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Development of subcellular fractionation techniques to determine the intracellular cobalamin transit in vitro and the impact of lysosomal dysfunction

Conference Paper


Abstract


  • 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.

Publication Date


  • 2013

Citation


  • Zhao, H., Ruberu, K. R., Li, H. Y. & Garner, B. (2013). Development of subcellular fractionation techniques to determine the intracellular cobalamin transit in vitro and the impact of lysosomal dysfunction. 33rd Meeting of the Australian Neuroscience Society: Program, Abstracts & List of Registrants (pp. 129-129). Australia: ANS.

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/311

Start Page


  • 129

End Page


  • 129

Place Of Publication


  • Australia

Abstract


  • 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.

Publication Date


  • 2013

Citation


  • Zhao, H., Ruberu, K. R., Li, H. Y. & Garner, B. (2013). Development of subcellular fractionation techniques to determine the intracellular cobalamin transit in vitro and the impact of lysosomal dysfunction. 33rd Meeting of the Australian Neuroscience Society: Program, Abstracts & List of Registrants (pp. 129-129). Australia: ANS.

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/311

Start Page


  • 129

End Page


  • 129

Place Of Publication


  • Australia