What is the significance of microtubules in the context of myogenesis

However, its epitope could not be mapped so far Randles et al. To address this issue, we now included -tubulin in all figures regarding centrosome activity. While -tubulin levels at centrosomes were affected by myogenin expression, the signal-to-noise ratio of -tubulin was still better than those of centriole markers like Cep and Centrin in our system and therefore allowed a reliable identification of centrosomes.

Due to the extended critique on Figure 6 from all three reviewers, we will omit the current Figure 6. Instead we focused on providing more data on the initiation of ncMTOC formation.

Previously, it has been reported that overexpression of nesprin-1 is sufficient in myoblasts to recruit the PACT domain and small amounts of endogenous PCM-1 Espigat-Georger et al. However, this recruitment was inefficient.

A more detailed study of AKAP6 domains and mutants is part of our recently accepted manuscript. The focus of the present manuscript was the upstream regulation of ncMTOC induction, where we show that myogenin preferentially activates the promoters of nesprin-1 and AKAP6 which in turn recruit centrosomal proteins and enable microtubule nucleation from the nuclear envelope.

We thank the reviewer for this feedback. The focus of this manuscript is to elucidate the initiation of ncMTOC formation in vertebrate cells, as mechanisms that initiate the switch from centrosomal to non-centrosomal MTOCs during differentiation of vertebrate cells are unknown.

The only mechanistic insight into ncMTOC induction has been gained by studying Drosophila identifying the transcription factor trachealess to be required for ncMTOC formation Brodu et al. The major finding of our study is the identification of the first transcription factor sufficient for induction of an ncMTOC and centrosomal MTOC attenuation. This is not the scope of this manuscript and has been addressed in detail in the published companion manuscript.

In addition, we show that myogenin directly regulates AKAP6 expression. Finally, we also demonstrate that overexpression of AKAP6 and nesprin-1 is sufficient to recuirt endogenous centrosomal proteins. Here, we show that overexpression of AKAP6 and nesprin-1 is sufficient to recuirt endogenous centrosomal proteins.

Yet, the more important finding is that myogenin expression in fibroblasts was sufficient to initiate the formation of a functional ncMTOC. We thank the reviewer for this suggestion. To clarify this issue, we performed chromatin immunoprecipitation and luciferase experiments, which demonstrate that myogenin binds and activates the nesprin-1 promoter in NIH3T3 fibroblasts. We thank the reviewer for pointing out the missing references to existing literature and apologize for it.

We changed Figure 1 and the corresponding section in the manuscript to clarify this issue. While we did not include all four suggested graphs, the new Figure 1 shows the percentage of C2C12 nuclei positive for MyoD, myogenin, nesprin-1, and PCM The reviewer is right in the point that the amount of PCM-1 at the centrosome also changes during the normal cell cycle.

The analysis of PCM-1 was chosen, as it is an integral part of centriolar satellites that play an important role in the transport of centrosomal proteins and centrosome assembly Prosser and Pelletier, J Cell Sci. For example, it has been shown that PCM-1 depletion impairs recruitment of ninein and Pcnt to the centrosome and disturbs microtubule organization Dammermann and Merdes, J Cell Biol. To address this issue, we included the information regarding PCM-1 in the text and expanded our analysis by analyzing microtubule regrowth as well as the centrosomal proteins pericentrin and AKAP9, the latter of which has been shown to be essential for microtubule nucleation in muscle cells Espigat-Georger et al.

Using chromatin immunoprecipitation and luciferase assays we added data to show that myogenin preferentially binds and activates the promoter of the muscle-specific -isoform of AKAP6. Together with the finding that depletion of AKAP6 results in a loss of centrosomal proteins from the nuclear envelope while nesprin-1 remains unaffected, this indicates that AKAP6 acts as an adapter between the nuclear envelope anchor nesprin-1 and centrosomal proteins.

This issues are beyond the scope of this manuscript and have been addressed in the published companion manuscript.

The focus of the manuscript is the initiation of ncMTOC. Therefore, the manuscript end now on demonstrating that myogenin binds and activates the promoters of AKAP6 and nesprin-1a and that overexpresiion of AKAP6 and nesprin-1a is sufficient to induce the recruitment of endogenous centrosomal proteins to the nuclear envelope. In order to address the raised questions regarding nuclear positioning, live cell imaging needs to be established which is beyond the scope of this study. Yet, we have added several data to provide further insides into the role of AKAP6 in nuclear positioning see answers below.

Our data show, that AKAP6 depletion abolished microtubule growth from the nuclear envelope and resulted consequently in defects in nuclear positioning. To address this issue we have assessed the intensity of detyronisated microtubules a marker for stable microtubules.

Our data reveal that AKAP depletion resulted in a reduction of detyronisated microtubules. Early nucleus positioning in myotubes after fusion can be divided in two sub-processes. First, nuclei that cluster in the center of the newly formed myotube are pushed apart to spread along the length of the myotube.

Second, nuclei are aligned on the long axis of the cell. This process is mediated by microtubule-associated motors that are at least in part recruited to the nuclear envelope via PCM1 Espigat-Georger et al. We quantified defects in nuclei spreading i. To clarify this issue, we included a more detailed description in the text and a model of how AKAP6 depletion affects nuclear positioning in Figure 6. In addition, we confirmed that AKAP6 depletion impaired nuclear envelope localization of the dynein activator pglued to the nuclear envelope in myotubes.

Thank you very much for raising this important point. As detailed below, we have performed the suggested experiments to clarify the regulation of MTOC activity at the nuclear envelope and the centrosome and added a paragraph to the discussion starting at line To address this issue, we have analyzed PCM-1 staining patterns in myogenin-depleted differentiating C2C The results are now described in lines and representative images are shown in Figure 1—figure supplement 2.

Importantly, in myogenin-depleted cultures, PCM-1 remains localized in a centrosomal pattern in cells with highly nesprinpositive nuclei. This suggests that endogenous myogenin contributes to the attenuation of the centrosome during NE-MTOC formation, which is in agreement with the observation that ectopic myogenin expression in fibroblast is sufficient to attenuate the centrosome.

We are not sure what experiment the reviewer suggests. We assume that the reviewer would like additional controls regarding the specificity of the anti-myogenin antibody or other data that substantiate our conclusion that myogenin binds to the promoter regions of Syne1 or Akap6.

To address this issue, we have included an intronic region of Syne1 or Akap6 as negative control as well as a promoter region of Desmin as positive control for the specificity of the myogenin antibody in the ChIP experiments.

The results of these new experiments, presented in Figure 2 — supplemental figure 2 and Figure 4 — supplemental figure 1 and described in lines and lines , further show that myogenin binds the promoter regions of Syne1 and Akap6. As positive control, we show that depletion of MyoD also reduces myogenin levels, which is consistent with literature as well as our findings that MyoD induces myogenin expression in fibroblasts.

These results have been added as panel C to Figure 2 — supplemental figure 1 and described in lines This is in accordance with our previous finding that overexpression of MyoD in this cell line induces myogenin expression as well as PCM-1 recruitment to the nuclear envelope Figure 2 — supplemental figure 1.

In order to address the relevance of myogenin in this system, we have repeated the experiments in the presence of siRNA targeting myogenin. In addition to adding new data to the manuscript, we have included a paragraph in the Discussion regarding the relative contributions of MyoD versus myogenin in the regulation of MTOC assembly at the nuclear envelope lines Furthermore, we adapted the model in Figure 8. The new data are shown in Figure 7 — supplement 1 and described in the text starting at line As requested, we have added information in regards to the relevance of the NE-MTOC and our data in muscle function and disease to the Introduction lines and Discussion lines Information regarding the roles of the different MTOC proteins has been added to the Introduction lines Thank you for this recommendation.

To assess the specificity of the ChIP experiments, we have performed additional experiments. For details, see response to essential revisions, point 3. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. We acknowledge the Platform for Immortalization of Human Cells at the Institute de Myologie, Paris for the generation and distribution of immortalized human myoblasts.

This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited. Article citation count generated by polling the highest count across the following sources: Crossref , PubMed Central , Scopus. Moreover, ectopic expression of AKAP6 in epithelial cells is sufficient to recruit endogenous centrosomal proteins. Finally, AKAP6 is required for cardiomyocyte hypertrophy and osteoclast bone resorption activity.

Collectively, we decipher the MTOC at the nuclear envelope as a bi-layered structure generating two pools of microtubules with AKAP6 as a key organizer. B-cell receptor BCR -mediated antigen internalization and presentation are essential for humoral memory immune responses.

Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here, we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane PM permeabilization, a process that requires BCR signaling and non-muscle myosin II activity.

B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte CM proliferation have been identified as potential candidates for functional heart repair following myocardial infarction. However, it is unclear whether the effects of these mitogens are species-specific and how they directly compare in the same cardiac setting. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues generated from NRVMs, a constitutively active mutant form of the human gene Erbb2 cahErbb2 was the most potent tested mitogen.

Persistent expression of cahErbb2 induced CM proliferation, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of Erk signaling.

Cited 0 Views Annotations Open annotations. The current annotation count on this page is being calculated. Cite this article as: eLife ;e doi: Figure 1 with 2 supplements see all. Download asset Open asset. Figure 2 with 3 supplements see all. Download elifefig2-data2-v2.

Download elifefig2-data3-v2. Figure 3. Figure 4 with 2 supplements see all. Figure 4—source data 1 Raw files and uncropped gels for Figure 4C. Download elifefig4-data2-v2. Figure 5 with 3 supplements see all. Download elifefig5-data2-v2. Figure 6 with 1 supplement see all. Figure 6—source data 1 Underlying data for graphs in Figure 6C. Figure 7 with 1 supplement see all. Figure 8. Key resources table. Morris Randles et al. The following previously published data sets were used.

Transcriptome changes during the differentiation of myoblasts into myotubes. Molecular Cell Biology 16 — Nat Genet 25 — Braun T Gautel M Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasis Nature Reviews. Molecular Cell Biology 12 — Buckingham M Rigby PW Gene regulatory networks and transcriptional mechanisms that control myogenesis Developmental Cell 28 — Holt I Specific localization of nesprinalpha2, the short isoform of nesprin-1 with a KASH domain, in developing, fetal and regenerating muscle, using a new monoclonal antibody BMC Cell Biology 17 Khodjakov A Rieder CL Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression The Journal of Cell Biology — Mature myotubes of day-8 muscle cultures were filled with well-aligned striated myofibrils.

They co-localized with MTs which were rich and overlapping in cylindrical myotube that it was hard to identify a single MT. Immunofluorescent micrographs of myotubes from day-3 muscle culture showing double staining of anti-tubulin a, b and anti—MHC c, d. Arrow in a pointing to an adhering myoblast.

Compare a with c , b with d , striated myofibrils are emerging in these developing myotubes where NITs are numerous and are orientated in precedence. The number of nuclei in such myosacs varied from several to over Those nuclei bend to cluster in central area of myosacs and commonly assume a ring-like arrangement. Those myosacs lack myofibrils but possess numerous MTs organized in radiating pattern.

When TPA-myosacs were transforred to normal medium, myofibrillogenesis recovered in more or less synchronized manner in flattened myosacs where bho detailed distribution of both structures was easier to be seen. These myofibril-depleted myosacs began to synthesize their myofibrillar proteins and reassembled them into numerous long, well-aligned myofibrils.

The influence of MTs on myofibrillogenesis was followed by allowing TPA-myosacs to recover in coloemid - or taxol containing medium in comparison with the recovery in normal medium in a time course from 3 hr. Numerous fine, long MTs radiated out from perinuclear area to cytoplasm, and a number of them approached to cell border.

Intense tubulin fluorescence concentrated in the central area of cytoplasm Fig. Striated myofibrils were dissociated into MHC-positive amorphous patches and were gradually depleted 7 Micrograph not shown. In 48hr. Most of these MHC-patehes disappeared in 72 hr. Anti-tubulin staining. Remarkable co-localization between MTs in e and myofibrils in f is shown.

Thin arrows in e and f pointing to a growth tip where only a few faintly stained MHC filameats ended at the base broad arrow while numerous MTs approached to the end thin arrow. Eoth are bipolarly arranged but no sign of further e1ongation. By 3 hr. MT was still in a radiating distribution but displaying a tendency to elongate Fig. During 16—24 hr. Most of the cytoplasmic MTs were distributed parallel to longitudinal axis and were specially prominent in cytoplasmic elongating process Fig.

A few nascent MHC-positive filaments as a sign of recovering myofibrillogenesis were shown. Amorphous MHC-patched remnants sometimes co-existed in early recovering myosacs Fig.

By 48 hr: recovery, myosacs became further elongated, resembling normal day-3 myotubes, but were more flattened. MTs were long, fine and numerous. They ran parallel to myotubular longitudinal axis, and extended to the very ends of growth tips far beyond the extension of growing myofibrils. Newly formed MHC-myofibrils, some non-striated and filamentous while some striated, were increased in number and length.

They co-localized with pre-existing MTs and ended at the base of growth tips Fig. This phenomenum, as well as that in normal developing myotubes, suggested that MTs might play roles as scaffold to support and guide the bipolar elongation of developing myofibrils.

By 3 day recovery, myotubes were comparable to those in day-7 to day-8 normal cultures. Co-localization of myofibrils and MTs was conspicuous in the belly of myobubes. However, MTs extended farther than myofibrils at growth tips. Noteworthy was the programmed myofibrillogenesis in recovering myosacs in taxol medium did proceed on schedule as well as those recovered in normal medium.

For further recovery in taxol medium, myosacs did not elongate properly. Though myofibrils became striated, increased in number, and contracted Occasionally, they did not elongate properly. EM investigation revealed that normal myofibrillar crystal-hexagonal transverse structures were interspersed by abundant MTs which intermingled or interdigitated with MHC thick filaments Fig. After 6 day exposure in taxol medium, myofibrils looked like mature normal myofibrils with clearcut striations Fig.

They co-localized with MT streaks, both were bipolarly orientated in myosacs. In summary, TPA-myosacs never elongated and recovered to normal myotube shape even they stayed in taxol medium as long as 6 days. Myofibrillogenesis in the absence of polymerized MTs was followed at the time course from 2 hr.

As shown in Fig. Double staining with anti-tubulin and anti- MHC antibodies revealed that instead of polymerized MTs, diffused tubulin fluorescence was distributed in the cytoplasm.

Tangled, non-striated MHC-positive filaments emerged in 24 hr. By 48 hr. Previous studies on the MAP6 KO mouse model were focused on neuronal function and shed light on numerous impairments [ 27 , 28 , 31 ], but no impact on skeletal muscle was demonstrated so far.

We have here characterized defects in muscle cell structure and function due to the absence of MAP6 proteins and shown that these animals experience muscle weakness with mild muscle atrophy. As muscle contraction was triggered in this study by a direct stimulation of muscle sarcolemma, it can be concluded that these mice have an intrinsic muscle dysfunction. We cannot exclude a small contribution of motor neuron dysfunction as the cause of the weakness; nevertheless, there was no sign of denervation.

It is noteworthy that the observed reduction of the mechanical performance in MAP6 KO mice is comparable to that reported in other mouse models for congenital myopathies [ 54 , 55 , 56 ], arguing that the absence of MAP6 has a direct deleterious impact on muscle function.

The general structure of the MAP6 KO mice muscle fibers was not profoundly affected, but rearrangements in microtubules and SR organization were observed. A higher density of transverse microtubules was observed, suggesting a disorganization of the thick transverse bundles of microtubules.

The EM analysis also revealed modifications of the SR which may be the result of the microtubule network disorganization, since both structures are in direct contact [ 57 , 58 ]. Although subtle, these modifications of the subcellular structure of the muscle fibers may have important consequences. The stabilization of some microtubule bundles by another MAP oMAP4 was for instance shown to play a role in muscle cell differentiation [ 8 ].

Modifications of SR structure have been observed in EDL muscles from calsequestrin-1 KO mice [ 59 ] or triadin-junctin double KO mice [ 52 ] and have been associated to defects in the contraction properties of the muscle fiber. The consequences of MAP6 deletion on the skeletal muscle function were further analyzed by calcium imaging.

Contraction of skeletal muscles is triggered by series of events leading to massive calcium efflux from SR cisternae via the intracellular RyR1 calcium channel. This altered coupling is probably very transient because when observed in EM, both RyR1- and DHPR-containing membranes respectively SR terminal cisternae and TT were normally associated, and both proteins were normally expressed.

Interestingly, it was demonstrated that MAP6 protein can stabilize microtubules against millimolar calcium concentration [ 60 ], a condition achieved only after muscle cell stimulation. It is therefore possible that the absence of MAP6 may alter the RyR1-DHPR crosstalk only during depolarization, when the cytosolic concentration of calcium rises, which would explain why no other variations are observed outside of muscle fiber stimulation.

Recently, it was also shown that microtubule detyrosination could play an important role in muscle contraction efficiency by modulating the production of reactive oxygen species and calcium transient [ 3 , 50 , 51 ]. The alterations observed in calcium release during excitation-contraction coupling are probably underlying the muscle weakness of the MAP6 KO mice.

The initial characterization of these mice showed several behavioral defects, among which a larger time spent standing still or walking at the expense of grooming and feeding [ 28 ].

Our demonstration that MAP6 KO mice also have a muscle weakness could explain in part this altered activity. Moreover, several abnormalities of the MAP6 KO mice were partially reverted using neuroleptics, leading to the proposal of this mouse line as a model for schizophrenia [ 28 ].

Interestingly, Chlorpromazine, a neuroleptic molecule used to revert MAP6 KO phenotype, was shown to increase the force in isolated muscle fibers at low concentration, by enhancing depolarization-induced calcium release [ 61 ]. A recent study also concluded to a correlative relationship between schizophrenia and muscle weakness in human patients [ 62 ]. It is therefore possible that together with a central nervous system dysfunction, skeletal muscle weakness contributes to the schizophrenia phenotype in the MAP6 KO mice.

Although several studies showed major roles of MAPs in muscle cell differentiation and function [ 4 , 8 , 13 ], our report points for the first time to a defect in the contraction properties of adult muscle due to the absence of a MAP.

The molecular bases for muscle weakness due to the absence of MAP6 probably rely on an alteration of the excitation-contraction coupling and calcium release, although we could not pinpoint a precise molecular mechanism so far. The subtle reorganization of the muscle fiber microtubule network could account for this excitation-contraction coupling alteration, but the involvement of other regulatory mechanisms could not be excluded as MAP6 proteins have been shown to interact with other proteins of the cytoskeleton like actin [ 23 ] or with Golgi elements [ 24 , 25 ].

In conclusion, our work emphasizes that MAP6 deletion in mice leads to brain alterations as well as skeletal muscle defects that contributes to the mice schizophrenia-like phenotype.

Building the Neuronal Microtubule Cytoskeleton. Sparrow JC, Schock F. The initial steps of myofibril assembly: integrins pave the way. Nat Rev Mol Cell Biol. Microtubules underlie dysfunction in duchenne muscular dystrophy. Sci Signal. Article Google Scholar.

Dystrophin is a microtubule-associated protein. J Cell Biol. Microtubules that form the stationary lattice of muscle fibers are dynamic and nucleated at Golgi elements. The organization of the Golgi complex and microtubules in skeletal muscle is fiber type-dependent.

J Neurosci. A muscle-specific variant of microtubule-associated protein 4 MAP4 is required in myogenesis. A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation. Straube A, Merdes A. EB3 regulates microtubule dynamics at the cell cortex and is required for myoblast elongation and fusion.

Curr Biol. Microtubule plus-end binding protein EB1 is necessary for muscle cell differentiation, elongation and fusion. J Cell Sci. Organelle positioning in muscles requires cooperation between two KASH proteins and microtubules. Nuclear movement during myotube formation is microtubule and dynein dependent and is regulated by Cdc42, Par6 and Par3.

EMBO Rep. MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function. Purification and assay of cold-stable microtubules and STOP protein. Methods Enzymol. Webb BC. A validated calibration method for hydrogen peroxide vapour sensors. Cloning, expression, and properties of the microtubule-stabilizing protein STOP. Nonneuronal isoforms of STOP protein are responsible for microtubule cold stability in mammalian fibroblasts.

Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells. EMBO J. STOP proteins are responsible for the high degree of microtubule stabilization observed in neuronal cells. MAP6-F is a temperature sensor that directly binds to and protects microtubules from cold-induced depolymerization.

J Biol Chem. Non-microtubular localizations of microtubule-associated protein 6 MAP6. PLoS One. Google Scholar. Microtubule-associated protein 6 mediates neuronal connectivity through Semaphorin 3E-dependent signalling for axonal growth. Nat Commun. The suppression of brain cold-stable microtubules in mice induces synaptic defects associated with neuroleptic-sensitive behavioral disorders.

Genes Dev. J Neurochem. Schizophr Bull. Microtubule stabilizer ameliorates synaptic function and behavior in a mouse model for schizophrenia. Biol Psychiatry. Post-pubertal emergence of alterations in locomotor activity in stop null mice. Rather, we believe that LKB1-induced microtubule destabilization could precede microtubule stabilization effected by EB1, EB3, and MAP4, and that a combination of stabilizing and destabilizing factors is likely to be needed to fine-tune the changes in microtubule organization and membrane events in differentiating myoblast elongation and fusion.

Based on the known downstream functions of LKB1 and the known changes required for myotube formation, there are several mechanisms by which LKB1 could facilitate the differentiation process. Our study establishes microtubule destabilization and reduced cell fusion by LKB1, although it does not rule out other contributions of LKB1 to the process. LKB1 is likely to destabilize microtubules indirectly, as it does not bind to myoblast microtubules in vitro data not shown.

Its role is likely to be associated with LKB1 kinase activity, because LKB1 substrate phosphorylation happens early by the first day of differentiation. There are several LKB1 substrates that could mediate its role in differentiation, and our work does not determine which substrate s are involved.

However, in contrast to our model, in which the ultimate effect of LKB1 activation was to destabilize microtubules, AMPK was shown to increase the rate of microtubule polymerization, an effect that would be expected to stabilize microtubules, in Vero cells [45].

Additional LKB1 substrates must also be tested. LKB1 could also reduce microtubule stability by suppressing tubulin expression, as lysates from cells with excess LKB1 contained less tubulin than control lysates, and lysates from cells with LKB1 RNAi showed progressive increases in tubulin levels. Further analysis of this possibility is also needed. Successful myoblast polarization has been proposed to be a prerequisite for fusion competence [14].

LKB1 has roles in both cell polarization and microtubule destabilization, and the degree to which these roles are separable is not completely clear.

Thus, LKB1 could be required for myoblast fusion through its effects on microtubules, cell polarization, or both, and its regulation of the two could bifurcate at many regulation points, or could be coupled. While our study does not address this level of mechanistic detail, it does suggest a useful model system in which these questions could be tested.

Muscle differentiation is not just important during embryogenesis. Muscle undergoes continuous regeneration, through the differentiation of satellite cells to myoblasts, and subsequent myoblast differentiation into myotubes and muscle fibers [48]. When muscles are injured, satellite cells are mobilized for repair by activating new fiber formation.

Thus, insights into muscle differentiation might prove useful for enhancing muscle homeostasis and repair in adults. Development of small molecules to manipulate LKB1 activity would be helpful for further investigating and altering the temporal control of differentiation. Another implication of our study is that transient use of microtubule destabilizing drugs might be useful for synchronizing muscle differentiation in vitro or in vivo.

LKB1 has been shown to play a role in the differentiation of neurites [30]. Now with our study showing a role in the differentiation of myoblasts, it would be interesting to see whether LKB1 could promote differentiation of other cell types as well. In a mouse model of the PJS cancer syndrome, LKB1 deletion in myofibroblasts appears to be sufficient to cause a polyposis syndrome [49]. This raises the possibility that introduction of myofibroblasts with wild-type LKB1 expression might play a role in preventing tumorigenesis in these patients.

Taxol and Nocodazole were purchased from Sigma Aldrich St. All other chemicals were from Sigma unless otherwise noted. Differentiation was induced when cells reached near-confluence using a standard media switch assay.

This induced differentiation within 3—4 days, depending on confluence at the time of the switch and the passage number. These were both matched between controls and other manipulations in all experiments. For immunofluorescence experiments, cells were plated on glass coverslips and allowed to adhere in growth media before differentiation was induced. They were fixed by immersion in ice-cold methanol and processed for immunofluorescence as described below.

The washout time was counted from the first PBS wash to fixation. Controls from different experiments are not comparable because of pre-warming of PBS and media in some experiments. Microtubules formed upon washout of Nocodazole were imaged by tubulin immunofluorescence.

Asters formed from the elongation of microtubules outward in all directions from the cell's centrosome. The diameter of each aster was measured by drawing a line across the aster at its maximum diameter in a single focal plane using the region measurements tool in MetaMorph software. Because the asters were small and the cells flat, a single focal plane contained the entire length of the microtubules.

Differentiation media was changed as needed without re-addition of virus. Random regions of the well were imaged to avoid bias. Cells were permeabilized in Tris-buffered saline TBS with 0. Primary and secondary antibodies were diluted in block solution and incubated for 1 hour at room temperature. Coverslips were mounted in media containing 0. All low-power fields were selected randomly to avoid bias. Overexpression of LKB1 and growth in differentiation media further increased this phosphorylation over and above that seen with trypsinization.

Adenovirus-infected cells were lysed by washing with PBS and scraping directly into sample buffer, followed by bath sonication to shear DNA and loading of equal lysate volume. Blots were multiply probed for proteins of different masses without stripping.

Microtubule destabilization is more conducive to differentiation than is microtubule stabilization. A Phase contrast images at differentiation day 2 in the presence of drugs and day 3 one day after drug washout show that both Taxol and Nocodazole cause cell rounding and prevent myoblast fusion, but washout of Nocodazole is associated with more substantial cell elongation than washout of Taxol.

B Corresponding immunofluorescence images were done on cells fixed 6 hours following drug washout. Insets show higher magnification images of multinucleate cells in controls and Nocodazole treated cultures, and cells with single nuclei in Taxol treated cells.

This shows that cells treated with both drugs express myosin heavy chain MHC, red , but only cells treated with Nocodazole show substantial cell fusion, even at 6 hours following washout. C Fusion index from the same time point as shown in B.

This showed that 60 percent of control cells expressing myosin heavy chain had fused, while only 24 percent of Taxol treated and 40 percent of Nocodazole treated cells had fused. Analyzed the data: JST. Wrote the paper: JST. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Background Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. Findings We find that a transient decrease in microtubule stabilization early during myoblast differentiation precedes the ultimate microtubule stabilization seen in differentiated myotubes.

Conclusions Reduced microtubule stability precedes myoblast differentiation and the associated ultimate microtubule stabilization seen in myotubes. Introduction Muscle fibers form in the developing embryo through the fusion of myoblasts into multinucleate myotubes. Results Microtubule stabilization prevents myoblast differentiation Differentiated myotubes show dramatically stabilized microtubules. Download: PPT. Figure 1. Forced microtubule stabilization prevents differentiation.