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Scientific Reports Jun 2024The proteasome-associated deubiquitinase USP14 is a potential drug target. Using an inducible USP14 knockout system in colon cancer cells, we found that USP14 depletion...
The proteasome-associated deubiquitinase USP14 is a potential drug target. Using an inducible USP14 knockout system in colon cancer cells, we found that USP14 depletion impedes cellular proliferation, induces cell cycle arrest, and leads to a senescence-like phenotype. Transcriptomic analysis revealed altered gene expression related to cell division and cellular differentiation. USP14 knockout cells also exhibited changes in morphology, actin distribution, and expression of actin cytoskeletal components. Increased ubiquitin turnover was observed, offset by upregulation of polyubiquitin genes UBB and UBC. Pharmacological inhibition of USP14 with IU1 increased ubiquitin turnover but did not affect cellular growth or morphology. BioGRID data identified USP14 interactors linked to actin cytoskeleton remodeling, DNA damage repair, mRNA splicing, and translation. In conclusion, USP14 loss in colon cancer cells induces a transient quiescent cancer phenotype not replicated by pharmacologic inhibition of its deubiquitinating activity.
Topics: Humans; Cellular Senescence; Colorectal Neoplasms; Cell Proliferation; Ubiquitin Thiolesterase; Cell Line, Tumor; Phenotype; Proteasome Endopeptidase Complex; Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Ubiquitin
PubMed: 38844605
DOI: 10.1038/s41598-024-63791-5 -
Biophysics Reviews Jun 2024Cell migration is a fundamental process for life and is highly dependent on the dynamical and mechanical properties of the cytoskeleton. Intensive physical and... (Review)
Review
Cell migration is a fundamental process for life and is highly dependent on the dynamical and mechanical properties of the cytoskeleton. Intensive physical and biochemical crosstalk among actin, microtubules, and intermediate filaments ensures their coordination to facilitate and enable migration. In this review, we discuss the different mechanical aspects that govern cell migration and provide, for each mechanical aspect, a novel perspective by juxtaposing two complementary approaches to the biophysical study of cytoskeletal crosstalk: live-cell studies (often referred to as top-down studies) and cell-free studies (often referred to as bottom-up studies). We summarize the main findings from both experimental approaches, and we provide our perspective on bridging the two perspectives to address the open questions of how cytoskeletal crosstalk governs cell migration and makes cells move.
PubMed: 38840976
DOI: 10.1063/5.0198119 -
Langmuir : the ACS Journal of Surfaces... Jun 2024Actin, found in all eukaryotic cells as globular (G) or filamentous (F) actin, undergoes polymerization, with G-actin units changing shape to become F-actin. Thermal...
Actin, found in all eukaryotic cells as globular (G) or filamentous (F) actin, undergoes polymerization, with G-actin units changing shape to become F-actin. Thermal proteins, or proteinoids, are created by heating amino acids (160-200 °C), forming polymeric chains. These proteinoids can swell in an aqueous solution at around 50 °C, producing hollow microspheres filled with a solution, exhibiting voltage spikes. Our research explores the signaling properties of proteinoids, actin filaments, and hybrid networks combining actin and proteinoids. Proteinoids replicate brain excitation dynamics despite lacking specific membranes or ion channels. We investigate enhancing conductivity and spiking by using pure actin, yielding improved coordination in networks compared with individual filaments or proteinoids. Temperature changes (20 short-peptide supramolecular C to 80 °C) regulate conduction states, demonstrating external control over emergent excitability in protobrain systems. Adding actin to proteinoids reduces spike timing variability, providing a more uniform feature distribution. These findings support theoretical models proposing cytoskeletal matrices for functional specification in synthetic protocell brains, promoting stable interaction complexity. The study concludes that life-like signal encoding can emerge spontaneously within biological polymer scaffolds, incorporating abiotic chemistry.
Topics: Actin Cytoskeleton; Microspheres; Actins; Temperature; Animals
PubMed: 38837748
DOI: 10.1021/acs.langmuir.4c01107 -
BioRxiv : the Preprint Server For... Jun 20242',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an abundant constituent of central nervous system non-compact myelin, frequently used as a marker antigen for...
2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an abundant constituent of central nervous system non-compact myelin, frequently used as a marker antigen for myelinating cells. The catalytic activity of CNPase, the 3'-hydrolysis of 2',3'-cyclic nucleotides, is well characterised , but the function of CNPase remains unclear. CNPase interacts with the actin cytoskeleton to counteract the developmental closure of cytoplasmic channels that travel through compact myelin; its enzymatic activity may be involved in adenosine metabolism and RNA degradation. We developed a set of high-affinity nanobodies recognizing the phosphodiesterase domain of CNPase, and the crystal structures of each complex show that the five nanobodies have distinct epitopes. One of the nanobodies bound deep into the CNPase active site and acted as an inhibitor. Moreover, the nanobodies were characterised in imaging applications and as intrabodies, expressed in mammalian cells, such as primary oligodendrocytes. Fluorescently labelled nanobodies functioned in imaging of teased nerve fibers and whole brain tissue sections, as well as super-resolution microscopy. These anti-CNPase nanobodies provide new tools for structural and functional biology of myelination, including high-resolution imaging of nerve tissue.
PubMed: 38826303
DOI: 10.1101/2024.05.25.595513 -
Molecules and Cells Jun 2024The actin-based cytoskeleton is considered a fundamental driving force for cell differentiation and development. Destrin (Dstn), a member of the actin-depolymerizing...
The actin-based cytoskeleton is considered a fundamental driving force for cell differentiation and development. Destrin (Dstn), a member of the actin-depolymerizing factor family, regulates actin dynamics by treadmilling actin filaments and increasing globular actin pools. However, the specific developmental roles of dstn have yet to be fully elucidated. Here, we investigated the physiological functions of dstn during early embryonic development using Xenopus laevis as an experimental model organism. dstn is expressed in anterior neural tissue and neural plate during Xenopus embryogenesis. Depleting dstn promoted morphants with short body axes and small heads. Moreover, dstn inhibition extended the neural plate region, impairing cell migration and distribution during neurulation. In addition to the neural plate, dstn knockdown perturbed neural crest cell migration. Our data suggest new insights for understanding the roles of actin dynamics in embryonic neural development, simultaneously presenting a new challenge for studying the complex networks governing cell migration involving actin dynamics.
Topics: Animals; Cell Movement; Xenopus laevis; Destrin; Embryonic Development; Xenopus Proteins; Neural Crest; Neurogenesis; Neural Plate; Actins; Gene Expression Regulation, Developmental
PubMed: 38825188
DOI: 10.1016/j.mocell.2024.100076 -
JACS Au May 2024The actin cytoskeleton and its elaborate interplay with the plasma membrane participate in and control numerous biological processes in eukaryotic cells. Malfunction of...
The actin cytoskeleton and its elaborate interplay with the plasma membrane participate in and control numerous biological processes in eukaryotic cells. Malfunction of actin networks and changes in their dynamics are related to various diseases, from actin myopathies to uncontrolled cell growth and tumorigenesis. Importantly, the biophysical and mechanical properties of actin and its assemblies are deeply intertwined with the biological functions of the cytoskeleton. Novel tools to study actin and its associated biophysical features are, therefore, of prime importance. Here we develop a new approach which exploits fluorescence lifetime imaging microscopy (FLIM) and environmentally sensitive fluorophores termed molecular rotors, acting as quantitative microviscosity sensors, to monitor dynamic viscoelastic properties of both actin structures and lipid membranes. In order to reproduce a minimal actin cortex in synthetic cell models, we encapsulated and attached actin networks to the lipid bilayer of giant unilamellar vesicles (GUVs). Using a cyanine-based molecular rotor, DiSC(3), we show that different types of actin bundles are characterized by distinct packing, which can be spatially resolved using FLIM. Similarly, we show that a lipid bilayer-localized molecular rotor can monitor the effects of attaching cross-linked actin networks to the lipid membrane, revealing an increase in membrane viscosity upon actin attachment. Our approach bypasses constraints associated with existing methods for actin imaging, many of which lack the capability for direct visualization of biophysical properties. It can therefore contribute to a deeper understanding of the role that actin plays in fundamental biological processes and help elucidate the underlying biophysics of actin-related diseases.
PubMed: 38818078
DOI: 10.1021/jacsau.4c00237 -
The Journal of Biological Chemistry May 2024Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested...
Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC's specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC's membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC's own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.
PubMed: 38815864
DOI: 10.1016/j.jbc.2024.107423 -
Communications Biology May 2024The cytoskeleton is a complex network of interconnected biopolymers consisting of actin filaments, microtubules, and intermediate filaments. These biopolymers work in...
The cytoskeleton is a complex network of interconnected biopolymers consisting of actin filaments, microtubules, and intermediate filaments. These biopolymers work in concert to transmit cell-generated forces to the extracellular matrix required for cell motility, wound healing, and tissue maintenance. While we know cell-generated forces are driven by actomyosin contractility and balanced by microtubule network resistance, the effect of intermediate filaments on cellular forces is unclear. Using a combination of theoretical modeling and experiments, we show that vimentin intermediate filaments tune cell stress by assisting in both actomyosin-based force transmission and reinforcement of microtubule networks under compression. We show that the competition between these two opposing effects of vimentin is regulated by the microenvironment stiffness. These results reconcile seemingly contradictory results in the literature and provide a unified description of vimentin's effects on the transmission of cell contractile forces to the extracellular matrix.
Topics: Microtubules; Actomyosin; Vimentin; Mechanotransduction, Cellular; Humans; Extracellular Matrix; Animals
PubMed: 38811770
DOI: 10.1038/s42003-024-06366-4 -
Journal of Virology May 2024Rabies virus (RABV) is highly lethal and triggers severe neurological symptoms. The neuropathogenic mechanism remains poorly understood. Ras-related C3 botulinum toxin...
Rabies virus (RABV) is highly lethal and triggers severe neurological symptoms. The neuropathogenic mechanism remains poorly understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a Rho-GTPase that is involved in actin remodeling and has been reported to be closely associated with neuronal dysfunction. In this study, by means of a combination of pharmacological inhibitors, small interfering RNA, and specific dominant-negatives, we characterize the crucial roles of dynamic actin and the regulatory function of Rac1 in RABV infection, dominantly in the viral entry phase. The data show that the RABV phosphoprotein interacts with Rac1. RABV phosphoprotein suppress Rac1 activity and impedes downstream Pak1-Limk1-Cofilin1 signaling, leading to the disruption of F-actin-based structure formation. In early viral infection, the EGFR-Rac1-signaling pathway undergoes a biphasic change, which is first upregulated and subsequently downregulated, corresponding to the RABV entry-induced remodeling pattern of F-actin. Taken together, our findings demonstrate for the first time the role played by the Rac1 signaling pathway in RABV infection and may provide a clue for an explanation for the etiology of rabies neurological pathogenesis.IMPORTANCEThough neuronal dysfunction is predominant in fatal rabies, the detailed mechanism by which rabies virus (RABV) infection causes neurological symptoms remains in question. The actin cytoskeleton is involved in numerous viruses infection and plays a crucial role in maintaining neurological function. The cytoskeletal disruption is closely associated with abnormal nervous symptoms and induces neurogenic diseases. In this study, we show that RABV infection led to the rearrangement of the cytoskeleton as well as the biphasic kinetics of the Rac1 signal transduction. These results help elucidate the mechanism that causes the aberrant neuronal processes by RABV infection and may shed light on therapeutic development aimed at ameliorating neurological disorders.
PubMed: 38809020
DOI: 10.1128/jvi.00606-24 -
Frontiers in Cellular and Infection... 2024is a uniquely adapted human pathogen and the etiological agent of gonorrhea, a sexually transmitted disease. has developed numerous mechanisms to avoid and actively...
is a uniquely adapted human pathogen and the etiological agent of gonorrhea, a sexually transmitted disease. has developed numerous mechanisms to avoid and actively suppress innate and adaptive immune responses. successfully colonizes and establishes topologically distinct colonies in human macrophages and avoids phagocytic killing. During colonization, manipulates the actin cytoskeleton to invade and create an intracellular niche supportive of bacterial replication. The cellular reservoir(s) supporting bacterial replication and persistence in gonorrhea infections are poorly defined. The manner in which gonococci colonize macrophages points to this innate immune phagocyte as a strong candidate for a cellular niche during natural infection. Here we investigate whether nutrients availability and immunological polarization alter macrophage colonization by . Differentiation of macrophages in pro-inflammatory (M1-like) and tolerogenic (M2-like) phenotypes prior to infection reveals that can invade macrophages in all activation states, albeit with lower efficiency in M1-like macrophages. These results suggest that during natural infection, bacteria could invade and grow within macrophages regardless of the nutrients availability and the macrophage immune activation status.
Topics: Neisseria gonorrhoeae; Macrophages; Humans; Nutrients; Gonorrhea; Macrophage Activation; Host-Pathogen Interactions
PubMed: 38808065
DOI: 10.3389/fcimb.2024.1384611