The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions
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eLife assessment
This manuscript presents valuable findings that bring together two important topics in cell biology: the function of membrane contact sites and cell migration. The authors describe a role of the ER tether protein VAP-A in focal adhesion dynamics and cell motility. Although the authors present solid evidence to support some of the main claims of the paper, some of the other claims would benefit from stronger experimental support. Nonetheless, this paper will be of interest to those cell biologists and biophysicists working on adhesion, migration, and membrane contact site biology.
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Abstract
Cell motility processes highly depend on the membrane distribution of Phosphoinositides, giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane contact sites serve as platforms for direct lipid exchange and calcium fluxes between two organelles. Here, we show that VAPA, an ER transmembrane contact site tether, plays a crucial role during cell motility. CaCo2 adenocarcinoma epithelial cells depleted for VAPA exhibit several collective and individual motility defects, disorganized actin cytoskeleton and altered protrusive activity. During migration, VAPA is required for the maintenance of PI(4)P and PI(4,5)P2 levels at the plasma membrane, but not for PI(4)P homeostasis in the Golgi and endosomal compartments. Importantly, we show that VAPA regulates the dynamics of focal adhesions (FA) through its MSP domain, is essential to stabilize and anchor ventral ER-PM contact sites to FA, and mediates microtubule-dependent FA disassembly. To conclude, our results reveal unknown functions for VAPA-mediated membrane contact sites during cell motility and provide a dynamic picture of ER-PM contact sites connection with FA mediated by VAPA.
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eLife assessment
This manuscript presents valuable findings that bring together two important topics in cell biology: the function of membrane contact sites and cell migration. The authors describe a role of the ER tether protein VAP-A in focal adhesion dynamics and cell motility. Although the authors present solid evidence to support some of the main claims of the paper, some of the other claims would benefit from stronger experimental support. Nonetheless, this paper will be of interest to those cell biologists and biophysicists working on adhesion, migration, and membrane contact site biology.
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Reviewer #1 (Public Review):
Siegfried et al. study a very interesting and timely topic in cell biology: the connection between ER-PM membrane contact sites (MCS) and cell migration. In brief, the authors use the polarized epithelial model cell line (CACO-2) to study this process. They routinely compare parental cells (Control) with a clonal CACO-2 cell line knocked out (KO) for the ER tether protein VAP-A. They convincingly show that KO cells move faster but in a less directional manner, leading to slower monolayer migration. Interestingly, they showed that KO cells have larger focal adhesions (FAs), a phenotype that was reverted upon expression of the wild-type VAP-A but not of a VAP-A mutant (VAP-A-KDMD, mutation in the MSP domain) defective in binding to FFAT-containing partner proteins. Some observations regarding the role of …
Reviewer #1 (Public Review):
Siegfried et al. study a very interesting and timely topic in cell biology: the connection between ER-PM membrane contact sites (MCS) and cell migration. In brief, the authors use the polarized epithelial model cell line (CACO-2) to study this process. They routinely compare parental cells (Control) with a clonal CACO-2 cell line knocked out (KO) for the ER tether protein VAP-A. They convincingly show that KO cells move faster but in a less directional manner, leading to slower monolayer migration. Interestingly, they showed that KO cells have larger focal adhesions (FAs), a phenotype that was reverted upon expression of the wild-type VAP-A but not of a VAP-A mutant (VAP-A-KDMD, mutation in the MSP domain) defective in binding to FFAT-containing partner proteins. Some observations regarding the role of VAP-A's MSP domain on the regulation of the actin cytoskeleton, although the evidence for this was incomplete. Furthermore, VAP-A depletion was shown to have an impact on PI(4,5)P2 levels at the PM (but not on PI(4)P levels at the Golgi membranes), to stabilize the dynamics of ER-PM MCS, and to increase FA lifetime by decreasing FA disassembly rate. Finally, they showed that there is a correlation between the appearance of ER-PM MCS at FAs with FA disassembly, however, how VAP-A plays a role in this effect is unclear. The authors put their findings in the context of the literature in the field to propose a working hypothesis by which VAP-A at ER-PM MCS could impact FA dynamics and cell motility.
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Reviewer #2 (Public Review):
In this study, the authors assessed the role of the ER protein VAPA in cell migration and regulation of focal adhesions dynamics. The authors used CRISPR/Cas9 knock-out of VAPA in Caco-2 cells. They demonstrate that VAPA KO cells have slower migration capacity which is linked to a slower FA disassembly rate. Interestingly, the VAPA KO cells don't show any defects of PI4P level at endosomes nor at the Golgi complex but have a decreased PI4,5P2 level, probably linked to the redundant function of VAPB at endosomes and Golgi while VAPA might be solely responsible for effects on migration.
The results provided by the authors support their conclusions. The experiments performed are well carried out. The VAPA KO cells used in this study are originating from a clonal population but the authors used rescue …
Reviewer #2 (Public Review):
In this study, the authors assessed the role of the ER protein VAPA in cell migration and regulation of focal adhesions dynamics. The authors used CRISPR/Cas9 knock-out of VAPA in Caco-2 cells. They demonstrate that VAPA KO cells have slower migration capacity which is linked to a slower FA disassembly rate. Interestingly, the VAPA KO cells don't show any defects of PI4P level at endosomes nor at the Golgi complex but have a decreased PI4,5P2 level, probably linked to the redundant function of VAPB at endosomes and Golgi while VAPA might be solely responsible for effects on migration.
The results provided by the authors support their conclusions. The experiments performed are well carried out. The VAPA KO cells used in this study are originating from a clonal population but the authors used rescue experiments expressing the VAPA wild-type of the KDMD mutant to demonstrate the role of VAPA in the phenotype. In addition, appropriate and careful quantifications are provided with the different experiments, strengthening the conclusions. The data provided in this manuscript suggest a role for the ER-resident membrane contact protein VAPA in cell migration potentially independent of lipid homeostasis.
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Reviewer #3 (Public Review):
In this manuscript, the authors examine the role of VAPA in focal adhesion (FA) turnover and cell motility via effects on ER-PM contact site functions. The authors show that VAPA KO CaCo2 cells form larger FA and have aberrant migration behavior and spreading. Those cells show lower levels of PI(4,5)P2 at PM, but no change in PI(4)P at Golgi and endosomes. PI(4)P is not tested at the PM. The authors show that VAPA KO cells have a similar number but less stable GFP-MAPPER positive ER-PM contact sites as compared to control cells. In contrast, FA are more stable over time in VAPA KO. The authors also aimed to evaluate GFP-MAPPER proximity with vinculin spots and concluded that ER-PM contacts partially overlap with FA, whereas they are more distant in VAPA KO. Thus, a correlation between stable contact sites …
Reviewer #3 (Public Review):
In this manuscript, the authors examine the role of VAPA in focal adhesion (FA) turnover and cell motility via effects on ER-PM contact site functions. The authors show that VAPA KO CaCo2 cells form larger FA and have aberrant migration behavior and spreading. Those cells show lower levels of PI(4,5)P2 at PM, but no change in PI(4)P at Golgi and endosomes. PI(4)P is not tested at the PM. The authors show that VAPA KO cells have a similar number but less stable GFP-MAPPER positive ER-PM contact sites as compared to control cells. In contrast, FA are more stable over time in VAPA KO. The authors also aimed to evaluate GFP-MAPPER proximity with vinculin spots and concluded that ER-PM contacts partially overlap with FA, whereas they are more distant in VAPA KO. Thus, a correlation between stable contact sites near FA and FA disassembly likely exists. From this set of data, the authors suggest that VAPA has a key role at ER-PM contacts near FA by mediating lipid transfer, which ultimately enables internalization of integrins and FA disassembly.
The approach in the paper is innovative and interesting because VAPA is a major tether at contact sites and the link between contact sites and cytoskeleton dynamics and cell motility remains little explored. This can potentially lead to significant advances in the field. The experiments presented are technically well executed, but most of the results and hypotheses arising from VAPA KO cells are not tested by rescue experiments with exogenous VAPA and VAPA mutants. Although the proposed role for VAPA might fit with the data, the final model is not experimentally tested and is thus highly speculative. The role of VAPA at ER-PM contact sites near FA, and the direct link between VAPA, PI(4,5P)2, and FA disassembly, are not established. VAPA is not shown at ER-PM contacts in the manuscript. Some controls are missing and statistics must be improved. In summary, this work seems to be on the right track, but looks quite preliminary.
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