An isoform-specific function of Cdc42 in regulating mammalian Exo70 during axon formation

The brain-specific isoform of the GTPase Cdc42 is shown to promote axon formation by interacting with the exocyst tethering complex and increasing exocytosis downstream of its activator Arhgef7.

1. Why do Cdc42u and Cdc42b interact differentially with ArhGEF7 and Exo70 although the splice variants are structurally extremely similar? A likely candidate could be the distinguishing palmytoylation and correspondingly a different membrane localization. It would be therefore interesting to investigate the presence of Cdc42u, Cdc42b, TC10, and Arhgef7 in lipid rafts in the presence and absence of IGF1. Will palmitoylation inhibitors abrogate the function of Cdc42b and TC10? 2. It would be also interesting to see whether inhibition of Cdc42b and TC10 have additive effects.

Referee #3 Review
Report for Author: Ravindram and Puschel present clear evidence that the function of Cdc42 in regulating exocytosis is conserved in mammals and is specific of Cdc42b. The study is mostly based in biochemical analyses that are well designed and controlled. Each experiment provides strong evidence for the conclusions that are drawn. Although the study is important to the specific field of axon formation/growth, it lacks a broad biological significance and the novelty is somehow limited. In this respect, it would be better suited for a more specialized audience. Thank you for submitting your manuscript entitled "An isoform-specific function of Cdc42 in regulating mammalian Exo70 during axon formation" to Life Science Alliance. We invite you to submit a revised manuscript addressing the Reviewer comments.
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--By submitting a revision, you attest that you are aware of our payment policies found here: https://www.life-sciencealliance.org/copyright-license-fee B. MANUSCRIPT ORGANIZATION AND FORMATTING: Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** Authors' Response to Reviewers December 7, 2022 We thank the reviewers for their helpful comments and have modified the manuscript according to their suggestions.
Referee #1: First, it is highly commendable that an effort was made to tackle such a fundamentally important problem in neuronal polarity. The authors used a robust approach to test their hypothesis, supplemented with a blend of primary culture experiments that are well executed and for the most part, the data is nicely presented. .... Basically, we just learned with this manuscript that the effect of Cdc42 in axon elongation is isoformspecific.
We thank the reviewer for the positive comments but respectfully disagree with the notion that our manuscript only shows "that the effect of Cdc42 in axon elongation is isoformspecific". While the highly conserved Cdc42 regulates cell polarity in yeast and Drosophila through the exocyst complex, it was thought that -surprisingly -this is not the case in mammals. The vertebrate-specific Cdc42 isoforms have been known for a long time but it remained unclear how they perform different functions. Our manuscript identifies a molecular mechanism that explains how Cdc42b mediates its previously unrecognized function in regulating exocytosis. The results show that the interaction with the exocyst is conserved in mammals but specific to the Cdc42b isoform.
Minor points to be considered are listed below: 1. The summary is mostly describing not the results from this manuscript. Only the last five lines described the actual results from this study 1) We modified the abstract as suggested 2. Some paragraphs in the results section should be considered as discussion (e.g., the first paragraph of page 6).
2) The manuscript was modified as suggested and the paragraph was moved to the discussion at the end of the manuscript.
Referee #2: Ravindran and Püschel reveal that the brain-specific isoform of Cdc42, Cdc42b, is efficiently activated by Arhgef7 and contributes to axon formation via binding to Exo70, similar to TC10. In contrast, the ubiquitously expressed splice variant of Cdc42, Cdc42u, is not able to efficiently work in this pathway. The experiments presented are meaningful, well executed, and properly interpreted. The result are interesting, but what needs to be elucidated more is the underlying mechanism.
Major comments: 1. Why do Cdc42u and Cdc42b interact differentially with ArhGEF7 and Exo70 although the splice variants are structurally extremely similar? A likely candidate could be the distinguishing palmytoylation and correspondingly a different membrane localization. It would be therefore interesting to investigate the presence of Cdc42u, Cdc42b, TC10, and Arhgef7 in lipid rafts in the presence and absence of IGF1. Will palmitoylation inhibitors abrogate the function of Cdc42b and TC10?
1) The reviewer suggested that the differential interaction of Cdc42 isoforms with Exo70 could depend on palmitoylation and mediate the localization to lipid rafts. Our results (Fig.  3A, B) show that both Cdc42 isoforms can be activated by Arhgef7, indicating that palmitoylation is not required for their interaction with Arhgef7. A previous study already demonstrated that a palmitoylation inhibitor blocks axon formation similar to the knockdown of TC10 and Cdc42 (Mukai et al., 2015). Therefore, we tested if palmitoylation is required for the membrane recruitment of Exo70 to establish a link between palmitoylation, axon formation and Exo70. The results (new Fig 4C-E; paragraph 2 on p. 8) show that the treatment of neurons with the palmitoylation inhibitor 2-BP blocks the preferential localization of Exo70 to a single neurite and reduces its membrane-proximal localization of Exo70 indicating that palmitoylation is required for neuronal polarization and Exo70 function. It has also been shown that palmitoylation directs GTPases including TC10 and Cdc42b to specific membrane domains often called lipid rafts (Watson et al., 2001(Watson et al., . 2003Wirth et al., 2013). The localization to lipid rafts is assayed biochemically by determining the distribution of membrane proteins to detergent resistant membrane fractions (DRMs) after extraction, sucrose density gradient centrifugation and Western blot (Klotzsch and Schütz, 2013). The presence of palmitoylated GTPases in DRMs has been demonstrated before but this result does not provide evidence for their localization to specific lipid domains in native membranes in living cells (Klotzsch and Schütz, 2013;Levental et al., 2020;Sezgin et al. 2017). The physiological relevance of DRM localization is also discussed controversially. The pathway that promotes axon formation acts in the nascent axon and differences in the localization to specific membrane domains will be restricted to the growth cone (Dupraz et al., 2009). Thus, any changes in the distribution to the DRMs will be limited to a small fraction of Cdc42 and difficult to detect. The isoform-specific antibodies also do not work well enough in Western blot to reliably detect endogenous levels of Cdc42 in neurons. To address the role of Cdc42 palmitoylation we, therefore, mutated the cysteine residues C188 and C189 at the C-terminus individually and in combination. C189 is palmitoylated and C188 is modified by prenylation or palmitoylation (Wirth et al., 2013). The function of the mutants was analyzed by their ability to restore axon formation after knockdown of Arhgef7 (new Fig S2E and F; last paragraph on p. 7). Mutation of a single cysteine did not impair the ability to rescue the Arhgef7 knockdown. This rescue indicates that neither cysteine is essential for Cdc42b function but does not exclude the possibility that palmitoylation is required for its function since both residues can be palmitoylated (Wirth et al., 2013). Mutation of both cysteines abolished the ability of Cdc42b to rescue the Arhgef7 knockout but also prevents membrane localization (Wirth et al., 2013). Taken together, these results indicate that membrane localization is essential but not the modification of a specific cysteine residue. An ultrastructural analysis of Exo70/Cdc42 complexes is required to elucidate the molecular details of the interaction but this would go beyond the scope of this study.
2. It would be also interesting to see whether inhibition of Cdc42b and TC10 have additive effects.
2) We analyzed the combined knockdown of both Cdc42b and TC10 as suggested. Our results (new Fig S3K and L; first paragraph on p. 8) show that the combined knockdown has a stronger effect than the single knockdowns, indicating that they may substitute for each other to some extent.
We think that our results are relevant not only for the question how axon formation is regulated. The function of Cdc42b is not restricted to axon formation and it has also been implicated in regulating activity-dependent plasticity of dendritic spines and synapses (Kang et al., 2008;Moutin et al., 2016;Wirth et al., 2022). It was thought that the highly conserved Cdc42 regulates cell polarity in yeast and Drosophila, but not in mammals, through the exocyst complex. The vertebrate-specific Cdc42 isoforms have been known for a long time but their relevance remained unclear. Our manuscript identifies a molecular mechanism that explains how Cdc42b mediates its previously unrecognized function in regulating exocytosis through the exocyst. This regulation Cdc42 is conserved in mammals but specific to the Cdc42b isoform. Thank you for submitting your revised manuscript entitled "An isoform-specific function of Cdc42 in regulating mammalian Exo70 during axon formation". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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