scholarly journals The dynamic instability of actin filament barbed ends

2021 ◽  
Vol 220 (4) ◽  
Author(s):  
Guillaume Romet-Lemonne ◽  
Antoine Jégou
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Molecular Mechanisms ◽  
Dynamic Instability ◽  
Filament Networks ◽  
In Cells

The turnover of actin filament networks in cells has long been considered to reflect the treadmilling behavior of pure actin filaments in vitro, where only the pointed ends depolymerize. Newly discovered molecular mechanisms challenge this notion, as they provide evidence of situations in which growing and depolymerizing barbed ends coexist.

scholarly journals Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells

2005 ◽  
Vol 16 (2) ◽  
pp. 649-664 ◽  
Author(s):  
Pirta Hotulainen ◽  
Eija Paunola ◽  
Maria K. Vartiainen ◽  
Pekka Lappalainen
Keyword(s):  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Depolymerizing Factor ◽  
Cytoskeletal Dynamics ◽  
Nonmuscle Cells ◽  
In Cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling “old” actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

scholarly journals Mechanisms of actin disassembly

2013 ◽  
Vol 24 (15) ◽  
pp. 2299-2302 ◽  
Author(s):  
William Brieher
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Depolymerization ◽  
Filament Dynamics ◽  
Physiological Demands ◽  
In Cells

The actin cytoskeleton is constantly assembling and disassembling. Cells harness the energy of these turnover dynamics to drive cell motility and organize cytoplasm. Although much is known about how cells control actin polymerization, we do not understand how actin filaments depolymerize inside cells. I briefly describe how the combination of imaging actin filament dynamics in cells and using in vitro biochemistry progressively altered our views of actin depolymerization. I describe why I do not think that the prevailing model of actin filament turnover—cofilin-mediated actin filament severing—can account for actin filament disassembly detected in cells. Finally, I speculate that cells might be able to tune the mechanism of actin depolymerization to meet physiological demands and selectively control the stabilities of different actin arrays.

scholarly journals Novel cryo-electron tomography structure of Arp2/3 complex in cells reveals mechanisms of branch formation

2020 ◽  
Author(s):  
Florian Fäßler ◽  
Georgi Dimchev ◽  
Victor-Valentin Hodirnau ◽  
William Wan ◽  
Florian KM Schur
Keyword(s):  
Actin Filament ◽  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Branch Formation ◽  
Complex Structural ◽  
Filament Networks ◽  
Subtomogram Averaging ◽  
Resolution Structure ◽  
In Cells

AbstractThe actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Our structure indicates a central role for the ArpC3 subunit in stabilizing the active conformation and suggests that in the branch junction relocation of the ArpC5 N-terminus and the C-terminal tail of Arp3 is important to fix Arp2 and Arp3 in an actin dimer-like conformation. Notably, our model of the branch junction in cells significantly differs from the previous in vitro branch junction model.

scholarly journals Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly

2020 ◽  
Author(s):  
Hugo Wioland ◽  
Stéphane Frémont ◽  
Bérengère Guichard ◽  
Arnaud Echard ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Post Translational Modification ◽  
Single Filament ◽  
In Cells

ABSTRACTProteins of the ADF/cofilin family play a central role in the disassembly of actin filaments, and their activity must be tightly regulated in cells. Recently, the oxidation of actin filaments by the enzyme MICAL1 was found to amplify the severing action of cofilin through unclear mechanisms. Two essential factors normally prevent filament disassembly: the inactivation of cofilin by phosphorylation, and the protection of filaments by tropomyosins, but whether actin oxidation might interfere with these safeguard mechanisms is unknown. Using single filament experiments in vitro, we found that actin filament oxidation by MICAL1 increases, by several orders of magnitude, both cofilin binding and severing rates, explaining the dramatic synergy between oxidation and cofilin for filament disassembly. Remarkably, we found that actin oxidation bypasses the need for cofilin activation by dephosphorylation. Indeed, non-activated, phosphomimetic S3D-cofilin binds and severs oxidized actin filaments rapidly, in conditions where non-oxidized filaments are unaffected. Finally, tropomyosin Tpm1.8 loses its ability to protect filaments from cofilin severing activity when actin is oxidized by MICAL1. Together, our results show that MICAL1-induced oxidation of actin filaments suppresses their physiological protection from the action of cofilin. We propose that in cells, direct post-translational modification of actin filaments by oxidation is a way to trigger their severing, in spite of being decorated by tropomyosin, and without requiring the activation of cofilin.

scholarly journals miR-182-5p and miR-378a-3p regulate ferroptosis in I/R-induced renal injury

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Chenguang Ding ◽  
Xiaoming Ding ◽  
Jin Zheng ◽  
Bo Wang ◽  
Yang Li ◽  
...  
Keyword(s):  
Cell Death ◽  
Renal Injury ◽  
Molecular Mechanisms ◽  
Ischemia Reperfusion ◽  
Kidney Injury ◽  
Luciferase Reporter ◽  
Renal Tubular Cell ◽  
In Cells

Abstract Renal tubular cell death is the key factor of the pathogenesis of ischemia/reperfusion (I/R) kidney injury. Ferroptosis is a type of regulated cell death (RCD) found in various diseases. However, the underlying molecular mechanisms related to ferroptosis in renal I/R injury remain unclear. In the present study, we investigated the regulatory role of microRNAs on ferroptosis in I/R-induced renal injury. We established the I/R-induced renal injury model in rats, and H/R induced HK-2 cells injury in vitro. CCK-8 was used to measure cell viability. Fe2+ and ROS levels were assayed to evaluate the activation of ferroptosis. We performed RNA sequencing to profile the miRNAs expression in H/R-induced injury and ferroptosis. Western blot analysis was used to detect the protein expression. qRT-PCR was used to detect the mRNA and miRNA levels in cells and tissues. We further used luciferase reporter assay to verify the direct targeting effect of miRNA. We found that ischemia/reperfusion-induced ferroptosis in rat’s kidney. We identified that miR-182-5p and miR-378a-3p were upregulated in the ferroptosis and H/R-induced injury, and correlates reversely with glutathione peroxidases 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression in renal I/R injury tissues, respectively. In vitro studies showed that miR-182-5p and miR-378a-3p induced ferroptosis in cells. We further found that miR-182-5p and miR-378a-3p regulated the expression of GPX4 and SLC7A11 negatively by directly binding to the 3′UTR of GPX4 and SLC7A11 mRNA. In vivo study showed that silencing miR-182-5p and miR-378a-3p alleviated the I/R-induced renal injury in rats. In conclusion, we demonstrated that I/R induced upregulation of miR-182-5p and miR-378a-3p, leading to activation of ferroptosis in renal injury through downregulation of GPX4 and SLC7A11.

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.

scholarly journals Microtubule minus-end stability is dictated by the tubulin off-rate

2019 ◽  
Vol 218 (9) ◽  
pp. 2841-2853 ◽  
Author(s):  
Claire Strothman ◽  
Veronica Farmer ◽  
Göker Arpağ ◽  
Nicole Rodgers ◽  
Marija Podolski ◽  
...  
Keyword(s):  
Dynamic Instability ◽  
In Vitro Reconstitution ◽  
Primary Determinant ◽  
Mean Size ◽  
The Mean ◽  
The Difference ◽  
Insight Into ◽  
Dynamic Organization ◽  
In Cells

Dynamic organization of microtubule minus ends is vital for the formation and maintenance of acentrosomal microtubule arrays. In vitro, both microtubule ends switch between phases of assembly and disassembly, a behavior called dynamic instability. Although minus ends grow slower, their lifetimes are similar to those of plus ends. The mechanisms underlying these distinct dynamics remain unknown. Here, we use an in vitro reconstitution approach to investigate minus-end dynamics. We find that minus-end lifetimes are not defined by the mean size of the protective GTP-tubulin cap. Rather, we conclude that the distinct tubulin off-rate is the primary determinant of the difference between plus- and minus-end dynamics. Further, our results show that the minus-end–directed kinesin-14 HSET/KIFC1 suppresses tubulin off-rate to specifically suppress minus-end catastrophe. HSET maintains its protective minus-end activity even when challenged by a known microtubule depolymerase, kinesin-13 MCAK. Our results provide novel insight into the mechanisms of minus-end dynamics, essential for our understanding of microtubule minus-end regulation in cells.

scholarly journals The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

2016 ◽  
Vol 27 (11) ◽  
pp. 1821-1833 ◽  
Author(s):  
Yujie Li ◽  
Jenna R. Christensen ◽  
Kaitlin E. Homa ◽  
Glen M. Hocky ◽  
Alice Fok ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Actin Filament ◽  
Actin Filaments ◽  
Biochemical Properties ◽  
Ring Formation ◽  
Cross Linking ◽  
Contractile Ring ◽  
Mixed Polarity ◽  
Dividing Cells

The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction.

scholarly journals α5β1 integrin recycling promotes Arp2/3-independent cancer cell invasion via the formin FHOD3

2015 ◽  
Vol 210 (6) ◽  
pp. 1013-1031 ◽  
Author(s):  
Nikki R. Paul ◽  
Jennifer L. Allen ◽  
Anna Chapman ◽  
Maria Morlan-Mairal ◽  
Egor Zindy ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Invasion ◽  
Molecular Mechanisms ◽  
Cancer Metastasis ◽  
Cancer Cell Invasion ◽  
Coupling Protein ◽  
Α5β1 Integrin ◽  
In Cells

Invasive migration in 3D extracellular matrix (ECM) is crucial to cancer metastasis, yet little is known of the molecular mechanisms that drive reorganization of the cytoskeleton as cancer cells disseminate in vivo. 2D Rac-driven lamellipodial migration is well understood, but how these features apply to 3D migration is not clear. We find that lamellipodia-like protrusions and retrograde actin flow are indeed observed in cells moving in 3D ECM. However, Rab-coupling protein (RCP)-driven endocytic recycling of α5β1 integrin enhances invasive migration of cancer cells into fibronectin-rich 3D ECM, driven by RhoA and filopodial spike-based protrusions, not lamellipodia. Furthermore, we show that actin spike protrusions are Arp2/3-independent. Dynamic actin spike assembly in cells invading in vitro and in vivo is regulated by Formin homology-2 domain containing 3 (FHOD3), which is activated by RhoA/ROCK, establishing a novel mechanism through which the RCP–α5β1 pathway reprograms the actin cytoskeleton to promote invasive migration and local invasion in vivo.

scholarly journals Spreading of perturbations in myosin group kinetics along actin filaments

2019 ◽  
Vol 116 (35) ◽  
pp. 17336-17344 ◽  
Author(s):  
Zsombor Balassy ◽  
Anne-Marie Lauzon ◽  
Lennart Hilbert
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Nearest Neighbor ◽  
Global Changes ◽  
Chain Model ◽  
Mechanical Coupling ◽  
In Vitro Motility ◽  
Local Perturbations ◽  
Nearest Neighbor Coupling

Global changes in the state of spatially distributed systems can often be traced back to perturbations that arise locally. Whether such local perturbations grow into global changes depends on the system geometry and the spatial spreading of these perturbations. Here, we investigate how different spreading behaviors of local perturbations determine their global impact in 1-dimensional systems of different size. Specifically, we assessed sliding arrest events in in vitro motility assays where myosins propel actin, and simulated the underlying mechanochemistry of myosins that bind along the actin filament. We observed spontaneous sliding arrest events that occurred more frequently for shorter actin filaments. This observation could be explained by spontaneous local arrest of myosin kinetics that stabilizes once it spreads throughout an entire actin filament. When we introduced intermediate concentrations of the actin cross-linker filamin, longer actin was arrested more frequently. This observation was reproduced by simulations where filamin binding induces persistent local arrest of myosin kinetics, which subsequently spreads throughout the actin filament. A spin chain model with nearest-neighbor coupling reproduced key features of our experiments and simulations, thus extending to other linear systems with nearest-neighbor coupling the following conclusions: 1) perturbations that are persistent only once they spread throughout the system are more effective in smaller systems, and 2) perturbations that are persistent upon their establishment are more effective in larger systems. Beyond these general conclusions, our work also provides a theoretical model of collective myosin kinetics with a finite range of mechanical coupling along the actin filament.

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