scholarly journals Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kei Yamamoto ◽  
Haruko Miura ◽  
Motohiko Ishida ◽  
Yusuke Mii ◽  
Noriyuki Kinoshita ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Tissue Mechanics ◽  
Light Illumination ◽  
Contractile Ring ◽  
Cortical Tension ◽  
Regulatory Light Chains ◽  
Actomyosin Contractility ◽  
Nonmuscle Myosin Ii ◽  
Wide Range ◽  
Subcellular Level

AbstractActomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, subcellular dynamics of actomyosin contractility underlying such processes remains elusive. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility at the subcellular level. The system, named OptoMYPT, combines a protein phosphatase 1c (PP1c)-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination is sufficient to induce dephosphorylation of myosin regulatory light chains and a decrease in actomyosin contractile force in mammalian cells and Xenopus embryos. The OptoMYPT system is further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We find that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system provides opportunities to understand cellular and tissue mechanics.

scholarly journals Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis

2021 ◽  
Author(s):  
Kei Yamamoto ◽  
Haruko Miura ◽  
Motohiko Ishida ◽  
Satoshi Sawai ◽  
Yohei Kondo ◽  
...  
Keyword(s):  
Traction Force ◽  
Tissue Mechanics ◽  
Light Illumination ◽  
Type I ◽  
Contractile Ring ◽  
Cortical Tension ◽  
Regulatory Light Chains ◽  
Actomyosin Contractility ◽  
Nonmuscle Myosin Ii ◽  
Wide Range

Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, it is still unknown how actomyosin contractility generates force and maintains cellular morphology. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility. The system, named OptoMYPT, combines a catalytic subunit of the type I phosphatase-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination was sufficient to induce dephosphorylation of myosin regulatory light chains and decrease in traction force at the subcellular level. The OptoMYPT system was further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We found that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system will provide new opportunities to understand cellular and tissue mechanics.

scholarly journals Mechanical Forces during Lymph Node Expansion Govern Fibroblastic Reticular Network Remodeling

2021 ◽  
Author(s):  
Harry L Horsnell ◽  
Robert J Tetley ◽  
Henry de Belly ◽  
Spiros Makris ◽  
Agnesska C Benjamin ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Lymph Node ◽  
Adaptive Immune Response ◽  
Tissue Mechanics ◽  
Mechanical Forces ◽  
Cortical Tension ◽  
Lectin Receptor ◽  
Actomyosin Contractility ◽  
The Impact ◽  
Fibroblastic Stroma

After immunogenic challenge, the lymph node rapidly increases in cellularity making space for infiltrating and dividing lymphocytes, expanding the tissue. In the early phases of expansion, the underlying fibroblastic stroma, which organises the lymph node, undergoes elongation and stretching. This is followed by the initiation of fibroblastic stroma proliferation as inflammation proceeds. In the steady state, fibroblastic reticular cells (FRCs) tightly wrap an interconnected network of extracellular matrix fibres. The initial physical deformability of the lymph node is in part determined by Podoplanin (PDPN) signalling in FRCs and is modulated by dendritic cells expressing C-type lectin receptor 2 (CLEC-2). However, the mechanisms changing tissue and cellular mechanical forces of the fibroblastic stroma and the triggers for FRC proliferation and growth are unknown. We examined the contributions of FRC actomyosin contractility and extracellular matrix to lymph node tissue tension. Further, we directly tested the impact of tissue mechanics on the kinetics of lymph node expansion. We show using laser ablation that the FRC network is under mechanical tension generated by actomyosin contractility and that tension changes throughout the course of immunogenic challenge. We find that CLEC-2/PDPN signalling alters the cell intrinsic mechanical state of FRCs by reducing cortical tension and increasing FRC size. We found that FRC network tension is a critical cue in controlling lymph node expansion gating the initiation of FRC proliferation. Together this study demonstrates mechanical forces are generated and sensed through the FRC network to determine lymph node expansion required for an adaptive immune response.

Evaluation of the nucleopolyhedrovirus of Anticarsia gemmatalis as a vector for gene therapy in mammals

2020 ◽  
Vol 20 ◽  
Author(s):  
Cintia N. Parsza ◽  
Diego L. Mengual Gómez ◽  
Jorge Alejandro Simonin ◽  
Mariano Nicolás Belaich ◽  
Pablo Daniel Ghiringhelli
Keyword(s):  
Gene Therapy ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Autographa Californica ◽  
Anticarsia Gemmatalis ◽  
Agricultural Pests ◽  
Mammalian Cell Lines ◽  
Delivery Vector ◽  
Wide Range ◽  
Insect Pathogens

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV) has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals, because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Objective/Method: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative of non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

scholarly journals Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 481
Author(s):  
Gemma G. Martínez-García ◽  
Raúl F. Pérez ◽  
Álvaro F. Fernández ◽  
Sylvere Durand ◽  
Guido Kroemer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Mammalian Cells ◽  
Tissue Homeostasis ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Cardiac Damage ◽  
Wide Range ◽  
First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

scholarly journals Hybrid ZnO/MoS2 Core/Sheath Heterostructures for Photoelectrochemical Water Splitting

Applied Nano ◽  
2021 ◽  
Vol 2 (3) ◽  
pp. 148-161
Author(s):  
Katerina Govatsi ◽  
Aspasia Antonelou ◽  
Labrini Sygellou ◽  
Stylianos G. Neophytides ◽  
Spyros N. Yannopoulos
Keyword(s):  
Visible Light ◽  
Wide Band ◽  
Nanowire Arrays ◽  
Maximum Efficiency ◽  
Light Illumination ◽  
Wide Band Gap ◽  
Long Term Stability ◽  
Visible Light Illumination ◽  
Nanowire Surface ◽  
Wide Range

The rational synthesis of semiconducting materials with enhanced photoelectrocatalytic efficiency under visible light illumination is a long-standing issue. ZnO has been systematically explored in this field, as it offers the feasibility to grow a wide range of nanocrystal morphology; however, its wide band gap precludes visible light absorption. We report on a novel method for the controlled growth of semiconductor heterostructures and, in particular, core/sheath ZnO/MoS2 nanowire arrays and the evaluation of their photoelectrochemical efficiency in oxygen evolution reaction. ZnO nanowire arrays, with a narrow distribution of nanowire diameters, were grown on FTO substrates by chemical bath deposition. Layers of Mo metal at various thicknesses were sputtered on the nanowire surface, and the Mo layers were sulfurized at low temperature, providing in a controlled way few layers of MoS2, in the range from one to three monolayers. The heterostructures were characterized by electron microscopy (SEM, TEM) and spectroscopy (XPS, Raman, PL). The photoelectrochemical properties of the heterostructures were found to depend on the thickness of the pre-deposited Mo film, exhibiting maximum efficiency for moderate values of Mo film thickness. Long-term stability, in relation to similar heterostructures in the literature, has been observed.

scholarly journals A novel human Smad4 mutation is involved in papillary thyroid carcinoma progression

2011 ◽  
Vol 19 (1) ◽  
pp. 39-55 ◽  
Author(s):  
Sonia D'Inzeo ◽  
Arianna Nicolussi ◽  
Caterina Francesca Donini ◽  
Massimo Zani ◽  
Patrizia Mancini ◽  
...  
Keyword(s):  
Papillary Thyroid Carcinoma ◽  
Thyroid Carcinoma ◽  
Mammalian Cells ◽  
Transforming Growth Factor ◽  
Human Cancer ◽  
Papillary Thyroid ◽  
Tgfβ Signaling ◽  
Strong Reduction ◽  
Mesenchymal Transition ◽  
Wide Range

Smad proteins are the key effectors of the transforming growth factor β (TGFβ) signaling pathway in mammalian cells. Smad4 plays an important role in human physiology, and its mutations were found with high frequency in wide range of human cancer. In this study, we have functionally characterized Smad4 C324Y mutation, isolated from a nodal metastasis of papillary thyroid carcinoma. We demonstrated that the stable expression of Smad4 C324Y in FRTL-5 cells caused a significant activation of TGFβ signaling, responsible for the acquisition of transformed phenotype and invasive behavior. The coexpression of Smad4 C324Y with Smad4 wild-type determined an increase of homo-oligomerization of Smad4 with receptor-regulated Smads and a lengthening of nuclear localization. FRTL-5 clones overexpressing Smad4 C324Y showed a strong reduction of response to antiproliferative action of TGFβ1, acquired the ability to grow in anchorage-independent conditions, showed a fibroblast-like appearance and a strong reduction of the level of E-cadherin, one crucial event of the epithelial–mesenchymal transition process. The acquisition of a mesenchymal phenotype gave the characteristics of increased cellular motility and a significant reduction in adhesion to substrates such as fibronectin and laminin. Overall, our results demonstrate that the Smad4 C324Y mutation plays an important role in thyroid carcinogenesis and can be considered as a new prognostic and therapeutic target for thyroid cancer.

scholarly journals Adhesion-dependent and Contractile Ring-independent Equatorial Furrowing during Cytokinesis in Mammalian Cells

2005 ◽  
Vol 16 (8) ◽  
pp. 3865-3872 ◽  
Author(s):  
Masamitsu Kanada ◽  
Akira Nagasaki ◽  
Taro Q.P. Uyeda
Keyword(s):  
Mammalian Cells ◽  
Rat Kidney ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Cellular Slime Mold ◽  
Animal Cells ◽  
Contractile Ring ◽  
Normal Rat ◽  
Cellular Slime ◽  
Coated Surfaces

Myosin II-dependent contraction of the contractile ring drives equatorial furrowing during cytokinesis in animal cells. Nonetheless, myosin II-null cells of the cellular slime mold Dictyostelium divide efficiently when adhering to substrates by making use of polar traction forces. Here, we show that in the presence of 30 μM blebbistatin, a potent myosin II inhibitor, normal rat kidney (NRK) cells adhering to fibronectin-coated surfaces formed equatorial furrows and divided in a manner strikingly similar to myosin II-null Dictyostelium cells. Such blebbistatin-resistant cytokinesis was absent in partially detached NRK cells and was disrupted in adherent cells if the advance of their polar lamellipodia was disturbed by neighboring cells. Y-27632 (40 μM), which inhibits Rho-kinase, was similar to 30 μM blebbistatin in that it inhibited cytokinesis of partially detached NRK cells but only prolonged furrow ingression in attached cells. In the presence of 100 μM blebbistatin, most NRK cells that initiated anaphase formed tight furrows, although scission never occurred. Adherent HT1080 fibrosarcoma cells also formed equatorial furrows efficiently in the presence of 100 μM blebbistatin. These results provide direct evidence for adhesion-dependent, contractile ring-independent equatorial furrowing in mammalian cells and demonstrate the importance of substrate adhesion for cytokinesis.

Nocodazole and cytochalasin D induce tetraploidy in mammalian cells

1984 ◽  
Vol 246 (1) ◽  
pp. C154-C156 ◽  
Author(s):  
G. W. Zieve
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Cytochalasin D ◽  
Microtubule Assembly ◽  
Reversible Inhibitor ◽  
Cleavage Furrow ◽  
Synchronized Cells ◽  
Cells In Culture ◽  
Wide Range

Nocodazole, a rapidly reversible inhibitor of microtubule assembly is useful for preparing mammalian cells synchronized at all stages of mitosis. When synchronized cells are allowed to progress through mitosis in the presence of cytochalasin D, the cleavage furrow is inhibited and dikaryon cells are formed. These cells become homogeneous populations of stable mononuclear tetraploid cells after the following cell division. This procedure is applicable to a wide range of mammalian cells in culture.

scholarly journals Use of peroxiredoxin for preconditioning of heterotopic heart transplantation in a rat

2020 ◽  
Vol 22 (2) ◽  
pp. 158-164
Author(s):  
N. V. Grudinin ◽  
V. K. Bogdanov ◽  
M. G. Sharapov ◽  
N. S. Bunenkov ◽  
N. P. Mozheiko ◽  
...  
Keyword(s):  
Heart Transplantation ◽  
Reperfusion Injury ◽  
Mammalian Cells ◽  
Ischemia Reperfusion Injury ◽  
Isolated Heart ◽  
Ischemia Reperfusion ◽  
Signal Transmission ◽  
Redox Homeostasis ◽  
Heterotopic Heart Transplantation ◽  
Wide Range

Peroxiredoxin 6 (Prdx6) is an antioxidant enzyme in the human body that performs a number of important functions in the cell. Prdx6 restores a wide range of peroxide substrates, thus playing a leading role in maintaining redox homeostasis in mammalian cells. In addition to peroxidase activity, Prdx6 has an activity of phospholipase A2, thus taking part in membrane phospholipid metabolism. Due to its peroxidase and phospholipase activity, Prdx6 participates in intracellular and intercellular signal transmission, thereby facilitating the initiation of regenerative processes in the cell, suppression of apoptosis and activation of cell proliferation. Given the functions performed, Prdx6 can effectively deal with oxidative stress caused by various factors, including ischemia-reperfusion injury. On an animal model of rat heterotopic heart transplantation, we showed the cardioprotective potential of exogenous recombinant Prdx6, introduced before transplantation and subsequent reperfusion injury of the heart. It has been demonstrated that exogenous Prdx6 effectively alleviates the severity of ischemia-reperfusion injury of the heart by 2–3 times, providing normalization of its structural and functional state during heterotopic transplantation. The use of recombinant Prdx6 can be an effective approach in preventing/alleviating ischemia-reperfusion injury of the heart, as well as in maintaining an isolated heart during transplantation.

scholarly journals Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells

2002 ◽  
Vol 156 (1) ◽  
pp. 87-100 ◽  
Author(s):  
Toshiro Ohta ◽  
Russell Essner ◽  
Jung-Hwa Ryu ◽  
Robert E. Palazzo ◽  
Yumi Uetake ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Amino Acid Residues ◽  
Protein Overexpression ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Centrosomal Protein ◽  
Wide Range ◽  
Spindle Microtubules

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.

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