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1.
Fig. 1.

Fig. 1. From: The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments.

Schematic representation of desmin and nebulin muscle proteins. A desmin intermediate filament is formed by a head, pre-coil (PCD) and tail regions interrupted by a central α-helical rod (coil 1A, coil 1B and coil 2), and it is further subdivided by linkers (L1 and L12). Nebulin consists of an acidic N-terminal region, 185 modules (M1–M185) organized as single repeats (M1–M8, M163–M185) or seven-module super-repeats (M9–M162), and a serine-rich SH3 C-terminal region (Labeit and Kolmerer, 1995).

Gloria M. Conover, et al. J Cell Sci. 2011 October 15;124(20):3464-3476.
2.
Fig. 10.

Fig. 10. From: The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments.

Intensity analysis of phalloidin-stained myofibrils in cells expressing GFP–desmin-K190A. Intensity profiles (black lines) of Texas-Red- and phalloidin-stained actin filaments in cells expressing GFP (A), GFP–desmin (B) and GFP–desmin-coil-1B (C) show peaks at the Z-discs where adjacent thin filaments overlap, and clearly detectable lower intensity staining (gaps) at the H-zones, in the middle of the sarcomere. By contrast, intensity profiles (red lines) of phalloidin staining in cells expressing GFP–desmin-K190A (B) and GFP–coil-1B-K190A (C) show irregularities in phalloidin staining with less intense staining at the M-line, continuous F-actin staining in some sarcomeres and an inconsistent increase in intensity at the Z-discs compared with profiles of GFP–desmin cells.

Gloria M. Conover, et al. J Cell Sci. 2011 October 15;124(20):3464-3476.
3.
Fig. 4.

Fig. 4. From: The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments.

Desmin coil 1B K190A mutant binds to C-terminal nebulin with reduced binding capacity compared with desmin coil 1B. The binding profiles of biotinylated desmin coil 1B (151–263) and its mutant K190A were compared on microtiter plates containing immobilized nebulin modules M160–M170 or nebulin modules M160–M164 using ELISA assays. Binding was assayed by a colorimetric reaction measured at absorbance at 405 nm. (A) Desmin coil 1B binds to nebulin M160–M170 with a Bmax≈3.5 and a Kd≈35 nM, whereas desmin coil 1B K190A binds with a Kd≈88 nM with a lowered Bmax≈2.2. (B) Desmin coil 1B binds to nebulin M160–M164 with a saturation Bmax≈3.3 and a Kd≈14 nM, whereas desmin coil 1B K190A binds with a Kd ≈ 34 nM with a lowered Bmax ≈ 1.9. Values are the mean of triplicate samples ± s.d.

Gloria M. Conover, et al. J Cell Sci. 2011 October 15;124(20):3464-3476.
4.
Fig. 3.

Fig. 3. From: The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments.

Desmin Lys190 is important for the interaction of desmin coil 1B and nebulin M160–M170. (A) An alanine substitution of mouse desmin Lys190 in desmin coil 1B (residues 185–191: DLQRLKAKLQEEI) decreases the binding of nebulin M160–170 to P5. By contrast, other desmin peptides with single alanine exchanges (D164A, E166A, N170A and R172A) in P4 and (K185A, Q187A and E195A) in P5 do not interfere with their interaction with nebulin M160–M170. (B) Blastp and ClustalW alignment analysis of desmin P5 reveal a high conservation of residues in this peptide among the different species analyzed (human, rodents, ruminants, chick, fish and frog). Alignment shows regions of conserved amino acids (red, 100%; blue, 70–99%; green, 50–69%; yellow, 30–49%; white, <29%).

Gloria M. Conover, et al. J Cell Sci. 2011 October 15;124(20):3464-3476.
5.
Fig. 7.

Fig. 7. From: The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments.

Expression of desmin K190A dramatically perturbs nebulin Z-disc localization in skeletal myocytes. (A) Quantification of the percentage of total chick myotubes with striated C-terminal nebulin staining at the Z-discs. Values are means ± s.d. of three independent experiments. Assembly of Z-disc nebulin in myotubes expressing mutant desmin K190A is nearly undetectable. Student's t-test was used to determine significance (**P<0.01) of GFP–coil-1B-K190A nebulin Z-disc assembly compared with GFP or GFP-coil-1B. (B) Transfected myotubes were stained with antibodies against C-terminal nebulin, 8 days after transfection. GFP–coil-1B assembles in a striated pattern at the Z-disc with the C-terminal region of nebulin (c, arrows indicate colocalization). By contrast, GFP–coil-1B-K190A demonstrate little or no assembly at the Z-disc (g–i), with a concomitant decrease in assembled nebulin. In most myotubes expressing GFP alone, C-terminal epitopes of nebulin are localized at the Z-discs (a–c). Scale bar: 10 μm.

Gloria M. Conover, et al. J Cell Sci. 2011 October 15;124(20):3464-3476.
6.
Fig. 8.

Fig. 8. From: The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments.

Expression of GFP–desmin-K190A reduces the assembly of the C-terminus of nebulin at the Z-discs in cardiomyocytes. (A) Quantification of the percentage of total cardiomyocytes with nebulin at the Z-discs in cells expressing WT GFP–desmin or GFP–coil-1B, in comparison to the distribution of Z-disc nebulin in cells expressing the mutant desmin K190A counterparts. Student's t-test was used to determine significance (**P<0.01) of alterations in the distribution of the C-terminus of nebulin in cells expressing GFP–desmin proteins nebulin compared with expression of GFP alone. (B) Cardiomyocytes were stained with antibodies against nebulin modules M176–M181 and α-actinin, 7 days after transfection. Expression of GFP–desmin-K190A results in a significant decrease in the localization of Z-disc-associated nebulin. Nebulin staining is less intense and more punctuate in cells expressing mutant desmin compared with cells expressing non-mutated desmin coil 1B, where nebulin striations colocalize with GFP–coil-1B (compare bottom row with middle row). GFP control transfected cardiomyocytes display a mostly striated nebulin distribution (a–c). Scale bar: 10 μm.

Gloria M. Conover, et al. J Cell Sci. 2011 October 15;124(20):3464-3476.
7.
Fig. 6.

Fig. 6. From: The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments.

Expression of desmin K190A perturbs the localization of endogenous Z-disc desmin. (A) The percentage of total cardiomyocytes with Z-disc-associated desmin was quantified in untransfected cells and in cells expressing non-mutated and K190A versions of GFP-tagged desmin full-length or coil 1B proteins. Z-disc localization was determined by colocalization with α-actinin. Shown are the average counts ± s.d. from three experiments. More than 300 cells were counted per condition per experiment. The endogenous distribution of desmin is significantly different in cells expressing mutant GFP–desmin-K190A (or coil 1B) compared with GFP alone or untransfected cells. Student's t-test was used to determine significance (*P<0.05, **P<0.01) of GFP–desmin proteins compared with GFP alone. (B) Untransfected cardiomyocyte show a typical striated endogenous desmin Z-disc staining pattern (red) (a). Cells expressing various GFP–desmin proteins (green) were co-labeled for endogenous desmin (red) (b–f) and α-actinin (not shown). As expected, striated endogenous desmin Z-disc staining is present in cells expressing GFP alone (b). Aggregates in cells expressing GFP–desmin-K190A (d) and GFP–coil-1B-K190A (f) colocalize with endogenous desmin (arrowheads); in comparison, non-mutated desmin proteins show mostly striated Z-disc staining (arrows). Scale bar: 10 μm.

Gloria M. Conover, et al. J Cell Sci. 2011 October 15;124(20):3464-3476.
8.
Fig. 2.

Fig. 2. From: The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments.

Multiple sites within desmin associate with C-terminal nebulin. (A) A desmin peptide array with overlapping 13 aa peptides spanning the entire open reading frame was incubated with GST-tagged nebulin M160–M170 (1 μg/ml), and bound nebulin was detected. Nebulin M160–M170 binds to eight desmin fragments (P1–P8) corresponding to the following desmin mouse residues: P1 (21–53) and P2 (57–77) within desmin head; P3 (137–149) within desmin coil 1A; P4 (171–173) and P5 (185–197) within desmin coil 1B; P6 (297–316) and P7 (337–357) within desmin coil 2B; and P8 (441–453) within the desmin tail. (B) Individual GST-tagged desmin fragments (head, coil 1B, coil 2B and tail) bound to glutathione beads were incubated with His–nebulin M160–M164 in GST pull-down assays. (+) Indicates addition of His–nebulin M160–M164; (-) indicates that no nebulin fragment was added. The bands below the desmin head band are probably degradation products because the desmin head is prone to degradation (Baron et al., 2004). In this assay, each of the desmin fragments tested was capable of binding nebulin M160–M164 as detected by western blot analysis. The residue numbers in parentheses correspond to human desmin.

Gloria M. Conover, et al. J Cell Sci. 2011 October 15;124(20):3464-3476.
9.
Fig. 9.

Fig. 9. From: The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments.

Expression of desmin K190A results in disorganization of the actin thin filaments. Myocytes expressing GFP (a), WT GFP–desmin (e,m) and GFP–desmin-K190A (i,q) were incubated in relaxing buffer and stained for F-actin (b,f,j,n,r) and α-actinin (c,g,k,o,s). Control myocytes expressing GFP display striated actin filament staining (b) with visible gaps in staining at the H-zones and unperturbed α-actinin staining (c). Full-length GFP–desmin is organized in its typical mesh-like fashion as small filaments (e) termed ‘squiggles’ (Goldman et al., 2008) and GFP–coil-1B is organized as striations at the Z-discs, with less-intense staining at the M-lines (m); these cells display striated actin filament (f and n) and α-actinin (g and o) staining patterns. By contrast, GFP–desmin-K190A is distributed as small dot-like aggregates (i), with nonstriated phalloidin staining in the identical myofibrils (j). Interestingly, two assembly patterns were detected in myocytes expressing GFP–coil-1BK190A. In most cells, GFP–coil-1B-K190A is organized at the Z-discs, with organized F-actin staining containing well-defined H-zone gaps (q–t). By contrast, in a minority of cells (~25%), GFP–coil-1B-K190A organizes into aggregates with visibly disorganized F-actin (data not shown), similarly to that observed in cells expressing GFP–desmin-K190A (j). Arrowheads show Z-disc localization, arrows show discontinuous staining and asterisks show M-line localization. Scale bar: 10 μm.

Gloria M. Conover, et al. J Cell Sci. 2011 October 15;124(20):3464-3476.
10.
Fig. 5.

Fig. 5. From: The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments.

Expression of desmin K190A does not significantly perturb the distribution of α-actinin in cardiomyocytes. (A) The assembly patterns of GFP-tagged desmin or coil 1B and its K190A mutants were quantified according to the degree of detectable striated Z-disc desmin staining. Groups were defined as: Z-disc (>80% of the cytosol contained Z-disc desmin assembly); mixed (presence of some aggregates of desmin and Z-disc desmin assembly); and aggregated (>80% of the cytosol contained aggregates of desmin). Our analysis shows that the majority of cells expressing mutant desmin K190A exhibit an increase in aggregated desmin with a corresponding decrease in Z-disc localization compared with their non-mutated counterparts (compare gray and spotted bars). Significance was determined using Student's t-test comparing full-length desmin (or coil 1B) with the mutants (*P<0.05). (B) Cardiomyocytes expressing various GFP–desmin proteins were stained for α-actinin (red). Images demonstrate the colocalization of α-actinin and GFP–desmin (a–c) or GFP–coil-1B (g–i). Cells expressing WT (data not shown) or mutant desmin K190A exhibiting aggregated (d–f,j–l) assembly show an unaltered distribution of α-actinin, indicating that the expression of GFP–desmin-K190A does not influence the assembly of α-actinin. More than 300 cells were counted per condition in three independent transfection experiments. Scale bar: 10 μm.

Gloria M. Conover, et al. J Cell Sci. 2011 October 15;124(20):3464-3476.

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