In this work, we have established a stable Arp2/3-depleted cell line that allowed us to study random and directional cell motility in the absence of lamellipodia. The depletion of Arp2/3 complex causes striking changes in cell morphology, motility and global focal adhesion geometry. Furthermore our study also reveals the essential role of lamellipodia in fibroblast haptotaxis, but not chemotaxis.
Establishment of a stable Arp2/3 depleted cell line
It is widely believed that Arp2/3 complex is required for viability in eukaryotic organisms, however our data suggest that this is not strictly true in mammalian cells in culture. It is worth considering why we were able to recover stable cell lines depleted of >98% of Arp2/3 complex. First, we quickly separated the cells as clones after they were infected with the shRNA-expressing lentiviruses through the use of fluorescent protein reporters rather than relying on drug resistance. Any selective growth advantage conferred by less than full knockdown is cancelled out under these circumstances. Although we initially had some concerns about using clones, these were put to rest by the microinjection rescue experiment that indicated that these cells were still capable of making lamellipodia within minutes of the re-introduction of the Arp2/3 complex. Second, we depleted two essential subunits (p34Arc and Arp2) to create a “fail-safe” condition where loss of either shRNA conferred no advantage due to the continued absence of another essential subunit. Third, the complexity of mammalian cells may have been working in our favor. Simpler eukaryotes may be strictly dependent on Arp2/3 activity for viability-associated processes such as nutrient uptake, while mammalian cells have a multitude of partially redundant pathways. Finally, we performed these experiments in the Ink4a/Arf
-deficient background. These genes are well known as tumor suppressors that arrest cell growth upon oncogenic insult (Kim and Sharpless, 2006
). Our results indicate that cells harboring an intact Arf gene are much more sensitive to the loss of Arp2/3 complex, suggesting that the deranged actin cytoskeleton resulting from the loss of Arp2/3 activity may trigger an Arf-dependent growth arrest pathway.
The relationship between Arp2/3 complex and lamellipodia
The most striking morphological change we observed in the Arp2/3 complex depleted cells was the disappearance of lamellipodia. We report several lines of evidence to support the idea that the phenotypic loss of lamellipodia is due to a specific loss of Arp2/3 complex activity. Both shRNAs used to deplete p34Arc and Arp2 respectively could be fully rescued by co-expression of RNAi-resistant versions of these genes. Pharmacological inhibition of Arp2/3 complex by CK-666 produced a similar, although slightly milder phenotype as the double depletion of p34Arc and Arp2. Importantly, this compound had no further effect on the 2xKD cells, indicating that any residual Arp2/3 activity was not contributing to the residual motility. Furthermore, lamellipodia could be recovered in the 2xKD cells within minutes of microinjection of bovine Arp2/3 complex. Therefore, the 2xKD cells are not only useful in the study of Arp2/3 complex dependent functions, but also serve as useful tools to dissect the functional role of lamellipodia in cell motility.
Previous studies have shown that the actin networks within lamellipodia contain short, branched filaments nucleated by Arp2/3 complex (Svitkina and Borisy, 1999
), consistent with observations of networks generated with purified Arp2/3 complex and actin (Mullins et al., 1998
). Recently, this model has been called into question based on an alternate electron microscopy protocol (Urban et al., 2010
). Using this technique, mostly long actin filaments were observed in the lamellipodia. Our results with the cryo-shadowing technique are entirely consistent with the notion of highly branched networks of short actin filaments in lamellipodia as originally observed by platinum replica EM (Svitkina and Borisy, 1999
). Further evidence for the role of Arp2/3 complex in branch generation comes from the apparent lack of these structures in cells with near total depletion of Arp2/3 complex.
In the absence of branched actin generated by the Arp2/3 complex, more filopodial protrusions form on the periphery of cells. 2xKD cells engage in a form of filopodia-dependent cell motility, in which new flat cell surface area is created by “filling the gap” between adjacent filopodia. The filopodia of 2xKD cells protrude and bend until they become anchored to the underlying matrix, at which time the membrane between two filopodia gradually fills-in. When myosin II was inhibited in the 2xKD cells using blebbistatin, we observed increased filopodia number and “filling the gap” behavior, and a proportional increase in cell speed. This suggests that the Arp2/3 depleted cells rely on this filopodia-initiated “filling the gap” motility to migrate. This form of motility is relatively inefficient compared to motility using lamellipodia; however, efficiency of motility does not necessarily relate to the ability of cells to sense or respond to directional cues.
The role of lamellipodia in chemotaxis
Chemotaxis is required for numerous physiological processes and has been the subject of intense study for well over a century. The most successful model systems have been the social amoeba Dictyostelium
and amoeboid cells of the haematopoietic system such as neutrophils (Parent, 2004
). Studies of chemotaxis in amoeboid cells and other cell types have largely focused on the signal transduction cascades that connect cell surface receptors to the polymerization of actin at the protrusive leading edge. As lamellipodia are the actin-rich, protrusive organelle in fibroblasts, we fully anticipated that depletion of Arp2/3 complex and the resulting loss of lamellipodia would have a profound impact on fibroblast chemotaxis. To our surprise, depletion of Arp2/3 complex has no effect whatsoever on fibroblast chemotaxis up shallow gradients of PDGF, other than affecting the speed at which the cells crawl. Although stimulated actin assembly may still be important for chemotactic response, this assembly clearly does not involve the Arp2/3 complex in this cell type. However, other forms of actin assembly such as those that lead to the increased filopodia on the 2xKD cells may be important for chemotaxis. It is also worth noting that our results may not translate to amoeboid cell chemotaxis, which may require Arp2/3 activity. Future studies will focus on testing the role of alternate actin assembly pathways in fibroblast chemotaxis and testing the generality of our findings for other cell types.
Although the normal chemotaxis of Arp2/3-depleted fibroblasts is surprising, several studies point toward a more complex picture of chemotaxis than previously appreciated. Transient depletion of either the Cdc42 or Rac1 small GTPase or both does not affect the ability of fibroblasts to respond to gradients of PDGF (Monypenny et al., 2009
). Similar to our cells, these depletions affect the morphology of the leading edge without affecting chemotaxis, although some non-GTPase dependent activation of Arp2/3 cannot be excluded. Another enzyme that was initially thought to be crucial for chemotaxis, PI-3 Kinase, is also not strictly required for chemotaxis of Dictyostelium
amoebae based on genetic studies (Hoeller and Kay, 2007
) or fibroblast chemotaxis towards PDGF based on pharmacological inhibition of this enzyme (Melvin et al., 2011
). Together with our results, these studies highlight the complexity of chemotactic mechanisms and the insufficiency of current models in explaining this process.
The role of lamellipodia in sensing/responding to changes in ECM
Our results with the Arp2/3 depleted cells reveal the importance of Arp2/3 complex and lamellipodia in sensing and responding to the changes in the ECM. Several groups have reported that cells exhibit a biphasic motility response to variable extracellular matrix concentration with fast migration occurring at intermediate ECM concentration and slower migration occurring at low and high ECM concentrations (DiMilla et al., 1993
; Gupton and Waterman-Storer, 2006
). The molecular basis of this biphasic response is thought to involve spatiotemporal feedback between actomyosin and focal adhesion systems (Gupton and Waterman-Storer, 2006
); however, the relationship between this response and specific cellular architecture is unclear. Our data indicate that Arp2/3 and lamellipodia are required for the biphasic response of cells to variable ECM. Stated another way, the adhesions formed by the filopodial protrusions on the 2xKD cells are insufficient to allow cells to sense or respond to these differences in ECM concentration. This suggests that the adhesions formed within lamellipodia have qualitatively different properties that allow the whole cell to coordinate global motility.
Cells not only can regulate velocity when migrating on different concentrations of ECM, but also can sense and respond to ECM gradients and migrate in a directional way. This process, termed haptotaxis, requires cells to 1) sense differences of ECM concentration/engagment across a single cell, 2) polarize cytoskeletal and motility machinery, and 3) migrate up the gradient. Our data show that Arp2/3 depletion and subsequent loss of lamellipodia completely ablates haptotaxis. Importantly, this was true not only on gradients of fibronectin, but also on gradients of laminin or vitronectin, ECMs that require different integrin heterodimers. One outstanding question about the role of lamellipodia in haptotaxis is whether this structure is involved in sensing the gradient or responding to the gradient or both. Considering the intact PDGF chemotactic response without Arp2/3 complex, the loss of haptotaxis appears to be a specific effect rather than a general defect in direction sensing. This remarkable difference in the need for lamellipodia between chemotaxis and haptotaxis suggests that cells use very different mechanisms to sense and respond to these different directional cues.
Spatial organization of cell-matrix adhesions and global cell motility
Although focal adhesions have been intensively studied for decades, the processes that spatially organize these structures in an ensemble manner across the whole cell are poorly understood. Our results indicate that lamellipodia play a major role in bringing spatial coherence to focal adhesion formation. Without lamellipodia, focal adhesions are poorly aligned to each other, which may explain why these cells migrate slowly. An open question is how lamellipodia promote the alignment of focal adhesions. One possibility is that the retrograde flow of actin networks in lamellipodia, which is itself spatially coherent over 0.5–5 µm length scales, could promote the alignment of the adhesions that form and mature within this flow field. However, how the alignment of focal adhesions within lamellipodia contribute to global alignment is less clear. Since the rear of the cell was once the front the cell, this alignment may reflect the history of these adhesions as born within previous lamellipodia. How cells manage to define a new axis of focal adhesion alignment upon turning will be the subject of future studies.
Our observations also provide a conceptual framework for linking events occurring at small length scales such as the formation of branches by the Arp2/3 complex (~10 nm) to whole cell motility at much longer length scales (~100 µm) (). Branched actin networks generated by the Arp2/3 complex are the main component of the lamellipodial cytoskeleton. As we have shown in this paper, lamellipodia are critical for the alignment of focal adhesions. Cells with aligned focal adhesions will have aligned stress fibers attached to those adhesions. Since stress fibers are the main contractile structure of fibroblasts, cells with aligned focal adhesions will have more coherent contractility. We postulate that this coherent contractility will contribute directly to efficient whole cell migration. This overall notion is consistent with our observation that a strong relationship exists between the fibronectin concentration the cells are plated on, the global alignment of focal adhesions, and their increased cell speed. Future studies will focus on testing this hypothesis in context of other 2D and 3D motility events.