Cultured Skin Fibroblasts Derived from Patients with Mucolipidosis 4 Are Auto-Fluorescent

Abstract

ABSTRACT: Mucolipidosis 4 (ML4) is an autosomal recessive disorder with both lipid and mucopolysaccharide storage. The disease is characterized by severe visual impairment and psychomotor retardation. In our effort to find a phenotypic marker for ML4 fibroblasts, living cells were stained with fluorescent compounds. The staining pattern in cells was complicated by auto-fluorescence. A careful study revealed that auto-fluorescence by itself was a sufficient marker for viable ML4 fibroblasts. ML4 cells in cultures obtained from four unrelated patients contain auto-fluorescent material. Auto-fluorescence was noted over a wide range of excitation wavelengths from ∼365 to ∼546 nm. The most intense fluorescence was observed in the lower wave-length range. Cultured fibroblasts from normal individuals or obligate ML4 heterozygotes did not fluoresce under adequately controlled culture conditions. High passage number or inadequate feeding caused a small proportion of fibroblasts obtained from normal individuals to auto-fluoresce. The auto-fluorescent material co-localized with phase-dense inclusion bodies, shown to be lysosomes by staining with LAMP-ab. These findings imply that fluorescence may relate to the specific compound(s) stored in the lysosomes. In a comparative study, neuronal ceroid lipofuscinosis fibroblasts were also fluorescent. Fibroblasts from other diseases such as Gaucher disease and glycogenosis type 2 did not show any fluorescence. These findings are currently used in our functional cloning strategy for determining the gene involved in MLA.

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