scholarly journals The structural basis of actin filament branching by the Arp2/3 complex

2008 ◽  
Vol 180 (5) ◽  
pp. 887-895 ◽  
Author(s):  
Isabelle Rouiller ◽  
Xiao-Ping Xu ◽  
Kurt J. Amann ◽  
Coumaran Egile ◽  
Stephan Nickell ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Actin Filaments ◽  
Electron Tomography ◽  
Leading Edge ◽  
Structural Basis ◽  
Actin Monomer ◽  
Related Protein ◽  
Cooperative Interactions ◽  
Motile Cells ◽  
Branch Formation

The actin-related protein 2/3 (Arp2/3) complex mediates the formation of branched actin filaments at the leading edge of motile cells and in the comet tails moving certain intracellular pathogens. Crystal structures of the Arp2/3 complex are available, but the architecture of the junction formed by the Arp2/3 complex at the base of the branch was not known. In this study, we use electron tomography to reconstruct the branch junction with sufficient resolution to show how the Arp2/3 complex interacts with the mother filament. Our analysis reveals conformational changes in both the mother filament and Arp2/3 complex upon branch formation. The Arp2 and Arp3 subunits reorganize into a dimer, providing a short-pitch template for elongation of the daughter filament. Two subunits of the mother filament undergo conformational changes that increase stability of the branch. These data provide a rationale for why branch formation requires cooperative interactions among the Arp2/3 complex, nucleation-promoting factors, an actin monomer, and the mother filament.

scholarly journals Late endosomes promote microglia migration via cytosolic translocation of immature protease cathD

2020 ◽  
Vol 6 (50) ◽  
pp. eaba5783
Author(s):  
Yi-Jun Liu ◽  
Ting Zhang ◽  
Daxiao Cheng ◽  
Junhua Yang ◽  
Sicong Chen ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Adenosine Triphosphate ◽  
Actin Filaments ◽  
Cathepsin D ◽  
Leading Edge ◽  
Organelle Transport ◽  
Actin Monomer ◽  
Late Endosomes ◽  
Cytoskeletal Dynamics ◽  
Cytoskeleton Remodeling

Organelle transport requires dynamic cytoskeleton remodeling, but whether cytoskeletal dynamics are, in turn, regulated by organelles remains elusive. Here, we demonstrate that late endosomes, a type of prelysosomal organelles, facilitate actin-cytoskeleton remodeling via cytosolic translocation of immature protease cathepsin D (cathD) during microglia migration. After cytosolic translocation, late endosome–derived cathD juxtaposes actin filaments at the leading edge of lamellipodia. Suppressing cathD expression or blocking its cytosolic translocation impairs the maintenance but not the initiation of lamellipodial extension. Moreover, immature cathD balances the activity of the actin-severing protein cofilin to maintain globular-actin (G-actin) monomer pool for local actin recycling. Our study identifies cathD as a key lysosomal molecule that unconventionally contributes to actin cytoskeleton remodeling via cytosolic translocation during adenosine triphosphate–evoked microglia migration.

scholarly journals Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia

1999 ◽  
Vol 145 (5) ◽  
pp. 1009-1026 ◽  
Author(s):  
Tatyana M. Svitkina ◽  
Gary G. Borisy
Keyword(s):  
Actin Filaments ◽  
Leading Edge ◽  
Actin Network ◽  
Actin Depolymerizing Factor ◽  
Nucleation Model ◽  
Branch Formation ◽  
Cell Front ◽  
Dendritic Organization ◽  
Pointed End

The leading edge (∼1 μm) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 μm of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181–6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.

scholarly journals Leading edge stability in motile cells is an emergent property of branched actin network growth

2020 ◽  
Author(s):  
Rikki M. Garner ◽  
Julie A. Theriot
Keyword(s):  
High Speed ◽  
Noise Suppression ◽  
Animal Cell ◽  
Leading Edge ◽  
Lateral Spreading ◽  
Actin Network ◽  
Biological Noise ◽  
Network Growth ◽  
Motile Cells ◽  
Branch Formation

AbstractAnimal cell migration is predominantly driven by the coordinated, yet stochastic, polymerization of thousands of nanometer-scale actin filaments across micron-scale cell leading edges. It remains unclear how such inherently noisy processes generate robust cellular behavior. We employed high-speed, high-resolution imaging of migrating neutrophil-like HL-60 cells to explore the fine-scale dynamic shape fluctuations that emerge and relax throughout the process of leading edge maintenance. We then developed a minimal stochastic model of the leading edge that is able to reproduce this stable relaxation behavior. Remarkably, we find that lamellipodial stability naturally emerges from the interplay between branched actin network growth and leading edge shape – with no additional feedback required – based on a synergy between membrane-proximal branching and lateral spreading of filaments. These results thus demonstrate a novel biological noise-suppression mechanism based entirely on system geometry. Furthermore, our model suggests that the Arp2/3-mediated ∼70-80º branching angle optimally smooths lamellipodial shape, addressing its long-mysterious conservation from protists to mammals.One sentence summaryAn experimental and computational investigation of fluctuation dynamics at the leading edge of motile cells demonstrates that the specific angular geometry of Arp2/3-mediated actin network branch formation lies at the core of a successful biological noise-suppression strategy.

scholarly journals Cofilin Regulates Filopodial Structure and Flexibility in Neuronal Growth Cones

2021 ◽  
Author(s):  
Ryan K. Hylton ◽  
Jessica Heebner ◽  
Michael Grillo ◽  
Matthew T Swulius
Keyword(s):  
Electron Tomography ◽  
Leading Edge ◽  
Growth Cones ◽  
Axonal Guidance ◽  
Neuronal Growth ◽  
Cellular Targets ◽  
Actin Networks ◽  
Motile Cells ◽  
Cryo Electron Tomography ◽  
Neuronal Growth Cones

Filopodia are actin-rich cytoskeletal protrusions at the leading edge of motile cells. In neuronal growth cones they function as antennae, guiding axonal growth toward the appropriate cellular targets. Proper brain development relies on robust axonal guidance mechanisms, so it is imperative to understand how the actin cytoskeleton functions in remodeling to meet the demands of growth cone exploration. Here we show by cryo-electron tomography and fluorescence imaging that filopodia in neuronal growth cones switch between fascin-linked and cofilin-decorated states, and that this transition regulates the exclusion of fascin from the cofilactin bundle at the filopodial base by hyper-twisting individual filaments and rearranging their packing. Additionally, we show that cofilactin bundles contribute to the flexibility of filopodial actin networks, thus, likely regulating the efficiency of targeted neurite outgrowth.

scholarly journals Pivotal role of VASP in Arp2/3 complex–mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility

2001 ◽  
Vol 155 (1) ◽  
pp. 89-100 ◽  
Author(s):  
Justin Skoble ◽  
Victoria Auerbuch ◽  
Erin D. Goley ◽  
Matthew D. Welch ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Monomer ◽  
Wild Type ◽  
Binding Region ◽  
Actin Nucleation ◽  
Branch Formation

The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding. Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation. These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L. monocytogenes. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility.

scholarly journals Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

1998 ◽  
Vol 9 (4) ◽  
pp. 841-852 ◽  
Author(s):  
R. Dyche Mullins ◽  
Joseph F. Kelleher ◽  
James Xu ◽  
Thomas D. Pollard
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Analytical Ultracentrifugation ◽  
Acanthamoeba Castellanii ◽  
Leading Edge ◽  
Actin Binding ◽  
Cross Linking ◽  
Motile Cells ◽  
Mechanism Of Interaction ◽  
Cross Links

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motileAcanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.

Vaccinia virus: a model system for actin-membrane interactions

1996 ◽  
Vol 109 (7) ◽  
pp. 1739-1747 ◽  
Author(s):  
S. Cudmore ◽  
I. Reckmann ◽  
G. Griffiths ◽  
M. Way
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Leading Edge ◽  
Virus Particles ◽  
Virion Incorporation ◽  
Motile Cells ◽  
Nucleation Sites ◽  
Tail Assembly

Our understanding of the interactions between the actin cytoskeleton and cellular membranes at the molecular level is rudimentary. One system that offers an opportunity to examine these interactions in greater detail is provided by vaccinia virus, which induces the nucleation of actin tails from the outer membrane surrounding the virion. To further understand the mechanism of their formation and how they generate motility, we have examined the structure of these actin tails in detail. Actin filaments in vaccinia tails are organized so they splay out at up to 45 degrees from the centre of the tail and are up to 0.74 micron in length, which is considerably longer than those reported in the Listeria system. Actin filaments show unidirectional polarity with their barbed filament ends pointing towards the surface of the virus particle. Rhodamine-actin incorporation experiments show that the first stage of tail assembly involves a polarized recruitment of G-actin, and not pre-formed actin filaments, to the membrane surrounding the virion. Incorporation of actin into the tail only occurs by nucleation from the viral surface, suggesting filament ends in the tail are blocked against further actin addition. As virus particles fuse with the plasma membrane during the extention of projections, actin nucleation sites previously in the viral membrane become localized to the plasma membrane, where they are able to nucleate actin polymerization in a manner analogous to the leading edge of motile cells.

scholarly journals Peering deeply inside the branch

2008 ◽  
Vol 180 (5) ◽  
pp. 853-855 ◽  
Author(s):  
Liang Cai ◽  
James E. Bear
Keyword(s):  
Actin Filaments ◽  
Electron Tomography ◽  
Related Protein ◽  
Detailed Structure ◽  
Computational Docking ◽  
Cell Biol

The actin-related protein 2/3 (Arp2/3) story has captivated the cytoskeleton community for over a decade. Not only does this complex nucleate new actin filaments, but it also anchors them into a dendritic meshwork that is used in many cellular contexts such as lamellipodial protrusion, endosome rocketing, and the movement of pathogens. One key piece of this puzzle that has been missing is a detailed structure of the Arp2/3-actin branch. Using electron tomography and computational docking, Rouiller et al. (Rouiller, I., X.-P. Xu, K.J. Amann, C. Egile, S. Nickell, D. Nicastro, R. Li, T.D. Pollard, N. Volkmann, and D. Hanein. 2008. J. Cell Biol. 180:887–895) present an elegant and intriguing structure of the Arp2/3 complex–mediated actin branch.

Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties

1992 ◽  
Vol 50 (1) ◽  
pp. 510-511
Author(s):  
Amy M. McGough ◽  
Robert Josephs
Keyword(s):  
Electron Microscopy ◽  
Elastic Properties ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Projection Algorithm ◽  
Structural Basis ◽  
Iterative Approximation ◽  
Membrane Skeletons

The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).

Modulated polarization microscopy displays the dynamics of cytoskeletal structures in living, motile cells

1995 ◽  
Vol 53 ◽  
pp. 902-903
Author(s):  
J. R. Kuhn ◽  
M. Poenie
Keyword(s):  
Mitotic Spindle ◽  
Actin Filaments ◽  
Cell Shape ◽  
Dynamic Structure ◽  
Living Cells ◽  
Cytoskeletal Proteins ◽  
Polarization Microscopy ◽  
Video Enhancement ◽  
Ultrastructural Studies ◽  
Motile Cells

Cell shape and movement are controlled by elements of the cytoskeleton including actin filaments an microtubules. Unfortunately, it is difficult to visualize the cytoskeleton in living cells and hence follow it dynamics. Immunofluorescence and ultrastructural studies of fixed cells while providing clear images of the cytoskeleton, give only a static picture of this dynamic structure. Microinjection of fluorescently Is beled cytoskeletal proteins has proved useful as a way to follow some cytoskeletal events, but long terry studies are generally limited by the bleaching of fluorophores and presence of unassembled monomers.Polarization microscopy has the potential for visualizing the cytoskeleton. Although at present, it ha mainly been used for visualizing the mitotic spindle. Polarization microscopy is attractive in that it pro vides a way to selectively image structures such as cytoskeletal filaments that are birefringent. By combing ing standard polarization microscopy with video enhancement techniques it has been possible to image single filaments. In this case, however, filament intensity depends on the orientation of the polarizer and analyzer with respect to the specimen.

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