The Arp2/3 complex promotes periodic removal of Pak1-mediated negative feedback to facilitate anticorrelated Cdc42 oscillations
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Abstract
The conserved GTPase Cdc42 is a major regulator of polarized growth in most eukaryotes. Cdc42 periodically cycles between active and inactive states at sites of polarized growth. These periodic cycles are caused by positive feedback and time-delayed negative feedback loops. In the bipolar yeast S. pombe , both growing ends must regulate Cdc42 activity. At each cell end, Cdc42 activity recruits the Pak1 kinase which prevents further Cdc42 activation thus establishing negative feedback. It is unclear how Cdc42 activation returns to the end after Pak1-dependent negative feedback. Using genetic and chemical perturbations, we find that disrupting branched actin-mediated endocytosis disables Cdc42 reactivation at the cell ends. With our experimental data and mathematical models, we show that endocytosis-dependent Pak1 removal from the cell ends allows the Cdc42 activator Scd1 to return to that end to enable reactivation of Cdc42. Moreover, we show that Pak1 elicits its own removal via activation of endocytosis. In agreement with these observations, our model and experimental data show that in each oscillatory cycle, Cdc42 activation increases followed by an increase in Pak1 recruitment at that end. These findings provide a deeper insight into the self-organization of Cdc42 regulation and reveal previously unknown feedback with endocytosis in the establishment of cell polarity.
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The differential regulation between actin structures allows us to specifically target and deplete each structure for our investigations.
Very cool! I love studies taking advantage of this differential regulation to assay how different parts of the actin cytoskeleton contribute to cellular functions!
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I wonder if the intensities of Cdc42 are different in these various treatment conditions? for example, it looks like there's lots more signal in the for3 mutants, but this could just be the images shown here. Additionally, the changes in the level of nuclear localization here are interesting!
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indicating that linear actin cables do not facilitate anticorrelation between the two ends
Maybe it's just the individual cells in the image, but the oscillations, while still present, do look somewhat different in the for3 mutants. For example, according to the red arrows, the frequency of oscillations between the DMSO treated and for3 mutants do not look the same in Figure 1A. I agree with Cameron that showing something like supplemental figure 1A would be super helpful, because with that you can really see the difference between DMSO treated/for3 mutants and CK-666 or LatA treated cells.
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Pak1 activity promotes successful internalization of endocytic vesicles
Very cool! Did you see Pak1 at endocytic sites along the side of the cell? If not, were the success rates similar at the side & at the tip of the cell? It would be neat if the Myo1 in the tip patches are phosphorylated and the Myo1 in the side patches aren't. I know there are a lot of other factors that could contribute to differences in side & tip patch internalization (membrane curvature/hardness, crowdedness, etc) but could be interesting to see!
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Similar to CK-666 treated cells, myo1Δ mutants showed a decrease in Scd1-mNG levels at the cell ends (Fig.5C, D)
Are these images maximum intensity projections or just single z-planes? If its a max intensity, it looks like scd1 might go to endocytic sites in the absence of Myo1?!
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Moreover, in keeping with previous reports, Scd1-mNG levels at the cell ends increases in pak1-ts mutant at permissive temperature (Fig.4A, B)
Oh wow! The nuclear localization of Scd1 in CK-666 goes away in the pak1-ts mutant!!!
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In CK-666 treated cells, in addition to decrease in levels of Scd1-mNG, we also observe bipolar localization in only 37% of cells. This suggests that in the absence of branched actin, Scd1 localization is restricted possibly due to the disruption of its oscillation.
Very cool! It also looks like there is more localization of Scd1 in the nucleus in CK-666 treated cells! Is this true across the dataset or just in the representative images?
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To this end, Cdc42 oscillations were analyzed in four conditions: DMSO, Lat-A, for3Δ, and CK-666. DMSO treatment served as a control (Fig.1A, B and S1A, B).
I found the S1 figure VERY helpful in understanding these claims. You've got such great signal that it is hard to see the differences the in polar signal intensities through the fluorescent images alone. I think it could be a lot easier to interpret with the temporal quantification from S1 shown in the main figure
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The data from each cell was then analyzed to find the correlation coefficient between fluorescent signals of both ends within the cell
Can you expand on how this analysis is done?
Was there a consistently sized ROI used in each cell? The LatA treated cells seemed to favor CRIB localization on the side of the cell instead of the proper tip so I'm curious what fraction of the image goes into the analysis.
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Red arrow heads mark the site of Cdc42 activation
Can you provide more context on how you know this is the site of Cdc42 activation when there is dense CRIB-3xGFP localization on both ends of the cell? and in many places theres no visible fluctuation from the earlier cells to the marked cells
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