Lamin A/C mutants disturb sumo1 localization and sumoylation in vitro and in vivo.

A-type lamins A and C are nuclear intermediate filament proteins in which mutations have been implicated in multiple disease phenotypes commonly known as laminopathies. A few studies have implicated sumoylation in the regulation of A-type lamins. Sumoylation is a post-translational protein modification that regulates a wide range of cellular processes through the attachment of small ubiquitin-related modifier (sumo) to various substrates. Here we showed that laminopathy mutants result in the mislocalization of sumo1 both in vitro (C2C12 cells overexpressing mutant lamins A and C) and in vivo (primary myoblasts and myopathic muscle tissue from the Lmna(H222P/H222P) mouse model). In C2C12 cells, we showed that the trapping of sumo1 in p.Asp192Gly, p.Gln353Lys, and p.Arg386Lys aggregates of lamin A/C correlated with an increased steady-state level of sumoylation. However, lamin A and C did not appear to be modified by sumo1. Our results suggest that mutant lamin A/C alters the dynamics of sumo1 and thus misregulation of sumoylation may be contributing to disease progression in laminopathies

Ubc9 co-localizes with wild-type and mutant lamin A/C.

<p>Confocal microscopy images of nuclei of C2C12 cells expressing wild-type and mutant lamin A-CFP, DsRed2-lamin C and ubc9-GFP. The lamin constructs used as well as the associated pathology are indicated at the top. Lamin A and lamin C images are represented in one panel as they co-localize in all cells. Cells were visualized by wide-field fluorescence microscopy with excitation wavelengths of 434 nm for lamin A-CFP, 558 nm for DsRed2-lamin C, and 588 nm for ubc9-GFP. Each picture presented is representative of the most commonly observed phenotype.</p

Sumo1 localization is disrupted in soleus muscle sections from <i>Lmna</i>^H222P mice.

<p>Fluorescent microscopy images of soleus muscle cross sections from <i>Lmna</i>^+/+ and <i>Lmna</i>^H222P/H222P mice. Top row sections were stained for sumo1 (green). Bottom row sections were stained with only secondary antibody to show background signal of anti-mouse-568 nm antibody (green). All sections were counterstained for DAPI (blue). Each picture presented is representative of the most commonly observed phenotype. Scale bar represents 10 µm. White arrow highlights one cell with nuclear aggregation of sumo1.</p

Quantitative analysis of colocalization of sumo1 and mutant lamin A and C.

<p>Quantitative colocalization analysis of sumo1 and lamin A and C was performed using the ImageJ- 1.46r software. Thresholded Manders coefficients tM1 and tM2 and the percentage of pixels intensity above threshold colocalized (% Ch1 or Ch2 int > thresh) in each of the two channels (Ch1 for sumo (green fluorescence) and Ch2 for lamin A and C (red fluorescence) were calculated using the threshold algorithm of Costes et al (2004) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045918#pone.0045918-Costes1" target="_blank">[43]</a>.</p

Mutant lamin A/C expression increases levels of sumoylated proteins.

<p>Western blot analysis of sumo1 in nuclear protein harvested from C2C12 untransfected (UNT) cells or transfected with wild-type (WT) or mutant lamin C-CFP and sumo1-YFP harvested with NEM. Blots were re-probed for GapDH as a loading control.</p

Lamin A and C are not sumoylated by sumo1.

<p>(A–C) Western blots of C2C12 nuclear proteins: (A) Untransfected (UNT) or sumo1-YFP transfected, or triple transfected wild-type lamin A-CFP, DsRed2-lamin C and sumo1-YFP probed for lamin A/C. NEM was included with harvesting of selected samples to stabilize sumo conjugation. (B) Western blot A stripped and reprobed for the reversibly sumoylated protein, SP3. (C) UNT or triple transfected wild-type and mutant DsRed2-lamin C, wild-type sumo1-YFP and wild-type Ubc9-HA probed for lamin A/C. (D–E) Immunoprecipitation of endogenous and exogenous lamin A and C. (D) Nuclear extracts of sumo1-HA transfected C2C12 cells were immunoprecipitated using anti-lamin A/C antibody and western blotting was performed for lamin A/C. (E) Nuclear extracts of lamin A-CFP, lamin C-CFP, or empty CFP vector transfected COS7 cells were immunoprecipitated using anti-GFP tag antibody and western blotting was performed for the GFP tags. All protein was harvested in the presence of NEM. Emerin, a known lamin A/C binding partner, was included as a positive control for immunoprecipitation.</p

Protein Kinase C Alpha Cellular Distribution, Activity, and Proximity with Lamin A/C in Striated Muscle Laminopathies

Striated muscle laminopathies are cardiac and skeletal muscle conditions caused by mutations in the lamin A/C gene (LMNA). LMNA codes for the A-type lamins, which are nuclear intermediate filaments that maintain the nuclear structure and nuclear processes such as gene expression. Protein kinase C alpha (PKC-&alpha;) interacts with lamin A/C and with several lamin A/C partners involved in striated muscle laminopathies. To determine PKC-&alpha;&rsquo;s involvement in muscular laminopathies, PKC-&alpha;&rsquo;s localization, activation, and interactions with the A-type lamins were examined in various cell types expressing pathogenic lamin A/C mutations. The results showed aberrant nuclear PKC-&alpha; cellular distribution in mutant cells compared to WT. PKC-&alpha; activation (phos-PKC-&alpha;) was decreased or unchanged in the studied cells expressing LMNA mutations, and the activation of its downstream targets, ERK 1/2, paralleled PKC-&alpha; activation alteration. Furthermore, the phos-PKC-&alpha;-lamin A/C proximity was altered. Overall, the data showed that PKC-&alpha; localization, activation, and proximity with lamin A/C were affected by certain pathogenic LMNA mutations, suggesting PKC-&alpha; involvement in striated muscle laminopathies

Sumo1 localization is disturbed by endogenous p.<i>His222Pro</i> mutant lamin A/C in primary mouse myoblasts.

<p>Fluorescent microscopy images of non-transfected and sumo1-YFP transfected <i>Lmna</i>^+/+ (WT) and <i>Lmna</i>^H222P/H222P primary myoblast nuclei. (A) Untransfected myoblasts immunostained for endogenous sumo-1 (green). (B) Sumo1-YFP transfected myoblasts expressing YFP-tagged sumo1 (green). All myoblasts were counterstained for DAPI (blue). Each picture presented is representative of the most commonly observed phenotype.</p