Deciphering the sub-Golgi localization of glycosyltransferases via 3D super-resolution imaging.

The Golgi apparatus, a crucial organelle involved in protein processing, including glycosylation, exhibits complex sub-structures, i.e., cis-, medial, and trans-cisternae. This study investigated the distribution of glycosyltransferases within the Golgi apparatus of mammalian cells via 3D super-resolution imaging. Focusing on human glycosyltransferases involved in N-glycan modification, we found that even enzymes presumed to coexist in the same Golgi compartment exhibit nuanced variations in localization. By artificially making their N-terminal regions [composed of a cytoplasmic, transmembrane, and stem segment (CTS)] identical, it was possible to enhance the degree of their colocalization, suggesting the decisive role of this region in determining the sub-Golgi localization of enzymes. Ultimately, this study reveals the molecular codes within CTS regions as key determinants of glycosyltransferase localization, providing insights into precise control over the positioning of glycosyltransferases, and consequently, the interactions between glycosyltransferases and substrate glycoproteins as cargoes in the secretory pathway. This study advances our understanding of Golgi organization and opens avenues for programming the glycosylation of proteins for clinical applications.Key words: Golgi apparatus, glycosyltransferase, 3D super-resolution imaging, N-glycosylation.

Episodic transient deformation revealed by the analysis of multiple GNSS networks in the Noto Peninsula, central Japan.

Since November 30, 2020, an intense seismic swarm and transient deformation have been continuously observed in the Noto Peninsula, central Japan, which is a non-volcanic/geothermal area far from major plate boundaries. We modeled transient deformation based on a combined analysis of multiple Global Navigation Satellite System (GNSS) observation networks, including one operated by a private sector company (SoftBank Corp.), relocated earthquake hypocenters, and tectonic settings. Our analysis showed a total displacement pattern over 2 years shows horizontal inflation and uplift of up to ~ 70 mm around the source of the earthquake swarm. In the first 3 months, the opening of the shallow-dipping tensile crack had an estimated volumetric increase of ~ 1.4 × 107 m3 at a depth of ~ 16 km. Over the next 15 months, the observed deformation was well reproduced by shear-tensile sources, which represent an aseismic reverse-type slip and the opening of a southeast-dipping fault zone at a depth of 14-16 km. We suggest that the upwelling fluid spread at a depth of ~ 16 km through an existing shallow-dipping permeable fault zone and then diffused into the fault zone, triggering a long-lasting sub-meter aseismic slip below the seismogenic depth. The aseismic slip further triggered intense earthquake swarms at the updip.

Improved secretion of glycoproteins using an N-glycan-restricted passport sequence tag recognized by cargo receptor.

MCFD2 and ERGIC-53, which are the products of causative genes of combined factor V and factor VIII deficiency, form a cargo receptor complex responsible for intracellular transport of these coagulation factors in the early secretory pathway. In this study, using an NMR technique, we successfully identified an MCFD2-binding segment from factor VIII composed of a 10 amino acid sequence that enhances its secretion. This prompted us to examine possible effects of attaching this sequence to recombinant glycoproteins on their secretion. We found that the secretion level of recombinant erythropoietin was significantly increased simply by tagging it with the passport sequence. Our findings not only provide molecular basis for the intracellular trafficking of coagulation factors and their genetic deficiency but also offer a potentially useful tool for increasing the production yields of recombinant glycoproteins of biopharmaceutical interest.

Red fluorescent cAMP indicator with increased affinity and expanded dynamic range.

cAMP is one of the most important second messengers in biological processes. Cellular dynamics of cAMP have been investigated using a series of fluorescent indicators; however, their sensitivity was sub-optimal for detecting cAMP dynamics at a low concentration range, due to a low ligand affinity and/or poor dynamic range. Seeking an indicator with improved detection sensitivity, we performed insertion screening of circularly permuted mApple, a red fluorescent protein, into the cAMP-binding motif of PKA regulatory subunit Iα and developed an improved cAMP indicator named R-FlincA (Red Fluorescent indicator for cAMP). Its increased affinity (Kd = 0.3 µM) and expanded dynamic range (860% at pH 7.2) allowed the detection of subtle changes in the cellular cAMP dynamics at sub-µM concentrations, which could not be easily observed with existing indicators. Increased detection sensitivity also strengthened the advantages of using R-FlincA as a red fluorescent indicator, as it permits a series of applications, including multi-channel/function imaging of multiple second messengers and combinatorial imaging with photo-manipulation. These results strongly suggest that R-FlincA is a promising tool that accelerates cAMP research by revealing unobserved cAMP dynamics at a low concentration range.

Along-trench variation in seafloor displacements after the 2011 Tohoku earthquake.

The 2011 Tohoku-oki earthquake was the largest earthquake ever observed with seafloor geodetic techniques in and around its source region. Large crustal deformation associated with both the coseismic rupture and the rapid postseismic deformation has been reported. However, these observations are insufficient to describe the postseismic deformation processes occurring around the broad rupture area. We report the first results of seafloor Global Positioning System and acoustic ranging (GPS-A) observations based on repeated campaign surveys conducted over nearly 4 years using the extended GPS-A network deployed along the Japan Trench in September 2012. The observed postseismic displacement rates (DRs) show evident spatial variation along the trench: (i) distinct landward DRs in the large coseismic slip area [primary rupture area (PRA)], evidencing the predominance of viscoelastic relaxation; (ii) remarkable trenchward DRs in the south of the PRA, indicating rapid afterslip; and (iii) slight trenchward DRs in the north of the PRA. These features provide great insights into constructing a more complete model of viscoelastic relaxation, and they also indicate spatial variation of afterslip and fault locking along the plate interface with clear spatial resolution, providing invaluable information for the improvement of seismic hazard assessment.

Nontrivial Effect of the Color-Exchange of a Donor/Acceptor Pair in the Engineering of Förster Resonance Energy Transfer (FRET)-Based Indicators.

Genetically encoded indicators driven by the Förster resonance energy transfer (FRET) mechanism are reliable tools for live imaging. While the properties of FRET-based indicators have been improved over the years, they often suffer from a poor dynamic range due to the lack of comprehensive understanding about how to apply an appropriate strategy to optimize the FRET parameters. One of the most successful optimizations is the incorporation of circularly permuted fluorescent proteins (cpFPs). To better understand the effects of this strategy, we systematically investigated the properties of the indicators by utilizing a set of FRET backbones consisting of native or one of the most effective cp variants (cp173FPs) with considerations of their order. As a result, the ordering of donor and acceptor FPs, which has been ignored in previous studies, was found to significantly affect the dynamic range of indicators. By utilizing these backbones, we succeeded in improving a cGMP indicator with 3.6-fold increased dynamic range and in generating an ultrasensitive cAMP indicator capable of environmental imaging, demonstrating the practical importance of the ordering of donors and acceptors in the engineering of FRET-based indicators.

Prevalence of viscoelastic relaxation after the 2011 Tohoku-oki earthquake.

After a large subduction earthquake, crustal deformation continues to occur, with a complex pattern of evolution. This postseismic deformation is due primarily to viscoelastic relaxation of stresses induced by the earthquake rupture and continuing slip (afterslip) or relocking of different parts of the fault. When postseismic geodetic observations are used to study Earth's rheology and fault behaviour, it is commonly assumed that short-term (a few years) deformation near the rupture zone is caused mainly by afterslip, and that viscoelasticity is important only for longer-term deformation. However, it is difficult to test the validity of this assumption against conventional geodetic data. Here we show that new seafloor GPS (Global Positioning System) observations immediately after the great Tohoku-oki earthquake provide unambiguous evidence for the dominant role of viscoelastic relaxation in short-term postseismic deformation. These data reveal fast landward motion of the trench area, opposing the seaward motion of GPS sites on land. Using numerical models of transient viscoelastic mantle rheology, we demonstrate that the landward motion is a consequence of relaxation of stresses induced by the asymmetric rupture of the thrust earthquake, a process previously unknown because of the lack of near-field observations. Our findings indicate that previous models assuming an elastic Earth will have substantially overestimated afterslip downdip of the rupture zone, and underestimated afterslip updip of the rupture zone; our knowledge of fault friction based on these estimates therefore needs to be revised.

Involvement of p114-RhoGEF and Lfc in Wnt-3a- and dishevelled-induced RhoA activation and neurite retraction in N1E-115 mouse neuroblastoma cells.

The Wnt-induced planar cell polarity (PCP) signaling pathway is essential for polarized cell migration and morphogenesis. Dishevelled (Dvl) and its binding protein Daam1 mediate RhoA activation in this pathway. WGEF, a member of the Rho-guanine nucleotide exchange factor (Rho-GEF) family, was shown to play a role in Wnt-induced RhoA activation in Xenopus embryos. However, it has remained unknown which member(s) of a Rho-GEF family are involved in Wnt/Dvl-induced RhoA activation in mammalian cells. Here we identified p114-RhoGEF and Lfc (also called GEF-H1) as the Rho-GEFs responsible for Wnt-3a-induced RhoA activation in N1E-115 mouse neuroblastoma cells. We screened for Rho-GEF-silencing short-hairpin RNAs (shRNAs) that are capable of suppressing Dvl-induced neurite retraction in N1E-115 cells and found that p114-RhoGEF and Lfc shRNAs, but not WGEF shRNA, suppressed Dvl- and Wnt-3a-induced neurite retraction. p114-RhoGEF and Lfc shRNAs also inhibited Dvl- and Wnt-3a-induced RhoA activation, and p114-RhoGEF and Lfc proteins were capable of binding to Dvl and Daam1. Additionally, the Dvl-binding domains of p114-RhoGEF and Lfc inhibited Dvl-induced neurite retraction. Our results suggest that p114-RhoGEF and Lfc are critically involved in Wnt-3a- and Dvl-induced RhoA activation and neurite retraction in N1E-115 cells.

Artificial control of subtype-specific platelet-derived growth factor-receptor signaling.

Platelet-derived growth factor (PDGF) signaling controls various physiological functions via two receptor subtypes: PDGF receptor (PDGFR) alpha and PDGFRbeta. Nevertheless, our understanding of their roles is limited because of a lack of pharmacological tools to discriminate subtype-specific signaling. We developed a chimeric receptor by combining ligand-binding-domain truncated PDGFRbeta with anti-fluorescein single chain antibody, expecting the control of PDGFRbeta-specific signaling by oligomerized fluorescein as an artificial agonist. Results show that calcium mobilization, Cdc42 activation, and cell migration were elicited specifically by the artificial ligand in cells expressing the chimeric receptor. Our method is expected to be useful to understand the subtype-specific roles of PDGFRs in various cellular functions.

Identification of multiple actin-binding sites in cofilin-phosphatase Slingshot-1L.

Slingshot-1L (SSH1L) is a phosphatase that specifically dephosphorylates and activates cofilin, an actin-severing and -depolymerizing protein. SSH1L binds to and is activated by F-actin in vitro, and co-localizes with F-actin in cultured cells. We examined the F-actin-binding activity, F-actin-mediated phosphatase activation, and subcellular distribution of various mutants of SSH1L. We identified three sites involved in F-actin binding of SSH1L: Trp-458 close to the C-terminus of the phosphatase domain, an LHK motif in the N-terminal region, and an LKR motif in the C-terminal region. These sites play unique roles in the control of subcellular localization and F-actin-mediated activation of SSH1L.

A pathway of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilin in lamellipodia.

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, we show that cofilin-phosphatase activity of SSH1L increases approximately 10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. We also provide evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1beta induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin-rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1beta and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm.

Differential activities, subcellular distribution and tissue expression patterns of three members of Slingshot family phosphatases that dephosphorylate cofilin.

BACKGROUND: Cofilin, a key regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 by LIM-kinases and is reactivated by dephosphorylation by a family of protein phosphatases, termed Slingshot (SSH). RESULTS: We have identified two novel isoforms of SSHs, termed SSH-2L and SSH-3L and characterized them in comparison with SSH-1L that was previously reported. SSH-1L and SSH-2L, but not SSH-3L, tightly bound to and co-localized with actin filaments. When expressed in cultured cells, SSH-1L, SSH-2L and SSH-3L decreased the level of Ser-3-phosphorylated cofilin (P-cofilin) in cells and suppressed LIM-kinase-induced actin reorganization, although SSH-3L was less effective than SSH-1L and SSH-2L. In cell-free assays, SSH-1L and SSH-2L efficiently dephosphorylated P-cofilin, whereas SSH-3L did do so only weakly. Using deleted mutants of SSH-1L and SSH-2L, we found that the N-terminal and C-terminal extracatalytic regions are critical for cofilin-phosphatase and F-actin-binding activities, respectively. In situ hybridization analyses revealed characteristic patterns of expression of each of the mouse Ssh genes in both neuronal and non-neuronal tissues; in particular, expression of Ssh-3 in epithelial tissues is evident. CONCLUSION: SSH-1L, SSH-2L and SSH-3L have the potential to dephosphorylate P-cofilin, but subcellular distribution, F-actin-binding activity, specific phosphatase activity and expression patterns significantly differ, which suggests that they have related but distinct functions in various cellular and developmental events.