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Subcellular Localization of Matrin 3 Containing Mutations Associated with ALS and Distal Myopathy.

Gallego-Iradi MC, Clare AM, Brown HH, Janus C, Lewis J, Borchelt DR - PLoS ONE (2015)

Bottom Line: Using CHO and human H4 neuroglioma cell models, we find that ALS/myopathy mutations do not produce profound changes in the localization of the protein.Notably, over-expression of mutant Matrin 3 did not induce the formation of obvious large inclusion-like structures in either the cytoplasm or nucleus.Our findings indicate that mutations in Matrin 3 that are associated with ALS and myopathy do not dramatically alter the normal localization of the protein or readily induce inclusion formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, McKnight Brain Institute, University of Florida, Gainesville, Florida, United States of America; Santa Fe HealthCare Alzheimer's Disease Research Center, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT

Background: Mutations in Matrin 3 [MATR3], an RNA- and DNA-binding protein normally localized to the nucleus, have been linked to amyotrophic lateral sclerosis (ALS) and distal myopathies. In the present study, we have used transient transfection of cultured cell lines to examine the impact of different disease-causing mutations on the localization of Matrin 3 within cells.

Results: Using CHO and human H4 neuroglioma cell models, we find that ALS/myopathy mutations do not produce profound changes in the localization of the protein. Although we did observe variable levels of Matrin 3 in the cytoplasm either by immunostaining or visualization of fluorescently-tagged protein, the majority of cells expressing either wild-type (WT) or mutant Matrin 3 showed nuclear localization of the protein. When cytoplasmic immunostaining, or fusion protein fluorescence, was seen in the cytoplasm, the stronger intensity of staining or fluorescence was usually evident in the nucleus. In ~80% of cells treated with sodium arsenite (Ars) to induce cytoplasmic stress granules, the nuclear localization of WT and F115C mutant Matrin 3 was not disturbed. Notably, over-expression of mutant Matrin 3 did not induce the formation of obvious large inclusion-like structures in either the cytoplasm or nucleus.

Conclusions: Our findings indicate that mutations in Matrin 3 that are associated with ALS and myopathy do not dramatically alter the normal localization of the protein or readily induce inclusion formation.

No MeSH data available.


Related in: MedlinePlus

Matrin 3 is not a major component of stress granules.H4 cells were transiently co-transfected with expression plasmids for G3BP1-mCherry (red) and Matrin 3-YFP (WT and F115C). At 24 h post-transfection, one set of cells were treated with sodium arsenite [100μM] for 45 minutes to induce stress granules, which were visualized by the localization of G3BP1-mCherry. WT and F115C Matrin 3 were found to be localized to both the nucleus and cytoplasm when stress granules are present. Untreated H4 cells (-) co-expressing G3BP1-mCherry and Matrin 3-YFP also show both nuclear and cytoplasmic localization. DAPI marks the nucleus (blue). The exposure times for all images in each channel were identical; all of the images in the red channel were exposed for a shorter period than the images in the green channel.
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pone.0142144.g005: Matrin 3 is not a major component of stress granules.H4 cells were transiently co-transfected with expression plasmids for G3BP1-mCherry (red) and Matrin 3-YFP (WT and F115C). At 24 h post-transfection, one set of cells were treated with sodium arsenite [100μM] for 45 minutes to induce stress granules, which were visualized by the localization of G3BP1-mCherry. WT and F115C Matrin 3 were found to be localized to both the nucleus and cytoplasm when stress granules are present. Untreated H4 cells (-) co-expressing G3BP1-mCherry and Matrin 3-YFP also show both nuclear and cytoplasmic localization. DAPI marks the nucleus (blue). The exposure times for all images in each channel were identical; all of the images in the red channel were exposed for a shorter period than the images in the green channel.

Mentions: To further investigate whether Matrin 3 may associate with stress granules, we use Ars to induce stress granule formation in cells that has been co-transfected with G3BP1-mCherry and Matrin 3-YFP. After several pilot studies (not shown), we chose conditions in which the ratio of plasmid DNA for Matrin 3-YFP to G3BP1-mCherry was set a 3 to 1, and the cells were exposed to 100 μM Ars for 45 minutes at 37°C. As was the case in the experiments described above, in all cells, the most intense fluorescence for Matrin 3-YFP was localized to the nucleus, under all conditions (Fig 5). In cells expressing the WT-Matrin 3-YFP construct that were exposed to Ars, we were able to identify clear examples in which the Matrin 3-YFP protein remained restricted to the nucleus (Fig 5). Conversely, in cells that were not exposed to Ars, but co-expressing the two fusion proteins, we occasionally observed cells in which the Matrin 3-YFP appeared to be distributed as cytoplasmic puncta (Fig 5). In cells expressing the F115C-Matrin 3-YFP mutant, we similarly observed Ars-treated cells in which mutant Matrin 3 was completely localized to the nucleus (Fig 5). There were rare examples of cells that displayed cytoplasmic F115C-Matrin 3 in structures that resembled the structures marked by G3BP1-mCherry (Fig 5). However, a pattern of similar distribution of mutant Matrin could also be found in a subset of cells that had not been exposed to Ars and had no obvious G3BP1-mCherry stress granules (Fig 5). This latter finding confirmed our observations above when untagged F115C-Matin 3 was co-expressed with G3BP1-mCherry. We conclude that co-expression of G3BP1-mCherry with Matrin 3 induces changes in Matrin 3 localization in a subset of cells, with the pattern of cytoplasmic Matrin localization resembling stress granules. Table 2 shows the results of quantification of data from multiple transfections with WT and F115C-Matrin 3 after treatment with Ars, indicating that mutant F115C-Matrin 3 is not more prone to be localized to these structures than WT protein. Based on the data in hand, we conclude that both WT and mutant Matrin 3 can form cytoplasmic structures that resemble stress granules, but there is no clear pattern of localization to indicate that the protein is actively involved in stress granule formation.


Subcellular Localization of Matrin 3 Containing Mutations Associated with ALS and Distal Myopathy.

Gallego-Iradi MC, Clare AM, Brown HH, Janus C, Lewis J, Borchelt DR - PLoS ONE (2015)

Matrin 3 is not a major component of stress granules.H4 cells were transiently co-transfected with expression plasmids for G3BP1-mCherry (red) and Matrin 3-YFP (WT and F115C). At 24 h post-transfection, one set of cells were treated with sodium arsenite [100μM] for 45 minutes to induce stress granules, which were visualized by the localization of G3BP1-mCherry. WT and F115C Matrin 3 were found to be localized to both the nucleus and cytoplasm when stress granules are present. Untreated H4 cells (-) co-expressing G3BP1-mCherry and Matrin 3-YFP also show both nuclear and cytoplasmic localization. DAPI marks the nucleus (blue). The exposure times for all images in each channel were identical; all of the images in the red channel were exposed for a shorter period than the images in the green channel.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4631352&req=5

pone.0142144.g005: Matrin 3 is not a major component of stress granules.H4 cells were transiently co-transfected with expression plasmids for G3BP1-mCherry (red) and Matrin 3-YFP (WT and F115C). At 24 h post-transfection, one set of cells were treated with sodium arsenite [100μM] for 45 minutes to induce stress granules, which were visualized by the localization of G3BP1-mCherry. WT and F115C Matrin 3 were found to be localized to both the nucleus and cytoplasm when stress granules are present. Untreated H4 cells (-) co-expressing G3BP1-mCherry and Matrin 3-YFP also show both nuclear and cytoplasmic localization. DAPI marks the nucleus (blue). The exposure times for all images in each channel were identical; all of the images in the red channel were exposed for a shorter period than the images in the green channel.
Mentions: To further investigate whether Matrin 3 may associate with stress granules, we use Ars to induce stress granule formation in cells that has been co-transfected with G3BP1-mCherry and Matrin 3-YFP. After several pilot studies (not shown), we chose conditions in which the ratio of plasmid DNA for Matrin 3-YFP to G3BP1-mCherry was set a 3 to 1, and the cells were exposed to 100 μM Ars for 45 minutes at 37°C. As was the case in the experiments described above, in all cells, the most intense fluorescence for Matrin 3-YFP was localized to the nucleus, under all conditions (Fig 5). In cells expressing the WT-Matrin 3-YFP construct that were exposed to Ars, we were able to identify clear examples in which the Matrin 3-YFP protein remained restricted to the nucleus (Fig 5). Conversely, in cells that were not exposed to Ars, but co-expressing the two fusion proteins, we occasionally observed cells in which the Matrin 3-YFP appeared to be distributed as cytoplasmic puncta (Fig 5). In cells expressing the F115C-Matrin 3-YFP mutant, we similarly observed Ars-treated cells in which mutant Matrin 3 was completely localized to the nucleus (Fig 5). There were rare examples of cells that displayed cytoplasmic F115C-Matrin 3 in structures that resembled the structures marked by G3BP1-mCherry (Fig 5). However, a pattern of similar distribution of mutant Matrin could also be found in a subset of cells that had not been exposed to Ars and had no obvious G3BP1-mCherry stress granules (Fig 5). This latter finding confirmed our observations above when untagged F115C-Matin 3 was co-expressed with G3BP1-mCherry. We conclude that co-expression of G3BP1-mCherry with Matrin 3 induces changes in Matrin 3 localization in a subset of cells, with the pattern of cytoplasmic Matrin localization resembling stress granules. Table 2 shows the results of quantification of data from multiple transfections with WT and F115C-Matrin 3 after treatment with Ars, indicating that mutant F115C-Matrin 3 is not more prone to be localized to these structures than WT protein. Based on the data in hand, we conclude that both WT and mutant Matrin 3 can form cytoplasmic structures that resemble stress granules, but there is no clear pattern of localization to indicate that the protein is actively involved in stress granule formation.

Bottom Line: Using CHO and human H4 neuroglioma cell models, we find that ALS/myopathy mutations do not produce profound changes in the localization of the protein.Notably, over-expression of mutant Matrin 3 did not induce the formation of obvious large inclusion-like structures in either the cytoplasm or nucleus.Our findings indicate that mutations in Matrin 3 that are associated with ALS and myopathy do not dramatically alter the normal localization of the protein or readily induce inclusion formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, McKnight Brain Institute, University of Florida, Gainesville, Florida, United States of America; Santa Fe HealthCare Alzheimer's Disease Research Center, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT

Background: Mutations in Matrin 3 [MATR3], an RNA- and DNA-binding protein normally localized to the nucleus, have been linked to amyotrophic lateral sclerosis (ALS) and distal myopathies. In the present study, we have used transient transfection of cultured cell lines to examine the impact of different disease-causing mutations on the localization of Matrin 3 within cells.

Results: Using CHO and human H4 neuroglioma cell models, we find that ALS/myopathy mutations do not produce profound changes in the localization of the protein. Although we did observe variable levels of Matrin 3 in the cytoplasm either by immunostaining or visualization of fluorescently-tagged protein, the majority of cells expressing either wild-type (WT) or mutant Matrin 3 showed nuclear localization of the protein. When cytoplasmic immunostaining, or fusion protein fluorescence, was seen in the cytoplasm, the stronger intensity of staining or fluorescence was usually evident in the nucleus. In ~80% of cells treated with sodium arsenite (Ars) to induce cytoplasmic stress granules, the nuclear localization of WT and F115C mutant Matrin 3 was not disturbed. Notably, over-expression of mutant Matrin 3 did not induce the formation of obvious large inclusion-like structures in either the cytoplasm or nucleus.

Conclusions: Our findings indicate that mutations in Matrin 3 that are associated with ALS and myopathy do not dramatically alter the normal localization of the protein or readily induce inclusion formation.

No MeSH data available.


Related in: MedlinePlus