Modifications of the golgi apparatus inSaccharomyces cerevisiae lacking microtubules

Rambourg, A.; Gachet, Emmanuel; Clermont, Y.; Képès, François

doi:10.1002/(sici)1097-0185(199610)246:2<162::aid-ar2>3.0.co;2-3

Cited by 14 publications

(4 citation statements)

References 22 publications

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“…However, small organelles typical of yeast such as the different Golgi compartments or the various classes of endosomes, but also vesicular carriers, cannot be easily seen because of the small difference in contrast between membranes and cytosol. Nevertheless, these conventional EM approaches can be very useful to monitor apparent morphological changes such as, for example, the proliferation of the ER, Golgi apparatus expansions, endosome enlargements or autophagosome formation (15–19). Another general limitation of these two conventional EM protocols is that only a limited number of proteins can be labelled with immunological reactions because fixation conditions, dehydration procedures, resin properties and the resin polymerization temperature have cumulative denaturing effects on epitopes.…”

Section: Resultsmentioning

confidence: 99%

“…Because of its structure and composition, yeast S. cerevisiae has proven to be a challenge for electron microscopists. Nevertheless, some EM procedures to analyse this unicellular organism at an ultrastructural level have been developed and allow the study of certain physiological aspects of this eukaryote (15–19). Permanganate fixation followed by embedding in the Spurr’s resin is one of them and the preparations are of good quality because of the excellent membrane contrasts (Figure 1A–C).…”

Section: Discussionmentioning

confidence: 99%

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A Cryosectioning Procedure for the Ultrastructural Analysis and the Immunogold Labelling of Yeast Saccharomyces cerevisiae

Griffith

Mari

Mazière

et al. 2008

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Yeast Saccharomyces cerevisiae has been a crucial model system for the study of a multitude of cellular processes because of its amenability to genetics, molecular biology and biochemical procedures. By contrast, the morphological analysis of this organism by immunoelectron microscopy (IEM) has remained in a primordial phase preventing researchers to routinely incorporate this technique into their investigations. Here, in addition to simple but detailed protocols to perform conventional electron microscopy (EM) on plastic embedded sections, we present a new IEM procedure adapted from the Tokuyasu method to prepare cryosections from mildly fixed cells. This novel approach allows an excellent cell preservation and the negatively stained membranes create superb contrast that leads to a unique resolution of the yeast morphology. This, plus the optimal preservation of the epitopes, permits combined localization studies with a fine resolution of protein complexes, vesicular carriers and organelles at an ultrastructural level. Importantly, we also show that this cryo-immunogold protocol can be combined with highpressure freezing and therefore cryofixation can be employed if difficulties are encountered to immobilize a particular structure with chemical fixation. This new IEM technique will be a valuable tool for the large community of scientists using yeast as a model system, in particular for those studying membrane transport and dynamics.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A Cryosectioning Procedure for the Ultrastructural Analysis and the Immunogold Labelling of Yeast Saccharomyces cerevisiae

Griffith

Mari

Mazière

et al. 2008

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“…The universal dependence of the integrity and movement of intracellular organelles on the cytoskeleton (26)(27)(28)(29)(30)(31)(32) implies that The trees were generated as described for Fig.1). The accession numbers are: Giardia Sar1 AAM83404; Arf1 S29008; Ran AAA21426; Rab2 EAA42348; Rab11 AAO49245; Rac EAA40663; Dictyostelium Sar1 AAO51740; Arf1 CAA03896; Ran S35619; Rab1 AAC37385; Rab5 AAO51496; Rab11 AAA80149; Rac AAC37389; Ras S31985; Arabidopsis SRb BAB09661; Arf1 AAF79587; Sar1 D86224; Ran AAB58478; Rac T48862; Rab11 T04539; Rab5 T06157; Rab6 T50814; Trypanosoma Arf1 AAF82562; Ras CAA05774; Rab5 AAC46991; Rab11 AAF70820; Plasmodium Sar1 AAF06723; Ran P38545; Rab6 AAF27978; Paramecium Rac AAA57056.…”

Section: Membrane Tubulation As a Means Of Generating The First Endommentioning

confidence: 99%

Small GTPases and the evolution of the eukaryotic cell

2003

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The origin of eukaryotes is one of the major challenges of evolutionary cell biology. Other than the endosymbiotic origin of mitochondria and chloroplasts, the steps leading to eukaryotic endomembranes and endoskeleton are poorly understood. Ras-family small GTPases are key regulators of cytoskeleton dynamics, vesicular trafficking and nuclear function. They are specific for eukaryotes and their expansion probably traces the evolution of core eukaryote features. The phylogeny of small GTPases suggests that the first endomembranes to evolve during eukaryote evolution had secretory, and not phagocytic, function. Based on the reconstruction of putative roles for ancestral small GTPases, a hypothetical scenario on the origins of the first endomembranes, the nucleus, and phagocytosis is presented.

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“…The role of microtubules in vesicle transport, organelle movement, and maintenance is well established in mammalian cell systems (reviewed by Bloom and Goldstein, 1998). In yeast, however, there are few studies implicating microtubules in vesicle transport: microtubules may be involved in autophagosome travel to the vacuole (Lang et al, 1998), and Golgi fragmentation has been reported in cells with disrupted microtubules (Rambourg et al, 1996). Any involvement with Luv1p/Rki1p with microtubules in transport is unclear and awaits further investigation.…”

Section: Luv1p and Microtubulesmentioning

confidence: 99%

Luv1p/Rki1p/Tcs3p/Vps54p, a Yeast Protein That Localizes to the Late Golgi and Early Endosome, Is Required for Normal Vacuolar Morphology.

Conboy

Cyert

2000

MBoC

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We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H ) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 ϫ g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.

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Modifications of the golgi apparatus inSaccharomyces cerevisiae lacking microtubules

Cited by 14 publications

References 22 publications

A Cryosectioning Procedure for the Ultrastructural Analysis and the Immunogold Labelling of Yeast Saccharomyces cerevisiae

A Cryosectioning Procedure for the Ultrastructural Analysis and the Immunogold Labelling of Yeast Saccharomyces cerevisiae

Small GTPases and the evolution of the eukaryotic cell

Luv1p/Rki1p/Tcs3p/Vps54p, a Yeast Protein That Localizes to the Late Golgi and Early Endosome, Is Required for Normal Vacuolar Morphology.

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