Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations

Gavin S Wilkie,Nadia Korfali,Selene K Swanson,Poonam Malik,Vlastimil Srsen,Dzmitry G Batrakou,Jose De Las Heras,Nikolaj Zuleger,Alastair R.W Kerr,Laurence Florens,Eric C Schirmer

doi:10.1074/mcp.m110.003129

Abstract

Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights.

Highlights

The nuclear envelope (NE)1 is an impenetrable membrane barrier between the nucleus and the cytoplasm perforated by
The three favored molecular mechanisms to explain NE disease pathology are mechanical instability from disruption of Lamin-cytoskeleton interactions, altered expression of genes regulated from the nuclear periphery, and disabling of the cell cycle/stem cell maintenance [6, 7]
To further purify NEs prior to mass spectrometry analysis, NE fractions were extracted with 1% ␤-octyl glucoside and 400 mM NaCl or with 0.1 N NaOH (Fig. 1A), which enrich for proteins associated with the insoluble intermediate filament Lamin polymer and integral membrane proteins, respectively

Summary

Muscle Nuclear Envelope Proteome

ONM proteins [15], which in turn connect the NE to the cytoskeleton [16, 17]. Mutations in the LMNA gene encoding Lamins A and C cause forms of Emery-Dreifuss muscular dystrophy (EDMD) [18, 19], limb-girdle muscular dystrophy (LGMD-1B) [20], and dilated cardiomyopathy with conduction defect (CMD1A) [21], which each affect different muscle groups, all are often associated with cardiac conduction defects. Nesprins have been shown to connect to cytoskeletal proteins [16, 17, 24, 25] Both Lamins and NETs involved in connecting Lamins to the cytoskeleton can cause muscle disease in humans. Postulating that other, more muscle-specific proteins might contribute to NE-cytoskeleton interactions, we sought to determine whether additional NETs could be found in the NE proteome of skeletal muscle. TMEM214 and the NET WFS1 have a unique distribution at the base of the spindle poles in mitosis similar to that recently reported for NET5/TMEM201/Samp1 [30] Some of these newly identified muscle NETs could potentially contribute to cytoskeletal defects in NE muscle diseases

EXPERIMENTAL PROCEDURES

Spectral FDR ϭ

RESULTS

Mm liver

DISCUSSION