scholarly journals Actin filaments regulate microtubule growth at the centrosome

2018 ◽  
Author(s):  
Daisuke Inoue ◽  
Dorian Obino ◽  
Francesca Farina ◽  
Jérémie Gaillard ◽  
Christophe Guerin ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Steric Hindrance ◽  
Actin Filaments ◽  
Network Architecture ◽  
Lymphocyte Activation ◽  
Local Network ◽  
Actin Network ◽  
In Vitro Reconstitution ◽  
Microtubule Growth

AbstractThe centrosome is the main microtubule-organizing centre. It also organizes a local network of actin filaments. However, the precise function of the actin network at the centrosome is not well understood. Here we show that increasing densities of actin filaments at the centrosome of lymphocytes were correlated with reduced amounts of microtubules. Furthermore, lymphocyte activation resulted in centrosomal-actin disassembly and an increase in microtubule number. To further investigate the direct crosstalk between actin and microtubules at the centrosome, we performed in vitro reconstitution assays based on (i) purified centrosomes and (ii) on the co-micropatterning of microtubule seeds and actin filaments. The two assays demonstrated that actin filaments perturb microtubule growth by steric hindrance. Finally, we showed that cell adhesion and spreading leads to lower densities of centrosomal actin thus resulting in higher microtubule growth. Hence we propose a novel mechanism by which the number of centrosomal microtubules is regulated by cell adhesion and actin-network architecture.

scholarly journals A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Johanna Funk ◽  
Felipe Merino ◽  
Matthias Schaks ◽  
Klemens Rottner ◽  
Stefan Raunser ◽  
...  
Keyword(s):  
Structural Biology ◽  
Actin Filaments ◽  
Actin Network ◽  
Essential Factor ◽  
Actin Networks ◽  
Capping Protein ◽  
Cellular Processes ◽  
In Vitro Reconstitution ◽  
Interference Mechanism

AbstractHeterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP’s C-terminal “tentacle” extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the β tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes—capping and nucleation—in branched actin network assembly.

scholarly journals Cross-linkers at growing microtubule ends generate forces that drive actin transport

2021 ◽  
Author(s):  
Celine Alkemade ◽  
Harmen Wierenga ◽  
Vladimir A. Volkov ◽  
Magdalena Preciado-López ◽  
Anna Akhmanova ◽  
...  
Keyword(s):  
Friction Force ◽  
Actin Filaments ◽  
Active Networks ◽  
Filament Length ◽  
Transport Time ◽  
Cellular Processes ◽  
In Vitro Reconstitution ◽  
Microtubule Growth ◽  
Actin Remodelling

The actin and microtubule cytoskeletons form active networks in the cell that can contract and remodel, resulting in vital cellular processes as cell division and motility. Motor proteins play an important role in generating the forces required for these processes, but more recently the concept of passive cross-linkers being able to generate forces has emerged. So far, these passive cross-linkers have been studied in the context of separate actin and microtubule systems. Here, we show that cross-linkers also allow actin and microtubules to exert forces on each other. More specifically, we study single actin filaments that are cross-linked to growing microtubule ends, using in vitro reconstitution, computer simulations, and a minimal theoretical model. We show that microtubules can transport actin filaments over large (micrometer-range) distances, and find that this transport results from two antagonistic forces arising from the binding of cross-linkers to the overlap between the actin and microtubule filaments. The cross-linkers attempt to maximize the overlap between the actin and the tip of the growing microtubules, creating an affinity-driven forward condensation force, and simultaneously create a competing friction force along the microtubule lattice. We predict and verify experimentally how the average transport time depends on the actin filament length and the microtubule growth velocity, confirming the competition between a forward condensation force and a backward friction force. In addition, we theoretically predict and experimentally verify that the condensation force is of the order of 0.1pN. Thus, our results reveal a new mechanism for local actin remodelling by growing microtubules.

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.

In Vitro Reconstitution of Dynamic Co-organization of Microtubules and Actin Filaments in Emulsion Droplets

2019 ◽  
pp. 53-75 ◽  
Author(s):  
Kim J. A. Vendel ◽  
Celine Alkemade ◽  
Nemo Andrea ◽  
Gijsje H. Koenderink ◽  
Marileen Dogterom
Keyword(s):  
Actin Filaments ◽  
Emulsion Droplets ◽  
In Vitro Reconstitution

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 Myosin-driven fragmentation of actin filaments triggers contraction of a disordered actin network

2018 ◽  
Author(s):  
Kyohei Matsuda ◽  
Takuya Kobayashi ◽  
Mitsuhiro Sugawa ◽  
Yurika Koiso ◽  
Yoko Y. Toyoshima ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Actin Filaments ◽  
Atp Hydrolysis ◽  
Myosin Ii ◽  
Network Connectivity ◽  
Experimental System ◽  
Actin Network ◽  
Atp Concentration ◽  
Time Required

AbstractThe dynamic cytoskeletal network is responsible for cell shape changes and cell division. The actin-based motor protein myosin II drives the remodeling of a highly disordered actin-based network and enables the network to perform mechanical work such as contraction, migration and adhesion. Myosin II forms bipolar filaments that self-associate via their tail domains. Such myosin minifilaments generate both extensile and compressive forces that pull and push actin filaments, depending on the relative position of myosin and actin filaments in the network. However, it remains unclear how the mechanical properties of myosin II that rely on the energy of ATP hydrolysis spontaneously contract the disordered actin network. Here, we used a minimal in vitro reconstituted experimental system consisting of actin, myosin, and a cross-linking protein, to gain insights into the molecular mechanism by which myosin minifilaments organize disordered actin networks into contractile states. We found that contracted cluster size and time required for the onset of network contraction decreased as ATP concentration decreased. Contraction velocity was negatively correlated with ATP concentrations. Reduction of ATP concentration caused fragmentation of actin filaments by myosin minifilament. We also found that gelsolin, a Ca2+-regulated actin filament-severing protein, induced contraction of a mechanically stable network, implying that fragmentations of actin filaments in the network weaken the intra-network connectivity and trigger contraction. Our findings reveal that the disordered actin network contraction can be controlled by fragmentation of actin filaments, highlighting the molecular mechanism underlying the myosin motor-severing activities, other than the sliding tensile and compressive stress in the disordered actin network.

scholarly journals Involvement of ZO-1 in Cadherin-based Cell Adhesion through Its Direct Binding to α Catenin and Actin Filaments

1997 ◽  
Vol 138 (1) ◽  
pp. 181-192 ◽  
Author(s):  
Masahiko Itoh ◽  
Akira Nagafuchi ◽  
Seiji Moroi ◽  
Shoichiro Tsukita
Keyword(s):  
Cell Adhesion ◽  
Dissociation Constant ◽  
Actin Filaments ◽  
Full Length ◽  
Amino Terminal ◽  
Adhesion Sites ◽  
Direct Binding ◽  
Carboxyl Terminal ◽  
E Cadherin

ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell–cell adhesion sites. In good agreement with these observations, N-ZO-1 was specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/α, β catenin complex. In contrast, C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with α catenin, but not to that with β catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and α catenin was ∼0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of ∼10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with α catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system.

scholarly journals EB3-informed dynamics of the microtubule stabilizing cap during stalled growth

2021 ◽  
Author(s):  
Maurits Kok ◽  
Florian Huber ◽  
Svenja-Marei Kalisch ◽  
Marileen Dogterom
Keyword(s):  
Growth Velocity ◽  
Hydrolysis Rate ◽  
Exact Relation ◽  
Gtp Hydrolysis ◽  
Lifetime Distributions ◽  
Microtubule Stability ◽  
In Vitro Reconstitution ◽  
1D Model ◽  
Microtubule Growth

Microtubule stability is known to be governed by a stabilizing GTP/GDP-Pi cap, but the exact relation between growth velocity, GTP hydrolysis and catastrophes remains unclear. We investigate the dynamics of the stabilizing cap through in vitro reconstitution of microtubule dynamics in contact with micro-fabricated barriers, using the plus-end binding protein GFP-EB3 as a marker for the nucleotide state of the tip. The interaction of growing microtubules with steric objects is known to slow down microtubule growth and accelerate catastrophes. We show that the lifetime distributions of stalled microtubules, as well as the corresponding lifetime distributions of freely growing microtubules, can be fully described with a simple phenomenological 1D model based on noisy microtubule growth and a single EB3-dependent hydrolysis rate. This same model is furthermore capable of explaining both the previously reported mild catastrophe dependence on microtubule growth rates and the catastrophe statistics during tubulin washout experiments.

scholarly journals Septin 2/6/7 complexes tune microtubule plus end growth and EB1 binding in a concentration- and filament-dependent manner

2019 ◽  
Author(s):  
Konstantinos Nakos ◽  
Megan R. Radler ◽  
Elias T. Spiliotis
Keyword(s):  
Membrane Trafficking ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Live Cells ◽  
Dependent Manner ◽  
In Vitro Reconstitution ◽  
In Trans ◽  
In Cis ◽  
Precise Role

AbstractSeptins are filamentous GTP-binding proteins, which affect microtubule (MT) dependent functions including membrane trafficking and cell division, but their precise role in MT dynamics is poorly understood. Here, in vitro reconstitution of MT dynamics with SEPT2/6/7, the minimal subunits of septin heteromers, shows that SEPT2/6/7 has a biphasic concentration-dependent effect on MT growth. Lower concentrations of SEPT2/6/7 enhance MT plus end growth and elongation, while higher and intermediate concentrations inhibit and pause plus end growth, respectively. We show that SEPT2/6/7 has a 1.5-fold preference for GTP-over GDP-bound MT lattice, and competes with EB1 for binding to GTPγS-stabilized MTs, which mimic the EB1-preferred GDP-Pi state of polymerized tubulin. Strikingly, SEPT2/6/7 triggers EB1 dissociation from plus end tips in cis by binding to the MT lattice and in trans when MT plus ends collide with SEPT2/6/7 filaments. At these intersections, SEPT2/6/7 filaments were more potent barriers than actin filaments in pausing MT growth and dissociating EB1 in vitro and in live cells. These data demonstrate that SEPT2/6/7 complexes and filaments can directly impact MT plus end growth and the tracking of plus end-binding proteins, and thereby may facilitate the capture of MT plus ends at intracellular sites of septin enrichment.Highlight Summary for eTOCKnowledge of septin roles in MT dynamics is poor and confounded by knockdown studies. Here, in vitro reconstitution assays show concentration-dependent effects of SEPT2/6/7 on MT plus end growth, pausing and EB1 tracking. We found that SEPT2/6/7 filaments are potent than actin in pausing MT growth and dissociating EB1 from intersecting plus ends.

A206 Attempt at in vitro reconstitution of actin filaments and liposomes

2007 ◽  
Vol 2007.18 (0) ◽  
pp. 87-88
Author(s):  
Yukiko MARUOKA ◽  
Taiji ADACHI ◽  
Junko SUNAGA ◽  
Masaki HOJO
Keyword(s):  
Actin Filaments ◽  
In Vitro Reconstitution
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