The adherens junction proteins α-catenin, vinculin and VASP cooperate to promote actin assembly
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Abstract
The cohesion of tissues requires that cells establish cell-cell junctions. Cells contact each other by forming Arp2/3-dependent lamellipodia before they initiate the formation of cadherin-based adherens junctions (AJs). Maturing AJs then assemble actin under force though the formation of a mechanosensitive complex comprising the actin-binding proteins α-catenin, vinculin and VASP, which individually act on the nucleation, elongation and organisation of actin filaments in different ways. However, the activity of the ternary complex that these actin-regulatory proteins form has not been investigated due to the difficulty of assembling this complex in vitro in the absence of force. Here, we first designed mutants of these proteins that interact independently of force. We then studied their activity by combining actin polymerization kinetics in fluorescence spectroscopy with observation of single actin filaments in TIRF microscopy. Our results reveal how α-catenin, vinculin and VASP combine their activities in a complex to inhibit Arp2/3-mediated branching, stimulate the nucleation and elongation of linear actin filaments from profilin-actin and crosslink these filaments into bundles. These findings shed light on the molecular mechanisms by which actin regulators synergistically control the transition of actin architecture and dynamics that accompanies the formation and maturation of AJs.
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Finally, our biochemical study must be placed in the context of cell-cell junctions
I think it would be useful to talk again about the effect of the different constructs that you used in the context of what that means biologically in the cell.
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We also observed, when the three proteins were combined, an increase in the density and length of actin filaments produced in the presence of profilin
I think this is obviously quite visible in the images, but it would be interesting to see a quantification of filament length in each case and also the occurrence of these bright bundle-like regions. I know you do this more precisely in the next section but I think this would also help set that section up a little bit.
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Observation of the details of single filament elongation revealed that it was regularly interrupted by marked pauses, reflecting capping events of the barbed ends by V1ab4 and Δmod
It would be super cool to see some images along with these graphs looking at single filament elongation.
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actin dynamics observed in cells
I am interested what this would look like in cells! Maybe a next step?
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Representative epifluorescence images of Alexa-488 actin filament bundles observed alone and in the presence of all the combinations of Δmod, V1ab4 and VASP. Conditions: 1.5 μM G-actin (2%Alexa-488-labeled), 0.25 μM Δmod, 0.25 μM V1ab4 and 0.25 μM VASP. Scale bar = 15 μm.
These are amazing!
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Kinetics of actin polymerization were measured in the presence of 2 μM G-actin (10% pyrene-labelled), 50 nM Arp2/3, 100 nM VCA, 5 μM Δmod, 5 μM V1ab4 and 3 μM VASP, in conditions that allow nucleation and elongation (50 mM KCl).
Actin + Arp2/3 + VCA + delta-mod (cyan) is interesting - it looks like nucleation is delayed, but then is able to rapidly at least approach the same level as Arp2/3 + VCA. I'd love to hear some more thoughts about this.
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These findings shed light on the molecular mechanisms by which actin regulators synergistically control the transition of actin architecture and dynamics that accompanies the formation and maturation of AJs.
This is a very interesting paper looking out how different actin binding proteins work together to regulate actin in specific functions!
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The direct observation of this reaction in TIRF microscopy revealed a progressive reorganization of the branched actin network by Δmod, V1ab4 and VASP
I think it would go a long way to support your concluding hypotheses if you did a quantification of branching for Figure 6C because it is a little hard to make out with the density of the filaments and because it is so important for your conclusions.
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The fact that VASP and α-catenin/Δmod, which are not known to interact directly, stimulated actin assembly in a synergistic manner was not expected, but suggests that these two ABPs cooperate when combined in the same protein complex.
It would be interesting to look at direct binding between these proteins, especially because they are mutants that might not behave quite as expected.
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These findings shed light on the molecular mechanisms by which actin regulators synergistically control the transition of actin architecture and dynamics that accompanies the formation and maturation of AJs.
This is a very interesting paper looking out how different actin binding proteins work together to regulate actin in specific functions!
-
Finally, our biochemical study must be placed in the context of cell-cell junctions
I think it would be useful to talk again about the effect of the different constructs that you used in the context of what that means biologically in the cell.
-
actin dynamics observed in cells
I am interested what this would look like in cells! Maybe a next step?
-
The fact that VASP and α-catenin/Δmod, which are not known to interact directly, stimulated actin assembly in a synergistic manner was not expected, but suggests that these two ABPs cooperate when combined in the same protein complex.
It would be interesting to look at direct binding between these proteins, especially because they are mutants that might not behave quite as expected.
-
The direct observation of this reaction in TIRF microscopy revealed a progressive reorganization of the branched actin network by Δmod, V1ab4 and VASP
I think it would go a long way to support your concluding hypotheses if you did a quantification of branching for Figure 6C because it is a little hard to make out with the density of the filaments and because it is so important for your conclusions.
-
Kinetics of actin polymerization were measured in the presence of 2 μM G-actin (10% pyrene-labelled), 50 nM Arp2/3, 100 nM VCA, 5 μM Δmod, 5 μM V1ab4 and 3 μM VASP, in conditions that allow nucleation and elongation (50 mM KCl).
Actin + Arp2/3 + VCA + delta-mod (cyan) is interesting - it looks like nucleation is delayed, but then is able to rapidly at least approach the same level as Arp2/3 + VCA. I'd love to hear some more thoughts about this.
-
Representative epifluorescence images of Alexa-488 actin filament bundles observed alone and in the presence of all the combinations of Δmod, V1ab4 and VASP. Conditions: 1.5 μM G-actin (2%Alexa-488-labeled), 0.25 μM Δmod, 0.25 μM V1ab4 and 0.25 μM VASP. Scale bar = 15 μm.
These are amazing!
-
We also observed, when the three proteins were combined, an increase in the density and length of actin filaments produced in the presence of profilin
I think this is obviously quite visible in the images, but it would be interesting to see a quantification of filament length in each case and also the occurrence of these bright bundle-like regions. I know you do this more precisely in the next section but I think this would also help set that section up a little bit.
-
Observation of the details of single filament elongation revealed that it was regularly interrupted by marked pauses, reflecting capping events of the barbed ends by V1ab4 and Δmod
It would be super cool to see some images along with these graphs looking at single filament elongation.
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