Arf6 Regulates Endocytosis and Angiogenesis by Promoting Filamentous Actin Assembly
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Abstract
Clathrin-mediated endocytosis (CME) is a process vital to angiogenesis as well as general vascular homeostasis. In pathologies where supraphysiological growth factor signaling underlies disease etiology, such as in diabetic retinopathy and solid tumors, strategies to limit chronic growth factor signaling by way of CME have been shown to have tremendous clinical value. ADP ribosylation factor 6 (Arf6) is a small GTPase that promotes the assembly of actin necessary for CME. In its absence, growth factor signaling is greatly diminished, which has been shown to ameliorate pathological signaling input in diseased vasculature. However, it is less clear if there are bystander effects related to loss of Arf6 on angiogenic behaviors. Our goal was to provide a analysis of Arf6’s function in angiogenic endothelium, focusing on its role in lumenogenesis as well as its relation to actin and CME. We found that Arf6 localized to both filamentous actin and sites of CME in 2-dimensional culture. Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, and this may be the primary driver underlying gross dysmorphogenesis during angiogenic sprouting in its absence. Our findings highlight that endothelial Arf6 is a potent mediator of both actin regulation and CME.
Article activity feed
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β1-integrin
The integrin localization looks weird compared to the control too (at least in this image), do you think hat loss of Arf6 is affecting trafficking or polarity or something?
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To confirm that Arf6 promoted actin polymerization, we compared the amounts of globular (G), or monomeric, actin to F-actin between groups by differential centrifugation.
Could we get a little more information about this experiment? Is it in vitro? Is it just actin from lysate? What is the buffer composition? Do you have some sort of control to show that you started with the same amount of actin in each case?
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This data suggests that having the ability to polymerize branched actin is necessary for protein internalization from the plasma membrane
Cool! But this isn't true for all proteins correct? For example, it looks like VEGFR2 doesn't require the Arp2/3 complex, which is interesting, especially because Arf6 does seem to be required. Any hypotheses about this?
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This finding suggests that Arf6 is preferentially recruited to sites of active actin polymerization.
Couldn't there still be active actin polymerization by formins and the like in other parts of the cell? I think you could add that the active actin polymerization is Arp2/3 complex-mediated.
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actin protein mCherry-Arp2
Is this the actin-related protein Arp2 that is a part of the Arp2/3 complex? I think this is an important distinction that you might take advantage of and discuss a little bit more here.
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Loss of Arf6 significantly increased the amount of clathrin in sprouts as compared with controls
Does loss of Arf6 cause differences in Arp2/3 complex recruitment to clathrin sites?
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Pitstop
Could be related to off-target effects of PitStop2
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thinner network of filaments with an abundance of small actin accumulations leading to a generally disorganized appearance in the actin architecture compared with controls
This is really interesting! I'd love to know more about the mechanism behind this phenotype.
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(CK-666)
I think DMSO is generally a fine control, but you might consider CK-689 (the inactive version of CK-666) just because some of these are so close.
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Figure
Beautiful images throughout the paper!
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Western blot of membrane isolations treated with scramble (Scram) or Arf6 siRNA (si)
Is there some sort of control you could include here to show that you loaded the same amount of total protein? Maybe a coomassie dye or something?
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Acute inhibition of Arp2/3 significantly reduced the colocalization of Arf6 with clathrin
This is interesting. I mentioned this before, but I'm curious about the opposite experiment. If you lose Arf6 function, does the Arp2/3 complex still colocalize with clathrin? This could help iron out the mechanism of Arf6 in this situation.
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Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, and this may be the primary driver underlying gross dysmorphogenesis during angiogenic sprouting in its absence.
Really cool work!
-
Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, and this may be the primary driver underlying gross dysmorphogenesis during angiogenic sprouting in its absence.
Really cool work!
-
(CK-666)
I think DMSO is generally a fine control, but you might consider CK-689 (the inactive version of CK-666) just because some of these are so close.
-
Western blot of membrane isolations treated with scramble (Scram) or Arf6 siRNA (si)
Is there some sort of control you could include here to show that you loaded the same amount of total protein? Maybe a coomassie dye or something?
-
Figure
Beautiful images throughout the paper!
-
Acute inhibition of Arp2/3 significantly reduced the colocalization of Arf6 with clathrin
This is interesting. I mentioned this before, but I'm curious about the opposite experiment. If you lose Arf6 function, does the Arp2/3 complex still colocalize with clathrin? This could help iron out the mechanism of Arf6 in this situation.
-
Pitstop
Could be related to off-target effects of PitStop2
-
This data suggests that having the ability to polymerize branched actin is necessary for protein internalization from the plasma membrane
Cool! But this isn't true for all proteins correct? For example, it looks like VEGFR2 doesn't require the Arp2/3 complex, which is interesting, especially because Arf6 does seem to be required. Any hypotheses about this?
-
To confirm that Arf6 promoted actin polymerization, we compared the amounts of globular (G), or monomeric, actin to F-actin between groups by differential centrifugation.
Could we get a little more information about this experiment? Is it in vitro? Is it just actin from lysate? What is the buffer composition? Do you have some sort of control to show that you started with the same amount of actin in each case?
-
thinner network of filaments with an abundance of small actin accumulations leading to a generally disorganized appearance in the actin architecture compared with controls
This is really interesting! I'd love to know more about the mechanism behind this phenotype.
-
β1-integrin
The integrin localization looks weird compared to the control too (at least in this image), do you think hat loss of Arf6 is affecting trafficking or polarity or something?
-
This finding suggests that Arf6 is preferentially recruited to sites of active actin polymerization.
Couldn't there still be active actin polymerization by formins and the like in other parts of the cell? I think you could add that the active actin polymerization is Arp2/3 complex-mediated.
-
Loss of Arf6 significantly increased the amount of clathrin in sprouts as compared with controls
Does loss of Arf6 cause differences in Arp2/3 complex recruitment to clathrin sites?
-
actin protein mCherry-Arp2
Is this the actin-related protein Arp2 that is a part of the Arp2/3 complex? I think this is an important distinction that you might take advantage of and discuss a little bit more here.
-
