Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips ofNicotiana andArabidopsis

Kiss, John Z.; Giddings, T. H.; Staehelin, L. Andrew; Sack, Fred D.

doi:10.1007/bf01322639

Cited by 112 publications

(62 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An entire animal can be immobilized in amorphic ice nearly instantaneously, thus its ultrastructure can be examined with minimal structural perturbation. At neuromuscular junctions, the ability of this fixation technique to preserve labile structures is apparent from the vast microtubule network observed in neuronal profiles: microtubules often depolymerize during traditional chemical fixation (Kiss et al, 1990;McMenamin et al, 2003;Rostaing et al, 2004).…”

Section: Ultrastructural Preservation By High-pressure Freezing C Elmentioning

confidence: 99%

UNC-13 and UNC-10/Rim Localize Synaptic Vesicles to Specific Membrane Domains

et al. 2006

View full text Add to dashboard Cite

Synaptic vesicles undergo a maturation step, termed priming, in which they become competent to fuse with the plasma membrane. To morphologically define the site of vesicle priming and identify fusion-competent synaptic vesicles, we combined a rapid physical-fixation technique with immunogold staining and high-resolution morphometric analysis at Caenorhabditis elegans neuromuscular junctions. In these presynaptic terminals, a subset of synaptic vesicles contact the plasma membrane within ϳ100 nm of a presynaptic dense projection. UNC-13, a protein required for vesicle priming, localizes to this same region of the plasma membrane. In an unc-13 null mutant, few synaptic vesicles contact the plasma membrane, suggesting that membrane-contacting synaptic vesicles represent the morphological correlates of primed vesicles. Interestingly, a subpopulation of membrane-contacting vesicles, located within 30 nm of a dense projection, are unperturbed in unc-13 mutants. We show that UNC-10/Rim, a protein implicated in presynaptic plasticity, localizes to dense projections and that loss of UNC-10/Rim causes an UNC-13-independent reduction in membrane-contacting synaptic vesicles within 30 nm of the dense projections. Our data together identify a discrete domain for vesicle priming within 100 nm of dense projections and further suggest that UNC-10/Rim and UNC-13 separately contribute to the membrane localization of synaptic vesicles within this domain.

show abstract

Section: Ultrastructural Preservation By High-pressure Freezing C Elmentioning

confidence: 99%

UNC-13 and UNC-10/Rim Localize Synaptic Vesicles to Specific Membrane Domains

et al. 2006

View full text Add to dashboard Cite

show abstract

“…For the electron microscopy studies, nontransgenic, mGFP5-ER, GFP:TGBp2, and GFP:TGBp3 transgenic tobacco leaf segments were fixed using the Balzer (Manchester, NH) HPM010 high-pressure freezing machine located at the Oklahoma Medical Research Center, according to standard protocols (Kiss et al, 1990). Leaf pieces were excised, washed with distilled, deionized water, and fitted into 0.4-mm freezer hats (Ted Pella, Reading, CA) filled with lecithin solution (100 mg mL 21 in chloroform).…”

Section: Fixation Lr White Embedding and Immunolabeling Of Plant Mamentioning

confidence: 99%

The Potato Virus X TGBp2 Movement Protein Associates with Endoplasmic Reticulum-Derived Vesicles during Virus Infection

et al. 2005

View full text Add to dashboard Cite

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virusinfected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.Plant viruses use the cellular endomembrane system to support virus replication (Schaad et al., 1997;Reichel and Beachy, 1998;Carette et al., 2002aCarette et al., , 2002bSchwartz et al., 2002). Among positive-strand RNA viruses, the viral replicase is typically anchored to membranes associated with the endoplasmic reticulum (ER), the endocytic pathway, or cellular organelles (Rubino and Russo, 1998;Mas and Beachy, 1999;Rubino et al., 2001;Schwartz et al., 2002Schwartz et al., , 2004. Many viral replicationassociated proteins cause reorganization of cellular membranes with the purpose of creating centers that protect the viral replication complexes (VRCs) from cellular nucleases and host defenses (Schwartz et al., 2002(Schwartz et al., , 2004Ding et al., 2004). Specifically, the replication complexes of plant viruses belonging to the genera Potyvirus, Comovirus, Dianthovirus, Pecluvirus, and Bromovirus cause proliferation and vesiculation of either the perinuclear or the cortical ER (Schaad et al., 1997;Dunoyer et al., 2002;Lee and Ahlquist, 2003). The replicases of cowpea mosaic virus and red clover necrotic mosaic virus induce ER proliferation (Carette et al., 2002a(Carette et al., , 2002bTurner et al., 2004). The best-studied example is the brome mosaic virus (BMV) 1a replicase, which localizes to the ER and induces membrane i...

show abstract

“…Nevertheless, in the last years, HPF-FS has been successfully applied to plant tissues. Besides, tissue devoid of large intercellular spaces, such as nodules, ovules, anthers, roots, and stems (Ding et al, 1991;Hess, 2003;Hippe et al, 1989;Kiss et al, 1990;Lee et al, 2000;Studer et al, 1992;Thijssen et al, 1997) also leaf material, has been analyzed (Otegui et al, 2005;Pfeiffer et al, 2003;Tiedemann et al, 1998). Rarely, the complex and dynamic structure of chloroplasts was investigated in specimen prepared by HPF-FS (Bourett et al, 1999;Pfeiffer and Krupinska, 2005).…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, other replacement media including water (Craig and Staehelin, 1988) or salt solutions (Meindl et al, 1992) were used instead. Occasionally, cryoprotectans such as sucrose (Ding et al, 1992;Kandasamy et al, 1991) and dextran (Kiss et al, 1990) were also used. In other studies, MeOH (Lee et al, 2000;Rodríguez-Gá lvez and Mendgen, 1995;Welter et al, 1988) was added to the replacement medium.…”

Section: Introductionmentioning

confidence: 99%

“…To ensure the rapid effect of the pressure and a high conductivity for heat removal during the freezing process, the air in the specimen holder's free space and the air within the intercellular spaces within the leaf tissues has to be replaced by a liquid medium (Kiss et al, 1990;Welter et al, 1988). For filling up the space between the sample and the aluminum platelets, 1-hexadecene has been frequently used for a variety of plant tissues, such as root nodules (Studer et al, 1992), root tips (Ding et al, 1991), anthers (Hess, 1994), and leaves (Kaneko and Walther, 1995).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Chloroplast ultrastructure in leaves ofUrtica dioica L. analyzed after high-pressure freezing and freeze-substitution and compared with conventional fixation followed by room temperature dehydration

Pfeiffer¹,

Krupinska²

2005

Microsc. Res. Tech.

View full text Add to dashboard Cite

In this article, we report on the adaptation of high-pressure freezing and freeze-substitution (HPF-FS) for ultrastructural analysis of leaf tissue with special emphasis on chloroplasts. To replace the gas in the intercellular spaces, a mixture of water and methanol (MeOH) was employed. We compared three different supplements for FS--osmiumtetroxide, uranyl acetate, and safranin--with regard to the preservation of the ultrastructure of chloroplasts and other cellular compartments. The results show that (i) replacement of air within intercellular spaces by 8% (v/v) MeOH has no influence on the ultrastructure of the chloroplasts, (ii) undulation of membranes frequently observed after conventional preparation of specimens does not occur during chemical fixation but during room temperature dehydration, and (iii) uranyl acetate or osmium tetroxide employed during FS are not superior over safranin.

show abstract

Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips ofNicotiana andArabidopsis

Cited by 112 publications

References 39 publications

UNC-13 and UNC-10/Rim Localize Synaptic Vesicles to Specific Membrane Domains

UNC-13 and UNC-10/Rim Localize Synaptic Vesicles to Specific Membrane Domains

The Potato Virus X TGBp2 Movement Protein Associates with Endoplasmic Reticulum-Derived Vesicles during Virus Infection

Chloroplast ultrastructure in leaves ofUrtica dioica L. analyzed after high-pressure freezing and freeze-substitution and compared with conventional fixation followed by room temperature dehydration

Contact Info

Product

Resources

About