Identification of novel proteins controlling persistence of cell migration – NovProtMig

We have identified two new proteins that associate differently with the WAVE complex depending on whether the pathway is activated or not, or whether it is activated but the feedback controls are abrogated by inhibiting the fibers forming the cell skeleton. Surprisingly, these new proteins associate with the proteins forming the shell of the WAVE regulatory complex, but without WAVE, indicating that these proteins take the place of WAVE within this new complex, which we have called the «WAVE shell complex«.

We have purified the WAVE shell complex in order to determine its composition and structure, thanks to direct observation by electron microscopy, which in the best case will allow us to obtain an atom-by-atom resolution of this new molecular machine. We have already revealed the role of the WAVE shell complex in migration persistence. We are seeking to understand the role of the WAVE shell complex in directed migration. Indeed, random cell migration can be biased by the rigidity and composition of the substrate. Cells migrate to the hardest areas and the densest matrix fibers, such as collagen. One of these two new proteins is mutated in cancers, particularly of the uterus, ovaries and breast. The mutated forms are no longer able to associate with the WAVE shell complex and regulate cell migration, confirming the importance of migration persistence in cancer progression.

Our study, therefore, provides hope for finding ways to interfere with the formation of metastases for the greatest benefit of patients with these cancers.

Wang Y, Boutillon A, Fokin AI, Chiapetta G, Vinh J, David NB, Gautreau AM*, Polesskaya A*. 2021. The WAVE shell complex, a novel molecular machine controlling migration persistence. In preparation.

This study, which involves all 3 partners of our consortium, is very likely to become a seminal paper of the field. This is the most direct outcome of the NovProtMig ANR grant.

Most normal cells migrate using actin rich protrusions to crawl on their substrate. Cell migration needs to be simultaneously efficient and responsive to guidance cues. Here we tackle the major challenge of understanding how protrusions can be sustained, or, conversely, quickly retracted in order to pause migration and to change direction. Positive and negative feedback loops, in which polymerized actin modulates its own polymerization, have been demonstrated by modelers to be responsible for this versatile regulation over time. Despite their importance, these signaling circuits are rarely dissected at the molecular level. Our project aims at identifying novel components of actin feedback loops, to characterize their role on cell migration, both in vitro and in vivo, and to dissect the mechanisms by which they control cell persistence.

Actin feedbacks imply that the branched actin network from the protrusion is sensed and that it signals back to the major regulators of actin polymerization. Feedback sensors will be identified through their proximity to the Arp2/3 complex, which creates the branched actin junctions. For this purpose, so called proximity biotinylation assays will be combined to differential proteomics to identify Arp2/3 neighboring proteins in WT cells compared to Rac knock-out cells that are unable to form actin rich protrusions. Feedback effectors will be identified through their differential association with the Arp2/3 activating WAVE complex or the Arp2/3 inhibiting Arpin protein, when actin polymerization is inhibited or not. As an alternative to these proteomic screens, we have also already identified 9 novel Rac effectors through multiple yeast two-hybrid screens using different cDNA libraries.

Our candidates will be tested for their functional importance in cell migration both in vitro for fast quantitative screening and in vivo for physiological relevance. The zebrafish gastrula will be used as a model system allowing straightforward functional genetic approaches and excellent 3D imaging. Our in vitro and in vivo models cover single cell and collective migration, 'random walks' and directed migrations. Using a validated strategy and custom made softwares, we will analyze the best proxies for efficient migration and cell persistence using autocorrelation functions of cell speed and directional migration. Inactivation of our hits through RNAi, morpholinos, and in a second step CRISPR-Cas9, will allow us to focus on the two hits from our screens having the greatest impact on these parameters in vitro and in vivo.

The effect of the two selected hits on membrane protrusion and cytoskeleton dynamics will be analyzed on fast frame rate movies. Dynamic localization of our hit proteins in protrusions will be addressed using GFP fusion proteins. Cross-correlations between these different parameters will be specifically looked for to examine for example if our hits support protrusion or rather appear right before membrane retraction. Finally, we will examine the specific contribution of the hit proteins in migration feedbacks. To this end, we will analyze, using a biosensor, how Rac is activated in response to a localized and transient dose of active Rac delivered through optogenetics. Second, we will measure hysteresis in cell migration, i.e. for how long directional persistence is maintained once a biophysical stimulus that guide migration is switched off. These direct measurements of feedbacks, in wild-type and loss of function situations, will unequivocally place our hits in positive or negative feedback loops.