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. We show that constitutively active Rab10 arrests the receptor within Rab5-positive early endosomes and significantly hinders the resensitization of M4-mediated Ca2+ signaling. Mechanistically, M4 binds to Rab10-GTP, which requires the motif 386RKKRQMAA393 (R386-A393) within the third intracellular loop. Moreover, Rab10-GTP inactivates Arf6 by recruiting the Arf6 GTPase-activating protein, ACAP1. Strikingly, deletion of the motif R386-A393 causes M4 to bypass the control by Rab10 and switch to the Rab4-facilitated fast recycling pathway, thus reusing the receptor. Therefore, Rab10 couples the cargo sorting and membrane trafficking regulation through cycle between GTP-bound and GDP-bound state. Our findings suggest a model that Rab10 binds to the M4 like a molecular brake and controls the receptor's transport through endosomes, thus modulating the signaling, and this regulation is specific among the mAChR subtypes.

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. Here we screened the effect on the ARF-6-associated CIE recycling endosomal tubules for all the SNX members in Caenorhabditis elegans (C. elegans). We identified SNX-3 as an essential factor for generation of the recycling tubules. The loss of SNX-3 abolishes the interconnected tubules in the intestine of C. elegans. Consequently, the surface and total protein levels of the recycling CIE protein hTAC are strongly decreased. Unexpectedly, depletion of the retromer components VPS-26/-29/-35 has no similar effect, implying that the retromer trimer is dispensable in this process. We determined that hTAC is captured by the ESCRT complex and transported into the lysosome for rapid degradation in snx-3 mutants. Interestingly, EEA-1 is increasingly recruited on early endosomes and localized to the hTAC-containing structures in snx-3 mutant intestines. We also showed that SNX3 and EEA1 compete with each other for binding to phosphatidylinositol-3-phosphate enriching early endosomes in Hela cells. Our data demonstrate for the first time that PX domain-only C. elegans SNX-3 organizes the tubular endosomes for efficient recycling and retrieves the CIE cargo away from the maturing sorting endosomes by competing with EEA-1 for binding to the early endosomes. However, our results call into question how SNX-3 couples the cargo capture and membrane remodeling in the absence of the retromer trimer complex.

Lamin A/C and Vimentin as a Coordinated Regulator during Amoeboid Migration in Microscale Confined Microenvironments.

Cell migration occurs in confined microenvironments, which plays a vital role in the process of tumor metastasis. However, it is challenging to study their behaviors in vivo. Here we developed a cell squeeze system that can be scaled down to micrometers to mimic native physical confined microenvironments, wherein degrees of surface adhesion and mechanical constraints could be manipulated in order to investigate cell-migrating behaviors. Based on the microscale cell squeeze system, we found the synergistic role of lamin A/C and vimentin in cell transition and migration under strong confinement. The dynamic variations in lamin A/C and vimentin expression establish a positive feedback loop in response to confinement, effectively promoting amoeboid migration by modulating nuclear deformability while ensuring cell viability. This work shed light on modulating cell response to microenvironments by altering the expression of lamin A/C and/or vimentin, which may be a more efficient way of inhibiting cancer metastasis.

The diverse roles and dynamic rearrangement of vimentin during viral infection.

Epidemics caused by viral infections pose a significant global threat. Cytoskeletal vimentin is a major intermediate filament (IF) protein, and is involved in numerous functions, including cell signaling, epithelial-mesenchymal transition, intracellular organization and cell migration. Vimentin has important roles for the life cycle of particular viruses; it can act as a co-receptor to enable effective virus invasion and guide efficient transport of the virus to the replication site. Furthermore, vimentin has been shown to rearrange into cage-like structures that facilitate virus replication, and to recruit viral components to the location of assembly and egress. Surprisingly, vimentin can also inhibit virus entry or egress, as well as participate in host-cell defense. Although vimentin can facilitate viral infection, how this function is regulated is still poorly understood. In particular, information is lacking on its interaction sites, regulation of expression, post-translational modifications and cooperation with other host factors. This Review recapitulates the different functions of vimentin in the virus life cycle and discusses how they influence host-cell tropism, virulence of the pathogens and the consequent pathological outcomes. These insights into vimentin-virus interactions emphasize the importance of cytoskeletal functions in viral cell biology and their potential for the identification of novel antiviral targets.

WIP-1 and DBN-1 promote scission of endocytic vesicles by bridging actin and Dynamin-1 in the C. elegans 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 in vivo live-imaging techniques, we identified a novel set of molecules underlying CCP scission in the multicellular organism Caenorhabditis elegans 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 C. elegans homolog of WASP/N-WASP (WSP-1) and is mediated by direct binding to G-actin. Moreover, the cortactin-binding domain of WIP-1 serves as the binding interface for DBN-1 (also known in other organisms as Abp1), another actin-binding protein. We demonstrate that the interaction between DBN-1 and F-actin is essential for Dynamin-1 (DYN-1) recruitment at endocytic sites. In addition, the recycling regulator RME-1, a homolog of mammalian Eps15 homology (EH) domain-containing proteins, is increasingly recruited at the arrested endocytic intermediates induced by F-actin loss or DYN-1 inactivation, which further stabilizes the tubular endocytic intermediates. Our study provides new insights into the molecular network underlying F-actin participation in the scission of CCPs.This article has an associated First Person interview with the first author of the paper.

Active FHOD1 promotes the formation of functional actin stress fibers.

The formin FHOD1 acts as a nucleating, capping and bundling protein of actin filaments. In cells, release from the C-terminal diaphanous autoregulatory domain (DAD) of FHOD1 stimulates the protein into the active form. However, the cellular physiological relevance of active form FHOD1 and the phenotypic regulation by FHOD1 depletion are not completely understood. Here, we show that in contrast with the cytosolic diffused expression of auto-inhibited FHOD1, active FHOD1 by C-terminal truncation was recruited into all three types of actin stress fibers in human osteosarcoma cells. Notably, the recruited active FHOD1 was more incorporated with myosin II than α-actinin, and associated with both naïve and mature focal adhesions. Active FHOD1 displayed faster turnover than actin molecules on ventral stress fibers. Moreover, we witnessed the emergence of active FHOD1 from the cell periphery, which subsequently moved centripetally together with transverse arcs. Furthermore, FHOD1 knockdown resulted in defective maturation of actomyosin bundles and subsequently longer non-contractile dorsal stress fibers, whereas the turnover of both actin and myosin II were maintained normally. Importantly, the loss of FHOD1 led to slower actin centripetal flow, resulting in abnormal cell spreading and migration defects. Taken together, these results reveal a critical role of FHOD1 in temporal- and spatial- control of the morphology and dynamics of functional actin stress fibers during variable cell behavior.

Unidirectional Regulation of Vimentin Intermediate Filaments to Caveolin-1.

Both the mechanosensitive vimentin cytoskeleton and endocytic caveolae contribute to various active processes such as cell migration, morphogenesis, and stress response. However, the crosstalk between these two systems has remained elusive. Here, we find that the subcellular expression between vimentin and caveolin-1 is mutual exclusive, and vimentin filaments physically arrest the cytoplasmic motility of caveolin-1 vesicles. Importantly, vimentin depletion increases the phosphorylation of caveolin-1 on site Tyr14, and restores the compromised cell migration rate and directionality caused by caveolin-1 deprivation. Moreover, upon hypo-osmotic shock, vimentin-knockout recovers the reduced intracellular motility of caveolin-1 vesicles. In contrary, caveolin-1 depletion shows no effect on the expression, phosphorylation (on sites Ser39, Ser56, and Ser83), distribution, solubility, and cellular dynamics of vimentin filaments. Taken together, our data reveals a unidirectional regulation of vimentin to caveolin-1, at least on the cellular level.

ABHD16A Negatively Regulates the Palmitoylation and Antiviral Function of IFITM Proteins.

Interferon-inducible transmembrane (IFITM) proteins are small homologous proteins that are encoded by the interferon-stimulated genes (ISGs), which can be strongly induced by interferon (IFN) and provide resistance to invasion by a variety of viral pathogens. However, the exact molecular mechanisms underlying this function have remained elusive. The antiviral activity of IFITMs from different species depends on S-palmitoylation at conserved cysteine residues. However, specific enzymes involved in the dynamic palmitoylation cycle of IFITMs, especially depalmitoylase, have not yet been reported. Here, we demonstrate that α/-hydrolase domain-containing 16A (ABHD16A) is a depalmitoylase and a negative regulator of IFITM protein that can catalyze the depalmitoyl reaction of S-palmitoylated IFITM proteins, thereby decreasing their antiviral activities on RNA viruses. Using the acyl-PEGyl exchange gel shift (APEGS) assay, we identified ABHD16A proteins from humans, pigs, and mice that can directly participate in the palmitoylation/depalmitoylation cycles of IFITMs in the constructed abhd16a-/- cells and ABHD16A-overexpressing cells. Furthermore, we showed that ABHD16A functions as a regulator of subcellular localization of IFITM proteins and is related to the immune system. It is tempting to suggest that pharmacological intervention in IFITMs and ABHD16A can be achieved either through controlling their expression or regulating their activity, thereby providing a broad-spectrum therapeutic strategy for animal viral diseases. IMPORTANCE IFITM protein is the cells first line of antiviral defense that blocks early stages of viral replication; the underlying mechanism might be associated with the proper distribution in cells. The palmitoylation/depalmitoylation cycle can dynamically regulate protein localization, stability, and function. This work is the first one that found the critical enzyme that participates in the palmitoylation/depalmitoylation cycle of IFITM, and this type of palmitoyl loss may be an essential regulation mode for balancing the antiviral functions of the IFN pathway. These findings imply that the pharmacological intervention in IFITM and ABHD16A, either through controlling their expression or regulating their activities, could provide a broad-spectrum therapeutic strategy for animal viral diseases and complications linked to interferon elevation.

Actin nucleator formins regulate the tension-buffering function of caveolin-1.

Both the mechanosensitive actin cytoskeleton and caveolae contribute to active processes such as cell migration, morphogenesis, and vesicular trafficking. Although distinct actin components are well studied, how they contribute to cytoplasmic caveolae, especially in the context of mechano-stress, has remained elusive. Here, we identify two actin-associated mobility stereotypes of caveolin-1 (CAV-1)-marked intracellular vesicles, which are characterized as 'dwelling' and 'go and dwelling'. In order to exploit the reason for their distinct dynamics, elongated actin-associated formin functions are perturbed. We find drastically decreased density, increased clustering, and compromised motility of cytoplasmic CAV-1 vesicles resulting from lacking actin nucleator formins by both chemical treatment and RNA silencing of formin genes. Furthermore, hypo-osmosis-stimulated diminishing of CAV-1 is dramatically intensified upon blocking formins. The clustering of CAV-1 vesicles when cells are cultured on soft substrate is also aggravated under formin inhibition condition. Together, we reveal that actin-associated formins are essential for maintaining the dynamic organization of cytoplasmic CAV-1 and importantly its sensitivity upon mechanical challenge. We conclude that tension-controlled actin formins act as a safety valve dampening excessive tension on CAV-1 and safeguarding CAV-1 against mechanical damage.

Cell migration orchestrates migrasome formation by shaping retraction fibers.

Migrasomes are recently discovered vesicle-like structures on retraction fibers of migrating cells that have been linked with transfer of cellular contents, shedding of unwanted materials, and information integration. However, whether and how the cell migration paradigm regulates migrasome formation is not clear. Here, we report that there are significantly fewer migrasomes in turning cells compared with straight persistently migrating cells. The major insight underlying this observation is that as the cells elongate, their rear ends become narrower, subsequently resulting in fewer retraction fibers during impersistent migration. In addition to migration persistence, we reveal that migration speed positively corelates with migrasome formation, owing to the derived length of retraction fibers. Substantiating our hypothesis, genetically removing vimentin compromises cell migration speed and persistence and leads to fewer migrasomes. Together, our data explicate the critical roles of two cell migration patterns, persistence and speed, in the control of migrasome formation by regulating retraction fibers.

[GAO Shu-zhong's clinical experience in "seeking yin from yang needling method" based on "qi street" and "four seas" theories].

The paper introduces professor GAO Shu-zhong's understanding on "seeking yin from yang needling method" and its clinical application on the basis of "qi street" and "four seas" theories. Through professor GAO's clinical practice for years, he integrates and extendes the theories of "seeking yin from yang", "qi street" and "four seas" in Huangdi Neijing (The Yellow Emperor's Inner Classic). In this specific acupuncture method, in reference with the theories of "qi street" and "four seas", acupuncture is exerted on yang part of body, e.g. the back and lumber region to treat the diseases of yin parts, e.g. the chest and abdomen, which is differentiated as yin-yang imbalance in pathogenesis. In order to fully explain the clinical curative effect of "seeking yin from yang needling method", the common diseases in clinic, e.g. the disorders of heart, spleen and stomach systems, as well as the gynecology are taken as examples in the paper.

Feedback-Driven Mechanisms Between Phosphorylated Caveolin-1 and Contractile Actin Assemblies Instruct Persistent Cell Migration.

The actin cytoskeleton and membrane-associated caveolae contribute to active processes, such as cell morphogenesis and motility. How these two systems interact and control directional cell migration is an outstanding question but remains understudied. Here we identified a negative feedback between contractile actin assemblies and phosphorylated caveolin-1 (CAV-1) in migrating cells. Cytoplasmic CAV-1 vesicles display actin-associated motilities by sliding along actin filaments or/and coupling to do retrograde flow with actomyosin bundles. Inhibition of contractile stress fibers, but not Arp2/3-dependent branched actin filaments, diminished the phosphorylation of CAV-1 on site Tyr14, and resulted in substantially increased size and decreased motility of cytoplasmic CAV-1 vesicles. Reciprocally, both the CAV-1 phospho-deficient mutation on site Tyr14 and CAV-1 knockout resulted in dramatic AMPK phosphorylation, further causing reduced active level of RhoA-myosin II and increased active level of Rac1-PAK1-Cofilin, consequently led to disordered contractile stress fibers and prominent lamellipodia. As a result, cells displayed depolarized morphology and compromised directional migration. Collectively, we propose a model in which feedback-driven regulation between actin and CAV-1 instructs persistent cell migration.

Efficacy and safety of acupuncture for adenomyosis: A protocol for systematic review and meta-analysis.

BACKGROUND: Adenomyosis (AM) is a disease in which the endometrium (including glands and stroma) invades the myometrium and grows. The main clinical symptoms include menorrhagia, dysmenorrhea, chronic pelvic pain, metrorrhagia, and dyspareunia, which will seriously affect the physical and mental health of patients, and most of which occur in women of childbearing age. Acupuncture, as a special external treatment of Traditional Chinese medicine, has shown good effects in the treatment of adenomyosis. At present, there is a lack of systematic review on acupuncture in the treatment of adenomyosis. We conduct this study to evaluate the efficacy and safety of acupuncture in the treatment of adenomyosis. METHODS: We will search Chinese and English databases: Medline, Pubmed, EMBASE, Cochrane library, China National Knowledge Infrastructure (CNKI), Chinese Scientific and Journal Database, Wan Fang database (Wanfang), Chinese Biomedical Literature Database (CBM) to identify articles of randomized clinical trials of acupuncture for adenomyosis. All above electronic databases will be searched from inception to September 30, 2021. RevMan 5.3 software will be used to conduct this systematic review. No language and publication status restrictions will be applied. RESULTS: The study will prove the efficacy and safety of acupuncture for adenomyosis. CONCLUSION: We plan to submit this systematic review to a peer-reviewed journal. TRIAL REGISTRATION NUMBER: CRD42021277136.

Myosin-18B Promotes Mechanosensitive CaMKK2-AMPK-VASP Regulation of Contractile Actin Stress Fibers.

Actin stress fibers guide cell migration and morphogenesis. During centripetal flow, actin transverse arcs fuse accompanied by the formation of myosin II stacks to generate mechanosensitive actomyosin bundles. However, whether myosin II stack formation plays a role in cell mechano-sensing has remained elusive. Myosin-18B is a "glue" molecule for assembling myosin II stacks. By examining actin networks and traction forces, we find that cells abolishing myosin-18B resemble Ca2+∕calmodulin-dependent kinase kinase 2 (CaMKK2)-defective cells. Inhibition of CaMKK2 activity reverses the strong actin network to thin filaments in myosin-18B-overexpressing cells. Moreover, AMP-activated protein kinase (AMPK) activation is able to relieve the thin stress fibers by myosin-18B knockout. Importantly, lack of myosin-18B compromises AMPK-vasodilator-stimulated phosphoprotein and RhoA-myosin signaling, thereby leading to defective persistent migration, which can be rescued only by full-length and C-extension-less myosin-18B. Together, these results reveal a critical role of myosin-18B in the mechanosensitive regulation of migrating cells.

Graphene oxide coating core-shell silver sulfide@mesoporous silica for active targeted dual-mode imaging and chemo-photothermal synergistic therapy against tumors.

The accurate treatment of tumors with the help of multimodality imaging is of great significance. Herein, a novel multifunctional probe combining active targeted fluorescent imaging (FL)/photoacoustic imaging (PA) and chemo-photothermal therapy for tumors has been designed. Targeting molecule folate (FA) modified graphene oxide (GO) was used to coat core-shell silver sulfide@mesoporous silica (QD@Si) while antitumoral doxorubicin (DOX) was loaded in mesoporous channels by electrostatic adhesion, and a delivery system (QD@Si-D/GO-FA) for active targeted dual-mode imaging and synergistic chemo-photothermal for tumors was successfully obtained. Experiments showed the cell survival rate was 76.3 ± 4.6% when the probe concentration reached 0.5 mg mL-1, and demonstrated that the probe had good biocompatibility. In vivo and in vitro results indicated that this probe could be used for active targeted FL/PA for tumors with overexpressed FA receptors. The temperature of the tumor rose to 63.5 °C under laser irradiation, and the tumor could be killed more effectively after combination with chemotherapy, which was caused by exfoliation of GO from QD@Si-D/GO-FA after irradiation. These results showed that the probe had great potential for application in oncology and is expected to provide evidence for early diagnosis and treatment of tumors.