Lens membrane fraction associated intermediate filaments of different aged rats

Fleschner, C.R.

doi:10.1076/ceyr.24.4.296.8415

Cited by 2 publications

(2 citation statements)

References 40 publications

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“… 26 Immunohistochemical studies indicate vimentin, a major type III IF protein, is expressed in the lens epithelium and fiber cells, although the expression pattern changes based on lens age and region. 4 , 27 , 28 Our results indicate vimentin is most abundant in the very outer fiber cells of the DF region and its abundance decreases in the RZ and further into the lens. Because we analyzed the plasma membrane fraction, this decrease in vimentin signal at the RZ could be due to either reduced vimentin expression, increased solubility of the protein, or decreased association with the plasma membrane.…”

Section: Discussionmentioning

confidence: 63%

Spatially Directed Proteomics of the Human Lens Outer Cortex Reveals an Intermediate Filament Switch Associated With the Remodeling Zone

Wenke

Schey

2016

Invest. Ophthalmol. Vis. Sci.

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PurposeTo quantify protein changes in the morphologically distinct remodeling zone (RZ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics.MethodsLightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (WGA-TRITC). Morphology directed laser capture microdissection (LCM) was used to isolate tissue from four distinct regions of human lens outer cortex: differentiating zone (DF), RZ, transition zone (TZ), and inner cortex (IC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, filensin, and phakinin. Peptides from proteins of interest were quantified using multiple reaction monitoring (MRM) mass spectrometry and isotopically-labeled internal peptide standards.ResultsResults revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the RZ. Several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. Targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the DF to the IC, while filensin, phakinin, and brain acid soluble protein 1 (BASP1) increase significantly at the RZ.ConclusionsMass spectrometry-compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. Results reveal dramatic cytoskeletal protein changes associated with the RZ, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human RZ.

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Section: Discussionmentioning

confidence: 63%

Spatially Directed Proteomics of the Human Lens Outer Cortex Reveals an Intermediate Filament Switch Associated With the Remodeling Zone

Wenke

Schey

2016

Invest. Ophthalmol. Vis. Sci.

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“…Crystallins account for approximately 90% of the extrinsic protein, with the remainder comprising cytoskeletal and other proteins (Cenedella and Fleschner 1992). A comparison of the cytoskeletal components vimentin, phakinin, and filensin in the NSMF and 25/45 fractions showed that these proteins differed quantitatively but not qualitatively between the two membrane fractions in both bovine and rat lenses (Fleschner 1998, 2002). …”

Section: Introductionmentioning

confidence: 98%

Monoclonal antibody 10A5 recognizes an antigen unique to the water-insoluble 25/45 membrane fraction of the rat ocular lens

et al. 2013

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The water-insoluble 25/45 fraction and non-sedimenting membrane fraction (NSMF) are two membrane preparations isolated from the ocular lens. The fractions are postulated to represent distinct subdomains of the lens with unique functions. However, attempts to distinguish between the two fractions by detecting proteins present in one fraction but absent from other have been unsuccessful. In this study, we exploited the ability of the mouse immune system to detect antigenic differences between the 25/45 fraction and NSMF isolated from the lenses of 20-day-old rats. We generated a monoclonal antibody (MAb 10A5) that reacts with a ganglioside-like antigen that is present in the 25/45 fraction but absent from the NSMF. Restriction of the antigen to the 25/45 fraction in 20-day-old animals supports the hypothesis that the 25/45 fraction and NSMF represent different subdomains within the ocular lens.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-500) contains supplementary material, which is available to authorized users.

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Lens membrane fraction associated intermediate filaments of different aged rats

Cited by 2 publications

References 40 publications

Spatially Directed Proteomics of the Human Lens Outer Cortex Reveals an Intermediate Filament Switch Associated With the Remodeling Zone

Spatially Directed Proteomics of the Human Lens Outer Cortex Reveals an Intermediate Filament Switch Associated With the Remodeling Zone

Monoclonal antibody 10A5 recognizes an antigen unique to the water-insoluble 25/45 membrane fraction of the rat ocular lens

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