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Ultrastructural imaging of endocytic sites in Saccharomyces cerevisiae by transmission electron microscopy and immunolabeling.

ABSTRACT: Defining the ultrastructure of endocytic sites and localization of endocytic proteins in Saccharomyces cerevisiae by immunoelectron microscopy is central in understanding the mechanisms of membrane deformation and scission during endocytosis. We show that an improved sample preparation protocol based on high-pressure freezing, freeze substitution, and low-temperature embedding allows us to maintain the cellular fine structure and to immunolabel green fluorescent protein-tagged endocytic proteins or actin in the same sections. Using this technique we analyzed the stepwise deformation of endocytic membranes and immunolocalized the endocytic proteins Abp1p, Sla1p, Rvs167p, and actin, and were able to draw a clear ultrastructural distinction between endocytic sites and eisosomes by immunolocalizing Pil1p. In addition to defining the geometry and the fine structure of budding yeast endocytic sites, we observed associated actin filaments forming a cage-like meshwork around the endocytic membrane.

SUBMITTER: Buser C

PROVIDER: S-EPMC4113337 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Ultrastructural imaging of endocytic sites in Saccharomyces cerevisiae by transmission electron microscopy and immunolabeling.

Buser Christopher C Drubin David G DG

Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada 20130305 2

Defining the ultrastructure of endocytic sites and localization of endocytic proteins in Saccharomyces cerevisiae by immunoelectron microscopy is central in understanding the mechanisms of membrane deformation and scission during endocytosis. We show that an improved sample preparation protocol based on high-pressure freezing, freeze substitution, and low-temperature embedding allows us to maintain the cellular fine structure and to immunolabel green fluorescent protein-tagged endocytic proteins ...[more]

PMID: 23458500

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Project description:BackgroundHeterozygous de novo variants in the gene SAMD9 cause the complex multisystem disorder, MIRAGE syndrome. Patients are characterised by myelodysplasia, infections, growth restriction, adrenal insufficiency, gonadal dysfunction and enteropathies. Pathogenic variants in SAMD9 are gain-of-function and enhance its role as a growth repressor, leading to growth restriction of many tissues. Two studies have reported changes in skin fibroblasts derived from MIRAGE patients, more specifically identifying enlarged endosomes. We have also previously shown subtle changes in endosome size in patients' fibroblasts compared to controls. However, these variations in endosomes were not as marked as those described in the literature.MethodsWe have performed an observational study using transmission electron microscopy (TEM) in a larger number of cells derived from three patients' fibroblasts to assess ultrastructure morphology compared to control images.ResultsConsistent changes were observed in cell organelles in all patient samples. In particular, increased endosomal activity was detected, characterised by augmented pinocytosis and vesicle budding, increased endosome number, as well as by large lysosomes and endosomes. Endoplasmic reticulum was also prominent. Mitochondria appeared enlarged in selected cells, possibly due to cellular stress. Cell nuclei did not display major differences compared to controls.ConclusionsTEM is a powerful tool to investigate morphological features of tissues and cell organelles, although TEM data could be affected by sample preparation methodology, therefore potentially explaining the variability between independent studies, and its analysis can be dependent on the experience of the researcher. The increased endosomal activity we have observed in patients' fibroblasts could indicate that SAMD9 regulates endocytosis of receptors, acting as an endosome fusion facilitator, or in lysosomal activation. However, the precise mechanism(s) by which SAMD9 regulates cell growth is still not fully understood, and further studies are needed to elucidate its pathogenic pathway and develop therapeutic approaches to support patients.

Dataset Information

Ultrastructural imaging of endocytic sites in Saccharomyces cerevisiae by transmission electron microscopy and immunolabeling.

Publications

Ultrastructural imaging of endocytic sites in Saccharomyces cerevisiae by transmission electron microscopy and immunolabeling.

Similar Datasets

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