Cooperative actin filament nucleation by the Arp2/3 complex and formins maintains the homeostatic cortical array in Arabidopsis epidermal cells
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Abstract
Precise control over how and where actin filaments are created leads to the construction of unique cytoskeletal arrays within a common cytoplasm. Actin filament nucleators are key players in this activity and include the conserved actin-related protein 2/3 (Arp2/3) complex as well as a large family of formins. In some eukaryotic cells, these nucleators compete for a common pool of actin monomers and loss of one favors the activity of the other. To test whether this mechanism is conserved, we combined the ability to image single filament dynamics in the homeostatic cortical actin array of living Arabidopsis (Arabidopsis thaliana) epidermal cells with genetic and/or small molecule inhibitor approaches to stably or acutely disrupt nucleator activity. We found that Arp2/3 mutants or acute CK-666 treatment markedly reduced the frequency of side-branched nucleation events as well as overall actin filament abundance. We also confirmed that plant formins contribute to side-branched filament nucleation in vivo. Surprisingly, simultaneous inhibition of both classes of nucleator increased overall actin filament abundance and enhanced the frequency of de novo nucleation events by an unknown mechanism. Collectively, our findings suggest that multiple actin nucleation mechanisms cooperate to generate and maintain the homeostatic cortical array of plant epidermal cells.
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left-skewed
There are a couple of these graphs where we're looking at the distribution of data and there's mention that there's more of a skew in a sample compared to another, but sometimes this is really difficult to see in the graphs. It might be useful to split out arp2 mutants vs arpc2 mutants so that we're comparing 2 instead of 4. It might also be useful if you could include some measure of skew, especially for the ones where you think there might be a difference in skew.
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Simultaneous Inhibition of the Arp2/3 Complex and Formins Enhances de novo Actin Filament Nucleation in Arabidopsis thaliana
What a cool paper! I think papers dealing with different proteins interacting with a single pool of actin are so interesting. Also super awesome that you validate CK-666 in these cells and start dealing with potential off-target effects of SMIFH2 in these cells - so much added value to the field!
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CK-666 (0, 1, 5, 10, 50, and 100 µM)
I think you do a great job here and in the next figure validating CK-666 (definitely showing that there isn't added effect in the Arp2/3 complex mutants is great!), but you might consider adding CK-689 as an inactive control just to cover all your bases.
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In addition, hypocotyls of arp2-1 and arpc2 were significantly shorter than wild-type siblings at the same time points
It would be interesting to see specific data points here instead of bars for these graphs. Based on the representative images, it looks like there could be a couple population with different lengths, especially in the arpc2 hypocotyls. If that's the case, it would also be interesting to hear why that might be.
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demonstrated the effectiveness
Do we know that SMIFH2 broadly inhibit all of the formins in Arabidopsis?
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We found that side-branched filaments in arp2-1 cells were significantly longer (Fig. 4 C) and filament lifetime was prolonged (Fig. 4 D) compared to the side-branched filaments in wild-type cells
Interesting, is the hypothesis that some of these formins that nucleate filaments off the side of existing filaments are responsible for these specific branched filaments?
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We found that side-branched filaments in arp2-1 cells were significantly longer (Fig. 4 C) and filament lifetime was prolonged (Fig. 4 D) compared to the side-branched filaments in wild-type cells
Interesting, is the hypothesis that some of these formins that nucleate filaments off the side of existing filaments are responsible for these specific branched filaments?
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CK-666 (0, 1, 5, 10, 50, and 100 µM)
I think you do a great job here and in the next figure validating CK-666 (definitely showing that there isn't added effect in the Arp2/3 complex mutants is great!), but you might consider adding CK-689 as an inactive control just to cover all your bases.
-
Simultaneous Inhibition of the Arp2/3 Complex and Formins Enhances de novo Actin Filament Nucleation in Arabidopsis thaliana
What a cool paper! I think papers dealing with different proteins interacting with a single pool of actin are so interesting. Also super awesome that you validate CK-666 in these cells and start dealing with potential off-target effects of SMIFH2 in these cells - so much added value to the field!
-
demonstrated the effectiveness
Do we know that SMIFH2 broadly inhibit all of the formins in Arabidopsis?
-
-
left-skewed
There are a couple of these graphs where we're looking at the distribution of data and there's mention that there's more of a skew in a sample compared to another, but sometimes this is really difficult to see in the graphs. It might be useful to split out arp2 mutants vs arpc2 mutants so that we're comparing 2 instead of 4. It might also be useful if you could include some measure of skew, especially for the ones where you think there might be a difference in skew.
-
In addition, hypocotyls of arp2-1 and arpc2 were significantly shorter than wild-type siblings at the same time points
It would be interesting to see specific data points here instead of bars for these graphs. Based on the representative images, it looks like there could be a couple population with different lengths, especially in the arpc2 hypocotyls. If that's the case, it would also be interesting to hear why that might be.
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