DAAM1 Is a Formin Required for Centrosome Re-Orientation during Cell Migration

BACKGROUND: Disheveled-associated activator of morphogenesis 1 (DAAM1) is a formin acting downstream of Wnt signaling that is important for planar cell polarity. It has been shown to promote proper cell polarization during embryonic development in both Xenopus and Drosophila. Importantly, DAAM1 binds to Disheveled (Dvl) and thus functions downstream of the Frizzled receptors. Little is known of how DAAM1 is localized and functions in mammalian cells. We investigate here how DAAM1 affects migration and polarization of cultured cells and conclude that it plays a key role in centrosome polarity. METHODOLOGY/PRINCIPAL FINDINGS: Using a specific antibody to DAAM1, we find that the protein localizes to the acto-myosin system and co-localizes with ventral myosin IIB-containing actin stress fibers. These fibers are particularly evident in the sub-nuclear region. An N-terminal region of DAAM1 is responsible for this targeting and the DAAM1(1-440) protein can interact with myosin IIB fibers independently of either F-actin or RhoA binding. We also demonstrate that DAAM1 depletion inhibits Golgi reorientation in wound healing assays. Wound-edge cells exhibit multiple protrusions characteristic of unpolarized cell migration. Finally, in U2OS cells lines stably expressing DAAM1, we observe an enhanced myosin IIB stress fiber network which opposes cell migration. CONCLUSIONS/SIGNIFICANCE: This work highlights the importance of DAAM1 in processes underlying cell polarity and suggests that it acts in part by affecting the function of acto-myosin IIB system. It also emphasizes the importance of the N-terminal half of DAAM1. DAAM1 depletion strongly blocks centrosomal re-polarization, supporting the concept that DAAM1 signaling cooperates with the established Cdc42 associated polarity complex. These findings are also consistent with the observation that ablation of myosin IIB but not myosin IIA results in polarity defects downstream of Wnt signaling. The structure-function analysis of DAAM1 in cultured cells parallels more complex morphological events in the developing embryo

A Genome-Wide Association Study of Diabetic Kidney Disease in Subjects With Type 2 Diabetes

Identification of sequence variants robustly associated with predisposition to diabetic kidney disease (DKD) has the potential to provide insights into the pathophysiological mechanisms responsible. We conducted a genome-wide association study (GWAS) of DKD in type 2 diabetes (T2D) using eight complementary dichotomous and quantitative DKD phenotypes: the principal dichotomous analysis involved 5,717 T2D subjects, 3,345 with DKD. Promising association signals were evaluated in up to 26,827 subjects with T2D (12,710 with DKD). A combined (T1D+T2D) GWAS was performed using complementary data available for subjects with T1D, which, with replication samples, involved up to 40,340 diabetic subjects (and 18,582 DKD cases). Analysis of specific DKD phenotypes identified a novel signal near GABRR1 (rs9942471, p=4.5×10-8) associated with 'microalbuminuria' in European T2D cases. However, no replication of this signal was observed in Asian subjects with T2D, or in the equivalent T1D analysis. There was only limited support, in this substantially enlarged analysis, for association at previously-reported DKD signals, except for those at UMOD and PRKAG2, both associated with 'EGFR'. We conclude that, despite challenges in addressing phenotypic heterogeneity, access to increased sample sizes will continue to provide more robust inference regarding risk-variant discovery for DKD.</p

Liquid Biopsies for Cancer Genotyping: Coming of Age?

Abstract Precision oncology is predicated on optimal molecular profiling that is “fit for purpose” to identify therapeutic vulnerabilities. Liquid biopsies may compensate for inadequate genotyping, but remain less sensitive and specific compared with tissue biopsies. The liquid biopsy toolbox is poised to expand through novel assays and insights from longitudinal profiling. See related article by Sugimoto et al., p. xxx </jats:sec

AP-1B facilitates endocytosis during cell migration

ABSTRACTThe epithelial cell-specific clathrin adaptor AP-1B has a well-established role in polarized sorting of cargos to the basolateral membrane. Here we demonstrate a novel function for AP-1B during collective cell migration of epithelial sheets. We show that AP-1B colocalized with β1 integrin in focal adhesions during cell migration using confocal microscopy and total internal reflection fluorescence (TIRF) microscopy on fixed specimens. Further, AP-1B labeling in cell protrusion was distinct from labeling for the canonical endocytic adaptor complex AP-2. Using stochastic optical reconstruction microscopy (STORM) and live TIRF imaging we identified numerous AP-1B-coated structures at or close to the plasma membrane in cell protrusions. Importantly, immuno-electron microscopy (EM) showed AP-1B in clathrin-coated pits and budding vesicles at the plasma membrane during cell migration. Our data therefore established a novel function for AP-1B in endocytosis. We further show that β1 integrin was dependent on AP-1B and its co-adaptor, autosomal recessive hypercholesterolemia protein (ARH), for sorting to the basolateral membrane. Notably, we found that expression of AP-1B (and ARH) slowed epithelial-cell migration, and qRT-PCR analysis of human epithelial-derived cell lines revealed a loss of AP-1B expression in highly metastatic cancer cells indicating that AP-1B-facilitated endocytosis during cell migration might be an anti-cancer mechanism.</jats:p

1661-P: NHG-KTPH Monogenic Diabetes Registry: Understanding Monogenic Diabetes in a Multiethnic Asian Population in Singapore

Introduction: Monogenic diabetes (also known as maturity-onset diabetes of the young or MODY) is a rare genetic disorder with an undetermined prevalence in Singapore. The NHG-KTPH Monogenic Diabetes Registry was set up in mid-2017 to capture longitudinal data of individuals genetically diagnosed with monogenic diabetes to study their prevalence and disease trajectories. Methods: Patients with clinical phenotype suggestive of monogenic diabetes i.e., diabetes onset =&amp;lt;45 years old, BMI &amp;lt;32.5 kg/m2 and GAD autoantibody-negative were recruited and subjected to genetic testing comprising of next-generation 16-MODY gene panel sequencing, mt.3242A&amp;gt;G TaqMan genotyping and multiplex ligation-dependent probe amplification (MLPA) for 4 common MODY genes. Genetic variants annotated as likely pathogenic/pathogenic according to ACMG guidelines were validated using Sanger sequencing. Individuals with such variant(s) were included in the Registry. EQ-5D-5L instrument was also administered for quality of life assessment. Results: Among 171 patients who were subjected to MODY genetic testing, 26 (15%) were found to harbor likely pathogenic/pathogenic variants. 93% of these variants were found in common MODY genes (HNF1A, HNF4A, GCK, HNF1B, MT-TL1) that had important clinical implications. Biomarkers such as C-peptide, hs-CRP and HDL were significantly different in those with likely pathogenic/pathogenic variants than those without. Discussion: Monogenic diabetes is present in 15% of our study participants with young-onset diabetes, which is greater than the reported 5% in UK. Clinical demographics and biomarkers can be further evaluated to improve the pick-up rate of monogenic diabetes among young-onset diabetes, with specific calibration applied to different subtypes of monogenic diabetes. This will improve cost-effectiveness of genetic testing and facilitate implementation of precision medicine for individuals with monogenic diabetes. Disclosure S. Ang: None. C. Tan: None. Y. Kon: None. L. Xia: None. S. Lim: None. </jats:sec