Arp2/3 complex-mediated actin polymerisation occurs on specific pre-existing networks in cells and requires spatial restriction to sustain functional lamellipod extension.
The classical Arp2/3-mediated dendritic network defines the cytoskeleton at the leading edge of crawling cells, and it is generally assumed that Arp2/3-mediated actin polymerization generates the force necessary to extend lamellipods. Our previous work suggested that successful lamellipod extension required not only free barbed ends for actin polymerization but also a proper ultrastructural organization of the cytoskeleton. To further explore the structural role of the Arp2/3 complex-mediated networks in lamellipod morphology and function, we performed a detailed analysis of the ultrastructure of the Arp2/3-mediated networks, using the WA domains of Scar and WASp to generate mislocalised Arp2/3 networks in vivo, and to reconstruct de novo Arp2/3-mediated actin nucleation and polymerization on extracted cytoskeletons. We present here evidence that spatially unrestricted Arp2/3-mediated networks are intrinsically three-dimensional and multilayered by nature and, as such, cannot sustain significant polarized extension. Furthermore, such networks polymerize only at preferred locations in extracted cells, corresponding to pre-existing Arp2/3 networks, suggesting that the specific molecular organization of the actin cytoskeleton, in terms of structure and/or biochemical composition, dictates the location of Arp2/3 complex-mediated actin polymerization. We propose that successful lamellipod extension depends not only on localized actin polymerization mediated through local signalling, but also on spatial restriction of the Arp2/3 complex-mediated nucleation of actin polymerization, both in terms of location within the cell and ultrastructural organization of the resulting network.
Shao
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Division of Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom.
Forge
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Munro
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Bailly
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D051376:Actin-Related Protein 2-3 Complex / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000737:chemistry; D045744:Cell Line, Tumor; D003599:Cytoskeleton / Q000187:drug effects* / Q000648:ultrastructure; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000473:pathology; D008845:Microinjections; D018482:Muscle, Skeletal / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000502:physiology*; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012235:Rhodamines; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D005456:Fluorescent Dyes; D011108:Polymers; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D051316:Wiskott-Aldrich Syndrome Protein; C009434:tetramethylrhodamine isothiocyanate; D004815:Epidermal Growth Factor; D005982:Glutathione Transferase; D016650:Fluorescein-5-isothiocyanate