Charisse M. Orme

Dual-mode of insulin action controls GLUT4 vesicle exocytosis

Insulin releases an intracellular brake and promotes fusion pore expansion to translocate GLUT4 vesicles, and switches vesicle trafficking between distinct exocytic circuits.

A Large-Scale Screen for Mutagen-Sensitive Loci in Drosophila

In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212–mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314–mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215ZIII-2059, that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination. At least one allele of each new gene identified in this study is available at the Bloomington Stock Center.

Endoproteolytic Cleavage of TUG Protein Regulates GLUT4 Glucose Transporter Translocation

Background: GLUT4 glucose transporters are trapped and sequestered intracellularly in adipocytes by TUG. Results: Insulin stimulates TUG cleavage, which separates regions of TUG that bind GLUT4 and Golgi matrix proteins. Cleavage is required for highly insulin-responsive GLUT4 translocation. Conclusion: TUG proteolysis liberates GLUT4 trapped at the Golgi matrix. Significance: Endoproteolytic cleavage is a novel biochemical mechanism for insulin action to regulate glucose uptake. To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18-kDa ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kDa and also as a 54-kDa form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake.

The Ubiquitin Regulatory X (UBX) Domain-containing Protein TUG Regulates the p97 ATPase and Resides at the Endoplasmic Reticulum-Golgi Intermediate Compartment

Background: TUG controls a specialized membrane trafficking pathway in adipocytes but is widely expressed. Results: TUG converts p97 ATPase hexamers into monomers and controls Golgi membrane dynamics. Conclusion: Localized disassembly of p97 hexamers may control a generalized trafficking pathway in eukaryotic cells. Significance: Understanding how TUG and p97 function provides insight into regulation of both generalized and specialized membrane trafficking. p97/VCP is a hexameric ATPase that is coupled to diverse cellular processes, such as membrane fusion and proteolysis. How p97 activity is regulated is not fully understood. Here we studied the potential role of TUG, a widely expressed protein containing a UBX domain, to control mammalian p97. In HEK293 cells, the vast majority of TUG was bound to p97. Surprisingly, the TUG UBX domain was neither necessary nor sufficient for this interaction. Rather, an extended sequence, comprising three regions of TUG, bound to the p97 N-terminal domain. The TUG C terminus resembled the Arabidopsis protein PUX1. Similar to the previously described action of PUX1 on AtCDC48, TUG caused the conversion of p97 hexamers into monomers. Hexamer disassembly was stoichiometric rather than catalytic and was not greatly affected by the p97 ATP-binding state or by TUG N-terminal regions in vitro. In HeLa cells, TUG localized to the endoplasmic reticulum-to-Golgi intermediate compartment and endoplasmic reticulum exit sites. Although siRNA-mediated TUG depletion had no marked effect on total ubiquitylated proteins or p97 localization, TUG overexpression caused an accumulation of ubiquitylated substrates and targeted both TUG and p97 to the nucleus. A physiologic role of TUG was revealed by siRNA-mediated depletion, which showed that TUG is required for efficient reassembly of the Golgi complex after brefeldin A removal. Together, these data support a model in which TUG controls p97 oligomeric status at a particular location in the early secretory pathway and in which this process regulates membrane trafficking in various cell types.

Capillary Malformation—Arteriovenous Malformation Syndrome: Review of the Literature, Proposed Diagnostic Criteria, and Recommendations for Management

Capillary malformation–arteriovenous malformation syndrome is an autosomal dominant disorder caused by mutations in the RASA1 gene and characterized by multiple small, round to oval capillary malformations with or without arteriovenous malformations. Ateriovenous malformations occur in up to one‐third of patients and may involve the brain and spine. Although making the diagnosis is straightforward in some patients, there are other patients for whom diagnostic criteria may be helpful in their evaluation. Here we review the literature regarding capillary malformation−arteriovenous malformation syndrome, propose diagnostic criteria, and discuss the care of patients with this condition.

Sorting Out Diabetes

Altered trafficking of storage vesicles that harbor a glucose transport protein in muscle and fat tissue may contribute to diabetes. Early during the development of type 2 diabetes, insulins ability to stimulate the cellular uptake of glucose from the blood is compromised (1). Muscle is the main tissue responsible for this absorption, and insulin enhances glucose movement into muscle cells through the GLUT4 transporter at the cell surface (2, 3). This hallmark action of insulin is conserved in vertebrates, and the molecular machinery by which it occurs is thought to be similar among mammals. On page 1192 of this issue, Vassilopoulos et al. (4) identify a key protein that mediates insulin action in humans, but not in mice, a distinction with potential implications for understanding glucose metabolism and diabetes pathophysiology.

Clinically amyopathic dermatomyositis in a patient with an extramedullary plasmacytoma of the tongue

patient. To our knowledge, in the dermatological published work, there have been no previous reports of NF1 presenting with spinal deformity accompanying dural ectasia. Neuroradiological methods are important in evaluating patients with NF1 to detect possible dural and spinal anomalies. Systemic manifestations other than skin lesions could be a presenting manifestation in patients with NF1, as in our patient. Evaluation of NF1 is especially important in patients with spinal dural ectasia, because, although rare, it is relatively common in patients with NF1.

Eschar Formation from Testosterone Patch

A 78-year-old man presented for his yearly skin exam and was noted to have an oval-shaped, green-black eschar on his midback. A pink, atrophic scar was located nearby, in addition to a medicated patch of similar size.