The G55R mutation increases the ability of 4R tau but not 3R tau to nucleate microtubule assembly.

<p>(A) Microtubule assembly in reactions containing a 1∶30 tau:tubulin dimer molar ratio were assayed by light scattering as a function of time. (B) Co-sedimentation assays demonstrate that the G55R mutation does not affect the ability of tau to assemble MT mass at steady-state, nor does it affect the ability of tau to bind to microtubules. Statistical significance was determined by comparing each mutant to its corresponding WT using two-tailed t-tests. Data in both panels represent the mean ± SEM from three independent experiments.</p

A. The family tree of the affected family shows the pattern of inheritance.

<p>The proband is the black oval on the left side of the figure (II:1), marked with an arrow. Tau haplotypes of sequenced individuals are also noted. “aoo” corresponds to age of onset; “aod” corresponds to age of death; black filling indicates persons possessing the G55R mutation; gray filling corresponds to diagnosed dementia of unknown origin (presumed to be G55R but inadequate medical records exist). Proband's son III:1 (from first marriage) is 36 years old and a carrier of G55R. Proband's second son III:2 (from second marriage) is 31 and also a G55R carrier. The other two sons (III:3 and III:4; from the second marriage) are not G55R carriers and are 29 and 28 years old. <b>B. The tau sequence in the region of the G55R mutation is extremely highly conserved across species lines.</b> The glycine at position 55 is completely conserved in seven species ranging from humans to lizards. Color coding emphasizes conserved nature of acidic (red), basic (blue), hydrophilic/polar (orange), hydrophobic (green) and proline (peach) positions.</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

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

blue look-out

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

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

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

Schematic map of the six CNS tau isoforms.

<p>Exons 2 (E2), 3 (E3) and 10 (E10) are alternatively spliced to generate all six possible combinations. Arrowheads denote the position of the G55R mutation, present in four of the six isoforms. R1, R2, R3 and R4 denote the four imperfect repeats in the MT binding region.</p