Skip to main content
NCBI home page
The FASEB Journal logoLink to The FASEB Journal
. 2010 Apr;24(4):1284–1295. doi: 10.1096/fj.09-136309

Myoferlin is required for insulin-like growth factor response and muscle growth

Alexis R Demonbreun *, Avery D Posey †, Konstantina Heretis ‡, Kayleigh A Swaggart §, Judy U Earley ‡, Peter Pytel ‖, Elizabeth M McNally *,†,‡,§,1
PMCID: PMC2845429  PMID: 20008164

Abstract

Insulin-like growth factor (IGF) is a potent stimulus of muscle growth. Myoferlin is a membrane-associated protein important for muscle development and regeneration. Myoferlin-null mice have smaller muscles and defective myoblast fusion. To understand the mechanism by which myoferlin loss retards muscle growth, we found that myoferlin-null muscle does not respond to IGF1. In vivo after IGF1 infusion, control muscle increased myofiber diameter by 25%, but myoferlin-null muscle was unresponsive. Myoblasts cultured from myoferlin-null muscle and treated with IGF1 also failed to show the expected increase in fusion to multinucleate myotubes. The IGF1 receptor colocalized with myoferlin at sites of myoblast fusion. The lack of IGF1 responsiveness in myoferlin-null myoblasts was linked directly to IGF1 receptor mistrafficking as well as decreased IGF1 signaling. In myoferlin-null myoblasts, the IGF1 receptor accumulated into large vesicular structures. These vesicles colocalized with a marker of late endosomes/lysosomes, LAMP2, specifying redirection from a recycling to a degradative pathway. Furthermore, ultrastructural analysis showed a marked increase in vacuoles in myoferlin-null muscle. These data demonstrate that IGF1 receptor recycling is required for normal myogenesis and that myoferlin is a critical mediator of postnatal muscle growth mediated by IGF1.—Demonbreun, A. R., Posey, A. D., Heretis, K., Swaggart, K. A., Earley, J. U., Pytel, P., McNally, E. M. Myoferlin is required for insulin-like growth factor response and muscle growth.

Keywords: myoblast fusion, receptor trafficking, lysosome, signaling

Muscle growth relies on the fusion of singly nucleated myoblasts to the multinucleate syncytium of individual mature myofibers. Insulin-like growth factor (IGF) 1 is a potent mediator of tissue and cell growth and is especially critical for muscle growth. IGF1 undergoes splicing to produce distinct forms with both paracrine and endocrine action (1). Of the multiple splice forms, several have been characterized for their effect in specifically promoting myoblast proliferation, fusion, and myofiber growth (2,3,4).

Mice lacking IGF1 have a high postnatal death rate related to muscle defects (5, 6), and surviving IGF1-null mice are small. Correspondingly, mice overexpressing IGF1 are nearly 25% larger than control mice (7) with an increase in skeletal fiber number and an increase in fiber cross sectional area (8). Because of its effects on growth of muscle, IGF1 is being investigated for its use to treat muscle disorders and as a target for increasing normal muscle growth and performance. IGF1s bind the type 1 IGF receptor (IGF1R), a receptor serine threonine kinase that, in turn, stimulates a cascade of signaling responses. In response to ligand binding, the IGF1R is phosphorylated and activates the MAP kinase-dependent signaling (MAPK) and the PI3K/AKT pathways. These pathways have been confirmed independently to regulate myoblast proliferation, differentiation, fusion, and ultimately muscle hypertrophy (9,10,11).

The ferlin proteins dysferlin and myoferlin are membrane-associated proteins implicated in muscle membrane repair and growth (12). Mutations in the dysferlin gene lead to muscular dystrophy characterized by defective membrane repair and an accumulation of intracellular vesicles (13). Dysferlin and myoferlin are highly related proteins. Myoferlin is expressed highly in prefusion myoblasts during muscle development and is up-regulated in skeletal muscle on injury. Mice lacking myoferlin have defects in the late stage of myogenesis, when myoblasts fuse to myotubes to augment their size. Myoferlin-null mice have reduced muscle mass and myofibers with a reduced cross-sectional area compared to normal (14). It was shown recently that myoferlin interacts with the vesicle recycling protein EHD2 (15). Mutations in the EHD family of proteins lead to delayed recycling of internalized vesicles back to the plasma membrane (16). Like EHD mutants, the loss of myoferlin results in defective recycling seen as an accumulation of transferrin in the perinuclear endocytic recycling compartment in the myoblasts (15).

The small muscle mass and reduced myofiber size in myoferlin-null muscle suggested a defect in muscle growth similar to that produced from manipulation of IGF1 or its receptor. We found that myoferlin-null muscle was completely unresponsive to IGF1. Because of the link to vesicle and receptor recycling, we examined IGF1R trafficking and found normal cell surface levels but abnormal internalization in myoferlin-null myoblasts. We found that myoferlin-null muscle lacks IGF1-induced signaling. Further examination showed that myoferlin-null myoblasts accumulate lysosomal structures and that IGF1Rs are targeted to these structures. These data demonstrate the importance of IGF1R trafficking in myoblast fusion and the role of myoferlin in mediating normal recycling of IGF1R to the plasma membrane during muscle growth and the necessity of this interaction for muscle growth.

MATERIALS AND METHODS

IGF1 treatment of mice

Myoferlin-null mice were described previously (14). Alzet pumps (Model 1002; Fisher Scientific, Pittsburgh, PA, USA) were filled with IGF1 (2 mg/kg of Long™ R3 IGF1; SAFC Biosciences, Lenexa, KS, USA) for intraperitoneal delivery, as described previously (17). Evans Blue dye was added to monitor flow and delivery. Mice were treated for 28 d. Harvested muscle was sectioned at 7 μm and immunostained as described previously (18). Cross-sectional area was analyzed using Image J (U.S. National Institutes of Health, Bethesda, MD, USA). Over 1000 fibers were measured from ≥2 animals of each genotype and treatment scheme. Statistical analysis was performed using Prism (GraphPad, La Jolla, CA, USA).

Primary myoblast cultures

Primary myoblasts were isolated from neonatal wild-type and myoferlin-null pups (postnatal days 0–3) as described previously (14, 15). All tissue culture media and sera used were from Invitrogen (Carlsbad, CA, USA). Cells were grown in F-10 Ham’s supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin/actinomycin (PSA) in 7% CO2. Differentiation medium contained DMEM, 2% horse serum, and 1% PSA.

IGF1 treatment of cultured myoblasts

Myoblasts (1.5×105) were plated, and after 24 h, growth medium was replaced with differentiation medium with or without 5 ng/ml IGF1 (Long R3 IGF1). For IGF1R blocking experiments, 3 × 105 myoblasts were plated, and after 24 h, differentiation medium with no IGF, 5 ng of IGF1, or 5 ng IGF1 plus 1 μg/ml of the IGF1R-blocking antibody (GR11L; Calbiochem, San Diego, CA, USA) was added to the cultures. After 48 h, cells were fixed and stained with anti-desmin antibody (Sigma). For each culture, the number of nuclei within each myotube was analyzed by immunofluorescence microscopy in 5 or 6 random fields.

For cell-signaling experiments, myoblasts (2×105) were plated in a 6-well dish. After 24 h, medium was replaced with growth medium without bFGF. After an additional 24 h, medium containing 50 ng/ml of IGF1 was added. Cells were incubated with IGF1 for varying times, then lysed in the presence of sodium orthovanidate at 1 μM.

Immunoblotting

Proteins transferred to membranes were immunoblotted with anti-myoferlin (MYOF3; 1:2000) (14), goat anti-EHD2 (1:10,000; ab23935; Abcam, Cambridge, MA, USA), anti-IGF1β receptor (1:2000; sc-713; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-MAPK (1:3000; 9102; Cell Signaling), rabbit anti-phosphoMAPK (1:1500; 9101S; Cell Signaling), rabbit anti-AKT (1:3000; 4685; Cell Signaling), rabbit anti-phospho AKT (1:1500; 9271S; Cell Signaling), anti-dsRed antibody (1:1000; 632496; Clontech, Palo Alto, CA, USA), anti-GFP antibody (1:1000; sc-9996; Santa Cruz), anti-Rab 11 (1:250; 610656; BD Transduction Laboratories, San Jose, CA, USA), anti-EEA1 (1:250; ab2900; Abcam,), anti-LC3B (1:200; ab48394; Abcam), or anti-LAMP2 (1:300; ab13524; Abcam). Secondary antibodies, goat anti-rabbit, donkey anti-rat, or donkey anti-goat HRP (Jackson ImmunoResearch, West Grove, PA, USA), were used at a dilution of 1:5000. Blocking and antibody incubations were done in Starting Block T20 (Invitrogen). ECL-Plus chemiluminescence (Amersham Pharmacia, Piscataway, NJ, USA) and Kodak Biomax MS film (Eastman Kodak, Rochester, NY, USA) were used for detection.

Immunostaining and microscopy

Primary myoblasts and C2C12 cells were grown on coverslips, fixed, and stained. Antibodies were used as follows: anti-MYOF3 (14) at 1:200, anti-EHD2 at 1:500 (ab23935; Abcam), anti-IGF-1Rα at 1:200 (sc-712; Santa Cruz), and anti-LAMP2 (ab13524; Abcam) at 1:800, anti-γ sarcoglycan at 1:200 (18). Donkey anti-rabbit Alexa488 and donkey anti-goat Alexa 594 were used at 1:2000 (Molecular Probes, Eugene, OR, USA). Donkey anti-Rat cy3 and goat anti-rabbit cy3 were used at 1:3000 (Jackson Immunologicals, West Grove, PA, USA). Blocking and antibody incubations were done in 1× PBS and 5% fetal bovine serum. Coverslips were mounted using ProLong Gold with DAPI. Images were captured using either a Zeiss Axiophot microscope and Axiovision software (Carl Zeiss, Oberkochen, Germany) or a Leica SP2 scanning laser confocal microscope and Leica LCS confocal software (Leica Microsystems, Wetzlar, Germany). Background was subtracted from each channel using Image J. Colocalization was analyzed using the Manders’ coefficient plug-in in Image J (19). Particle size, particle number, and fiber cross-sectional area were calculated using particle analysis in Image J (20).

Immunoprecipitation

C2C12 cells (5×105 cells; CRL-1772; ATCC) were plated and transfected with GFP-tagged EHD2 and dsRed tagged myoferlin using lipofectamine and Plus reagent (Invitrogen). After 24 h post-transfection, cells were lysed in coimmunoprecipitation buffer (150 mM NaCl; 50 mM Tris HCl, pH 7.4; 0.15% CHAPS with Complete Mini Protease Inhibitor cocktail, Roche, Indianapolis, IN, USA). Protein G sepharose beads (200 μl; Amersham Pharmacia) were blocked for 1 h with 20 μg/ml BSA in coimmunoprecipitation buffer, preincubated for 30 min with antibody, then incubated for 4 h with 50 μg of protein. Beads were washed, and the supernatant was loaded on a 10% acrylamide gel. Gels were transferred to PVDF membrane (Millipore, Bedford, MA, USA).

I125 IGF1 internalization

Myoblasts (2×105) were plated on 40-mm gelatinized plates. Cells were incubated with Krebs-Ringer phosphate HEPES binding buffer (KRPHBB), pH 7.5, containing 5.2 mM KCl, 1.4 mM CaCl2, 128 mM NaCl, 30 mM HEPES, 10 mM Na2HPO4, 1.4 mM MgSO4, and 1% bovine serum albumin, for 1 h at 37°C. KRPHBB was replaced with KRPHBB (pH 7.5) containing 1:1000 dilution of I125IGF1 (IM172; Amersham Lifesciences). Cells were incubated at 37°C with I125IGF1 for 0 to 120 min, rinsed, and washed in KRPHBB (pH 3.5) for 10 min at 37°C to remove surface-bound IGF1. This supernatant was analyzed for surface-bound IGF1 by γ-counting. Cells were rinsed, then solubilized with 0.4 N NaOH for 30 min on ice. Cell lysates were analyzed by scintillation counting to determine internalized IGF1. All values were standardized against protein content. The internalization rate constant Ke, represented by the slope of the line correlating the internalized to surface bound IGF, was calculated by linear regression (21, 22).

Electron microscopy

Cells (2×105) were seeded on glass coverslips. After 24 h, cells were rinsed and fixed in 2.5% glutaraldahyde. Cells were postfixed in 1% OsO4 for 1 h at 4°C. Cells were rinsed, dehydrated in ethanol, and infiltrated overnight. Embedded cultures were sectioned and stained with 1% uranyl acetate followed by lead citrate. Samples were photographed on a Tecnai electron microscope (FEI, Hillsboro, OR, USA). Whole muscle from 12-wk-old mice was fixed and visualized similarly.

RESULTS

Myoferlin-null muscle is unresponsive to IGF1

We treated wild-type mice and myoferlin-null mice with IGF1 for 4 wk using a protocol previously shown to increase myofiber size (17). In postnatal life, muscle response to IGF1 is thought to rely greatly on myoblast fusion to existing myofibers, a process we refer to as myoaugmentation (4, 23). After 4 wk of IGF1 treatment at 2 mg/kg delivered intraperitoneally, wild-type mice increased myofiber diameter in quadriceps muscle by 23%, while myoferlin-null mice were unresponsive to IGF1 exposure (Fig. 1A, B; P<0.0001). At baseline, myoferlin-null myofibers are smaller than wild-type, consistent with what was shown previously (14). The increase in myofiber area was seen for wild-type quadriceps muscle (Fig. 1A) and for gastrocnemius and soleus muscles (data not shown) and was similarly absent in myoferlin-null animals.

Figure 1.

Figure 1.

Open in a new tab

Myoferlin muscle does not respond to IGF1. Myoferlin-null (mko) and strain-matched wild-type (wt) mice aged 8 wk were treated with IGF1 via intraperitoneal delivery for 4 wk. A) Images of representative quadriceps muscles outlined with a membrane associated antibody (anti-γ-sarcoglycan). B) Normal quadriceps muscle showed a 25% increase in myofiber diameter, consistent with previously reported results (17). Myoferlin-null muscle has smaller myofibers at baseline, and these fibers did not enlarge after IGF1 exposure (n>1000 fibers from n=2 mice for myoferlin-null and strain-matched control wild-type fibers; P<0.0001). C) Primary myoblasts were cultured from neonatal myoferlin-null and control mice and exposed to IGF1 in culture. Myotube number and size were increased after IGF1 treatment in wild-type control myoblasts, but no increase was noted in myoferlin-null cells treated with IGF1 (n=4 myoferlin-null myoblast cultures, n=5 control, strain-matched wild-type myoblasts cultures, all cultures isolated from independent animals). Desmin staining is shown to demonstrate the multinucleate nature of myotubes. White boxes highlight the size and number of nuclei per myotube. D) Percentage increase, averaged for multiple experiments in C, bottom panel. Scale bars = 10 μm.

To confirm that this effect is related to myoblast fusion, we treated freshly isolated and cultured myoblasts from wild-type and myoferlin-null mice with IGF1. Myoblasts were subject to serum starvation to induce differentiation, and IGF1 was added to half the cultures, while the remainder were untreated. IGF1 increased myoblast fusion to myotubes in wild-type myoblasts by 25% (Fig. 1C, D), while myoferlin-null myoblasts were unresponsive to IGF1 treatment (Fig. 1C). IGF1 response, or lack thereof, was independent of cell density, since myoblast cultures were cultured at higher and lower density with identical results (data not shown). These results were repeated for myoferlin-null and control cultures from multiple animals in independent cultures (n=4 animals for myoferlin null; n=5 animals for wild type).

Reduced IGF1R internalization in myoferlin-null cells

Since myoferlin-null myoblasts are unresponsive to IGF1 treatment, we examined surface IGF1 binding and IGF1 internalization using radiolabeled 125I-IGF1 (21, 22). Myoblasts were isolated from myoferlin-null and strain-matched neonatal mice and plated at equal density and purity. Cells were incubated with 125I-IGF1 for specified periods. Surface-bound 125I-IGF1 was collected, and following that, myoblasts were lysed to collect internalized 125I-IGF1. Surface-bound and internalized 125I-IGF1 was measured in ≥3 independent cultures from 3 different animals of each genotype, and radioactive counts were normalized to protein content. After adding 125I-IGF1, surface 125I-IGF1 was not significantly different between myoferlin-null and control cells, indicating that IGF1R density at the cell surface is unchanged (Fig. 2A). At early time points, internalized 125I-IGF1 was similar between myoferlin-null and control cells. However, at 60–120 min, internalized 125I-IGF1 was significantly less in myoferlin-null myoblasts compared to control (Fig. 2B). Plotting these data over time demonstrates the reduction in the slope of the line for internalized 125I-IGF1 in myoferlin-null myoblasts compared to control myoblasts (Fig. 2C). These data indicate a late defect in 125I-IGF1 internalization where a reduction of internalized 125I-IGF1 in myoferlin-null myoblasts exists. This timing is consistent with the phase in which IGF1Rs recycle to plasma membrane (24). The reduction of internalized 125I-IGF1 implicates redirection of the IGF1R to a degradation pathway since 125I-IGF1 did not accumulate over time.

Figure 2.

Figure 2.

Open in a new tab

Myoferlin-null myoblasts internalize 125I-IGF1 abnormally. Surface-bound and internalized 125I-IGF1 was determined from myoferlin-null (mko) and wild-type (wt) control myoblasts. Independent cultures of myoblasts were treated for specified time points with 125I-IGF1. A, B) Surface bound (A) and internalized 125I-IGF1 (B) were measured and compared. C) Ratio of surface to internalized 125I-IGF1 was determined at each time point. Surface binding is comparable between myoferlin and wild-type control myoblasts from time 0 to 30 min. At 60 and 120 min, less internalized 125I-IGF1 was found in myoferlin-null myoblasts compared to control, consistent with a later delay in processing and recycling 125I-IGF1. These data are consistent with abnormal trafficking after the initial phase of endocytosis. Slope of the line indicates the reduced rate of 125I-IGF1 internalization in myoferlin-null myoblasts. *P < 0.03; **P < 0.002.

Myoferlin and IGF1R colocalize with EHD2 in fusing myoblasts

Vesicles accumulate at the site of myoblast fusion (25), but the protein cargo carried by these vesicles is not well understood. Since IGFs have been implicated in muscle growth (9), we hypothesized that receptors for IGF1 may localize to these vesicles. We studied localization of myoferlin and the β subunit of the IGF1R in C2C12 myoblasts undergoing fusion. Myoferlin and IGF1R associated at sites of myoblast fusion (Fig. 3A). We recently showed that EHD2, a protein that mediates receptor recycling, was decreased in myoferlin-null cells (15). We found that EHD2 and myoferlin also demonstrated significant colocalization in myoblasts (Fig. 3A) and that EHD2 and IGF1R colocalize in normal myoblasts (Fig. 3A). Results were similar with antibodies directed to the IGF1α receptor subunit (data not shown). The association of myoferlin, EHD2, and IGF1R was confirmed by determining the Mander’s overlap coefficient (19). Values for each pairwise comparison are as follows: EHD2:IGF1R 0.72, MYOF:EHD2 0.68, MYOF:IGF1R 0.72 (n=15 cells for each comparison). Immunoprecipitation of dsRed-tagged myoferlin, GFP-tagged EHD2, and endogenous IGF1R-β subunit by the IGF1R-β antibody were detected (Fig. 3B), demonstrating that these proteins complex with each other in myoblasts.

Figure 3.

Figure 3.

Open in a new tab

Colocalization of IGF1R, myoferlin, and EHD2. A) Antibodies to IGF1α receptor, EHD2, IGF1β receptor, and myoferlin were used on undifferentiated C2C12 myoblasts to examine colocalization. DAPI staining identifies nuclei. Arrows indicate regions of colocalization near membranes. Insets: colocalization at a higher magnification. B) Immunoprecipitation with the anti-IGF1β receptor pulled down myoferlin, IGF1R, and EHD2, as demonstrated by blotting with the anti-dsRed, anti-IGF1β receptor, and anti-GFP antibody to visualize myoferlin-dsRed, IGF1R β subunit, and EHD2-GFP, respectively. Right lane represents beads without antibody as a negative control.

IGF receptor levels are normal in myoferlin-null muscle

Wild-type and myoferlin-null skeletal muscle lysates were immunoblotted with anti-IGF receptor, and the results were quantified (Fig. 4A, B). Myoferlin skeletal muscle contains equivalent amounts of the IGF1R compared to wild type. Similarly, the IGF1R was normal from cultured wild-type and myoferlin-null myoblasts (Fig. 4C, D). These data provide more evidence that the fusion defect and lack of IGF1 response of myoferlin-null cells are not due to inadequate amounts of IGF1R but to another mechanism, such as defective receptor recycling caused by the loss of myoferlin.

Figure 4.

Figure 4.

Open in a new tab

Normal IGF1R levels in myoferlin-null mouse muscle. A, B) Lysates from wild-type and myoferlin-null skeletal muscle were immunoblotted for IGF1R. Myoferlin-null skeletal muscle expresses equivalent levels of the IGF1R as wild-type muscle. This finding was quantified by density analysis of the IGF1R bands from 3 independent animals. C, D) Wild-type and myoferlin-null neonatal myoblasts were isolated, and whole-cell lysates were immunoblotted for IGF1R. Myoferlin-null and wild-type myoblasts express similar levels of IGF1R, quantified by density analysis of the IGF1R bands from ≥4 independent cultures.

IGF-receptor-neutralizing antibodies block IGF response in wild-type but not myoferlin-null myoblasts

IGF1 promotes muscle growth through binding the IGF1R. Mice lacking the IGF1R have decreased muscle mass (5, 6). Furthermore, blocking the IGF1R inhibits the IGF1 hypertrophic effect (26). To confirm that the lack of IGF1 response in the myoferlin-null myoblasts relates to an IGF1R-mediated response, isolated myoblasts from wild-type and myoferlin-null mice were induced to differentiate in the presence of IGF1 or IGF1 plus anti-IGF1R blocking antibody (3, 27). The addition of the IGF1R blocking antibody prevents the IGF1 ligand from binding the IGF1R. As expected, exposure to IGF1 increased the fusion of myoblasts to myotubes in wild-type cultures. The presence of an IGF receptor blocking antibody abrogated this response in wild-type but not myoferlin-null cells (Fig. 5). This finding was quantified by counting the number of myotubes per condition containing 6 or more nuclei (P=0.03). These results were repeated for myoferlin-null and control cultures from multiple animals in independent cultures (n=2 and 3, respectively). These data imply that myoferlin plays an active role in regulating the IGF1R response.

Figure 5.

Figure 5.

Open in a new tab

IGF-receptor-neutralizing antibodies are effective in wild-type but not myoferlin-null myoblasts. Wild-type and myoferlin-null neonatal myoblasts were isolated and exposed to differentiation medium without IGF1, with IGF1, or with IGF1 plus an anti-IGF1R antibody for 48 h. Myotube number and size were increased with IGF1 treatment in wild-type control myoblasts. The IGF1 hypertrophic effect was abolished by the addition of IGF1R-blocking antibody. This finding was statistically significant, as shown by the graph of the number of myotubes containing ≥6 nuclei. No increase in myotube number or size was noted in myoferlin-null cells treated with IGF1, and the addition of IGF1R-blocking antibody did not alter myoblast fusion. At least 1600 myotubes were analyzed from cultures isolated from ≥2 independent animals/genotype. *P < 0.03.

Reduced IGF1 induced MAPK and AKT signaling in myoferlin-null myoblasts

We next examined whether IGF1 induced signaling was defective in myoferlin-null muscle. Both the AKT and the MAPK pathways are activated in IGF1-mediated growth, differentiation, and repair (22). Wild-type and myoferlin-null myoblasts were cultured and stimulated with 50 ng/ml of IGF1. Within 20–40 min after IGF1 exposure, normal myoblasts displayed a marked increase in phosphorylated AKT and MAPK (Fig. 6A–F). In contrast, myoferlin-null myoblasts lacked the transient increase in AKT and MAPK activity post-IGF1 stimulation. Furthermore, myoferlin-null myoblasts have a reduced maximum MAPK and AKT phosphorylation level (Fig. 6C, F).

Figure 6.

Figure 6.

Open in a new tab

Myoferlin-null myoblasts have reduced IGF1-induced MAPK and AKT signaling. Wild-type (w, wt) and myoferlin-null (m, mko) myoblasts were serum-starved, followed by incubation with IGF1 for 0, 5, 15, 45, or 120 min. Cell lysates were immunoblotted with anti-phosphorylated AKT (pAKT), anti-AKT, anti-phosphorylated MAPK (pMAPK), or anti-MAPK. A–F) Representative immunoblots illustrate a decrease in amplitude of the IGF1 mediated signaling cascade in myoferlin-null myoblasts (A, D). pAKT (B) and AKT (E) pMAPK and MAPK bands were quantified by densitometry and are expressed graphically as a percentage of maximum phosphorylation. Bar graphs illustrate the decrease in AKT (C) and MAPK (F) phosphorylation levels at the time of maximum signaling in myoferlin-null myoblasts compared to wild-type myoblasts. G) Wild-type and myoferlin newborn whole-muscle lysates (n=3 each) were immunoblotted with anti-pMAPK or anti-MAPK. Representative blots show increased MAPK and decreased pMAPK levels in myoferlin-null muscle relative to wild-type. Actin is shown as a loading control for all blots.

To examine kinase signaling in vivo (Fig. 6G), we isolated muscle from newborn mice (d 0) since this period is associated with pronounced growth. Unphosphorylated MAPK was up-regulated 2.7-fold in myoferlin-null muscle compared to wild-type muscle (P<0.0001), and a 1.8-fold decrease occurred in phosphorylated MAPK in myoferlin-null muscle compared with wild-type (P=0.04). The ratio of phosphorylated to unphosphorylated MAPK in newborn myoferlin-null skeletal muscle was reduced 5-fold compared with wild-type skeletal muscle (P<0.0001). The up-regulation of MAPK in myoferlin-null mice may represent a compensatory mechanism for the defect in the phosphorylation cascade. These data show that not only does myoferlin-null muscle fail to respond to IGF1 but also that IGF1-mediated signaling is similarly defective in the absence of myoferlin.

Myoferlin-null myoblasts accumulate IGF1R vesicles that colocalize with LAMP2-positive autolysosomes

Since myoferlin-null myoblasts do not recycle IGF1R properly and IGF1 signaling is altered, we examined the IGF1R localization after IGF1 stimulation. Myoferlin-null and wild-type myoblasts were grown in serum-free medium and stimulated with IGF1 for 0, 5, or 60 min. Myoblasts were fixed and then costained with IGF1R antibodies and anti-LAMP2, a marker of autolysosomes/late endosomes/lysosomes. Confocal imaging and Image J analysis showed myoferlin-null myoblasts accumulate large IGF1R aggregates in their cytoplasm on IGF1 stimulation. These aggregates were never seen in wild-type myoblasts. Furthermore, these IGF1R aggregates colocalized with the lysosomal marker LAMP2 (Fig. 7), and this was seen only in myoferlin-null cells. Myoferlin is required for normal endocytic recycling of the IGF1R, and in the absence of myoferlin, this receptor is shuttled into the lysosomal degradation pathway. The accumulation of vesicles in this pathway removes IGF1 from its normal recycling circuit and renders cells unable to signal and respond to IGF1.

Figure 7.

Figure 7.

Open in a new tab

IGF1 treatment results in enlarged IGF1R aggregates that colocalize with LAMP2-positive vesicles in the absence of myoferlin. Wild-type (wt) and myoferlin-null (mko) primary myoblasts were treated with IGF1 for 0, 5, or 60 min and then fixed and stained with anti-LAMP2 (red) and IGF1R (green). Images were acquired identically for control and myoferlin-null myoblasts and show that myoferlin-null myoblasts contained large IGF1R aggregates that colocalize with LAMP2-positive vesicles (yellow) with 5 and 60 min of IGF1 stimulation. Insets: magnified views of IGF1R aggregates colocalized with LAMP2 structures in myoferlin-null myoblasts; no aggregates were seen in wild-type cells. Scale bars = 10 μm.

Myoferlin-null myoblasts accumulate LAMP2-positive vesicles

To investigate the nature of lysosome accumulation in myoferlin-null myoblasts, we studied the expression of lysosome associated membrane protein (LAMP2), a protein implicated in lysosomal maturation and autophagy (28). Myoferlin-null and wild-type myoblasts were stained with an anti-LAMP2 antibody. Myoferlin-null myoblasts had a two-fold increase in the number of LAMP2 vesicles as compared to wild-type myoblasts (P<0.001) (Fig. 8A, C). Furthermore, these LAMP2-positive vesicles were enlarged in myoferlin-null myoblasts (P=0.008) (Fig. 8B). Myoblasts isolated from both neonatal mice and adult mice displayed this lysosomal accumulation (data not shown). Immunoblotting of whole cell lysates from myoferlin-null and wild-type myoblasts with an anti-LAMP2 antibody demonstrated that myoferlin-null myoblasts had a 3-fold increase in LAMP2 protein content as compared to wild-type myoblasts (Fig. 8D). In Fig. 8D, we also analyzed early endosome antigen 1 (EEA1) and Rab 11 but found no change. Microtubule-associated protein 1 light chain 3 (LC3), a marker of autophagosomes, was also evaluated but was not altered in expression nor in conversion to the active form. We further studied the size and number of LC3-positive vesicles in wild-type and myoferlin-null myoblasts to determine whether the autophagic pathway was perturbed; however no difference was noted between wild-type and myoferlin-null myoblasts (data not shown). LC3 is implicated in autophagy that involves double-membrane vesicles found before fusion with lysosomes. Thus, the lack of accumulated double-walled vesicles and normal LC3 suggests that myoferlin-null cells have increased lysosomal content without a clear increase in typical early autophagy.

Figure 8.

Figure 8.

Open in a new tab

Myoferlin-null myoblasts accumulate enlarged LAMP2 vesicles. A) Wild-type (wt) and myoferlin-null (mko) primary myoblasts were stained with LAMP2. DIC and fluorescent images were acquired identically for control and myoferlin-null myoblasts. B, C) Size (B) and number (C) of LAMP2-positive vesicles were increased in myoferlin-null myoblasts. D) Immunoblotting of wild-type and myoferlin-null myoblast whole-cell lysates shows increased LAMP2 levels in myoferlin-null myoblasts, while protein levels of Rab11, LC3, and EEA1 remain equal. Actin is shown as a loading control. Scale bars = 10 μm.

Myoferlin-null myoblasts accumulate enlarged autolysosomes

Examination of the ultrastructure of myoferlin-null and wild-type myoblasts by electron microscopy revealed large vacuolar structures in myoferlin-null cells (Fig. 9A–C). The appearance of these vacuoles is consistent with autolysosomes, single-membrane bound vacuoles containing degraded cytoplasmic contents (29). Myoferlin-null myoblasts had a 6-fold enlargement of these structures and 2-fold increase in total number of autolysosomes per cell (Fig. 9D, F). The average diameter of the autolysosomes in myoferlin-null myoblasts increased to 2000 nm compared with the 600 nm average diameter of the autolysosomes in wild-type cells. The average wild-type autolysosome diameter was comparable to prior reports (30). Furthermore, a 6-fold increase in the number of autolysosomes containing electron-dense cellular material in myoferlin-null myoblasts was found compared to those in wild-type myoblasts (Fig. 9E). The accumulation of cellular material in autolysosomes is consistent with autophagy, and a model where reduced recycling of vesicles to the plasma membrane leads to accumulation of vesicles in alternative pathways, allowing vesicle degradation. Finally, we noticed a 2-fold increase in the number of resting dense core lysosomes in the myoferlin-null myoblasts compared with wild-type myoblasts. Double-walled vesicles, representing early autophagy, were scarcely seen in myoblasts of either genotype.

Figure 9.

Figure 9.

Open in a new tab

Accumulation of autolysosomes in myoferlin-null myoblasts. Wild-type (wt) and myoferlin-null (mko) myoblasts were grown in serum-free medium for 2 h, fixed, and processed for electron microscopy. A, B) Representative images from wild-type (A) and myoferlin-null myoblasts (B). Myoferlin-null myoblasts have a marked increase in enlarged vesicles whose appearance is consistent with autolysosomes. C) High-magnification view from a myoferlin-null myoblast shows an autolysosome containing cellular material. D) Total number of vesicles in mko myoblasts was increased compared to wt; P < 0.0001. E) Total number of autolysosomes with cellular material was increased compared to wt; P < 0.001. F) Area of autophagic vacuoles was 12 times larger in mko myoblasts compared to wt; P < 0.0001.

An increase in LAMP2 staining was also evident in mature myoferlin muscle compared to wild-type (Fig. 10A). The increase in LAMP2 staining in myoferlin-null muscle was evident in both IGF1-treated and untreated muscle (data not shown). LAMP2 staining was punctate, and the puncta were more discrete after IGF1 treatment in both mutant and wild-type muscle (data not shown). Electron microscopy of mature muscle from myoferlin-null mice similarly displayed an increase in vesicular structures compared to wild-type (Fig. 10B). The vesicles were seen between sarcomeres scattered throughout the muscle cytoplasm and are consistent with an effect in mature myofibers beyond myoblast fusion, similar to what is seen in dysferlin deficiency (31).

Figure 10.

Figure 10.

Open in a new tab

Accumulation of enlarged autolysosomes in myoferlin-null muscle. A) Low-magnification (left panel) and high-magnification (right panel) views of wild-type (wt) and myoferlin-null (mko) muscle stained with LAMP2. Images were acquired identically for control and mko myoblasts and show that the size of and number of the LAMP2-positive vesicles were increased in mko myoblasts and were located between sarcomeres. B) Low-magnification (left panel) and high-magnification (right panel) views of wt and mko muscle, with many enlarged vacuoles present only in the mko muscle.

DISCUSSION

IGF1 is a potent mediator of skeletal muscle growth and repair. The lack of IGF1 can cause a wide range of defects, from perinatal lethality to aging-related atrophy (5, 6, 32). Because IGF1 is intimately involved in the development and maintenance of muscle, IGF1 has become an important clinical target. The anabolic effects of IGF1 can be achieved through in vivo overexpression of IGF1 using adenovirus or by direct infusion of IGF1 (17, 33). Blocking endogenous IGF1 with neutralizing IGF1 antibodies at the time of muscle injury results in reduced muscle cross-sectional area as well as number of regenerating myofibers (26). Like IGF1-manipulated animals, myoferlin-null myoblasts have decreased myofiber diameter as well as defective myofiber repair (14). In this study, we link myoferlin directly to IGF receptor activity and trafficking, demonstrating that the absence of myoferlin causes a redirection from a recycling pathway to a lysosomal pathway.

Myoferlin and the related protein dysferlin are membrane-associated proteins found at the plasma membrane and associated with intracellular vesicles (13, 14). Mutations in dysferlin cause muscular dystrophy associated with defective membrane resealing, a calcium-dependent process where intracellular vesicles are recruited to the sites of membrane disruption (13). Both myoferlin and dysferlin contain ≥6 C2 domains. C2 domains are independently folding domains found in proteins associated with membranes and implicated in vesicle trafficking. The C2 domains from synaptotagmins have been well studied and are thought to regulate the calcium sensitivity of vesicle fusion seen with fast exocytosis at nerve terminals. The first C2 domain of both myoferlin and dysferlin, C2A, binds to phospholipids in the presence of calcium, and thus is well positioned to mediate vesicle trafficking directly (34).

Myoferlin’s second C2 domain, C2B, directly binds to EHD2 (15), and EHD proteins have been implicated in both clathrin-mediated endocytosis and endocytic recycling (35). Myoferlin-null myoblasts have delayed recycling of the transferrin receptor, seen as reduced trafficking of transferrin and accumulation of abnormal perinuclear aggregates of transferrin (15). These data are consistent with other reports that EHD2 is involved in internalization and/or exit from the endocytic recycling compartment (36).

Taken together with our current data, we propose that myoferlin and EHD2 are responsible for vesicular trafficking and that the cargo being translocated includes the IGF receptor (Fig. 11). Moreover, the proper intracellular transfer of the IGF receptor is linked tightly to its signaling activity and effectiveness. Many membrane receptors are involved in regulating the process of myoblast fusion in muscle growth, including the interleukin-4 (IL-4) and mannose-6-phosphate receptors (MR) (37, 38). Skeletal muscle from mice lacking IL-4, the IL-4 receptor, or MR has a decreased cross sectional area resulting from a lack of myoblast fusion, similar to what is seen in myoferlin-null muscle (37, 38). Thus, we hypothesize that proper recycling of other surface-bound receptors beyond the IGF1R by myoferlin is crucial for proper myoblast fusion and muscle growth.

Figure 11.

Figure 11.

Open in a new tab

Model for the role of myoferlin in receptor recycling. A) On ligand binding, membrane-bound receptors such as IGF1R are internalized, sorted, and shuttled back to the cell surface. Once recycled back to the plasma membrane, these receptors may function in further rounds of ligand binding and potential local amplification of response. Myoferlin (green) binds directly to EHD2 and functions in a complex with the IGF receptor. B) Lack of myoferlin is associated with reduced recycling and redirects the majority of vesicles that are normally recycled back to the plasma membrane toward the lysosomal degradation pathway, resulting in a dampened response of the activated receptors.

Recent work on a myoferlin homologue, otoferlin, links the ferlin family to endocytic recycling and vesicle transport. Mutations in otoferlin result in a recessive disorder, DFNB9 (39). Otoferlin is expressed in a wide range of tissues and is highly expressed in the cochlea of the ear in the auditory inner hair cell (39). To function, this specialized inner hair cell has a high level of neurotransmitter release, resulting in a high level of synaptic vesicle exocytosis. Mice lacking otoferlin display the characteristic deafness of DNFB9 patients due to abolished synaptic exocytosis in the inner hair cell (40). Otoferlin was linked further to vesicle trafficking through colocalization and direct interactions with the endosomal protein EEA1, the Golgi protein GM130, and the Rab GTPase Rab8 (40, 41). Rab8 is known to regulate secretory trafficking in the trans-Golgi network as well as endosomal sorting and recycling (42), and it is hypothesized that the otoferlin/Rab8 mediates this pathway. These data reinforce a substantive role for ferlin proteins in vesicle trafficking with diverse biological consequences.

Our findings indicate that myoferlin-IGF receptor intracellular transport is important for muscle growth that arises from myoblast fusion to myotubes or myoaugmentation. Myoblast fusion is dependent on both extracellular and intracellular signaling cascades. The downstream IGF1-signaling cascades, MAPK and PI3K/AKT, are activated after the IGF1R/ligand complex is internalized via clathrin-mediated endocytosis (22, 43). IGF1Rs are transported selectively down two pathways. After internalization, the majority of receptors are recycled to the plasma membrane while a smaller fraction of IGF receptors are targeted for degradation through the late endosome/lysosome in an ∼80/20 ratio, respectively (44). This allows the majority of the IGF1Rs in the cell to be reused where they may prolong IGF1 binding/signaling and potentially amplify local IGF1 response. In vivo, IGF1 is secreted maximally by injured muscle 3 d post-trauma (45). After that, IGF1 expression declines until it is no longer expressed at 10 d postinjury (45). Myoferlin is expressed similarly with high expression levels 3–5 d postinjury (14). This expression profile would allow myoferlin to be available readily for protein–protein interactions that would facilitate IGF1 vesicle transfer as well as promote myoblast fusion. Although myoferlin is less highly expressed in mature myofibers, its presence in myofibers suggests myofiber growth independent of myoblast fusion may be similarly affected in the absence of myoferlin.

The loss of myoferlin caused intracellular vesicular accumulation. With IGF1 stimulation, IGF receptors colocalized into large vesicles with the lysosomal protein LAMP2. Further analysis demonstrated lysosome accumulation in both myoferlin-null myoblasts and mature skeletal muscle. These lysosomes resemble and are in the same location as the lysosomes seen in muscle storage diseases such as Pompe disease (46). Pompe disease (OMIM 232200), a storage disease caused by deficiency in the glycogen-degrading enzyme acid α-1,4-glucosidase (acid maltase), leads to progressive accumulation of glycogen and enlarged lysosomes in muscle. LAMP1 is a target of acid maltase activity, and in the absence of normal post-translational modification, lysosome function is impaired (47). Pompe disease is associated with muscle weakness due to an accumulation of enlarged lysosomes and undigestable protein aggregates. Muscle samples from patients with Pompe disease also display increased high-molecular-weight ubiquitinated proteins (46), and we have noted a similar finding in myoferlin-null muscle (unpublished results).

Myoferlin displays calcium-dependent phospholipid binding and the ability to interact directly with components of the recycling machinery, and thus is well suited to mediate trafficking of many receptors. Moreover, of the 6 ferlin proteins, at least 5 (dysferlin, myoferlin, fer1l4, ferl5, and fer1l6) are expressed in myoblasts or muscle (48, 49). We anticipate that ferlin-mediated receptor recycling is equally critical to both developmental myogenesis and regeneration and that other ferlin proteins, such as dysferlin, fer1l4, ferl5, and fer1l6, are available to mediate these processes in muscle as well as other growth-responsive tissues.

Acknowledgments

This work was supported by U.S. National Institutes of Health (NIH) grant NS047726, the Muscular Dystrophy Association, and the Jain Foundation. A.R.D. was supported by NIH grant T32HL007381.

References

  1. Barton E R. The ABCs of IGF-I isoforms: impact on muscle hypertrophy and implications for repair. Appl Physiol Nutr Metab. 2006;31:791–797. doi: 10.1139/h06-054. [DOI] [PubMed] [Google Scholar]
  2. Musaro A, McCullagh K, Paul A, Houghton L, Dobrowolny G, Molinaro M, Barton E R, Sweeney H L, Rosenthal N. Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat Genet. 2001;27:195–200. doi: 10.1038/84839. [DOI] [PubMed] [Google Scholar]
  3. Yang S Y, Goldspink G. Different roles of the IGF-I Ec peptide (MGF) and mature IGF-I in myoblast proliferation and differentiation. FEBS Lett. 2002;522:156–160. doi: 10.1016/s0014-5793(02)02918-6. [DOI] [PubMed] [Google Scholar]
  4. Coleman M E, DeMayo F, Yin K C, Lee H M, Geske R, Montgomery C, Schwartz R J. Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J Biol Chem. 1995;270:12109–12116. doi: 10.1074/jbc.270.20.12109. [DOI] [PubMed] [Google Scholar]
  5. Liu J P, Baker J, Perkins A S, Robertson E J, Efstratiadis A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r) Cell. 1993;75:59–72. [PubMed] [Google Scholar]
  6. Powell-Braxton L, Hollingshead P, Warburton C, Dowd M, Pitts-Meek S, Dalton D, Gillett N, Stewart T A. IGF-I is required for normal embryonic growth in mice. Genes Dev. 1993;7:2609–2617. doi: 10.1101/gad.7.12b.2609. [DOI] [PubMed] [Google Scholar]
  7. Musaro A, McCullagh K J, Naya F J, Olson E N, Rosenthal N. IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1. Nature. 1999;400:581–585. doi: 10.1038/23060. [DOI] [PubMed] [Google Scholar]
  8. Liao L, Dearth R K, Zhou S, Britton O L, Lee A V, Xu J. Liver-specific overexpression of the insulin-like growth factor-I enhances somatic growth and partially prevents the effects of growth hormone deficiency. Endocrinology. 2006;147:3877–3888. doi: 10.1210/en.2005-1537. [DOI] [PubMed] [Google Scholar]
  9. Mourkioti F, Rosenthal N. IGF-1, inflammation and stem cells: interactions during muscle regeneration. Trends Immunol. 2005;26:535–542. doi: 10.1016/j.it.2005.08.002. [DOI] [PubMed] [Google Scholar]
  10. Coolican S A, Samuel D S, Ewton D Z, McWade F J, Florini J R. The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J Biol Chem. 1997;272:6653–6662. doi: 10.1074/jbc.272.10.6653. [DOI] [PubMed] [Google Scholar]
  11. Rommel C, Bodine S C, Clarke B A, Rossman R, Nunez L, Stitt T N, Yancopoulos G D, Glass D J. Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat Cell Biol. 2001;3:1009–1013. doi: 10.1038/ncb1101-1009. [DOI] [PubMed] [Google Scholar]
  12. Doherty K R, McNally E M. Repairing the tears: dysferlin in muscle membrane repair. Trends Mol Med. 2003;9:327–330. doi: 10.1016/s1471-4914(03)00136-9. [DOI] [PubMed] [Google Scholar]
  13. Bansal D, Miyake K, Vogel S S, Groh S, Chen C C, Williamson R, McNeil P L, Campbell K P. Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature. 2003;423:168–172. doi: 10.1038/nature01573. [DOI] [PubMed] [Google Scholar]
  14. Doherty K R, Cave A, Davis D B, Delmonte A J, Posey A, Earley J U, Hadhazy M, McNally E M. Normal myoblast fusion requires myoferlin. Development. 2005;132:5565–5575. doi: 10.1242/dev.02155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Doherty K R, Demonbreun A R, Wallace G Q, Cave A, Posey A D, Heretis K, Pytel P, McNally E M. The endocytic recycling protein EHD2 interacts with myoferlin to regulate myoblast fusion. J Biol Chem. 2008;283:20252–20260. doi: 10.1074/jbc.M802306200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. George M, Ying G, Rainey M A, Solomon A, Parikh P T, Gao Q, Band V, Band H. Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans. BMC Cell Biol. 2007;8:3. doi: 10.1186/1471-2121-8-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lynch G S, Cuffe S A, Plant D R, Gregorevic P. IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice. Neuromuscul Disord. 2001;11:260–268. doi: 10.1016/s0960-8966(00)00192-9. [DOI] [PubMed] [Google Scholar]
  18. McNally E M, Duggan D, Gorospe J R, Bonnemann C G, Fanin M, Pegoraro E, Lidov H G, Noguchi S, Ozawa E, Finkel R S, Cruse R P, Angelini C, Kunkel L M, Hoffman E P. Mutations that disrupt the carboxyl-terminus of gamma-sarcoglycan cause muscular dystrophy. Hum Mol Genet. 1996;5:1841–1847. doi: 10.1093/hmg/5.11.1841. [DOI] [PubMed] [Google Scholar]
  19. Manders E E M. Measurement of co-localisation of objects in dual-colour confocal images. J Microsc. 1993;169:375–382. doi: 10.1111/j.1365-2818.1993.tb03313.x. [DOI] [PubMed] [Google Scholar]
  20. Abramoff M D. Image processing with Image J. Biophotonics International. 2004;11:36–42. [Google Scholar]
  21. Wiley H S, Cunningham D D. A steady state model for analyzing the cellular binding, internalization and degradation of polypeptide ligands. Cell. 1981;25:433–440. doi: 10.1016/0092-8674(81)90061-1. [DOI] [PubMed] [Google Scholar]
  22. Chow J C, Condorelli G, Smith R J. Insulin-like growth factor-I receptor internalization regulates signaling via the Shc/mitogen-activated protein kinase pathway, but not the insulin receptor substrate-1 pathway. J Biol Chem. 1998;273:4672–4680. doi: 10.1074/jbc.273.8.4672. [DOI] [PubMed] [Google Scholar]
  23. Schmid C, Steiner T, Froesch E R. Preferential enhancement of myoblast differentiation by insulin-like growth factors (IGF I and IGF II) in primary cultures of chicken embryonic cells. FEBS Lett. 1983;161:117–121. doi: 10.1016/0014-5793(83)80742-x. [DOI] [PubMed] [Google Scholar]
  24. Zapf A, Hsu D, Olefsky J M. Comparison of the intracellular itineraries of insulin-like growth factor-I and insulin and their receptors in Rat-1 fibroblasts. Endocrinology. 1994;134:2445–2452. doi: 10.1210/endo.134.6.8194471. [DOI] [PubMed] [Google Scholar]
  25. Kalderon N, Gilula N B. Membrane events involved in myoblast fusion. J Cell Biol. 1979;81:411–425. doi: 10.1083/jcb.81.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lefaucheur J P, Sebille A. Muscle regeneration following injury can be modified in vivo by immune neutralization of basic fibroblast growth factor, transforming growth factor beta 1 or insulin-like growth factor I. J Neuroimmunol. 1995;57:85–91. doi: 10.1016/0165-5728(94)00166-l. [DOI] [PubMed] [Google Scholar]
  27. Nguyen T T, Sheppard A M, Kaye P L, Noakes P G. IGF-I and insulin activate mitogen-activated protein kinase via the type 1 IGF receptor in mouse embryonic stem cells. Reproduction. 2007;134:41–49. doi: 10.1530/REP-06-0087. [DOI] [PubMed] [Google Scholar]
  28. Eskelinen E L, Illert A L, Tanaka Y, Schwarzmann G, Blanz J, Von Figura K, Saftig P. Role of LAMP-2 in lysosome biogenesis and autophagy. Mol Biol Cell. 2002;13:3355–3368. doi: 10.1091/mbc.E02-02-0114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Eskelinen E L. Fine structure of the autophagosome. Methods Mol Biol. 2008;445:11–28. doi: 10.1007/978-1-59745-157-4_2. [DOI] [PubMed] [Google Scholar]
  30. Bakker A C, Webster P, Jacob W A, Andrews N W. Homotypic fusion between aggregated lysosomes triggered by elevated [Ca2+]i in fibroblasts. J Cell Sci. 1997;110:2227–2238. doi: 10.1242/jcs.110.18.2227. [DOI] [PubMed] [Google Scholar]
  31. Cenacchi G, Fanin M, De Giorgi L B, Angelini C. Ultrastructural changes in dysferlinopathy support defective membrane repair mechanism. J Clin Pathol. 2005;58:190–195. doi: 10.1136/jcp.2004.018978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Giovannini S, Marzetti E, Borst S E, Leeuwenburgh C. Modulation of GH/IGF-1 axis: potential strategies to counteract sarcopenia in older adults. Mech Ageing Dev. 2008;129:593–601. doi: 10.1016/j.mad.2008.08.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Barton-Davis E R, Shoturma D I, Sweeney H L. Contribution of satellite cells to IGF-I induced hypertrophy of skeletal muscle. Acta Physiol Scand. 1999;167:301–305. doi: 10.1046/j.1365-201x.1999.00618.x. [DOI] [PubMed] [Google Scholar]
  34. Davis D B, Doherty K R, Delmonte A J, McNally E M. Calcium-sensitive phospholipid binding properties of normal and mutant ferlin C2 domains. J Biol Chem. 2002;277:22883–22888. doi: 10.1074/jbc.M201858200. [DOI] [PubMed] [Google Scholar]
  35. Grant B D, Caplan S. Mechanisms of EHD/RME-1 protein function in endocytic transport. Traffic. 2008;9:2043–2052. doi: 10.1111/j.1600-0854.2008.00834.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Guilherme A, Soriano N A, Bose S, Holik J, Bose A, Pomerleau D P, Furcinitti P, Leszyk J, Corvera S, Czech M P. EHD2 and the novel EH domain binding protein EHBP1 couple endocytosis to the actin cytoskeleton. J Biol Chem. 2004;279:10593–10605. doi: 10.1074/jbc.M307702200. [DOI] [PubMed] [Google Scholar]
  37. Horsley V, Jansen K M, Mills S T, Pavlath G K. IL-4 acts as a myoblast recruitment factor during mammalian muscle growth. Cell. 2003;113:483–494. doi: 10.1016/s0092-8674(03)00319-2. [DOI] [PubMed] [Google Scholar]
  38. Jansen K M, Pavlath G K. Mannose receptor regulates myoblast motility and muscle growth. J Cell Biol. 2006;174:403–413. doi: 10.1083/jcb.200601102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yasunaga S, Grati M, Cohen-Salmon M, El-Amraoui A, Mustapha M, Salem N, El-Zir E, Loiselet J, Petit C. A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness. Nat Genet. 1999;21:363–369. doi: 10.1038/7693. [DOI] [PubMed] [Google Scholar]
  40. Roux I, Safieddine S, Nouvian R, Grati M, Simmler M C, Bahloul A, Perfettini I, Le Gall M, Rostaing P, Hamard G, Triller A, Avan P, Moser T, Petit C. Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse. Cell. 2006;127:277–289. doi: 10.1016/j.cell.2006.08.040. [DOI] [PubMed] [Google Scholar]
  41. Heidrych P, Zimmermann U, Bress A, Pusch C M, Ruth P, Pfister M, Knipper M, Blin N. Rab8b GTPase, a protein transport regulator, is an interacting partner of otoferlin, defective in a human autosomal recessive deafness form. Hum Mol Genet. 2008;17:3814–3821. doi: 10.1093/hmg/ddn279. [DOI] [PubMed] [Google Scholar]
  42. Henry L, Sheff D R. Rab8 regulates basolateral secretory, but not recycling, traffic at the recycling endosome. Mol Biol Cell. 2008;19:2059–2068. doi: 10.1091/mbc.E07-09-0902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Furlanetto R W. Receptor-mediated endocytosis and lysosomal processing of insulin-like growth factor I by mitogenically responsive cells. Endocrinology. 1988;122:2044–2053. doi: 10.1210/endo-122-5-2044. [DOI] [PubMed] [Google Scholar]
  44. Mukherjee S, Tessema M, Wandinger-Ness A. Vesicular trafficking of tyrosine kinase receptors and associated proteins in the regulation of signaling and vascular function. Circ Res. 2006;98:743–756. doi: 10.1161/01.RES.0000214545.99387.e3. [DOI] [PubMed] [Google Scholar]
  45. Edwall D, Schalling M, Jennische E, Norstedt G. Induction of insulin-like growth factor I messenger ribonucleic acid during regeneration of rat skeletal muscle. Endocrinology. 1989;124:820–825. doi: 10.1210/endo-124-2-820. [DOI] [PubMed] [Google Scholar]
  46. Raben N, Hill V, Shea L, Takikita S, Baum R, Mizushima N, Ralston E, Plotz P. Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease. Hum Mol Genet. 2008;17:3897–3908. doi: 10.1093/hmg/ddn292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Meikle P J, Yan M, Ravenscroft E M, Isaac E L, Hopwood J J, Brooks D A. Altered trafficking and turnover of LAMP-1 in Pompe disease-affected cells. Mol Genet Metab. 1999;66:179–188. doi: 10.1006/mgme.1998.2800. [DOI] [PubMed] [Google Scholar]
  48. Anderson L V, Davison K, Moss J A, Young C, Cullen M J, Walsh J, Johnson M A, Bashir R, Britton S, Keers S, Argov Z, Mahjneh I, Fougerousse F, Beckmann J S, Bushby K M. Dysferlin is a plasma membrane protein and is expressed early in human development. Hum Mol Genet. 1999;8:855–861. doi: 10.1093/hmg/8.5.855. [DOI] [PubMed] [Google Scholar]
  49. Davis D B, Delmonte A J, Ly C T, McNally E M. Myoferlin, a candidate gene and potential modifier of muscular dystrophy. Hum Mol Genet. 2000;9:217–226. doi: 10.1093/hmg/9.2.217. [DOI] [PubMed] [Google Scholar]

Articles from The FASEB Journal are provided here courtesy of The Federation of American Societies for Experimental Biology

RESOURCES