Nuclei of Non-Muscle Cells Bind Centrosome Proteins upon Fusion with Differentiating Myoblasts

Background

In differentiating myoblasts, the microtubule network is reorganized from a centrosome-bound, radial array into parallel fibres, aligned along the long axis of the cell. Concomitantly, proteins of the centrosome relocalize from the pericentriolar material to the outer surface of the nucleus. The mechanisms that govern this relocalization are largely unknown.

Methodology

In this study, we perform experiments in vitro and in cell culture indicating that microtubule nucleation at the centrosome is reduced during myoblast differentiation, while nucleation at the nuclear surface increases. We show in heterologous cell fusion experiments, between cultures of differentiating mouse myoblasts and human cells of non-muscular origin, that nuclei from non-muscle cells recruit centrosome proteins once fused with the differentiating myoblasts. This recruitment still occurs in the presence of cycloheximide and thus appears to be independent of new protein biosynthesis.

Conclusions

Altogether, our data suggest that nuclei of undifferentiated cells have the dormant potential to bind centrosome proteins, and that this potential becomes activated during myoblast differentiation.     

Expression and functional roles of angiopoietin-2 in skeletal muscles

Background

Angiopoietin-1 (ANGPT1) and angiopoietin-2 (ANGPT2) are angiogenesis factors that modulate endothelial cell differentiation, survival and stability. Recent studies have suggested that skeletal muscle precursor cells constitutively express ANGPT1 and adhere to recombinant ANGPT1 and ANGPT2 proteins. It remains unclear whether or not they also express ANGPT2, or if ANGPT2 regulates the myogenesis program of muscle precursors. In this study, ANGPT2 regulatory factors and the effects of ANGPT2 on proliferation, migration, differentiation and survival were identified in cultured primary skeletal myoblasts. The cellular networks involved in the actions of ANGPT2 on skeletal muscle cells were also analyzed.

Methodology/Principal Findings

Primary skeletal myoblasts were isolated from human and mouse muscles. Skeletal myoblast survival, proliferation, migration and differentiation were measured in-vitro in response to recombinant ANGPT2 protein and to enhanced ANGPT2 expression delivered with adenoviruses. Real-time PCR and ELISA measurements revealed the presence of constitutive ANGPT2 expression in these cells. This expression increased significantly during myoblast differentiation into myotubes. In human myoblasts, ANGPT2 expression was induced by H₂O₂, but not by TNFα, IL1β or IL6. ANGPT2 significantly enhanced myoblast differentiation and survival, but had no influence on proliferation or migration. ANGPT2-induced survival was mediated through activation of the ERK1/2 and PI-3 kinase/AKT pathways. Microarray analysis revealed that ANGPT2 upregulates genes involved in the regulation of cell survival, protein synthesis, glucose uptake and free fatty oxidation.

Conclusion/Significance

Skeletal muscle precursors constitutively express ANGPT2 and this expression is upregulated during differentiation into myotubes. Reactive oxygen species exert a strong stimulatory influence on muscle ANGPT2 expression while pro-inflammatory cytokines do not. ANGPT2 promotes skeletal myoblast survival and differentiation. These results suggest that muscle-derived ANGPT2 production may play a positive role in skeletal muscle fiber repair.     

Absence of Functional Leptin Receptor Isoforms in the POUND (Leprdb/lb) Mouse Is Associated with Muscle Atrophy and Altered Myoblast Proliferation and Differentiation

Objective

Leptin receptors are abundant in human skeletal muscle, but the role of leptin in muscle growth, development and aging is not well understood. Here we utilized a novel mouse model lacking all functional leptin receptor isoforms (POUND mouse, Lepr^db/lb) to determine the role of leptin in skeletal muscle.

Methods and Findings

Skeletal muscle mass and fiber diameters were examined in POUND mice, and primary myoblast cultures were used to determine the effects of altered leptin signaling on myoblast proliferation and differentiation. ELISA assays, integrated pathway analysis of mRNA microarrays, and reverse phase protein analysis were performed to identify signaling pathways impacted by leptin receptor deficiency. Results show that skeletal muscle mass and fiber diameter are reduced 30–40% in POUND mice relative to wild-type controls. Primary myoblast cultures demonstrate decreased proliferation and decreased expression of both MyoD and myogenin in POUND mice compared to normal mice. Leptin treatment increased proliferation in primary myoblasts from muscles of both adult (12 months) and aged (24 months) wild-type mice, and leptin increased expression of MyoD and myogenin in aged primary myoblasts. ELISA assays and protein arrays revealed altered expression of molecules associated with the IGF-1/Akt and MAPK/MEK signaling pathways in muscle from the hindlimbs of mice lacking functional leptin receptors.

Conclusion

These data support the hypothesis that the adipokine leptin is a key factor important for the regulation of skeletal muscle mass, and that leptin can act directly on its receptors in peripheral tissues to regulate cell proliferation and differentiation.     

The cortical cytoskeletal network and cell-wall dynamics in the unicellular charophycean green alga Penium margaritaceum

Background and Aims

Penium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division.

Methods

Microtubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy.

Key Results

The cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division.

Conclusions

The cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre-division wall expansion and the zone where a cross wall will form during cytokinesis. This suggests that prior to the evolution of land plants, a dynamic, cortical cytoskeletal array similar to a pre-prophase band had evolved in the charophytes. However, an interesting variation on the cortical band theme is present in Penium, where two satellite microtubule bands are produced at the onset of cell expansion, each of which is destined to become an IMB in the two daughter cells after cytokinesis. These unique cytoskeletal components demonstrate the close temporal control and highly coordinated cytoskeletal dynamics of cellular development in Penium.     

Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays

Background and Aims

The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-β-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs.

Methods

Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy.

Results

Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes.

Conclusions

The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule reorganization and may define microtubule ring disposition.     

GSK3 regulates mitotic chromosomal alignment through CRMP4

Background

Glycogen Synthase Kinase 3 (GSK3) has been implicated in regulating chromosomal alignment and mitotic progression but the physiological substrates mediating these GSK3-dependent effects have not been identified. Collapsin Response Mediator Protein 4 (CRMP4) is a cytosolic phosphoprotein known to regulate cytoskeletal dynamics and is a known physiological substrate of GSK3. In this study, we investigate the role of CRMP4 during mitosis.

Methodology and Principal Findings

Here we demonstrate that during mitosis CRMP4 phosphorylation is regulated in a GSK3-dependent manner. We show that CRMP4 localizes to spindle microtubules during mitosis and loss of CRMP4 disrupts chromosomal alignment and mitotic progression. The effect of CRMP4 on chromosomal alignment is dependent on phosphorylation by GSK3 identifying CRMP4 as a critical GSK3 substrate during mitotic progression. We also provide mechanistic data demonstrating that CRMP4 regulates spindle microtubules consistent with its known role in the regulation of the microtubule cytoskeleton.

Conclusion and Significance

Our findings identify CRMP4 as a key physiological substrate of GSK3 in regulating chromosomal alignment and mitotic progression through its effect on spindle microtubules.     

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

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.     

Expression Levels of a Kinesin-13 Microtubule Depolymerase Modulates the Effectiveness of Anti-Microtubule Agents

Background

Chemotheraputic drugs often target the microtubule cytoskeleton as a means to disrupt cancer cell mitosis and proliferation. Anti-microtubule drugs inhibit microtubule dynamics, thereby triggering apoptosis when dividing cells activate the mitotic checkpoint. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs); however, we lack a comprehensive understanding about how anti-microtubule agents functionally interact with MAPs. In this report, we test the hypothesis that the cellular levels of microtubule depolymerases, in this case kinesin-13 s, modulate the effectiveness of the microtubule disrupting drug colchicine.

Methodology/Principal Findings

We used a combination of RNA interference (RNAi), high-throughput microscopy, and time-lapse video microscopy in Drosophila S2 cells to identify a specific MAP, kinesin-like protein 10A (KLP10A), that contributes to the efficacy of the anti-microtubule drug colchicine. KLP10A is an essential microtubule depolymerase throughout the cell cycle. We find that depletion of KLP10A in S2 cells confers resistance to colchicine-induced microtubule depolymerization to a much greater extent than depletion of several other destabilizing MAPs. Using image-based assays, we determined that control cells retained 58% (±2%SEM) of microtubule polymer when after treatment with 2 µM colchicine for 1 hour, while cells depleted of KLP10A by RNAi retained 74% (±1%SEM). Likewise, overexpression of KLP10A-GFP results in increased susceptibility to microtubule depolymerization by colchicine.

Conclusions/Significance

Our results demonstrate that the efficacy of microtubule destabilization by a pharmacological agent is dependent upon the cellular expression of a microtubule depolymerase. These findings suggest that expression levels of Kif2A, the human kinesin-13 family member, may be an attractive biomarker to assess the effectiveness of anti-microtubule chemotherapies. Knowledge of how MAP expression levels affect the action of anti-microtubule drugs may prove useful for evaluating possible modes of cancer treatment.     

Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

Background

TRPV4 and the cellular cytoskeleton have each been reported to influence cellular mechanosensitive processes as well as the development of mechanical hyperalgesia. If and how TRPV4 interacts with the microtubule and actin cytoskeleton at a molecular and functional level is not known.

Methodology and Principal Findings

We investigated the interaction of TRPV4 with cytoskeletal components biochemically, cell biologically by observing morphological changes of DRG-neurons and DRG-neuron-derived F-11 cells, as well as functionally with calcium imaging. We find that TRPV4 physically interacts with tubulin, actin and neurofilament proteins as well as the nociceptive molecules PKCε and CamKII. The C-terminus of TRPV4 is sufficient for the direct interaction with tubulin and actin, both with their soluble and their polymeric forms. Actin and tubulin compete for binding. The interaction with TRPV4 stabilizes microtubules even under depolymerizing conditions in vitro. Accordingly, in cellular systems TRPV4 colocalizes with actin and microtubules enriched structures at submembranous regions. Both expression and activation of TRPV4 induces striking morphological changes affecting lamellipodial, filopodial, growth cone, and neurite structures in non-neuronal cells, in DRG-neuron derived F11 cells, and also in IB4-positive DRG neurons. The functional interaction of TRPV4 and the cytoskeleton is mutual as Taxol, a microtubule stabilizer, reduces the Ca²⁺-influx via TRPV4.

Conclusions and Significance

TRPV4 acts as a regulator for both, the microtubule and the actin. In turn, we describe that microtubule dynamics are an important regulator of TRPV4 activity. TRPV4 forms a supra-molecular complex containing cytoskeletal proteins and regulatory kinases. Thereby it can integrate signaling of various intracellular second messengers and signaling cascades, as well as cytoskeletal dynamics. This study points out the existence of cross-talks between non-selective cation channels and cytoskeleton at multiple levels. These cross talks may help us to understand the molecular basis of the Taxol-induced neuropathic pain development commonly observed in cancer patients.     

Low Glucose but Not Galactose Enhances Oxidative Mitochondrial Metabolism in C2C12 Myoblasts and Myotubes

Background

Substituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes.

Methodology/Principal Findings

We measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels.

Conclusions/Significance

Our results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological concentrations of glucose cause mitochondrial dysfunction in C2C12 myoblasts and myotubes.     

Remodeling lipid metabolism and improving insulin responsiveness in human primary myotubes

Objective

Disturbances in lipid metabolism are strongly associated with insulin resistance and type 2 diabetes (T2D). We hypothesized that activation of cAMP/PKA and calcium signaling pathways in cultured human myotubes would provide further insight into regulation of lipid storage, lipolysis, lipid oxidation and insulin responsiveness.

Methods

Human myoblasts were isolated from vastus lateralis, purified, cultured and differentiated into myotubes. All cells were incubated with palmitate during differentiation. Treatment cells were pulsed 1 hour each day with forskolin and ionomycin (PFI) during the final 3 days of differentiation to activate the cAMP/PKA and calcium signaling pathways. Control cells were not pulsed (control). Mitochondrial content, ¹⁴C lipid oxidation and storage were measured, as well as lipolysis and insulin-stimulated glycogen storage. Myotubes were stained for lipids and gene expression measured.

Results

PFI increased oxidation of oleate and palmitate to CO₂ (p<0.001), isoproterenol-stimulated lipolysis (p = 0.01), triacylglycerol (TAG) storage (p<0.05) and mitochondrial DNA copy number (p = 0.01) and related enzyme activities. Candidate gene and microarray analysis revealed increased expression of genes involved in lipolysis, TAG synthesis and mitochondrial biogenesis. PFI increased the organization of lipid droplets along the myofibrillar apparatus. These changes in lipid metabolism were associated with an increase in insulin-mediated glycogen storage (p<0.001).

Conclusions

Activation of cAMP/PKA and calcium signaling pathways in myotubes induces a remodeling of lipid droplets and functional changes in lipid metabolism. These results provide a novel pharmacological approach to promote lipid metabolism and improve insulin responsiveness in myotubes, which may be of therapeutic importance for obesity and type 2 diabetes.     

Activation of Wnt/β-Catenin Signaling Increases Insulin Sensitivity through a Reciprocal Regulation of Wnt10b and SREBP-1c in Skeletal Muscle Cells

Background

Intramyocellular lipid accumulation is strongly related to insulin resistance in humans, and we have shown that high glucose concentration induced de novo lipogenesis and insulin resistance in murin muscle cells. Alterations in Wnt signaling impact the balance between myogenic and adipogenic programs in myoblasts, partly due to the decrease of Wnt10b protein. As recent studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, we hypothesized that activation of Wnt signaling could play a crucial role in muscle insulin sensitivity.

Methodology/Principal Findings

Here we demonstrate that SREBP-1c and Wnt10b display inverse expression patterns during muscle ontogenesis and regeneration, as well as during satellite cells differentiation. The Wnt/β-catenin pathway was reactivated in contracting myotubes using siRNA mediated SREBP-1 knockdown, Wnt10b over-expression or inhibition of GSK-3β, whereas Wnt signaling was inhibited in myoblasts through silencing of Wnt10b. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in contracting myotubes and to activate the Wnt/β-catenin pathway. Conversely, silencing Wnt10b in myoblasts induced SREBP-1c protein expression, suggesting a reciprocal regulation. Stimulation of the Wnt/β-catenin pathway i) drastically decreased SREBP-1c protein and intramyocellular lipid deposition in myotubes; ii) increased basal glucose transport in both insulin-sensitive and insulin-resistant myotubes through a differential activation of Akt and AMPK pathways; iii) restored insulin sensitivity in insulin-resistant myotubes.

Conclusions/Significance

We conclude that activation of Wnt/β-catenin signaling in skeletal muscle cells improved insulin sensitivity by i) decreasing intramyocellular lipid deposition through downregulation of SREBP-1c; ii) increasing insulin effects through a differential activation of the Akt/PKB and AMPK pathways; iii) inhibiting the MAPK pathway. A crosstalk between these pathways and Wnt/β-catenin signaling in skeletal muscle opens the exciting possibility that organ-selective modulation of Wnt signaling might become an attractive therapeutic target in regenerative medicine and to treat obese and diabetic populations.     

Galactose enhances oxidative metabolism and reveals mitochondrial dysfunction in human primary muscle cells

Background

Human primary myotubes are highly glycolytic when cultured in high glucose medium rendering it difficult to study mitochondrial dysfunction. Galactose is known to enhance mitochondrial metabolism and could be an excellent model to study mitochondrial dysfunction in human primary myotubes. The aim of the present study was to 1) characterize the effect of differentiating healthy human myoblasts in galactose on oxidative metabolism and 2) determine whether galactose can pinpoint a mitochondrial malfunction in post-diabetic myotubes.

Methodology/Principal Findings

Oxygen consumption rate (OCR), lactate levels, mitochondrial content, citrate synthase and cytochrome C oxidase activities, and AMPK phosphorylation were determined in healthy myotubes differentiated in different sources/concentrations of carbohydrates: 25 mM glucose (high glucose (HG)), 5 mM glucose (low glucose (LG)) or 10 mM galactose (GAL). Effect of carbohydrates on OCR was also determined in myotubes derived from post-diabetic patients and matched obese non-diabetic subjects. OCR was significantly increased whereas anaerobic glycolysis was significantly decreased in GAL myotubes compared to LG or HG myotubes. This increased OCR in GAL myotubes occurred in conjunction with increased cytochrome C oxidase activity and expression, as well as increased AMPK phosphorylation. OCR of post-diabetic myotubes was not different than that of obese non-diabetic myotubes when differentiated in LG or HG. However, whereas GAL increased OCR in obese non-diabetic myotubes, it did not affect OCR in post-diabetic myotubes, leading to a significant difference in OCR between groups. The lack of an increase in OCR in post-diabetic myotubes differentiated in GAL was in relation with unaltered cytochrome C oxidase activity levels or AMPK phosphorylation.

Conclusions/Significance

Our results indicate that differentiating human primary myoblasts in GAL enhances aerobic metabolism. Because this cell culture model elicited an abnormal response in cells from post-diabetic patients, it may be useful in further studies of the molecular mechanisms of mitochondrial dysfunction.     

Atmospheric Oxygen Tension Slows Myoblast Proliferation via Mitochondrial Activation

Background

Mitochondrial activity inhibits proliferation and is required for differentiation of myoblasts. Myoblast proliferation is also inhibited by the ∼20% oxygen level used in standard tissue culture. We hypothesize that mitochondrial activity would be greater at hyperoxia (20% O₂) relative to more physiological oxygen (5% O₂).

Methodology/Principal Findings

Murine primary myoblasts from isolated myofibres and conditionally immortalized H-2K myoblasts were cultured at 5% and 20% oxygen. Proliferation, assayed by cell counts, EdU labeling, and CFSE dilution, was slower at 20% oxygen. Expression of MyoD in primary myoblasts was delayed at 20% oxygen, but myogenicity, as measured by fusion index, was slightly higher. FACS-based measurement of mitochondrial activity indicators and luminometric measurement of ATP levels revealed that mitochondria exhibited greater membrane potential and higher levels of Reactive Oxygen Species (ROS) at 20% oxygen with concomitant elevation of intracellular ATP. Mitochondrial mass was unaffected. Low concentrations of CCCP, a respiratory chain uncoupler, and Oligomycin A, an ATP synthase inhibitor, each increased the rate of myoblast proliferation. ROS were investigated as a potential mechanism of mitochondrial retrograde signaling, but scavenging of ROS levels by N-acetyl-cysteine (NAC) or α-Phenyl-N-tert-butylnitrone (PBN) did not rescue the suppressed rate of cell division in hyperoxic conditions, suggesting other pathways. Primary myoblasts from older mice showed a slower proliferation than those from younger adult mice at 20% oxygen but no difference at 5% oxygen.

Conclusions/Significance

These results implicate mitochondrial regulation as a mechanistic explanation for myoblast response to oxygen tension. The rescue of proliferation rate in myoblasts of aged mice by 5% oxygen suggests a major artefactual component to age-related decline of satellite cell proliferation in standard tissue culture at 20% oxygen. It lends weight to the idea that these age-related changes result at least in part from environmental factors rather than characteristics intrinsic to the satellite cell.     

Cellular Contractility Requires Ubiquitin Mediated Proteolysis

Background

Cellular contractility, essential for cell movement and proliferation, is regulated by microtubules, RhoA and actomyosin. The RhoA dependent kinase ROCK ensures the phosphorylation of the regulatory Myosin II Light Chain (MLC) Ser19, thereby activating actomyosin contractions. Microtubules are upstream inhibitors of contractility and their depolymerization or depletion cause cells to contract by activating RhoA. How microtubule dynamics regulates RhoA remains, a major missing link in understanding contractility.

Principal Findings

We observed that contractility is inhibited by microtubules not only, as previously reported, in adherent cells, but also in non-adhering interphase and mitotic cells. Strikingly we observed that contractility requires ubiquitin mediated proteolysis by a Cullin-RING ubiquitin ligase. Inhibition of proteolysis, ubiquitination and neddylation all led to complete cessation of contractility and considerably reduced MLC Ser19 phosphorylation.

Conclusions

Our results imply that cells express a contractility inhibitor that is degraded by ubiquitin mediated proteolysis, either constitutively or in response to microtubule depolymerization. This degradation seems to depend on a Cullin-RING ubiquitin ligase and is required for cellular contractions.     

An intact centrosome is required for the maintenance of polarization during directional cell migration

Background

Establishing and maintaining polarization is critical during cell migration. It is known that the centrosome contains numerous proteins whose roles of organizing the microtubule network range include nucleation, stabilization and severing. It is not known whether the centrosome is necessary to maintain polarization. Due to its role as the microtubule organizing center, we hypothesize that the centrosome is necessary to maintain polarization in a migrating cell. Although there have been implications of its role in cell migration, there is no direct study of the centrosome''s role in maintaining polarization. In this study we ablate the centrosome by intracellular laser irradiation to understand the role of the centrosome in two vastly different cell types, human osteosarcoma (U2OS) and rat kangaroo kidney epithelial cells (PtK). The PtK cell line has been extensively used as a model for cytoskeletal dynamics during cell migration. The U2OS cell line serves as a model for a complex, single migrating cell.

Methodology/Principal Findings

In this study we use femtosecond near-infrared laser irradiation to remove the centrosome in migrating U2OS and PtK2 cells. Immunofluorescence staining for centrosomal markers verified successful irradiation with 94% success. A loss of cell polarization is observed between 30 and 90 minutes following removal of the centrosome. Changes in cell shape are correlated with modifications in microtubule and actin organization. Changes in cell morphology and microtubule organization were quantified revealing significant depolarization resulting from centrosome irradiation.

Conclusions/Significance

This study demonstrates that the centrosome is necessary for the maintenance of polarization during directed cell migration in two widely different cell types. Removal of the centrosome from a polarized cell results in the reorganization of the microtubule network into a symmetric non-polarized phenotype. These results demonstrate that the centrosome plays a critical role in the maintenance of cytoskeletal asymmetry during cell migration.