Invited Lecture

Karen L. Anderson 1 Praveen Suraneni 2 Mark F. Swift 1 Christopher Page 1 Mandy E.W. Janssen 1,4 Thomas D. Pollard 3 Rong Li 2 Niels Volkmann 1 Dorit Hanein 1
1Bioinformatics and Structural Biology Program, Sanford–Burnham Medical Research Institute, La Jolla, CA, USA
2Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
3Departments of Molecular, Cellular and Developmental Biology, of Cell Biology and of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
4Chemistry & Biochemistry and Molecular Biology, University of California, San Diego, La Jolla, CA, USA

Cells employ protrusive leading edges to navigate and promote their migration in diverse physiological environments. Classical models of leading edge protrusion rely on a treadmilling dendritic actin network that undergoes continuous assembly nucleated by the Arp2/3 complex, forming ruffling lamellipodia. Although the dendritic nucleation model has been rigorously evaluated in several computational studies. Experimental evidence demonstrating a critical role for Arp2/3 in the generation of protrusive actin structures and cell motility has been far from clear. Most components of the pathway have been probed for their relevance by RNA interference or dominant-negative constructs. However, given that the Arp2/3 complex nucleates actin at nanomolar concentrations, even a dramatic knockdown could still leave behind a level sufficient to fully or partially support Arp2/3-complex dependent functions.

Our recent work characterizes newly developed fibroblasts cell lines completely lacking functional Arp2/3 complex. We tested the impact Arp2/3-complex function on these genetically matched cells with and without Arp2/3 complex using single cell spreading assays, wound healing assays, long-time single cell motility tracking, chemotaxis assays, and fluorescence imaging with confocal or structured illumination microscopy [1,2]. In the absence of functional Arp2/3 complex, the fibroblasts were unable to extend lamellipodia but generated dynamic leading edges composed primarily of filopodia-like protrusions (FLPs), with formin proteins (mDia1 and mDia2) concentrated near their tips. These mutant fibroblasts maintained an ability to move but exhibited a strong defect in persistent directional migration in both wound healing and chemotaxis assays [1,2]. Here, we will highlight our advances in determining the molecular-level organization of the actin networks of these cells through an integrated approach that employs electron cryo-tomography of whole mammalian cells in conjunction with correlative light microscopy.


[1] Suraneni P, Rubinstein B, Unruh JR, Durnin M, Hanein, D, and Li R (2012). The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration. J Cell Biol. 197, 239.

[2] Suraneni P, Fogelson B, Rubinstein B, Noguera P, Volkmann N, Hanein D, Mogilner A, Li R (2015). A mechanism of leading edge protrusion in the absence of Arp2/3 complex. Mol Biol Cell. 26:901-912.

[3] This work is supported by NIGMS grants P01 GM066311 and P01 GM098412.

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