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 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 Active cargo positioning in antiparallel transport networks

2019 ◽  
Author(s):  
Mathieu Richard ◽  
Carles Blanch-Mercader ◽  
Hajer Ennomani ◽  
Wenxiang Cao ◽  
Enrique M. De La Cruz ◽  
...  
Keyword(s):  
Transport Properties ◽  
Molecular Motors ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Actin Network ◽  
Cargo Transport ◽  
Stochastic Transport ◽  
Mixed Polarity ◽  
Micro Patterns

ABSTRACTCytoskeletal filaments assemble into dense parallel, antiparallel or disordered networks, providing a complex environment for active cargo transport and positioning by molecular motors. The interplay between the network architecture and intrinsic motor properties clearly affects transport properties but remains poorly understood. Here, by using surface micro-patterns of actin polymerization, we investigate stochastic transport properties of colloidal beads in antiparallel networks of overlapping actin filaments. We found that 200-nm beads coated with myosin-Va motors displayed directed movements towards positions where the net polarity of the actin network vanished, accumulating there. The bead distribution was dictated by the spatial profiles of local bead velocity and diffusion coefficient, indicating that a diffusion-drift process was at work. Remarkably, beads coated with heavy mero-myosin-II motors showed a similar behavior. However, although velocity gradients were steeper with myosin II, the much larger bead diffusion observed with this motor resulted in less precise positioning. Our observations are well described by a three-state model, in which active beads locally sense the net polarity of the network by frequently detaching from and reattaching to the filaments. A stochastic sequence of processive runs and diffusive searches results in a biased random walk. The precision of bead positioning is set by the gradient of net actin polarity in the network and by the run length of the cargo in an attached state. Our results unveiled physical rules for cargo transport and positioning in networks of mixed polarity.Significance statementCellular functions rely on small groups of molecular motors to transport their cargoes throughout the cell along polar filaments of the cytoskeleton. Cytoskeletal filaments self-assemble into dense networks comprising intersections and filaments of mixed polarity, challenging directed motor-based transport. Using micro-patterns of actin polymerization in-vitro, we investigated stochastic transport of colloidal beads in antiparallel networks of overlapping actin filaments. We found that beads coated with myosin motors sensed the net polarity of the actin network, resulting in active bead positioning to regions of neutral polarity with a precision depending on the motor type. A theoretical description of our experimental results provides the key physical rules for cargo transport and positioning in filament networks of mixed polarity.

scholarly journals Novel Myosin Heavy Chain Kinase Involved in Disassembly of Myosin II Filaments and Efficient Cleavage in MitoticDictyostelium Cells

2002 ◽  
Vol 13 (12) ◽  
pp. 4333-4342 ◽  
Author(s):  
Akira Nagasaki ◽  
Go Itoh ◽  
Shigehiko Yumura ◽  
Taro Q.P. Uyeda
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Myosin Ii ◽  
Kinase Domain ◽  
Cleavage Furrow ◽  
Wild Type ◽  
Daughter Cells ◽  
Cleavage Process ◽  
Interphase Cells ◽  
Myosin Heavy Chain Kinase

We have cloned a full-length cDNA encoding a novel myosin II heavy chain kinase (mhckC) from Dictyostelium. Like other members of the myosin heavy chain kinase family, themhckC gene product, MHCK C, has a kinase domain in its N-terminal half and six WD repeats in the C-terminal half. GFP-MHCK C fusion protein localized to the cortex of interphase cells, to the cleavage furrow of mitotic cells, and to the posterior of migrating cells. These distributions of GFP-MHCK C always corresponded with that of myosin II filaments and were not observed in myosin II-null cells, where GFP-MHCK C was diffusely distributed in the cytoplasm. Thus, localization of MHCK C seems to be myosin II-dependent. Cells lacking the mhckC gene exhibited excessive aggregation of myosin II filaments in the cleavage furrows and in the posteriors of the daughter cells once cleavage was complete. The cleavage process of these cells took longer than that of wild-type cells. Taken together, these findings suggest MHCK C drives the disassembly of myosin II filaments for efficient cytokinesis and recycling of myosin II that occurs during cytokinesis.

scholarly journals Contribution of Filopodia to Cell Migration: A Mechanical Link between Protrusion and Contraction

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Fei Xue ◽  
Deanna M. Janzen ◽  
David A. Knecht
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Temporal Distribution ◽  
Leading Edge ◽  
Distal Portion ◽  
Actin Binding ◽  
Actin Networks ◽  
Motile Cells ◽  
A Cell

Numerous F-actin containing structures are involved in regulating protrusion of membrane at the leading edge of motile cells. We have investigated the structure and dynamics of filopodia as they relate to events at the leading edge and the function of the trailing actin networks. We have found that although filopodia contain parallel bundles of actin, they contain a surprisingly nonuniform spatial and temporal distribution of actin binding proteins. Along the length of the actin filaments in a single filopodium, the most distal portion contains primarily T-plastin, while the proximal portion is primarily bound byα-actinin and coronin. Some filopodia are stationary, but lateral filopodia move with respect to the leading edge. They appear to form a mechanical link between the actin polymerization network at the front of the cell and the myosin motor activity in the cell body. The direction of lateral filopodial movement is associated with the direction of cell migration. When lateral filopodia initiate from and move toward only one side of a cell, the cell will turn opposite to the direction of filopodial flow. Therefore, this filopodia-myosin II system allows actin polymerization driven protrusion forces and myosin II mediated contractile force to be mechanically coordinated.

scholarly journals Myosin-II activity generates a dynamic steady state with continuous actin turnover in a minimal actin cortex

2018 ◽  
Author(s):  
Sonal ◽  
Kristina A. Ganzinger ◽  
Sven K. Vogel ◽  
Jonas Mücksch ◽  
Philipp Blumhardt ◽  
...  
Keyword(s):  
Steady State ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Fine Tuning ◽  
Cellular Functions ◽  
Actin Cortex ◽  
Actin Turnover ◽  
Cytoskeletal Dynamics

ABSTRACTDynamic reorganization of the actomyosin cytoskeleton allows a fine-tuning of cell shape that is vital to many cellular functions. It is well established that myosin-II motors generate the forces required for remodeling the cell surface by imparting contractility to actin networks. An additional, less understood, role of myosin-II in cytoskeletal dynamics is believed to be in the regulation of actin turnover; it has been proposed that myosin activity increases actin turnover in various cellular contexts, presumably by contributing to disassembly. In vitro reconstitution of actomyosin networks has confirmed the role of myosin in actin network disassembly, but factors such as diffusional constraints and the use of stabilized filaments have thus far limited the observation of myosin-assisted actin turnover in these networks. Here, we present the reconstitution of a minimal dynamic actin cortex where actin polymerization is catalyzed on the membrane in the presence of myosin-II activity. We demonstrate that myosin activity leads to disassembly and redistribution in this simplified cortex. Consequently, a new dynamic steady state emerges in which actin filaments undergo constant turnover. Our findings suggest a multi-faceted role of myosin-II in fast remodeling of the eukaryotic actin cortex.

scholarly journals The Myosin IXb Motor Activity Targets the Myosin IXb RhoGAP Domain as Cargo to Sites of Actin Polymerization

2007 ◽  
Vol 18 (4) ◽  
pp. 1507-1518 ◽  
Author(s):  
Frank van den Boom ◽  
Heiko Düssmann ◽  
Katharina Uhlenbrock ◽  
Marouan Abouhamed ◽  
Martin Bähler
Keyword(s):  
Motor Activity ◽  
Actin Polymerization ◽  
Fluorescence Recovery After Photobleaching ◽  
Rho Gtpase ◽  
Tail Region ◽  
Lysine Residues ◽  
Motor Region ◽  
Necessary And Sufficient ◽  
Loop 2

Myosin IXb (Myo9b) is a single-headed processive myosin that exhibits Rho GTPase-activating protein (RhoGAP) activity in its tail region. Using live cell imaging, we determined that Myo9b is recruited to extending lamellipodia, ruffles, and filopodia, the regions of active actin polymerization. A functional motor domain was both necessary and sufficient for targeting Myo9b to these regions. The head domains of class IX myosins comprise a large insertion in loop2. Deletion of the large Myo9b head loop 2 insertion abrogated the enrichment in extending lamellipodia and ruffles, but enhanced significantly the enrichment at the tips of filopodia and retraction fibers. The enrichment in the tips of filopodia and retraction fibers depended on four lysine residues C-terminal to the loop 2 insertion and the tail region. Fluorescence recovery after photobleaching and photoactivation experiments in lamellipodia revealed that the dynamics of Myo9b was comparable to that of actin. The exchange rates depended on the Myo9b motor region and motor activity, and they were also dependent on the turnover of F-actin. These results demonstrate that Myo9b functions as a motorized RhoGAP molecule in regions of actin polymerization and identify Myo9b head sequences important for in vivo motor properties.

scholarly journals A Minus-End–directed Kinesin with Plus-End Tracking Protein Activity Is Involved in Spindle Morphogenesis

2005 ◽  
Vol 16 (4) ◽  
pp. 1584-1592 ◽  
Author(s):  
J. Christian Ambrose ◽  
Wuxing Li ◽  
Adam Marcus ◽  
Hong Ma ◽  
Richard Cyr
Keyword(s):  
Motor Activity ◽  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Null Mutant ◽  
Mitotic Spindles ◽  
Protein Activity ◽  
Interphase Cells ◽  
Motor Activities ◽  
Spindle Function

Diverse kinesin motor proteins are involved in spindle function; however, the mechanisms by which they are targeted to specific sites within spindles are not well understood. Here, we show that a fusion between yellow fluorescent protein (YFP) and a minus-end–directed Kinesin-14 (C-terminal family) from Arabidopsis, ATK5, localizes to mitotic spindle midzones and regions rich in growing plus-ends within phragmoplasts. Notably, in Arabidopsis interphase cells, YFP::ATK5 localizes to microtubules with a preferential enrichment at growing plus-ends; indicating ATK5 is a plus-end tracking protein (+TIP). This +TIP activity is conferred by regions outside of the C-terminal motor domain, which reveals the presence of independent plus-end tracking and minus-end motor activities within ATK5. Furthermore, mitotic spindles of atk5 null mutant plants are abnormally broadened. Based on these data, we propose a model in which ATK5 uses plus-end tracking to reach spindle midzones, where it then organizes microtubules via minus-end–directed motor activity.

Formation of neutrophil extracellular traps requires actin cytoskeleton rearrangements

Blood ◽  
2022 ◽  
Author(s):  
Evelien G.G. Sprenkeler ◽  
Anton T.J. Tool ◽  
Stefanie Henriet ◽  
Robin van Bruggen ◽  
Taco W. Kuijpers
Keyword(s):  
Actin Polymerization ◽  
Nuclear Dna ◽  
Myosin Ii ◽  
Neutrophil Extracellular Traps ◽  
Actin Dynamics ◽  
Polymerization Process ◽  
Effector Cells ◽  
Extracellular Traps ◽  
Cytoskeleton Rearrangements ◽  
Cellular Components

Neutrophils are important effector cells in the host defense against invading micro-organisms. One of the mechanisms they employ to eliminate pathogens is the release of neutrophil extracellular traps (NETs). Although NET release and subsequent cell death known as NETosis have been intensively studied, the cellular components and factors determining or facilitating the formation of NETs remain incompletely understood. Using various actin polymerization and myosin II modulators on neutrophils from healthy individuals, we show that intact F-actin dynamics and myosin II function are essential for NET formation when induced by different stimuli, i.e. phorbol 12-myristate 13-acetate, monosodium urate crystals and Candida albicans. The role of actin polymerization in NET formation could not be explained by the lack of reactive oxygen species production or granule release, which were normal or enhanced under the given conditions. Neutrophils from patients with very rare inherited actin polymerization defects by either ARPC1B- or MKL1-deficiency also failed to show NETosis. We found that upon inhibition of actin dynamics there is a lack of translocation of NE to the nucleus, which may well explain the impaired NET formation. Collectively, our data illustrate the essential requirement of an intact and active actin polymerization process, as well as active myosin II to enable the release of nuclear DNA by neutrophils during NET formation.

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