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

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.

Fig 1. 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.

Fig 2. Overexpression of WT and mutant Matrin 3 in CHO cells.

Cells were transiently transfected with expression plasmids for WT Matrin 3 and 4 ALS/myopathy variants; S85C, F115C, P154S, T622A. After 24 hours the cells were fixed and immunostained α-Matrin primary antibody (same as Fig 1). Most of the Matrin 3 immunoreactivity was located in the nucleus (red) but a few cells show cytoplasmic staining, particularly for the S85C and F115C variants. DAPI (blue) marks the nucleus. The exposure times for all images was identical.

Fig 3. Co-expression of G3BP1-mCherry with Matrin3 in H4 cells.

Cells were co-transfected with G3BP1-mCherry (RED) and WT or F115C Matrin3 and treated with arsenite (Ars) to induce stress granule formation at 24 h post-transfection. After 45 min exposure to Ars, the cells were fixed and immunostained with Matrin3 antibodies. There was no obvious co-localization of Matrin3 immunoreactivity with G3BP1-labeled stress granules. In cells that were not treated with ARS, we observed rarely cells that had Matrin3 immunoreactivity in the cytoplasm in the absence of stress granules. DAPI (blue) shows nuclear cell staining.

Fig 4. 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.

Fig 5. 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.