New Method for Quantitation of Lipid Droplet Volume From Light Microscopic Images With an Application to Determination of PAT Protein Density on the Droplet Surface.
J Histochem Cytochem
; 66(6): 447-465, 2018 06.
Article
em En
| MEDLINE
| ID: mdl-29361239
ABSTRACT
Determination of lipid droplet (LD) volume has depended on direct measurement of the diameter of individual LDs, which is not possible when LDs are small or closely apposed. To overcome this problem, we describe a new method in which a volume-fluorescence relationship is determined from automated analysis of calibration samples containing well-resolved LDs. This relationship is then used to estimate total cellular droplet volume in experimental samples, where the LDs need not be individually resolved, or to determine the volumes of individual LDs. We describe quantitatively the effects of various factors, including image noise, LD crowding, and variation in LD composition on the accuracy of this method. We then demonstrate this method by utilizing it to address a scientifically interesting question, to determine the density of green fluorescent protein (GFP)-tagged Perilipin-Adipocyte-Tail (PAT) proteins on the LD surface. We find that PAT proteins cover only a minority of the LD surface, consistent with models in which they primarily serve as scaffolds for binding of regulatory proteins and enzymes, but inconsistent with models in which their major function is to sterically block access to the droplet surface.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Processamento de Imagem Assistida por Computador
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Microscopia Confocal
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Gotículas Lipídicas
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Perilipinas
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article