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Method for ultrastructural fine details of plant cuticles by transmission electron microscopy.

ABSTRACT: This method for plant cuticles was adapted from spores and pollen procedure. It already enabled to prepare ultrathin plant cuticle sections of 60-70 nanometers of thickness, in high numbers as a routine, and allowed statistical measurements as any other macro- and/or micro-features. It provides through transmission electron microscope clean and contrasted observations of layers and sublayers of the cuticle, at a magnification over 100,000 times. It has been used efficiently for taxonomy, signature of environment and evolution topics since the end of the nineties (Gaëtan Guignard, 2019), however it is detailed here step by step for the first time, and illustrated. It can serve in the future for many purposes, like other taxonomical comparisons, palaeoreconstructions and evolutionary considerations. Value of the protocol•This method provides ultrathin plant cuticle sections of 60-70 nanometers of thickness•Details of layers and sublayers of the cuticle are easily observed•High numbers of sections allows statistical measurements as a routine.

SUBMITTER: Guignard G

PROVIDER: S-EPMC8374509 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Method for ultrastructural fine details of plant cuticles by transmission electron microscopy.

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