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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

Endogenous immunostaining for Matrin 3 in untransfected cell lines.Human brain neuroglioma H4, Human Embryonic Kidney (HEK293), Chinese Hamster Ovary (CHO), Mouse Neuroblastoma (N2a), and Mouse embryonic fibroblast (3T3) were stained with α-Matrin primary antibody (ab 151714, Abcam) 1:200 overnight (Matrin 3 in red and DAPI in blue). The exposure time was set by the intensity of signal in the H4 cells. All other images were exposed to the same length of time to provide an estimation of immunostaining intensity.
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pone.0142144.g001: Endogenous immunostaining for Matrin 3 in untransfected cell lines.Human brain neuroglioma H4, Human Embryonic Kidney (HEK293), Chinese Hamster Ovary (CHO), Mouse Neuroblastoma (N2a), and Mouse embryonic fibroblast (3T3) were stained with α-Matrin primary antibody (ab 151714, Abcam) 1:200 overnight (Matrin 3 in red and DAPI in blue). The exposure time was set by the intensity of signal in the H4 cells. All other images were exposed to the same length of time to provide an estimation of immunostaining intensity.

Mentions: Matrin 3 is a highly conserved protein, and to our knowledge there are no antibodies that can distinguish the human protein from Matrin 3 expressed by any other species. Thus, we initially screened various human and non-human cell lines to identify those with the lowest endogenous expression of Matrin 3 protein, allowing easy detection of any expressed human Matrin 3. Five cell lines commonly used in transfection studies were examined, including human H4 neuroglioma, HEK293 cells, Chinese Hamster Ovary (CHO) cells, mouse neuroblastoma N2a cells, and mouse 3T3 fibroblast cells. The weakest immunostaining with Matrin 3 antibodies was observed in CHO cells (Fig 1). By contrast, human H4 cells showed the strongest staining. In all cell lines examined, all Matrin 3 immunoreactivity was confined to the nucleus.


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)

Endogenous immunostaining for Matrin 3 in untransfected cell lines.Human brain neuroglioma H4, Human Embryonic Kidney (HEK293), Chinese Hamster Ovary (CHO), Mouse Neuroblastoma (N2a), and Mouse embryonic fibroblast (3T3) were stained with α-Matrin primary antibody (ab 151714, Abcam) 1:200 overnight (Matrin 3 in red and DAPI in blue). The exposure time was set by the intensity of signal in the H4 cells. All other images were exposed to the same length of time to provide an estimation of immunostaining intensity.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142144.g001: Endogenous immunostaining for Matrin 3 in untransfected cell lines.Human brain neuroglioma H4, Human Embryonic Kidney (HEK293), Chinese Hamster Ovary (CHO), Mouse Neuroblastoma (N2a), and Mouse embryonic fibroblast (3T3) were stained with α-Matrin primary antibody (ab 151714, Abcam) 1:200 overnight (Matrin 3 in red and DAPI in blue). The exposure time was set by the intensity of signal in the H4 cells. All other images were exposed to the same length of time to provide an estimation of immunostaining intensity.
Mentions: Matrin 3 is a highly conserved protein, and to our knowledge there are no antibodies that can distinguish the human protein from Matrin 3 expressed by any other species. Thus, we initially screened various human and non-human cell lines to identify those with the lowest endogenous expression of Matrin 3 protein, allowing easy detection of any expressed human Matrin 3. Five cell lines commonly used in transfection studies were examined, including human H4 neuroglioma, HEK293 cells, Chinese Hamster Ovary (CHO) cells, mouse neuroblastoma N2a cells, and mouse 3T3 fibroblast cells. The weakest immunostaining with Matrin 3 antibodies was observed in CHO cells (Fig 1). By contrast, human H4 cells showed the strongest staining. In all cell lines examined, all Matrin 3 immunoreactivity was confined to the nucleus.

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