The actin binding protein profilin 1 localizes inside mitochondria and is critical for their function
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Abstract
The monomer-binding protein profilin 1 (PFN1) plays a crucial role in actin polymerization. However, mutations in PFN1 are also linked to hereditary amyotrophic lateral sclerosis, resulting in a broad range of cellular pathologies which cannot be explained by its primary function as a cytosolic actin assembly factor. This implies that there are important, undiscovered roles for PFN1 in cellular physiology. Here we screened knockout cells for novel phenotypes associated with PFN1 loss of function and discovered that mitophagy was significantly upregulated. Indeed, despite successful autophagosome formation, fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells accumulate depolarized, dysmorphic mitochondria with altered metabolic properties. Surprisingly, we also discovered that PFN1 is present inside mitochondria and provide evidence that mitochondrial defects associated with PFN1 loss are not caused by reduced actin polymerization in the cytosol. These findings suggest a previously unrecognized role for PFN1 in maintaining mitochondrial integrity and highlight new pathogenic mechanisms that can result from PFN1 dysregulation.
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When expressed in PFN1 KO cells, these mutants would sometimes form aggregates inside of mitochondria, causing them to swell and enlarge
This is really interesting, and I'm curious how often these aggregates show up for the different mutants.
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these results suggest a novel function for PFN1 in regulating mitochondria
This paper is so cool and such a fun read! I found the data that showed PFN1 in the mitochondria (figure 6) especially interesting and the mutant data throughout particularly convincing. I'm looking forward to following this story and learning more about PFN1 and how it affects mitochondria!
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These data show that PFN1 is not located on the surface of the OMM, but rather inside the mitochondria membrane.
This experiment and this data is so cool!!! I am curious if you think actin is also present inside the mitochondria?
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G) Quantification of Parkin foci in PFN1 cells expressing GFP, GFP-PFN1 and GFP-PFN1R88E and the ALS associated mutants M114T, E117G and G118V, showing that rescue was only possible with functional PFN1.
Are the stars here signifying significance between each mutant and the GFP control or the PFN1 rescue?
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Figure 1.
In Figures 1 and 2, I don't think you mention what the stars represent. I'm guessing that they are the usual significance cutoffs, but you might add them in the legends!
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but not mutants deficient in binding actin (Fig. 4M) or those associated with ALS
M114T looks a bit more promising than the others. I'd be interesting to know more about how this mutant is different!
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Decreasing the amount of polymerized actin in control cells by 40-50% with a low overnight dose of Latrunculin A (10-20 nM) (Cisterna et al 2023, in preparation) to approximate the loss of actin caused by PFN1 KO cells, did not result in more mitochondria being delivered to lysosomes (Fig. 2E) or cause an increase in the formation of Parkin foci (Fig. S2).
Does decreasing the amount of polymerized actin further eventually result in mitochondrial defects and activation of mitophagy? In the discussion you mentioned that this process doesn't actually require that much actin so I wonder if the 50-60% that's left is actually sufficient. Additionally, because profilin has such a complex role in actin function (polymerization promotion, monomer sequestering, ATP hydrolysis promoting), I imagine that it is probably quite difficult to mimic …
Decreasing the amount of polymerized actin in control cells by 40-50% with a low overnight dose of Latrunculin A (10-20 nM) (Cisterna et al 2023, in preparation) to approximate the loss of actin caused by PFN1 KO cells, did not result in more mitochondria being delivered to lysosomes (Fig. 2E) or cause an increase in the formation of Parkin foci (Fig. S2).
Does decreasing the amount of polymerized actin further eventually result in mitochondrial defects and activation of mitophagy? In the discussion you mentioned that this process doesn't actually require that much actin so I wonder if the 50-60% that's left is actually sufficient. Additionally, because profilin has such a complex role in actin function (polymerization promotion, monomer sequestering, ATP hydrolysis promoting), I imagine that it is probably quite difficult to mimic what loss of profilin would look like in this way. Is there some other cellular function that is affected by loss of profilin function that you could use as a readout to show that this is affecting cells in a similar way to loss of profilin?
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Interestingly, the mutants used for this assay have varying effects on PFN1’s ability to operate as an actin assembly factor, from complete to partial loss of function
I think that the mutant data throughout is quite convincing since they have varying actin polymerization activity. It could be really useful for digging into this more to know a little more about these mutants. Do they block binding to other proteins, affect overall profilin folding or expression, or affect other functions that we know of?
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nterestingly, RNA-seq analysis identified significant changes in expression of genes associated with lysosome/endosome systems and autophagy21 upon the loss of PFN1 expression
I'm interested in knowing a bit more about this expression dataset in the context of this work. Do actin and related proteins express at normal levels in PFN1 KO cells?
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unpainted
unpaired?
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(Fig. 2D)
should this be 2F and 2G?
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(Fig. 2C)
should this read Fig. 2D, or 2E where this is quantified?
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This is a super interesting set of results, particularly the localization inside mitos! Do you think that the mitochondrial phenotype in PFN1 knockouts is due to spontaneous nucleation of actin filaments within mitos? Also I may have missed if you checked this but do you think the decreased f-actin in PFN1 KOs as shown in figure 4H is from degradation of actin as seen in this other system (https://doi.org/10.1073/pnas.1721935115) or is that already known for these PFN1 KOs in these cells?
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