RESUMEN
Fatty acids (FAs) have diverse functions in cellular activities. The intracellular distribution of FAs is critical for their functions. Imaging of FAs by time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been achieved. However, TOF-SIMS images of FAs so far do not have subcellular distribution due to inadequate sample preparation methods. In this study, we developed a chemical fixation method using glutaraldehyde (GA) with uranyl acetate (UA), which preserved cellular structure and intracellular FA distribution well. Combining GA+UA fixation with sputtering-based methods and unroofing-based methods, respectively, we successfully imaged intracellular lipids with the subcellular distribution.
Asunto(s)
Ácidos Grasos , Espectrometría de Masa de Ion Secundario , GlutaralRESUMEN
The plasma membrane (PM) serves multiple functions to support cell activities with its heterogeneous molecular distribution. Fatty acids (FAs) are hydrophobic components of the PM whose saturation and length determine the membrane's physical properties. The FA distribution contributes to the PM's lateral heterogeneity. However, the distribution of PM FAs is poorly understood. Here, we proposed the FA cluster hypothesis, which suggested that FAs on the PM exist as clusters. By the optogenetic tool translocating the endoplasmic reticulum (ER), we were able to manipulate the distribution of PM FAs. We used time-of-flight combined secondary ion mass spectrometry (TOF-SIMS) to image PM FAs and discovered that PM FAs were presented and distributed as clusters and are also manipulated as clusters. We also found the existence of multi-FA clusters formed by the colocalization of more than one FA. Our optogenetic tool also decreased the clustering degree of FA clusters and the formation probability of multi-FA clusters. This research opens up new avenues and perspectives to study PM heterogeneity from an FA perspective. This research also suggests a possible treatment for diseases caused by PM lipid aggregation and furnished a convenient tool for therapeutic development.
Asunto(s)
Ácidos Grasos , Espectrometría de Masa de Ion Secundario , Ácidos Grasos/metabolismo , Espectrometría de Masa de Ion Secundario/métodos , Optogenética , Membrana Celular/metabolismo , Diagnóstico por ImagenRESUMEN
X-ray powder diffractometry/Rietveld refinement was employed to estimate the purity of several chrysotile powders for calibrating standards. α-Corundum powder was mixed into each chrysotile sample as an internal standard. X-ray diffractometry was performed on these mixtures, and the mass fractions of amorphous and impurity phase content were calculated using Rietveld refinement. The chrysotile samples had 56.7-92.0% crystalline purity. X-ray diffraction intensities of chrysotile (002) from the samples showed good correlation with the crystalline purity data. In differential thermal analysis results, the exothermal peak intensities were found to be directly proportional to crystalline contents for crystalline purity lower than 80%. Any chrysotile sample can be used as a standard material for quantitative determination upon correction of the crystalline purity, which can be estimated using the proposed method.