Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.

Both the cytoskeletal intermediate filaments (IFs) and cytoplasmic caveolae contribute to active processes such as cell migration, morphogenesis and vesicular trafficking, but the interplay between these two systems has remained elusive. Here, we find that vimentin and nestin IFs interact with caveolae central component caveolin-1 (CAV-1) and importantly, restrain the intracellular trafficking of CAV-1 positive vesicles by serving as a physical barrier. Consequently, CAV-1 vesicles show less density and mobility in vimentin IFs enriched region, which is a substrate stiffness independent process. Moreover, depletion of vimentin IFs releases the slow movement proportion of CAV-1 positive vesicles and thus increases their cytoplasmic dynamics, whereas the expression of caveolae-associated protein CAV-1, CAV-2 and cavin-1 were unaffected. Collectively, these results reveal a negative role of IFs in regulating the trafficking of intracellular CAV-1 vesicles in live cells.

A variety of intracellular bacteria modulate the host cytoskeleton to establish subcellular niches for replication. However, the role of intermediate filaments, which are crucial for mechanical strength and resilience of the cell, and in bacterial vacuole preservation remains unclear. Here, we show that Salmonella effector SopB reorganizes the vimentin network to form cage-like structures that surround Salmonella-containing vacuoles (SCVs). Genetic removal of vimentin markedly disrupts SCV organization, significantly reduces bacterial replication and cell death. Mechanistically, SopB uses its N-terminal Cdc42-binding domain to interact with and activate Cdc42 GTPase, which in turn recruits vimentin around SCVs. A high-content imaging-based screening identified that MEK1/2 inhibition led to vimentin dispersion. Our work therefore elucidates the signaling axis SopB-Cdc42-MEK1/2 as mobilizing host vimentin to maintain concrete SCVs and identifies a mechanism contributing to Salmonella replication. Importantly, Trametinib, a clinically-approved MEK1/2 inhibitor identified in the screen, displayed significant anti-infection efficacy against Salmonella both in vitro and in vivo, and may provide a therapeutic option for treating drug-tolerant salmonellosis.

Eukaryotic cells have diverse protrusive and contractile actin filament structures, which compete with one another for a limited pool of actin monomers. Numerous actin-binding proteins regulate the dynamics of actin structures, including tropomodulins (Tmods), which cap the pointed end of actin filaments. In striated muscles, Tmods prevent actin filaments from overgrowing, whereas in non-muscle cells, their function has remained elusive. Here, we identify two Tmod isoforms, Tmod1 and Tmod3, as key components of contractile stress fibers in non-muscle cells. Individually, Tmod1 and Tmod3 can compensate for one another, but their simultaneous depletion results in disassembly of actin-tropomyosin filaments, loss of force-generating stress fibers, and severe defects in cell morphology. Knockout-rescue experiments reveal that Tmod's interaction with tropomyosin is essential for its role in the stabilization of actin-tropomyosin filaments in cells. Thus, in contrast to their role in muscle myofibrils, in non-muscle cells, Tmods bind actin-tropomyosin filaments to protect them from depolymerizing, not elongating. Furthermore, loss of Tmods shifts the balance from linear actin-tropomyosin filaments to Arp2/3 complex-nucleated branched networks, and this phenotype can be partially rescued by inhibiting the Arp2/3 complex. Collectively, the data reveal that Tmods are essential for the maintenance of contractile actomyosin bundles and that Tmod-dependent capping of actin-tropomyosin filaments is critical for the regulation of actin homeostasis in non-muscle cells.

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.

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.

The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work, little is known how exocyst function is controlled. To identify regulators for exocyst function, we performed a targeted RNA interference (RNAi) screen in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. We identified seven kinase and seven phosphatase genes that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. We show that in line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic soluble NSF attachment protein receptor (SNARE) proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the phosphatase protein phosphatase 2A (PP2A) phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular green fluorescent protein signal for the same proteins. Double RNAi experiments for par-1, pkc-3, and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in postembryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that atypical protein kinase C kinase and phosphatase PP2A regulate exocyst-dependent, insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process.

Cell adhesion, morphogenesis, mechanosensing, and muscle contraction rely on contractile actomyosin bundles, where the force is produced through sliding of bipolar myosin II filaments along actin filaments. The assembly of contractile actomyosin bundles involves registered alignment of myosin II filaments and their subsequent fusion into large stacks. However, mechanisms underlying the assembly of myosin II stacks and their physiological functions have remained elusive. Here, we identified myosin-18B, an unconventional myosin, as a stable component of contractile stress fibers. Myosin-18B co-localized with myosin II motor domains in stress fibers and was enriched at the ends of myosin II stacks. Importantly, myosin-18B deletion resulted in drastic defects in the concatenation and persistent association of myosin II filaments with each other and thus led to severely impaired assembly of myosin II stacks. Consequently, lack of myosin-18B resulted in defective maturation of actomyosin bundles from their precursors in osteosarcoma cells. Moreover, myosin-18B knockout cells displayed abnormal morphogenesis, migration, and ability to exert forces to the environment. These results reveal a critical role for myosin-18B in myosin II stack assembly and provide evidence that myosin II stacks are important for a variety of vital processes in cells.

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.

HCV cell-culture system uses hepatoma-derived cell lines for efficient virus propagation. Tumor cells cultured in glucose undergo active aerobic glycolysis, but switch to oxidative phosphorylation for energy production when cultured in galactose. Here, we investigated whether modulation of glycolysis in hepatocytes affects HCV infection. We showed HCV release, but not entry, genome replication or virion assembly, is significantly blocked when cells are cultured in galactose, leading to accumulation of intracellular infectious virions within multivesicular body (MVB). Blockade of the MVB-lysosome fusion or treatment with pro-inflammatory cytokines promotes HCV release in galactose. Furthermore, we found this glycometabolic regulation of HCV release is mediated by MAPK-p38 phosphorylation. Finally, we showed HCV cell-to-cell transmission is not affected by glycometabolism, suggesting that HCV cell-to-supernatant release and cell-to-cell transmission are two mechanistically distinct pathways. In summary, we demonstrated glycometabolism regulates the efficiency and route of HCV release. We proposed HCV may exploit the metabolic state in hepatocytes to favor its spread through the cell-to-cell transmission in vivo to evade immune response.

In fluorescence microscopy, computational algorithms have been developed to suppress noise, enhance contrast, and even enable super-resolution (SR). However, the local quality of the images may vary on multiple scales, and these differences can lead to misconceptions. Current mapping methods fail to finely estimate the local quality, challenging to associate the SR scale content. Here, we develop a rolling Fourier ring correlation (rFRC) method to evaluate the reconstruction uncertainties down to SR scale. To visually pinpoint regions with low reliability, a filtered rFRC is combined with a modified resolution-scaled error map (RSM), offering a comprehensive and concise map for further examination. We demonstrate their performances on various SR imaging modalities, and the resulting quantitative maps enable better SR images integrated from different reconstructions. Overall, we expect that our framework can become a routinely used tool for biologists in assessing their image datasets in general and inspire further advances in the rapidly developing field of computational imaging.

The actin and intermediate filament cytoskeletons contribute to numerous cellular processes, including morphogenesis, cytokinesis and migration. These two cytoskeletal systems associate with each other, but the underlying mechanisms of this interaction are incompletely understood. Here, we show that inactivation of vimentin leads to increased actin stress fiber assembly and contractility, and consequent elevation of myosin light chain phosphorylation and stabilization of tropomyosin-4.2 (see Geeves et al., 2015). The vimentin-knockout phenotypes can be rescued by re-expression of wild-type vimentin, but not by the non-filamentous 'unit length form' vimentin, demonstrating that intact vimentin intermediate filaments are required to facilitate the effects on the actin cytoskeleton. Finally, we provide evidence that the effects of vimentin on stress fibers are mediated by activation of RhoA through its guanine nucleotide exchange factor GEF-H1 (also known as ARHGEF2). Vimentin depletion induces phosphorylation of the microtubule-associated GEF-H1 on Ser886, and thereby promotes RhoA activity and actin stress fiber assembly. Taken together, these data reveal a new mechanism by which intermediate filaments regulate contractile actomyosin bundles, and may explain why elevated vimentin expression levels correlate with increased migration and invasion of cancer cells.

The exocyst complex is required for cell polarity regulation and the targeting and tethering of transport vesicles to the plasma membrane. The complex is structurally well conserved, however, the functions of individual subunits and their regulation is poorly understood. Here we characterize the mutant phenotypes for the exocyst complex genes exoc-7 (exo70) and exoc-8 (exo84) in Caenorhabditis elegans. The mutants display pleiotropic behavior defects that resemble those observed in cilia mutants (slow growth, uncoordinated movement, defects in chemo-, mechano- and thermosensation). However, no obvious morphological defects in cilia were observed. A targeted RNAi screen for small GTPases identified eleven genes with enhanced phenotypes when combined with exoc-7, exoc-8 single and exoc-7;exoc-8 double mutants. The screen verified previously identified functional links between the exocyst complex and small GTPases and, in addition, identified several novel potential regulators of exocyst function. The exoc-8 and exoc-7;exoc-8 mutations caused a significant size increase in the rab-10 RNAi-induced endocytic vacuoles in the intestinal epithelial cells. In addition, exoc-8 and exoc-7;exoc-8 mutations resulted in up-regulation of RAB-10 expression and affected the accumulation of endocytic marker proteins in these cells in response to rab-10 RNAi. The findings identify novel, potential regulators for exocyst function and show that exoc-7 and exoc-8 are functionally linked to rab-10 in endosomal trafficking in intestinal epithelial cells in C. elegans.

Protein-protein interactions govern almost all cellular functions. These complex networks of stable and transient associations can be mapped by affinity purification mass spectrometry (AP-MS) and complementary proximity-based labeling methods such as BioID. To exploit the advantages of both strategies, we here design and optimize an integrated approach combining AP-MS and BioID in a single construct, which we term MAC-tag. We systematically apply the MAC-tag approach to 18 subcellular and 3 sub-organelle localization markers, generating a molecular context database, which can be used to define a protein's molecular location. In addition, we show that combining the AP-MS and BioID results makes it possible to obtain interaction distances within a protein complex. Taken together, our integrated strategy enables the comprehensive mapping of the physical and functional interactions of proteins, defining their molecular context and improving our understanding of the cellular interactome.

Ras homologous guanosine triphosphatases (RhoGTPases) control several cellular functions, including cytoskeletal actin remodeling and cell migration. Their activities are downregulated by GTPase-activating proteins (GAPs). Although RhoGTPases are implicated in bone remodeling and osteoclast and osteoblast function, their significance in human bone health and disease remains elusive. Here, we report defective RhoGTPase regulation as a cause of severe, early-onset, autosomal-dominant skeletal fragility in a three-generation Finnish family. Affected individuals (n = 13) presented with multiple low-energy peripheral and vertebral fractures despite normal bone mineral density (BMD). Bone histomorphometry suggested reduced bone volume, low surface area covered by osteoblasts and osteoclasts, and low bone turnover. Exome sequencing identified a novel heterozygous missense variant c.652G>A (p.G218R) in ARHGAP25, encoding a GAP for Rho-family GTPase Rac1. Variants in the ARHGAP25 5' untranslated region (UTR) also associated with BMD and fracture risk in the general population, across multiple genomewide association study (GWAS) meta-analyses (lead variant rs10048745). ARHGAP25 messenger RNA (mRNA) was expressed in macrophage colony-stimulating factor (M-CSF)-stimulated human monocytes and mouse osteoblasts, indicating a possible role for ARHGAP25 in osteoclast and osteoblast differentiation and activity. Studies on subject-derived osteoclasts from peripheral blood mononuclear cells did not reveal robust defects in mature osteoclast formation or resorptive activity. However, analysis of osteosarcoma cells overexpressing the ARHGAP25 G218R-mutant, combined with structural modeling, confirmed that the mutant protein had decreased GAP-activity against Rac1, resulting in elevated Rac1 activity, increased cell spreading, and membrane ruffling. Our findings indicate that mutated ARHGAP25 causes aberrant Rac1 function and consequently abnormal bone metabolism, highlighting the importance of RhoGAP signaling in bone metabolism in familial forms of skeletal fragility and in the general population, and expanding our understanding of the molecular pathways underlying skeletal fragility. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Myosin is a diverse superfamily of motor proteins responsible for actin-based motility and contractility in eukaryotic cells. Myosin-18 family, including myosin-18A and myosin-18B, belongs to an unconventional class of myosin, which lacks ATPase motor activity, and the investigations on their functions and molecular mechanisms in vertebrate development and diseases have just been initiated in recent years. Myosin-18A is ubiquitously expressed in mammalian cells, whereas myosin-18B shows strong enrichment in striated muscles. Myosin-18 family is important for cell motility, sarcomere formation, and mechanosensing, mostly by interacting with other cytoskeletal proteins and cellular apparatus. Myosin-18A participates in several intracellular transport processes, such as Golgi trafficking, and has multiple roles in focal adhesions, stress fibers, and lamellipodia formation. Myosin-18B, on the other hand, participates in actomyosin alignment and sarcomere assembly, thus relating to cell migration and muscle contractility. Mutations of either Myo18a or Myo18b cause cardiac developmental defects in mouse, emphasizing their crucial role in muscle development and cardiac diseases. In this review, we revisit the discovery history of myosin-18s and summarize the evolving understanding of the molecular functions of myosin-18A and myosin-18B, with an emphasis on their separate yet closely related functions in cell motility and contraction. Moreover, we discuss the diseases tightly associated with myosin-18s, especially cardiovascular defects and cancer, as well as highlight the unanswered questions and potential future research perspectives on myosin-18s.

Lipid droplets (LDs) are dynamic lipid storage organelles that can be degraded by autophagy machinery to release neutral lipids, a process called lipophagy. However, specific receptors and regulation mechanisms for lipophagy remain largely unknown. Here, we identify that ATG14, the core unit of the PI3KC3-C1 complex, also targets LD and acts as an autophagic receptor that facilitates LD degradation. A negative regulator, Syntaxin18 (STX18) binds ATG14, disrupting the ATG14-ATG8 family members interactions and subverting the PI3KC3-C1 complex formation. Knockdown of STX18 activates lipophagy dependent on ATG14 not only as the core unit of PI3KC3-C1 complex but also as the autophagic receptor, resulting in the degradation of LD-associated anti-viral protein Viperin. Furthermore, coronavirus M protein binds STX18 and subverts the STX18-ATG14 interaction to induce lipophagy and degrade Viperin, facilitating virus production. Altogether, our data provide a previously undescribed mechanism for additional roles of ATG14 in lipid metabolism and virus production.

Structured illumination microscopy (SIM) has become a widely used tool for insight into biomedical challenges due to its rapid, long-term, and super-resolution (SR) imaging. However, artifacts that often appear in SIM images have long brought into question its fidelity, and might cause misinterpretation of biological structures. We present HiFi-SIM, a high-fidelity SIM reconstruction algorithm, by engineering the effective point spread function (PSF) into an ideal form. HiFi-SIM can effectively reduce commonly seen artifacts without loss of fine structures and improve the axial sectioning for samples with strong background. In particular, HiFi-SIM is not sensitive to the commonly used PSF and reconstruction parameters; hence, it lowers the requirements for dedicated PSF calibration and complicated parameter adjustment, thus promoting SIM as a daily imaging tool.

Vimentin has been implicated in wound healing, inflammation, and cancer, but its functional contribution to intestinal diseases is poorly understood. To study how vimentin is involved during tissue injury and repair of simple epithelium, we induced colonic epithelial cell damage in the vimentin null (Vim-/-) mouse model. Vim-/- mice challenged with dextran sodium sulfate (DSS) had worse colitis manifestations than wild-type (WT) mice. Vim-/- colons also produced more reactive oxygen and nitrogen species, possibly contributing to the pathogenesis of gut inflammation and tumorigenesis than in WT mice. We subsequently describe that CD11b+ macrophages served as the mainly cellular source of reactive oxygen species (ROS) production via vimentin-ROS-pSTAT3-interleukin-6 inflammatory pathways. Further, we demonstrated that Vim-/- mice did not develop colitis-associated cancer model upon DSS treatment spontaneously but increased tumor numbers and size in the distal colon in the azoxymethane/DSS model comparing with WT mice. Thus, vimentin has a crucial role in protection from colitis induction and tumorigenesis of the colon.

Mechanical stress following injury regulates the quality and speed of wound healing. Improper mechanotransduction can lead to impaired wound healing and scar formation. Vimentin intermediate filaments control fibroblasts' response to mechanical stress and lack of vimentin makes cells significantly vulnerable to environmental stress. We previously reported the involvement of exosomal vimentin in mediating wound healing. Here we performed in vitro and in vivo experiments to explore the effect of wide-type and vimentin knockout exosomes in accelerating wound healing under osmotic stress condition. Our results showed that osmotic stress increases the size and enhances the release of exosomes. Furthermore, our findings revealed that exosomal vimentin enhances wound healing by protecting fibroblasts against osmotic stress and inhibiting stress-induced apoptosis. These data suggest that exosomes could be considered either as a stress modifier to restore the osmotic balance or as a conveyer of stress to induce osmotic stress-driven conditions.