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. 2007 Aug 7;17(15):1318-25.
doi: 10.1016/j.cub.2007.06.058. Epub 2007 Jul 19.

EB3 regulates microtubule dynamics at the cell cortex and is required for myoblast elongation and fusion

Anne Straube  1 , Andreas Merdes
Affiliations

EB3 regulates microtubule dynamics at the cell cortex and is required for myoblast elongation and fusion

Anne Straube et al. Curr Biol. .

Abstract

During muscle differentiation, myoblasts elongate and fuse into syncytial myotubes [1]. An early event during this process is the remodeling of the microtubule cytoskeleton, involving disassembly of the centrosome and, crucially, the alignment of microtubules into a parallel array along the long axis of the cell [2-5]. To further our understanding on how microtubules support myogenic differentiation, we analyzed the role of EB1-related microtubule-plus-end-binding proteins. We demonstrate that EB3 [6] is specifically upregulated upon myogenic differentiation and that knockdown of EB3, but not that of EB1, prevents myoblast elongation and fusion into myotubes. EB3-depleted cells show disorganized microtubules and fail to stabilize polarized membrane protrusions. Using live-cell imaging, we show that EB3 is necessary for the regulation of microtubule dynamics and microtubule capture at the cell cortex. Expression of EB1/EB3 chimeras on an EB3-depletion background revealed that myoblast fusion depends on two specific amino acids in the calponin-like domain of EB3, whereas the interaction sites with Clip-170 and CLASPs are dispensable. Our results suggest that EB3-mediated microtubule regulation at the cell cortex is a crucial step during myogenic differentiation and might be a general mechanism in polarized cell elongation.

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Figures

Figure 1
Figure 1
EB Proteins during Myogenic Differentiation (A) Morphological changes and reorganization of the microtubule cytoskeleton. Immunofluorescence of C2C12 cells that were shifted to differentiation for the times indicated and stained for α-tubulin (red), PCM-1 (green), and DAPI (blue) is shown. The scale bar represents 20 μm. (B) Reverse-transcription polymerase chain reaction (RT-PCR) of EB1, EB2, EB3, and α-tubulin from RNA isolated from C2C12 myoblasts that were cultured under differentiation conditions for the times indicated. (C) Western blots of total cell lysates of C2C12 cells that were cultured under differentiation conditions for the times indicated and probed with antibodies against EB1, EB2, EB3, and α-tubulin. (D) C2C12 myoblasts, cultured under differentiation conditions for 41 hr, 3 days after transfection with shRNA constructs against luciferase (control), EB3, and EB1, coexpressing GFP-tubulin. The scale bar represents 50 μm. (E) Average of the mean cell length in four to seven cell-elongation experiments after 19–54 hr under differentiation conditions. The average cell size of undifferentiated myoblasts is included for comparison. Error bars represent the standard deviation (SD). (F) Myoblast fusion after 54 hr differentiation. GFP-tubulin (white) highlights cells that were transfected with the shRNA constructs (control and EB3); DNA staining is shown in blue. Clusters of nuclei (arrows) indicate myoblast fusion in both fields of view. Note that cells transfected with EB3 shRNAs do not participate in fusion. The scale bar represents 100 μm. (G) Quantification of myoblast-fusion efficiency after 49–54 hr under differentiation conditions. Because of variations between experiments, the efficiency of cell fusion (percentage of cells containing two or more nuclei) was set to 100% for control cells in each experiment. Columns represent the average of three to four experiments, and error bars represent the SD.
Figure 2
Figure 2
Expression of Myogenic Markers Is Not Affected after EB3 Depletion (A) Western-blot analysis of 20,000 GFP-expressing cells after fluorescence-activated cell sorting (FACS), 3 days after transfection with shRNA constructs. Blots were probed with antibodies against EB1, EB3, α-tubulin, myogenin, Glu-tubulin, and embryonic myosin. Positions of molecular weight markers are indicated on the left. (B) Immunofluorescence staining for embryonic myosin in EB3-depletion experiments after 55 hr in differentiation conditions. In the top panel, GFP-tubulin (white) highlights a transfected cell; in the bottom panel, embryonic myosin staining (white) of the same group of cells is shown. DNA is shown in blue. Scale bars represent 20 μm. (C) Immunolocalization of PCM-1 (green) after 55 hr in differentiation conditions. GFP-tubulin (red) highlights transfected cells with either control (upper row) or EB3 shRNAs (lower row). DNA is shown in blue. Scale bars represent 20 μm.
Figure 3
Figure 3
Microtubule Behavior at the Cell Cortex is Altered in EB3-Depleted Cells (A) Microtubule organization in C2C12 cells 3 days after transfection with shRNA constructs and after 52 hr under differentiation conditions. GFP-tubulin highlights the microtubule cytoskeleton in transfected cells. The insets show the cell edges of differentiating myoblasts after 49 hr under differentiation conditions. Scale bars represent 20 μm, and 2 μm in the insets. (B) Microtubule organization in C2C12 cells 3 days after cotransfection of the EB3 shRNA construct and rescue constructs for full-length EB3 or EB1. GFP-tubulin highlights the microtubule cytoskeleton in transfected cells. Scale bars represent 20 μm. (C) Images taken from time-lapse movies of GFP-tubulin in shRNA transfected cells that were shifted to differentiation for 49 hr. The time in seconds is indicated in the upper-right corner. Arrows highlight newly appearing bumps in microtubules that reach the cell cortex and continue growth. Asterisks indicate the relaxation of such bumps. The scale bar represents 2 μm. See the Supplemental Data for the respective movies. (D) Quantification of the time microtubule tips spent at or close to the cortex (maximum of 2 μm away). Columns represent data obtained from 93–129 microtubules in five to eight cells, and error bars indicate SD. Microtubule tips spend significantly less time close to the cortex in EB3-depleted compared to control cells (analysis of variance [ANOVA]: p = 0.0003). (E) Quantification of the time microtubules continue to grow after reaching the cell cortex, resulting in the introduction of bends into microtubules. Columns represent data obtained from 38–64 microtubules in four to eight cells, and error bars indicate SD.
Figure 4
Figure 4
Rescue of EB3 Depletion (A) Quantification of myoblast fusion efficiency after 54–55 hr under differentiation conditions. The efficiency of cell fusion (percentage cells containing two or more nuclei) was set to 1.0 for control cells in each experiment (ranging from 8% to 34% fused cells), and relative values are shown for all other constructs. Columns represent an average of three to four experiments, and error bars indicate the SD. The columns representing values not significantly different from those of control cells (ANOVA: p > 0.05) are shown in light blue, whereas values not significantly different from those of EB3 deletion are shown in orange (ANOVA: p > 0.05). A partial rescue (which falls in neither of these categories, with p http://www.proteinexplorer.org). (E) Surface maps of N-terminal domains (amino acids 1–130) of EB1 and EB3 were created with SYBYL 6.9.1 on the basis of PDB files 1PA7 and 1WYO. The four surface-exposed amino acid changes are highlighted in pink and red. Note that the surface structure changes as Y32 in EB3 fills a deep cleft present in EB1, and amino acids H29 and D26 emanate from the surface of the molecule.
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