Direct Arp2/3-vinculin binding is essential for cell spreading, but only on compliant substrates and in 3D

Tadamoto Isogai, Kevin M. Dean, Philippe Roudot, Qiuyan Shao, Justin D. Cillay, Erik. S. Welf, Meghan K. Driscoll, Shaina P. Royer, Nikhil Mittal, Bo-Jui Chang, Sangyoon J. Han, Reto Fiolka, Gaudenz Danuser

Preprint posted on 4 September 2019

2D or 3D: Cells mould themselves.

Selected by Ankita Jha


The actin cytoskeleton is actively remodeled in response to differential extra-cellular matrix (ECM) cues. Thus, the architectural and the chemical composition of ECM can regulate complex cell shapes. How cells respond to the different ECM environment are one of the major questions in the field. It is also becoming clear that response of cells to mechanochemical changes encountered in2D and 3D environments occurs through adhesion complexes with different mechanisms in place (Doyle and Yamada et al 2015).

Major findings:

To understand how cells respond to different ECM environments, i.e. on stiff 2D planar and 3D reconstituted soft environments, the authors performed a CRISPR-Cas9 knock-out screen of actin binding proteins (ABPs) and then characterized the cell shapes after cells were plated on the planar surfaces like glass (2D) and also in 3D collagen matrices. Knock-out of actin nucleators FMNL1 and Arp3 revealed difference in the spreading between 2D and 3D. In order to understand if the architecture of the fibrillar collagen or the stiffness of the ECM is important for the spreading of the cells, authors looked at the spreading of cells on the collagen coated on the glass and also silicone-based substrates with similar stiffness like that of collagen but without any fibrils. In both cases Arp3 knock out showed reduced spreading, which suggested that spreading is a combination of stiffness and architectural features. In 3D, cells show elongated adhesion compared to Arp3 KO cells, whereas in 2D no such differences were seen. Authors suggest the presence of a compensatory pathways in 2D, and also show that a phospho-specific Arp3-vinculin interaction is important for cells to spread in soft, 3D collagen environments.

What I like about the pre-print:

This work along with others shed light on the how cells sense their micro-environment. Cells on 2D form more lamellopodial like protrusions compared to 3D. It is also very clear from the experiments that cells respond to the difference in the stiffness of the substrate, which is uniform in 2D and also to the architectural make of the 3D like the fibers of collagen. It is becoming increasingly clear that the cells mechano-sensing on the 2D substrates involves dynamic interactions between vinculin, talin and actin filaments generating a molecular clutch (Hu et al., 2007). It is not clear if there are difference in the mechano-sensing mechanisms between 2D and 3D, and we have only begun to understand the differences that might exist.

What’s next and my questions to the authors:

Cells in 2D largely depend on the ECM stiffness and those in 3D depend on the fibril architecture, cross-linking, ECM adhesion ligand density and pore size of the fibrils. This makes it difficult to compare between 2D and 3D ECM. There lies a large conundrum about the 3D matrix where the overall macro gel stiffness differs from the fiber stiffness that cells witness in 3D (Guthold et al. 2007). I wonder how authors plan to answer this, if the difference in the spreading difference they see between 2D and 3D is because of the fibril stiffness that is seen by the cells? Vinculin knock-out in MEFs have been shown to change morphology in 3D but not in 2D (Thievessen et al., 2015). It will be interesting to see if the binding partners of vinculin changes based on the conformation it shows in 2D versus 3D. This could be a result of the Rho/Rac activity in the cells (Deakin et al 2011). Vinculin mutant where it does not interact with Arp3 show diminished lamellipodia and spreading on fibronectin in 2D (DeMali et al, 2002) similar to what authors have in 3D. It would be interesting to see if this could be a combination of the available adhesive ligands in 2D and stiffness together effects the spreading of cells. Authors mention at the different mechanistic pathways and mechanisms in 2D and 3D, it would be great if any of the of the components of these pathways were tested in future. For example, does the clutch model exist in 3D?


Deakin, Nicholas O., and Christopher E. Turner. 2011. “Distinct Roles for Paxillin and Hic-5 in Regulating Breast Cancer Cell Morphology, Invasion, and Metastasis.” Molecular Biology of the Cell 22 (3): 327–41.

DeMali, Kris A., Christy A. Barlow, and Keith Burridge. 2002. “Recruitment of the Arp2/3 Complex to Vinculin: Coupling Membrane Protrusion to Matrix Adhesion.” The Journal of Cell Biology 159 (5): 881–91.

Doyle, Andrew D., and Kenneth M. Yamada. 2016. “Mechanosensing via Cell-Matrix Adhesions in 3D Microenvironments.” Experimental Cell Research 343 (1): 60–66.

Guthold, M., W. Liu, E. A. Sparks, L. M. Jawerth, L. Peng, M. Falvo, R. Superfine, R. R. Hantgan, and S. T. Lord. 2007. “A Comparison of the Mechanical and Structural Properties of Fibrin Fibers with Other Protein Fibers.” Cell Biochemistry and Biophysics 49 (3): 165–81.

Hu, Ke, Lin Ji, Kathryn T. Applegate, Gaudenz Danuser, and Clare M. Waterman-Storer. 2007. “Differential Transmission of Actin Motion within Focal Adhesions.” Science 315 (5808): 111–15.

Thievessen, Ingo, Nikta Fakhri, Julian Steinwachs, Viola Kraus, R. Scott McIsaac, Liang Gao, Bi-Chang Chen, et al. 2015. “Vinculin Is Required for Cell Polarization, Migration, and Extracellular Matrix Remodeling in 3D Collagen.” FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology 29 (11): 4555–67.

Tags: cellspreading, extracellularmatrix

Posted on: 15 September 2019 , updated on: 24 September 2019


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Author's response

Tadamoto Isogai, Kevin M. Dean,Gaudenz Danuser shared

We would like to thank Ankita for writing this prelight and for sharing our enthusiasms for this work. We would like to clarify that the intent of the study was not necessary to study how cells mechanically sense the extracellular matrix, but using genetic perturbations experiments to study how the actin cytoskeleton responds and contributes to cellular response to varying ECM conditions (i.e. stiff planar 2D or soft 3D gels). In this study we focused on how cell spreading is affected on 2D and 3D environments.

The knockout of the Arp2/3 complex had the most prominent effects in 3D cell spreading. The surprise, however, comes from the stark contrast of phenotype observed between 2D and 3D conditions. With further detailed analyses, we further highlight that the direct interaction of Arp2/3 with vinculin is essential for vinculin function during cell spreading in 3D (or soft substrates). Intriguingly, the actin-vinculin interaction, which has been thought to play a more dominant role based on extensive studies on 2D substrates, is more dispensable compared to the Arp2/3-vinculin interaction in 3D. The spreading defects that Dr. DeMali and colleagues reported back in 2002 measures the circularity of the cells (De Mali et al., 2002), yet both vinculin KO cells as well as the KO cells reconstituted with the Arp2/3-binding deficient vinculin mutant still spread in our view.

In conclusion, although we here focus on the Arp2/3-vinculin interaction, we want to stress that this is just one example of what we believe many other molecular interactions that are specifically required for a cellular process such as cell spreading in an ECM context-dependent manner. Thus, we are excited to re-evaluate molecular requirements in apparently established pathways to unveil their critical functions specific to the ECM context.



DeMali, Kris A., Christy A. Barlow, and Keith Burridge. 2002. “Recruitment of the Arp2/3 Complex to Vinculin: Coupling Membrane Protrusion to Matrix Adhesion.” The Journal of Cell Biology 159 (5): 881–91.

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