scholarly journals Homophilic and heterophilic cadherin bond rupture forces in homo- or hetero-cellular systems measured by AFM based SCFS

2020 ◽  
Author(s):  
Prem Kumar Viji Babu ◽  
Ursula Mirastschijski ◽  
Gazanfer Belge ◽  
Manfred Radmacher
Keyword(s):  
Single Molecule ◽  
Actin Filaments ◽  
Adherens Junctions ◽  
Bond Formation ◽  
Force Spectroscopy ◽  
Cellular Systems ◽  
Bond Rupture ◽  
Extracellular Region ◽  
Tensile Forces

AbstractCadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. Single molecule force spectroscopy experiments in in-vitro experiments can reveal the cadherin-cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin homophilic and heterophilic binding in their native conformational and functional state in living cells has rarely been done. Here, we used Atomic Force Microscopy (AFM) based Single cell force Spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N-, OB- and E-cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e. between same type of cells and between cells of different type. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E cadherin homophilic bindings are stronger than N/OB, E/N and E/OB heterophilic bindings. Disassembly of intracellular actin filaments reduces the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements. Whereas, myosin inactivation particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins homophilic bonds.

scholarly journals Homophilic and heterophilic cadherin bond rupture forces in homo- or hetero-cellular systems measured by AFM-based single-cell force spectroscopy

2021 ◽  
Author(s):  
Prem Kumar Viji Babu ◽  
Ursula Mirastschijski ◽  
Ganzanfer Belge ◽  
Manfred Radmacher
Keyword(s):  
Single Cell ◽  
Single Molecule ◽  
Actin Filaments ◽  
Adherens Junctions ◽  
Bond Formation ◽  
Force Spectroscopy ◽  
Cellular Systems ◽  
Bond Rupture ◽  
Extracellular Region ◽  
Cell Force

AbstractCadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. In-vitro single molecule force spectroscopy experiments can reveal cadherin–cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin-presenting cell homophilic and heterophilic binding in the proteins’ native conformational and functional states in living cells has rarely been done. Here, we used atomic force microscopy (AFM) based single-cell force spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N- (neural), OB- (osteoblast) and E- (epithelial) cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e., between cells and cadherins of the same and different types. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E-cadherin homophilic binding events are stronger than N/OB heterophilic binding events. Disassembly of intracellular actin filaments affects the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements, but particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins’ homophilic bonds.

scholarly journals Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity

2020 ◽  
pp. jbc.RA120.015863
Author(s):  
Venukumar Vemula ◽  
Tamás Huber ◽  
Marko Ušaj ◽  
Beáta Bugyi ◽  
Alf Mansson
Keyword(s):  
Motor Activity ◽  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Actin Binding ◽  
In Vitro Motility Assay ◽  
Cooperative Effects ◽  
Myosin Motor ◽  
Filament Dynamics

Actin is a major intracellular protein with key functions in cellular motility, signaling and structural rearrangements. Its dynamic behavior, such as polymerisation and depolymerisation of actin filaments in response to intra- and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin-binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

Specific responses of axons and dendrites to cytoskeleton perturbations: an in vitro study

1993 ◽  
Vol 104 (2) ◽  
pp. 433-443 ◽  
Author(s):  
F. Lafont ◽  
M. Rouget ◽  
A. Rousselet ◽  
C. Valenza ◽  
A. Prochiantz
Keyword(s):  
Surface Tension ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
In Vitro Study ◽  
Axonal Growth ◽  
Vitro Study ◽  
Tensile Forces ◽  
The Asymmetry

Several factors can influence the development of axons and dendrites in vitro. Some of these factors modify the adhesion of neurons to their substratum. We have previously shown that the threshold of neuron-substratum adhesion necessary for initiation and elongation of dendrites is higher than that required for axonal growth. To explain this difference we propose that, in order to antagonize actin-driven surface tension, axons primarily rely on the compression forces of microtubules whereas dendrites rely on adhesion. This model was tested by seeding the cells in conditions allowing the development either of axons or of axons and dendrites, then adding cytochalasin B or nocodazole 1 hour or 24 hours after plating. The addition of cytochalasin B, which depolymerizes actin filaments and thus reduces actin-tensile forces, increases the length of both axons and dendrites, indicating that both axons and dendrites have to antagonize surface tension in order to elongate. The addition of nocodazole, which acts primarily on microtubules, slightly reduces dendrite elongation and totally abolishes axonal growth. Similar results are obtained when the drugs are added 1 or 24 hours after plating, suggesting that the same mechanisms are at work both in initiation and in elongation. Finally, we find that in the presence of cytochalasin B axons adopt a curly morphology, a fact that could be explained by the importance of tensile forces in antagonizing the asymmetry created by polarized microtubules presenting a uniform minus/plus orientation.

scholarly journals Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Shashank Shekhar ◽  
Johnson Chung ◽  
Jane Kondev ◽  
Jeff Gelles ◽  
Bruce L. Goode
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Binding Protein ◽  
Actin Dynamics ◽  
Actin Network ◽  
Cellular Mechanisms ◽  
Actin Networks ◽  
Binding Event

AbstractCellular actin networks can be rapidly disassembled and remodeled in a few seconds, yet in vitro actin filaments depolymerize slowly over minutes. The cellular mechanisms enabling actin to depolymerize this fast have so far remained obscure. Using microfluidics-assisted TIRF, we show that Cyclase-associated protein (CAP) and Cofilin synergize to processively depolymerize actin filament pointed ends at a rate 330-fold faster than spontaneous depolymerization. Single molecule imaging further reveals that hexameric CAP molecules interact with the pointed ends of Cofilin-decorated filaments for several seconds at a time, removing approximately 100 actin subunits per binding event. These findings establish a paradigm, in which a filament end-binding protein and a side-binding protein work in concert to control actin dynamics, and help explain how rapid actin network depolymerization is achieved in cells.

Single Molecule Force Spectroscopy Maps to Study Receptors Clustering

2001 ◽  
Vol 7 (S2) ◽  
pp. 860-861
Author(s):  
R. Bhatia ◽  
N. Almqvist ◽  
S. Banerjee ◽  
G. Primbs ◽  
N. Desai ◽  
...  
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Single Molecule ◽  
Regional Distribution ◽  
Cellular Membrane ◽  
Angiogenic Factor ◽  
Extracellular Region ◽  
Force Volume ◽  
Endothelial Growth Factor

An atomic force microscope (AFM) allows molecular resolution imaging of hydrated specimens. However, it is often limited in providing identity of the imaged structures, especially in a complex system such as a cellular membrane. Cell surface macromolecules such as ion channels and receptors serve as the interface between the cytoplasm and the extracellular region and toward which many regulatory signals are directed. Their density, distribution and clustering are key spatial features influencing effective and proper physiological responses. We used a method that uses AFM “force-volume maps” to identify and map regional distribution as well as ligand-, or antibody-induced real-time clustering of receptors on the cell surface. This technique also allows simultaneous imaging of the resultant changes in cellular micromechanical properties, such as elasticity and cytoskeletal reorganization of the cell. As an appropriate physiological sample, we have examined spatial distribution and real-time clustering of VEGFR, the receptor for vascular endothelial growth factor which is an important angiogenic factor in human and animal tissues.We have used AFM probes conjugated with anti-VEGFR-antibody (anti-Flk-1 antibody) to examine binding (or unbinding) forces between VEGF-R2 (Flk-1) in both in vitro as well as in live endothelial cells. A quantal set of binding and unbinding forces was measured between the antibody conjugated to the AFM tip and purified VEGFRs adsorbed on to a mica surface (Fig 1). The unbinding force varied between 60 and 240 pN and was a multiple of discrete quantized strength of approximately 60 pN (Figure 1B).

scholarly journals Ligand-specific binding forces of LFA-1 and Mac-1 in neutrophil adhesion and crawling

2018 ◽  
Vol 29 (4) ◽  
pp. 408-418 ◽  
Author(s):  
Ning Li ◽  
Hao Yang ◽  
Manliu Wang ◽  
Shouqin Lü ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Adhesion Molecules ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Specific Binding ◽  
Bond Rupture ◽  
Dynamic Force ◽  
Lymphocyte Function ◽  
Underlying Mechanisms ◽  
Distinct Features

Lymphocyte function–associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1) and their counterreceptors such as intercellular cell adhesion molecules (ICAM-1 and ICAM-2), junctional adhesion molecules (JAM-A, JAM-C), and receptors for advanced glycation end products (RAGE) are crucial for promoting polymorphonuclear leukocyte (neutrophil, PMN) recruitment. The underlying mechanisms of ligand-specific bindings in this cascade remain incompletely known. We compared the dynamic force spectra for various LFA-1/Mac-1–ligand bonds using single-molecule atomic force microscopy (AFM) and tested their functions in mediating PMN recruitment under in vitro shear flow. Distinct features of bond rupture forces and lifetimes were uncovered for these ligands, implying their diverse roles in regulating PMN adhesion on endothelium. LFA-1 dominates PMN adhesion on ICAM-1 and ICAM-2, while Mac-1 mediates PMN adhesion on RAGE, JAM-A, and JAM-C, which is consistent with their bond strength. All ligands can trigger PMN spreading and polarization, in which Mac-1 seems to induce outside-in signaling more effectively. LFA-1–ICAM-1 and LFA-1/Mac-1–JAM-C bonds can accelerate PMN crawling under high shear stress, presumably due to their high mechanical strength. This work provides new insight into basic molecular mechanisms of physiological ligands of β2 integrins in PMN recruitment.

scholarly journals Mechanical stability of bivalent transition metal complexes analyzed by single-molecule force spectroscopy

2015 ◽  
Vol 11 ◽  
pp. 817-827 ◽  
Author(s):  
Manuel Gensler ◽  
Christian Eidamshaus ◽  
Maurice Taszarek ◽  
Hans-Ulrich Reissig ◽  
Jürgen P Rabe
Keyword(s):  
Transition Metal Complexes ◽  
Single Molecule ◽  
Mechanical Stability ◽  
Force Spectroscopy ◽  
Model Systems ◽  
Bond Rupture ◽  
Aqueous Environment ◽  
Single Molecule Force Spectroscopy ◽  
Bivalent Transition Metal ◽  
Rupture Behavior

Multivalent biomolecular interactions allow for a balanced interplay of mechanical stability and malleability, and nature makes widely use of it. For instance, systems of similar thermal stability may have very different rupture forces. Thus it is of paramount interest to study and understand the mechanical properties of multivalent systems through well-characterized model systems. We analyzed the rupture behavior of three different bivalent pyridine coordination complexes with Cu2+ in aqueous environment by single-molecule force spectroscopy. Those complexes share the same supramolecular interaction leading to similar thermal off-rates in the range of 0.09 and 0.36 s−1, compared to 1.7 s−1 for the monovalent complex. On the other hand, the backbones exhibit different flexibility, and we determined a broad range of rupture lengths between 0.3 and 1.1 nm, with higher most-probable rupture forces for the stiffer backbones. Interestingly, the medium-flexible connection has the highest rupture forces, whereas the ligands with highest and lowest rigidity seem to be prone to consecutive bond rupture. The presented approach allows separating bond and backbone effects in multivalent model systems.

scholarly journals Actin turnover–dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

2006 ◽  
Vol 175 (6) ◽  
pp. 947-955 ◽  
Author(s):  
Takushi Miyoshi ◽  
Takahiro Tsuji ◽  
Chiharu Higashida ◽  
Maud Hertzog ◽  
Akiko Fujita ◽  
...  
Keyword(s):  
Single Molecule ◽  
Actin Filaments ◽  
Dissociation Rate ◽  
Actin Network ◽  
Lim Kinase ◽  
Capping Protein ◽  
Filament Dynamics ◽  
Actin Turnover ◽  
Kinetics Of

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s−1, respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.

scholarly journals Biased localization of actin binding proteins by actin filament conformation

2020 ◽  
Author(s):  
Andrew R Harris ◽  
Pamela Jreij ◽  
Brian Belardi ◽  
Andreas Bausch ◽  
Daniel A Fletcher
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Conformational Changes ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Critical Role ◽  
Actin Binding ◽  
Cell Physiology ◽  
Physical Constraints

ABSTRACTThe assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1-CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells, and we demonstrate that the affinity of CH1-CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs, physical constraints, and other binding proteins. These findings suggest that conformational heterogeneity of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.

scholarly journals Biased localization of actin binding proteins by actin filament conformation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Andrew R. Harris ◽  
Pamela Jreij ◽  
Brian Belardi ◽  
Aaron M. Joffe ◽  
Andreas R. Bausch ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Conformational Changes ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Critical Role ◽  
Actin Binding ◽  
Cell Physiology ◽  
Binding Domains

AbstractThe assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1–CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells. We demonstrate that the binding kinetics of CH1–CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins. These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.

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