Myosin VI in the nucleus of neurosecretory PC12 cells: Stimulation-dependent nuclear translocation and interaction with nuclear proteins^*

<p>Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a “bait” identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes <i>via</i> interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.</p

Histogram of helix bending angle at residue 348.

<p>WT helix is naturally bent, about 175°, in a dimer coiled-coil conformation. For the Q348P mutation the maximum of the helix bending angle is nearly the same, about 172°, but a long tail of this plot with a local maximum at 158° indicates a high flexibility of helix at this residue in the mutant structure. The structure of A357-E359del also demonstrates a higher flexibility than WT.</p

Two Desmin Gene Mutations Associated with Myofibrillar Myopathies in Polish Families

<div><p>Desmin is a muscle-specific intermediate filament protein which forms a network connecting the sarcomere, T tubules, sarcolemma, nuclear membrane, mitochondria and other organelles. Mutations in the gene coding for desmin (<i>DES</i>) cause skeletal myopathies often combined with cardiomyopathy, or isolated cardiomyopathies. The molecular pathomechanisms of the disease remain ambiguous. Here, we describe and comprehensively characterize two <i>DES</i> mutations found in Polish patients with a clinical diagnosis of desminopathy. The study group comprised 16 individuals representing three families. Two mutations were identified: a novel missense mutation (Q348P) and a small deletion of nine nucleotides (A357_E359del), previously described by us in the Polish population. A common ancestry of all the families bearing the A357_E359del mutation was confirmed. Both mutations were predicted to be pathogenic using a bioinformatics approach, including molecular dynamics simulations which helped to rationalize abnormal behavior at molecular level. To test the impact of the mutations on <i>DES</i> expression and the intracellular distribution of desmin muscle biopsies were investigated. Elevated desmin levels as well as its atypical localization in muscle fibers were observed. Additional staining for M-cadherin, α-actinin, and myosin heavy chains confirmed severe disruption of myofibrill organization. The abnormalities were more prominent in the Q348P muscle, where both small atrophic fibers as well large fibers with centrally localized nuclei were observed. We propose that the mutations affect desmin structure and cause its aberrant folding and subsequent aggregation, triggering disruption of myofibrils organization.</p></div

Electron microscopy analysis of the longitudinal sections of probands's muscle biopsies.

<p>Bars, 1 µm.</p

Quantification of desmin content in muscle by immunoblotting.

<p>Equal volumes of homogenates (A) and supernatants (B) from control (C1-C4), patient IV:2, ZP family (P1, A357_E359del mutation), and patient III:3, DP family (P2, Q348P mutation) muscle were subjected to SDS polyacrylamide gel electrophoresis, blotted on nitrocellulose membrane and probed with anti-desmin and anti-GAPDH antibodies, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115470#s3" target="_blank">Materials and Methods</a>. Lower panels in A and B, densitometric analyses of desmin content in the examined muscles. For controls, the data are presented as mean ± SEM for n = 4.</p

Pedigree diagram of family ZP.

<p>Open diamond (IV:17). indicates sex unknown. Open triangle (V:4) indicates miscarriage. The other symbols indications as above. Family ZP consisted of 52 members, five of which participated in the study. Genetic analyses were performed for individuals III:7, IV:2, IV:4, IV:5, and IV:14.</p

Alignment of WT (blue), point-mutated (yellow) and deletion (purple) structures of desmin dimer after a 30ns MD simulation.

<p>Residue 348 is colored green in both helices.</p

blue look-out

blue look-out na bad look-out; a bad prospecta bad look-out; a bad prospectNot usedNot usedWithdrawnWithdrawn but no stam

Distribution of desmin in muscle with A357_E359del mutation.

<p>Transverse sections of the muscle biopsy of the patient were stained with anti-desmin, anti-M-cadherin and anti-slow myosin heavy chain antibodies, as indicated in the figure. Right panels, ∼2x magnification of the regions marked in left panels. These are 0.8-µm images of the center of transverses muscle section obtained with a Leica confocal microscope. Other details as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115470#s3" target="_blank">Materials and Methods</a> section. Bars, 50 µm.</p