Characterization of lens fiber cell triton insoluble fraction reveals ERM (ezrin, radixin, moesin) proteins as major cytoskeletal-associated proteins

Abstract

To understand lens fiber cell elongation- and differentiation-associated cytoskeletal remodeling, here we identified and characterized the major protein components of lens fiber cell Triton X-100 insoluble fraction by mass spectrometry and immunoblot analysis. This analysis identified spectrin, filensin, vimentin, tubulin, phakinin, and beta-actin as major cytoskeletal proteins in the lens fibers. Importantly, ezrin, radixin, and moesin (ERM), heat-shock cognate protein 70, and beta/gamma-crystallins were identified as major cytoskeletal-associated proteins. ERM proteins were confirmed to exist as active phosphorylated forms that exhibited intense distribution in the organelle free-zone fibers. Furthermore, ERM protein phosphorylation was found to be dramatically reduced in Rho GTPase-targeted transgenic mouse lenses. These data identify the ERM proteins, which cross-link the plasma membrane and actin, as major and stable cytoskeletal-associated proteins in lens fibers, and indicate a potential role(s) for the ERMs in fiber cell actin cytoskeletal and membrane organization.

Figure 1

SDS-polyacrylamide gel electrophoretic separation and MALDI-TOF-TOF mass spectrometry-based identification of proteins in the triton insoluble fractions of mouse lens fibers. To identify the major proteins of the lens fiber cell triton insoluble fraction, tissues were processed as described in Methods and separated by SDS-PAGE, using gels containing 5.5, 8 and 12% acrylamide. Gels were stained with GelCode blue and distinctly separated protein bands excised, subjected to in-gel tryptic digestion and MALDI-TOF-TOF MS analysis. Representative photographs of the GelCode stained SDS-PAGE gels are shown. The MALDI-TOF-TOF based protein identity is indicated next to the corresponding protein band on the gels.

Figure 2

Immunological identification of the ERM proteins and Hsc-70 in lens fiber cell triton-insoluble fractions. For an independent confirmation of the MALDI-TOF-TOF MS-based protein identification results, immunoblot analysis was carried out to identify specific proteins using polyclonal antibodies. Different amounts of lens fiber cell triton insoluble protein fractions obtained from both mouse and porcine were immnoblotted for ERM (panel A) and Hsc-70 (panel B) proteins using phospho-threonine-specific anti- ERM and anti-Hsc 70 antibodies, respectively.

Figure 3

Distribution of phosphorylated ERM proteins in the neonatal mouse lens and in cultured mouse lens epithelial cells. A. To determine the distribution pattern of activated (phosphorylated) ERM proteins in the lens, P1 mouse lens cryosections (sagittal and equatorial planes) were immunostained with polyclonal antibody raised against phospho-specific ERM protein in conjunction with Alexa Fluor 488 conjugated secondary antibody. Immunofluorescence images were captured using confocal microscopy at 10x (upper panels) and 63X (lower panels) magnification. Arrows and arrowheads indicate staining at the lens fiber cell lateral membrane and lens sutures, respectively. Asterisks in upper panels depict the area that is shown at higher magnification in lower panels. Epi: Epithelium, CF: Cortical fibers and NF: Nuclear fibers. B. Distribution of phospho-ERMs in the cultured mouse lens epithelial cells. The cell apical region and cell surface protrusions including filopodia and microvilli exhibit specific localization of phospho-ERM proteins (red immunofluorescence staining).

Figure 3

Distribution of phosphorylated ERM proteins in the neonatal mouse lens and in cultured mouse lens epithelial cells. A. To determine the distribution pattern of activated (phosphorylated) ERM proteins in the lens, P1 mouse lens cryosections (sagittal and equatorial planes) were immunostained with polyclonal antibody raised against phospho-specific ERM protein in conjunction with Alexa Fluor 488 conjugated secondary antibody. Immunofluorescence images were captured using confocal microscopy at 10x (upper panels) and 63X (lower panels) magnification. Arrows and arrowheads indicate staining at the lens fiber cell lateral membrane and lens sutures, respectively. Asterisks in upper panels depict the area that is shown at higher magnification in lower panels. Epi: Epithelium, CF: Cortical fibers and NF: Nuclear fibers. B. Distribution of phospho-ERMs in the cultured mouse lens epithelial cells. The cell apical region and cell surface protrusions including filopodia and microvilli exhibit specific localization of phospho-ERM proteins (red immunofluorescence staining).

Figure 4

Phosphorylation status of ERMs in Rho GTPase targeted transgenic lenses. Transgenic mouse lenses (one day old) expressing either C3-exoenzyme, an inactivator of RhoA, B and C, or expressing Rho GDP dissociation inhibitor (Rho GDIα), a negative regulator of Rho, Rac and Cdc42, were analyzed for changes in ERM protein phosphorylation by immunoblot analysis. The water insoluble fractions (100,000×g pellets) derived from the pooled lenses of transgenic and wild type littermates, were dissolved in urea sample buffer and equal amounts of protein from both transgenic and wild type specimens were subjected to analysis. Actin in the 800×g supernatants of the lens homogenates was immunoblotted to confirm equality of protein loading.