Protein Analysis and Molecular Imaging of Concentric Shells of the Lens

Abstract

Purpose: : "Zones of discontinuity" describe the concentric shells of differentiating fiber cells in lenses observed using a slit lamp (Koretz et al. 1994.Vision Res 34:2955). Our interest is in determining the molecular basis for the concentric shells resulting from normal development and growth in transparent mouse lenses.

Methods: : C57BL/6 mouse and ICR/f rat lenses were frozen at liquid nitrogen temperatures and cryosectioned. Sections for fluorescence cytochemistry were then fixed with 0.75% paraformaldehyde and stained with selected protein stains or antibodies to structural lens proteins and crystallins. Separate frozen sections were placed on conductive carbon tape and robotically spotted with matrix for imaging by MALDI mass spectrometry. Spatial resolution achieved was 200µm.

Results: : Each antibody or protein label stained a specific pattern of concentric shells in mouse lenses. The αB crystallin was observed in a deep cortical shell and shells labeled for γ crystallin were observed throughout the lens. The pattern of shells labeled for N-cadherin, beta catenin and actin were similar. Shells for the intermediate filaments CP49 and filensin were similar, as expected.A new sample preparation permitted analysis of concentric shells of alpha crystallins using MALDI MS imaging. Shells of truncated crystallins were observed in older fiber cell layers in both mouse and rat lenses. Peripheral shells contained large molecular weight truncation products. Smaller molecular weight truncation products (e.g. due to loss of more than 25 C-terminal residues of αB crystallin) were found centrally.

Conclusions: : Previously used for larger animal lenses, novel MALDI-TOF mass spectrometric imaging can characterize protein distributions in rat and mouse lenses having radial dimensions of only 4.8mm and 2mm. MS imaging and immunocytochemistry confirmed the presence of concentric shells in mouse lenses and demonstrated the feasibility for use of MS imaging for high resolution cellular proteomics during development and maintenance of lens transparency.

Keywords: crystallins • proteomics • differentiation