ABSTRACT
Clarification of the cytotoxic function of T cells is crucial for understanding human immune responses and immunotherapy procedures. Here, we report a high-throughput Bessel oblique plane microscopy (HBOPM) platform capable of 3D live imaging and phenotyping of chimeric antigen receptor (CAR)-modified T-cell cytotoxicity against cancer cells. The HBOPM platform has the following characteristics: an isotropic subcellular resolution of 320 nm, large-scale scouting over 400 interacting cell pairs, long-term observation across 5 hours, and quantitative analysis of the Terabyte-scale 3D, multichannel, time-lapse image datasets. Using this advanced microscopy platform, several key subcellular events in CAR-T cells are captured and comprehensively analyzed; these events include the instantaneous formation of immune synapses and the sustained changes in the microtubing morphology. Furthermore, we identify the actin retrograde flow speed, the actin depletion coefficient, the microtubule polarization and the contact area of the CAR-T/target cell conjugates as essential parameters strongly correlated with CAR-T-cell cytotoxic function. Our approach will be useful for establishing criteria for quantifying T-cell function in individual patients for all T-cell-based immunotherapies.
Subject(s)
Imaging, Three-Dimensional , Immunotherapy, Adoptive , Microtubules , Receptors, Chimeric Antigen , T-Lymphocytes , Humans , Receptors, Chimeric Antigen/metabolism , Receptors, Chimeric Antigen/immunology , Imaging, Three-Dimensional/methods , Immunotherapy, Adoptive/methods , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Microtubules/metabolism , Cell Line, Tumor , Immunological Synapses/immunology , Immunological Synapses/metabolism , Cytotoxicity, Immunologic , Actins/metabolism , Microscopy/methods , PhenotypeABSTRACT
Purpose: The purpose of this study was to evaluate the safety and efficacy of topical losartan in the therapeutic treatment of established corneal scaring fibrosis at 1 month after alkali burn in rabbits. Methods: Standardized alkali burns were performed in 1 eye of 24 rabbits with 0.75N NaOH for 15 seconds. Corneas were allowed to heal and develop scaring of the cornea for 1 month. Twelve eyes per group were treated with 50 µL of topical 0.8 mg/mL losartan in balanced salt solution (BSS), pH 7.0, and 12 eyes were treated with vehicle BSS 6 times per day. Six corneas were analyzed at 1 week or 1 month in each group. Standardized slit lamp photographs were obtained at the end point for each cornea and opacity was quantitated using ImageJ. Corneoscleral rims were cryofixed in optimum cutting temperature (OCT) solution and combined duplex immunohistochemistry for myofibroblast marker alpha-smooth muscle actin (α-SMA), mesenchymal cell marker vimentin, and TUNEL assay for apoptosis was performed on all corneas. Results: Topical losartan was effective in the treatment of established stromal fibrosis following alkali burn injury to the rabbit cornea. Stromal myofibroblast density was decreased and stromal cell apoptosis was increased (included both α-SMA-positive myofibroblasts and α-SMA-negative, vimentin-positive cells) at both 1 week and 1 month in the topical losartan-treated compared with vehicle-treated groups. Conclusions: Topical losartan is effective in the treatment of established stromal fibrosis in rabbits. Most myofibroblasts disappear from the stroma within the first month of losartan treatment. Longer treatment with topical losartan is needed to allow time for corneal fibroblast regeneration of the epithelial basement membrane (in coordination with epithelial cells) and the removal of disordered extracellular matrix produced by myofibroblasts.
Subject(s)
Burns, Chemical , Eye Burns , Fibrosis , Losartan , Animals , Rabbits , Losartan/pharmacology , Losartan/administration & dosage , Losartan/therapeutic use , Fibrosis/drug therapy , Burns, Chemical/drug therapy , Burns, Chemical/pathology , Eye Burns/drug therapy , Eye Burns/pathology , Eye Burns/chemically induced , Disease Models, Animal , Apoptosis/drug effects , Angiotensin II Type 1 Receptor Blockers/administration & dosage , Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin II Type 1 Receptor Blockers/therapeutic use , Sodium Hydroxide , Corneal Diseases/drug therapy , Corneal Diseases/pathology , Ophthalmic Solutions/therapeutic use , Ophthalmic Solutions/administration & dosage , Cornea/drug effects , Cornea/pathology , In Situ Nick-End Labeling , Myofibroblasts/drug effects , Myofibroblasts/pathology , Actins/metabolism , Male , Corneal Stroma/drug effects , Corneal Stroma/pathology , Corneal Stroma/metabolism , Administration, Topical , Vimentin/metabolism , Wound Healing/drug effectsABSTRACT
Subretinal fibrosis is a major untreatable cause of poor outcomes in neovascular age-related macular degeneration. Mouse models of subretinal fibrosis all possess a degree of invasiveness and tissue damage not typical of fibrosis progression. This project characterises JR5558 mice as a model to study subretinal fibrosis. Fundus and optical coherence tomography (OCT) imaging was used to non-invasively track lesions. Lesion number and area were quantified with ImageJ. Retinal sections, wholemounts and Western blots were used to characterise alterations. Subretinal lesions expand between 4 and 8 weeks and become established in size and location around 12 weeks. Subretinal lesions were confirmed to be fibrotic, including various cell populations involved in fibrosis development. Müller cell processes extended from superficial retina into subretinal lesions at 8 weeks. Western blotting revealed increases in fibronectin (4 wk and 8 wk, p < 0.001), CTGF (20 wks, p < 0.001), MMP2 (12 wks and 20 wks p < 0.05), αSMA (12 wks and 20 wks p < 0.05) and GFAP (8 wk and 12 wk, p ≤ 0.01), consistent with our immunofluorescence results. Intravitreal injection of Aflibercept reduced subretinal lesion growth. Our study provides evidence JR5558 mice have subretinal fibrotic lesions that grow between 4 and 8 weeks and confirms this line to be a good model to study subretinal fibrosis development and assess treatment options.
Subject(s)
Disease Models, Animal , Fibrosis , Retina , Tomography, Optical Coherence , Animals , Mice , Tomography, Optical Coherence/methods , Retina/pathology , Retina/metabolism , Receptors, Vascular Endothelial Growth Factor/metabolism , Fibronectins/metabolism , Ependymoglial Cells/metabolism , Ependymoglial Cells/pathology , Connective Tissue Growth Factor/metabolism , Connective Tissue Growth Factor/genetics , Macular Degeneration/pathology , Macular Degeneration/metabolism , Matrix Metalloproteinase 2/metabolism , Intravitreal Injections , Glial Fibrillary Acidic Protein/metabolism , Actins/metabolism , Mice, Inbred C57BL , Recombinant Fusion ProteinsABSTRACT
Bombesin receptor-activated protein (BRAP), encoded by the C6orf89 gene in humans, is expressed in various cells with undefined functions. BC004004, the mouse homologue of C6orf89, has been shown to play a role in bleomycin-induced pulmonary fibrosis through the use of a BC004004 gene knockout mouse (BC004004-/-). In this study, we investigated the potential involvement of BRAP in renal fibrosis using two mouse models: unilateral ureteral obstruction (UUO) and type 2 diabetes mellitus induced by combination of a high-fat diet (HFD) and streptozocin (STZ). BRAP or its homologue was expressed in tubular epithelial cells (TECs) in the kidneys of patients with chronic kidney disease (CKD) and in BC004004+/+ mice. Compared to control mice, BC004004-/- mice exhibited attenuated renal injury and renal fibrosis after UUO or after HFD/STZ treatment. Immunohistochemistry and immunoblot analyses of the kidneys of BC004004+/+ mice after UUO surgery showed a more significant decrease in E-cadherin expression and a more significant increase in both α smooth muscle actin (α-SMA) and vimentin expression compared to BC004004-/- mice. Additionally, stimulation with transforming growth factor-ß1 (TGF-ß1) led to a more significant decrease in E-cadherin expression and a more significant increase in α-SMA and vimentin expression in isolated TECs from BC004004+/+ than in those from BC004004-/- mice. These results suggest that an enhanced epithelial-mesenchymal transition (EMT) process occurred in TECs in BC004004+/+ mice during renal injury, which might contribute to renal fibrosis. The loss of the BRAP homologue in BC004004-/- mice suppressed EMT activation in kidneys and contributed to the suppression of fibrosis during renal injury.
Subject(s)
Fibrosis , Animals , Mice , Male , Humans , Epithelial-Mesenchymal Transition , Mice, Knockout , Ureteral Obstruction/pathology , Ureteral Obstruction/complications , Kidney/pathology , Kidney/metabolism , Transforming Growth Factor beta1/metabolism , Actins/metabolism , Mice, Inbred C57BL , Cadherins/metabolism , Cadherins/genetics , Renal Insufficiency, Chronic/pathology , Renal Insufficiency, Chronic/metabolism , Renal Insufficiency, Chronic/genetics , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Diabetes Mellitus, Type 2/geneticsABSTRACT
AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.
Subject(s)
Actins , Dendrites , Hippocampus , Neuronal Plasticity , Polymerization , Receptors, AMPA , Animals , Receptors, AMPA/metabolism , Actins/metabolism , Rats , Neuronal Plasticity/physiology , Dendrites/metabolism , Hippocampus/metabolism , Hippocampus/cytology , Protein Transport , Neurons/metabolism , Cells, Cultured , ExocytosisABSTRACT
Rationale: Cardiomyocytes (CMs) undergo dramatic structural and functional changes in postnatal maturation; however, the regulatory mechanisms remain greatly unclear. Cypher/Z-band alternatively spliced PDZ-motif protein (ZASP) is an essential sarcomere component maintaining Z-disc stability. Deletion of mouse Cypher and mutation in human ZASP result in dilated cardiomyopathy (DCM). Whether Cypher/ZASP participates in CM maturation and thereby affects cardiac function has not been answered. Methods: Immunofluorescence, transmission electron microscopy, real-time quantitative PCR, and Western blot were utilized to identify the role of Cypher in CM maturation. Subsequently, RNA sequencing and bioinformatics analysis predicted serum response factor (SRF) as the key regulator. Rescue experiments were conducted using adenovirus or adeno-associated viruses encoding SRF, both in vitro and in vivo. The molecular mechanisms were elucidated through G-actin/F-actin fractionation, nuclear-cytoplasmic extraction, actin disassembly assays, and co-sedimentation assays. Results: Cypher deletion led to impaired sarcomere isoform switch and morphological abnormalities in mitochondria, transverse-tubules, and intercalated discs. RNA-sequencing analysis revealed significant dysregulation of crucial genes related to sarcomere assembly, mitochondrial metabolism, and electrophysiology in the absence of Cypher. Furthermore, SRF was predicted as key transcription factor mediating the transcriptional differences. Subsequent rescue experiments showed that SRF re-expression during the critical postnatal period effectively rectified CM maturation defects and notably improved cardiac function in Cypher-depleted mice. Mechanistically, Cypher deficiency resulted in the destabilization of F-actin and a notable increase in G-actin levels, thereby impeding the nuclear localisation of myocardin-related transcription factor A (MRTFA) and subsequently initiating SRF transcription. Conclusion: Cypher/ZASP plays a crucial role in CM maturation through actin-mediated MRTFA-SRF signalling. The linkage between CM maturation abnormalities and the late-onset of DCM is suggested, providing further insights into the pathogenesis of DCM and potential treatment strategies.
Subject(s)
Actins , Cardiomyopathy, Dilated , Myocytes, Cardiac , Serum Response Factor , Signal Transduction , Trans-Activators , Animals , Myocytes, Cardiac/metabolism , Serum Response Factor/metabolism , Serum Response Factor/genetics , Mice , Actins/metabolism , Trans-Activators/metabolism , Trans-Activators/genetics , Cardiomyopathy, Dilated/metabolism , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/pathology , Sarcomeres/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Humans , Mice, KnockoutABSTRACT
Cell migration through complex 3D environments relies on the interplay between actin and microtubules. A new study shows that, when cells pass through narrow constrictions, CLASP-dependent microtubule stabilisation at the cell rear controls actomyosin contractility to enable nuclear translocation and preserve cell integrity.
Subject(s)
Cell Movement , Microtubules , Microtubules/metabolism , Microtubules/physiology , Cell Movement/physiology , Actomyosin/metabolism , Actins/metabolism , AnimalsABSTRACT
The fundamental question of how forces are generated in a motile cell, a lamellipodium, and a comet tail is the subject of this note. It is now well established that cellular motility results from the polymerization of actin, the most abundant protein in eukaryotic cells, into an interconnected set of filaments. We portray this process in a continuum mechanics framework, claiming that polymerization promotes a mechanical swelling in a narrow zone around the nucleation loci, which ultimately results in cellular or bacterial motility. To this aim, a new paradigm in continuum multi-physics has been designed, departing from the well-known theory of Larché-Cahn chemo-transport-mechanics. In this note, we set up the theory of network growth and compare the outcomes of numerical simulations with experimental evidence.
Subject(s)
Actins , Cell Movement , Actins/metabolism , Models, Biological , Actin Cytoskeleton/metabolism , Pseudopodia/metabolism , Pseudopodia/physiology , Biomechanical Phenomena , PolymerizationABSTRACT
MICAL proteins represent a unique family of actin regulators crucial for synapse development, membrane trafficking, and cytokinesis. Unlike classical actin regulators, MICALs catalyze the oxidation of specific residues within actin filaments to induce robust filament disassembly. The potent activity of MICALs requires tight control to prevent extensive damage to actin cytoskeleton. However, the molecular mechanism governing MICALs' activity regulation remains elusive. Here, we report the cryo-EM structure of MICAL1 in the autoinhibited state, unveiling a head-to-tail interaction that allosterically blocks enzymatic activity. The structure also reveals the assembly of C-terminal domains via a tripartite interdomain interaction, stabilizing the inhibitory conformation of the RBD. Our structural, biochemical, and cellular analyses elucidate a multi-step mechanism to relieve MICAL1 autoinhibition in response to the dual-binding of two Rab effectors, revealing its intricate activity regulation mechanisms. Furthermore, our mutagenesis study of MICAL3 suggests the conserved autoinhibition and relief mechanisms among MICALs.
Subject(s)
Actins , Cryoelectron Microscopy , Mixed Function Oxygenases , Humans , Actins/metabolism , Mixed Function Oxygenases/metabolism , Mixed Function Oxygenases/chemistry , Microfilament Proteins/metabolism , Microfilament Proteins/genetics , Microfilament Proteins/chemistry , Protein Binding , Actin Cytoskeleton/metabolism , Models, Molecular , rab GTP-Binding Proteins/metabolism , rab GTP-Binding Proteins/genetics , Cytoskeletal Proteins/metabolism , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Protein Domains , CalponinsABSTRACT
Adipose tissue is a dynamic regulatory organ that has profound effects on the overall health of patients. Unfortunately, inconsistencies in human adipose tissues are extensive and multifactorial, including large variability in cellular sizes, lipid content, inflammation, extracellular matrix components, mechanics, and cytokines secreted. Given the high human variability, and since much of what is known about adipose tissue is from animal models, we sought to establish correlations and patterns between biological, mechanical, and epidemiological properties of human adipose tissues. To do this, twenty-six independent variables were cataloged for twenty patients, which included patient demographics and factors that drive health, obesity, and fibrosis. A factorial analysis for mixed data (FAMD) was used to analyze patterns in the dataset (with BMI > 25), and a correlation matrix was used to identify interactions between quantitative variables. Vascular endothelial growth factor A (VEGFA) and actin alpha 2, smooth muscle (ACTA2) gene expression were the highest loadings in the first two dimensions of the FAMD. The number of adipocytes was also a key driver of patient-related differences, where a decrease in the density of adipocytes was associated with aging. Aging was also correlated with a decrease in overall lipid percentage of subcutaneous tissue, with lipid deposition being favored extracellularly, an increase in transforming growth factor-ß1 (TGFß1), and an increase in M1 macrophage polarization. An important finding was that self-identified race contributed to variance between patients in this study, where Black patients had significantly lower gene expression levels of TGFß1 and ACTA2. This finding supports the urgent need to account for patient ancestry in biomedical research to develop better therapeutic strategies for all patients. Another important finding was that TGFß induced factor homeobox 1 (TGIF1), an understudied signaling molecule, which is highly correlated with leptin signaling, was correlated with metabolic inflammation. Furthermore, this study draws attention to what we define as "extracellular lipid droplets", which were consistently found in collagen-rich regions of the obese adipose tissues evaluated here. Reduced levels of TGIF1 were correlated with higher numbers of extracellular lipid droplets and an inability to suppress fibrotic changes in adipose tissue. Finally, this study indicated that M1 and M2 macrophage markers were correlated with each other and leptin in patients with a BMI > 25. This finding supports growing evidence that macrophage polarization in obesity involves a complex, interconnecting network system rather than a full switch in activation patterns from M2 to M1 with increasing body mass. Overall, this study reinforces key findings in animal studies and identifies important areas for future research, where human and animal studies are divergent. Understanding key drivers of human patient variability is required to unravel the complex metabolic health of unique patients.
Subject(s)
Subcutaneous Fat , Humans , Subcutaneous Fat/metabolism , Male , Female , Middle Aged , Adult , Adipocytes/metabolism , Obesity/metabolism , Obesity/pathology , Actins/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/genetics , AgedABSTRACT
Inflammation plays a crucial role in progression of fibrosis. Epoxyeicosatrienoic acids (EET) have multiple protective effects in different diseases, but their ability to inhibit the development of LPS-induced fibrosis remains unknown. The potential therapeutic effects of 11,12-EET were studied in in vitro model of LPS-induced fibrosis. Mouse embryonic fibroblast cells NIH/3T3 were pre-incubated with 1 µM 11,12-EET and/or a structural analogue and selective EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid before exposing to LPS. The effect of EET was evaluated by the protein and mRNA expression of NF-κB, collagens I and III, and α-smooth muscle actin by Western blotting and quantitative reverse transcription PCR, respectively. LPS provoked inflammation and fibrosis-like changes accompanied by elevated expression of NF-κB and collagens in NIH/3T3 cells. We also studied the effects of 11,12-EET on the A2AR and PI3K/Akt signaling pathways in intact and LPS-treated NIH/3T3 cells. 11,12-EET prevented inflammation and fibrosis-like changes through up-regulation of A2AR and PI3K/Akt signaling pathways. Our findings demonstrate the potential antifibrotic effects of 11,12-EET, which can be natural antagonists of tissue fibrosis.
Subject(s)
8,11,14-Eicosatrienoic Acid , Fibrosis , Lipopolysaccharides , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , Animals , Mice , Lipopolysaccharides/toxicity , Signal Transduction/drug effects , NIH 3T3 Cells , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/genetics , Fibrosis/drug therapy , Phosphatidylinositol 3-Kinases/metabolism , 8,11,14-Eicosatrienoic Acid/analogs & derivatives , 8,11,14-Eicosatrienoic Acid/pharmacology , NF-kappa B/metabolism , Actins/metabolism , Actins/genetics , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathologyABSTRACT
Myosin motors perform many fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Motor activity control within the cell involves on/off switches, however, few examples are known of how myosins regulate speed or processivity and fine-tune their activity to a specific cellular task. Here, we describe a phosphorylation event for myosins of class VI (MYO6) in the motor domain, which accelerates its ATPase activity leading to a 4-fold increase in motor speed determined by actin-gliding assays, single molecule mechanics and stopped flow kinetics. We demonstrate that the serine/threonine kinase DYRK2 phosphorylates MYO6 at S267 in vitro. Single-molecule optical-tweezers studies at low load reveal that S267-phosphorylation results in faster nucleotide-exchange kinetics without change in the working stroke of the motor. The selective increase in stiffness of the acto-MYO6 complex when proceeding load-dependently into the nucleotide-free rigor state demonstrates that S267-phosphorylation turns MYO6 into a stronger motor. Finally, molecular dynamic simulations of the nucleotide-free motor reveal an alternative interaction network within insert-1 upon phosphorylation, suggesting a molecular mechanism, which regulates insert-1 positioning, turning the S267-phosphorylated MYO6 into a faster motor.
Subject(s)
Molecular Dynamics Simulation , Myosin Heavy Chains , Phosphorylation , Myosin Heavy Chains/metabolism , Myosin Heavy Chains/genetics , Kinetics , Protein Serine-Threonine Kinases/metabolism , Nucleotides/metabolism , Humans , Animals , Protein Domains , Protein-Tyrosine Kinases/metabolism , Actins/metabolismABSTRACT
Long-term synaptic plasticity is typically associated with morphological changes in synaptic connections. However, the molecular mechanisms coupling functional and structural aspects of synaptic plasticity are still poorly defined. The catalytic activity of type I phosphoinositide-3-kinase (PI3K) is required for specific forms of synaptic plasticity, such as NMDA receptor-dependent long-term potentiation (LTP) and mGluR-dependent long-term depression (LTD). On the other hand, PI3K signaling has been linked to neuronal growth and synapse formation. Consequently, PI3Ks are promising candidates to coordinate changes in synaptic strength with structural remodeling of synapses. To investigate this issue, we targeted individual regulatory subunits of type I PI3Ks in hippocampal neurons and employed a combination of electrophysiological, biochemical and imaging techniques to assess their role in synaptic plasticity. We found that a particular regulatory isoform, p85α, is selectively required for LTP. This specificity is based on its BH domain, which engages the small GTPases Rac1 and Cdc42, critical regulators of the actin cytoskeleton. Moreover, cofilin, a key regulator of actin dynamics that accumulates in dendritic spines after LTP induction, failed to do so in the absence of p85α or when its BH domain was overexpressed as a dominant negative construct. Finally, in agreement with this convergence on actin regulatory mechanisms, the presence of p85α in the PI3K complex determined the extent of actin polymerization in dendritic spines during LTP. Therefore, this study reveals a molecular mechanism linking structural and functional synaptic plasticity through the coordinate action of PI3K catalytic activity and a specific isoform of the regulatory subunits.
Subject(s)
Actin Depolymerizing Factors , Actins , Dendritic Spines , Hippocampus , Long-Term Potentiation , Animals , Dendritic Spines/metabolism , Long-Term Potentiation/physiology , Actins/metabolism , Hippocampus/metabolism , Hippocampus/cytology , Actin Depolymerizing Factors/metabolism , Rats , rac1 GTP-Binding Protein/metabolism , Synapses/metabolism , Polymerization , cdc42 GTP-Binding Protein/metabolism , Neuronal Plasticity/physiology , Phosphatidylinositol 3-Kinases/metabolism , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Class Ia Phosphatidylinositol 3-Kinase/genetics , Neurons/metabolism , Signal Transduction , Mice , Cells, CulturedABSTRACT
Mature neurons have stable dendritic architecture, which is essential for the nervous system to operate correctly. The ability to undergo structural plasticity, required to support adaptive processes like memory formation, is still present in mature neurons. It is unclear what molecular and cellular processes control this delicate balance between dendritic structural plasticity and stabilization. Failures in the preservation of optimal dendrite structure due to atrophy or maladaptive plasticity result in abnormal connectivity and are associated with various neurological diseases. Vascular endothelial growth factor D (VEGFD) is critical for the maintenance of mature dendritic trees. Here, we describe how VEGFD affects the neuronal cytoskeleton and demonstrate that VEGFD exerts its effects on dendrite stabilization by influencing the actin cortex and reducing microtubule dynamics. Further, we found that during synaptic activity-induced structural plasticity VEGFD is downregulated. Our findings revealed that VEGFD, acting on its cognate receptor VEGFR3, opposes structural changes by negatively regulating dendrite growth in cultured hippocampal neurons and in vivo in the adult mouse hippocampus with consequences on memory formation. A phosphoproteomic screening identified several regulatory proteins of the cytoskeleton modulated by VEGFD. Among the actin cortex-associated proteins, we found that VEGFD induces dephosphorylation of ezrin at tyrosine 478 via activation of the striatal-enriched protein tyrosine phosphatase (STEP). Activity-triggered structural plasticity of dendrites was impaired by expression of a phospho-deficient mutant ezrin in vitro and in vivo. Thus, VEGFD governs the equilibrium between stabilization and plasticity of dendrites by acting as a molecular brake of structural remodeling.
Subject(s)
Dendrites , Hippocampus , Neuronal Plasticity , Signal Transduction , Animals , Dendrites/metabolism , Mice , Hippocampus/metabolism , Hippocampus/cytology , Mice, Inbred C57BL , Cells, Cultured , Cytoskeleton/metabolism , Male , Neurons/metabolism , Neurons/cytology , Actins/metabolism , Phosphorylation , Microtubules/metabolismABSTRACT
The mechanism underlying the preferential and cooperative binding of cofilin and the expansion of clusters toward the pointed-end side of actin filaments remains poorly understood. To address this, we conducted a principal component analysis based on available filamentous actin (F-actin) and C-actin (cofilins were excluded from cofilactin) structures and compared to monomeric G-actin. The results strongly suggest that C-actin, rather than F-ADP-actin, represented the favourable structure for binding preference of cofilin. High-speed atomic force microscopy explored that the shortened bare half helix adjacent to the cofilin clusters on the pointed end side included fewer actin protomers than normal helices. The mean axial distance (MAD) between two adjacent actin protomers along the same long-pitch strand within shortened bare half helices was longer (5.0-6.3 nm) than the MAD within typical helices (4.3-5.6 nm). The inhibition of torsional motion during helical twisting, achieved through stronger attachment to the lipid membrane, led to more pronounced inhibition of cofilin binding and cluster formation than the presence of inorganic phosphate (Pi) in solution. F-ADP-actin exhibited more naturally supertwisted half helices than F-ADP.Pi-actin, explaining how Pi inhibits cofilin binding to F-actin with variable helical twists. We propose that protomers within the shorter bare helical twists, either influenced by thermal fluctuation or induced allosterically by cofilin clusters, exhibit characteristics of C-actin-like structures with an elongated MAD, leading to preferential and cooperative binding of cofilin.
Subject(s)
Actin Depolymerizing Factors , Actins , Protein Binding , Actins/metabolism , Actins/chemistry , Actin Depolymerizing Factors/metabolism , Actin Depolymerizing Factors/chemistry , Microscopy, Atomic Force , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/chemistry , Protein Conformation , Models, Molecular , AnimalsABSTRACT
Many cytoskeletal networks consist of individual filaments that are organized into elaborate higher-order structures. While it is appreciated that the size and architecture of these networks are critical for their biological functions, much of the work investigating control over their assembly has focused on mechanisms that regulate the turnover of individual filaments through size-dependent feedback. Here, we propose a very different, feedback-independent mechanism to explain how yeast cells control the length of their actin cables. Our findings, supported by quantitative cell imaging and mathematical modeling, indicate that actin cable length control is an emergent property that arises from the cross-linked and bundled organization of the filaments within the cable. Using this model, we further dissect the mechanisms that allow cables to grow longer in larger cells and propose that cell length-dependent tuning of formin activity allows cells to scale cable length with cell length. This mechanism is a significant departure from prior models of cytoskeletal filament length control and presents a different paradigm to consider how cells control the size, shape, and dynamics of higher-order cytoskeletal structures.
Subject(s)
Cytoskeleton , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolism , Cytoskeleton/metabolism , Actins/metabolism , Actin Cytoskeleton/metabolism , Models, Biological , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The morphology of dendritic spines, the postsynaptic compartment of most excitatory synapses, decisively modulates the function of neuronal circuits as also evident from human brain disorders associated with altered spine density or morphology. Actin filaments (F-actin) form the backbone of spines, and a number of actin-binding proteins (ABP) have been implicated in shaping the cytoskeleton in mature spines. Instead, only little is known about the mechanisms that control the reorganization from unbranched F-actin of immature spines to the complex, highly branched cytoskeleton of mature spines. Here, we demonstrate impaired spine maturation in hippocampal neurons upon genetic inactivation of cyclase-associated protein 1 (CAP1) and CAP2, but not of CAP1 or CAP2 alone. We found a similar spine maturation defect upon overactivation of inverted formin 2 (INF2), a nucleator of unbranched F-actin with hitherto unknown synaptic function. While INF2 overactivation failed in altering spine density or morphology in CAP-deficient neurons, INF2 inactivation largely rescued their spine defects. From our data we conclude that CAPs inhibit INF2 to induce spine maturation. Since we previously showed that CAPs promote cofilin1-mediated cytoskeletal remodeling in mature spines, we identified them as a molecular switch that control transition from filopodia-like to mature spines.
Subject(s)
Cytoskeletal Proteins , Dendritic Spines , Formins , Hippocampus , Microfilament Proteins , Dendritic Spines/metabolism , Animals , Mice , Formins/metabolism , Formins/genetics , Microfilament Proteins/metabolism , Microfilament Proteins/genetics , Cytoskeletal Proteins/metabolism , Cytoskeletal Proteins/genetics , Hippocampus/metabolism , Hippocampus/cytology , Cells, Cultured , Neurons/metabolism , Actins/metabolism , Actin Cytoskeleton/metabolism , Mice, Knockout , Humans , Carrier ProteinsABSTRACT
We study the dynamic structure of lipid domain inclusions embedded within a phase-separated reconstituted lipid bilayer in contact with a swarming flow of gliding filamentous actin. Passive circular domains transition into highly deformed morphologies that continuously elongate, rotate, and pinch off into smaller fragments, leading to a dynamic steady state with ≈23× speedup in the relaxation of the intermediate scattering function compared with passive membrane domains driven by purely thermal forces. To corroborate experimental results, we develop a phase-field model of the lipid domains with two-way coupling to the Toner-Tu equations. We report phase domains that become entrained in the chaotic eddy patterns, with oscillating waves of domains that correlate with the dominant wavelengths of the Toner-Tu flow fields.
Subject(s)
Lipid Bilayers , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Actins/metabolism , Models, Biological , Membrane Microdomains/metabolismABSTRACT
The interior of cellular nuclei, the nucleoplasm, is a crowded fluid that is pervaded by protein-decorated DNA polymers, the chromatin. Due to the complex architecture of chromatin and a multitude of associated nonequilibrium processes, e.g., DNA repair, the nucleoplasm can be expected to feature nontrivial material properties and hence anomalous transport phenomena. Here, we have used single-particle tracking on nuclear actin rods to probe such transport phenomena. Our analysis reveals that short actin rods in the nucleus show an intermittent, antipersistent subdiffusion with clear signatures of fractional Brownian motion. Moreover, the diffusive motion is heterogeneous with clear signatures of an intermittent switching of trajectories between at least two different mobilities, most likely due to transient associations with chromatin. In line with this interpretation, hyperosmotic stress is seen to stall the motion of nuclear actin rods, whereas hypo-osmotic conditions yield a reptationlike motion. Our data highlights the heterogeneity of transport in the nucleoplasm that needs to be taken into account for an understanding of nucleoplasmic organization and the mechanobiology of nuclei.
Subject(s)
Actins , Cell Nucleus , Chromatin , Diffusion , Actins/metabolism , Chromatin/metabolism , Cell Nucleus/metabolism , Animals , Models, Biological , Osmotic PressureABSTRACT
Eukaryotic cells have been evolving for billions of years, giving rise to wildly diverse cell forms and functions. Despite their variability, all eukaryotic cells share key hallmarks, including membrane-bound organelles, heavily regulated cytoskeletal networks and complex signaling cascades. Because the actin cytoskeleton interfaces with each of these features, understanding how it evolved and diversified across eukaryotic phyla is essential to understanding the evolution and diversification of eukaryotic cells themselves. Here, we discuss what we know about the origin and diversity of actin networks in terms of their compositions, structures and regulation, and how actin evolution contributes to the diversity of eukaryotic form and function.