Par3 cooperates with Sanpodo for the assembly of Notch clusters following asymmetric division of Drosophila sensory organ precursor cells
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Evaluation Summary:
This study reveals the existence of a novel Notch-containing signaling hub, organized by Sanpodo and Par3, that operates in Notch receptor signaling during cell fate decisions in the peripheral nervous system of Drosophila. These Notch clusters are modulated by components of the Notch signaling pathway, and are proposed to reinforce Notch signaling by concentrating ligands and receptors. These findings are highly relevant to different areas of biology including membrane biology, cytokinesis, PAR polarity, Notch signaling and cell fate decisions making.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
In multiple cell lineages, Delta-Notch signalling regulates cell fate decisions owing to unidirectional signalling between daughter cells. In Drosophila pupal sensory organ lineage, Notch regulates the intra-lineage pIIa/pIIb fate decision at cytokinesis. Notch and Delta that localise apically and basally at the pIIa-pIIb interface are expressed at low levels and their residence time at the plasma membrane is in the order of minutes. How Delta can effectively interact with Notch to trigger signalling from a large plasma membrane area remains poorly understood. Here, we report that the signalling interface possesses a unique apico-basal polarity with Par3/Bazooka localising in the form of nano-clusters at the apical and basal level. Notch is preferentially targeted to the pIIa-pIIb interface, where it co-clusters with Bazooka and its cofactor Sanpodo. Clusters whose assembly relies on Bazooka and Sanpodo activities are also positive for Neuralized, the E3 ligase required for Delta activity. We propose that the nano-clusters act as snap buttons at the new pIIa-pIIb interface to allow efficient intra-lineage signalling.
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Author Response:
Reviewer #1:
The evolutionary conserved Notch receptor cell-cell communication pathway is required in cell fate decisions in many vertebrate and invertebrate cells. In Drosophila, Notch controls (among others) the cell fate decision of the sensory organ precursor cell, SOP. SOPs divides asymmetrically to give rise to an anterior and a posterior cell, pIIb and pIIa, respectively, which ultimately result in the formation of a bristle. In a recent paper form the Schweisguth lab (Trylinsky et al., 2017) is was shown that Notch is found both apical and basal of the midbody at the pIIa/pIIb interface during cytokinesis, and that it is mainly the basal pool of Notch that contributes to signaling.
Houssin et al. now asked how polarity and signaling proteins involved are distributed during cytokinesis and how this …
Author Response:
Reviewer #1:
The evolutionary conserved Notch receptor cell-cell communication pathway is required in cell fate decisions in many vertebrate and invertebrate cells. In Drosophila, Notch controls (among others) the cell fate decision of the sensory organ precursor cell, SOP. SOPs divides asymmetrically to give rise to an anterior and a posterior cell, pIIb and pIIa, respectively, which ultimately result in the formation of a bristle. In a recent paper form the Schweisguth lab (Trylinsky et al., 2017) is was shown that Notch is found both apical and basal of the midbody at the pIIa/pIIb interface during cytokinesis, and that it is mainly the basal pool of Notch that contributes to signaling.
Houssin et al. now asked how polarity and signaling proteins involved are distributed during cytokinesis and how this distribution could impact on Notch signaling and hence fate decision. The authors show that during cytokinesis of the SOP several polarity determinants are re-distributed. Bazooka /Par3 becomes enriched at the pIIa/pIIb interface, where it occurs in nano-clusters, both apical and basal to the midbody, while aPKC remains in the apical compartment. Bazooka co-localizes with Notch, Sanpodo, Delta and Neuralised (Neur) in these clusters. In the absence of baz, both the apical and the lateral Notch-positive clusters are decreased in intensity and the number of lateral clusters is reduced at the pIIa/pIIb interface. Strikingly, this only slightly reduces the signaling activity of Notch. Formation of the Baz-Notch clusters depend on the Notch-cofactor Sanpodo: in its absence, the lateral Baz-Notch clusters do not assemble, suggesting that Sanpodo supports Notch signaling by promoting lateral clusters. From the data the authors conclude that the Notch/Baz/Spdo/Neur clusters represent the signaling units at the pIIa/pIIb interface.
Major strengths and weaknesses
The authors performed a very detailed analysis to further dissect how Notch signaling at the pIIa/pIIb interface is controlled. They used state-of-the-art live-cell imaging of tagged proteins in wild-type and mutant animals and applied careful statistical analyses of their data. Thereby, they provide a novel link between the role of the polarity protein Bazooka in clustering Notch, and how the particular redistribution of Bazooka/Notch in clusters on the lateral membrane during cytokinesis of the SOP organize putative signaling hubs.
However, in the discussion the authors fall somewhat short to substantiate their main conclusion that these clusters "represent signaling units at the pIIa/pIIb interface." (line 560). First, although in the absence of Baz the number and size of Notch clusters are decreased, Notch signaling is only slightly affected.
Second, no suggestion for any molecular mechanism is provided as to how Baz may organize these clusters, e.g. about the molecular interaction between Baz and Spdo, both of which are required to cluster Notch.
We have not tested experimentally the putative molecular interaction between Baz and Spdo. As also explained in the discussion, we postulate several hypotheses regarding the mode of action of Baz (e.g. positioning of Notch/Spdo clusters, exocyst receptor, physical interaction with Notch/Spdo, regulation of Serrate activity). By way of comparison, although it is accepted that Baz, by assembling into nanoscopic clusters, regulates the repositioning of Cadherin-Catenin clusters at apico-lateral sites for AJ spot assembly (McGill et al., 2009), the underlying molecular mechanisms have not yet been characterised to our knowledge. Thus, understanding the mechanism of action of Baz is a study in itself, which we believe is beyond the scope of this work.
And finally, the fact that the clusters are similar in composition apical and basal to the midbody does not help to support (or disprove) the conclusions put forward in Trylinsky et al., 2017, showing that Notch signaling mainly occurs by the lateral clusters.
From the work published in (Trylinski and Schweisguth, 2017) and (Bellec et al, 2021) there is no question that both apical and basal pools of Notch contribute to signalling following asymmetric division of the SOP.
The novelty of this study is to describe the function of Baz in Notch signalling, on the one hand, and the function of Baz in the assembly of the Delta, Neuralized/Notch, Spdo clusters, which we hypothesised would constitute Notch signalling units, at the apical as well as the lateral interface. Our findings on Baz/Notch/Spdo clusters further support the notion that signalling can occur from both sites, albeit likely not to same extent, as the apical pool has a short life compared with the lateral ones.
Reviewer #2:
Sensory organ precursor cells of the fly are a strong model system to understand the spatio- temporal regulation of Notch signalling in the context of cell fate regulation. Different signalling competent pools of Notch have been identified previously at the newly formed membrane that separates the two SOP daughters. It is unclear how for instance the Notch signalling pools are restricted to localize exclusively to this membrane.
This study now takes a closer look at one of the Notch pools and finds that SOPs known to remodel PAR-dependent polarity at the beginning of mitosis, seem to remodel polarity once more, this time later, around anaphase when the new membrane is formed. This remodelling is evident with the assembly of intriguing Par3/Baz containing clusters that strikingly co-localize with Notch as well as other members of the Notch signalling pathway. Baz cluster formation is independent of Notch, but negatively regulated by Numb and Neuralized. Notch in turn depends on Baz to some extend to localize to the clusters. The study proposes that the Baz dependent clusters form a "snap button" type of platform to cluster Notch and facilitate directed Notch signalling, which is an interesting idea.
The concept is relevant, especially as the dependency on PAR regulation provides an angle for future research to address the question why Notch accumulates only at the interphase of pIIa/b, but not at other interfaces with other neighbours in the future. The Baz clusters are well-described and the experiments to dissect their origin, dependency and impact on Notch well-designed.
The signalling relevance of the different Notch pools is extremely challenging to address. This has been attempted in the past by the authors and redone in this study. Despite the fact that the sensitivity of these assays is notoriously noisy, the observed tendencies of signalling measured by nuclear Notch levels in the relevant cells support their model. Relevance of the Baz dependent Notch pool appears to be a likely possibility. The fact that this clusters are modulated by Numb, Delta, Neuralized ans Sanpodo are in contrast in strong favour that the here described Baz clusters are under control of this system and relevant.
The study is a little imbalanced in the use of quantification, the phenotypes appear admittedly often evident and convincing, but would need to be backed up by more thorough quantification. Clarity of figures, legends and writing could be strengthened.
We thank the reviewer for her/his constructive comments. In the revised manuscript, we have now quantified all the experiments, and added statistical tests where they were missing. We have also taken care to amend the legends and the body of the text to clarify the points raised by the reviewer.
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Reviewer #3 (Public Review):
Previously published work had shown that small clusters of N protein can be observed at the newly forming interface between pIIa and pIIb and that these clusters are the source of nuclear N intracellular domain (NICD) following N activation by its ligand Delta (Dl). In this manuscript the authors have used live imaging of fluorescently tagged proteins to study the contribution of several regulators of apical-basal cell polarity and of N signaling to the formation of these N clusters. Their analysis revealed differences in the localization of polarity and junction markers between the SOP daughters and surrounding epithelial cells, confirming that the pIIa/pIIb interface has special features different from epidermal epithelial cells. They found that the polarity regulator Bazooka (Baz), the homolog of …
Reviewer #3 (Public Review):
Previously published work had shown that small clusters of N protein can be observed at the newly forming interface between pIIa and pIIb and that these clusters are the source of nuclear N intracellular domain (NICD) following N activation by its ligand Delta (Dl). In this manuscript the authors have used live imaging of fluorescently tagged proteins to study the contribution of several regulators of apical-basal cell polarity and of N signaling to the formation of these N clusters. Their analysis revealed differences in the localization of polarity and junction markers between the SOP daughters and surrounding epithelial cells, confirming that the pIIa/pIIb interface has special features different from epidermal epithelial cells. They found that the polarity regulator Bazooka (Baz), the homolog of mammalian Par3, colocalizes with N in the clusters and is required for efficient cluster formation. Two other polarity regulators, Crumbs (Crb) and atypical protein kinase C (aPKC) do not colocalize with N and Baz in the clusters but are found in intracellular vesicles in pIIa and pIIb, in contrast to the surrounding epithelial cells where they localize at the apical plasma membrane. Two regulators of N signaling, Neuralized (Neur) and Sanpodo (Spdo) also localize in the clusters together with N and Baz, whereas the N ligand Dl and the negative regulator of N signaling, Numb (Nb) are not detectable in the clusters.
To test the functional contribution of the proteins mentioned above to formation of the clusters at the pIIa/pIIb interface, the function of the corresponding genes was eliminated by mutation, RNA interference, or ubiquitin-mediated degradation (degradFP). The authors found that Baz and Spdo are required for efficient cluster formation, while Neur, Dl and Nb are negative regulators of cluster formation. In the absence of these three proteins, N clusters are more frequent and show brighter fluorescence signals.
The data also confirm the intimate connection between N signaling and cell polarity. Nonethless, the data do not reveal a novel mechanism but rather describe a cell-biological phenomenon in greater detail than before. Thus, the data are certainly of great interest to specialists in the field, but it is less clear whether they will have a greater impact in the general cell biology or signaling community.
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Reviewer #2 (Public Review):
Sensory organ precursor cells of the fly are a strong model system to understand the spatio- temporal regulation of Notch signalling in the context of cell fate regulation. Different signalling competent pools of Notch have been identified previously at the newly formed membrane that separates the two SOP daughters. It is unclear how for instance the Notch signalling pools are restricted to localize exclusively to this membrane.
This study now takes a closer look at one of the Notch pools and finds that SOPs known to remodel PAR-dependent polarity at the beginning of mitosis, seem to remodel polarity once more, this time later, around anaphase when the new membrane is formed. This remodelling is evident with the assembly of intriguing Par3/Baz containing clusters that strikingly co-localize with Notch as …
Reviewer #2 (Public Review):
Sensory organ precursor cells of the fly are a strong model system to understand the spatio- temporal regulation of Notch signalling in the context of cell fate regulation. Different signalling competent pools of Notch have been identified previously at the newly formed membrane that separates the two SOP daughters. It is unclear how for instance the Notch signalling pools are restricted to localize exclusively to this membrane.
This study now takes a closer look at one of the Notch pools and finds that SOPs known to remodel PAR-dependent polarity at the beginning of mitosis, seem to remodel polarity once more, this time later, around anaphase when the new membrane is formed. This remodelling is evident with the assembly of intriguing Par3/Baz containing clusters that strikingly co-localize with Notch as well as other members of the Notch signalling pathway. Baz cluster formation is independent of Notch, but negatively regulated by Numb and Neuralized. Notch in turn depends on Baz to some extend to localize to the clusters. The study proposes that the Baz dependent clusters form a "snap button" type of platform to cluster Notch and facilitate directed Notch signalling, which is an interesting idea.
The concept is relevant, especially as the dependency on PAR regulation provides an angle for future research to address the question why Notch accumulates only at the interphase of pIIa/b, but not at other interfaces with other neighbours in the future. The Baz clusters are well-described and the experiments to dissect their origin, dependency and impact on Notch well-designed.
The signalling relevance of the different Notch pools is extremely challenging to address. This has been attempted in the past by the authors and redone in this study. Despite the fact that the sensitivity of these assays is notoriously noisy, the observed tendencies of signalling measured by nuclear Notch levels in the relevant cells support their model. Relevance of the Baz dependent Notch pool appears to be a likely possibility. The fact that this clusters are modulated by Numb, Delta, Neuralized ans Sanpodo are in contrast in strong favour that the here described Baz clusters are under control of this system and relevant.
The study is a little imbalanced in the use of quantification, the phenotypes appear admittedly often evident and convincing, but would need to be backed up by more thorough quantification. Clarity of figures, legends and writing could be strengthened.
-
Reviewer #1 (Public Review):
The evolutionary conserved Notch receptor cell-cell communication pathway is required in cell fate decisions in many vertebrate and invertebrate cells. In Drosophila, Notch controls (among others) the cell fate decision of the sensory organ precursor cell, SOP. SOPs divides asymmetrically to give rise to an anterior and a posterior cell, pIIb and pIIa, respectively, which ultimately result in the formation of a bristle. In a recent paper form the Schweisguth lab (Trylinsky et al., 2017) is was shown that Notch is found both apical and basal of the midbody at the pIIa/pIIb interface during cytokinesis, and that it is mainly the basal pool of Notch that contributes to signaling.
Houssin et al. now asked how polarity and signaling proteins involved are distributed during cytokinesis and how this distribution …
Reviewer #1 (Public Review):
The evolutionary conserved Notch receptor cell-cell communication pathway is required in cell fate decisions in many vertebrate and invertebrate cells. In Drosophila, Notch controls (among others) the cell fate decision of the sensory organ precursor cell, SOP. SOPs divides asymmetrically to give rise to an anterior and a posterior cell, pIIb and pIIa, respectively, which ultimately result in the formation of a bristle. In a recent paper form the Schweisguth lab (Trylinsky et al., 2017) is was shown that Notch is found both apical and basal of the midbody at the pIIa/pIIb interface during cytokinesis, and that it is mainly the basal pool of Notch that contributes to signaling.
Houssin et al. now asked how polarity and signaling proteins involved are distributed during cytokinesis and how this distribution could impact on Notch signaling and hence fate decision. The authors show that during cytokinesis of the SOP several polarity determinants are re-distributed. Bazooka /Par3 becomes enriched at the pIIa/pIIb interface, where it occurs in nano-clusters, both apical and basal to the midbody, while aPKC remains in the apical compartment. Bazooka co-localizes with Notch, Sanpodo, Delta and Neuralised (Neur) in these clusters. In the absence of baz, both the apical and the lateral Notch-positive clusters are decreased in intensity and the number of lateral clusters is reduced at the pIIa/pIIb interface. Strikingly, this only slightly reduces the signaling activity of Notch. Formation of the Baz-Notch clusters depend on the Notch-cofactor Sanpodo: in its absence, the lateral Baz-Notch clusters do not assemble, suggesting that Sanpodo supports Notch signaling by promoting lateral clusters. From the data the authors conclude that the Notch/Baz/Spdo/Neur clusters represent the signaling units at the pIIa/pIIb interface.
Major strengths and weaknesses
The authors performed a very detailed analysis to further dissect how Notch signaling at the pIIa/pIIb interface is controlled. They used state-of-the-art live-cell imaging of tagged proteins in wild-type and mutant animals and applied careful statistical analyses of their data. Thereby, they provide a novel link between the role of the polarity protein Bazooka in clustering Notch, and how the particular redistribution of Bazooka/Notch in clusters on the lateral membrane during cytokinesis of the SOP organize putative signaling hubs.
However, in the discussion the authors fall somewhat short to substantiate their main conclusion that these clusters "represent signaling units at the pIIa/pIIb interface." (line 560). First, although in the absence of Baz the number and size of Notch clusters are decreased, Notch signaling is only slightly affected. Second, no suggestion for any molecular mechanism is provided as to how Baz may organize these clusters, e.g. about the molecular interaction between Baz and Spdo, both of which are required to cluster Notch. And finally, the fact that the clusters are similar in composition apical and basal to the midbody does not help to support (or disprove) the conclusions put forward in Trylinsky et al., 2017, showing that Notch signaling mainly occurs by the lateral clusters.
-
Evaluation Summary:
This study reveals the existence of a novel Notch-containing signaling hub, organized by Sanpodo and Par3, that operates in Notch receptor signaling during cell fate decisions in the peripheral nervous system of Drosophila. These Notch clusters are modulated by components of the Notch signaling pathway, and are proposed to reinforce Notch signaling by concentrating ligands and receptors. These findings are highly relevant to different areas of biology including membrane biology, cytokinesis, PAR polarity, Notch signaling and cell fate decisions making.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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