Donghoon M. Lee

JunB transcription factor maintains skeletal muscle mass and promotes hypertrophy

Decreasing JunB expression causes muscle atrophy, whereas overexpression induces hypertrophy and blocks atrophy via myostatin inhibition and regulation of atrogin-1 and MuRF expression via FoxO3.

SIRT1 Protein, by Blocking the Activities of Transcription Factors FoxO1 and FoxO3, Inhibits Muscle Atrophy and Promotes Muscle Growth

Background: SIRT1 regulates the activity of FoxO transcription factors and protects tissues from diverse insults. Results: Upon fasting, SIRT1 levels fall in skeletal muscle, but SIRT1 overexpression deacetylates FoxO3 and thus inhibits induction of atrophy-related genes. Conclusion: SIRT1 overexpression blocks muscle atrophy induced by fasting and denervation and in fed mice promotes hypertrophy. Significance: SIRT1 activity is important in the regulation of muscle size. In several cell types, the protein deacetylase SIRT1 regulates the activities of FoxO transcription factors whose activation is critical in muscle atrophy. However, the possible effects of SIRT1 on the activity of FoxOs in skeletal muscle and on the regulation of muscle size have not been investigated. Here, we show that after food deprivation, SIRT1 levels fall dramatically in type II skeletal muscles (tibialis anterior), which show marked atrophy, unlike in the liver (where SIRT1 rises) or heart or the soleus, a type I muscle (where SIRT1 is unchanged). Maintenance of high SIRT1 levels by electroporation in mouse muscle inhibits markedly the muscle wasting induced by fasting as well as by denervation, and these protective effects require its deacetylase activity. SIRT1 overexpression reduces muscle wasting by blocking the activation of FoxO1 and 3. It thus prevents the induction of key atrogenes, including the muscle-specific ubiquitin ligases, atrogin1 and MuRF1, and multiple autophagy (Atg) genes and the increase in overall proteolysis. In normal muscle, SIRT1 overexpression by electroporation causes rapid fiber hypertrophy without, surprisingly, activation of the PI3K-AKT signaling pathway. Thus, SIRT1 activation favors postnatal muscle growth, and its fall appears to be critical for atrophy during fasting. Consequently, SIRT1 activation represents an attractive possible pharmacological approach to prevent muscle wasting and cachexia.

Trim32 reduces PI3K–Akt–FoxO signaling in muscle atrophy by promoting plakoglobin–PI3K dissociation

By promoting dissociation of the desmosomal component plakoglobin from PI3K, the ubiquitin ligase Trim32 reduces PI3K–Akt–FoxO signaling in normal and atrophying muscle, potentially contributing to insulin resistance and catabolic disorders.

A Modifier Screen for Bazooka/PAR-3 Interacting Genes in the Drosophila Embryo Epithelium

Background The development and homeostasis of multicellular organisms depends on sheets of epithelial cells. Bazooka (Baz; PAR-3) localizes to the apical circumference of epithelial cells and is a key hub in the protein interaction network regulating epithelial structure. We sought to identify additional proteins that function with Baz to regulate epithelial structure in the Drosophila embryo. Methodology/Principal Findings The baz zygotic mutant cuticle phenotype could be dominantly enhanced by loss of known interaction partners. To identify additional enhancers, we screened molecularly defined chromosome 2 and 3 deficiencies. 37 deficiencies acted as strong dominant enhancers. Using deficiency mapping, bioinformatics, and available single gene mutations, we identified 17 interacting genes encoding known and predicted polarity, cytoskeletal, transmembrane, trafficking and signaling proteins. For each gene, their loss of function enhanced adherens junction defects in zygotic baz mutants during early embryogenesis. To further evaluate involvement in epithelial polarity, we generated GFP fusion proteins for 15 of the genes which had not been found to localize to the apical domain previously. We found that GFP fusion proteins for Drosophila ASAP, Arf79F, CG11210, Septin 5 and Sds22 could be recruited to the apical circumference of epithelial cells. Nine of the other proteins showed various intracellular distributions, and one was not detected. Conclusions/Significance Our enhancer screen identified 17 genes that function with Baz to regulate epithelial structure in the Drosophila embryo. Our secondary localization screen indicated that some of the proteins may affect epithelial cell polarity by acting at the apical cell cortex while others may act through intracellular processes. For 13 of the 17 genes, this is the first report of a link to baz or the regulation of epithelial structure.

Polarized E-cadherin endocytosis directs actomyosin remodeling during embryonic wound repair

Clathrin, dynamin, and ARF6 accumulate around wounds in Drosophila embryos in a calcium- and actomyosin-dependent manner and drive polarized E-cadherin endocytosis, which is necessary for actomyosin remodeling during wound repair.

An Arf-GEF Regulates Antagonism between Endocytosis and the Cytoskeleton for Drosophila Blastoderm Development

BACKGROUND Actin cytoskeletal networks push and pull the plasma membrane (PM) to control cell structure and behavior. Endocytosis also regulates the PM and can be promoted or inhibited by cytoskeletal networks. However, endocytic regulation of the general membrane cytoskeleton is undocumented. RESULTS Here, we provide evidence for endocytic inhibition of actomyosin networks. Specifically, we find that Steppke, a cytohesin Arf-guanine nucleotide exchange factor (GEF), controls initial PM furrow ingression during the syncytial nuclear divisions and cellularization of the Drosophila embryo. Acting at the tips of ingressing furrows, Steppke promotes local endocytic events through its Arf-GEF activity and in cooperation with the AP-2 clathrin adaptor complex. These Steppke activities appear to reduce local Rho1 protein levels and ultimately restrain actomyosin networks. Without Steppke, Rho1 pathways linked to actin polymerization and myosin activation abnormally expand the membrane cytoskeleton into taut sheets emanating perpendicularly from the furrow tips. These expansions lead to premature cellularization and abnormal expulsions of nuclei from the forming blastoderm. Finally, consistent with earlier reports, we also find that actomyosin activity can act reciprocally to inhibit the endocytosis at furrow tips. CONCLUSIONS We propose that Steppke-dependent endocytosis keeps the cytoskeleton in check as early PM furrows form. Specifically, a cytohesin Arf-GEF-Arf G protein-AP-2 endocytic axis appears to antagonize Rho1 cytoskeletal pathways to restrain the membrane cytoskeleton. However, as furrows lengthen during cellularization, the cytoskeleton gains strength, blocks the endocytic inhibition, and finally closes off the base of each cell to form the blastoderm.

Muscle Wasting in Fasting Requires Activation of NF-κB and Inhibition of AKT/Mechanistic Target of Rapamycin (mTOR) by the Protein Acetylase, GCN5.

NF-κB is best known for its pro-inflammatory and anti-apoptotic actions, but in skeletal muscle, NF-κB activation is important for atrophy upon denervation or cancer. Here, we show that also upon fasting, NF-κB becomes activated in muscle and is critical for the subsequent atrophy. Following food deprivation, the expression and acetylation of the p65 of NF-κB on lysine 310 increase markedly in muscles. NF-κB inhibition in mouse muscles by overexpression of the IκBα superrepressor (IκBα-SR) or of p65 mutated at Lys-310 prevented atrophy. Knockdown of GCN5 with shRNA or a dominant-negative GCN5 or overexpression of SIRT1 decreased p65K310 acetylation and muscle wasting upon starvation. In addition to reducing atrogene expression, surprisingly inhibiting NF-κB with IκBα-SR or by GCN5 knockdown in these muscles also enhanced AKT and mechanistic target of rapamycin (mTOR) activities, which also contributed to the reduction in atrophy. These new roles of NF-κB and GCN5 in regulating muscle proteolysis and AKT/mTOR signaling suggest novel approaches to combat muscle wasting.

Atrogin1/MAFbx: What Atrophy, Hypertrophy, and Cardiac Failure Have in Common

See related article, pages 161–171 Proteins in cardiac and skeletal muscle cells, as in other cells, are continually being synthesized and degraded back to their constituent amino acids. Protein turnover in cardiac myocytes utilizes the same proteolytic systems as other eukaryotic cells: the ubiquitin-proteasome pathway, which catalyzes the rapid degradation of misfolded and regulatory proteins, and the lysosomal-autophagic system, which degrades organelles and aggregated proteins. These systems are of major importance in determining cardiac size and functional capacity. The overall rates of proteolysis in a cell and the degradation of individual components are precisely regulated. For example, cardiac hypertrophy occurs when overall rates of protein synthesis exceed overall rates of protein degradation; conversely cells decrease in mass when degradation rates exceed synthesis, as occurs in skeletal muscle with disuse, fasting, and many systemic diseases, including cardiac failure. In addition, the levels of individual proteins, whether they are enzymes, transcription factors, or components of the sarcomere, are determined in large part by their rates of ubiquitin-mediated degradation. In this issue of Circulation Research , Usui et al1 demonstrate how a single ubiquitination enzyme can have major effects on cardiac growth and function. In this pathway, proteins are targeted for degradation by the 26S proteasome by covalent attachment of a chain of ubiquitin molecules. This multistep pathway first involves the activation of the small protein ubiquitin by an enzyme, E1, which then transfers the highly reactive ubiquitin to one of the cells many ubiquitin-conjugating enzymes, E2s. A ubiquitin protein ligase, E3, then binds the protein substrate and the ubiquitin-E2 and catalyzes the formation of a chain of ubiquitins on the protein. Different E2-E3 pairs function in the degradation of different proteins, and the specificity of the E3s for specific groups of proteins provides exquisite selectivity to this degradation process. The content …

Coordinating the cytoskeleton and endocytosis for regulated plasma membrane growth in the early Drosophila embryo.

Plasma membrane organization is under the control of cytoskeletal networks and endocytic mechanisms, and a growing literature is showing how closely these influences are interconnected. Here, we review how plasma membranes are formed around individual nuclei of the syncytial Drosophila embryo. Specifically, we outline the pathways that promote and maintain the growth of pseudocleavage and cellularization furrows, as well as specific pathways that keep furrow growth in check. This system has become important for studies of actin regulators, such as Rho1, Diaphanous, non-muscle myosin II and Arp2/3, and endocytic regulators, such as a cytohesin Arf-GEF (Steppke), clathrin, Amphiphysin and dynamin. More generally, it provides a model for understanding how cytoskeletal-endocytic cross-talk regulates the assembly of a cell.

Germ Cell Segregation from the Drosophila Soma Is Controlled by an Inhibitory Threshold Set by the Arf-GEF Steppke

Germline cells segregate from the soma to maintain their totipotency, but the cellular mechanisms of this segregation are unclear. The Drosophila melanogaster embryo forms a posterior group of primordial germline cells (PGCs) by their division from the syncytial soma. Extended plasma membrane furrows enclose the PGCs in response to the germ plasm protein Germ cell-less (Gcl) and Rho1–actomyosin activity. Recently, we found that loss of the Arf-GEF Steppke (Step) leads to similar Rho1-dependent plasma membrane extensions but from pseudocleavage furrows of the soma. Here, we report that the loss of step also leads to premature formation of a large cell group at the anterior pole of the embryo . These anterior cells lacked germ plasm, but budded and formed at the same time as posterior PGCs, and then divided asynchronously as PGCs also do. With genetic analyses we found that Step normally activates Arf small G proteins and antagonizes Rho1–actomyosin pathways to inhibit anterior cell formation. A uniform distribution of step mRNA around the one-cell embryo cortex suggested that Step restricts cell formation through a global control mechanism. Thus, we examined the effect of Step on PGC formation at the posterior pole. Reducing Gcl or Rho1 levels decreased PGC numbers, but additional step RNAi restored their numbers. Reciprocally, GFP–Step overexpression induced dosage- and Arf-GEF-dependent loss of PGCs, an effect worsened by reducing Gcl or actomyosin pathway activity. We propose that a global distribution of Step normally sets an inhibitory threshold for Rho1 activity to restrict early cell formation to the posterior.