Insulin receptor (IR) signaling is central to normal metabolic control and is dysregulated in metabolic diseases such as type 2 diabetes. We report here that IR is incorporated into dynamic clusters at the plasma membrane, in the cytoplasm and in the nucleus of human hepatocytes and adipocytes. Insulin stimulation promotes further incorporation of IR into these dynamic clusters in insulin-sensitive cells but not in insulin-resistant cells, where both IR accumulation and dynamic behavior are reduced. Treatment of insulin-resistant cells with metformin, a first-line drug used to treat type 2 diabetes, can rescue IR accumulation and the dynamic behavior of these clusters. This rescue is associated with metformin’s role in reducing reactive oxygen species that interfere with normal dynamics. These results indicate that changes in the physico-mechanical features of IR clusters contribute to insulin resistance and have implications for improved therapeutic approaches.

Guanine nucleotide exchange factors (GEFs) are enzymes that promote the activation of GTPases through GTP loading. Whole exome sequencing has identified rare variants in GEFs that are associated with disease, demonstrating that GEFs play critical roles in human development. However, the consequences of these rare variants can only be understood through measuring their effects on cellular activity. Here, we provide a detailed user-friendly protocol for purification and fluorescence-based analysis of the two GEF domains within the protein, Trio. This analysis offers a straight-forward, quantitative tool to test the activity of GEF domains on their respective GTPases, as well as utilize high-throughput screening to identify regulators and inhibitors. This protocol can be adapted for characterization of other Rho family GEFs. Such analyses are crucial for the complete understanding of the roles of GEF genetic variants in human development and disease.

OBJECTIVELumbar disc herniation (LDH) in the pediatric population is rare and exhibits unique characteristics compared with adult LDH. There are limited data regarding the safety and efficacy of minimally invasive surgery (MIS) using tubular retractors in pediatric patients with LDH. Here, the outcomes of MIS tubular microdiscectomy for the treatment of pediatric LDH are evaluated.METHODSTwelve consecutive pediatric patients with LDH were treated with MIS tubular microdiscectomy at the authors’ institution between July 2011 and October 2015. Data were gathered from retrospective chart review and from mail or electronic questionnaires. The Macnab criteria and the Oswestry Disability Index (ODI) were used for outcome measurements.RESULTSThe mean age at surgery was 17 ± 1.6 years (range 13–19 years). Seven patients were female (58%). Prior to surgical intervention, 100% of patients underwent conservative treatment, and 50% had epidural steroid injections. Preoperative low-back and leg pain, positive straight leg raise, and myotomal leg weakness were noted in 100%, 83%, and 67% of patients, respectively. The median duration of symptoms prior to surgery was 9 months (range 1–36 months). The LDH level was L5–S1 in 75% of patients and L4–5 in 25%. The mean ± SD operative time was 90 ± 21 minutes, the estimated blood loss was ≤ 25 ml in 92% of patients (maximum 50 ml), and no intraoperative or postoperative complications were noted at 30 days. The median hospital length of stay was 1 day (range 0–3 days). The median follow-up duration was 2.2 years (range 0–5.8 years). One patient experienced reherniation at 18 months after the initial operation and required a second same-level MIS tubular microdiscectomy to achieve resolution of symptoms. Of the 11 patients seen for follow-up, 10 patients (91%) reported excellent or good satisfaction according to the Macnab criteria at the last follow-up. Only 1 patient reported a fair level of satisfaction by using the same criteria. Seven patients completed an ODI evaluation at the last follow-up. For these 7 patients, the mean ODI low-back pain score was 19.7% (SEM 2.8%).CONCLUSIONSTo the authors’ knowledge, this is the longest outcomes study and the largest series of pediatric patients with LDH who were treated with MIS microdiscectomy using tubular retractors. These data suggest that MIS tubular microdiscectomy is safe and efficacious for pediatric LDH. Larger prospective cohort studies with longer follow-up are needed to better evaluate the long-term efficacy of MIS tubular microdiscectomy versus other open and MIS techniques for the treatment of pediatric LDH.

Rho family GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacteriumOrientia tsutsugamushi. A proteomics-based OtDUB interaction screen identified numerous potential host interactors, including the Rho GTPases Rac1 and Cdc42. We discovered a domain in OtDUB with Rac1/Cdc42 GEF activity (OtDUBGEF), with higher activity toward Rac1 in vitro. While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of OtDUBGEFalone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, with a unique topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF domains. Structure-guided mutational analyses identified residues critical for activity and a mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 preferentially in cells, and expression of the OtDUBGEFalone alters cell morphology. Cumulatively, this work reveals a bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to induce changes in cytoskeletal structure.

17Rho family GTPases regulate an array of cellular processes and are often modulated by 18 pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor 19 (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia 20 tsutsugamushi. A proteomics-based OtDUB interaction screen identified numerous potential host 21 interactors, including the Rho-GTPases Rac1 and Cdc42. We discovered a new domain in 22OtDUB with Rac1/Cdc42 GEF activity (OtDUB GEF ), with higher activity toward Rac1 in vitro. 23While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of 24 OtDUB GEF alone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, 25 with a distinct topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF 26 domains. Structure-guided mutational analyses identified residues critical for activity and a novel 27 mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 28 preferentially in cells, and expression of the OtDUB GEF alone alters cell morphology. 29Cumulatively, this work reveals a novel bacterial GEF within the multifunctional OtDUB that 30 co-opts host Rac1 signaling to evoke changes in cytoskeletal structure. 31 motif important for folding and structural integrity) or the SopE family (SopE, SopE2, and 55 BopE). These bacterial effectors share no sequence or structural homology to eukaryotic Rho 56 GEFs, which predominantly belong to the Dbl homology (DH) family of GEFs typically formed 57 by a six-helix bundle with an elongated, kinked "chaise lounge" fold. 8,9 Rather, bacterial effector 58GEFs adopt a characteristic compact V-shaped fold, yet activate the Rho GTPases via the same 59 contact regions in the GTPases that are crucial for nucleotide exchange by DH-family GEFs. 10 60While substantial effort has been exerted in detailing the molecular determinants of bacterial 61 GEF activities and specificities, no bacterial effector GEFs have been identified outside of the 62 WxxxE/SopE-like families. 63Recently, we identified and characterized a putative effector protein, OtDUB, from the 64 obligate intracellular bacterium that causes scrub typhus, Orientia tsutsugamushi. Despite 65 extensive characterization of the OtDUB deubiquitylase (DUB) domain (residues 1-259), the 66 function of the extensive C-terminal region, encompassing more than 1000 amino acids, 67 remained elusive. 11 Here, we report that the OtDUB encodes a novel GEF domain. Using 68 biochemical, structural, and cellular methods, we demonstrate that the OtDUB GEF predominantly 69 activates Rac1 in vitro and in vivo, and that its three-dimensional structure differs drastically in 70 topological and helical arrangement from both the WxxxE and SopE effector GEFs. We further 71 determined the OtDUB GEF :Rac1 complex crystal structure, which reveals that despite its 72 divergence in primary sequence and topology, the OtDUB GEF has convergently evolved the V-73 shaped fold of bacterial GEFs and interacts with ...