Biology subjects

Tran, Q. D.

Publications and source records attributed to Tran, Q. D..

11 recordsLinked to original sources

bioRxiv2025

Vimentin promotes actin assembly by stabilizing ATP-actin subunits at the barbed end

Vimentin intermediate filaments play essential roles in maintaining cell integrity and regulating numerous cellular functions. In particular, vimentin cooperates with the actin cytoskeleton in key cellular processes that rely on actin dynamics, such as migration, division, and mechanosensing. While there is evidence that these two cytoskeletal components interact in cells, the underlying molecular mechanisms are only partially understood. Actin and vimentin can interact through biochemical signaling pathways or via cross-linkers, but whether they engage in a direct protein-protein interaction has remained controversial, in part because such interactions are difficult to isolate and characterize in cells. Using in vitro reconstitution coupled to theoretical modeling, and total internal reflection fluorescence microscopy to monitor the elongation of single actin filaments, we show that vimentin promotes actin elongation by stabilizing actin subunits at the barbed end in a dose-dependent manner. Strikingly, this effect depends on the nucleotide state of actin, as the acceleration is only observed for the elongation from ATP-actin, and not ADP-actin monomers. We further establish that neither the vimentin tail nor head domains are required for this effect, and both filamentous and non-filamentous vimentin enhance actin elongation. Finally, we find that vimentin promotes the nucleation of actin filaments. Consistently, magnetic pull-down assays demonstrate a direct interaction between vimentin and ATP-actin monomers. Altogether, these findings identify vimentin as an unexpected new actor in the regulation of actin dynamics at the barbed end and bring new insights into the functional role of vimentin through cytoskeletal crosstalk.

biophysics↗

bioRxiv2024

Supervised non-negative matrix factorization on cell-free DNA fragmentomic features enhances early cancer detection

BackgroundCell-free circulating DNA (cfDNA) fragments exhibit non-random patterns in their length (FLEN), end-motif (EM), and distance to nucleosome position (ND). While these cfDNA features have shown promise as inputs for machine learning and deep learning models in early cancer detection, most studies utilize them as raw inputs, overlooking the potential benefits of pre-processing to extract cancer-specific features. This study aims to enhance cancer detection accuracy by developing a novel approach to feature extraction from cfDNA fragmentomics. MethodsWe implemented a supervised non-negative matrix factorization (SNMF) algorithm to generate embedding vectors capturing cancer-specific signals within cfDNA fragmentomic features. These embeddings served as input for a machine learning model to classify cancer patients from healthy individuals. ResultsWe validated our framework using two datasets: an in-house cohort of 431 cancer patients and 442 healthy individuals (dataset 1), and a published cohort comprising 90 hepatocellular carcinoma (HCC) patients and 103 individuals with cirrhosis or hepatitis B (dataset 2). In dataset 1, we achieved an AUC of 94% in pan-cancer detection. In dataset 2, our framework achieved an AUC of 100% for HCC vs healthy classification, 99% for HCC vs non-HCC patients classification, and 96% for identifying HCC patients among a mixed group of non-HCC patients and healthy donors. ConclusionThis study demonstrates the efficiency of SNMF-transformed features in improving both pan-cancer detection and specific HCC detection. Our approach offers a significant advancement in leveraging cfDNA fragmentomics for early cancer detection, potentially enhancing diagnostic accuracy in clinical settings.

bioinformatics↗

bioRxiv2024

Rapid microfluidic perfusion system enables controlling dynamics of intracellular pH regulated by Na+/H+ exchanger NHE1

pH regulation of eukaryotic cells is of crucial importance and influences different mechanisms including chemical kinetics, buffer effects, metabolic activity, membrane transport and cell shape parameters. In this study, we develop a microfluidic system to rapidly and precisely control a continuous flow of ionic chemical species to acutely challenge the intracellular pH regulation mechanisms and confront predictive models. We monitor the intracellular pH dynamics in real-time using pH-sensitive fluorescence imaging and establish a robust mathematical tool to translate the fluorescence signals to pH values. By varying flow rate across the cells and duration for rinsing process, we manage to tweak the dynamics of intracellular pH from a smooth recovery to either an overshooting state, where the pH goes excitedly to a maximum value before decreasing to a plateau, or an undershooting state where the pH is unable to recover to [~] 7. We believe our findings will provide more insight into intracellular regulatory mechanisms and promote the possibility of exploring cellular behavior in the presence of strong gradients or fast changes in homogeneous conditions.

bioengineering↗

bioRxiv2024

Continuous self-repair protects vimentin intermediate filaments from fragmentation

Intermediate filaments are key regulators of cell mechanics. Vimentin, a type of intermediate filament expressed in mesenchymal cells and involved in migration, forms a dense network in the cytoplasm that is constantly remodeling through filament transport, elongation/shortening, and subunit exchange. While it is known that filament elongation involves end-to-end annealing, the reverse process of filament shortening by fragmentation remains unclear. Here, we use a combination of in vitro reconstitution, probed by fluorescence imaging and AFM, with theoretical modeling to uncover the molecular mechanism involved in filament breakage. We first show that vimentin filaments are composed of two populations of subunits, half of which are exchangeable and half immobile. We also show that the exchangeable subunits are tetramers. Furthermore, we reveal a mechanism of continuous filament self-repair, where a soluble pool of vimentin tetramers in equilibrium with the filaments is essential to maintain filament integrity. Filaments break due to local fluctuations in the number of tetramers per cross-section, induced by the constant subunit exchange. We determine that a filament tends to break if approximately four tetramers are removed from the same filament cross-section. Finally, we analyze the dynamics of association/dissociation and fragmentation to estimate the binding energy of a tetramer to a complete versus a partially disassembled filament. Our results provide a comprehensive description of vimentin turnover and reveal the link between subunit exchange and fragmentation. SIGNIFICANCE STATEMENTIntermediate filaments such as vimentin are key contributors to cell mechanics. These filaments are continuously assembled and disassembled by the cell as it grows and moves. While the assembly process is relatively well characterized, here we investigate their much less understood disassembly. We find that the filaments continuously exchange their constitutive proteins with the surrounding solution throughout their length and when too many subunits are removed, the filament breaks. This process sharply differs from the length regulation mechanisms known to occur in other types of filaments involved in the regulation of cell mechanics. Importantly, we also identify two distinct populations of subunits within the filaments, which raises new questions about vimentin intermediate filament structure.

biophysics↗

bioRxiv2023

Reconstitution of cytolinker-mediated crosstalk between actin and vimentin

Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical properties of each filament type individually have been studied extensively by cell-free reconstitution. By contrast, the interactions between the three cytoskeletal networks are relatively unexplored. They are coupled via crosslinkers of the plakin family such as plectin. These are challenging proteins for reconstitution because of their giant size and multidomain structure. Here we engineer a recombinant actin-vimentin crosslinker protein called ACTIF that provides a minimal model system for plectin, recapitulating its modular design with actin-binding and intermediate filament-binding domains separated by a coiled-coil linker for dimerisation. We show by fluorescence and electron microscopy that ACTIF has a high binding affinity for vimentin and actin and creates mixed actin-vimentin bundles. Rheology measurements show that ACTIF-mediated crosslinking strongly stiffens actin-vimentin composites. Finally, we demonstrate the modularity of this approach by creating an ACTIF variant with the intermediate filament binding domain of Adenomatous Polyposis Coli. Our protein engineering approach provides a new cell-free system for the biophysical characterization of intermediate filament-binding crosslinkers and for understanding the mechanical synergy between actin and vimentin in mesenchymal cells. HIGHLIGHTSO_LIEngineering of a recombinant actin-vimentin crosslinker called ACTIF with the plectin intermediate filament binding domain (IFBD), calponin homology domains that mediate actin binding, and a coiled-coil linker. C_LIO_LIACTIF crosslinks F-actin and vimentin and mediates their co-localization in vitro. C_LIO_LIACTIF has a binding affinity for vimentin that is about 500 times higher than for F-actin. C_LIO_LIACTIF forms composite bundles of F-actin and vimentin filaments. C_LIO_LIComposite F-actin/vimentin networks stiffen upon crosslinking with ACTIF. C_LIO_LIThe design of actin-vimentin crosslinker is modular, as other IFBDs like APCn2 can also be used. C_LI

biochemistry↗

bioRxiv2022

Vimentin intermediate filament assembly is a reversible process

Networks of intermediate filaments (IFs) need to constantly reorganize to fulfil their functions at different locations within the cell. The mechanism of IF assembly is well described and involves filament end-to-end annealing. By contrast, the mechanisms involved in IF disassembly are far less understood. In vitro, IFs are assumed to be very stable and their disassembly negligible. IF fragmentation has been observed in many cell types, but it has been suggested to be associated with active processes such as IF post-translational modifications. In this article, we uncover the contribution of filament spontaneous fragmentation in the assembly dynamics of type III vimentin IF using a combination of in vitro reconstitution probed by fluorescence imaging and theoretical modeling. We first show that vimentin assembly at low concentrations results in an equilibrium between filament annealing and fragmentation at times [≥]24 hours. At higher concentrations, entanglements kinetically trap the system out of equilibrium, and we show that this trapping is reversible upon dilution. Taking into account both fragmentation and entanglement, we estimate that the mean bond breaking time is [~]18 hours. This translates into a mean breaking time of [~] 5 hours for a 1 m long filament, which is a relevant time scale for IF reorganization in live cells. Finally, we provide direct evidence through dual-color imaging that filament fragmentation and annealing coexist during assembly. By showing that IF fragmentation can occur without cofactors or post-translational modifications, our study provides new insights into the physical understanding of the IF length regulation.

biophysics↗

bioRxiv2021

Molecular organization and mechanics of single vimentin filaments revealed by super-resolution imaging

Intermediate filaments (IF) are involved in key cellular functions including polarization, migration, and protection against large deformations. These functions are related to their remarkable ability to extend without breaking, a capacity that should be determined by the molecular organization of subunits within filaments. However, this structure-mechanics relationship remains poorly understood at the molecular level. Here, using super-resolution microscopy (SRM), we show that vimentin filaments exhibit a ~49 nm axial repeat both in cells and in vitro. As unit-length-filaments (ULFs) were measured at ~59 nm, this demonstrates a partial overlap of ULFs during filament assembly. Using an SRM-compatible stretching device, we also provide evidence that the extensibility of vimentin is due to the unfolding of its subunits and not to their sliding, thus establishing a direct link between the structural organization and its mechanical properties. Overall, our results pave the way for future studies of IF assembly, mechanical and structural properties in cells.

biophysics↗

bioRxiv2021

Identification of DAXX As A Restriction Factor Of SARS-CoV-2 Through A CRISPR/Cas9 Screen

Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identified DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX was sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricted infection. In contrast with most of its previously described antiviral activities, DAXX-mediated restriction of SARS-CoV-2 was independent of the SUMOylation pathway. SARS-CoV-2 infection triggered the re-localization of DAXX to cytoplasmic sites and promoted its degradation. Mechanistically, this process was mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.

microbiology↗

bioRxiv2021

Cooperation of two opposite flagella allows high-speed swimming and active turning in zoospore

Phytophthora species cause diseases in a large variety of plants and represent a serious agricultural threat, leading, every year, to multibillion dollar losses. Infection occurs when these biflagellated zoospores move across the soil at their characteristic high speed and reach the roots of a host plant. Despite the relevance of zoospore spreading in the epidemics of plant diseases, characteristics of individual swimming of zoospores have not been fully investigated. It remains unknown about the characteristics of two opposite beating flagella during translation and turning, and the roles of each flagellum on zoospore swimming. Here, combining experiments and modeling, we show how these two flagella contribute to generate thrust when beating together, and identify the mastigonemes-attached anterior flagellum as the main source of thrust. Furthermore, we find that turning involves a complex active process, in which the posterior flagellum temporarily stops, while the anterior flagellum keeps on beating and changes its pattern from sinusoidal waves to power and recovery strokes, similar to Chlamydomonass breaststroke, to reorient its body to a new direction. Our study is a fundamental step towards a better understanding of the spreading of plant pathogens motile forms, and shows that the motility pattern of these biflagellated zoospores represents a distinct eukaryotic version of the celebrated "run-and-tumble" motility class exhibited by peritrichous bacteria.

biophysics↗

bioRxiv2021

Host PDZ-containing proteins targeted by SARS-Cov-2

Small linear motif targeting protein interacting domains called PDZ have been identified at the C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A and N showing significant interactions with dissociation constants values ranging from 3 M to 82 M. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Seven of the PDZ-containing proteins among binders of the SARS-CoV-2 proteins E, 3a or N affect significantly viral replication under knock-down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes.

biochemistry↗

bioRxiv2021

BRD2 inhibition blocks SARS-CoV-2 infection in vitro by reducing transcription of the host cell receptor ACE2

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.

cell biology↗