Development of subcellular fractionation techniques to determine the intracellular cobalamin transit in vitro and the impact of lysosomal dysfunction
Intracellular cobalamin (Cbl) utilization is critically dependent on its efficient transit through the lysosome and subsequent delivery to cytosol and mitochondria. We propose that age-related pathological processes may inhibit lysosomal function and impair intracellular Cbl transport. Purpose: To investigate alterations of intracellular [57Co]Cbl trafficking in subcellular organelles when lysosome function is interrupted it is essential to develop an optimal subcellular fractionation method to isolate pure lysosomes, mitochondria and cytosol. Methods: Approximately 100 million human SH-SY5Y neurons or HT1080 fibroblasts were labelled with [57Co]Cbl, homogenised using a ball-bearing homogeniser and the lysates fractionated using an Optiprep gradient and reagent kits from either Pierce or Sigma. [57Co] in each fraction was measured using a gamma counter and subcellular fractions were probed by western blotting. Results: Both protocols separated subcellular organelles to a certain extent. The Pierce method seemed to be superior, separating pure lysosomes from mitochondrial fractions without cytosol contamination. SH-SY5Y [57Co]Cbl lysosome / mitochondria / cytosol distribution was 5.2 + 0.4 / 13.2 + 0.6 / 81.6 + 0.8, respectively, and this was changed (all p < 0.01) to 54.2 + 1.9 / 7.0 + 0.9 / 35.5 + 2.7, respectively (all mean + SE, n = 3), when cells were treated for 48 h with 25 μM chloroquine (to increase lysosomal pH). Similar results were obtained using HT1080 fibroblasts. Conclusion: Development of subcellular fractionation methods provides a useful tool for investigating intracellular Cbl trafficking. This method can be adapted to study the impact of age- or pathology-related lysosomal dysfunction on intracellular [57Co]Cbl transport.
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