scholarly journals Myosin II isoforms promote internalization of spatially distinct clathrin-independent endocytosis cargoes through modulation of cortical tension downstream of ROCK2

2020 ◽  
pp. mbc.E20-07-0480
Author(s):  
Jessica Wayt ◽  
Alexander Cartagena-Rivera ◽  
Dipannita Dutta ◽  
Julie G. Donaldson ◽  
Clare M. Waterman
Keyword(s):  
Myosin Ii ◽  
Apical Cell ◽  
Cell Junctions ◽  
Intestinal Epithelial ◽  
Cortical Tension ◽  
Force Microscopy ◽  
Atomic Force ◽  
Caco2 Cells ◽  
Myosin Iia ◽  
Actomyosin Cytoskeleton

Although the actomyosin cytoskeleton has been implicated in clathrin-mediated endocytosis, a clear requirement for actomyosin in clathrin-independent endocytosis (CIE) has not been demonstrated. We discovered that the Rho-associated kinase ROCK2 is required for CIE of MHCI and CD59 through promotion of myosin II activity. Myosin IIA promoted internalization of MHCI and myosin IIB drove CD59 uptake in both HeLa and polarized Caco2 intestinal epithelial cells. In Caco2 cells, myosin IIA localized to the basal cortex and apical brush border and mediated MHCI internalization from the basolateral domain, while myosin IIB localized at the basal cortex and apical cell-cell junctions and promoted CD59 uptake from the apical membrane. Atomic force microscopy demonstrated that myosin IIB mediated apical epithelial tension in Caco2 cells. Thus, specific cargoes are internalized by ROCK2-mediated activation of myosin II isoforms to mediate spatial regulation of CIE, possibly by modulation of local cortical tension.

scholarly journals Nanomechanical properties of enucleated cells: contribution of the nucleus to the passive cell mechanics

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Yuri M. Efremov ◽  
Svetlana L. Kotova ◽  
Anastasia A. Akovantseva ◽  
Peter S. Timashev
Keyword(s):  
Mechanical Properties ◽  
Cell Mechanics ◽  
Normal Cells ◽  
Force Microscopy ◽  
Nanomechanical Properties ◽  
Atomic Force ◽  
Fibrosarcoma Cells ◽  
Actomyosin Cytoskeleton ◽  
Small Deformations

Abstract Background The nucleus, besides its functions in the gene maintenance and regulation, plays a significant role in the cell mechanosensitivity and mechanotransduction. It is the largest cellular organelle that is often considered as the stiffest cell part as well. Interestingly, the previous studies have revealed that the nucleus might be dispensable for some of the cell properties, like polarization and 1D and 2D migration. Here, we studied how the nanomechanical properties of cells, as measured using nanomechanical mapping by atomic force microscopy (AFM), were affected by the removal of the nucleus. Methods The mass enucleation procedure was employed to obtain cytoplasts (enucleated cells) and nucleoplasts (nuclei surrounded by plasma membrane) of two cell lines, REF52 fibroblasts and HT1080 fibrosarcoma cells. High-resolution viscoelastic mapping by AFM was performed to compare the mechanical properties of normal cells, cytoplasts, and nucleoplast. The absence or presence of the nucleus was confirmed with fluorescence microscopy, and the actin cytoskeleton structure was assessed with confocal microscopy. Results Surprisingly, we did not find the softening of cytoplasts relative to normal cells, and even some degree of stiffening was discovered. Nucleoplasts, as well as the nuclei isolated from cells using a detergent, were substantially softer than both the cytoplasts and normal cells. Conclusions The cell can maintain its mechanical properties without the nucleus. Together, the obtained data indicate the dominating role of the actomyosin cytoskeleton over the nucleus in the cell mechanics at small deformations inflicted by AFM.

Improved unroofing protocols for cryo-electron microscopy, atomic force microscopy and freeze-etching electron microscopy and the associated mechanisms

Microscopy ◽  
2020 ◽  
Vol 69 (6) ◽  
pp. 350-359
Author(s):  
Nobuhiro Morone ◽  
Eiji Usukura ◽  
Akihiro Narita ◽  
Jiro Usukura
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Cell Membrane ◽  
Apical Cell ◽  
Force Microscopy ◽  
Atomic Force ◽  
Cryo Electron Microscopy ◽  
Cytoplasmic Surface ◽  
A Cell ◽  
Freeze Etching

Abstract Unroofing, which is the mechanical shearing of a cell to expose the cytoplasmic surface of the cell membrane, is a unique preparation method that allows membrane cytoskeletons to be observed by cryo-electron microscopy, atomic force microscopy, freeze-etching electron microscopy and other methods. Ultrasound and adhesion have been known to mechanically unroof cells. In this study, unroofing using these two means was denoted sonication unroofing and adhesion unroofing, respectively. We clarified the mechanisms by which cell membranes are removed in these unroofing procedures and established efficient protocols for each based on the mechanisms. In sonication unroofing, fine bubbles generated by sonication adhered electrostatically to apical cell surfaces and then removed the apical (dorsal) cell membrane with the assistance of buoyancy and water flow. The cytoplasmic surface of the ventral cell membrane remaining on the grids became observable by this method. In adhesion unroofing, grids charged positively by coating with Alcian blue were pressed onto the cells, thereby tightly adsorbing the dorsal cell membrane. Subsequently, a part of the cell membrane strongly adhered to the grids was peeled from the cells and transferred onto the grids when the grids were lifted. This method thus allowed the visualization of the cytoplasmic surface of the dorsal cell membrane. This paper describes robust, improved protocols for the two unroofing methods in detail. In addition, micro-unroofing (perforation) likely due to nanobubbles is introduced as a new method to make cells transparent to electron beams.

scholarly journals Stiffness and heterogeneity of the pulmonary endothelial glycocalyx measured by atomic force microscopy

2011 ◽  
Vol 301 (3) ◽  
pp. L353-L360 ◽  
Author(s):  
Ryan O'Callaghan ◽  
Kathleen M. Job ◽  
Randal O. Dull ◽  
Vladimir Hlady
Keyword(s):  
Atomic Force Microscopy ◽  
Elastic Modulus ◽  
Indentation Depth ◽  
Cell Junctions ◽  
Endothelial Glycocalyx ◽  
Cell Junction ◽  
Microvascular Endothelial Cell ◽  
Cell Contact ◽  
Force Microscopy ◽  
Atomic Force

The mechanical properties of endothelial glycocalyx were studied using atomic force microscopy with a silica bead (diameter ∼18 μm) serving as an indenter. Even at indentations of several hundred nanometers, the bead exerted very low compressive pressures on the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx and allowed for an averaging of stiffness in the bead-cell contact area. The elastic modulus of BLMVEC glycocalyx was determined as a pointwise function of the indentation depth before and after enzymatic degradation of specific glycocalyx components. The modulus-indentation depth profiles showed the cells becoming progressively stiffer with increased indentation. Three different enzymes were used: heparinases III and I and hyaluronidase. The main effects of heparinase III and hyaluronidase enzymes were that the elastic modulus in the cell junction regions increased more rapidly with the indentation than in BLMVEC controls, and that the effective thickness of glycocalyx was reduced. Cytochalasin D abolished the modulus increase with the indentation. The confocal profiling of heparan sulfate and hyaluronan with atomic force microscopy indentation data demonstrated marked heterogeneity of the glycocalyx composition between cell junctions and nuclear regions.

Role for stress fiber contraction in surface tension development and stretch-activated channel regulation in C2C12 myoblasts

2008 ◽  
Vol 295 (1) ◽  
pp. C160-C172 ◽  
Author(s):  
Francesca Sbrana ◽  
Chiara Sassoli ◽  
Elisabetta Meacci ◽  
Daniele Nosi ◽  
Roberta Squecco ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Myosin Ii ◽  
Cell Stiffness ◽  
C2c12 Myoblasts ◽  
Tension Development ◽  
Force Microscopy ◽  
Atomic Force ◽  
Sphingosine 1 Phosphate ◽  
Membrane Stretching

Membrane-cytoskeleton interaction regulates transmembrane currents through stretch-activated channels (SACs); however, the mechanisms involved have not been tested in living cells. We combined atomic force microscopy, confocal immunofluorescence, and patch-clamp analysis to show that stress fibers (SFs) in C2C12 myoblasts behave as cables that, tensed by myosin II motor, activate SACs by modifying the topography and the viscoelastic (Young's modulus and hysteresis) and electrical passive (membrane capacitance, Cm) properties of the cell surface. Stimulation with sphingosine 1-phosphate to elicit SF formation, the inhibition of Rho-dependent SF formation by Y-27632 and of myosin II-driven SF contraction by blebbistatin, showed that not SF polymerization alone but the generation of tensional forces by SF contraction were involved in the stiffness response of the cell surface. Notably, this event was associated with a significant reduction in the amplitude of the cytoskeleton-mediated corrugations in the cell surface topography, suggesting a contribution of SF contraction to plasma membrane stretching. Moreover, Cm, used as an index of cell surface area, showed a linear inverse relationship with cell stiffness, indicating participation of the actin cytoskeleton in plasma membrane remodeling and the ability of SF formation to cause internalization of plasma membrane patches to reduce Cm and increase membrane tension. SF contraction also increased hysteresis. Together, these data provide the first experimental evidence for a crucial role of SF contraction in SAC activation. The related changes in cell viscosity may prevent SAC from abnormal activation.

Millisecond Conformational Dynamics of Skeletal Myosin II Power Stroke Studied by High-Speed Atomic Force Microscopy

ACS Nano ◽  
2020 ◽  
Author(s):  
Oleg S. Matusovsky ◽  
Noriyuki Kodera ◽  
Caitlin MacEachen ◽  
Toshio Ando ◽  
Yu-Shu Cheng ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
High Speed ◽  
Myosin Ii ◽  
Conformational Dynamics ◽  
Power Stroke ◽  
Force Microscopy ◽  
Atomic Force ◽  
Skeletal Myosin

scholarly journals Biomechanical and biochemical assessment of YB-1 expression in melanoma cells

2021 ◽  
Author(s):  
Anna Magdalena Cykowska ◽  
Ulf Krister Hofmann ◽  
Aadhya Tiwari ◽  
Corinna Kosnopfel ◽  
Rosa Riester ◽  
...  
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Proteome Analysis ◽  
Melanoma Cells ◽  
Cell Stiffness ◽  
Knock Out ◽  
Mesenchymal Transition ◽  
Force Microscopy ◽  
Atomic Force ◽  
Rearrangement Process ◽  
Actomyosin Cytoskeleton

Malignant melanoma is the most lethal form of skin cancer; its incidence has increased over the last five decades. Y-box binding protein 1 (YB-1) plays a prominent role in mediating metastatic behavior by promoting epithelial-to-mesenchymal transition (EMT) processes. Migratory melanoma cells exhibit two major phenotypes: elongated mesenchymal or rounded amoeboid. The actomyosin cytoskeleton is key in both phenotypes, but intermediate filaments also undergo a significant rearrangement process, switching from cytokeratin-rich to vimentin and nestin-rich network. In this study, we aimed to investigate to what extent YB-1 impacts the biomechanical (cell stiffness) and biochemical aspects of melanoma cells and their cytoskeleton. To this end, we subjected A375 YB-1 knock-out and parental cells to atomic force microscopy investigations (stiffness determination), immunolabelling, and proteome analysis. We found that YB-1 expressing cells were significantly stiffer compared to the corresponding YB-1 knock-out cell line. Proteomic analysis revealed that expression of YB-1 results in a strong co-expression of nestin, vimentin, fascin-1, and septin-9. In the YB-1 knock-out nestin was completely depleted, but zyxin was strongly upregulated. Collectively, our results showed that YB-1 knock-out acquires some characteristics of mesenchymal phenotype but lacks important markers of malignancy and invasiveness such as nestin or vimentin. We posit that there is an association of YB-1 expression with an amoeboid phenotype, which would explain the increased migratory capacity.

scholarly journals Cytoskeleton remodelling of confluent epithelial cells cultured on porous substrates

2015 ◽  
Vol 12 (103) ◽  
pp. 20141057 ◽  
Author(s):  
Jan Rother ◽  
Matthias Büchsenschütz-Göbeler ◽  
Helen Nöding ◽  
Siegfried Steltenkamp ◽  
Konrad Samwer ◽  
...  
Keyword(s):  
Pore Size ◽  
Imaging Techniques ◽  
Microscopy Imaging ◽  
Cortical Tension ◽  
Force Microscopy ◽  
Atomic Force ◽  
Force Volume ◽  
The Impact ◽  
Cells Cultured ◽  
Porous Substrates

The impact of substrate topography on the morphological and mechanical properties of confluent MDCK-II cells cultured on porous substrates was scrutinized by means of various imaging techniques as well as atomic force microscopy comprising force volume and microrheology measurements. Regardless of the pore size, ranging from 450 to 5500 nm in diameter, cells were able to span the pores. They did not crawl into the holes or grow around the pores. Generally, we found that cells cultured on non-porous surfaces are stiffer, i.e. cortical tension rises from 0.1 to 0.3 mN m −1 , and less fluid than cells grown over pores. The mechanical data are corroborated by electron microscopy imaging showing more cytoskeletal filaments on flat samples in comparison to porous ones. By contrast, cellular compliance increases with pore size and cells display a more fluid-like behaviour on larger pores. Interestingly, cells on pores larger than 3500 nm produce thick actin bundles that bridge the pores and thereby strengthen the contact zone of the cells.

Mapping specific adhesive interactions on living human intestinal epithelial cells with atomic force microscopy

2008 ◽  
Vol 22 (7) ◽  
pp. 2331-2339 ◽  
Author(s):  
A. Patrick Gunning ◽  
Stephen Chambers ◽  
Carmen Pin ◽  
Angela L. Man ◽  
Victor J. Morris ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Force Microscopy ◽  
Atomic Force ◽  
Human Intestinal Epithelial Cells ◽  
Adhesive Interactions

scholarly journals In vitro single-cell dissection revealing the interior structure of cable bacteria

2018 ◽  
Vol 115 (34) ◽  
pp. 8517-8522 ◽  
Author(s):  
Zaixing Jiang ◽  
Shuai Zhang ◽  
Lasse Hyldgaard Klausen ◽  
Jie Song ◽  
Qiang Li ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Long Range ◽  
Cell Junctions ◽  
Structural Basis ◽  
Bacterial Cells ◽  
Force Microscopy ◽  
Atomic Force ◽  
The Individual ◽  
Cell Cell

Filamentous Desulfobulbaceae bacteria were recently discovered as long-range transporters of electrons from sulfide to oxygen in marine sediments. The long-range electron transfer through these cable bacteria has created considerable interests, but it has also raised many questions, such as what structural basis will be required to enable micrometer-sized cells to build into centimeter-long continuous filaments? Here we dissected cable bacteria cells in vitro by atomic force microscopy and further explored the interior, which is normally hidden behind the outer membrane. Using nanoscale topographical and mechanical maps, different types of bacterial cell–cell junctions and strings along the cable length were identified. More important, these strings were found to be continuous along the bacterial cells passing through the cell–cell junctions. This indicates that the strings serve an important function in maintaining integrity of individual cable bacteria cells as a united filament. Furthermore, ridges in the outer membrane are found to envelop the individual strings at cell–cell junctions, and they are proposed to strengthen the junctions. Finally, we propose a model for the division and growth of the cable bacteria, which illustrate the possible structural requirements for the formation of centimeter-length filaments in the recently discovered cable bacteria.

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