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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: 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.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.

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

Expression of Matrin 3-YFP fusion proteins in CHO cells.Cells were transiently transfected with expression plasmids for Matrin 3-YFP fusion constructs (WT, S85C, F115C, P154S, and T622A). At 24 h post-transfection, the cells were fixed and the fluorescence was directly imaged. Nearly all of the Matrin 3-YFP fluorescence was localized to the nucleus. In a small minority of cells transfected with the S85C, we observed both nuclear and cytoplasmic localization. DAPI (blue) marks the nucleus. The exposure times for all images was identical.
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pone.0142144.g004: Expression of Matrin 3-YFP fusion proteins in CHO cells.Cells were transiently transfected with expression plasmids for Matrin 3-YFP fusion constructs (WT, S85C, F115C, P154S, and T622A). At 24 h post-transfection, the cells were fixed and the fluorescence was directly imaged. Nearly all of the Matrin 3-YFP fluorescence was localized to the nucleus. In a small minority of cells transfected with the S85C, we observed both nuclear and cytoplasmic localization. DAPI (blue) marks the nucleus. The exposure times for all images was identical.

Mentions: To confirm our immunostaining data, we generated a panel of constructs in which YFP was fused, in-frame, to the C-terminus of Matrin 3. As above, expression plasmids for these constructs were transiently-transfected into CHO cells, but in this case we directly imaged the YFP fusion protein. As we observed with untagged Matrin 3 expressed in CHO cells, most of the Matrin 3-YFP proteins showed nuclear localization, regardless of the mutation status (Fig 4). In a small percentage of cells, particularly cells expressing the S85C and F115C variants, there was visible fluorescence in the cytoplasm (Fig 4, arrow; Table 1). In cells in which cytoplasmic localization of Matrin 3-YFP was detected, the more intense signal was always seen in the nucleus (Fig 4).


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)

Expression of Matrin 3-YFP fusion proteins in CHO cells.Cells were transiently transfected with expression plasmids for Matrin 3-YFP fusion constructs (WT, S85C, F115C, P154S, and T622A). At 24 h post-transfection, the cells were fixed and the fluorescence was directly imaged. Nearly all of the Matrin 3-YFP fluorescence was localized to the nucleus. In a small minority of cells transfected with the S85C, we observed both nuclear and cytoplasmic localization. DAPI (blue) marks the nucleus. The exposure times for all images was identical.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142144.g004: Expression of Matrin 3-YFP fusion proteins in CHO cells.Cells were transiently transfected with expression plasmids for Matrin 3-YFP fusion constructs (WT, S85C, F115C, P154S, and T622A). At 24 h post-transfection, the cells were fixed and the fluorescence was directly imaged. Nearly all of the Matrin 3-YFP fluorescence was localized to the nucleus. In a small minority of cells transfected with the S85C, we observed both nuclear and cytoplasmic localization. DAPI (blue) marks the nucleus. The exposure times for all images was identical.
Mentions: To confirm our immunostaining data, we generated a panel of constructs in which YFP was fused, in-frame, to the C-terminus of Matrin 3. As above, expression plasmids for these constructs were transiently-transfected into CHO cells, but in this case we directly imaged the YFP fusion protein. As we observed with untagged Matrin 3 expressed in CHO cells, most of the Matrin 3-YFP proteins showed nuclear localization, regardless of the mutation status (Fig 4). In a small percentage of cells, particularly cells expressing the S85C and F115C variants, there was visible fluorescence in the cytoplasm (Fig 4, arrow; Table 1). In cells in which cytoplasmic localization of Matrin 3-YFP was detected, the more intense signal was always seen in the nucleus (Fig 4).

Bottom Line: 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.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.

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