of Ceratopteris flagella labelled with JIM8, a monoclonal antibody that identifies an unknown AGP epitope, showing specific localization on the plasmalemma. Bars = 0.5 µm for A-E; 0.1 µm for F-G.

Materials and Reagents

  1. Immunogold labeling of microtubules and microtubule organizing center proteins
    1. Scintillation vials with aluminum covered caps (Fisher Scientific, catalog number: 03-340-4B)
      Manufacturer: DWK Life Sciences, Kimble, catalog number: 7450320 .
    2. Pasteur pipette (Fisher Scientific, catalog number: 13-678-20C )
    3. Gelatin capsules (Electron Microscopy Sciences, catalog number: 70100 )
    4. 200 mesh nickel grids (Electron Microscopy Sciences, catalog number: EMS200-Ni )
    5. 300 mesh gold grids (Electron Microscopy Sciences, catalog number: EMS300-Au )
    6. Glass Petri dishes (Corning, catalog number: 3160-101 )
    7. 90 mm filter paper (GE Healthcare, catalog number: 1004-090
    8. Sterile filters (0.2 µm) (Corning, catalog number: 431212 )
    9. Parafilm (Sigma-Aldrich, Parafilm, catalog number: P7793 )
    10. Dental wax plate (Electron Microscopy Sciences, catalog number:   72670 )
    11. Glass slides (Fisher Scientific, catalog number: 12-544-1 )
    12. Microcentrifuge tubes, 1.5 ml natural (USA Scientific, catalog number: 1615-5500 )
    13. Ethylene dichloride (Electron Microscopy Sciences, catalog number: 13250 )
    14. LR white resin (Electron Microscopy Sciences, catalog number: 14383 )
    15. Primary antibodies: (see Table 1), centrin (Sigma-Aldrich, catalog number: ABE480 ); MPM-2 (EMD Millipore, catalog number: 05-368 )

      Table 1. Primary antibodies used to immunogold label microtubules, microtubule organizing center proteins, and carbohydrates and arabinogalactan proteins in extracellular matrices during spermatogenesis

      aPhaeoceros carolinianus (hornwort); bPhyscomitrella patens (moss); cAulacomnium palustre (moss); dCeratopteris richardii (fern), and eGinkgo biloba (seed plant).

    16. Secondary antibody: goat anti-mouse conjugated with gold (Millipore Sigma, catalog number: G7652 )
    17. Sorenson’s phosphate buffer, 0.2 M, pH 7.2 (Electron Microscopy Sciences , catalog number: 11600-10 )
    18. Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16120 )
    19. Osmium tetroxide (Electron Microscopy Sciences, catalog number: 19150 )
    20. PIPES buffer, 0.2 M, pH 7.2 (Sigma-Aldrich, catalog number: P6757 )
    21. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: B4287 )
    22. Uranyl acetate (Polyscience, catalog number: 21447 )
    23. Lead nitrate (Electron Microscopy Sciences, catalog number: 17900 )
    24. Sodium citrate (Electron Microscopy Sciences, catalog number: 21140 )
    25. 1 N NaOH (Electron Microscopy Sciences, catalog number: 21170-01 )
    26. 0.01 M phosphate buffer (pH 7.2) (see Recipes)
    27. 0.05 M phosphate buffer (pH 7.2) (see Recipes)
    28. 2.5% glutaraldehyde (see Recipes)
    29. 2% aqueous osmium tetroxide (see Recipes)
    30. 0.02 M phosphate buffer (pH 7.2) (see Recipes)
    31. Antibodies (see Recipes)
    32. 2% PBS/BSA (see Recipes)
    33. 0.05 M PIPES buffer (pH 7.2) (see Recipes)
    34. 2% uranyl acetate (see Recipes)
    35. 1 N NaOH (see Recipes)
    36. Reynold’s lead citrate (see Recipes)

  2. Immunogold labeling of spermatozoid matrix and cell wall constituents
    1. Scintillation vials with aluminum covered caps (Fisher Scientific, catalog number: 03-340-4B)
      Manufacturer: DWK Life Sciences, Kimble, catalog number: 7450320 .
    2. Pasteur pipette (Fisher Scientific, catalog number: 13-678-20C )
    3. 200 mesh nickel grids (Electron Microscopy Sciences, catalog number: EMS200-Ni )
    4. 300 mesh gold grids (Electron Microscopy Sciences, catalog number: EMS300-Au )
    5. 90 mm filter paper (GE Healthcare, catalog number: 1004-090
    6. Dental wax plate (Electron Microscopy Sciences, catalog number:  72670 )
    7. Glass Petri dish (Corning, catalog number: 3160-101 )
    8. Glass Slides (Fisher Scientific, catalog number: 12-544-1 )
    9. Gelatin capsules (Electron Microscopy Sciences, catalog number: 70100 )
    10. Microcentrifuge tubes, 1.5 ml natural (USA Scientific, catalog number: 1615-5500 )
    11. Parafilm (Sigma-Aldrich, Parafilm, catalog number: P7793 )
    12. Sterile filters (0.2 µm) (Corning, catalog number: 431212 )
    13. Ethylene dichloride (Electron Microscopy Sciences, catalog number: 13250 )
    14. LR white resin (Electron Microscopy Sciences, catalog number: 14383 )
    15. Primary antibodies (PlantProbes) (see Table 1)
    16. Secondary antibody: Goat-Anti-Rat IgG-gold (Sigma-Aldrich, catalog number: G7035 )
    17. Secondary antibody: goat anti-mouse conjugated with gold (Sigma-Aldrich, catalog number: G7652 )
    18. Sorensen’s phosphate buffer, 0.2 M, pH 7.2 (Electron Microscopy Sciences , catalog number: 11600-10 )
    19. Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16120 )
    20. Osmium tetroxide (Electron Microscopy Sciences, catalog number: 19150 )
    21. PIPES buffer, 0.2 M, pH 7.2 (Sigma-Aldrich, catalog number: P6757 )
    22. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: B4287 )
    23. Uranyl acetate (Polyscience, Inc., catalog number: 21447 )
    24. Lead nitrate (Electron Microscopy Sciences, catalog number: 17900 )
    25. Sodium citrate (Electron Microscopy Sciences, catalog number: 21140 )
    26. 1 N NaOH (Electron Microscopy Sciences, catalog number: 21170-01 )
    27. 0.01 M phosphate buffer (pH 7.2) (see Recipes)
    28. 0.05 M phosphate buffer (pH 7.2) (see Recipes)
    29. 2.5% glutaraldehyde (see Recipes)
    30. 2% aqueous osmium tetroxide (see Recipes)
    31. 0.02 M phosphate buffer (pH 7.2) (see Recipes)
    32. Antibodies (see Recipes)
    33. 2% PBS/BSA (see Recipes)
    34. 0.05 M PIPES buffer (pH 7.2) (see Recipes) (see Recipes)
    35. 2% uranyl acetate (see Recipes)
    36. 1 N NaOH (see Recipes)
    37. Reynold’s lead citrate (see Recipes)

Equipment

  1. Immunogold labeling of microtubules and microtubule organizing center proteins
    1. Bunsen burner (Fisher Scientific, catalog number: S12809 )
    2. -20 °C freezer
    3. M2100 Benchmark Tube Rocker (Benchmark Scientific Inc.)
    4. Oven (General Signal, model: Gravity convection )
    5. Water bath (Sheldon Manufacturing, model: SWB23 )
    6. Micropipette (Bioexpress, GeneMate, catalog number: P-4963-20 )
    7. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75004061 )
    8. Diamond knife (Diatome, specs: Ultra, 45°, 4 mm, Wet)
    9. Transmission electron microscope (Hitachi, model: HF7100 )

  2. Immunogold labeling of spermatozoid matrix and cell wall constituents
    1. Bunsen burner (Fisher Scientific, catalog number: S12809 )
    2. M2100 Benchmark Tube Rocker (Benchmark Scientific Inc.)
    3. Oven (General Signal, model: Gravity convection )
    4. Micropipette (Bioexpress, catalog number: P-4963-20 )
    5. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75004061 )
    6. Diamond knife (Diatome, specs: Ultra, 45°, 4 mm, Wet)
    7. Humid chamber (see Notes)
    8. Transmission electron microscope (Hitachi, model: HF7100 )

Procedure

  1. Immunogold labeling of microtubules and microtubule organizing center proteins (Vaughn and Bowling, 2008; Vaughn, 2013)
    Note: For each solution change described below, completely saturate samples by adding solution until it covers over the material in the vial/tube.
    1. Fix small pieces of several antheridia or whole gametophyte plants (if small, like those of ferns) in 2.5% glutaraldehyde in 0.05 M Sorenson’s phosphate buffer (pH 7.2) at room temperature. Scintillation vials with aluminum-covered caps are ideal for this process (alternatively, you can also use 1.5 ml microcentrifuge tubes). If parts of plants are used, do all cutting of samples in a drop of the glutaraldehyde solution.
    2. Wash in the same 0.05 M Sorenson’s buffer, 2-3 exchanges, 15 min each at 4 °C. To facilitate exchanging solutions without losing the small samples, the tip of the Pasteur pipette is heated over a Bunsen burner so that the tip aperture is down to about 1/3 of its original size. In this way, clean exchanges may be made so that the tiny samples are not sucked up into the pipette and lost during the exchanges.
    3. Post-fix tissue for 10 min in 2% (v/v) osmium.
    4. Serial dehydration: Remove the PIPES buffer and add 25% ethanol (in 0.05 M PIPES buffer) to the vial, increasing the percentage of ethanol by 25% increments until 75% ethanol is reached.
    5. Exchange the solution with 100% ethanol several times. These steps should be at 4 °C, and the vials transferred to a -20 °C freezer after the second 100% ethanol transfer.
    6. Add LR White resin (London Resin Company) to reach 25% plastic (in ethanol). Shake vial to mix plastic into the solvent and maintain at -20 °C for one day. Repeat this procedure by adding plastic to 50% for one day and then add plastic to 75% for another day. Exchange the solutions, putting 100% resin in the vial. Shake the vial and make sure the samples have sunk in the resin. Allow them to spend 24 h in 100% resin.
    7. Allow the vials to warm to room temperature and shake on a rocking platform for 24 h. This will allow for infiltration of the resin throughout the sample.
    8. Transfer the samples to gelatin capsules and fill the capsule with 100% resin forming a meniscus. Rotate the cap as it is inserted to create an oxygen-excluding shield. Place in a 50 °C oven and remove in ~2 d.
    9. Trim the samples and cut 100 nm sections (pale gold reflectance colors) with a diamond knife and fume the sections with ethylene dichloride to stretch the sections. Pick up the sections by touching the dull side of 300 mesh gold grids or 200 mesh nickel grids.
    10. Place mounted grids in a closed CLEAN Petri dish and dry for 1-2 h at room temperature or place the Petri dish on a slide warmer (45-50 °C) for 30 min.
    11. Float the grids, section-side down on 10 µl drops of the following solutions (all of which are filtered through 250 nm filters) and times:
      1. 2% BSA in 0.02 M sodium phosphate buffer (PBS/BSA; pH 7.2), 30 min (BLOCKING).
      2. On drops of primary antibodies or monoclonal antibodies diluted from 1:20 in 2% PBS/BSA for 3-4 h at room temperature.
    12. Rinse grids by placing them on drops of 2% BSA/PBS 4 x 3 min.
    13. Place grids on 5-15 µl drops of secondary gold-labeled antibody or Protein A/gold diluted 1/20-in 2% BSA/PBS in ‘humid chamber’ 1-3 h.
    14. Move grids to 5-10 µl drops of 0.02 M PBS for rinsing 4 x 3-5 min (FILTER and CENTRIFUGE).
    15. Rinse each grid with a jet of double distilled autoclaved water that has been filtered (0.2 µm) and let it dry.
    16. Post-stain the grids for 2 min in 2% uranyl acetate, wash in double distilled autoclaved water and post-stain in Reynold’s lead citrate. Wash in double distilled autoclaved water, dry and examine under transmission electron microscope.

  2. Immunogold labeling of spermatozoid matrix and cell wall constituents (Lopez and Renzaglia, 2014)
    1. Fix small pieces of antheridial tissue or whole gametophyte plants (if small, like those of ferns) in 2.5% glutaraldehyde in 0.05 M Sorenson’s phosphate buffer (pH 7.2) at room temperature. Scintillation vials with aluminum-covered caps are ideal for this process (alternatively, you can also use 1.5 ml microcentrifuge tubes). If parts of plants are used, do all cutting of samples in a drop of the glutaraldehyde solution.
    2. Wash in the same 0.05 M Sorenson’s buffer, 2-3 exchanges, 15 min each at 4 °C. To facilitate exchanging solutions without losing the small samples, the tip of the Pasteur pipette is heated over a Bunsen burner so that the tip aperture is down to about 1/3 of its original size. In this way, clean exchanges may be made so that the tiny samples are not sucked up into the pipette during the exchanges.
    3. Post-fix tissue for 10 min in 1% (v/v) osmium tetroxide (OsO4).
    4. Rinse tissue 3 x in distilled water (10 min each).
    5. Serial dehydration: Remove the water and add 25% ethanol (in water) to the vial, increasing the percentage of ethanol by 25% increments until 75% ethanol is reached.
    6. Exchange the solution 3 x with 100% ethanol.
    7. Add LR White resin (London Resin Company) to reach 25% plastic (in ethanol). Shake vial to mix plastic into the solvent and maintain at -20 °C for one day. Repeat this procedure by adding plastic to 50% for one day and then add plastic to 75% for another day. Exchange the solutions, putting 100% resin in the vial. Shake the vial and make sure the samples have sunk in the resin. Allow them to spend 24 h in 100% resin.
    8. Allow the vials to warm to room temperature and shake on a rocking platform for 24 h. This will allow for infiltration of the resin throughout the sample.
    9. Transfer the samples to gelatin capsules and fill the capsule with 100% resin forming a meniscus. Rotate the cap as it is inserted to create an oxygen-excluding shield. Place in a 50 °C oven and remove in ~2 d.
    10. Trim the samples and cut 90-100 nm sections (pale gold reflectance colors) with a diamond knife and fume the sections with ethylene dichloride to stretch the sections. Pick up the sections by touching the dull side of 300 mesh gold grids or 200 mesh nickel grids.
    11. Place mounted grids on a clean piece of filter paper in a closed CLEAN Petri dish and dry for 1-2 h at room temperature or place the Petri dish on a slide warmer (45-50 °C) for 30 min.
    12. Using a humid chamber (see Note 2) place a piece of dental wax on completely wet filter paper, float the grids, section-side down on 10 µl drops in the following solutions (all of which are centrifuged and filtered through 250 nm filters) and times:
      1. 2% BSA in 0.05 M sodium phosphate buffer (BSA/PBS; pH 7.2), overnight (BLOCKING).
      2. On drops of primary antibodies diluted from 1:20 in 2% BSA/PBS for 3-4 h at room temperature. Alternatively, grids can be left in primary antibody overnight at 4 °C.
    13. Rinse grids by placing them on drops of 2% BSA/PBS 4 x 3 min.
    14. Place grids on 10 µl drops of secondary gold-labeled antibody diluted 1/20 in 2% BSA/PBS in ‘humid chamber’ 1-3 h.
    15. Move grids to 10 µl drops of 0.02 M PBS (FILTER and CENTRIFUGE) for rinsing 4 x 3-5 min.
    16. Rinse each grid with a jet of double distilled autoclaved water that has been filtered (0.2 µm) and let it dry.
    17. Post-stain the grids for 2 min in 2% uranyl acetate, wash in double distilled autoclaved water and post-stain in Reynold’s lead citrate for 30 sec. Wash in double distilled autoclaved water, dry and examine under transmission electron microscope.

Notes

  1. Osmium tetroxide (OsO4) is a heavy fixative that stains lipids in membranous structures and vesicles. Additionally, the reduced heavy metal adds density and contrast to biological tissue.
  2. Humid chamber is a Petri dish, whose bottom is covered with a fresh, clean wet filter paper and cover is sealed with Parafilm if sitting overnight. Aliquots of BSA/PBS can be kept frozen until used. Centrifuge prior to use and store refrigerated (4 °C). 1-2% BSA/PBS can be divided in small aliquots (100-200 µl) for each time of labeling and kept at -20 °C, up to 6 months.
  3. The primary antibody in BSA/PBS should not be frozen and thawed repeatedly; it decreases the strength of enzyme.
  4. Use completely clean tools and Petri dishes during the labeling process.
  5. Millipore filter or centrifuge every single agent and only draw from supernatant, use sterile filtered (0.2 µm) water for rinses after PBS, lead citrate and uranyl acetate stains.
  6. Aliquots (40-50 µl) of primary antibody must be kept at -20 °C.
  7. Secondary antibody with gold particles must be kept at 4 °C, never place in freezer.
  8. After labeling agents are thawed (aliquots) do not freeze them again. They are good for maximum 3-4 days at 4 °C.
  9. Drop size for BSA/PBS as well as other liquids could be between 5 to 10 µl, if you are stopping for a step overnight use 10 µl.
  10. If you stop at any of the above steps (prior to putting grids in the secondary antibody) it should not be more than overnight and humid chamber should be sealed and kept in the refrigerator.
    Note: Grids should not be left in the secondary antibody overnight because it will increase the amount of background ‘noise’ (i.e., over labels).
  11. It is recommended to wick grids on a small piece of filter paper prior to moving them to a new solution and between different steps of the same rinses.
  12. Use a new piece of Parafilm (or rinse and wipe dental wax) each time you move grids to a new solution.
  13. Glass Petri dishes are preferred. Plastic creates static electricity and causes the grids to cling to the top of the dish.

Recipes

  1. 0.01 M phosphate buffer (pH 7.2)
    Add 5 parts 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 95 parts double distilled autoclaved water
  2. 0.05 M phosphate buffer (pH 7.2)
    Add 25 parts 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 75 parts double distilled autoclaved water
  3. 2.5% glutaraldehyde
    1. Add 1 part 10% glutaraldehyde to 1 part double distilled autoclaved water
    2. Add 1 part 0.20 M Sorensen’s phosphate buffer (pH 7.2) to 1 part double distilled autoclaved water
    3. Add 1 part 5% glutaraldehyde to 1 part 0.10 M Sorensen’s phosphate buffer (pH 7.2)
  4. 2% aqueous osmium tetroxide
    Add 1 part 4% aqueous osmium tetroxide to 1 part double distilled autoclaved water
  5. 0.02 M phosphate buffer (pH 7.2)
    Add 1 part 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 10 parts double distilled autoclaved water
  6. Antibodies
    Add 1 part antibody to 20 parts 2% PBS/BSA
  7. 2% PBS/BSA
    Add 0.20 g of bovine serum albumin to 10 ml 0.02 M phosphate buffer (pH 7.2)
  8. 0.05 M PIPES buffer (pH 7.2)
    Add 1 part 0.30 M PIPES buffer (pH 7.2) to 6 parts double distilled autoclaved water
  9. 2% uranyl acetate
    Add 1 g uranyl acetate in 50 ml double distilled autoclaved water
  10. 1 N NaOH
    Dissolve 40.00 g sodium hydroxide in water to make volume 1 L
  11. Reynold’s lead citrate
    1. Add 1.33 g lead nitrate and 1.76 g sodium citrate to 30 ml boiled double distilled autoclaved water that has cooled to room temperature and shake for 30 min
    2. Add 8 ml 1 N NaOH
    3. Add 12 ml double distilled autoclaved water

Acknowledgments

This research was supported by research grants (DEB-0322664, DEB-0423625, DEB0521177, and DEB-0228679) from the National Science Foundation as part of the Research Experience for Undergraduates and Assembling the Tree of Life Programs.
The Authors declare we have no conflicts of interest or competing interest.

References

  1. Davis, F. M., Tsao, T. Y., Fowler, S. K. and Rao, P. N. (1983). Monoclonal antibodies to mitotic cells. Proc Natl Acad Sci U S A 80(10): 2926-2930.
  2. Hepler, P. K. (1976). The blepharoplast of Marsilea: its de novo formation and spindle association. J Cell Sci 21(2): 361-390.
  3. Hoffman, J. C. and Vaughn, K. C. (1995). Using the developing spermatogenous cells of Ceratopteris to unlock the mysteries of the plant cytoskeleton. Int J Plant Sci 156(3): 346-358.
  4. Hoffman, J. C., Vaughn, K. C. and Joshi, H. C. (1994). Structural and immunocytochemical characterization of microtubule organizing centers in pteridophyte spermatogenous cells. Protoplasma 179(1): 46-60.
  5. Joshi, H. C., Palacios, M. J., McNamara, L. and Cleveland, D. W. (1992). γ-Tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation. Nature 356(6364): 80-83.
  6. Klink, V. P. and Wolniak, S. M. (2003). Changes in the abundance and distribution of conserved centrosomal, cytoskeletal and ciliary proteins during spermiogenesis in Marsilea vestita. Cell Motil Cytoskeleton 56 (1): 57-73.
  7. Knox, J. P., Linstead, P. J., King, J., Cooper, C., Roberts, K. (1990). Pectin esterification is spatially regulated both within cell walls and between developing tissues of root apices. Planta 181(4): 512-521.
  8. Lamport, D. T. and Várnai, P. (2013). Periplasmic arabinogalactan glycoproteins act as a calcium capacitor that regulates plant growth and development. New Phytol 197(1): 58-64.
  9. Liu, B., Joshi, H. C., Wilson, T. J., Silflow, C. D., Palevitz, B. A. and Snustad, D. P. (1994). gamma-Tubulin in Arabidopsis: gene sequence, immunoblot, and immunofluorescence studies. Plant Cell 6(2): 303-314.
  10. Liu, B., Marc, J., Joshi, H. C. and Palevitz, B. A. (1993). A gamma-tubulin-related protein associated with the microtubule arrays of higher plants in a cell cycle-dependent manner. J Cell Sci 104(4): 1217-1228.
  11. Lopez, R. A. and Renzaglia, K. S. (2014). Multiflagellated sperm cells of Ceratopteris richardii are bathed in arabinogalactan proteins throughout development. Am J Bot 101(12): 2052-2061.
  12. Lopez, R. A. and Renzaglia, K. S. (2017). The Ceratopteris (fern) developing motile gamete walls contain diverse polysaccharides but not pectin. Planta.
  13. Lopez-Swalls and Renee A. (2016). The special walls around gametes in Ceratopteris richardii and Aulacomnium palustre: using immunocytochemistry to expose structure, function, and development. Southern Illinois University Carbondale.
  14. Manandhar, G., Simerly, C., Salisbury, J. L. and Schatten, G., (1999). Centriole and centrin degeneration during mouse spermiogenesis. Cytoskeleton 43(2): 137-144.
  15. Marcus, S. E., Verhertbruggen, Y., Hervé, C., Ordaz-Ortiz, J. J., Farkas, V., Pedersen, H. L., Willats, W. G., Knox, J. P. (2008). Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls. BMC Plant Biol 8(1): 60.
  16. Meikle, P. J., Bonig, I., Hoogenraad, N. J., Clarke, A. E. and Stone, B. A. (1991). The location of (1→3)-β-glucans in the walls of pollen tubes of Nicotiana alata using a (1→3)-β-glucan-specific monoclonal antibody. Planta 185(1): 1-8.
  17. Murata, T. and Hasebe, M. (2007). Microtubule-dependent microtubule nucleation in plant cells. J Plant Res 120 (1): 73-78.
  18. Murata, T., Sonobe, S., Baskin, T. I., Hyodo, S., Hasezawa, S., Nagata, T., Horio, T. and Hasebe, M. (2005). Microtubule-dependent microtubule nucleation based on recruitment of gamma-tubulin in higher plants. Nat Cell Biol 7(10): 961-968.
  19. Oakley, B. R., Oakley, C. E., Yoon, Y. and Jung, M. K. (1990). γ-Tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans. Cell 61(7): 1289-1301.
  20. Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., von Schantz, L., Marcus, S. E., Andersen, M. C. and Field, R. (2012). Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research. J Biol Chem 287(47): 39429-39438.
  21. Pennell, R. I., Janniche, L., Kjellbom, P., Scofield, G. N., Peart, J. M. and Roberts, K. (1991). Developmental regulation of a plasma membrane arabinogalactan protein epitope in oilseed rape flowers. Plant Cell 3(12): 1317-1326
  22. Renzaglia, K. S. and Garbary, D. J. (2001). Motile male gametes of land plants: Diversity, development, and evolution. Crit Rev Sci 20(2):107-213.
  23. Satisbury, J. L. (1995). Centrin, centrosomes, and mitotic spindle poles. Curr Opin Cell Biol 7(1): 39-45.
  24. Showalter, A. M. (2001). Arabinogalactan-proteins: structure, expression and function. Cell Mol Life Sci 58(10): 1399-1417.
  25. Vandre, D. D., Davis, F. M., Rao, P. N. and Borisy, G. G. (1984). Phosphoproteins are components of mitotic microtubule organizing centers. Proc Natl Acad Sci U S A 81(14): 4439-4443.
  26. Vaughn, K. (2013). Immunocytochemical techniques. In: Immunocytochemistry of Plant Cells. Springer pp: 1-41.
  27. Vaughn, K. C. and Bowling, A. J. (2008). Recovery of microtubules on the blepharoplast of Ceratopteris spermatogenous cells after oryzalin treatment. Protoplasma 233 (3-4): 231-240.
  28. Vaughn, K. C. and Harper, J. D. (1998). Microtubule-organizing centers and nucleating sites in land plants. Int Rev Cytol 181: 75-149.
  29. Vaughn, K. C. and Renzaglia, K. S. (1993). Centrin in spermatogenesis of archegoniates. Am J Bot (Suppl.) 80(1): 58-66.
  30. Vaughn, K. C. and Renzaglia, K. S. (2006). Structural and immunocytochemical characterization of the Ginkgo biloba L. sperm motility apparatus. Protoplasma 227(2-4):165-173.
  31. Verhertbruggen, Y., Marcus, S. E., Haeger, A., Ordaz-Ortiz, J. J. and Knox, J. P. (2009). An extended set of monoclonal antibodies to pectic homogalacturonan. Carbohyd Res 344(14): 1858−186.
  32. Yates, E. A., Valdor, J-F., Haslam, S. M., Morris, H. R., Dell, A., Mackie, W. and Knox, J. P. (1996). Characterization of carbohydrate structural features recognized by anti-arabinogalactan-protein monoclonal antibodies. Glycobiology 6(2): 131-139.

简介

雄性配子(精子)是陆地植物生命周期中唯一产生的运动细胞。虽然没有开花和大多数含有锥体的植物,但在少量来源的分类群中发现运动细胞,包括苔藓植物(苔藓,苔草和horn)),蕨类植物(石膏植物和蕨类植物)和一些种子植物(银杏,苏铁)。在发育过程中,这些细胞发生深刻的变化,涉及生产运动装置,独特的微管(MT)阵列和一系列特定的细胞壁,这些细胞壁依次产生并与细胞分化同步。透射电子显微镜(TEM)中的免疫金标记提供了涉及该发育过程的大分子的确切位置和潜在功能的信息。具体而言,可能将表位定位于与涉及MT产生和功能的细胞内含物相关的蛋白质。在这些植物中的精子发生对于检查构成细胞外基质的碳水化合物和糖蛋白的差异表达也是理想的,所述细胞外基质与配子形状和运动装置发育中的戏剧性建筑变化相关。在这里我们提供使用单克隆抗体(MAbs)和透射电子显微镜中免疫金标记的方法来定位精子发育不可或缺的大分子。

【背景】动植物的陆地植物是惊人的多样化,鞭毛数量从2到4万以上(Renzaglia和Garbary,2001)。在一系列同心的有丝分裂分裂内,新生精细胞(精细胞)在一个动态和生长的细胞壁的范围内经历一系列的发育变化。当细胞器重新定位和成形时,产生复杂的运动装置并且鞭毛在细胞周围伸长。同步开发在一个单一的天竺鼠中,在一个单一的成熟阶段和不同的剖面中产生数百个细胞。

这种深刻的细胞分化涉及独特的MT阵列的发展,样条和鞭毛,从离散的微管组织中心(MTOCs),唯一含有中心粒的中心体在陆地植物散发。由于基因体,鞭毛及相关复合体在发育中的雄性配子中的独家出现,精子发生的研究揭示了MT阵列的结构,组成和发育变化的重要信息,因为它们涉及细胞周期,MTOC和细胞分化(Joshi等人,1992; Lui等人,1993; Vaughn和Renzaglia,1993; Hoffman等人,1994; Hoffman和Vaughn,1995; Vaughn和Harper,1998; Klink和Wolniak,2003; Vaughn和Renzaglia,2006; Vaughn和Bowling,2008; Vaughn,2013)。一个动态和灵活的细胞外基质是精子发生所必需的(Garbary and Renzaglia,2001; Lopez and Renzaglia,2014);因此植物中的精子发生提供了在发育过程中检查细胞壁变化的机会。

这次审查的目的是描述在陆地植物运动配子的发展中定位蛋白质,碳水化合物和糖蛋白的方法。这些研究中最有力的工具之一涉及到在TEM水平使用免疫金标记技术识别大分子表位的抗体(Vaughn,2013)。在这里,我们提供图像和简要的讨论结果使用免疫胶体金标记来检查涉及植物精子细胞发育的分子成分。提供了两个使用相同的材料和试剂以及设备的调查程序:程序A描述了微管相关蛋白质的方案和用于定位细胞壁成分的方法B.

步骤A:免疫金标记已经在理解蛋白质中心蛋白和微管蛋白在植物中的作用方面取得重要进展(图1A-1E)。 Centrin是一种在运动绿藻中首次发现的约20kDa的Ca结合蛋白(Satisbury,1995),其在鞭毛的过渡区中定位于星状模式,并且密集的纤维带(远端纤维)核到基底体。在精原细胞中,中心蛋白定位于特定的,看似不同的结构(图1A-1D)。运动装置的多层结构(MLS)而不是微管(MT)的板用识别中心蛋白的抗体强烈标记(Vaughn和Renzaglia,1993; Vaughn和Harper,1998)(图1A和1B)。在具有活动细胞的大多数植物的鞭毛中发生的过渡区域也用抗中心蛋白的抗体强烈标记,表明与绿藻中的基体装置中的类似区域同源(Vaughn和Renzaglia,1993; Hoffman等人, 1994; Vaughn和Harper,1998; Klink和Wolniak,2003; Vaughn和Renzaglia,2006)(图1C和1D)。在气管植物中,一种称为无定形区(AZ)的电子不透明周质型材料称为无定形区(AZ),沿着样条(MT带)的顶部延伸,将相邻鞭毛的基底体和标记与中心蛋白抗体连接(图1B)等,1994; Hoffman和Vaughn,1995)。因此AZ用作与绿藻中发现的那种相同种类的基体连接器。

这些定位表明中心蛋白有两种可能的功能,一种是MTOC蛋白,另一种是收缩蛋白。在MLS中,中心蛋白似乎参与MT成核和样条MT阵列的组织。在AZ和过渡区,中心体更可能涉及收缩功能。由于它位于基体的底部并且靠近样条MT阵列,所以AZ也可能参与MT的成核/组织。

γ微管蛋白是待发现的微管蛋白蛋白中的最后一个(Oakley等人,1990),其发生量比α和β微管蛋白低得多。在哺乳动物细胞中,γ微管蛋白局限于MT的末端,在那里它形成MT形成的模板(Joshi等人,1992)。相反,植物中的γ-微管蛋白沿着MT而不是仅在它们的末端发生(Liu等人,1993; Liu等人,1994; Hoffman等人1994年; Vaughn和Harper,1998年)。这些位点实际上是新的成核位点,因为植物MT形成比哺乳动物细胞更多的“杉树”或高度分枝的模式(Murata等人,2005; Murata和Hasebe,2007 )。

睑上皮细胞发生在蕨类植物的最后两个精子细胞分裂中,并作为这些分裂中的纺锤体极体以及基底体产生模板(Hepler,1976; Hoffman和Vaughn,1995)。为了确定其成核MT的能力,Vaughn和Bowling(2008)用有效的微管干扰剂oryzalin(ChemService Inc.,West Chester,PA)处理了Ceratopteris antheridia,导致全部丧失除稳定的MT阵列如鞭毛中的那些之外的微管。在这些经由oryzalin处理的细胞中,blepharoplast清晰可辨,但不含MT,并覆盖有在哺乳动物细胞中识别的MT模板(微管蛋白环复合物)的大小和结构的凹坑。

“”src
图1.免疫金标记基础小体和地上植物的鞭毛A.中央蛋白定位在片层带(ls)中,微管(mt)和基体(b) >黄Pha鱼(Phaeoceros carolinianus),horn 草。 B.在种子植物中,中心蛋白抗原决定簇定位于基底体插入的非晶区(az)和过渡区星状图(sp)的片层带(ls)中。 C. Ceratopteris 中的长星状图案的纵向切片标记有抗中心。箭头处可以看到星状图案的微弱轮廓。 D. Ceratopteris的基部横切面显示中心定位于星状模式和基底周围的无定形区域。 E.在角叉菜属精原细胞中,在用oryzalin处理后,γ微管蛋白(箭头)定位在blepharoplast周围。棒=0.1μm。

γ-微管蛋白抗体在经oryazalin处理的细胞中标记blepharoplast的外周(图1E)。当从结肠中清洗出oryzalin时,MT沿着这个凹陷表面很快被重新形成,这进一步表明了blephalastlast作为MTOC的能力。

在哺乳动物细胞中,中心粒被电子不透明材料包围,其中主轴MT发出。为了鉴定该种周围细胞物质的成分,将单克隆抗体(MAbs)制备成有丝分裂细胞,识别中心物质的MAbs不仅可用于哺乳动物细胞,而且还可用于其他物质,包括精原细胞。例如,MPM-2在精原细胞中识别磷酸化蛋白质表位(Davis等人,1983; Vandre等人,1984)。在没有眼袋的细胞中,该MAb在有丝分裂前立即识别核膜的表面(Hoffman等,1994; Klink和Wolniak,2003)。在所有植物细胞有丝分裂之前,这些是MT似乎被生产的地方。在具有blepharoplast的细胞中,这些抗体强烈地标记了该结构的内部,而不是边缘(Hoffman等人,1994; Vaughn和Bowling,2008)。有趣的是,随着blepharoplast开始重组,抗体的反应性丧失,centrin标记在周围细胞材料中增加。因此,随着形成不同的MT阵列,MTOC的蛋白质发生变化。

方法B:精子发生过程中序列基质的免疫金定位揭示了在发育过程中和整个系统发育过程中碳水化合物特异性单克隆抗体的差异标记。胼is体是蕨类植物精子发生的主要组成部分,特别是个体发育早期圆形精子增厚的壁。在这个阶段,运动器械起源于多层结构(MLS)和基体(图2A)。在这个加厚的蕨类植物墙壁中没有果胶(Lopez and Renzaglia,2017)。相比之下,苔藓具有类似的壁,当精子细胞变成圆形时沉积,但它没有胼and质,并且含有与JIM7 MAb一起定位的酯化果胶的分散聚集体(图2B)。到目前为止,苔藓中这种增厚的壁层中最丰富的多糖是半纤维素,其与LM15和LM25 MAb(图2C和2D)(Lopez-Swalls,2016)定位。

除了碳水化合物之外,参与植物精子发生的壁含有丰富而多样的阿拉伯半乳聚糖蛋白(AGPs)(图2E-2G)。 LM2单抗所识别的AGPs取代了苔藓精子细胞周围的半纤维素(图2E)。随着精子细胞成熟并开始发育鞭毛并呈螺旋状构象,在蕨类植物的质膜和厚厚的胼wall壁之间以及苔藓的质膜和半纤维质 - 果胶质壁之间形成柔性的外原质基质(图2F和2G)。基质不用抗标准细胞壁多糖表位(例如果胶,纤维素和半纤维素)产生的单克隆抗体进行标记。相反,识别AGPs糖残基的单克隆抗体大量标记了基质以及蕨类植物和苔藓精子细胞中延长鞭毛的质膜(图1F和1G)(Lopez and Renzaglia,2014)。这些结果与Yariv(结合和沉淀AGPs的试剂)的光和荧光显微镜检查和抑制剂实验结果表明,AGPs参与鞭毛的生长和定位。 AGPs作为钙调节剂通过结合和释放Ca2 +的含义(Lamport和Várnai,2013)是调节植物细胞发育的潜在机制,精子发生是其中理想的系统进一步追求这个假设。

“”src
图2.陆生植物中精原细胞壁的免疫金标记A.胼ose体在形成运动过程中围绕精虫Ceratopteris richardii的不均匀增厚的壁层中的定位包括前线粒体(am),多层结构(mls)和基体(b)的装置。 B.一个与Ceratopteris 精子细胞相似的增厚的壁层,由年轻的精子细胞沉积在苔藓中,Physcomitrella patens 。 JIM7单克隆抗体结合酯化的果胶抗原决定簇,光盘。青苔Aulacomnium palustre中幼年精细胞增厚的壁层中半纤维素的免疫金标记。 C.该壁层包含由LM15 MAb识别的丰富的木葡聚糖表位。 D.类似地,半乳聚糖葡聚糖表位(LM25 MAb)是这些增稠壁的丰富组分。注意:C中的精子细胞被固定在四氧化锇(OsO 4)中,与D中的精子细胞相比,这种细胞壁松散的纤维一致性未被固定在OsO 4 。)EG。免疫金标记的精子细胞壁中的阿拉伯半乳聚糖蛋白(AGPs)。 E.由LM2 MAb识别的AGP表位替换 P中精细胞周围壁中的半纤维素。藓。 F.JIM13,AGP的表位结构(β)-D-GlcpA1-(1,3)-α-D-GalpA-(1,2)-L-Rha的单克隆抗体在细胞外基质*)在 C开发期间围绕鞭毛。 richardii 。发育中的精细胞内可见微管带(mt),基体(b)和轮毂延伸(h)。 G.用JIM8标记的Ceratopteris 鞭毛的横切片,JIM8是识别未知AGP表位的单克隆抗体,显示在质膜上的特异性定位。 A-E棒=0.5μm;对于F-G为0.1μm。

关键字:阿拉伯半乳聚糖蛋白, 碳水化合物, 中心体蛋白, 细胞外基质, 鞭毛, γ微管蛋白, 免疫金标记, 微管组织中心, 透射电子显微镜检查

材料和试剂

  1. 免疫金标记微管和微管组织中心蛋白
    1. 带铝盖的闪烁瓶(Fisher Scientific,目录号:03-340-4B)
      制造商:DWK Life Sciences,Kimble,目录号:7450320。
    2. 巴斯德吸管(Fisher Scientific,目录号:13-678-20C)
    3. 明胶胶囊(电子显微镜科学,目录号:70100)
    4. 200目镍网(Electron Microscopy Sciences,目录号:EMS200-Ni)
    5. 300目金网格(电子显微镜科学,目录号:EMS300-Au)
    6. 玻璃培养皿(康宁,目录号:3160-101)
    7. 90毫米滤纸(GE Healthcare,目录号:1004-090) 
    8. 无菌过滤器(0.2μm)(Corning,目录号:431212)
    9. Parafilm(Sigma-Aldrich,Parafilm,目录号:P7793)
    10. 牙科蜡板(电子显微镜科学,目录号:72670)
    11. 玻璃载玻片(Fisher Scientific,目录号:12-544-1)
    12. 微量离心管,1.5毫升天然(美国科学,目录号:1615-5500)
    13. 二氯乙烷(电子显微镜科学,目录号:13250)
    14. LR白色树脂(Electron Microscopy Sciences,目录号:14383)
    15. 一抗(见表1),中心蛋白(Sigma-Aldrich,目录号:ABE480); MPM-2(EMD Millipore,目录号:05-368)

      表1.在精子发生过程中用于免疫金标记微管,微管组织中心蛋白质和胞外基质中的碳水化合物和阿拉伯半乳聚糖蛋白质的一级抗体

      角叉菜(hornwort);藓类小立碗藓(苔藓); Aulacomnium palu stre(moss); (蕨),和银杏(种子植物)。

    16. 第二抗体:与金结合的山羊抗小鼠(Millipore Sigma,目录号:G7652)
    17. Sorenson磷酸盐缓冲液,0.2M,pH7.2(Electron Microscopy Sciences,目录号:11600-10)
    18. 戊二醛(电子显微镜科学,目录号:16120)
    19. 四氧化锇(电子显微镜科学,目录号:19150)
    20. PIPES缓冲液,0.2M,pH7.2(Sigma-Aldrich,目录号:P6757)
    21. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:B4287)
    22. 乙酸铀酰(Polyscience,目录号:21447)
    23. 硝酸铅(电子显微镜科学,目录号:17900)
    24. 柠檬酸钠(电子显微镜科学,目录号:21140)
    25. 1N NaOH(Electron Microscopy Sciences,目录号:21170-01)
    26. 0.01 M磷酸盐缓冲液(pH 7.2)(见食谱)
    27. 0.05M磷酸盐缓冲液(pH7.2)(见食谱)
    28. 2.5%戊二醛(见食谱)
    29. 2%的四氧化锇水溶液(见食谱)
    30. 0.02 M磷酸盐缓冲液(pH 7.2)(见食谱)
    31. 抗体(见食谱)
    32. 2%PBS / BSA(见食谱)
    33. 0.05 M PIPES缓冲液(pH 7.2)(见食谱)
    34. 2%醋酸铀(见食谱)
    35. 1 N NaOH(见食谱)
    36. 雷诺的铅柠檬酸盐(见食谱)

  2. 免疫金标记的精子和基质细胞壁成分
    1. 带铝盖的闪烁瓶(Fisher Scientific,目录号:03-340-4B)
      制造商:DWK Life Sciences,Kimble,目录号:7450320。
    2. 巴斯德吸管(Fisher Scientific,目录号:13-678-20C)
    3. 200目镍网(Electron Microscopy Sciences,目录号:EMS200-Ni)
    4. 300目金网格(电子显微镜科学,目录号:EMS300-Au)
    5. 90毫米滤纸(GE Healthcare,目录号:1004-090) 
    6. 牙科蜡板(电子显微镜科学,目录号:72670)
    7. 玻璃培养皿(康宁,目录号:3160-101)
    8. 玻璃载玻片(Fisher Scientific,目录号:12-544-1)
    9. 明胶胶囊(电子显微镜科学,目录号:70100)
    10. 微量离心管,1.5毫升天然(美国科学,目录号:1615-5500)
    11. Parafilm(Sigma-Aldrich,Parafilm,目录号:P7793)
    12. 无菌过滤器(0.2μm)(Corning,目录号:431212)
    13. 二氯乙烷(电子显微镜科学,目录号:13250)
    14. LR白色树脂(Electron Microscopy Sciences,目录号:14383)
    15. 一抗( PlantProbes )(见表1)
    16. 二抗:羊抗鼠IgG-金(Sigma-Aldrich,目录号:G7035)
    17. 第二抗体:与金结合的山羊抗小鼠(Sigma-Aldrich,目录号:G7652)
    18. 索伦森的磷酸盐缓冲液,0.2M,pH7.2(Electron Microscopy Sciences,目录号:11600-10)
    19. 戊二醛(电子显微镜科学,目录号:16120)
    20. 四氧化锇(电子显微镜科学,目录号:19150)
    21. PIPES缓冲液,0.2M,pH7.2(Sigma-Aldrich,目录号:P6757)
    22. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:B4287)
    23. 乙酸铀酰(Polyscience,Inc.,目录号:21447)
    24. 硝酸铅(电子显微镜科学,目录号:17900)
    25. 柠檬酸钠(电子显微镜科学,目录号:21140)
    26. 1N NaOH(Electron Microscopy Sciences,目录号:21170-01)
    27. 0.01 M磷酸盐缓冲液(pH 7.2)(见食谱)
    28. 0.05M磷酸盐缓冲液(pH7.2)(见食谱)
    29. 2.5%戊二醛(见食谱)
    30. 2%的四氧化锇水溶液(见食谱)
    31. 0.02 M磷酸盐缓冲液(pH 7.2)(见食谱)
    32. 抗体(见食谱)
    33. 2%PBS / BSA(见食谱)
    34. 0.05 M PIPES缓冲液(pH 7.2)(见食谱)(见食谱)
    35. 2%醋酸铀(见食谱)
    36. 1 N NaOH(见食谱)
    37. 雷诺的铅柠檬酸盐(见食谱)

设备

  1. 免疫金标记微管和微管组织中心蛋白
    1. 本生灯(Fisher Scientific,产品目录号:S12809)
    2. -20°C冷冻机
    3. M2100 Benchmark Tube Rocker( Benchmark Scientific Inc.
    4. 烤箱(一般信号,型号:重力对流)
    5. 水浴(谢尔顿制造,型号:SWB23)
    6. 微量移液器(Bioexpress,GeneMate,目录号:P-4963-20)
    7. 离心机(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:75004061)
    8. 钻石刀(Diatome,规格:超,45°,4毫米,湿)
    9. 透射电子显微镜(日立,型号:HF7100)

  2. 免疫金标记的精子和基质细胞壁成分
    1. 本生灯(Fisher Scientific,产品目录号:S12809)
    2. M2100 Benchmark Tube Rocker( Benchmark Scientific Inc.
    3. 烤箱(一般信号,型号:重力对流)
    4. 微量移液器(Bioexpress,目录号:P-4963-20)
    5. 离心机(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:75004061)
    6. 钻石刀(Diatome,规格:超,45°,4毫米,湿)
    7. 潮湿的房间(见注)
    8. 透射电子显微镜(日立,型号:HF7100)

程序

  1. 免疫金标记微管和微管组织中心蛋白(Vaughn和Bowling,2008; Vaughn,2013)
    注意:对于下面描述的每种溶液变化,通过添加溶液使样品完全饱和直到覆盖样品瓶/管中的材料。
    1. 在0.05M Sorenson's磷酸盐缓冲液(pH7.2)中的2.5%戊二醛中,在室温下固定小块的几个antheridia或整个配子体植物(如果小,如蕨类植物)。带铝盖的闪烁瓶是这个过程的理想选择(或者,您也可以使用1.5毫升微量离心管)。如果使用部分植物,则在一滴戊二醛溶液中切割样品。
    2. 在相同的0.05M Sorenson缓冲液中洗涤2-3次,每次在4℃下15分钟。为了便于在不损失小样本的情况下交换解决方案,巴斯德移液管的尖端通过本生灯加热,使得尖端孔径降低到其原始尺寸的大约三分之一。通过这种方式,可以进行清洁的交流,以便在交换过程中,微小的样品不会被吸入移液管中并丢失。

    3. 2%(v / v)锇后固定组织10分钟
    4. 连续脱水:取出PIPES缓冲液,加入25%乙醇(在0.05M PIPES缓冲液中)到小瓶中,以25%的增量增加乙醇的百分比,直到达到75%乙醇。
    5. 用100%乙醇交换溶液数次。这些步骤应该在4°C,并在第二次100%乙醇转移后将小瓶转移到-20°C冷冻箱中。
    6. 加入LR白色树脂(伦敦树脂公司)以达到25%塑料(在乙醇中)。摇动小瓶将塑料混合到溶剂中,并保持在-20°C一天。重复此步骤,将塑料添加至50%一天,然后再将塑料添加至75%一天。交换解决方案,把100%的树脂放入小瓶中。摇动小瓶,确保样品沉入树脂中。让他们在100%树脂中度过24小时。
    7. 让小瓶升温至室温,并在摇摆平台上摇动24小时。这将允许整个样品渗透树脂。
    8. 将样品转移至明胶胶囊,并用100%树脂填充胶囊形成弯月面。旋转盖子插入,以创建一个不含氧气的屏蔽。放入一个50°C的烤箱中,并在〜2天后取出。
    9. 修剪样品,并用钻石刀切割100纳米(浅金色反射颜色),并用二氯乙烷烟雾部分来拉伸切片。
      通过触摸300目金网格或200目镍网格的钝面来拾取部分
    10. 将安装好的网格放入一个封闭的CLEAN培养皿中,室温下干燥1-2小时,或将培养皿置于载玻片加热器上(45-50°C)30分钟。
    11. 浮动网格,截面侧下降10μL下列解决方案(所有这些都是通过250纳米过滤器过滤)下降和时间:
      1. 在0.02M磷酸钠缓冲液(PBS / BSA; pH 7.2)中的2%BSA,30分钟(封闭)。
      2. 在2%PBS / BSA中1:20稀释的一抗或单克隆抗体滴在室温下3-4小时。
    12. 冲洗网格放置在2%BSA / PBS 4×3分钟的水滴。
    13. 将网格放置在5-15μl滴加二次金标记的抗体或蛋白质A /黄金稀释的1/20-in 2%BSA / PBS'潮湿室'1-3小时。
    14. 将网格移动到5-10μl的0.02 M PBS中,冲洗4 x 3-5 min(过滤和离心)。
    15. 用已经过滤(0.2微米)的双蒸蒸馏水冲洗每个网格,并使其干燥。
    16. 在2%乙酸铀酰中将网格染色2分钟,用双蒸压灭菌水洗涤,并在雷诺的柠檬酸铅中后期染色。在蒸馏水蒸馏水中洗涤,干燥并在透射电子显微镜下观察。

  2. 免疫金标记的精子基质和细胞壁成分(Lopez和Renzaglia,2014)
    1. 在0.05M Sorenson's磷酸盐缓冲液(pH7.2)中的2.5%戊二醛中,在室温下固定小块的花药组织或整个配子体植物(如果小的话,如蕨类植物)。带铝盖的闪烁瓶是这个过程的理想选择(或者,您也可以使用1.5毫升微量离心管)。如果使用部分植物,则在一滴戊二醛溶液中切割样品。
    2. 在相同的0.05M Sorenson缓冲液中洗涤2-3次,每次在4℃下15分钟。为了便于在不损失小样本的情况下交换解决方案,巴斯德移液管的尖端通过本生灯加热,使得尖端孔径降低到其原始尺寸的大约三分之一。通过这种方式,可以进行清洁的交换,以便在交换期间微小的样品不会被吸入移液管中。
    3. 在1%(v / v)四氧化锇(OsO 4)中固定组织10分钟。
    4. 用蒸馏水冲洗3次(每次10分钟)。
    5. 连续脱水:取出水并加入25%乙醇(在水中)到小瓶中,以25%的增量增加乙醇的百分比,直到达到75%的乙醇。
    6. 用100%乙醇交换溶液3次。
    7. 加入LR白色树脂(伦敦树脂公司)以达到25%塑料(在乙醇中)。摇动小瓶将塑料混合到溶剂中,并保持在-20°C一天。重复此步骤,将塑料添加至50%一天,然后再将塑料添加至75%一天。交换解决方案,把100%的树脂放入小瓶中。摇动小瓶,确保样品沉入树脂中。让他们在100%树脂中度过24小时。
    8. 让小瓶升温至室温,并在摇摆平台上摇动24小时。这将允许整个样品渗透树脂。
    9. 将样品转移至明胶胶囊,并用100%树脂填充胶囊形成弯月面。旋转盖子插入,以创建一个不含氧气的屏蔽。放入一个50°C的烤箱中,并在〜2天后取出。
    10. 修剪样品,并用钻石刀切割90-100nm的切片(浅金色反射色),用二氯乙烷使切片发烟以拉伸切片。
      通过触摸300目金网格或200目镍网格的钝面来拾取部分
    11. 将一个干净的滤纸放在封闭的CLEAN培养皿中,并在室温下干燥1-2小时,或将培养皿置于载玻片加热器(45-50°C)上30分钟。
    12. 使用潮湿的室(参见注2)将一块牙蜡放在完全湿润的滤纸上,将网格浮在上面,在下面的溶液中将样品滴下10μl(所有这些溶液均离心并通过250nm过滤器过滤)和时间:
      1. 在0.05M磷酸钠缓冲液(BSA / PBS; pH 7.2)中的2%BSA过夜(封闭)。
      2. 在2%BSA / PBS中1:20稀释的初级抗体滴在室温下3-4小时。或者,网格可以在4℃保持在一抗过夜。
    13. 冲洗网格放置在2%BSA / PBS 4×3分钟的水滴。
    14. 将网格放置在10μl1/2稀释的二次金标记抗体的2%BSA / PBS'湿润室'中1-3小时。
    15. 将网格移动到10μL0.02M PBS(FILTER and CENTRIFUGE)(用于冲洗4 x 3-5分钟)。
    16. 用已经过滤(0.2微米)的双蒸蒸馏水冲洗每个网格,并使其干燥。
    17. 将网格在2%乙酸铀酰中进行2分钟后染色,用双蒸压灭菌水洗涤,并在雷诺氏酸铅柠檬酸盐中染色30秒。在蒸馏水蒸馏水中洗涤,干燥并在透射电子显微镜下观察。

笔记

  1. 四氧化锇(OsO 4 4)是一种重要的固定剂,可以染色膜状结构和囊泡中的脂质。另外,减少的重金属增加生物组织的密度和对比度。
  2. 潮湿的房间是一个培养皿,其底部覆盖着一个新鲜,干净的湿滤纸,如果坐过夜,盖用石蜡密封。等份的BSA / PBS可以保持冷冻直到使用。使用前离心并冷藏(4°C)。每次标记1-2%BSA / PBS可分成小份(100-200μl),并保存在-20°C,最长可达6个月。
  3. BSA / PBS中的一抗不应反复冷冻和解冻;它会降低酶的强度。

  4. 在标签过程中使用完全干净的工具和培养皿
  5. Millipore过滤或离心每一种药物,只从上清液中提取,使用无菌过滤(0.2μm)水冲洗PBS,柠檬酸铅和醋酸铀酰染色液。

  6. 初级抗体的等分试样(40-50μl)必须保存在-20°C
  7. 含有金颗粒的二抗必须保持在4°C,切勿放在冰箱里。
  8. 标记试剂解冻后(等分试样)不要再冻结。
    在4°C时最多3-4天
  9. 对于BSA / PBS以及其他液体的液滴大小可以在5到10微升之间,如果你停下来一个晚上使用10微升。
  10. 如果您停止上述任何步骤(将网格放入二抗之前),不应超过一夜,潮湿的室应密封并放在冰箱中。
    注:网格不应该留在二抗中过夜,因为它会增加背景“噪音”(即标签)。
  11. 建议在将滤网移动到一个新的解决方案之前,并在同一个冲洗的不同步骤之间,在一小块滤纸上形成网格。

  12. 每次将网格移至新的解决方案时,请使用新的Parafilm(或冲洗并擦拭牙齿蜡)。
  13. 玻璃培养皿是首选。塑料创造静电并且导致栅格紧贴到盘子顶部。

食谱

  1. 0.01 M磷酸盐缓冲液(pH 7.2)

    加入5份0.20 M Sorenson磷酸盐缓冲液(pH 7.2)至95份双蒸压水
  2. 0.05M磷酸盐缓冲液(pH7.2)

    加入25份0.20M Sorenson's磷酸盐缓冲液(pH7.2)到75份双蒸压的高压灭菌水中
  3. 2.5%戊二醛
    1. 将1份10%戊二醛加入1份双蒸压蒸馏水中
    2. 将1份0.20M Sorensen's磷酸盐缓冲液(pH7.2)加入到1份双蒸蒸馏水中
    3. 将1份5%戊二醛加入到1份0.10M Sorensen's磷酸盐缓冲液(pH7.2)中
  4. 2%四氧化锇水溶液

    加1份4%四氧化锇水溶液到1份双蒸压蒸馏水中
  5. 0.02 M磷酸盐缓冲液(pH 7.2)
    将10份0.20M Sorenson's磷酸盐缓冲液(pH7.2)加入到10份双蒸压的高压灭菌水中
  6. 抗体
    添加1份抗体到20份2%PBS / BSA
  7. 2%PBS / BSA
    将0.20g牛血清白蛋白加入到10ml 0.02M磷酸盐缓冲液(pH7.2)中
  8. 0.05 M PIPES缓冲液(pH 7.2)

    加入1份0.30 M PIPES缓冲液(pH 7.2)至6份双蒸压蒸馏水
  9. 2%醋酸铀。
    在50毫升双蒸压蒸馏水中加入1克醋酸铀。
  10. 1 N NaOH
    将40.00克氢氧化钠溶于水中,使体积达到1升
  11. 雷诺的铅柠檬酸盐
    1. 加入1.33克硝酸铅和1.76克柠檬酸钠到30毫升煮沸的蒸馏水中,冷却到室温,摇30分钟。
    2. 加入8ml 1N NaOH
    3. 加入12毫升双蒸压蒸馏水

致谢

这项研究得到了国家科学基金会的研究经费支持(DEB-0322664,DEB-0423625,DEB0521177和DEB-0228679),作为大学生研究经验和组装生命之树计划的一部分。
作者声明我们没有利益冲突或利益冲突。

参考

  1. Davis,F.M.,Tsao,T.Y.,Fowler,S.K。和Rao,P.N。(1983)。 有丝分裂细胞的单克隆抗体 Proc Natl Acad Sci USA 80(10):2926-2930。
  2. Hepler,P.K。(1976)。 Marsilea的blephalastlast :它的 de novo
  3. Hoffman,J.C。和Vaughn,K.C。(1995)。 使用发育中的Ceratopteris精原细胞揭开奥秘植物细胞骨架.Int J Plant Sci 156(3):346-358。
  4. Hoffman,J.C。,Vaughn,K.C。和Joshi,H.C。(1994)。 蕨类植物精子细胞中微管组织中心的结构和免疫细胞化学表征原生质体
    179(1):46-60。
  5. Joshi,H.C.,Palacios,M.J.,McNamara,L。和Cleveland,D.W。(1992)。 γ-微管蛋白是细胞周期依赖性微管形核所需的中心体蛋白。自然 356(6364):80-83。
  6. Klink,V.P。和Wolniak,S.M。(2003)。 Marsilea精子发生过程中保守的中心体,细胞骨架和睫状体蛋白的丰度和分布的变化。 Cell Motil Cytoskeleton 56(1):57-73。
  7. Knox,J.P.,Linstead,P.J.,King,J.,Cooper,C.,Roberts,K。(1990)。 果胶酯化在根部顶端的细胞壁和发育组织之间都是空间调节的。 a> Planta 181(4):512-521。
  8. Lamport,D.T。和Várnai,P。(2013)。 周质阿拉伯半乳糖糖蛋白作为调节植物生长和发育的钙电容器。 > New Phytol 197(1):58-64。
  9. Liu,B.,Joshi,H.C.,Wilson,T.J.,Silflow,C.D。,Palevitz,B.A。和Snustad,D.P.(1994)。 拟南芥中的γ-微管蛋白:基因序列,免疫印迹和免疫荧光研究。植物细胞 6(2):303-314。
  10. Liu,B.,Marc,J.,Joshi,H.C。和Palevitz,B.A。(1993)。 与细胞周期依赖的高等植物微管阵列相关的γ-微管蛋白相关蛋白(J Cell Sci)104(4):1217-1228。
  11. Lopez,R.A。和Renzaglia,K.S。(2014)。 Ceratopteris richardii的多鞭毛细胞在整个发育过程中都浸泡在阿拉伯半乳聚糖蛋白中。 Am J Bot 101(12):2052-2061。
  12. Lopez,R.A。和Renzaglia,K.S。(2017)。 Ceratopteris (蕨类)发展动力配子墙含有不同的多糖,但不是果胶。
  13. Lopez-Swalls和Renee A.(2016)。 Ceratopteris richardii 和 南伊利诺伊大学卡本代尔。
  14. Manandhar,G.,Simerly,C.,Salisbury,J.L。和Schatten,G。(1999)。 小鼠精子发生过程中的中心粒和中心粒变性 细胞骨架 43 (2):137-144。
  15. Marcus,S.E.,Verhertbruggen,Y.,Hervé,C.,Ordaz-Ortiz,J.J.,Farkas,V.,Pedersen,H.L.,Willats,W.G.,Knox,J.P.(2008)。 Pectic homogalacturonan掩盖植物细胞壁中丰富的木葡聚糖抗原决定基。 BMC Plant Biol 8(1):60.
  16. Meikle,P.J.,Bonig,I.,Hoogenraad,N.J。,Clarke,A.E。和Stone,B.A。(1991)。 (1→3)-β-葡聚糖在花粉管壁上的位置(1→3)-β-葡聚糖特异性单克隆抗体的烟草(Nicotiana alata)。植物185(1):1-8。
  17. Murata,T。和Hasebe,M。(2007)。 植物细胞中微管依赖性微管成核植物研究(120)(1):73-78。
  18. Murata,T.,Sonobe,S.,Baskin,T. I.,Hyodo,S.,Hasezawa,S.,Nagata,T.,Horio,T。和Hasebe,M。(2005)。 基于在高等植物中募集γ-微管蛋白的微管依赖性微管成核 < em> Nat Cell Biol 7(10):961-968。
  19. Oakley,B.R.,Oakley,C.E.,Yoon,Y.和Jung,M.K。(1990)。 γ-微管蛋白是纺锤体的一个组成部分,对于微管功能至关重要。构巢曲霉(Aspergillus nidulans) 。 61(7):1289-1301。
  20. Pedersen,HL,Fangel,JU,McCleary,B.,Ruzanski,C.,Rydahl,MG,Ralet,MC,Farkas,V.,von Schantz,L.,Marcus,SE,Andersen,MC和Field, 2012)。 用于植物糖生物学和细胞壁研究的多功能高分辨寡糖微阵列 J Biol Chem 287(47):39429-39438。
  21. Pennell,R. I.,Janniche,L.,Kjellbom,P.,Scofield,G. N.,Peart,J. M.和Roberts,K。(1991)。 油菜花油中质膜阿拉伯半乳聚糖蛋白表位的发育调控 植物细胞 3(12):1317-1326
  22. Renzaglia,K. S.和Garbary,D. J.(2001)。 Crit Rev Rev <动植物雄性配子:多样性,发展和进化 Crit Rev Sci < / em> 20(2):107-213。
  23. Satisbury,J.L。(1995)。 中心体,中心体和有丝分裂纺锤体两极 Curr Opin Cell Biol < / em> 7(1):39-45。
  24. Showalter,A.M。(2001)。 Arabinogalactan-proteins:结构,表达和功能 Cell Mol Life Sc 58(10):1399-1417。
  25. Vandre,D. D.,Davis,F.M.,Rao,P.N。和Borisy,G.G。(1984)。 磷酸化蛋白是有丝分裂微管组织中心的组成部分。美国国家科学院院刊 81(14):4439-4443。
  26. Vaughn,K。(2013)。免疫细胞化学技术。在:植物细胞的免疫细胞化学。免疫组化技术斯普林格 pp:1-41。
  27. Vaughn,K.C。和Bowling,A.J。(2008)。 在oryzalin治疗后恢复角质形成细胞的角质细胞上的微管。 Protoplasma 233(3-4):231-240。
  28. Vaughn,K.C。和Harper,J.D。(1998)。 微生物组织中心和陆地植物成核点 Int Rev Cytol 181:75-149。
  29. Vaughn,K.C。和Renzaglia,K.S。(1993)。 Centrin在精子发生过程中的作用。 Am J Bot (Suppl。)80(1):58-66。
  30. Vaughn,K.C。和Renzaglia,K.S。(2006)。 银杏叶的结构和免疫细胞化学表征。精子活动器械。


    Protoplasma 227(2-4):165-173。

  31. Verhertbruggen,Y.,Marcus,S.E.,Haeger,A.,Ordaz-Ortiz,J.J。和Knox,J.P。(2009)。 延长的单克隆抗体对pectic homogalacturonan。 Carbohyd Res 344(14):1858-186。
  32. Yates,E.A.,Valdor,J-F。,Haslam,S.M.,Morris,H.R.,Dell,A.,Mackie,W。和Knox,J.P。(1996)。 抗阿拉伯半乳聚糖蛋白单克隆抗体识别的碳水化合物结构特征的表征 糖生物学 6(2):131-139。
  • 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: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Lopez, R. A., Mansouri, K., Henry, J. S., Flowers, N. D., Vaughn, K. C. and Renzaglia, K. S. (2017). Immunogold Localization of Molecular Constituents Associated with Basal Bodies, Flagella, and Extracellular Matrices in Male Gametes of Land Plants. Bio-protocol 7(21): e2599. DOI: 10.21769/BioProtoc.2599.
  2. Renzaglia, K. S., Villarreal, J. C., Piatkowski, B. T., Lucas, J. R. and Merced, A. (2017). Hornwort Stomata: Architecture and Fate Shared with 400-Million-Year-Old Fossil Plants without Leaves. Plant Physiol 174(2): 788-797.
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google 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。