Rudhira-mediated microtubule stability controls TGFβ signaling during mouse vascular development

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.

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 Figure 2 and 4 experiments?
(3) A model shown in Figure 5E needs improvement to grasp their findings easily.

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,

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.

(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.

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

(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.

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

Author response:

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, however, additional evidence to convincingly demonstrate the functional involvement of Rudhira in this process is highly appreciated. The work will be of broad interest to developmental biologists in general and molecular biologists in the field of growth factor signaling.

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 better 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; Shetty, 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.