scholarly journals Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability

2017 ◽  
Author(s):  
Alyson S. Smith ◽  
Roberta B. Nowak ◽  
Sitong Zhou ◽  
Michael Giannetto ◽  
David S. Gokhin ◽  
...  
Keyword(s):  
Motor Activity ◽  
Myosin Ii ◽  
Control Cell ◽  
Cell Types ◽  
Membrane Skeleton ◽  
Membrane Curvature ◽  
General Function ◽  
Actin Network ◽  
Actin Networks ◽  
Cell Shapes

AbstractThe biconcave disc shape and deformability of mammalian red blood cells (RBCs) relies upon the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network in RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by super-resolution fluorescence microscopy. NMIIA association with the membrane skeleton is ATP-dependent, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical stability. In addition, the NMIIA heavy and light chains are phosphorylatedin vivoin RBCs, indicating active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disc cell shape. As structures similar to the RBC membrane skeleton are conserved in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.Significance statementThe biconcave disc shape and deformability of the mammalian RBC is vital to its circulatory function, relying upon a 2D viscoelastic spectrin-F-actin network attached to the membrane. A role for myosin II (NMII) contractility in generating tension in this network and controlling RBC shape has never been tested. We show that NMIIA forms phosphorylated bipolar filaments in RBCs, which associate with F-actin at the membrane. NMIIA motor activity is required for interactions with the spectrin-F-actin network, and regulates RBC biconcave shape and deformability. These results provide a novel mechanism for actomyosin force generation at the plasma membrane, and may be applicable to other cell types such as neurons and polarized epithelial cells with a spectrin-F-actin-based membrane skeleton.

scholarly journals Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability

2018 ◽  
Vol 115 (19) ◽  
pp. E4377-E4385 ◽  
Author(s):  
Alyson S. Smith ◽  
Roberta B. Nowak ◽  
Sitong Zhou ◽  
Michael Giannetto ◽  
David S. Gokhin ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Motor Activity ◽  
Control Cell ◽  
Cell Types ◽  
Membrane Skeleton ◽  
Membrane Curvature ◽  
General Function ◽  
Actin Networks ◽  
Cell Shapes

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin–F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin–F-actin networks.

scholarly journals Nanoscale organization of Actin Filaments in the Red Blood Cell Membrane Skeleton

2021 ◽  
Author(s):  
Roberta B. Nowak ◽  
Haleh Alimohamadi ◽  
Kersi Pestonjamasp ◽  
Padmini Rangamani ◽  
Velia M. Fowler
Keyword(s):  
Blood Cell ◽  
Single Molecule ◽  
Red Blood Cell ◽  
Synthetic Data ◽  
Membrane Skeleton ◽  
Membrane Curvature ◽  
Data Sets ◽  
Actin Network ◽  
Computational Cluster ◽  
Planar Network

AbstractRed blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes interconnected by long spectrin molecules at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modelling of forces controlling RBC shape, membrane curvature and deformation, yet the nanoscale organization of F-actin nodes in the network in situ is not understood. Here, we examined F-actin distribution in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence (TIRF) and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200-300 nm apart interspersed with dimmer F-actin staining regions. Live cell imaging reveals dynamic lateral movements, appearance and disappearance of F-actin foci. Single molecule STORM imaging and computational cluster analysis of experimental and synthetic data sets indicate that individual filaments are non-randomly distributed, with the majority as multiple filaments, and the remainder sparsely distributed as single filaments. These data indicate that F-actin nodes are non-uniformly distributed in the spectrin-F-actin network and necessitate reconsideration of current models of forces accounting for RBC shape and membrane deformability, predicated upon uniform distribution of F-actin nodes and associated proteins across the micron-scale RBC membrane.

scholarly journals Patterned cortical tension mediated by N-cadherin controls cell geometric order in the Drosophila eye

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Eunice HoYee Chan ◽  
Pruthvi Chavadimane Shivakumar ◽  
Raphaël Clément ◽  
Edith Laugier ◽  
Pierre-François Lenne
Keyword(s):  
Adhesion Molecules ◽  
Myosin Ii ◽  
Control Cell ◽  
Cortical Tension ◽  
Indirect Control ◽  
Drosophila Eye ◽  
Cell Patterns ◽  
Cell Shapes ◽  
Contractile Forces

Adhesion molecules hold cells together but also couple cell membranes to a contractile actomyosin network, which limits the expansion of cell contacts. Despite their fundamental role in tissue morphogenesis and tissue homeostasis, how adhesion molecules control cell shapes and cell patterns in tissues remains unclear. Here we address this question in vivo using the Drosophila eye. We show that cone cell shapes depend little on adhesion bonds and mostly on contractile forces. However, N-cadherin has an indirect control on cell shape. At homotypic contacts, junctional N-cadherin bonds downregulate Myosin-II contractility. At heterotypic contacts with E-cadherin, unbound N-cadherin induces an asymmetric accumulation of Myosin-II, which leads to a highly contractile cell interface. Such differential regulation of contractility is essential for morphogenesis as loss of N-cadherin disrupts cell rearrangements. Our results establish a quantitative link between adhesion and contractility and reveal an unprecedented role of N-cadherin on cell shapes and cell arrangements.

scholarly journals Dynamic actin crosslinking governs the cytoplasm’s transition to fluid-like behavior

2019 ◽  
Author(s):  
Loïc Chaubet ◽  
Abdullah R. Chaudhary ◽  
Hossein K. Heris ◽  
Allen J. Ehrlicher ◽  
Adam G. Hendricks
Keyword(s):  
Viscoelastic Properties ◽  
Myosin Ii ◽  
Optical Trap ◽  
Living Cells ◽  
Actin Network ◽  
Wild Type ◽  
Actin Networks ◽  
Long Timescales

AbstractCells precisely control their mechanical properties to organize and differentiate into tissues. The architecture and connectivity of cytoskeletal filaments changes in response to mechanical and biochemical cues, allowing the cell to rapidly tune its mechanics from highly-crosslinked, elastic networks to weakly-crosslinked viscous networks. While the role of actin crosslinking in controlling actin network mechanics is well-characterized in purified actin networks, its mechanical role in the cytoplasm of living cells remains unknown. Here, we probe the frequency-dependent intracellular viscoelastic properties of living cells using multifrequency excitation and in situ optical trap calibration. At long timescales in the intracellular environment, we observe that the cytoskeleton becomes fluid-like. The mechanics are well-captured by a model in which actin filaments are dynamically connected by a single dominant crosslinker. A disease-causing point mutation (K255E) of the actin crosslinker α-actinin 4 (ACTN4) causes its binding kinetics to be insensitive to tension. Under normal conditions, the viscoelastic properties of wild type (WT) and K255E+/- cells are similar. However, when tension is reduced through myosin II inhibition, WT cells relax 3x faster to the fluid-like regime while K255E+/- cells are not affected. These results indicate that dynamic actin crosslinking enables the cytoplasm to flow at long timescales.

Nanoscale Dynamics of Actin Filaments in the Red Blood Cell Membrane Skeleton

2022 ◽  
Author(s):  
Roberta B. Nowak ◽  
Haleh Alimohamadi ◽  
Kersi Pestonjamasp ◽  
Padmini Rangamani ◽  
Velia M. Fowler
Keyword(s):  
Blood Cell ◽  
Single Molecule ◽  
Red Blood Cell ◽  
Actin Polymerization ◽  
Network Connectivity ◽  
Membrane Skeleton ◽  
Membrane Curvature ◽  
Actin Network ◽  
Node Distribution ◽  
Planar Network

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15-18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ is not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence (TIRF) and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200-300 nm apart interspersed with dimmer F-actin staining regions. Single molecule STORM imaging of Alexa-647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, non-random distribution of F-actin nodes. Treatment of RBCs with LatA and CytoD indicates F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape and membrane deformability.

scholarly journals Non-uniform distribution of myosin-mediated forces governs red blood cell membrane curvature through tension modulation

2019 ◽  
Author(s):  
Haleh Alimohamadi ◽  
Alyson S. Smith ◽  
Roberta B. Nowak ◽  
Velia M. Fowler ◽  
Padmini Rangamani
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Contractile Activity ◽  
Cell Types ◽  
Membrane Skeleton ◽  
Membrane Curvature ◽  
Force Density ◽  
Additional Contribution ◽  
Rbc Membrane ◽  
Applied Forces

AbstractThe biconcave disk shape of the mammalian red blood cell (RBC) is unique to the RBC and is vital for its circulatory function. Due to the absence of a transcellular cytoskeleton, RBC shape is determined by the membrane skeleton, a network of actin filaments cross-linked by spectrin and attached to membrane proteins. While the physical properties of a uniformly distributed actin network interacting with the lipid bilayer membrane have been assumed to control RBC shape, recent experiments reveal that RBC biconcave shape also depends on the contractile activity of nonmuscle myosin IIA (NMIIA) motor proteins. Here, we use the classical Helfrich-Canham model for the RBC membrane to test the role of heterogeneous force distributions along the membrane and mimic the contractile activity of sparsely distributed NMIIA filaments. By incorporating this additional contribution to the Helfrich-Canham energy, we find that the RBC biconcave shape depends on the ratio of forces per unit volume in the dimple and rim regions of the RBC. Experimental measurements of NMIIA densities at the dimple and rim validate our prediction that (a) membrane forces must be non-uniform along the RBC membrane and (b) the force density must be larger in the dimple than the rim to produce the observed membrane curvatures. Furthermore, we predict that RBC membrane tension and the orientation of the applied forces play important roles in regulating this force-shape landscape. Our findings of heterogeneous force distributions on the plasma membrane for RBC shape maintenance may also have implications for shape maintenance in different cell types.

scholarly journals Regulation of thickness of actomyosin cortex in well-spread cells by contractility and spread area

2017 ◽  
Author(s):  
Rinku Kumar ◽  
Bidisha Sinha
Keyword(s):  
Motor Activity ◽  
Total Internal Reflection ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Actin Network ◽  
Area Reduction ◽  
Interphase Cells ◽  
Spread Area ◽  
Actomyosin Cytoskeleton ◽  
Growing Population

AbstractThe contractile cortical actomyosin cytoskeleton (or cortex) in interphase cells confers rigidity to cells, but also lead to shape dynamics. Regulation of its thickness, although well studied in rounded cells, is less explored in well-spread cells. In this paper, we quantify the variations in thickness and study the contribution of actin polymerization, myosin II activity and spread area of cells. We report an increase in cortex thickness and its variations on disrupting actin network by actin depolymerizing agents or reducing contractility by inhibiting motor activity of myosin II. On spread area reduction by substrate micropatterning, we find reduced cell volume and increased mean & variability of thickness. To validate, we follow cells through de-adhesion with EDTA. The thickness of cortex increases (and oscillates) while the volume of cells reduces with 5-15 mins timescales. Moreover, total internal reflection fluorescence (TIRF) imaging reveals stress fibre dissolution and events of their buckling along with a growing population of micron-sized mobile filaments. We believe that the cytoskeleton responds to the loss of adhesion by contracting and fragmenting, hence leading to cortex thickening. Limiting volume reduction does not suppress cortex thickening on de-adhesion, suggesting that decreased traction stress may be primarily responsible for the cortex thickening.

scholarly journals Analysis of the local organization and dynamics of cellular actin networks

2013 ◽  
Vol 202 (7) ◽  
pp. 1057-1073 ◽  
Author(s):  
Weiwei Luo ◽  
Cheng-han Yu ◽  
Zi Zhao Lieu ◽  
Jun Allard ◽  
Alex Mogilner ◽  
...  
Keyword(s):  
Myosin Ii ◽  
Spatial Arrangement ◽  
Filamin A ◽  
Actin Network ◽  
Drift Diffusion ◽  
Superresolution Microscopy ◽  
Actin Networks ◽  
Latrunculin A ◽  
Diffusion Motion ◽  
Network Patterns

A ctin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm.

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