Abstract
BackgroundThe nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. The lamina is involved in nuclear structure, gene expression, and association of the cytoplasmic cytoskeleton with the nucleus. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Because mutations in lamina proteins have been linked to several human diseases affecting skeletal muscle, we examined NET expression during differentiation of C2C12 myoblasts. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation.ResultsUsing transcriptional microarray analysis, we found that expression of 6 of the NETs significantly increases during myoblast differentiation. We confirmed these results using quantitative RT-PCR, and furthermore, found that all 6 NETs are expressed at high levels in adult mouse skeletal muscle relative to 9 other tissues examined. Using epitope-tagged cDNAs, we determined that the 5 NETs we could analyze (NETs 9, 25, 32, 37 and 39) all target to the nuclear envelope in C2C12 cells. Furthermore, the 3 NETs that we could analyze by immunoblotting were highly enriched in nuclear envelopes relative to microsomal membranes purified from mouse liver. Database searches showed that 4 of the 6 up-regulated NETs contain regions of homology to proteins previously linked to signaling.ConclusionThis work identified 6 NETs that are predicted to have important functions in muscle development and/or maintenance from their expression patterns during myoblast differentiation and in mouse tissues. We confirmed that 5 of these NETs are authentic nuclear envelope proteins. Four members of this group have potential signaling functions at the NE, based on their sequence homologies.
Highlights
The nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane
Changes in nuclear envelope transmembrane proteins (NETs) expression during C2C12 differentiation We have examined the expression of the novel putative/ confirmed NETs in mouse C2C12 cells during differentiation, as a means of identifying proteins with a potential role in muscle development
C2C12 cells are an immortalized myoblast line derived from a stem cell ("satellite cell") of adult skeletal muscle involved in muscle regeneration, which expresses the myogenic specification markers MyoD and Myf5
Summary
The nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation. The nuclear envelope (NE), which forms the boundary of the nucleus in eukaryotic cells, compartmentalizes nuclear metabolism and helps to organize nuclear structure INM is lined by the nuclear lamina, a meshwork containing a polymer of the lamin intermediate filament proteins as well as other more minor polypeptides, including transmembrane proteins concentrated at the INM Lamins B1 and B2 are expressed in most somatic cells throughout development, whereas lamins A/C usually are expressed only at or following differentiation
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