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First published online 14 April 2009
doi: 10.1242/jcs.039255

Journal of Cell Science 122, 1401-1409 (2009)
Published by The Company of Biologists 2009
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Microtubule plus-end binding protein EB1 is necessary for muscle cell differentiation, elongation and fusion

Tan Zhang1,*, Kristien J. M. Zaal1, John Sheridan1, Amisha Mehta1, Gregg G. Gundersen2 and Evelyn Ralston1

1 Light-imaging Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA
2 Department of Anatomy and Cell Biology, Columbia University, New York, NY 10032, USA


Figure 1
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  		Fig. 1. EB1 is expressed throughout C2 differentiation. (A) Immunoblotting of extracts from C2 cultures in GM and FM for EB1, EB3, glu-tubulin and myogenin, with GAPDH as loading control, shows EB1 at the earliest time point, whereas EB3 is only detected after differentiation. Data shown here are representative of three different experiments. (B) Immunostaining of myoblasts in GM with mouse anti-EB1 shows the typical comet-like pattern at microtubule plus-ends, pointing from the center of the cell outwards. (C) Mitotic myoblasts, by contrast, show strong centrosomal EB1. In FM, both mononucleated cells (D) and multinucleated myotubes (E) show the comet-like pattern and a finer punctate staining along microtubules (arrow). The microtubule plus-end EB1 comets are uniformly oriented outwards at the myotube ends. Each image is the projection of a z-series of confocal images taken with identical imaging parameters. EB1 staining is shown in green; nuclei stained with Hoechst 33342 are shown in blue. Scale bars: 10 µm (B,D,E) and 5 µm (C).

 

Figure 2
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  		Fig. 2. Specific knockdown of EB1 with shRNAs inhibits C2 myotube formation. (A) Schematic representation of EB1 mRNA coding region with shRNA targeting sequences (sh2-WT, sh3-WT) used for knockdown of EB1. Silent mutations introduced in cDNAs used for rescue (sh2-SM, sh3-SM) are indicated in red. The lower bars show schematic representations of EB1 and EB1C proteins. (B) After 2 days in FM, extracts were prepared from pooled C2 cells stably transfected with shRNA-2 (C2-sh2), shRNA-3 (C2-sh3) or non-targeted control shRNA (C2-shC). Immunoblot from one of two independent experiments shows that the shRNAs are specific for EB1 because it is knocked down by both sh2 and sh3 whereas EB3 is unaffected. β-actin is shown as loading control. (C) Immunofluorescence staining of EB1 and EB3 in control and sh2-transfected C2 shows that EB3 is not affected in the EB1 KD cells (indicated by asterisks). Confocal images were taken with identical imaging parameters. (D) Phase-contrast images show that the level of fusion is reduced in C2-sh2 or C2-sh3 cells after 2 days in FM, compared with C2-shC. Scale bars: 10 µm (C) and 100 µm (D).

 

Figure 3
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  		Fig. 3. Differentiation, fusion and elongation are inhibited in EB1 KD C2 cell lines. (A) Extracts were prepared from the indicated stable EB1 KD and control cell lines after 2 days in FM. The level of EB1 and EB3, and cadherin, β-catenin, glu-tubulin and myogenin, which normally increase during muscle differentiation, were examined by immunoblotting, with β-actin as a loading control. (B) Representative phase-contrast images of C2-sh2F, C2-sh2L and C2-shCA cultured for 2 days in FM show practically complete inhibition of fusion in the EB1 KD cells. Scale bar: 100 µm. (C) Quantification of myogenin-positive cells from immunofluorescence (n=335, 350 and 345, respectively); myogenin expression from immunoblots was normalized to β-actin (n=3); fusion efficiency is shown as the percentage of nuclei in myotubes (n=3 and a total of 1125, 729 and 716 cells counted, respectively), and cell elongation as the average length of mononucleated cells (n=129, 122 and 115, respectively). In final graph, the boundaries of the boxes indicate the 25th and the 75th percentile, and the whiskers represent the minimum and maximum values respectively. The mean is shown by the straight line in the box. Values are mean ± s.d. Significant differences (unpaired Student's t-test): *P≤0.001.

 

Figure 4
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  		Fig. 4. The basal level of glu-tubulin microtubules is reduced in EB1 KD cells. (A) The level of glu-tubulin was measured by immunoblotting extracts from several stable EB1 KD cell lines (C2-sh2F, C2-sh2L and C2-sh3I) and control C2-shCA cells, all in GM. Relative glu-tubulin levels were normalized to GAPDH. Values are means ± s.d. (n=3). Significant differences (unpaired Student's t-test): *P≤0.0001. (B) Immunofluorescence staining of C2-sh2F and control C2 cells for glu-tubulin (red) and EB1 (green). To make the visualization of the weak fluorescence easier, the individual confocal images are shown with the gray scale inverted. The imaging parameters were identical for each antibody staining. These representative images show that microtubules containing glu-tubulin, except for those in the centrioles (arrowheads), are barely detectable in EB1 KD cells. In addition, primary cilia (arrows), which are abundant in glu-tubulin, are only detected in control C2. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar: 10 µm.

 

Figure 5
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  		Fig. 5. In the absence of EB1, the accumulation of β-catenin on the plasma membrane is reduced. (A) Immunofluorescence of EB1 and β-catenin is shown in control C2 cells in GM (top row) or FM (middle row), and in C2-sh2F after 1 day in FM (bottom row). As in Fig. 4, the gray scale is inverted and the imaging parameters were identical for each antibody staining. Notice that the β-catenin staining at the plasma membrane of C2-sh2F after 1 day in FM is patchy and weak compared with that in control cells. Scale bar: 10 µm. (B) Immunoprecipitation with anti-GFP from extracts of cultures transfected with either EB1-GFP or doubly transfected with GFP and GFP-f. Immunoblotting shows that cadherin and β-catenin coimmunoprecipitate with EB1-GFP. (C) Immunoprecipitation of endogenous EB1 from C2 cells in FM. Immunoblotting shows the presence of cadherin and β-catenin in the immunoprecipitates of anti-EB1-coated beads but not in that of anti-GFP coated beads. Immunoprecipitation efficiency was verified with mouse anti-EB1 [EB1(m)] or rabbit anti-EB1 [EB1(r)]. In addition, both antibody heavy (HC) and light (LC) chains were detected and used as loading controls.

 

Figure 6
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  		Fig. 6. EB1-GFP restores fusion of the EB1 KD cell lines. (A) Representative fluorescence images show that overexpression of EB1-GFP, EB3-GFP and GFP-f (as control) does not inhibit control C2-shCA elongation or fusion. (B) Representative fluorescence images show C2-sh2F EB1 KD cultures in FM 2 days after transfection with the same constructs. GFP-f-overexpressing cells show practically no elongation or fusion rescue. EB3 overexpression rescues cell elongation but only occasionally rescues fusion. EB1-GFP (with a silent mutation in the shRNA-2 targeting sequence) leads to restoration of both elongation and fusion. The bar graph shows the fusion index (percentage of nuclei in GFP-positive cells that are part of myotubes). Values are means ± s.d. (n=3 and a total of 2014, 1283 and 946 nuclei counted, respectively). Significant differences (unpaired Student's t-test): **P≤0.0001; *P<0.02. (C) Immunofluorescence staining shows that glu-tubulin microtubules are present in EB1-GFP-rescued myotubes. Scale bars: 50 µm (A,B) and 20 µm (C).

 

Figure 7
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  		Fig. 7. A model demonstrating the essential roles of EB1 during early C2 differentiation. Early in muscle differentiation, microtubules become stabilized and cadherin and β-catenin translocate from the cytoplasm to the plasma membrane. We have shown that EB1 is necessary for both events, but we do not know whether the two events are causally related. In the absence of EB1, differentiation is largely inhibited and later events, especially fusion, are blocked. Rescue experiments show that EB1 is necessary for the events of late differentiation, but we do not know whether it is sufficient. Since the level of EB3 becomes elevated at this stage, it is possible that EB3 also contributes.

 

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