XB-ART-38957Mol Biol Cell April 1, 2008; 19 (4): 1561-74.
Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells.
A role of Arp2/3 complex in lamellipodia is well established, whereas its roles in filopodia formation remain obscure. We addressed this question in neuronal cells, in which motility is heavily based on filopodia, and we found that Arp2/3 complex is involved in generation of both lamellipodia and filopodia in growth cones, and in neuritogenesis, the processes thought to occur largely in Arp2/3 complex-independent manner. Depletion of Arp2/3 complex in primary neurons and neuroblastoma cells by small interfering RNA significantly decreased the F-actin contents and inhibited lamellipodial protrusion and retrograde flow in growth cones, but also initiation and dynamics of filopodia. Using electron microscopy, immunochemistry, and gene expression, we demonstrated the presence of the Arp2/3 complex-dependent dendritic network of actin filaments in growth cones, and we showed that individual actin filaments in filopodia originated at Arp2/3 complex-dependent branch points in lamellipodia, thus providing a mechanistic explanation of Arp2/3 complex functions during filopodia formation. Additionally, Arp2/3 complex depletion led to formation of multiple neurites, erratic pattern of neurite extension, and excessive formation of stress fibers and focal adhesions. Consistent with this phenotype, RhoA activity was increased in Arp2/3 complex-depleted cells, indicating that besides nucleating actin filaments, Arp2/3 complex may influence cell motility by altering Rho GTPase signaling.
PubMed ID: 18256280
PMC ID: PMC2291425
Article link: Mol Biol Cell
Genes referenced: aagab actl6a actr2 actr3 aicda rho.2 rhoa
GO keywords: Arp2/3 protein complex
Article Images: [+] show captions
|Figure 1. Filopodia initiation in neuronal cells. (A–C) Phase-contrast time-lapse sequences of rat hippocampal neuron (A), differentiated B35 neuroblastoma cell (B), and Xenopus neuron (C). Large panels at left and right show the beginning and the end of the sequence, respectively. Montages in the middle show individual time frames for the boxed regions in the corresponding large panels (B, all; A and C, top) or for other time-lapse sequences (A and C, bottom). Arrows and arrowheads mark individual filopodia formed during the sequence. Filopodia initiation occurs from lamellipodia-like regions and is usually preceded by formation of a small bulge at the leading edge of a growth cone (A–C, top), or at the tip of a secondary neurite (C, bottom), or within a lateral protrusion (A and B, bottom). (D and E) Dynamics of YFP-actin in a growth cone (D) and a lateral protrusion (E) of B35 cells. A corresponding phase contrast sequence is shown for D. Cone-shaped condensation of actin fluorescence precedes filopodia formation (arrows and arrowheads). Time in minutes:seconds. Bars, 5 μm.|
|Figure 2. Arp2/3 complex in neuronal cells. (A–C) Localization of Arp2/3 complex (red) by immunostaining with p34-Arc antibody (A) or p16-Arc antibody (B and C) in hippocampal neurons after 4 d in culture (A), or differentiated B35 (B) or NG-108 neuroblastoma cells (C). (D) Expression of YFP-Arp3 in differentiated B35 cells on a background of depletion of endogenous Arp3. Actin (green) is stained by phalloidin (A–D). Boxed regions for A–C are enlarged at right. Arp2/3 complex is present at the periphery of growth cones and variably in the central domains of growth cones and in neuronal shafts. Bars, 2 μm (A, B, and D) or 5 μm (C).|
|Figure 3. Structural organization of actin network in neuronal growth cones. EM of differentiated B35 cells (A–C), differentiated NG-108 cells (D), Xenopus neurons (E), or hippocampal neurons (F). (A) Overview of a growth cone. (B and D–F) Periphery of growth cones contains branched actin filaments. Boxed regions are enlarged in insets (B) or below main panels (D–F); branched filaments are highlighted in green. (C) Immunogold (18 nm) staining of growth cones with p34-Arc antibody. Arrows point to gold particles located at branch points. Bars, 1 μm (A), 0.2 μm (B and D–F), or 0.1 μm (C).|
|Figure 4. Nascent filopodia contain branched filaments in their roots. Correlative EM of B35 cells. (A) Time-lapse sequence of a growth cone segment shows formation of nascent filopodia (arrows). Black arrow marks formation of a small bulge (0:14), which then forms a filopodium (0:20); the filopodium then becomes partially engulfed by an advancing lamellipodium (0:30–0:36). White arrow marks a small protrusion adjacent to a mature filopodium (0:36). (B) EM of the region shown in A overlaid with a cell contour (yellow line) from 0:14 frame, when the bulge first occurred. Numbered arrows point to corresponding regions in A (0:36); they contain filopodial bundles. Encircled region is enlarged in C. (C–E) High magnifications of nascent filopodia 1 and 2 show branched filaments in their roots (C and D, highlighted in green). Note, that branched filaments in the root of filopodium 2 (C) correspond to the bulge region (B), from which the filopodium emerged. Some actin filaments originated as branches enter the filopodial bundles. Time in minutes:seconds. Bars, 1 μm (A), 0.5 μm (B), or 0.2 μm (D and E).|
|Figure 6. EM of Arp2/3 complex knockdown growth cones of B35 cells. (A) Growth cone of p34-Arc siRNA-treated differentiated cell shows sparse actin network in lamellipodia and few remaining filopodial bundles (compare with Figure 3). (B) Enlarged boxed region from A shows filopodia and lamellipodia in detail. Branched filaments (green) are seen in the sparse lamellipodial network. Many filopodial filaments originate from branch points. Boxed regions in B are further enlarged at bottom without highlighting. (C) Inhibition of lamellipodia in growth cones of differentiated p34-Arc siRNA-treated cells, as revealed by quantification of the lamellipodium width (left), density of actin filament branches (middle), or density of gold particles after immunostaining with p16-Arc antibody (right) (n = 6 cells for each). Branch and immunogold densities are plotted against the distance from the leading edge. (D–F) Correlative EM of nascent filopodia in p34-Arc siRNA-treated differentiated B35 cells. (D) Time-lapse sequence of a growth cone shows formation of a small bulge at the end of the sequence (arrow). (E) EM of the region corresponding to the boxed area in E. Arrow marks a filopodial bundle formed at the position of the bulge. (F) Enlarged boxed region in E. Branched filaments (green) are seen in the root of this thin nascent filopodium. Time in minutes:seconds. Bars, 2 μm (A and D), 0.5 μm (B and E), or 0.2 μm (F).|
|Figure 10. Actin filament organization (A) and the mechanism of filopodia formation (B–D) in neuronal growth cones. (A) Actin filaments (black lines) at the periphery of growth cones are organized into bundles of long filaments in filopodia and branching networks in lamellipodia; these filaments do not form distinct sets, but lamellipodial filaments may branch off of the filopodial filaments and/or merge into filopodial bundles. (B) New filopodia usually form from interfilopodial networks that are rich in Arp2/3 complex (yellow hearts) and branching actin filaments (blue hearts). (C) Arp2/3 complex-nucleated filaments elongate persistently with an assistance of anti-capping molecules (purple rings) and converge to form a bulge-like protrusion at the site of an incipient filopodium. (D) Continued elongation of barbed ends with a concomitant bundling by a cross-linker (green crosses) results in filopodia formation.|
References [+] :
Adams, Roles of fascin in cell adhesion and motility. 2004, Pubmed