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The Journal of Cell Biology Sep 2023Apical constriction is a cell shape change that drives key morphogenetic events during development, including gastrulation and neural tube formation. The forces driving...
Apical constriction is a cell shape change that drives key morphogenetic events during development, including gastrulation and neural tube formation. The forces driving apical constriction are primarily generated through the contraction of apicolateral and/or medioapical actomyosin networks. In the Drosophila ventral furrow, the medioapical actomyosin network has a sarcomere-like architecture, with radially polarized actin filaments and centrally enriched non-muscle myosin II and myosin activating kinase. To determine if this is a broadly conserved actin architecture driving apical constriction, we examined actomyosin architecture during C. elegans gastrulation, in which two endodermal precursor cells internalize from the surface of the embryo. Quantification of protein localization showed that neither the non-muscle myosin II NMY-2 nor the myosin-activating kinase MRCK-1 is enriched at the center of the apex. Further, visualization of barbed- and pointed-end capping proteins revealed that actin filaments do not exhibit radial polarization at the apex. Our results demonstrate that C. elegans endodermal precursor cells apically constrict using a mixed-polarity actin filament network and with myosin and a myosin activator distributed throughout the network. Taken together with observations made in other organisms, our results demonstrate that diverse actomyosin architectures are used in animal cells to accomplish apical constriction.
Topics: Animals; Actin Cytoskeleton; Actomyosin; Caenorhabditis elegans; Constriction; Morphogenesis; Myosin Type II; Myosins; Caenorhabditis elegans Proteins
PubMed: 37351566
DOI: 10.1083/jcb.202302102 -
European Journal of Cell Biology Jun 2024While it is well-established that F-actin networks with specific organizations and dynamics are tightly regulated by distinct sets of associated actin-binding proteins...
While it is well-established that F-actin networks with specific organizations and dynamics are tightly regulated by distinct sets of associated actin-binding proteins (ABPs), how ABPs self-sort to particular F-actin networks remains largely unclear. We report that actin assembly factors Arp2/3 complex and formin Cdc12 tune the association of ABPs fimbrin Fim1 and tropomyosin Cdc8 to different F-actin networks in fission yeast. Genetic and pharmacological disruption of F-actin networks revealed that Fim1 is preferentially directed to Arp2/3-complex mediated actin patches, whereas Cdc8 is preferentially targeted to formin Cdc12-mediated filaments in the contractile ring. To investigate the role of Arp2/3 complex- and formin Cdc12-mediated actin assembly, we used four-color TIRF microscopy to observe the in vitro reconstitution of ABP sorting with purified proteins. Fim1 or Cdc8 alone bind similarly well to filaments assembled by either assembly factor. However, in 'competition' reactions containing both actin assembly factors and both ABPs, ∼2.0-fold more Fim1 and ∼3.5-fold more Cdc8 accumulates on Arp2/3 complex branch points and formin Cdc12-assembled actin filaments, respectively. These findings indicate that F-actin assembly factors Arp2/3 complex and formin Cdc12 help facilitate the recruitment of specific ABPs, thereby tuning ABP sorting and subsequently establishing the identity of F-actin networks in fission yeast.
Topics: Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Actin-Related Protein 2-3 Complex; Actin Cytoskeleton; Microfilament Proteins; Cell Cycle Proteins; Actins; Protein Transport; Cytoskeletal Proteins; Membrane Glycoproteins
PubMed: 38493594
DOI: 10.1016/j.ejcb.2024.151404 -
Toxins Dec 2023iota-toxin is composed of two separate proteins: a binding protein (Ib) that recognizes a host cell receptor and promotes the cellular uptake of a catalytic protein and... (Review)
Review
iota-toxin is composed of two separate proteins: a binding protein (Ib) that recognizes a host cell receptor and promotes the cellular uptake of a catalytic protein and (Ia) possessing ADP-ribosyltransferase activity that induces actin cytoskeleton disorganization. Ib exhibits the overall structure of bacterial pore-forming toxins (PFTs). Lipolysis-stimulated lipoprotein receptor (LSR) is defined as a host cell receptor for Ib. The binding of Ib to LSR causes an oligomer formation of Ib in lipid rafts of plasma membranes, mediating the entry of Ia into the cytoplasm. Ia induces actin cytoskeleton disruption via the ADP-ribosylation of G-actin and causes cell rounding and death. The binding protein alone disrupts the cell membrane and induces cytotoxicity in sensitive cells. Host cells permeabilized by the pore formation of Ib are repaired by a Ca-dependent plasma repair pathway. This review shows that the cellular uptake of iota-toxin utilizes a pathway of plasma membrane repair and that Ib alone induces cytotoxicity.
Topics: Animals; Chlorocebus aethiops; Clostridium perfringens; Biological Transport; Actins; Vero Cells; ADP Ribose Transferases
PubMed: 38133199
DOI: 10.3390/toxins15120695 -
PLoS Pathogens Aug 2023Ebola (EBOV) and Marburg viruses (MARV) cause severe hemorrhagic fever associated with high mortality rates in humans. A better understanding of filovirus-host...
Ebola (EBOV) and Marburg viruses (MARV) cause severe hemorrhagic fever associated with high mortality rates in humans. A better understanding of filovirus-host interactions that regulate the EBOV and MARV lifecycles can provide biological and mechanistic insight critical for therapeutic development. EBOV glycoprotein (eGP) and MARV glycoprotein (mGP) mediate entry into host cells primarily by actin-dependent macropinocytosis. Here, we identified actin-binding cytoskeletal crosslinking proteins filamin A (FLNa) and B (FLNb) as important regulators of both EBOV and MARV entry. We found that entry of pseudotype psVSV-RFP-eGP, infectious recombinant rVSV-eGP-mCherry, and live authentic EBOV and MARV was inhibited in filamin A knockdown (FLNaKD) cells, but was surprisingly enhanced in filamin B knockdown (FLNbKD) cells. Mechanistically, our findings suggest that differential regulation of macropinocytosis by FLNa and FLNb likely contributes to their specific effects on EBOV and MARV entry. This study is the first to identify the filamin family of proteins as regulators of EBOV and MARV entry. These findings may provide insight into the development of new countermeasures to prevent EBOV and MARV infections.
Topics: Humans; Filamins; Ebolavirus; Actins; Hemorrhagic Fever, Ebola; Marburgvirus; Glycoproteins
PubMed: 37585478
DOI: 10.1371/journal.ppat.1011595 -
Journal of Experimental & Clinical... Aug 2023Acute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse....
BACKGROUND
Acute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse. FLT3-ITD is retained in the endoplasmic reticulum (ER) and generates intrinsic proteotoxic stress. We devised a strategy based on proteotoxic stress, generated by the combination of low doses of the differentiating agent retinoic acid (R), the proteasome inhibitor bortezomib (B), and the oxidative stress inducer arsenic trioxide (A).
METHODS
We treated FLT3-ITD AML cells with low doses of the aforementioned drugs, used alone or in combinations and we investigated the induction of ER and oxidative stress. We then performed the same experiments in an in vitro co-culture system of FLT3-ITD AML cells and bone marrow stromal cells (BMSCs) to assess the protective role of the niche on AML blasts. Eventually, we tested the combination of drugs in an orthotopic murine model of human AML.
RESULTS
The combination RBA exerts strong cytotoxic activity on FLT3-ITD AML cell lines and primary blasts isolated from patients, due to ER homeostasis imbalance and generation of oxidative stress. AML cells become completely resistant to the combination RBA when treated in co-culture with BMSCs. Nonetheless, we could overcome such protective effects by using high doses of ascorbic acid (Vitamin C) as an adjuvant. Importantly, the combination RBA plus ascorbic acid significantly prolongs the life span of a murine model of human FLT3-ITD AML without toxic effects. Furthermore, we show for the first time that the cross-talk between AML and BMSCs upon treatment involves disruption of the actin cytoskeleton and the actin cap, increased thickness of the nuclei, and relocalization of the transcriptional co-regulator YAP in the cytosol of the BMSCs.
CONCLUSIONS
Our findings strengthen our previous work indicating induction of proteotoxic stress as a possible strategy in FLT3-ITD AML therapy and open to the possibility of identifying new therapeutic targets in the crosstalk between AML and BMSCs, involving mechanotransduction and YAP signaling.
Topics: Humans; Animals; Mice; Tretinoin; Cytoprotection; Disease Models, Animal; Mechanotransduction, Cellular; Proteotoxic Stress; Ascorbic Acid; Cell Death
PubMed: 37653435
DOI: 10.1186/s13046-023-02793-z -
Current Opinion in Cell Biology Apr 2024The cytoskeleton, comprising actin microfilaments, microtubules, and intermediate filaments, is crucial for cell motility and tissue integrity. While prior studies... (Review)
Review
The cytoskeleton, comprising actin microfilaments, microtubules, and intermediate filaments, is crucial for cell motility and tissue integrity. While prior studies largely focused on individual cytoskeletal networks, recent research underscores the interconnected nature of these systems in fundamental cellular functions like adhesion, migration, and division. Understanding the coordination of these distinct networks in both time and space is essential. This review synthesizes current findings on the intricate interplay between these networks, emphasizing the pivotal role of intermediate filaments. Notably, these filaments engage in extensive crosstalk with microfilaments and microtubules through direct molecular interactions, cytoskeletal linkers, and molecular motors that form molecular bridges, as well as via more complex regulation of intracellular signaling.
Topics: Intermediate Filaments; Cytoskeleton; Microtubules; Actin Cytoskeleton; Cell Movement; Actins
PubMed: 38359728
DOI: 10.1016/j.ceb.2024.102325 -
The Journal of Allergy and Clinical... Oct 2023The actin cytoskeleton has a crucial role in the maintenance of the immune homeostasis by controlling various cellular processes, including cell migration. Mutations in...
BACKGROUND
The actin cytoskeleton has a crucial role in the maintenance of the immune homeostasis by controlling various cellular processes, including cell migration. Mutations in TTC7A have been described as the cause of a primary immunodeficiency associated to different degrees of gut involvement and alterations in the actin cytoskeleton dynamics.
OBJECTIVES
This study investigates the impact of TTC7A deficiency in immune homeostasis. In particular, the role of the TTC7A/phosphatidylinositol 4 kinase type III α pathway in the control of leukocyte migration and actin dynamics.
METHODS
Microfabricated devices were leveraged to study cell migration and actin dynamics of murine and patient-derived leukocytes under confinement at the single-cell level.
RESULTS
We show that TTC7A-deficient lymphocytes exhibit an altered cell migration and reduced capacity to deform through narrow gaps. Mechanistically, TTC7A-deficient phenotype resulted from impaired phosphoinositide signaling, leading to the downregulation of the phosphoinositide 3-kinase/AKT/RHOA regulatory axis and imbalanced actin cytoskeleton dynamics. TTC7A-associated phenotype resulted in impaired cell motility, accumulation of DNA damage, and increased cell death in dense 3-dimensional gels in the presence of chemokines.
CONCLUSIONS
These results highlight a novel role of TTC7A as a critical regulator of lymphocyte migration. Impairment of this cellular function is likely to contribute to the pathophysiology underlying progressive immunodeficiency in patients.
Topics: Humans; Animals; Mice; Actins; Phosphatidylinositol 3-Kinases; Cell Death; Mutation; Cell Movement; DNA Damage; Proteins; 1-Phosphatidylinositol 4-Kinase
PubMed: 37390900
DOI: 10.1016/j.jaci.2023.06.016 -
Cell Jun 2024Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell...
Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin β-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.
Topics: Animals; Humans; Mice; Actin Cytoskeleton; Cell Adhesion; CHO Cells; Cricetulus; Integrins; Ligands; Protein Binding; Protein Conformation; Signal Transduction; Single Molecule Imaging; Talin
PubMed: 38772370
DOI: 10.1016/j.cell.2024.04.049 -
Biochemical Society Transactions Feb 2024The Arp2/3 complex, which generates both branched but also linear actin filaments via activation of SPIN90, is evolutionarily conserved in eukaryotes. Several factors... (Review)
Review
The Arp2/3 complex, which generates both branched but also linear actin filaments via activation of SPIN90, is evolutionarily conserved in eukaryotes. Several factors regulate the stability of filaments generated by the Arp2/3 complex to maintain the dynamics and architecture of actin networks. In this review, we summarise recent studies on the molecular mechanisms governing the tuning of Arp2/3 complex nucleated actin filaments, which includes investigations using microfluidics and single-molecule imaging to reveal the mechanosensitivity, dissociation and regeneration of actin branches. We also discuss the high-resolution cryo-EM structure of cortactin bound to actin branches, as well as the differences and similarities between the stability of Arp2/3 complex nucleated branches and linear filaments. These new studies provide a clearer picture of the stabilisation of Arp2/3 nucleated filaments at the molecular level. We also identified gaps in our understanding of how different factors collectively contribute to the stabilisation of Arp2/3 complex-generated actin networks.
Topics: Actin-Related Protein 2-3 Complex; Actins; Actin Cytoskeleton; Cytoskeleton
PubMed: 38288872
DOI: 10.1042/BST20230638 -
American Journal of Respiratory Cell... Nov 2023Prostaglandin E2 imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1-EP4)....
Prostaglandin E2 imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1-EP4). Gs-coupled EP2 and EP4 receptors are expressed on airway smooth muscle (ASM), yet their capacity to regulate the ASM contractile state remains subject to debate. We used EP2 and EP4 subtype-specific agonists (ONO-259 and ONO-329, respectively) in cell- and tissue-based models of human ASM contraction-magnetic twisting cytometry (MTC), and precision-cut lung slices (PCLSs), respectively-to study the EP2 and EP4 regulation of ASM contraction and signaling under conditions of histamine or methacholine (MCh) stimulation. ONO-329 was superior (<0.05) to ONO-259 in relaxing MCh-contracted PCLSs (log half maximal effective concentration [logEC]: 4.9 × 10 vs. 2.2 × 10; maximal bronchodilation ± SE, 35 ± 2% vs. 15 ± 2%). However, ONO-259 and ONO-329 were similarly efficacious in relaxing histamine-contracted PCLSs. Similar differential effects were observed in MTC studies. Signaling analyses revealed only modest differences in ONO-329- and ONO-259-induced phosphorylation of the protein kinase A substrates VASP and HSP20, with concomitant stimulation with MCh or histamine. Conversely, ONO-259 failed to inhibit MCh-induced phosphorylation of the regulatory myosin light chain (pMLC20) and the F-actin/G-actin ratio (F/G-actin ratio) while effectively inhibiting their induction by histamine. ONO-329 was effective in reversing induced pMLC20 and the F/G-actin ratio with both MCh and histamine. Thus, the contractile-agonist-dependent differential effects are not explained by changes in the global levels of phosphorylated protein kinase A substrates but are reflected in the regulation of pMLC20 (cross-bridge cycling) and F/G-actin ratio (actin cytoskeleton integrity, force transmission), implicating a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes.
Topics: Humans; Receptors, Prostaglandin E, EP2 Subtype; Actins; Histamine; Receptors, Prostaglandin E, EP4 Subtype; Dinoprostone; Muscle, Smooth; Lung; Cyclic AMP-Dependent Protein Kinases
PubMed: 37523713
DOI: 10.1165/rcmb.2022-0445OC