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 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 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.

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

Analysis of Cytoskeleton-Destabilizing Agents by Optimized Optical Navigation and AFM Force Measurements

2010 ◽  
Vol 18 (2) ◽  
pp. 34-37 ◽  
Author(s):  
A. Berquand ◽  
A. Holloschi ◽  
M. Trendelenburg ◽  
P. Kioschis
Keyword(s):  
Cancer Cells ◽  
Actin Filaments ◽  
Elastic Moduli ◽  
Ex Vivo ◽  
Optical Navigation ◽  
Force Measurements ◽  
Cellular Processes ◽  
Physical Interactions ◽  
Cellular Pathology

Mechanical properties of cells are determined by the dynamic behavior of the cytoskeleton and physical interactions with the environment. The cytoskeleton, composed of actin filaments, intermediate filaments, and microtubules, is vital for numerous key cellular processes, such as cell division, vesicle trafficking, cell contraction, cell motility, and cell signaling. There is increasing evidence that deregulation of cytoskeletal components like disassembly of actin and tubulin filaments is an important parameter in cellular pathology. Thus, significant alterations of the mechanical phenotype of the cell and its surrounding microenvironment are reported to be involved in aberrant cellular processes and successively contribute to onset and progression of diseases such as cancer, malaria, and possibly neurodegeneration. In vitro and ex vivo biomechanical studies have shown that cancer cells have significantly decreased elastic moduli than their normal counterparts, a characteristic that is attributed to the ability of cancer cells to metastasize or spread.

scholarly journals Calmodulin dissociation regulates brush border myosin I (110-kD-calmodulin) mechanochemical activity in vitro.

1990 ◽  
Vol 110 (4) ◽  
pp. 1137-1147 ◽  
Author(s):  
K Collins ◽  
J R Sellers ◽  
P Matsudaira
Keyword(s):  
Actin Filaments ◽  
Actin Binding ◽  
Filament Length ◽  
Myosin I ◽  
High Calcium ◽  
Hydrolysis Of ◽  
Mgatpase Activity ◽  
Low Rate ◽  
Actin Concentration

110-kD-calmodulin, when immobilized on nitrocellulose-coated coverslips, translocates actin filaments at a maximal rate of 0.07-0.1 micron/s at 37 degrees C. Actin activates MgATPase activity greater than 40-fold, with a Km of 40 microM and Vmax of 0.86 s-1 (323 nmol/min/mg). The rate of motility mediated by 110-kD-calmodulin is dependent on temperature and concentration of ATP, but independent of time, actin filament length, amount of enzyme, or ionic strength. Tropomyosin inhibits actin binding by 110-kD-calmodulin in MgATP and inhibits motility. Micromolar calcium slightly increases the rate of motility and increases the actin-activated MgATP hydrolysis of the intact complex. In 0.1 mM or higher calcium, motility ceases and actin-dependent MgATPase activity remains at a low rate not activated by increasing actin concentration. Correlated with these inhibitions of activity, a subset of calmodulin is dissociated from the complex. To determine if calmodulin loss is the cause of calcium inhibition, we assayed the ability of calmodulin to rescue the calcium-inactivated enzyme. Readdition of calmodulin to the nitrocellulose-bound, calcium-inactivated enzyme completely restores motility. Addition of calmodulin also restores actin activation to MgATPase activity in high calcium, but does not affect the activity of the enzyme in EGTA. These results demonstrate that in vitro 110-kD-calmodulin functions as a calcium-sensitive mechanoenzyme, a vertebrate myosin I. The properties of this enzyme suggest that despite unique structure and regulation, myosins I and II share a molecular mechanism of motility.

Actin Dynamics, Architecture, and Mechanics in Cell Motility

2014 ◽  
Vol 94 (1) ◽  
pp. 235-263 ◽  
Author(s):  
Laurent Blanchoin ◽  
Rajaa Boujemaa-Paterski ◽  
Cécile Sykes ◽  
Julie Plastino
Keyword(s):  
Mechanical Properties ◽  
Actin Filaments ◽  
Molecular Motor ◽  
Actin Dynamics ◽  
Tight Coupling ◽  
Actin Organization ◽  
Cellular Processes ◽  
Shock Absorbers ◽  
Cellular Environment

Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or “dashpots” (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

scholarly journals Enhancement of Actin-depolymerizing Factor/Cofilin-dependent Actin Disassembly by Actin-interacting Protein 1 Is Required for Organized Actin Filament Assembly in the Caenorhabditis elegans Body Wall Muscle

2006 ◽  
Vol 17 (5) ◽  
pp. 2190-2199 ◽  
Author(s):  
Kurato Mohri ◽  
Kanako Ono ◽  
Robinson Yu ◽  
Sawako Yamashiro ◽  
Shoichiro Ono
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Actin Depolymerizing Factor ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Cytoskeletal Dynamics ◽  
End Capping

Regulated disassembly of actin filaments is involved in several cellular processes that require dynamic rearrangement of the actin cytoskeleton. Actin-interacting protein (AIP) 1 specifically enhances disassembly of actin-depolymerizing factor (ADF)/cofilin-bound actin filaments. In vitro, AIP1 actively disassembles filaments, caps barbed ends, and binds to the side of filaments. However, how AIP1 functions in the cellular actin cytoskeletal dynamics is not understood. We compared biochemical and in vivo activities of mutant UNC-78 proteins and found that impaired activity of mutant UNC-78 proteins to enhance disassembly of ADF/cofilin-bound actin filaments is associated with inability to regulate striated organization of actin filaments in muscle cells. Six functionally important residues are present in the N-terminal β-propeller, whereas one residue is located in the C-terminal β-propeller, suggesting the presence of two separate sites for interaction with ADF/cofilin and actin. In vitro, these mutant UNC-78 proteins exhibited variable alterations in actin disassembly and/or barbed end-capping activities, suggesting that both activities are important for its in vivo function. These results indicate that the actin-regulating activity of AIP1 in cooperation with ADF/cofilin is essential for its in vivo function to regulate actin filament organization in muscle cells.

scholarly journals The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex

2013 ◽  
Vol 202 (2) ◽  
pp. 251-260 ◽  
Author(s):  
Sara Solinet ◽  
Kazi Mahmud ◽  
Shannon F. Stewman ◽  
Khaled Ben El Kadhi ◽  
Barbara Decelle ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Metastatic Potential ◽  
Actin Binding ◽  
Cell Cortex ◽  
Shape Transformation ◽  
Erm Proteins ◽  
Cellular Processes ◽  
Anaphase Onset

Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM’s role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. In this paper, we show that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. We identified two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs that mediated the association with microtubules. This ERM–microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs.

scholarly journals Human CLASP2 specifically regulates microtubule catastrophe and rescue

2018 ◽  
Vol 29 (10) ◽  
pp. 1168-1177 ◽  
Author(s):  
Elizabeth J. Lawrence ◽  
Göker Arpag˘ ◽  
Stephen R. Norris ◽  
Marija Zanic
Keyword(s):  
Growth Rates ◽  
Molecular Mechanisms ◽  
Direct Interaction ◽  
Microtubule Dynamics ◽  
Microtubule Associated Proteins ◽  
Cellular Processes ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Protein Components

Cytoplasmic linker-associated proteins (CLASPs) are microtubule-associated proteins essential for microtubule regulation in many cellular processes. However, the molecular mechanisms underlying CLASP activity are not understood. Here, we use purified protein components and total internal reflection fluorescence microscopy to investigate the effects of human CLASP2 on microtubule dynamics in vitro. We demonstrate that CLASP2 suppresses microtubule catastrophe and promotes rescue without affecting the rates of microtubule growth or shrinkage. Strikingly, when CLASP2 is combined with EB1, a known binding partner, the effects on microtubule dynamics are strongly enhanced. We show that synergy between CLASP2 and EB1 is dependent on a direct interaction, since a truncated EB1 protein that lacks the CLASP2-binding domain does not enhance CLASP2 activity. Further, we find that EB1 targets CLASP2 to microtubules and increases the dwell time of CLASP2 at microtubule tips. Although the temporally averaged microtubule growth rates are unaffected by CLASP2, we find that microtubules grown with CLASP2 display greater variability in growth rates. Our results provide insight into the regulation of microtubule dynamics by CLASP proteins and highlight the importance of the functional interplay between regulatory proteins at dynamic microtubule ends.

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.

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