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Protein Distribution during Human Erythroblast Enucleation In Vitro

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorti...

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Autores principales: Bell, Amanda J., Satchwell, Timothy J., Heesom, Kate J., Hawley, Bethan R., Kupzig, Sabine, Hazell, Matthew, Mushens, Rosey, Herman, Andrew, Toye, Ashley M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614867/
https://www.ncbi.nlm.nih.gov/pubmed/23565219
http://dx.doi.org/10.1371/journal.pone.0060300
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author Bell, Amanda J.
Satchwell, Timothy J.
Heesom, Kate J.
Hawley, Bethan R.
Kupzig, Sabine
Hazell, Matthew
Mushens, Rosey
Herman, Andrew
Toye, Ashley M.
author_facet Bell, Amanda J.
Satchwell, Timothy J.
Heesom, Kate J.
Hawley, Bethan R.
Kupzig, Sabine
Hazell, Matthew
Mushens, Rosey
Herman, Andrew
Toye, Ashley M.
author_sort Bell, Amanda J.
collection PubMed
description Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes.
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spelling pubmed-36148672013-04-05 Protein Distribution during Human Erythroblast Enucleation In Vitro Bell, Amanda J. Satchwell, Timothy J. Heesom, Kate J. Hawley, Bethan R. Kupzig, Sabine Hazell, Matthew Mushens, Rosey Herman, Andrew Toye, Ashley M. PLoS One Research Article Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes. Public Library of Science 2013-04-02 /pmc/articles/PMC3614867/ /pubmed/23565219 http://dx.doi.org/10.1371/journal.pone.0060300 Text en © 2013 Bell et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Bell, Amanda J.
Satchwell, Timothy J.
Heesom, Kate J.
Hawley, Bethan R.
Kupzig, Sabine
Hazell, Matthew
Mushens, Rosey
Herman, Andrew
Toye, Ashley M.
Protein Distribution during Human Erythroblast Enucleation In Vitro
title Protein Distribution during Human Erythroblast Enucleation In Vitro
title_full Protein Distribution during Human Erythroblast Enucleation In Vitro
title_fullStr Protein Distribution during Human Erythroblast Enucleation In Vitro
title_full_unstemmed Protein Distribution during Human Erythroblast Enucleation In Vitro
title_short Protein Distribution during Human Erythroblast Enucleation In Vitro
title_sort protein distribution during human erythroblast enucleation in vitro
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614867/
https://www.ncbi.nlm.nih.gov/pubmed/23565219
http://dx.doi.org/10.1371/journal.pone.0060300
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