Identification of green lineage osmotic stress pathways
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Abstract
Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the green alga Chlamydomonas reinhardtii to establish a foundational understanding of evolutionarily conserved osmotic-stress signaling pathways in the green lineage through transcriptomics, phosphoproteomics, and functional genomics approaches. Five genes acting across diverse cellular pathways were found to be essential for osmotic-stress tolerance in Chlamydomonas including cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs of these genes in the multicellular land plant Arabidopsis thaliana have conserved functional roles in stress tolerance and reveal a novel PROFILIN-dependent actin remodeling stage of acclimation that ensures cell survival and tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.
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(Extended data Fig. 10)
I think this should say Extended data Fig. 9
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Interestingly, visualization of actin dynamics in Chlamydomonas using Lifeact-NeonGreen, showed a similar response to osmotic stress with an increase in skewness
The control image here looks quite a bit different than typical actin localization in a wild-type cell (with a more prominent localization to what I am assuming is the cell apex relative to mid-cell actin). I'm wondering if this is specific to this/a cw strain or whether this is a non-representative image. Also curious what the larger number of cells looks like in NaCl as skewness is measured but the clear phenotype from this image appears to be an increase in actin bundling, just as in Arabidopsis. If this is representative, is there an increase in actin bundler expression or expression of formins in your Chlamy dataset? Lastly, for visualization, fixed cell phalloidin …
Interestingly, visualization of actin dynamics in Chlamydomonas using Lifeact-NeonGreen, showed a similar response to osmotic stress with an increase in skewness
The control image here looks quite a bit different than typical actin localization in a wild-type cell (with a more prominent localization to what I am assuming is the cell apex relative to mid-cell actin). I'm wondering if this is specific to this/a cw strain or whether this is a non-representative image. Also curious what the larger number of cells looks like in NaCl as skewness is measured but the clear phenotype from this image appears to be an increase in actin bundling, just as in Arabidopsis. If this is representative, is there an increase in actin bundler expression or expression of formins in your Chlamy dataset? Lastly, for visualization, fixed cell phalloidin staining of these cells might give you better resolution to identify the specific phenotypic consequences of osmotic stress with respect to the cytoskeletal rearrangement.
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Early cellular responses to osmotic stress involve shifts in the balance between endocytosis and exocytosis of proteins important for growth, such as cellulose synthase complexes
Using lifeact in this particular Chlamy strain, do you typically see apical actin puncta in untreated cells (which we think are endocytic pits, see https://doi.org/10.1101/2020.11.24.396002) and if so, are these quantifiably different in NaCl?
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Early cellular responses to osmotic stress involve shifts in the balance between endocytosis and exocytosis of proteins important for growth, such as cellulose synthase complexes
Using lifeact in this particular Chlamy strain, do you typically see apical actin puncta in untreated cells (which we think are endocytic pits, see https://doi.org/10.1101/2020.11.24.396002) and if so, are these quantifiably different in NaCl?
-
Interestingly, visualization of actin dynamics in Chlamydomonas using Lifeact-NeonGreen, showed a similar response to osmotic stress with an increase in skewness
The control image here looks quite a bit different than typical actin localization in a wild-type cell (with a more prominent localization to what I am assuming is the cell apex relative to mid-cell actin). I'm wondering if this is specific to this/a cw strain or whether this is a non-representative image. Also curious what the larger number of cells looks like in NaCl as skewness is measured but the clear phenotype from this image appears to be an increase in actin bundling, just as in Arabidopsis. If this is representative, is there an increase in actin bundler expression or expression of formins in your Chlamy dataset? Lastly, for visualization, fixed cell phalloidin …
Interestingly, visualization of actin dynamics in Chlamydomonas using Lifeact-NeonGreen, showed a similar response to osmotic stress with an increase in skewness
The control image here looks quite a bit different than typical actin localization in a wild-type cell (with a more prominent localization to what I am assuming is the cell apex relative to mid-cell actin). I'm wondering if this is specific to this/a cw strain or whether this is a non-representative image. Also curious what the larger number of cells looks like in NaCl as skewness is measured but the clear phenotype from this image appears to be an increase in actin bundling, just as in Arabidopsis. If this is representative, is there an increase in actin bundler expression or expression of formins in your Chlamy dataset? Lastly, for visualization, fixed cell phalloidin staining of these cells might give you better resolution to identify the specific phenotypic consequences of osmotic stress with respect to the cytoskeletal rearrangement.
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(Extended data Fig. 10)
I think this should say Extended data Fig. 9
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