BMC Molecular and Cell Biology

BackgroundLamins are major regulators of the spatial and functional organization of chromatin. Lamins at the nuclear periphery form the lamina that anchors heterochromatin to the nuclear envelope. A subpool of A-type lamins localizes in the nuclear interior, where they also bind to euchromatic genomic regions. A-type lamin properties and chromatin association are regulated by lamin-associated polypeptide 2alpha (LAP2). Here we systematically analyze, how LAP2 depletion affects chromatin organization, accessibility and gene expression on a genome-wide level. ResultsLAP2 depletion in mouse dermal fibroblasts positively and negatively affects chromatin accessibility and gene expression throughout the genome, which correlates with changes in chromatin association of A-type lamins and the nucleosomal remodeler proteins BRG1 and CHD4. In particular, A-type lamins bind to open chromatin regions close to BRG1 and CHD4 binding sites and deregulated genes, but do not directly accumulate on genes and BRG1 and CHD4-enriched sites. Unsupervised clustering of the datasets on LAP2-bound genomic regions confirms spreading of A-type lamins to active chromatin regions containing deregulated genes and an enrichment of chromatin remodelers on a subset of these genomic regions. ConclusionsLAP2 depletion in fibroblasts leads to a gross rearrangement of chromatin. Genome-wide chromatin reorganization is linked to spreading of A-type lamins to active chromatin regions and accompanied by a restriction of chromatin remodelers to a subset of active genomic regions. These changes correlate with changes in chromatin accessibility and gene expression throughout the genome, particularly in regions where lamin binding is gained in LAP2 knockout versus wildtype cells.

Tropomyosins are coiled-coil dimers that polymerize head-to-tail along actin filaments. They stabilize distinct filament populations and regulate the access of myosins and actin-binding proteins in both muscle and non-muscle contexts. Despite their central regulatory role, how filament length and isoform identity of different tropomyosin homologues might modulate actin affinity is not completely understood, especially across species. Here, we present a stepwise computational docking pipeline combining AlphaFold2-Multimer coiled-coil models, experimentally informed residue-level restraints, and pseudo-energy analysis via PPCheck to build and evaluate actin-tropomyosin co-polymer models for three isoforms: human TPM1 (hTPM1; 284 residues), human TPM4 (hTPM4; 248 residues), and Schizosaccharomyces pombe Cdc8 (SpCdc8; 161 residues). Interface energetics reveal a consistent hierarchy in which the shortest filament, SpCdc8, achieves the most stabilizing and residue-rich actin contacts, consistent with reduced cumulative geometric penalty along the actin helix. Among human isoforms, hTPM1 forms stronger interfaces with actin than hTPM4. The hTPM1-actin model also exhibits higher contact density and additional energetic hotspots, in agreement with the experimentally established slower exchange kinetics of TPM1 isoforms on actin filaments relative to TPM4. Hotspot mapping identifies conserved acidic residues at equivalent positions across all three isoforms, emphasizing the importance of electrostatic anchor points in maintaining interface integrity across diverse evolutionary contexts. Modeling of four temperature-sensitive SpCdc8 mutations (A18T, R21H, E31K and E129K) reveals that these substitutions substantially destabilize the coiled-coil dimer without significantly affecting actin interactions, suggesting that subtle regulatory failure arises from compromised longitudinal cable continuity rather than from direct loss of actin affinity. Taken together, our results support a hierarchical model of tropomyosin dimer stability, actin-tropomyosin recognition in which filament length imposes a geometric baseline on interface stability, onto which isoform-specific sequence evolution superimposes functional tuning. The tropomyosin homologues we studied appear to retain conserved electrostatic hotspots thereby providing a common structural scaffold across tissues and organisms.

Using a genetic screening approach based on an inducible gene-activating system and cell sorting, Down syndrome critical region 3 (DSCR3) was isolated as a gene whose overexpression increased cell size. Fibroblasts derived from individuals with Down syndrome (DS) exhibit elevated DSCR3 expression at both the mRNA and protein levels, correlating with increased cell volume compared to fibroblasts from healthy donors. Despite a slower proliferation rate, DS fibroblasts demonstrate higher basal and maximal mitochondrial respiration, suggesting enhanced metabolic activity associated with increased cell size. siRNA-mediated knockdown of DSCR3 reduces cell size in both DS and normal fibroblasts, indicating its general role in cell size regulation. As DSCR3 is a component of the retriever complex involved in endosomal cargo recycling, these findings position membrane protein trafficking as a novel module for cell size control.

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

The methyltransferase EZH2 (Enhancer of Zest Homolog 2) and the demethylase KDM6B (Lysine Demethylase 6B) have been associated with epithelial to mesenchymal transition (EMT) and poor prognosis in various cancers. These enzymes methylate and demethylate H3K27me3 and regulate distinct sets of genes controlling EMT induction, despite having opposite catalytic activities. This could be due to their recruitment or the modulation of their activity by partner proteins on specific loci. This work sought to identify proteins associated with chromatin and interacting with EZH2 or with KDM6B during EMT. To do so, co-immunoprecipitation and mass spectroscopy was used under TGF{beta} (Tumor growth factor {beta}) and TNF (Tumor necrosis factor ) treatment to induce EMT in A549 lung cancer cells. Surprisingly, numerous proteins related to focal adhesions were identified to interact with EZH2 or KDM6B. These proteins are part of a nuclear protein interaction network previously described as nucleo-adhesome. Among these proteins, TGFB1I1 (transforming growth factor induced peptide 1) and CSRP2 (cysteine and glycine rich protein 2) were further confirmed to interact with KDM6B in the nucleus and even more so during EMT. The target genes of these complexes were then sought by knocking down KDM6B, TGFB1I1 or CSRP2. Three genes (coding Integrin alpha 5, Laminin y2 and Matrix Metalloproteinase 9) were confirmed to be regulated by KDM6B, TGFB1I1 and CSRP2. These findings may have clinical relevance, as immunohistochemistry analyses performed on a cohort of lung cancer patients revealed increased nuclear localization of TGFB1I1 and CSRP2 in cells undergoing EMT.

Mechanical forces have a major effect on cell behavior. Most cells in vitro are grown under static conditions on hard tissue culture plastic, conditions that do not accurately reflect living tissues. The ability of cells to sense and respond to mechanical forces is essential for key biological processes, including development, proliferation, and migration. Disruption of the ability to respond to mechanical forces are known to be a critical factor in many diseases, including cardiovascular disease, progeria, and cancer. Here, we present the design, fabrication, and biological testing of a custom-built cell-stretching device that applies controlled biaxial strain to cells cultured on a polydimethylsiloxane (PDMS) membrane. We then used this device to examine how cells respond to strain. In response to biaxial strain, MCF-7 cells activated the mechanosensitive immediate early gene (IEX-1), with its expression increasing significantly after 1 and 3 hours of stretching. Cells exposed to mechanical strain also remodeled their cytoskeleton in a direction-dependent manner. Under uniaxial strain, actin filaments reoriented perpendicular to the stretch direction, whereas biaxially stretched cells do not promote directional reorientation, but instead appear to reinforce actin at the cell periphery. Similarly, cells under uniaxial strain exhibited changes in nuclear orientation and shape that were not observed under biaxial strain. Nuclear area remained unchanged in either strain condition. These results highlight that the biaxial stretcher can be used to apply strain to cells, and that cells respond differently to biaxial strain compared to what has been reported for uniaxial strain.

Kinesins are molecular motor proteins essential for organizing and remodeling the cytoskeleton during neuronal development and maintenance. One key regulator is kinesin family binding protein (KIFBP), which inhibits a subset of kinesins by blocking motor-microtubule interactions. Homozygous mutations in KIFBP cause Goldberg-Shprintzen Syndrome (GOSHS), a neurodevelopmental disorder characterized by intellectual disability, microcephaly, and axonal neuropathy. Although loss of KIFBP has been linked to reduced neurite length and microtubule disorganization, the specific kinesins underlying these phenotypes remain unclear. Here we use a CRISPR-Cas9 generated KIFBP knockout Neuro-2a cell line to demonstrate that KIFBP is required for neurite extension and use inducible GFP-KIFBP to define the KIFBP interactome during neuronal differentiation. Immunoprecipitation coupled with mass spectrometry identified both known and novel KIFBP-associated kinesins. Single molecule TIRF microscopy confirmed direct inhibition of a subset of kinesins that co-immunoprecipitated with KIFBP. Notably, we identified KIF5A and KIF18B as previously unrecognized regulatory targets with potential roles in neuronal development. Together, these findings establish Neuro-2a cells as a model for studying KIFBP function and provide new insight into the regulation of kinesin activity and cytoskeletal dynamics in neurons.

Epithelial cells in Hydra perform an unusual combination of functions: they divide continuously like adult stem cells while simultaneously executing the complex physiological tasks of differentiated epithelia. This challenges the traditional distinction between proliferative stem cells and terminally differentiated tissue, raising the question of how a single cell type integrates these opposing roles. Using electron microscopy, we examined morphological characteristics that define the stem-like and differentiated states of Hydras ectodermal and endodermal epithelial cells. Stemness is reflected by nuclear characteristics of active proliferation, including extensive euchromatin, large nucleoli, and the presence of nuage. However, differentiated epithelial cells exhibit strong apical-basal polarity, various endomembrane compartments for endocytosis and transport, specialized secretion mechanisms, and basal muscle processes with dense-core vesicles implicated in hormonal communication. Cryofixation improved ultrastructure preservation, elucidating the pleiomorphic configurations of complex intracellular channel systems traditionally presenting as singular vacuoles. This may shed new light on possible functions of this compartment. Taken together, Hydra epithelial cells combine ancient stem cell traits with highly specialized differentiated functions. This multifunctionality provides insight into the cellular organization of early-branching animals and suggests that multifunctional epithelia may represent an ancestral condition preceding the strict segregation of stem and differentiated cell lineages in bilaterians.

Uveal Melanoma (UM) is the most common eye cancer, with a metastatic mortality rate of 80%. Only 1-3% of patients have detectable UM at metastasis, and UM exhibits punctuated early growth. Doxycycline has recently been shown to inhibit metabolic processes exploited by cancer cells and reduce cancer cell growth in models of liver cancer. We hypothesized doxycycline may also be effective in UM and therefore tested doxycycline treatment in an eye organoid model of uveal melanoma. Using a stem cell line whereby BAP1 can be knocked down with a tetracycline-inducible system, we differentiated this line into a whole eye organoid model termed self-formed ectodermal autonomous multi-zone of ocular cells (SEAM). We found an enhanced proliferation in neural crest cells within the SEAM colonies. To identify the neural crest cells, we conducted single-cell RNA sequencing (scRNA-seq) analysis utilizing the Seurat R toolkit to pinpoint genes within neural crest clusters. To confirm the results of the in silico scRNA-seq analysis, genes with notable functions and differential expression in the neural crest cluster in relation to UM proliferation, angiogenesis, and oxidative phosphorylation were analyzed through immunofluorescence and RT-qPCR. Based on the scRNA-seq analysis, immunofluorescence, and RT-qPCR, the novel BAP1 KD (UM phenotype) model was found to replicate UM-relevant gene and protein expressions effectively, so the BAP1 KD (UM phenotype) was then treated with doxycycline to evaluate its effect on UM metastasis. Subsequent analysis found that doxycycline significantly inhibited UM growth, angiogenesis, and oxidative phosphorylation in the BAP1 KD (UM phenotype) model more than that of the control model, perhaps due to doxycycline targeting higher regions with more mitochondrial activity, indicating doxycyclines therapeutic potential in treating UM.

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

Transient plasma membrane domains called lipid rafts have emerged as important regulators of signal transduction. These territories are formed by lipid-lipid and lipid-protein interactions, and these local interactions can be scaffolded by resident lipid raft organizing protein family members. While roles for lipid rafts have been described for multiple signaling pathways in many contexts, their in vivo prevalence and role during embryonic development remains incompletely understood. Here we examined gene expression for the Raftlin family of lipid raft organizing proteins, Raftlin (RFTN1) and Raftlin-2 (RFTN2), over the course of early vertebrate development, with a focus on neural crest cell dynamics. By analyzing transcriptomic data across vertebrate species, we identified conserved patterns of RFTN1 and RFTN2 expression across species, where RFTN1 is broadly expressed at low levels, while RFTN2 is distinctly enriched in neural crest cells. We used fluorescent in situ hybridization to spatially define Raftlin gene expression patterns in the early avian embryo. Our results show that RFTN1 is broadly expressed with periods of enrichment in the developing paraxial mesoderm. In contrast, RFTN2 expression is strongly enriched in neural crest cells, beginning during specification and persisting through migration, with additional expression in both the cranial and intermediate mesoderm. Together, these patterns suggest that Raftlins may play important roles in regulating signaling during development with specific roles in somitogenesis and in neural crest and mesodermal cell migrations.

Long non-coding RNAs (lncRNAs) have attracted increasing interest because of their roles as modulators of tumor progression, acting either as oncogenic drivers or tumor suppressors, depending on the cellular context. LINC01133 has been implicated in regulation of multiple tumor-related mechanisms; however, its role in breast cancer, particularly in the triple-negative subtype, remains poorly characterized. In this study, we investigated the impact of LINC01133 depletion on malignant phenotypes and on the expression of migration- and invasion-associated genes using the Hs578T triple-negative breast cancer (TNBC) cell line, through comparative analyses of parental, control, and LINC01133-knockout cell lines, namely Hs578T_wt, Hs578T_ctr, and Hs578T_ko. Functional characterization included morphological analysis, growth assays, anchorage-independent colony formation, migration, invasion, and quantitative biomolecular experiments. Depletion of LINC01133 led to reduction of cell diameter, a significant increase in colony-forming capacity, and marked enhancement of migratory and invasive potential. At the molecular level, LINC01133 loss induced the expression of genes associated with extracellular matrix remodeling and cellular plasticity, including fibronectin, vimentin, integrins, FOXC1, and TWIST1, concomitant with reduced expression of ZEB1, TWIST2, and N-cadherin. Collectively, these data indicate that LINC01133 acts as a potential fine regulator of in vitro migration and invasion processes in TNBC, with its expression favoring a more asymptomatic mode of tumor progression, whereas its loss markedly enhances tumor malignancy.

Gasdermins (GSDMs) are a family of pore forming protein that trigger pyroptosis by permeabilizing cell membranes. Pyroptotic cells often release the proinflammatory cytokines interleukin-1{beta} (IL-1{beta}), and IL-18, thereby promoting an inflammatory response. GSDMs are typically cleaved by caspases or granzymes, which enable their translocation to the membrane. Here, we showed GSDMA and GSMDB localize to the cytoskeletal fraction of keratinocytes. Disruption of cell junctions causes gasdermin A and B (GSDMA and GSDMB) to translocate to the membrane fraction in the absence of cleavage. Cell junction disrupted keratinocytes release post-translationally modified keratins, but not IL-1{beta} or IL-18. These events depend on endocytic mechanisms associated with recycling of cell junctional proteins. Our study suggests that cell junction disruption can drive translocation of GSDMA and GSDMB from cytoskeleton to plasma membrane in keratinocytes, however there may be a subsequent trigger that causes the confirmational change allowing these gasdermins to form open pores.

Endothelial-to-mesenchymal transition (EndMT) has become a central mechanism in developmental biology, fibrosis, vascular disease, and cancer. We previously reported on an integrated signaling model in which TGF-{beta}2 induces EndMT through coordinated activation of Smad-dependent and Smad-independent signaling pathways converging on Snail, while GSK-3{beta} regulates Snail activity. We performed a systematic figure-by-figure reproducibility analysis of the original publication. Independent studies published between 2011 and 2026 were identified and curated according to predefined inclusion criteria. Each original experimental conclusion was evaluated for independent confirmation. In parallel, selected biochemical experiments were independently reproduced using newly acquired reagents and contemporary Western blot methodologies. Independent publications consistently reproduced each major mechanistic conclusion of the original study, including activation of Smad, ERK, PI3K/AKT, and p38 MAPK signaling, regulation of Snail expression, EndMT-associated marker switching, and GSK-3{beta}-dependent control of Snail activity. Independent laboratory experiments reproduced the principal biochemical findings using contemporary reagents and experimental workflows. The combined literature analysis and independent laboratory replication demonstrate that the mechanistic framework in our previous study has remained reproducible across multiple laboratories, endothelial cell types, disease models, and fifteen years of investigation. This work illustrates a complementary framework for assessing reproducibility that integrates direct experimental replication with cumulative independent validation.

BackgroundMechanical forces, particularly tensile stress, influence tumor progression by modulating cancer-associated fibroblast (CAF) behaviour and extracellular matrix remodelling, yet their role in oral cancer remains insufficiently defined. To our knowledge, this is the first study investigating in vitro tensile stress responses in CAF and normal fibroblasts (NF) derived from oral squamous cell carcinoma (OSCC). ObjectiveTo assess how tensile stress affects fibroblast gap closure, proliferation, metabolic activity, and paracrine signalling relevant to tumor-stroma interactions. MethodsCAFs and NFs were isolated from OSCC tissue and matched healthy mucosa and exposed to cyclic tensile strain (3%, 0.02 Hz, 96 h) using the FX-6000T system. Gap closure was assessed by wound-healing assay, proliferation by cell counting, metabolic activity by AlamarBlue, and paracrine effects on A549 tumor cell gap closure using fibroblast-conditioned supernatants. ResultsTensile loading significantly increased CAF gap closure capacity compared with both stimulated NFs (p<0.0001) and unstimulated CAF controls (p=0.0167). Metabolic activity showed a non-significant trend toward higher values in CAFs. Proliferation did not differ between groups up to 48 h, arguing against a major early contribution of proliferation to the observed group differences in the fibroblast scratch assays; however, because proliferation was not inhibited and was not quantified beyond 48 h, later time points should be interpreted conservatively as composite gap closure. Supernatants from mechanically stimulated CAFs increased A549 gap closure by [~]40% compared with non-stimulated controls (p=0.0485), whereas stimulated NFs did not enhance the tumor cells capacity to close the cell-free gap. ConclusionsTensile strain was associated with increased fibroblast gap closure and enhanced gap-closure-promoting paracrine effects of oral CAFs under the conditions tested. These exploratory findings support the concept that biomechanical cues can modulate CAF functional behaviour in OSCC. Given the limited cohort size and inter-patient variability, these results should be interpreted as hypothesis-generating and require confirmation in larger studies. Mechanistic pathways were not interrogated in this study and should be addressed in future work using more complex tumor-stroma models. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/737904v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@7fb458org.highwire.dtl.DTLVardef@192ee6org.highwire.dtl.DTLVardef@1562f54org.highwire.dtl.DTLVardef@13d3ebd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Down syndrome is a neurodevelopmental disorder caused by the trisomy of human chromosome 21 (T21). Down syndrome is associated with a wide range of variable clinical features, including congenital heart defects and slow wound healing; however, intellectual disability is ubiquitous and results, in part, from altered neuronal connectivity. Here, we used three sets of control and T21 human fibroblasts, and one set of human induced pluripotent stem cell (hiPSC)-derived cortical neurons, to examine whether changes in cellular morphology and motility are consistent across cell types in Down syndrome and to elucidate the underlying mechanisms. We found that fibroblast morphology is dysregulated in all T21 fibroblast lines. Using a transwell migration assay, cellular migration is decreased in two of the three T21 fibroblast lines. T21 hiPSC-derived cortical neurons also exhibit morphological defects, including a decrease in the length of the longest neurite and growth cone area. Because of these significant changes in morphology and motility in T21 cells, we examined proteins in the focal adhesion complex, which links the intracellular cytoskeleton to the extracellular matrix and directly controls these processes. Multiple proteins in the adhesion complex, including paxillin, vinculin, talin, and RACK1, are dysregulated in T21 fibroblasts and hiPSC-derived neurons, but these changes have high inter-individual variability. Taken together, these findings suggest that altered cellular morphology and motility are conserved features of Down syndrome that arise through heterogeneous alterations in adhesion networks. Thus, this work significantly contributes to the recent literature highlighting the need for personalized medicine in Down syndrome.

RASAL3 acts as a negative regulator of small cellular GTPases in hematopoietic cells. In immune cells, it primarily modulates the RAS/MAPK signaling pathway and affects cellular events including proliferation, differentiation, survival, and migration. Due to its inhibitory role in T cells, RASAL3 may represent a potential modulatory target for improving therapeutic strategies such as cell-based immunotherapy. However, most existing knowledge about RASAL3 function is derived from murine models, and its role in human T-cell signaling remains insufficiently characterized. To address this gap, we systematically investigated the function of RASAL3 in human primary T cells and T-cell line. For this purpose, we employed RASAL3 overexpression, CRISPR/Cas9-mediated deletion, and siRNA-mediated knockdown to thoroughly analyze the effects of RASAL3 on T-cell signaling, proliferation, and migration. Our data demonstrate that RASAL3 modulates primarily CDC42 and RAC1/RAC2 GTPases activity, SAPK/JNK phosphorylation, c-Fos and c-Jun expression, and IL-2 gene promoter activation. In addition, RASAL3 regulates actin polymerization and T-cell migration. Notably, loss of RASAL3 increases Jurkat T cells motility in vivo and potentiates their homing to the spleen. Collectively, these findings identify RASAL3 as an important regulator of human T-cell activation and motility and highlight its application potential for improving CAR-T cell therapy.

Deglycosylated (N153Q) dengue virus (DENV) mutant shows attenuated infection in a mouse model, mainly due to its increased antibody susceptibility. This is consistent with the neutralization assay showing human monoclonal antibodies 2D22 and C10 are more potent towards the mutant than the wild-type (WT) virus. Here, we compared the cryoEM structures of WT and mutant viruses complexed with these Fabs (2.7-3.2 [A] resolution). We observed increased occupancies for both Fabs on the mutant virus, suggesting higher accessibility of epitopes that were previously blocked by glycosylation. Using biolayer interferometry, we showed although the Fabs have slower binding rate to the mutant than WT virus, they also have slower dissociation rate. The slow dissociation rate might contribute to higher Fab occupancies, as once bound, they remain associated with the virus. N153Q mutant might be a good vaccine candidate, as important epitopes are made more accessible for stimulating highly potent antibodies.

RhoG is a member of the Rho-family of small GTPases, and is closely related to the canonical Rac1 GTPase, implicated in membrane trafficking, dorsal ruffling, macropinocytosis, and cell protrusion, but its activity has been difficult to visualize directly in living cells with high spatial and temporal resolution. Here, we developed and validated a genetically encoded, single-chain Forster resonance energy transfer (FRET) biosensor for RhoG based on a C-terminal full-length RhoG and an intramolecular RhoG-binding domain derived from ELMO1. The biosensor showed a robust dynamic range when comparing constitutively active and inactive RhoG mutants, responded appropriately to regulation by RhoGDI, GAPs, and GEFs, and detected growth factor-stimulated RhoG activation in live cells. Imaging in mouse embryonic fibroblasts revealed dynamic RhoG activation at leading-edge protrusions, dorsal ruffles, and forming pinocytic and macropinocytic structures. To define the signaling relationship between RhoG and its closely related family member Rac1, we combined the RhoG biosensor with a near-infrared Rac1 FRET biosensor for simultaneous live-cell imaging. Morphodynamic mapping showed that both RhoG and Rac1 activities were positively coupled to edge protrusion, with strongest correlations near the leading-edge, but their direct coupling varied with distance from the edge, indicating partial spatial decoupling within protrusive regions. Inhibition of Src-family kinases altered RhoG dynamics, strongly suppressed Rac1 coupling to protrusion, and inverted the normal positive correlation between RhoG and Rac1 activities. Signaling microdomain analysis further showed that Src inhibition selectively prolonged Rac1 microdomain lifetimes without significantly affecting RhoG domains. Together, these results establish a new biosensor for direct visualization of RhoG activity and reveal that RhoG and Rac1 are coordinated but spatially and temporally distinct components of protrusion-associated signaling networks, with Src-family kinases playing a central role in maintaining their normal coupling.

The NAD+-dependent histone deacetylase Sirt6 regulates transcription of multiple classes of genes, including those involved in metabolism, immune response, oxidative stress response, and development. Defining the Sirt6-regulated transcriptome is relevant to understanding the various important physiological roles of Sirt6, such as extending lifespan, maintaining metabolic health, and tumor suppression. Numerous studies have identified Sirt6 target genes, using both targeted and genome-wide approaches; however, consensus is limited and there has yet to be a systematic analysis of gene expression changes induced by altering Sirt6 levels. In the present study, we conducted a meta-analysis of 19 mammalian RNA-seq datasets in which Sirt6 levels were perturbed (knockout, knockdown, or overexpression). Our analyses included Gene Set Enrichment Analysis, pathway analysis of differentially expressed genes, and identification of individual differentially expressed genes. Our analysis identified consistent gene expression changes associated with lowering Sirt6 levels, including increased expression of immune response and ribosomal protein genes and reduced expression of lipid oxidation and oxidative phosphorylation genes. Extracellular Matrix and E2F target genes also had consistently increased expression upon Sirt6 reduction, highlighting novel regulation by Sirt6. To determine the conservation of gene regulation by Sirt6, we performed additional RNA-Seq meta-analysis on tissues from Drosophila melanogaster with Sirt6 deletion and overexpression. The fly datasets produced similar results to the mammal results, except for lipid oxidation genes, which were found to increase in Sirt6-low conditions. These results provide consensus about conserved and novel pathways transcriptionally regulated by Sirt6.