Abstract
Purpose:
Progressive destabilization and unfolding of proteins that self-assemble to form fibrils or aggregates are a hallmark of cellular degeneration during aging. Characterization of the mechanisms of cellular degeneration in vivo is limited by the difficulties of studying the dynamics of molecular aging and degeneration in animal models. The purpose of the current study is to express yellow fluorescent protein (YFP) amyloid fusion proteins in the transparent lens of the eye to follow the dynamics of their cellular distribution and progressive aggregation with age.
Methods:
Human cDNAs for alphaB crystallin (αB), betaB crystallin (βB) and amyloid beta (Aβ) (generously provided by J. Sullivan) fusion proteins were cloned into Invitrogen Gateway Tol2 destination vectors downstream of the αA crystallin promoter (generously provided by B. Link), in frame with the yellow fluorescent protein (YFP). The αA crystallin promoter drives vector constructs of human YFP fusion proteins specifically in zebrafish lens cells (Kurita et al. (2003) Dev Biol 255:113). Control and experimental vectors were injected into fertilized wild-type ABWIK zebrafish embryos at the one- or two-cell stage. At four to six days post-fertilization (dpf), the lenses of developing zebrafish were imaged in 3D using fluorescence or multiphoton microscopy.
Results:
YFP, αB-YFP and βB-YFP were observed in a pattern of alternating bright and dark layers throughout the lens. In contrast, amyloid Aβ-YFP was found primarily in cortical cells. Aβ-YFP was observed in bands of cortical fiber cells as bright condensed fluorescent aggregates. The cellular distribution was asymmetric and Aβ-YFP was not observed in every cell. Aβ-YFP was found in peripheral cells only and core cells were either protected against aggregate formation or did not express Aβ. The patterns of Aβ fluorescence changed with age.
Conclusions:
YFP-fusion proteins were successful for the expression of human proteins in the zebrafish lens in vivo using the αA crystallin promoter. The patterns of gene expression varied with the selected protein and with age. Supported by EY04542 from the NEI.
Keywords: 488 crystallins •
533 gene/expression •
660 proteins encoded by disease genes