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BioRxiv : the Preprint Server For... Mar 2024We present single-molecule labeling and localization microscopy (SMLLM) using dye-conjugated phalloidin to achieve enhanced superresolution imaging of filamentous actin...
We present single-molecule labeling and localization microscopy (SMLLM) using dye-conjugated phalloidin to achieve enhanced superresolution imaging of filamentous actin (F-actin). We demonstrate that the intrinsic phalloidin dissociation enables SMLLM in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-based SMLLM are better preservation of cellular structures sensitive to mechanical and shear forces during standard sample preparation and more consistent F-actin quantification at the nanoscale. In a proof-of-concept study, we employed SMLLM to super-resolve F-actin structures in U2OS and dendritic cells (DCs) and demonstrate more consistent F-actin quantification in the cell body and structurally delicate cytoskeletal proportions, which we termed membrane fibers, of DCs compared to direct stochastic optical reconstruction microscopy (STORM). Using DC2.4 mouse dendritic cells as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane fibers on the culture glass surface after lipopolysaccharide exposure. While our work demonstrates SMLLM for F-actin, the concept opens new possibilities for protein-specific single-molecule labeling and localization in the same step using commercially available reagents.
PubMed: 38496456
DOI: 10.1101/2024.03.04.583337 -
Methods in Molecular Biology (Clifton,... 2024Cytoskeletal dysregulation forms an important aspect of many neurodegenerative diseases such as Alzheimer's disease. Cytoskeletal functions require the dynamic activity...
Cytoskeletal dysregulation forms an important aspect of many neurodegenerative diseases such as Alzheimer's disease. Cytoskeletal functions require the dynamic activity of the cytoskeletal proteins-actin, tubulin, and the associated proteins. One of such important phenomena is that of actin remodeling, which helps the cell to migrate, navigate, and interact with extracellular materials. Podosomes are complex actin-rich cytoskeletal structures, abundant in proteins that interact and degrade the extracellular matrix, enabling cells to displace and migrate. The formation of podosomes requires extensive actin networks and remodeling. Here we present a novel immunofluorescence-based approach to study actin remodeling in neurons through the medium of podosomes.
Topics: Actins; Podosomes; Cytoskeleton; Cytoskeletal Proteins; Extracellular Matrix; Actin Cytoskeleton
PubMed: 38427242
DOI: 10.1007/978-1-0716-3662-6_18 -
Biochemical and Biophysical Research... Apr 2024Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the...
Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.
Topics: Humans; Osteoclasts; Podosomes; Actins; Cell Differentiation; Bone Resorption; CSK Tyrosine-Protein Kinase; Collagen; Oligopeptides
PubMed: 38402724
DOI: 10.1016/j.bbrc.2024.149636 -
Cell Reports Feb 2024The acidic metabolic byproducts within the tumor microenvironment (TME) hinder T cell effector functions. However, their effects on T cell infiltration remain largely...
The acidic metabolic byproducts within the tumor microenvironment (TME) hinder T cell effector functions. However, their effects on T cell infiltration remain largely unexplored. Leveraging the comprehensive The Cancer Genome Atlas dataset, we pinpoint 16 genes that correlate with extracellular acidification and establish a metric known as the "tumor acidity (TuAci) score" for individual patients. We consistently observe a negative association between the TuAci score and T lymphocyte score (T score) across various human cancer types. Mechanistically, extracellular acidification significantly impedes T cell motility by suppressing podosome formation. This phenomenon can be attributed to the reduced expression of methyltransferase-like 3 (METTL3) and the modification of RNA N-methyladenosine (mA), resulting in a subsequent decrease in the expression of integrin β1 (ITGB1). Importantly, enforced ITGB1 expression leads to enhanced T cell infiltration and improved antitumor activity. Our study suggests that modulating METTL3 activity or boosting ITGB1 expression could augment T cell infiltration within the acidic TME, thereby improving the efficacy of cell therapy.
Topics: Humans; Cell- and Tissue-Based Therapy; Hydrogen-Ion Concentration; Integrin beta1; Methyltransferases; Neoplasms; T-Lymphocytes; Tumor Microenvironment
PubMed: 38367240
DOI: 10.1016/j.celrep.2024.113796 -
Cichoric acid targets RANKL to inhibit osteoclastogenesis and prevent ovariectomy-induced bone loss.Phytotherapy Research : PTR Apr 2024Osteoporosis, a systemic metabolic bone disease, is characterized by the decline of bone mass and quality due to excessive osteoclast activity. Currently, drug-targeting...
BACKGROUND AND AIM
Osteoporosis, a systemic metabolic bone disease, is characterized by the decline of bone mass and quality due to excessive osteoclast activity. Currently, drug-targeting osteoclasts show promising therapy for osteoporosis. In this study, we investigated the effect of cichoric acid (CA) on receptor activator of nuclear kappa-B ligand (RANKL)-induced osteoclastogenesis and the bone loss induced by ovariectomy in mice.
EXPERIMENTAL PROCEDURE
Molecular docking technologies were employed to examine the interaction between CA and RANKL. CCK8 assay was used to evaluate the cell viability under CA treatment. TRAcP staining, podosome belt staining, and bone resorption assays were used to test the effect of CA on osteoclastogenesis and osteoclast function. Further, an OVX-induced osteoporosis mice model was employed to identify the effect of CA on bone loss using micro-CT scanning and histological examination. To investigate underlying mechanisms, network pharmacology was applied to predict the downstream signaling pathways, which were verified by Western blot and immunofluorescence staining.
KEY RESULTS
The molecular docking analysis revealed that CA exhibited a specific binding affinity to RANKL, engaging multiple binding sites. CA inhibited RANKL-induced osteoclastogenesis and bone resorption without cytotoxic effects. Mechanistically, CA suppressed RANKL-induced intracellular reactive oxygen species, nuclear factor-kappa B, and mitogen-activated protein kinase pathways, followed by abrogated nuclear factor activated T-cells 1 activity. Consistent with this finding, CA attenuated post-ovariectomy-induced osteoporosis by ameliorating osteoclastogenesis.
CONCLUSIONS AND IMPLICATIONS
CA inhibited osteoclast activity and bone loss by targeting RANKL. CA might represent a promising candidate for treating osteoclast-related diseases, such as osteoporosis.
Topics: Animals; Female; Humans; Mice; Bone Resorption; Caffeic Acids; Cell Differentiation; Mice, Inbred C57BL; Molecular Docking Simulation; NF-kappa B; Osteoclasts; Osteogenesis; Osteoporosis; Ovariectomy; RANK Ligand; Succinates
PubMed: 38358727
DOI: 10.1002/ptr.8141 -
The Journal of Cell Biology Mar 2024The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and...
The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and deadliest breast cancer subtype, triple-negative breast cancer (TNBC), but not in other subtypes. Expression of MAP1B was found to be highly correlated with poor prognosis. Depletion of MAP1B in TNBC cells impairs cell migration and invasion concomitant with a defect in tumorigenesis. We found that MAP1B interacts with key components for invadopodia formation, cortactin, and Tks5, the latter of which is a PtdIns(3,4)P2-binding and scaffold protein that localizes to invadopodia. We also found that Tks5 associates with microtubules and supports the association between MAP1B and α-tubulin. In accordance with their interaction, depletion of MAP1B leads to Tks5 destabilization, leading to its degradation via the autophagic pathway. Collectively, these findings suggest that MAP1B is a convergence point of the cytoskeleton to promote malignancy in TNBC and thereby a potential diagnostic and therapeutic target for TNBC.
Topics: Humans; Carcinogenesis; Cell Transformation, Neoplastic; Cortactin; Microtubule-Associated Proteins; Triple Negative Breast Neoplasms; MDA-MB-231 Cells; Adaptor Proteins, Vesicular Transport; Microtubules; Cytoskeleton; Female; Animals; Mice; Mice, Inbred BALB C; Podosomes; Tubulin
PubMed: 38353696
DOI: 10.1083/jcb.202303102 -
Bulletin of Mathematical Biology Feb 2024One of the most crucial and lethal characteristics of solid tumors is represented by the increased ability of cancer cells to migrate and invade other organs during the...
One of the most crucial and lethal characteristics of solid tumors is represented by the increased ability of cancer cells to migrate and invade other organs during the so-called metastatic spread. This is allowed thanks to the production of matrix metalloproteinases (MMPs), enzymes capable of degrading a type of collagen abundant in the basal membrane separating the epithelial tissue from the connective one. In this work, we employ a synergistic experimental and mathematical modelling approach to explore the invasion process of tumor cells. A mathematical model composed of reaction-diffusion equations describing the evolution of the tumor cells density on a gelatin substrate, MMPs enzymes concentration and the degradation of the gelatin is proposed. This is completed with a calibration strategy. We perform a sensitivity analysis and explore a parameter estimation technique both on synthetic and experimental data in order to find the optimal parameters that describe the in vitro experiments. A comparison between numerical and experimental solutions ends the work.
Topics: Humans; Podosomes; Gelatin; Extracellular Matrix; Models, Biological; Mathematical Concepts; Matrix Metalloproteinases; Neoplasm Invasiveness
PubMed: 38347328
DOI: 10.1007/s11538-024-01260-w -
Frontiers in Physiology 2024Bone health is controlled by the balance between bone formation by osteoblasts and degradation by osteoclasts. A disequilibrium in favor of bone resorption leads to...
Bone health is controlled by the balance between bone formation by osteoblasts and degradation by osteoclasts. A disequilibrium in favor of bone resorption leads to osteolytic diseases characterized by decreased bone density. Osteoclastic resorption is dependent on the assembly of an adhesion structure: the actin ring, also called podosome belt or sealing zone, which is composed of a unique patterning of podosomes stabilized by microtubules. A better understanding of the molecular mechanisms regulating the crosstalk between actin cytoskeleton and microtubules network is key to find new treatments to inhibit bone resorption. Evidence points to the importance of the fine tuning of the activity of the small GTPase RHOA for the formation and maintenance of the actin ring, but the underlying mechanism is not known. We report here that actin ring disorganization upon microtubule depolymerization is mediated by the activation of the RHOA-ROCK signaling pathway. We next show the involvement of GEF-H1, one of RHOA guanine exchange factor highly expressed in osteoclasts, which has the particularity of being negatively regulated by sequestration on microtubules. Using a CRISPR/Cas9-mediated GEF-H1 knock-down osteoclast model, we demonstrate that RHOA activation upon microtubule depolymerization is mediated by GEF-H1 release. Interestingly, although lower levels of GEF-H1 did not impact sealing zone formation in the presence of an intact microtubule network, sealing zone was smaller leading to impaired resorption. Altogether, these results suggest that a fine tuning of GEF-H1 through its association with microtubules, and consequently of RHOA activity, is essential for osteoclast sealing zone stability and resorption function.
PubMed: 38312316
DOI: 10.3389/fphys.2024.1342024 -
Bone Apr 2024Osteoclasts, the exclusive bone resorptive cells, are indispensable for bone remodeling. Hence, understanding novel signaling modulators regulating osteoclastogenesis is...
Osteoclasts, the exclusive bone resorptive cells, are indispensable for bone remodeling. Hence, understanding novel signaling modulators regulating osteoclastogenesis is clinically important. Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) is a master transcription factor in osteoclastogenesis, and binding of NF-κB p65 subunit to NFATc1 promoter is required for its expression. It is well-established that DNA binding activity of p65 can be regulated by various post-translational modifications, including S-nitrosation. Recent studies have demonstrated that S-nitrosoglutathione reductase (GSNOR)-mediated protein denitrosation participated in cell fate commitment by regulating gene transcription. However, the role of GSNOR in osteoclastogenesis remains unexplored and enigmatic. Here, we investigated the effect of GSNOR-mediated denitrosation of p65 on osteoclastogenesis. Our results revealed that GSNOR was up-regulated during osteoclastogenesis in vitro. Moreover, GSNOR inhibition with a chemical inhibitor impaired osteoclast differentiation, podosome belt formation, and bone resorption activity. Furthermore, GSNOR inhibition enhanced the S-nitrosation level of p65, precluded the binding of p65 to NFATc1 promoter, and suppressed NFATc1 expression. In addition, mouse model of lipopolysaccharides (LPS)-induced calvarial osteolysis was employed to evaluate the therapeutic effect of GSNOR inhibitor in vivo. Our results indicated that GSNOR inhibitor treatment alleviated the inflammatory bone loss by impairing osteoclast formation in mice. Taken together, these data have shown that GSNOR activity is required for osteoclastogenesis by facilitating binding of p65 to NFATc1 promoter via promoting p65 denitrosation, suggesting that GSNOR may be a potential therapeutic target in the treatment of osteolytic diseases.
Topics: Animals; Mice; Osteogenesis; Oxidoreductases; NFATC Transcription Factors; Osteoclasts; Bone Resorption; NF-kappa B; Cell Differentiation; Osteolysis; RANK Ligand; Aldehyde Oxidoreductases
PubMed: 38311303
DOI: 10.1016/j.bone.2024.117036 -
Biochimie Jul 2024Isoprenyl cysteine carboxyl methyltransferase (ICMT) catalyzes the last step of the prenylation pathway. Previously, we found that high ICMT levels enhance tumorigenesis...
Isoprenyl cysteine carboxyl methyltransferase (ICMT) catalyzes the last step of the prenylation pathway. Previously, we found that high ICMT levels enhance tumorigenesis in vivo and that its expression is repressed by the p53 tumor suppressor. Based on evidence suggesting that some ICMT substrates affect invasive traits, we wondered if this enzyme may promote metastasis. In this work, we found that ICMT overexpression enhanced lung metastasis in vivo. Accordingly, ICMT overexpression also promoted cellular functions associated with aggressive phenotypes such as migration and invasion in vitro. Considering that some ICMT substrates are involved in the regulation of actin cytoskeleton, we hypothesized that actin-rich structures, associated with invasion and metastasis, may be affected. Our findings revealed that ICMT enhanced the formation of invadopodia. Additionally, by analyzing cancer patient databases, we found that ICMT is overexpressed in several tumor types. Furthermore, the concurrent expression of ICMT and CTTN, which encodes a crucial component of invadopodia, showed a significant correlation with clinical outcome. In summary, our work identifies ICMT overexpression as a relevant alteration in human cancer that promotes the development of metastatic tumors.
Topics: Animals; Humans; Mice; Cell Line, Tumor; Cell Movement; Cortactin; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Podosomes; Protein Methyltransferases
PubMed: 38301884
DOI: 10.1016/j.biochi.2024.01.015