Cargando…

In Vivo Assembly of a Dictyostelium Lamin Mutant Induced by Light, Mechanical Stress, and pH

We expressed Dictyostelium lamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in lat...

Descripción completa

Detalles Bibliográficos
Autores principales: Grafe, Marianne, Hofmann, Phillip, Batsios, Petros, Meyer, Irene, Gräf, Ralph
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464662/
https://www.ncbi.nlm.nih.gov/pubmed/32759812
http://dx.doi.org/10.3390/cells9081834
_version_ 1783577415042203648
author Grafe, Marianne
Hofmann, Phillip
Batsios, Petros
Meyer, Irene
Gräf, Ralph
author_facet Grafe, Marianne
Hofmann, Phillip
Batsios, Petros
Meyer, Irene
Gräf, Ralph
author_sort Grafe, Marianne
collection PubMed
description We expressed Dictyostelium lamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-ΔNLSΔCLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of the Dictyostelium lamin, they are likely relevant also for wild-type lamin.
format Online
Article
Text
id pubmed-7464662
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-74646622020-09-04 In Vivo Assembly of a Dictyostelium Lamin Mutant Induced by Light, Mechanical Stress, and pH Grafe, Marianne Hofmann, Phillip Batsios, Petros Meyer, Irene Gräf, Ralph Cells Article We expressed Dictyostelium lamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-ΔNLSΔCLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of the Dictyostelium lamin, they are likely relevant also for wild-type lamin. MDPI 2020-08-04 /pmc/articles/PMC7464662/ /pubmed/32759812 http://dx.doi.org/10.3390/cells9081834 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Grafe, Marianne
Hofmann, Phillip
Batsios, Petros
Meyer, Irene
Gräf, Ralph
In Vivo Assembly of a Dictyostelium Lamin Mutant Induced by Light, Mechanical Stress, and pH
title In Vivo Assembly of a Dictyostelium Lamin Mutant Induced by Light, Mechanical Stress, and pH
title_full In Vivo Assembly of a Dictyostelium Lamin Mutant Induced by Light, Mechanical Stress, and pH
title_fullStr In Vivo Assembly of a Dictyostelium Lamin Mutant Induced by Light, Mechanical Stress, and pH
title_full_unstemmed In Vivo Assembly of a Dictyostelium Lamin Mutant Induced by Light, Mechanical Stress, and pH
title_short In Vivo Assembly of a Dictyostelium Lamin Mutant Induced by Light, Mechanical Stress, and pH
title_sort in vivo assembly of a dictyostelium lamin mutant induced by light, mechanical stress, and ph
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464662/
https://www.ncbi.nlm.nih.gov/pubmed/32759812
http://dx.doi.org/10.3390/cells9081834
work_keys_str_mv AT grafemarianne invivoassemblyofadictyosteliumlaminmutantinducedbylightmechanicalstressandph
AT hofmannphillip invivoassemblyofadictyosteliumlaminmutantinducedbylightmechanicalstressandph
AT batsiospetros invivoassemblyofadictyosteliumlaminmutantinducedbylightmechanicalstressandph
AT meyerirene invivoassemblyofadictyosteliumlaminmutantinducedbylightmechanicalstressandph
AT grafralph invivoassemblyofadictyosteliumlaminmutantinducedbylightmechanicalstressandph