eLife Assessment

This important work provides another layer of regulatory mechanism for TGF-beta signaling activity. The evidence supports the involvement of microtubules as a reservoir of Smad2/3 and additional evidence convincingly demonstrates functional involvement of Rudhira in this process. The work will be of board interest to developmental biologists in general and molecular biologists in the field of growth factor signaling.

Reviewer #1 (Public review):

Summary:

This manuscript aimed to study the role of Rudhira (also known as Breast Carcinoma Amplified Sequence 3), an endothelium-restricted microtubules-associated protein, in regulating of TGFβ signaling. The authors demonstrate that Rudhira is a critical signaling modulator for TGFβ signaling by releasing Smad2/3 from cytoskeletal microtubules and how that Rudhira is a Smad2/3 target gene. Taken together, the authors provide a model of how Rudhira contributes to TGFβ signaling activity to stabilize the microtubules, which is essential for vascular development.

Strengths:

The study used different methods and techniques to achieve aims and support conclusions, such as Gene Ontology analysis, functional analysis in culture, immunostaining analysis, and proximity ligation assay. This study provides unappreciated additional layer of TGFβ signaling activity regulation after ligand-receptor interaction.

Weaknesses:

(1) It is unclear how current findings provide a better understanding of Rudhira KO mice, which the authors published some years ago.

(2) Why do they use HEK cells instead of SVEC cells in Fig 2 and 4 experiments?

(3) A model shown in Fig 5E needs improvement to grasp their findings easily.

Comments on revised version:

The authors have adequately responded to the reviewers' concerns.

Reviewer #2 (Public review):

Summary:

It was first reported in 2000 that Smad2/3/4 are sequestered to microtubules in resting cells and TGF-β stimulation releases Smad2/3/4 from microtubules, allowing activation of the Smad signaling pathway. Although the finding was subsequently confirmed in a few papers, the underlying mechanism has not been explored. In the present study, the authors found that Rudhira/breast carcinoma amplified sequence 3 is involved in release Smad2/3 from microtubules in response to TGF-β stimulation. Rudhira is also induced by TGF-β and probably involved in stabilization of microtubules in the delayed phase after TGF-β stimulation. Therefore, Rudhira has two important functions downstream of TGF-β in the early as well as delayed phase.

Strengths:

This work aimed to address an unsolved question on one of the earliest events after TGF-β stimulation. Based on loss-of-function experiments, the authors identified Rudhira, as a key player that triggers Smad2/3 release from microtubules after TGF-β stimulation. This is an important first step for understanding the initial phase of Smad signaling activation.

Weaknesses:

Currently, the processes how Rudhira causes the release of Smad proteins from microtubules and how Rudhira is mobilized to microtubules in response to TGF-β remain unclear. The authors are expected to address these points experimentally in the future.

This reviewer is also afraid that some of the biochemical data lack appropriate controls and are not convincing enough.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary

This manuscript aimed to study the role of Rudhira (also known as Breast Carcinoma Amplified Sequence 3), an endothelium-restricted microtubules-associated protein, in regulating of TGFβ signaling. The authors demonstrate that Rudhira is a critical signaling modulator for TGFβ signaling by releasing Smad2/3 from cytoskeletal microtubules and how Rudhira is a Smad2/3 target gene. Taken together, the authors provide a model of how Rudhira contributes to TGFβ signaling activity to stabilize the microtubules, which is essential for vascular development.

Strengths

The study used different methods and techniques to achieve aims and support conclusions, such as Gene Ontology analysis, functional analysis in culture, immunostaining analysis, and proximity ligation assay. This study provides an unappreciated additional layer of TGFβ signaling activity regulation after ligand receptor interaction.

We thank the reviewer for acknowledging the importance of our study and providing a clear summary of our findings.

Weaknesses

(1) It is unclear how current findings provide a beVer understanding of Rudhira KO mice, which the authors published some years ago.

Our previous study demonstrated that Rudhira KO mice have a predominantly developmental cardiovascular phenotype that phenocopies TGFβ loss of function (Shetty, Joshi et al., 2018). Additionally, we found that at the molecular level, Rudhira regulates cytoskeletal organization (Jain et al., 2012; Joshi and Inamdar, 2019). Our current study builds upon these previous findings, showing an essential role of Rudhira in maintaining TGFβ signaling and controlling the microtubule cytoskeleton during vascular development. On one hand Rudhira regulates TGFβ signaling by promoting the release of Smads from microtubules, while on the other, Rudhira is a TGFβ target essential for stabilizing microtubules. Thus, our current study provides a molecular basis for Rudhira function in cardiovascular development.

(2) Why do they use HEK cells instead of SVEC cells in Figure 2 and 4 experiments?

Our earlier studies have characterized the role of Rudhira in detail using both loss and gain of function methods in multiple cell types (Jain et al., 2012; SheVy, Joshi et al., 2018; Joshi and Inamdar, 2019). As endothelial cells are particularly difficult to transfect, and because the function of Rudhira in promoting cell migration is conserved in HEK cells, it was practical and relevant to perform these experiments in HEK cells (Figures 2 and 4E).

(3) A model shown in Figure 5E needs improvement to grasp their findings easily.

We have modified Figure 5E for clarity.

Reviewer #2 (Public Review):

Summary

It was first reported in 2000 that Smad2/3/4 are sequestered to microtubules in resting cells and TGF-β stimulation releases Smad2/3/4 from microtubules, allowing activation of the Smad signaling pathway. Although the finding was subsequently confirmed in a few papers, the underlying mechanism has not been explored. In the present study, the authors found that Rudhira/breast carcinoma amplified sequence 3 is involved in the release of Smad2/3 from microtubules in response to TGF-β stimulation. Rudhira is also induced by TGF-β and is probably involved in the stabilization of microtubules in the delayed phase after TGF-β stimulation. Therefore, Rudhira has two important functions downstream of TGF-β in the early as well as delayed phase.

Strengths:

This work aimed to address an unsolved question on one of the earliest events after TGF-β stimulation. Based on loss-of-function experiments, the authors identified a novel and potentially important player, Rudhira, in the signal transmission of TGF-β.

We thank the reviewer for the critical evaluation and appreciation of our findings.

Weaknesses:

The authors have identified a key player that triggers Smad2/3 released from microtubules after TGF-β stimulation probably via its association with microtubules. This is an important first step for understanding the regulation of Smad signaling, but underlying mechanisms as well as upstream and downstream events largely remain to be elucidated.

We acknowledge that the mechanisms regulating cytoskeletal control of Smad signaling are far from clear, but these are out of scope of this manuscript. This manuscript rather focuses on Rudhira/Bcas3 as a pivot to understand vascular TGFβ signaling and microtubule connections.

(1) The process of how Rudhira causes the release of Smad proteins from microtubules remains unclear. The statement that "Rudhira-MT association is essential for the activation and release of Smad2/3 from MTs" (lines 33-34) is not directly supported by experimental data.

We agree with the reviewer’s comment. Although we provide evidence that the loss of Rudhira (and thereby deduced loss of Rudhira-MT association) prevents release of Smad2/3 from MTs (Fig 3C), it does not confirm the requirement of Rudhira-MT association for this. In light of this, we have modified the statement to ‘Rudhira associates with MTs and is essential for the activation and release of Smad2/3 from MTs”.

(2) The process of how Rudhira is mobilized to microtubules in response to TGF-β remains unclear.

Our previous study showed that Rudhira associates with microtubules, and preferentially binds to stable microtubules (Jain et al., 2012; Joshi and Inamdar, 2019). Since TGFβ stimulation is known to stabilize microtubules, we hypothesize that TGFβ stimulation increases Rudhira binding to stable microtubules. We have mentioned this in our revised manuscript.

(3) After Rudhira releases Smad proteins from microtubules, Rudhira stabilizes microtubules. The process of how cells return to a resting state and recover their responsiveness to TGF-β remains unclear.

We show that dissociation of Smads from microtubules is an early response and stabilization of microtubules is a late TGFβ response. However, we agree that the sequence of these molecular events has not been characterized in-depth in this or any other study, making it difficult to assign causal roles (eg. whether release of Smads from MTs is a pre-requisite for MT stabilization by Rudhira) or reversibility. However, the TGFβ pathway is auto regulatory, leading to increased turnover of receptors and Smads and increased expression of inhibitory Smads, which may recover responsiveness to TGFβ. Additionally, the still short turnover time of stable microtubules (several minutes to hours) may also promote quick return to resting state. We have discussed this in our revised manuscript.

Recommendations for the authors:

Reviewer #2 (Recommendations for The Authors):

(1) Overall: Duration of TGF-β stimulation in cell-based assays should be described in the legends for readers' convenience. Avoid simple bar graphs because sample numbers are only 3. A scaVer plot should be super-imposed.

Details added, as suggested. Duration of treatment is mentioned in Materials and methods section for figures 1C-D; 2A-B; 3; 4A-C; 5A-C; S2D; S3A-C; S4C, D. Bar graphs have been replaced with a bar + scatter plot. Note that, as the Excel file for data related to fig 4A was corrupted, we repeated the experiments to generate fresh data. Hence the graph had to be replaced. However, the result holds true as before.

(2) Figure 1A: This panel is too small. Gene names are almost invisible.

Modified for clarity.

(3) Figure 1B: Show TGFβRI expression by immunoblomng (re-probing) to verify that it is expressed in the rightmost lane.

TGFβRI overexpression was confirmed by qPCR in a replicate in the same experiment (Fig S2C).

(4) Figure 1C: Show expression of Rudhira. In addition, confirm the positions of molecular weight markers. Smad2 migrated slower than pSmad2.

Rudhira expression is shown in Fig S1B. Molecular weight markers have been corrected.

(5) Figure 3A: This panel shows a negative result that Smad2/3 fails to interact with Rudhira. A positive control, for example, Smad4, would make the data convincing.

Although it would be nice to have a positive control for interaction, we do not agree that a positive control of Smad4 is essential for our conclusion from this experiment, which is that ‘we were unable to detect an interaction between Rudhira and Smad2/3’.

(6) Fig. 3B: Show Rudhira blot. If possible, show that the Rudhira-MT association precedes Smad phosphorylation by a time course experiment. This is an important point but not experimentally demonstrated.

The interaction between Rudhira and microtubules with or without TGFβ is demonstrated by PLA (Fig 3E). Although important, the suggested time course experiments to assess the sequence of events are beyond the scope of this manuscript. 

(7) Figure 3E: Does the process require the type I receptor kinase activity or non-Smad signaling pathways?

Since TGFβ pathway is complex and is regulated at multiple steps, this possibility has not been tested and is beyond the scope of current study.

(8) Figure 4A: The authors did not examine if these elements are functional. Therefore, this panel can be presented as a supplementary figure.

As suggested, the panel has been moved to supplementary information.

(9) Figure 4E: The figure legend does not say that cells were TGF-β-stimulated. It remains unclear if Smad2 and Smad3 are involved in Rudhira expression as phosphorylated or non-phosphorylated forms. Therefore, the authors should show a pSmad2 blot. In the absence of TGF-β stimulation, Smad2 and Smad3 are expected to be sequestrated to microtubules and therefore not phosphorylated. In the case that cells were stimulated with TGF-β, show if Rudhira is induced by TGF-β in HEK293T cells. This is not shown in this manuscript.

This experiment was not performed under regulated conditions with or without TGFβ, hence the sensitivity to TGFβ could not be assessed. Cells were not stimulated with exogenous TGFβ, but cultured in regular medium with serum, which can have up to ~40 ng/ml of TGFβ (latent and active). Additionally, owing to severe depletion of Smad2 or Smad3 by shRNAs we expect sufficient loss of phospho-Smads2/3. 

(10) Figure S1A: Rudhira migrated at the position corresponding to 91 kD only in this panel.

Corrected the position of molecular weight marker.

(11) Line 205-206, "Since in vivo studies indicate that rudhira depletion severely affects the TGFβ pathway [11]": Refer to Reference 11. The paper does not say anything about TGFβ.

Reference corrected to Ref #14.

(12) Smad4 was previously reported to be sequestered to microtubules [Ref. 7]. Does Rudhira release Smad4 also?

This is an interesting point which could be followed up on our future studies.

(13) It would be nice if the authors examined how Rudhira causes the release of Smad2/3 from microtubules. Currently, it remains unclear whether the association of Rudhira to microtubules is required for the release of Smad2/3. Does a Rudhira mutant lacking microtubule binding fail to induce the release of Smad2/3 after TGF-β stimulation? If so, do Rudhira and Smad2/3 share the same binding site on microtubules? In that case, the mechanism can be regarded as "competitive".

This is a thoughtful experiment much beyond the scope of current manuscript. In our previous study we were able to localize the Tubulin binding sites of Rudhira primarily to its Bcas3 domain (Joshi and Inamdar, 2019), however the equivalent sites in Tubulin were not assessed. While MH2 domains of Smad2/3 bind β-tubulin, amino acids 114-243 of β-tubulin bind to Smad2/3 (Dai et al., 2007). A systematic study of these tripartite interactions including Rudhira would be an interesting follow up for our future study.