PMC

Fraction purity. A, Coomassie-stained gel of NE and microsome/SR muscle fractions analyzed and also of a separately isolated muscle mitochondrial fraction (Mito). B, the SR marker Calnexin was present in both the SR and NE fractions because the ONM is continuous with the SR and shares many proteins. In contrast, the SR fraction was completely free of NE-specific markers, the NET LAP2β and Lamins A/C. Loading was the same as for A with similar amounts of total protein loaded. C, the mitochondrial marker Porin was absent from NEs, whereas Lamins A/C were absent from mitochondria. Loading was the same as in A and B.

NE composition changes during muscle differentiation. Expression levels of several muscle NETs were tested in both C2C12 (mouse) and RD (human) myoblast to myotube differentiation systems. RNA extracted from both untreated myoblast and chemically differentiated myotube populations were subjected to RT-PCR for each NET, and expression levels were quantified. The relative change between myoblast and myotube populations was determined after first normalizing values to the control peptidylprolyl isomerase A (PPIA). Relative transcript levels for both human and mouse systems are shown, and error bars indicate standard deviations between three replicates for each differentiation system. Four NETs were induced similarly to positive differentiation controls, MYOG and MYH1.

Muscle NET alignment with microtubules. U2OS cells expressing various NET-GFP fusions were fixed and stained for microtubules. Sections from deconvolved images are shown for a focal plane at the upper nuclear surface. DAPI staining for DNA is in blue, microtubules are in green, and the NET is in red. In the right columns, the microtubules or NET are shown in grayscale. Microtubule filaments are observed crossing the nuclear surface in the SUN2 control-transfected cell, but the SUN2 staining on the surface is spotty, consistent with previous reports. The same was observed for RHBDD1 and most muscle NETs tested; however, KLHL31, which had a more filamentous appearance in , exhibited filaments also on the nuclear surface that partly tracked with the microtubule filaments (note both grayscale images for each and some yellow co-localization in the merge panel). TMEM214, although consistently yielding a much crisper and continuous rim at the nuclear periphery, also exhibited filamentous accumulation at the nuclear surface that exhibited even more overlap with microtubules. Scale bars, 10 μm.

Partial NET-microtubule associations are also observed in mitosis. In mitosis, the NE breaks down, and several NPC proteins and Lamins have previously been reported to partly assemble on the mitotic spindle. The negative control SUN2 and the new muscle-specific NET RHBDD1 were generally distributed throughout the mitotic cell but excluded from the spindle (white arrowheads point to one of the spindles in each cell). No particular accumulation or change in the NET distribution was observed with respect to the mitotic spindle. As a positive control, TMEM201 (NET5/Samp1) is shown as it has previously been shown to associate with the spindle. TMEM214 exhibited in most cells a strong accumulation at the base of the spindles close to the poles but was excluded from the poles themselves. In other cells, the distribution was even more focused on the spindle itself with significant co-localization with the microtubules. A third muscle NET, WFS1, consistently yielded the first phenotype of TMEM214; however, in many cases, an even more pronounced accumulation surrounding the spindle poles was observed. One spindle pole in each cell (except the prophase TMEM214 cell) is marked by an arrowhead. Scale bar, 10 μm.

Confirmation of NE residence for NETs by targeting tagged fusion proteins. A, NETs were tested as tagged fusions for NE targeting, which is determined by their enrichment in a rim at the limits of DNA staining. NET fusions are visualized in white in the left panels and in red in the right panels, whereas DNA is visualized in white in the right panels. NETs were sometimes expressed or targeted better in certain cell types: all panels shown are U2OS osteosarcoma cells except for TMEM38A in mouse C2C12 cells and TMEM70 and CKAP4 in HeLa cells. All micrographs are on the same scale except for CKAP4 with all scale bars at 10 μm. B and C, inner versus outer nuclear membrane targeting. Structured illumination microscopy can distinguish proteins in the INM from those in the ONM when co-stained with nuclear basket protein Nup153 and cytoplasmic filament protein Nup358. B, an ONM NET or ER/ONM protein (red) should be in the same plane with Nup358 (green) but should be external to Nup153 (green), and this is observed for the control ER protein Sec61β (upper schematic and images). Correspondingly, an INM NET should be in the same plane as Nup153 and internal to Nup358 as is observed for the control NET LAP2β (lower schematic and images). C, all new NETs tested appeared in the same plane of the INM with Nup153 and formed an internal ring to Nup358, indicating INM residence. Bars, 5 μm.

NET tissue specificity of expression. A, expression data were downloaded from the BioGPS transcriptome database that compared over 60 mouse tissues on microarrays. The median expression value was obtained over the complete set of tissues, and data for a subset of tissues are graphed relative to this median value. Of the novel muscle putative NETs represented on the arrays, only one was not expressed notably above the median value in either heart or skeletal muscle (LPCAT3) with six expressed more than 100-fold over the median. In many cases, the median value occurred at background levels of expression (see ), so NETs that tended to be expressed around the median in most tissues besides muscle are more uniquely expressed in muscle. Error bars are standard deviations from fluorescence values from multiple microarrays. B, quantitative real time PCR of the same NETs and the control CKM over a smaller set of tissues. The values were generated in relation to GAPDH expression in the same reactions, and each NET is graphed as the -fold expression over its levels in liver. Tissue-preferential expression was also observed for several NETs directly tested by qRT-PCR; however, expression was sometimes observed in tissues that were at background levels according to the transcriptome data. Of particular note, LPCAT3, the only NET that was not up-regulated in skeletal muscle or heart according to transcriptome data, was up-regulated in skeletal muscle more than 5-fold by qRT-PCR. The expression of these new muscle NETs in blood and liver was at low/background levels by both methods, consistent with their having been uniquely identified in the skeletal muscle NE. Error bars are standard deviations from 3 Q-RT PCR replicates.

Antibody staining confirms novel NET identifications. A, Western blots testing antibodies generated to novel muscle NETs TMEM38A, LOC203547, and POPDC2, control NET SUN2, and a NET previously identified in liver that appears to be much more abundant in muscle, TMEM201/NET5. The correct size for each NET is indicated by an asterisk. B, comparison of the relative amounts of proteins in ER (microsome) and NE fractions. Lamin A was used as a control for a NE-specific protein, whereas Calreticulin was used as a control for an ER protein. Western blots of the two fractions similarly loaded for total protein were quantified on a LI-COR Odyssey imaging system, and the percentage of the total signal between both microsome and NE lanes was plotted with NE signal in blue and microsome signal in yellow (three repeats were averaged). The new muscle NETs were more enriched in the NE fraction than was the control NET SUN2. C, cryosections of rat muscle stained with NET antibodies. NE staining was clearly observed for all NETs tested as determined by a rim around the DAPI-stained DNA. Leg muscle is shown for TMEM38A, and heart is shown for the other two NETs. Bars, 10 μm. D, antibody staining on pre-extracted cells. All cells shown are C2C12 mouse myoblasts except in the case of TMEM201 where HeLa cells are shown. Cells were pre-extracted with detergent (1% Triton X-100) to remove membranes and most soluble cytoplasmic material and then fixed and incubated with NET antibodies. A nuclear rim staining was observed for the control NET SUN2 and all novel NETs tested. The resistance to detergent pre-extraction is consistent with the INM localization indicated by OMX results in .

Cellular fractionation of rat muscle. A, method schematic. Nuclei were first prepared from diced and homogenized rat leg muscle and cleaned of contaminating cellular structures by centrifugation first on isopycnic gradients and then on sucrose to float contaminating membranes while pelleting the denser nuclei. Crude NEs were prepared by digesting/extracting nuclear contents from isolated nuclei. Before MudPIT analysis, these were further extracted with 1% β-octyl glucoside and 400 mm NaCl or with 0.1 n NaOH to enrich for proteins associated with the insoluble Lamin polymer and integral membrane proteins, respectively. Microsomes/sarcoplasmic reticulum preparations were generated separately and analyzed for comparison/subtraction as they contain most expected membrane contaminants of the NE. B, top panel, before running both gradients, the crude nuclear fractions contained many chunks of myofibrillar material (e.g. Z-bands; highlighted with white arrowheads) released during homogenization. Bottom panels, after both gradients, isolated nuclei were clean of these contaminants. Phase-contrast light microscope images are shown. Scale bar, 10 μm. C, enrichment for NEs by chromatin digestion. DAPI staining for DNA visualizes significant nuclear chromatin content in an isolated muscle nucleus (left panels) and the loss of most of this material after two rounds of digestion with DNase and RNase each followed by salt washes (two right panels). Fluorescence microscope images are shown. Scale bar, 5 μm. D, ultrastructure of isolated NEs. Electron micrographs of crude muscle NEs show that most material in the population has the characteristic double membrane structure of the NE with little contamination from single membrane vesicles (a likely SR single membrane vesicle contaminant is highlighted by the white arrowhead). Arrows point to positions of NPCs. These NEs were further salt/detergent- or alkali-extracted prior to analysis by MudPIT. After such treatment, no structure remains that can be readily discerned by EM with the characteristics of NEs. Scale bar, 0.2 μm.