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ELife Oct 2022In most eukaryotic cells, actin filaments assemble into a shell-like actin cortex under the plasma membrane, controlling cellular morphology, mechanics, and signaling....
In most eukaryotic cells, actin filaments assemble into a shell-like actin cortex under the plasma membrane, controlling cellular morphology, mechanics, and signaling. The actin cortex is highly polymorphic, adopting diverse forms such as the ring-like structures found in podosomes, axonal rings, and immune synapses. The biophysical principles that underlie the formation of actin rings and cortices remain unknown. Using a molecular simulation platform called MEDYAN, we discovered that varying the filament treadmilling rate and myosin concentration induces a finite size phase transition in actomyosin network structures. We found that actomyosin networks condense into clusters at low treadmilling rates or high myosin concentrations but form ring-like or cortex-like structures at high treadmilling rates and low myosin concentrations. This mechanism is supported by our corroborating experiments on live T cells, which exhibit ring-like actin networks upon activation by stimulatory antibody. Upon disruption of filament treadmilling or enhancement of myosin activity, the pre-existing actin rings are disrupted into actin clusters or collapse towards the network center respectively. Our analyses suggest that the ring-like actin structure is a preferred state of low mechanical energy, which is, importantly, only reachable at sufficiently high treadmilling rates.
Topics: Actins; Actomyosin; Cytoskeleton; Myosins; Actin Cytoskeleton
PubMed: 36269229
DOI: 10.7554/eLife.82658 -
Oncogene Jan 2023Dissemination of cancer cells from the primary tumor into distant body tissues and organs is the leading cause of death in cancer patients. While most clinical...
Dissemination of cancer cells from the primary tumor into distant body tissues and organs is the leading cause of death in cancer patients. While most clinical strategies aim to reduce or impede the growth of the primary tumor, no treatment to eradicate metastatic cancer exists at present. Metastasis is mediated by feet-like cytoskeletal structures called invadopodia which allow cells to penetrate through the basement membrane and intravasate into blood vessels during their spread to distant tissues and organs. The non-receptor tyrosine kinase Pyk2 is highly expressed in breast cancer, where it mediates invadopodia formation and function via interaction with the actin-nucleation-promoting factor cortactin. Here, we designed a cell-permeable peptide inhibitor that contains the second proline-rich region (PRR2) sequence of Pyk2, which binds to the SH3 domain of cortactin and inhibits the interaction between Pyk2 and cortactin in invadopodia. The Pyk2-PRR2 peptide blocks spontaneous lung metastasis in immune-competent mice by inhibiting cortactin tyrosine phosphorylation and actin polymerization-mediated maturation and activation of invadopodia, leading to reduced MMP-dependent tumor cell invasiveness. The native structure of the Pyk2-PRR2:cortactin-SH3 complex was determined using nuclear magnetic resonance (NMR), revealing an extended class II interaction surface spanning the canonical binding groove and a second hydrophobic surface which significantly contributes to ligand affinity. Using structure-guided design, we created a mutant peptide lacking critical residues involved in binding that failed to inhibit invadopodia maturation and function and consequent metastatic dissemination in mice. Our findings shed light on the specific molecular interactions between Pyk2 and cortactin and may lead to the development of novel strategies for preventing dissemination of primary breast tumors predicted at the time of diagnosis to be highly metastatic, and of secondary tumors that have already spread to other parts of the body.
Topics: Animals; Mice; Actins; Cell Line, Tumor; Cortactin; Focal Adhesion Kinase 2; Neoplasm Invasiveness; Podosomes; Breast Neoplasms
PubMed: 36258022
DOI: 10.1038/s41388-022-02481-w -
Blood Advances Dec 2022Mature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to...
Mature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to thrombocytopenia and increased risk of bleeding. Mice lacking the actin-regulatory proteins Profilin 1 (PFN1), Wiskott-Aldrich Syndrome protein (WASp), Actin Related Protein 2/3 complex (Arp2/3), or adhesion and degranulation-promoting adapter protein (ADAP) display thrombocytopenia and ectopic release of (pro)platelet-like particles into the BM compartment, pointing to an important axis of actin-mediated directional proplatelet formation. The mechanism underlying ectopic release in these mice is still not completely understood. However, we hypothesized that similar functional defects account for this observation. We analyzed WASp-, ADAP-, PFN1-, and ARPC2-knockout mice to determine the role of actin reorganization and integrin activation in directional proplatelet formation. ADAP-, ARPC2-, and PFN1-deficient MKs displayed reduced adhesion to collagen, defective F-actin organization, and diminished β1-integrin activation. WASp-deficient MKs showed the strongest reduction in the adhesion assay of collagen and altered F-actin organization with reduced podosome formation. Our results indicate that ADAP, PFN1, WASp, and ARP2/3 are part of the same pathway that regulates polarization processes in MKs and directional proplatelet formation into BM sinusoids.
Topics: Mice; Animals; Megakaryocytes; Actins; Thrombocytopenia; Mice, Knockout; Disease Models, Animal; Adaptor Proteins, Signal Transducing; Integrins
PubMed: 36251748
DOI: 10.1182/bloodadvances.2022007824 -
Physiological Genomics Nov 2022The vast majority of studies focusing on the effects of endurance exercise on hematological parameters and leukocyte gene expression were performed in adult men, so our...
The vast majority of studies focusing on the effects of endurance exercise on hematological parameters and leukocyte gene expression were performed in adult men, so our aim was to investigate these changes in young females. Four young (age 15.3 ± 1.3 yr) elite female athletes completed an exercise session, in which they accomplished the cycling and running disciplines of a junior triathlon race. Blood samples were taken immediately before the exercise, right after the exercise, and then 1, 2, and 7 days later. Analysis of cell counts and routine biochemical parameters were complemented by RNA sequencing (RNA-seq) to whole blood samples. The applied exercise load did not trigger remarkable changes in either cardiovascular or biochemical parameters; however, it caused a significant increase in the percentage of neutrophils and a significant reduction in the ratio of lymphocytes immediately after exercise. Furthermore, endurance exercise induced a characteristic gene expression pattern change in the blood transcriptome. Gene set enrichment analysis (GSEA) using the Reactome database revealed that the expression of genes involved in immune processes and neutrophil granulocyte activation was upregulated, whereas the expression of genes important in translation and rRNA metabolism was downregulated. Comparison of a set of immune cell gene signatures (ImSig) and our transcriptomic data identified 15 overlapping genes related to T-cell functions and involved in podosome formation and adhesion to the vessel wall. Our results suggest that RNA-seq to whole blood together with ImSig analysis are useful tools for the investigation of systemic responses to endurance exercise.
Topics: Male; Humans; Female; Adolescent; Transcriptome; Physical Endurance; Pilot Projects; Athletes; Running
PubMed: 36250559
DOI: 10.1152/physiolgenomics.00090.2022 -
PLoS Computational Biology Oct 2022Phagocytosis, the biological process in which cells ingest large particles such as bacteria, is a key component of the innate immune response. Fcγ receptor...
Phagocytosis, the biological process in which cells ingest large particles such as bacteria, is a key component of the innate immune response. Fcγ receptor (FcγR)-mediated phagocytosis is initiated when these receptors are activated after binding immunoglobulin G (IgG). Receptor activation initiates a signaling cascade that leads to the formation of the phagocytic cup and culminates with ingestion of the foreign particle. In the experimental system termed "frustrated phagocytosis", cells attempt to internalize micropatterned disks of IgG. Cells that engage in frustrated phagocytosis form "rosettes" of actin-enriched structures called podosomes around the IgG disk. The mechanism that generates the rosette pattern is unknown. We present data that supports the involvement of Cdc42, a member of the Rho family of GTPases, in pattern formation. Cdc42 acts downstream of receptor activation, upstream of actin polymerization, and is known to play a role in polarity establishment. Reaction-diffusion models for GTPase spatiotemporal dynamics exist. We demonstrate how the addition of negative feedback and minor changes to these models can generate the experimentally observed rosette pattern of podosomes. We show that this pattern formation can occur through two general mechanisms. In the first mechanism, an intermediate species forms a ring of high activity around the IgG disk, which then promotes rosette organization. The second mechanism does not require initial ring formation but relies on spatial gradients of intermediate chemical species that are selectively activated over the IgG patch. Finally, we analyze the models to suggest experiments to test their validity.
Topics: Actins; Immunoglobulin G; Macrophages; Phagocytosis; Receptors, IgG
PubMed: 36190993
DOI: 10.1371/journal.pcbi.1010092 -
Current Protein & Peptide Science 2022Exosome research is a current trend in functional proteomics as it provides important data on the pathophysiology and pathogenesis of diseases. The scientific outputs...
BACKGROUND
Exosome research is a current trend in functional proteomics as it provides important data on the pathophysiology and pathogenesis of diseases. The scientific outputs regarding these topics often only approach disease-protein/peptide/exosome or mechanismprotein/ peptide/exosome association. Approaching all three aspects could be the key to a better understanding of the pathophysiology and uncovering novel biomarkers for urogenital diseases. The focus of this work is to study exosome datasets to understand the possible role of underlying proteins in disease manifestation. We also attempt to link 4 different diseases that affect renal functions and are genetically inherited.
METHODS
For this purpose, the existing literature is consulted to understand the importance of exosomes in disease prediction, diagnosis and therapy. Available biotechnological methods of exosome analysis and the tools of proteomic analysis, data mining and visualization are discussed. The database PRIDE is selected to query the information of several datasets related to urinary exosome analysis.
RESULTS
We have obtained a list of 19 proteins/genes involved in the mentioned diseases. On this list, we found a proteomic fingerprint consisting of Rab-7a, PDCD6, and CDC42, among others, and we are exploring their biological significance and underlying processes.
CONCLUSION
APOA1, CD59, CD9, IGHG1, RAB7A, RAP1A, SEMG1 and SEMG2 are common in four urogenital diseases, and are involved in interactions with podosomes and endosomes, remodeling of chylomicrons, regulation of interleukin production, regulation of endopeptidase activity, and establishment of apical/basal polarity of epithelial cells.
Topics: Humans; Proteomics; Biomarkers; Exosomes; Urinalysis; Urogenital Diseases; Calcium-Binding Proteins; Apoptosis Regulatory Proteins
PubMed: 36177616
DOI: 10.2174/1389203723666220929155542 -
Cell Death & Disease Sep 2022Distant metastasis is the main cause of mortality in breast cancer patients. Using the breast cancer genomic data from The Cancer Genome Atlas (TCGA), we identified...
Distant metastasis is the main cause of mortality in breast cancer patients. Using the breast cancer genomic data from The Cancer Genome Atlas (TCGA), we identified brain specific Ca2.2 as a critical regulator of metastasis. Ca2.2 expression is significantly upregulated in breast cancer and its higher expression is inversely correlated with survival suggesting a previously unappreciated role of Ca2.2 in breast cancer. Ca2.2 is required for breast cancer migration, invasion, and metastasis. Interestingly, Ca2.2 promotes invadopodia formation and extracellular matrix (ECM) degradation through the stabilization of invadopodia component cortactin in a proteosome-dependent manner. Moreover, deubiquitinating enzyme USP43 mediated the functions of Ca2.2 in cortactin stabilization, invadopodia formation, ECM degradation, and metastasis. Interestingly, Ca2.2 upregulates USP43 expression through NFAT2 dephosphorylation and nuclear localization. Our study uncovered a novel pathway that regulates cortactin expression and invadopodia formation in breast cancer metastasis.
Topics: Breast Neoplasms; Calcium Channels, N-Type; Cell Line, Tumor; Cortactin; Deubiquitinating Enzymes; Extracellular Matrix; Female; Humans; NFATC Transcription Factors; Neoplasm Invasiveness; Podosomes
PubMed: 36137995
DOI: 10.1038/s41419-022-05174-0 -
Nature Reviews. Molecular Cell Biology Feb 2023Cell invasion into the surrounding extracellular matrix or across tissue boundaries and endothelial barriers occurs in both physiological and pathological scenarios such... (Review)
Review
Cell invasion into the surrounding extracellular matrix or across tissue boundaries and endothelial barriers occurs in both physiological and pathological scenarios such as immune surveillance or cancer metastasis. Podosomes and invadopodia, collectively called 'invadosomes', are actin-based structures that drive the proteolytic invasion of cells, by forming highly regulated platforms for the localized release of lytic enzymes that degrade the matrix. Recent advances in high-resolution microscopy techniques, in vivo imaging and high-throughput analyses have led to considerable progress in understanding mechanisms of invadosomes, revealing the intricate inner architecture of these structures, as well as their growing repertoire of functions that extends well beyond matrix degradation. In this Review, we discuss the known functions, architecture and regulatory mechanisms of podosomes and invadopodia. In particular, we describe the molecular mechanisms of localized actin turnover and microtubule-based cargo delivery, with a special focus on matrix-lytic enzymes that enable proteolytic invasion. Finally, we point out topics that should become important in the invadosome field in the future.
Topics: Podosomes; Actins; Extracellular Matrix; Microtubules; Proteolysis
PubMed: 36104625
DOI: 10.1038/s41580-022-00530-6 -
Small (Weinheim An Der Bergstrasse,... Sep 2022Precise timing of macrophage polarization plays a pivotal role in immunomodulation of tissue regeneration, yet most studies mainly focus on M2 macrophages for their...
Precise timing of macrophage polarization plays a pivotal role in immunomodulation of tissue regeneration, yet most studies mainly focus on M2 macrophages for their anti-inflammatory and regenerative effects while the essential proinflammatory role of the M1 phenotype on the early inflammation stage is largely underestimated. Herein, a superparamagnetic hydrogel capable of timely controlling macrophage polarization is constructed by grafting superparamagnetic nanoparticles on collagen nanofibers. The magnetic responsive hydrogel network enables efficient polarization of encapsulated macrophage to the M2 phenotype through the podosome/Rho/ROCK mechanical pathway in response to static magnetic field (MF) as needed. Taking advantage of remote accessibility of magnetic field together with the superparamagnetic hydrogels, a temporal engineered M1 to M2 transition course preserving the essential role of M1 at the early stage of tissue healing, as well as enhancing the prohealing effect of M2 at the middle/late stages is established via delayed MF switch. Such precise timing of macrophage polarization matching the regenerative process of injured tissue eventually leads to optimized immunomodulatory bone healing in vivo. Overall, this study offers a remotely time-scheduled approach for macrophage polarization, which enables precise manipulation of inflammation progression during tissue healing.
Topics: Bone Regeneration; Collagen; Humans; Hydrogels; Immunomodulation; Inflammation; Macrophages; Phenotype
PubMed: 36031402
DOI: 10.1002/smll.202203680 -
Military Medical Research Aug 2022Wear particles-induced osteolysis is a major long-term complication after total joint arthroplasty. Up to now, there is no effective treatment for wear particles-induced...
BACKGROUND
Wear particles-induced osteolysis is a major long-term complication after total joint arthroplasty. Up to now, there is no effective treatment for wear particles-induced osteolysis except for the revision surgery, which is a heavy psychological and economic burden to patients. A metabolite of gut microbiota, short chain fatty acids (SCFAs), has been reported to be beneficial for many chronic inflammatory diseases. This study aimed to investigate the therapeutic effect of SCFAs on osteolysis.
METHODS
A model of inflammatory osteolysis was established by applying CoCrMo alloy particles to mouse calvarium. After two weeks of intervention, the anti-inflammatory effects of SCFAs on wear particle-induced osteolysis were evaluated by Micro-CT analysis and immunohistochemistry staining. In vitro study, lipopolysaccharide (LPS) primed bone marrow-derived macrophages (BMDMs) and Tohoku Hospital Pediatrics-1 (THP-1) macrophages were stimulated with CoCrMo particles to activate inflammasome in the presence of acetate (C2), propionate (C3), and butyrate (C4). Western blotting, Enzyme-linked immunosorbent assay, and immunofluorescence were used to detect the activation of NLRP3 inflammasome. The effects of SCFAs on osteoclasts were evaluate by qRT-PCR, Western blotting, immunofluorescence, and tartrate-resistant acid phosphatase (TRAP) staining. Additionally, histone deacetylase (HDAC) inhibitors, agonists of GPR41, GPR43, and GPR109A were applied to confirm the underlying mechanism of SCFAs on the inflammasome activation of macrophages and osteoclastogenesis.
RESULTS
C3 and C4 but not C2 could alleviate wear particles-induced osteolysis with fewer bone erosion pits (P < 0.001), higher level of bone volume to tissue volume (BV/TV, P < 0.001), bone mineral density (BMD, P < 0.001), and a lower total porosity (P < 0.001). C3 and C4 prevented CoCrMo alloy particles-induced ASC speck formation and nucleation-induced oligomerization, suppressing the cleavage of caspase-1 (P < 0.05) and IL-1β (P < 0.05) stimulated by CoCrMo alloy particles. C3 and C4 also inhibited the generation of Gasdermin D-N-terminal fragment (GSDMD-NT) to regulate pyroptosis. Besides, C3 and C4 have a negative impact on osteoclast differentiation (P < 0.05) and its function (P < 0.05), affecting the podosome arrangement and morphologically normal podosome belts formation.
CONCLUSION
Our work showed that C3 and C4 are qualified candidates for the treatment of wear particle-induced osteolysis.
Topics: Alloys; Animals; Butyrates; Humans; Inflammasomes; Macrophages; Mice; Osteogenesis; Osteolysis; Propionates; Pyroptosis
PubMed: 35996168
DOI: 10.1186/s40779-022-00404-0