NDR2‐mediated Rabin8 phosphorylation is crucial for ciliogenesis by switching binding specificity from phosphatidylserine to Sec15

Primary cilia are antenna‐like sensory organelles protruding from the plasma membrane. Defects in ciliogenesis cause diverse genetic disorders. NDR2 was identified as the causal gene for a canine ciliopathy, early retinal degeneration, but its role in ciliogenesis remains unknown. Ciliary membranes are generated by transport and fusion of Golgi‐derived vesicles to the pericentrosome, a process requiring Rab11‐mediated recruitment of Rabin8, a GDP–GTP exchange factor (GEF) for Rab8, and subsequent Rab8 activation and Rabin8 binding to Sec15, a component of the exocyst that mediates vesicle tethering. This study shows that NDR2 phosphorylates Rabin8 at Ser‐272 and defects in this phosphorylation impair preciliary membrane assembly and ciliogenesis, resulting in accumulation of Rabin8‐/Rab11‐containing vesicles at the pericentrosome. Rabin8 binds to and colocalizes with GTP‐bound Rab11 and phosphatidylserine (PS) on pericentrosomal vesicles. The phospho‐mimetic S272E mutation of Rabin8 decreases affinity for PS but increases affinity for Sec15. These results suggest that NDR2‐mediated Rabin8 phosphorylation is crucial for ciliogenesis by triggering the switch in binding specificity of Rabin8 from PS to Sec15, thereby promoting local activation of Rab8 and ciliary membrane formation.     

Signaling mechanisms and functional roles of cofilin phosphorylation and dephosphorylation

Cofilin and actin-depolymerizing factor (ADF) are actin-binding proteins that play an essential role in regulating actin filament dynamics and reorganization by stimulating the severance and depolymerization of actin filaments. Cofilin/ADF are inactivated by phosphorylation at the serine residue at position 3 by LIM-kinases (LIMKs) and testicular protein kinases (TESKs) and are reactivated by dephosphorylation by the slingshot (SSH) family of protein phosphatases and chronophin. This review describes recent advances in our understanding of the signaling mechanisms regulating LIMKs and SSHs and the functional roles of cofilin phospho-regulation in cell migration, tumor invasion, mitosis, neuronal development, and synaptic plasticity. Accumulating evidence demonstrates that the phospho-regulation of cofilin/ADF is a key convergence point of cell signaling networks that link extracellular stimuli to actin cytoskeletal dynamics and that spatiotemporal control of cofilin/ADF activity by LIMKs and SSHs plays a crucial role in a diverse array of cellular and physiological processes. Perturbations in the normal control of cofilin/ADF activity underlie many pathological conditions, including cancer metastasis and neurological and cardiovascular disorders.     

Molecular Dynamics Calculations of Wild Type vs. Mutant Protein C: Relationship Between Binding Affinity to Endothelial Cell Protein C Receptor and Hereditary Disease

Abstract

Molecular dynamics simulations of the protein C γ-carboxyglutamic acid (Gla) domain and endothelial cell protein C receptor (EPCR) complex were performed to determine the effect of a hereditary disease, which results in a mutation (Gla 25 → Lys) in the protein C Gla domain. Our results suggest that the Gla 25 → Lys mutation causes a significant reduction in the binding force between protein C Gla domain and EPCR due to destabilization of the helix structure of EPCR and displacement of a Ca²⁺ ion.     

Anti‐adhesive property of P‐selectin glycoprotein ligand‐1 (PSGL‐1) due to steric hindrance effect

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is an adhesive molecule that is known to be a ligand for P‐selectin. An anti‐adhesive property of PSGL‐1 has not been previously reported. In this study, we show that PSGL‐1 expression is anti‐adhesive for adherent cells and we have elucidated the underlying mechanism. Overexpression of PSGL‐1 induced cell rounding and floating in HEK293T cells. Similar phenomena were demonstrated in other adherent cell lines with overexpression of PSGL‐1. PSGL‐1 overexpression inhibits access of antibodies to cell surface molecules such as integrins, HLA and CD25. Cells transfected with PSGL‐1 deletion mutants that lack a large part of the extracellular domain and chimeric construct expressing extracellular CD86 and intracellular PSGL‐1 only showed rounded morphology, but there are no floating cells. These results indicated that PSGL‐1 causes steric hindrance due to the extended structure of its extracellular domain that is highly O‐glycosylated, but intracellular domain also has some effect on cell rounding. This study implies that PSGL‐1 has Janus‐faced functions, being both adhesive and anti‐adhesive. J. Cell. Biochem. 114: 1271–1285, 2013. © 2013 Wiley Periodicals, Inc.     

Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.     

Why do ants shift their foraging from extrafloral nectar to aphid honeydew?

When aphids parasitize plants with extrafloral nectaries (EFNs) and aphid colony size is small, ants frequently use EFNs but hardly tend aphids. However, as the aphid colony size increases, ants stop using EFNs and strengthen their associations with aphids. Although the shift in ant behavior is important for determining the dynamics of the ant–plant–aphid interaction, it is not known why this shift occurs. Here, we test two hypotheses to explain the mechanism responsible for this behavioral shift: (1) Extrafloral nectar secretion changes in response to aphid herbivory, or (2) plants do not change extrafloral nectar secretion, but the total reward to ants from aphids will exceed that from EFNs above a certain aphid colony size. To judge which mechanism is plausible, we investigated secretion patterns of extrafloral nectar produced by plants with and without aphids, compared the amount of sugar supplied by EFNs and aphids, and examined whether extrafloral nectar or honeydew was more attractive to ants. Our results show that there was no inducible extrafloral secretion in response to aphid herbivory, but the sugar concentration in extrafloral nectar was higher than in honeydew, and more ant workers were attracted to an artificial extrafloral nectar solution than to an artificial aphid honeydew solution. These results indicate that extrafloral nectar is a more attractive reward than aphid honeydew per unit volume. However, even an aphid colony containing only two individuals can supply a greater reward to ants than EFNs. This suggests that the ant behavioral shift may be explained by the second hypothesis.     

Conformational Change of a Natto Mucin in Solution

The mucin obtained from a natto sample was found to be composed of 58 % of γ-polyglutamic acid and 40% of polysaccharide. The ratio of l- and d-glutamic acid was determined to be 58:42 using l-glutamic acid decarboxylase. The weight- and z-average molecular weight were 2.08 × 10⁵ and 2.22 × 10⁵, respectively. The distribution curve of the sedimentation coefficient showed a small heterogeneity. The mucin molecule was considered to be randomly coiled at pH 5.0 ~ 8.8 and to be a rod-like molecule in the lower pH region.     

Extra tyrosine in the carbohydrate-binding module of Irpex lacteus Xyn10B enhances its cellulose-binding ability

The xylanase (Xyn10B) that strongly adsorbs on microcrystalline cellulose was isolated from Driselase. The Xyn10B contains a Carbohydrate-binding module family 1 (CBM1) (IrpCBM_Xyn10B) at N-terminus. The canonical essential aromatic residues required for cellulose binding were conserved in IrpCBM_Xyn10B; however, its adsorption ability was markedly higher than that typically observed for the CBM1 of an endoglucanase from Trametes hirsuta (ThCBM_EG1). An analysis of the CBM-GFP fusion proteins revealed that the binding capacity to cellulose (7.8 μmol/g) and distribution coefficient (2.0 L/μmol) of IrpCBM_Xyn10B-GFP were twofold higher than those of ThCBM_EG1-GFP (3.4 μmol/g and 1.2 L/μmol, respectively), used as a reference structure. Besides the canonical aromatic residues (W24-Y50-Y51) of typical CBM1-containing proteins, IrpCBM_Xyn10B had an additional aromatic residue (Y52). The mutation of Y52 to Ser (IrpCBM_Y52S-GFP) reduced these adsorption parameters to 4.4 μmol/g and 1.5 L/μmol, which were similar to those of ThCBM_EG1-GFP. These results indicate that Y52 plays a crucial role in strong cellulose binding.     

Novel mutations of CDKN1C in Japanese patients with Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an imprinting-related human disease that is characterized by macrosomia, macroglossia, abdominal wall defects, and variable minor features. BWS is caused by several genetic/epigenetic alterations, such as loss of methylation at KvDMR1, gain of methylation at H19-DMR, paternal uniparental disomy of chromosome 11, CDKN1C mutations, and structural abnormalities of chromosome 11. CDKN1C is an imprinted gene with maternal preferential expression, encoding for a cyclin-dependent kinase (CDK) inhibitor. Mutations in CDKN1C are found in 40 % of familial BWS cases with dominant maternal transmission and in ~5 % of sporadic cases. In this study, we searched for CDKN1C mutations in 37 BWS cases that had no evidence for other alterations. We found five mutations—four novel and one known—from a total of six patients. Four were maternally inherited and one was a de novo mutation. Two frame-shift mutations and one nonsense mutation abolished the QT domain, containing a PCNA-binding domain and a nuclear localization signal. Two missense mutations occurred in the CDK inhibitory domain, diminishing its inhibitory function. The above-mentioned mutations were predicted by in silico analysis to lead to loss of function; therefore, we strongly suspect that such anomalies are causative in the etiology of BWS.