A Rab10-ACAP1-Arf6 GTPases cascade modulates M4 muscarinic acetylcholine receptor trafficking and signaling.

Membrane trafficking processes regulate the G protein-coupled receptor activity. The muscarinic acetylcholine receptors (mAChRs) are highly pursued drug targets for neurological diseases, but the cellular machineries that control the trafficking of these receptors remain largely elusive. Here, we revealed the role of the small GTPase Rab10 as a negative regulator for the post-activation trafficking of M4 mAChR and the underlying mechanism.

RAB21 controls autophagy and cellular energy homeostasis by regulating retromer-mediated recycling of SLC2A1/GLUT1.

The endosomal system maintains cellular homeostasis by coordinating multiple vesicular trafficking events, and the retromer complex plays a critical role in endosomal cargo recognition and sorting. Here, we demonstrate an essential role for the small GTPase RAB21 in regulating retromer-mediated recycling of the glucose transporter SLC2A1/GLUT1 and macroautophagy/autophagy. RAB21 depletion mis-sorts SLC2A1 to lysosomes and affects glucose uptake, thereby activating the AMPK-ULK1 pathway to increase autophagic flux.

[Effectiveness of total hip arthroplasty in patients with a history of hip preservation surgery with secondary osteoarthritis for developmental dysplasia of the hip].

Objective: To evaluate early to medium-term effectiveness of total hip arthroplasty (THA) in patients with a history of hip preservation surgery with secondary severe osteoarthritis for developmental dysplasia of the hip (DDH).

Methods: The clinical data of 25 DDH patients (31 hips) who had severe osteoarthritis after hip preservation surgery and received THA between September 2009 and March 2021 were retrospectively analyzed. There were 1 male and 24 females; the age ranged from 18 to 65 years, with an average age of 43 years; 8 hips were classified into Crowe type Ⅰ, 9 hips were type Ⅱ, 3 hips were type Ⅲ, and 11 hips were type Ⅳ.

SNX-3 mediates retromer-independent tubular endosomal recycling by opposing EEA-1-facilitated trafficking.

Early endosomes are the sorting hub on the endocytic pathway, wherein sorting nexins (SNXs) play important roles for formation of the distinct membranous microdomains with different sorting functions. Tubular endosomes mediate the recycling of clathrin-independent endocytic (CIE) cargoes back toward the plasma membrane. However, the molecular mechanism underlying the tubule formation is still poorly understood.

WIP-1 and DBN-1 promote scission of endocytic vesicles by bridging actin and Dynamin-1 in the intestine.

There has been a consensus that actin plays an important role in scission of the clathrin-coated pits (CCPs) together with large GTPases of the dynamin family in metazoan cells. However, the recruitment, regulation and functional interdependence of actin and dynamin during this process remain inadequately understood. Here, based on small-scale screening and live-imaging techniques, we identified a novel set of molecules underlying CCP scission in the multicellular organism We found that loss of Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP-1) impaired CCP scission in a manner that is independent of the homolog of WASP/N-WASP (WSP-1) and is mediated by direct binding to G-actin.

Sorting Out the Genetic Background of the Last Surviving South China Tigers.

The South China tiger (Panthera tigris amoyensis) is endemic to China and also the most critically endangered subspecies of living tigers. It is considered extinct in the wild and only about 150 individuals survive in captivity to date, whose genetic heritage, however, is ambiguous and controversial. Here, we conducted an explicit genetic assessment of 92 studbook-registered South China tigers from 14 captive facilities using a subspecies-diagnostic system in the context of comparison with other voucher specimens to evaluate the genetic ancestry and level of distinctiveness of the last surviving P.

Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor.

Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes.

The effect of the size of fluorescent dextran on its endocytic pathway.

Fluorescent dextrans are commonly used as macropinocytic probes to study the properties of endocytic cargoes; however, the effect of the size of dextrans on endocytic mechanisms has not been carefully analyzed. By using chemical and siRNA inhibition of individual endocytic pathways, we evaluated the internalization of two commonly used dextrans, Dex10 (dextran 10 kDa) and Dex70 (dextran 70 kDa), in mammalian HeLa cells and Caenorhabditis elegans coelomocytes. We revealed that Dex70 enters these two cell types predominantly via clathrin- and dynamin-independent and amiloride-sensitive macropinocytosis process; Dex10, on the other hand, enters the two cell types through clathrin-/dynamin-dependent micropinocytosis in addition to macropinocytosis.

SEC-10 and RAB-10 coordinate basolateral recycling of clathrin-independent cargo through endosomal tubules in Caenorhabditis elegans.

Despite the increasing number of regulatory proteins identified in clathrin-independent endocytic (CIE) pathways, our understanding of the exact functions of these proteins and the sequential manner in which they function remains limited. In this study, using the Caenorhabditis elegans intestine as a model, we observed a unique structure of interconnected endosomal tubules, which is required for the basolateral recycling of several CIE cargoes including hTAC, GLUT1, and DAF-4. SEC-10 is a subunit of the octameric protein complex exocyst.

Mechanisms of U87 astrocytoma cell uptake and trafficking of monomeric versus protofibril Alzheimer's disease amyloid-β proteins.

A significant hallmark of Alzheimer's disease is the formation of senile plaques in the brain due to the unbalanced levels of amyloid-beta (Aβ). However, although how Aβ is produced from amyloid precursor proteins is well understood, little is known regarding the clearance and metabolism of various Aβ aggregates from the brain. Similarly, little is known regarding how astrocytes internalize and degrade Aβ, although astrocytes are known to play an important role in plaque maintenance and Aβ clearance.

Tracking the down-regulation of folate receptor-α in cancer cells through target specific delivery of quantum dots coupled with antisense oligonucleotide and targeted peptide.

Based on the multivalent binding capability of streptavidin (SA) to biotin, a multifunctional quantum dot probe (QD-(AS-ODN+p160)) coupled with antisense oligonucleotide (AS-ODN) and peptide p160 is designed for real-time tracking of targeted delivery of AS-ODN and regulation of folate receptor-α (hFR-α) in MCF-7 breast cancer cells. Fluorescence spectra, capillary electrophoresis (CE) and dynamic light scattering (DLS) are used to characterize the conjugation of AS-ODN and p160 with quantum dots (QDs), DLS results confirm the well stability of the probe in aqueous media. Confocal imaging and quantitative flow cytometry show that QD-(AS-ODN+p160) is able to specifically target human breast cancer MCF-7 cells.

Analysis of variable sites between two complete South China tiger (Panthera tigris amoyensis) mitochondrial genomes.

In order to investigate the mitochondrial genome of Panthera tigris amoyensis, two South China tigers (P25 and P27) were analyzed following 15 cymt-specific primer sets. The entire mtDNA sequence was found to be 16,957 bp and 17,001 bp long for P25 and P27 respectively, and this difference in length between P25 and P27 occurred in the number of tandem repeats in the RS-3 segment of the control region. The structural characteristics of complete P.

Roles of AP-2 in clathrin-mediated endocytosis.

Background: The notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway.

Methodology/principal Findings: We report here the use of in vivo imaging data obtained by spinning-disk confocal microscopy to study the formation of clathrin-coated structures at the plasma membranes of BSC1 and HeLa cells depleted by RNAi of the clathrin adaptor, AP-2. Very few clathrin coats continue to assemble after AP-2 knockdown.

Silence of Synaptotagmin VII inhibits release of dense core vesicles in PC12 cells.

Synaptotagmin VII (Syt VII), which has a higher Ca(2+) affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca(2+)-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca(2+) sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells.

Fabrication of size-controllable ultrasmall-disk electrode: monitoring single vesicle release kinetics at tiny structures with high spatio-temporal resolution.

Size-controllable micron or nano-disk carbon fiber electrode (CFE) is prepared and demonstrated to be excellent for extra-cellular transmitter release detection at tiny structures and vesicle fusion kinetics analysis with high spatio-temporal resolution. An improved electrochemical etching procedure was employed, for the first time, to fabricate cylindrical fiber with controlled micron or nano-diameter. Afterwards, a facile insulation with polypropylene sheath was employed to completely insulate the whole body of the thinned fiber, and an ultrasmall-disk sensing area was finally produced by cutting of the insulated fibers.

Synaptotagmin IV regulates dense core vesicle (DCV) release in LbetaT2 cells.

Synaptotagmins (Syts) are calcium-binding proteins which are conserved from nematodes to humans. Fifteen Syts have been identified in mammalian species. Syt I is recognized as a Ca(2+) sensor for the synchronized release of synaptic vesicles in some types of neurons, but its role in the secretion of dense core vesicles (DCVs) remains unclear.

Synaptotagmin I and IX function redundantly in controlling fusion pore of large dense core vesicles.

Synaptotagmins (Syts) constitute a large family of at least 16 members and individual Syt isoforms exhibit distinct Ca(2+)-binding properties and subcellular localization. It remains to be demonstrated whether multiple Syt isoforms can function independently or cooperatively on certain type of vesicle. In the current study, we have developed NPY-pHluorin to specifically assess exocytosis of large dense core vesicles (LDCVs) and studied the requirement of Syt I and Syt IX for LDCV exocytosis in PC12 cells.

Differential properties of GTP- and Ca(2+)-stimulated exocytosis from large dense core vesicles.

Many cells utilize a GTP-dependent pathway to trigger exocytosis in addition to Ca(2+)-triggered exocytosis. However, little is known about the mechanism by which GTP triggers exocytosis independent of Ca(2+). We used dual-color evanescent field microscopy to compare the motion and fusion of large dense core vesicles stimulated by either mastoparan (Mas) in Ca(2+)-free conditions or high K(+) in the presence of Ca(2+).

Monitoring dopamine release from single living vesicles with nanoelectrodes.

Carbon fiber nanoelectrodes (tip diameter = ca. 100 nm) have been first used to monitor real-time dopamine release from single living vesicles of single rat pheochromocytoma (PC12) cells. The experiments show that active and inactive release sites exist on the surface of cells, and the spatial distributions have been differentiated even in the same active release zone.

Spectroscopic and voltammetric study on the binding of aluminium(III) to DNA.

Cyclic voltammetry (CV) and ultraviolet (UV) spectroscopy were used, for the first time, to study the interaction between aluminium(III) and calf thymus DNA under neutral pH conditions. Thus obtained data confirmed the existence of a relatively strong interaction between Al(III) and DNA. The binding site for aluminium(III) on DNA chains is not the bases, but the phosphate groups on the DNA backbones, the same as that for [Co(phen)3](3+/2+) that binds non-specifically and electrostatically to the deoxyribose phosphate backbone of DNA.

Investigation of DNA orientation on gold by EC-STM.

The immobilization of thiol-derivatized DNA on a Au (111) single crystal surface by self-assembly has been investigated by electrochemical scanning tunneling microscopy (EC-STM). Continuous potential-dependent orientation changes of double-stranded oligodeoxynucleotides (ODN) have been observed in a certain potential range from 200 to 600 mV (versus SCE). It is suggested that the DNA duplexes stand straight on the gold surface at potentials negative of the potential of zero charge (pzc) and then lay down on the surface when the potential shifts positively.