InteractiveFly: GeneBrief

Smad anchor for receptor activation: Biological Overview | References

Gene name - Smad anchor for receptor activation

Synonyms -

Cytological map position - 57E6-57E6

Function - signaling

Keywords - binds the PP1c phosphatase and its regulator Sds22 - both Notch and Delta traffic through Sara endosomes - involved in signalling bias between the pIIa-pIIb sibling cells - Sara endosomes are specifically targeted to the pIIa cell during sensory organ precursor division, mediating the transport of a pool of Notch and Delta that contribute to the activation of Notch in the pIIa - Uninflatable controls the asymmetric dispatch of the Sara endosomes

Symbol - Sara

FlyBase ID: FBgn0026369

Genetic map position - chr2R:21,481,927-21,487,107

NCBI classification - FYVE domain found in endofin and similar proteins; Smad anchor for receptor activation

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

During asymmetric division, fate assignation in daughter cells is mediated by the partition of determinants from the mother. In the fly sensory organ precursor cell, Notch signalling partitions into the pIIa daughter. Notch and its ligand Delta are endocytosed into Sara endosomes in the mother cell and they are first targeted to the central spindle, where they get distributed asymmetrically to finally be dispatched to pIIa. While the processes of endosomal targeting and asymmetry are starting to be understood, the machineries implicated in the final dispatch to pIIa are unknown. This study shows that Sara binds the PP1c phosphatase and its regulator Sds22. Sara phosphorylation on three specific sites functions as a switch for the dispatch: if not phosphorylated, endosomes are targeted to the spindle and upon phosphorylation of Sara, endosomes detach from the spindle during pIIa targeting (Loubery, 2017).

Asymmetric cell division plays many roles in development. In particular, stem cells divide asymmetrically to self-renew while also forming differentiated cells. Asymmetric cell division involves the specific partitioning of cell fate determinants (RNA, proteins or organelles) in one of the two sibling daughter cells. The Sensory Organ Precursor cells (SOPs) of the Drosophila notum are a model system of choice to unravel the molecular mechanisms of asymmetric cell division (Loubery, 2017).

The division of each SOP gives rise to a pIIa and a pIIb daughter cell and, after two more rounds of asymmetric cell divisions, to the four cells of the sensory organ: the outer cells (shaft and socket) are progeny of the pIIa, while the pIIb forms the inner cells (sheath and neuron) and a glial cell that rapidly undergoes apoptosis. The Notch signalling pathway controls cell fate determination in this system: a signalling bias between the pIIa-pIIb sibling cells is essential to obtain a correct lineage (Loubery, 2017).

The asymmetric dispatch of cell fate determinants during SOP division is governed by the polarity of the dividing cell. The Par complex (composed by the aPKC, Par-3 and Par-6 proteins) is the master regulator of the establishment of this polarity. Downstream the Par complex, Notch signalling is regulated by endocytosis and endosomal trafficking through four independent mechanisms: (1) The E3 Ubiquitin ligase Neuralized is segregated to the pIIb cell, where it induces the endocytosis and thereby the activation of the Notch ligand Delta; (2) Recycling endosomes accumulate in the perinuclear region of the pIIb cell, in which they enhance the recycling and activation of Delta; (3) The endocytic proteins α-adaptin and Numb are segregated to the pIIb cell, where they inhibit the Notch activator Sanpodo; (4) During SOP mitosis, Sara endosomes transport a signalling pool of Notch and Delta to the pIIa cell, where Notch can be activated. Asymmetric Sara endosomes have also been shown to operate in the larval neural stem cells (Coumailleau, 2009) as well as in the adult intestinal stem cells in flies (Montagne, 2014), where they also play a role during asymmetric Notch signalling. In fish, Sara endosomes mediate asymmetric cell fate assignation mediated by Notch during the mitosis of neural precursor of the spinal cord (Loubery, 2017).

Sara endosomes are a subpopulation of Rab5-positive early endosomes characterised by the presence of the endocytic protein Sara. Sara directly binds the lipid phosphatidyl-inositol-3-phosphate and both molecules are found at the surface of these endosomes (Bokel, 2006). A pulse-chase antibody uptake assay has been established to monitor the trafficking of endogenous internalised Notch and Delta and showed that both Notch and Delta traffic through Sara endosomes (Coumailleau, 2009; Loubery, 2014; Derivery, 2014). Furthermore, it was shown that Sara endosomes are specifically targeted to the pIIa cell during SOP division, mediating thus the transport of a pool of Notch and Delta that contribute to the activation of Notch in the pIIa. The Notch cargo and its Uninflatable binding partner are required for this asymmetric dispatch (Loubery, 2014). Targeting of Sara endosomes to the central spindle is mediated by a plus-end-directed kinesin, Klp98A (Derivery, 2015). The asymmetric distribution of endosomes at the central spindle results from a higher density of microtubules in pIIb with their plus ends pointed towards pIIa15 (Loubery, 2017).

This study shows that the Sara protein itself controls both the targeting and the final dispatch of Sara endosomes to the pIIa daughter cell. Sara binds and is a target of the PP1 phosphatase complex. The phosphorylation state of Sara functions as a switch that enables the targeting of Sara endosomes to the central spindle of the dividing SOP, and their subsequent detachment from the central spindle, which is necessary to allow their movement to the pIIa daughter cell (Loubery, 2017).

Previous work has shown that a subpopulation of Rab5 early endosomes positive for Sara are asymmetrically dispatched into the pIIa daughter cell during cytokinesis of the SOP. This was monitored by following in vivo either GFP-Sara or internalized Delta or Notch, which reach the Sara endosomes 20 min after their endocytosis in the mother cell. These vesicles were termed iDelta20' endosomes. In contrast, the pools of Notch in endosomal populations upstream or downstream of the Sara endosomes (that is, the Rab5 early endosomes with low Sara levels and the Rab7 late endosomes, respectively) were segregated symmetrically. Rab5 endosomes show different levels of Sara signal: by a progressive targeting of Sara to the Rab5 endosomes, Rab5 early endosomes mature into Sara endosomes. This prompts the question whether the levels of Sara in endosomes correlate indeed with their asymmetric behaviour (Loubery, 2017).

To study the relationship between the levels of Sara in endosomes and their targeting to the spindle, Matlab codes were written to perform automatic 3D-tracking of the Sara endosomes. Sara endosomes were detected by monitoring a GFP-Sara fusion, which was overexpressed through the UAS/Gal4 system. This way, the position of the endosomes, their displacement towards and away from the central spindle was monitored as well as the levels of Sara. In addition, the position was detected automatically of the Pon cortical crescent, which forecasts the side of the cell that will become the pIIb cell (Loubery, 2017).

The localization of endosomes was studied with respect to a 2 μm-wide box centered in the central spindle during SOP mitosis. The enrichment was measured of endosomes in this central spindle as a function of time. Two phases were observed in the movement of the endosomes during mitosis: (1) targeting to the central spindle and (2) departure into the pIIa cell. The endosomes are progressively accumulating in the central spindle area from the end of metaphase (~450 s before abscission) through anaphase and during cytokinesis until they are enriched at the central spindle by about 10-fold at 250 s before abscission (Loubery, 2017).

Subsequently, the endosomes depart from the central spindle area into the pIIa cell. By fitting an exponential decay to the profile of abundance of the endosomes at the central spindle, the characteristic residence time of the endosomes at the central spindle was measured after the recruitment phase: after recruitment, endosomes remain at the central spindle 98±9.8 s before they depart into one of the daughter cells, preferentially the pIIa cell (Loubery, 2017).

To address a potential role of Sara on central spindle targeting and asymmetric segregation, the behaviour was tracked and quantified of the endosomes in a Sara loss of function mutant (Sara12) and in conditions of Sara overexpression in the SOP (Neur-Gal4; UAS-GFP-Sara). In Sara12 SOPs, targeting of iDelta20' endosomes to the cleavage plane is severely impaired. Consistent with the fact that the asymmetric dispatch of endosomes to pIIa requires first their targeting to the central spindle as previously shown, in Sara12 SOPs the dispatch to the pIIa daughter is strongly affected. A slight bias (60% pIIa targeting) is, however, retained in the mutant, consistent with a previous report (Loubery, 2017).

Conversely, overexpression of Sara increases targeting to the central spindle. In these conditions, Sara is found not only in Rab5 endosomes, but also in Rab7 late endosomes as well as in the Rab4 recycling endosomes. Correlating with this, Rab4, Rab5 and Rab7 endosomes, which are not all recruited to the central spindle in wild-type conditions, are now targeted to the central spindle upon Sara overexpression and are asymmetrically targeted (Loubery, 2017).

Furthermore, consistent with the correlation that is observed between the levels of Sara at the endosomes and their displacement towards the cleavage plane, quantification of central spindle targeting of the Sara endosomes upon its overexpression shows that targeting of the endosomes to the cleavage plane is increased by a factor of 2.5 in these conditions. These observations indicate that Sara plays a crucial role on the targeting of the endosomes to the spindle and the subsequent dispatch of the Notch/Delta containing endosomes to pIIa. Does this play a role during Notch-dependent asymmetric cell fate assignation? (Loubery, 2017).

Sara function contributes to cell fate assignation through asymmetric Notch signalling, but this activity is redundantly covered by Neuralized. Neuralized E3 Ubiquitin ligase does play an essential role during the endocytosis and activation of the Notch ligand Delta. Therefore, during larval development, Neuralized is essential for Notch-mediated lateral inhibition in the proneural clusters, which leads to the singling-out of SOP cells from the proneural clusters. Later, during pupal development, Neuralized appears as a cortical crescent in the pIIb side of the dividing SOPs, thereby biasing Delta activation in the pIIb cell and asymmetric activation of Notch in pIIa6 (Loubery, 2017).

Consistently, a partial loss of function of Neuralized by RNAi interference in the centre of the notum (Pnr>NeurRNAi Control) showed lateral inhibition defects in the proneural clusters, causing the appearance of supernumerary SOPs as well as asymmetric Notch signalling defects in the SOP lineage, leading to supernumerary neurons and loss of the external shaft/socket cells in the lineage. The remaining Neuralized activity in this partial loss of function condition allows many sensory organs (more than forty in the centre of the notum) to perform asymmetric cell fate assignation and to develop, as in wild type, into structures containing at least the two external cells (Loubery, 2017).

In Pnr>NeurRNAi, Sara12/Df(2R)48 transheterozygote mutants, the number of supernumerary SOPs is increased by 35% with respect to the Pnr>NeurRNAi controls (668±38 versus 498±52). This indicates that during lateral inhibition, Sara endosomes contributes to Notch signalling. This general role of Sara is uncovered when the Neuralized activity during Notch signalling is compromised (Loubery, 2017).

In the case of Neuralized, its localization to the anterior cortex biases Notch signalling to be elicited in the pIIa cell. This is the same in the case of Sara endosomes: asymmetric dispatch of Sara endosomes also biases Notch signalling to pIIa10. Indeed, in Pnr>NeurRNAi, Sara12/Df(2R)48 transheterozygote mutants, the number of bristles (external shaft/socket cells) in the notum is strongly reduced at the expense of supernumerary neurons compared to the Pnr>NeurRNAi controls. This indicates that Notch-dependent asymmetric cell fate assignation in the SOP lineage is synergistically affected in the Sara/Neuralized mutant. This implies that the SOP lineages which still could generate bristles with lower levels of Neuralized function in Pnr>NeurRNAi need Sara function to perform asymmetric cell fate assignation: in Pnr>NeurRNAi, Sara12/Df(2R)48 and Pnr>NeurRNAi, Sara12/Sara1 transheterozygote mutants, these lineages failed to perform asymmetric signalling, causing the notum to be largely bald. Therefore, Sara contributes to Notch signalling and asymmetric cell fate assignation, as observed in conditions in which other redundant systems for asymmetric Notch signalling are compromised (Loubery, 2017).

Both Neuralized and Sara play general roles in Notch signalling: they are both involved in lateral inhibition at early stages and, at later stages, in asymmetric cell fate assignation. Indeed, both Neuralized and Sara mutants show early defects in lateral inhibition and, accordingly, they show supernumerary SOPs. In addition, Neuralized and Sara mutant conditions also show defective Notch signalling during cell fate assignation in the SOP lineage and therefore cause the transformation of the cells in the lineage into neurons. In this later step, Notch signalling is asymmetric. The possibility that both Sara and Neuralized play key roles in ensuring the asymmetric nature of this signalling event is only correlative: in the case of Neuralized, it is enriched in the anterior cortex of the cell, which will give rise to pIIb; in the case of Sara, (1) both Delta and Notch are cargo of these endosomes, (2) cleaved Notch is seen in the pIIa endosomes and (3) Sara endosomes are dispatched asymmetrically to pIIa10. It is tantalizing to conclude that the asymmetric localization of these two proteins mediate the asymmetric nature of Notch signalling in the SOP lineage, but further assays will be necessary to unambiguously address this issue. Clonal analysis is unfortunately a too slow assay to sort out the specific requirement of these cytosolic factors (Sara and Neuralized) in the pIIa versus the pIIb cell (Loubery, 2017).

Sara mediates the targeting of Notch/Delta containing endosomes to the central spindle and could contributes to Notch-mediated asymmetric signalling in the SOP lineage. What machinery controls in turn the Sara-dependent targeting of endosomes to the central spindle? Previous proteomic studies uncovered bona fide Sara-binding factors, including the Activin pathway R-Smad, Smox17 and the beta subunit of the PP1c serine-threonine phosphatase (PP1β(9C)). In an IP/Mass Spectrometry approach, those interactions were confirmed and in addition to PP1β(9C), two of the other three Drosophila isoforms of PP1c: PP1α(87B) and PP1α(96A) were found. Furthermore, the PP1c regulatory subunit Sds22 was found, suggesting that Sara binds the full serine-threonine PP1 phosphatase complex. The interaction with Sds22 was confirmed by immunoprecipitation of overexpressed Sds22-GFP and western blot detection of endogenous Sara in the immunoprecipitate (Loubery, 2017).

Prompted by these results, whether the PP1 complex plays a role in the asymmetric targeting of the Sara endosomes was explored by manipulating the activity of Sds22, the common regulatory unit in all the complexes containing the different PP1 isoforms. Sds22 was overexpressed specifically during SOP mitosis, by driving Sds22-GFP under the Neur-Gal4 driver with temporal control by the Gal80ts system. In SOPs where PP1-dependent dephosphorylation is enhanced by overexpressing Sds22, the Sara endosomes fail to be dispatched asymmetrically toward the pIIa daughter cell (Loubery, 2017).

The role of PP1-dependent dephosphorylation in the SOP was examined by knocking down Sds22 (through a validated Sds22-RNAi). Loss of function Sds22 did also affect the asymmetric targeting of endosomes. These data uncover a key role for phosphorylation and PP1-dependent dephosphorylation as a switch that contributes to the asymmetric targeting of Sara during asymmetric cell division (Loubery, 2017).

The observations raise the question of which is the step in the asymmetric dispatch of the endosomes that is controlled by the levels of phosphorylation: central spindle targeting, central spindle detachment or targeting to the pIIa cell? PP1/Sds22-dependent dephosphorylation controls a plethora of mitotic events, including mitotic spindle morphogenesis, cortical relaxation in anaphase, epithelial polarity and cell shape, Aurora B activity and kinetochore-microtubule interactions as well as metabolism, protein synthesis, ion pumps and channels. Therefore, to establish the specific event during the asymmetric dispatch of Sara endosomes that is controlled by PP1/Sds22 dephosphorylation, focus was placed on the phosphorylation state of Sara itself and its previously identified phosphorylation sites. This allowed specific interference with this phosphorylation event and thereby untangle it from other cellular events also affected by dephosphorylation (Loubery, 2017).

PP1/Sds22 was shown to bind Sara. It has previously been shown that mammalian Sara itself is phosphorylated at multiple sites and that the level of this Sara phosphorylation is independent on the level of TGF-beta signalling. Three phosphorylation sites have been identified at position S636, at position S709, and at position S774 in Sara protein and these sites were confirmed by Mass Spectrometry of larval tissue expressing GFP-Sara. Phosphorylation of Sara had been previously reported to be implicated in BMP signalling during wing development. However, the role of these three phosphorylation sites during asymmetric division are to date unknown (Loubery, 2017).

ProQ-Diamond phospho-staining of immunoprecipitated GFP-Sara confirmed that Sara is phosphorylated. To test whether PP1/Sds22 controls the phosphorylation state of Sara, ProQ-Diamond stainings of GFP-Sara were performed with and without down-regulation of Sds22. Downregulating Sds22 induced a 40%-increase in the normalized quantity of phosphorylated Sara, showing that PP1/Sds22 does control the phosphorylation state of Sara (Loubery, 2017).

To study the role of Sara phosphorylation during asymmetric targeting of the endosomes, the mitotic behaviour of the endosomes was analyzed in conditions of overexpression of mutant versions of Sara where (1) the three phosphorylated Serines (at position S636, S709, and S774) were substituted by Alanine (phosphorylation defective: GFP-Sara3A) or (2) the PP1 interaction was abolished by an F678A missense mutation in the PP1 binding domain (hyper-phosphorylated: GFP-SaraF678A). Neither mutation affects the general levels of abundance of the Sara protein in SOPs, the targeting of Sara itself to the endosomes, nor the residence time of Sara in endosomes as determined by FRAP experiments. Also, the targeting dynamics of internalized Delta to endosomes are not affected in these mutants (Loubery, 2017).

Upon overexpression of GFP-Sara3A in SOPs, the rate of targeting of the endosomes to the central spindle is greatly increased. In addition, GFP-Sara3A shows impaired departure from the spindle: while the residence time of Sara endosomes at the central spindle after their recruitment is around 100 s in wild type, GFP-Sara3A endosomes stay at the spindle significantly longer (151±21 s). In GFP-Sara3A endosomes, impaired departure leads to defective asymmetric targeting to the pIIa cell while, in wild type, departure from the central spindle occurs well before abscission, in the GFP-Sara3A condition, endosomes that did not depart are caught at the spindle while abscission occurs. These data indicate that the endosomal targeting to the central spindle is greatly favoured when these three sites in Sara are dephosphorylated and suggest that the departure from the microtubules of the central spindle requires that the endosomes are disengaged by phosphorylation of Sara (Loubery, 2017).

Loss of Sara phosphorylation in these sites impairs disengagement from the central spindle. Conversely, impairing Sara binding to the PP1 phosphatase results in defective targeting to the central spindle. Indeed, when binding of Sara to the PP1/Sds22 phosphatase is impaired in the GFP-SaraF678A overexpressing SOP mutants, Sara endosomes fail to be targeted to the spindle. Mistargeted away from the central spindle, the GFP-SaraF678A endosomes fail thereby to be asymmetrically targeted to the pIIa cell. Loss and gain of function phenotypes of the Phosphatase regulator Sds22 during endosomal spindle targeting support the role of Sara phosphorylation during targeting to the central spindle microtubules suggested by the GFP-Sara3A and GFP-SaraF678A experiments (Loubery, 2017).

What are the functional consequences on signalling of impaired phosphorylation/dephosphorylation in Sara mutants? The presence of Sara in endosomes is itself essential for Notch signalling. Sara loss of function mutants show a phenotype in SOP specification (supernumerary SOPs) as well as during fate determination within the SOP lineage (all cells in the lineage acquire a neural fate). In addition, this study showed that Sara is also essential for the targeting of endosomes to the spindle: in the absence of Sara, endosomes fail to move to the spindle in the SOP. They are therefore dispatched symmetrically, but those endosomes do not mediate Notch signalling. As a consequence, both daughters fail to perform Notch signalling in sensitized conditions in which Neuralized is compromised. The result is a Notch loss of function phenotype: the whole lineage differentiates into neurons (Loubery, 2017).

In both Sara3A and SaraF678A mutants, because of reasons that are different in the two cases (either they do not go to the spindle or their departure from the spindle is impaired), functional Sara endosomes are dispatched symmetrically. In contrast to the situation in the Sara loss of function mutant, those endosomes are functional Sara signalling endosomes, which can mediate Notch signalling in both cells. Therefore, these mutations are consistently shown to cause a gain of function Sara signalling phenotype: supernumerary sockets are seen in the lineages (88% of the lineages for Sara3A and 82% of the lineages for SaraF678A). A milder version of this phenotype can be also seen by overexpressing wild-type Sara (34% of the lineages) consistent again with some gain of function Notch signalling phenotype when Sara concentrations are elevated. In summary, this implies that the 3A and F678A mutations impair the phosphorylation state of Sara (with consequences in targeting), but not its function in Notch signalling (Loubery, 2017).

These results indicate that Sara itself plays a key, rate limiting role on the asymmetric targeting of the endosomes by controlling the targeting to the spindle and its departure. Maturation of the early endosomes by accumulating PI(3)P leads to accumulation of the PI(3)P-binding protein Sara to this vesicular compartment. At the endosome, the phosphorylation state of Sara indeed determines central spindle targeting and departure: in its default, dephosphorylated state, Sara is essential to engage the endosomes with the mitotic spindle. Phosphorylation of Sara disengages the endosomes from the central spindle allowing the asymmetric departure into the pIIa cell (Loubery, 2017).

Polarized endosome dynamics by spindle asymmetry during asymmetric cell division

During asymmetric division, fate determinants at the cell cortex segregate unequally into the two daughter cells. It has recently been shown that Sara (Smad anchor for receptor activation) signalling endosomes in the cytoplasm also segregate asymmetrically during asymmetric division (Coumailleau, 2009; Loubéry, 2014). Biased dispatch of Sara endosomes mediates asymmetric Notch/Delta signalling during the asymmetric division of sensory organ precursors in Drosophila. In flies, this has been generalized to stem cells in the gut and the central nervous system, and, in zebrafish, to neural precursors of the spinal cord. However, the mechanism of asymmetric endosome segregation is not understood. This study shows that the plus-end kinesin motor Klp98A targets Sara endosomes to the central spindle, where they move bidirectionally on an antiparallel array of microtubules. The microtubule depolymerizing kinesin Klp10A and its antagonist Patronin generate central spindle asymmetry. This asymmetric spindle, in turn, polarizes endosome motility, ultimately causing asymmetric endosome dispatch into one daughter cell. This mechanism was demonstrated by inverting the polarity of the central spindle by polar targeting of Patronin using nanobodies (single-domain antibodies). This spindle inversion targets the endosomes to the wrong cell. These data uncover the molecular and physical mechanism by which organelles localized away from the cellular cortex can be dispatched asymmetrically during asymmetric division (Derivery, 2015).

Klp98A was first identified as the kinesin mediating Sara endosome motility during sensory organ precursor (SOP) division. Klp98A is the Drosophila homologue of mammalian KIF16B, an early endosomal kinesin containing a phosphatidylinositol 3-phosphate-binding PX domain. Indeed, Klp98A localizes to Sara-positive early endosomes (Derivery, 2015).

During SOP division, Klp98A-GFP-positive Sara endosomes segregate to the pIIa daughter, but not the pII (Coumailleau, 2009; Loubéry, 2014). Sara endosomes were monitored by following Delta 20 min after internalization (iDelta20) through an improved antibody internalization assay. iDelta20 parallels Sara endosome dynamics in the controls and mutants studied here (in vivo and primary cultures. Like KIF16B, purified Klp98A binds specifically to phosphatidylinositol 3-phosphate and is a plus-end-directed motor whose velocity is 0.76 ± 0.02 microm s-1 (Derivery, 2015).

To study Klp98A function, deletions within the motor domain (Klp98AΔ6, Klp98AΔ7 and Klp98AΔ8, 6, 7 and 8-base-pair deletions, respectively) and a clean coding sequence deletion (Klp98AΔ47). Except Klp98AΔ6, all are protein nulls. In Klp98A-, Sara endosomes move diffusively. Therefore, Klp98A mediates Sara endosome motility (Derivery, 2015).

In wild-type cells, Sara endosomes move on microtubules to the Pavarotti-positive central spindle and, late in cytokinesis, to pIIa. Spindle microtubule plus-ends are oriented towards the equator, explaining central spindle endosomal targeting by a plus-end motor. Indeed, Sara endosome central spindle targeting fails in Klp98A- mutants. Importantly, in Klp98A- mutants and upon RNAi-mediated Klp98A knockdown, endosomes are symmetrically dispatched (Derivery, 2015).

Klp98A-mediated motility contributes to cell fate assignation through asymmetric Notch signalling, but this activity is redundantly covered by Neuralized and Numb (Fürthauer, 2009). Indeed, Klp98A-;pnr > neurRNAidouble mutants show a synergistic fate assignation phenotype: the notum is largely void of bristles. Conversely, Klp98A;Numb double mutants strongly suppress the diagnostic Numb- multiple socket phenotype. Therefore, having established the role of Klp98A motility in Notch signalling, this study focused on the mechanisms orchestrating asymmetric motility (Derivery, 2015).

Central spindle targeting of Sara endosomes precedes asymmetric segregation to pIIa. Focus was therefore placed on Sara endosome motility with respect to the central spindle reference frame. The central spindle is composed of the Pavarotti-positive core (containing antiparallel microtubules) plus the microtubules emanating from it. The Pavarotti core was automatically tracked, defining a 2D cartesian reference frame whose origin is the Pavarotti centroid and whose x axis is the pIIb-pIIa axis. This also defines a Pavarotti width (PW) and length (PL, the length of the microtubule antiparallel array (Derivery, 2015).

Sara endosomes were tracked with respect to this reference frame (with 160 nm accuracy. Automatic tracking and spatio-temporal registration provided a large data set (2,897 traces) from which a spatio-temporal density plot of endosomes at the central spindle was generated. For 500 s, endosomes remain mostly within the Pavarotti region. Remarkably, at the central spindle, motility along the x axis is bidirectional. Motility along the y axis merely follows PW contraction, consistent with motility along central spindle microtubules, parallel to the x axis. Velocities are similar towards pIIa and pIIb and slower than in vitro, possibly due to crowding by microtubule-associated proteins (Derivery, 2015).

Confinement within the Pavarotti region and bidirectional movement are both consistent with a plus-end motor switching direction on antiparallel microtubules. On single microtubules, Klp98A-bound quantum dots always maintain their directionality when resuming after a pause. It was asked whether Klp98A could switch direction in an antiparallel bundle. In an in vitro reconstitution assay, Klp98A-bound quantum dots move bidirectionally within antiparallel MAP65-1-mediated microtubule arrays. 68% tracks change direction after pausing. Therefore, Klp98A supports bidirectional motility in antiparallel array (Derivery, 2015).

Notably, in vivo, bidirectional endosome motility is asymmetric: the residence time in pIIa is 1.8-fold longer than in pIIb. Consistently, the spatio-temporal density plot is asymmetric. Furthermore, tracks overshoot beyond the Pavarotti region more frequently into pIIa (Derivery, 2015).

Eventually, endosomes depart from central spindle microtubules into the cytoplasm and therefore move also on the y axis. The longer pIIa residence time and higher pIIa overshoot frequency make this final departure asymmetric, explaining the biased segregation into pIIa. Therefore, asymmetric endosome motility at the central spindle underlies asymmetric dispatch to pIIa (Derivery, 2015).

It was then asked whether the central spindle itself is asymmetric. Using Pavarotti spatio-temporal registration, an 'average cell' was generated to map the densities of the microtubule markers Jupiter and SiR-tubulin (microtubule markers), Patronin (Goodwin, 2010) (minus-end), and Pavarotti (plus-ends/antiparallel overlap). This 'average cell' reveals a polarity map of the central spindle consistent with electron microscopy reports: plus-ends are in the middle and minus-ends on the outer side. Microtubule densities in general, and Patronin in particular, are ~20% higher on the pIIb side. This asymmetry depends on Par complex activity, and is absent in neighbouring cells dividing symmetrically, but this seems independent of central spindle or endosomal asymmetry (Derivery, 2015).

Microtubule asymmetry builds up during anaphaseB, concomitant with biased endosome motility, while, earlier, the metaphase spindle is symmetrical. During anaphaseB, the central spindle shrinks by microtubule depolymerization through depolymerizing kinesins like Klp10A, among other factors. Depolymerization dynamics are asymmetric: microtubule loss is faster in pIIa. This could be explained by Patronin enrichment in the central spindle pIIb outer side where it binds to minus-ends, counteracting Klp10A-mediated depolymerization (Goodwin, 2010; Wang, 2013; Hendershott, 2014; Jiang, 2014; Derivery, 2015).

Indeed, Klp10A/Patronin control asymmetric microtubule depolymerization: their depletion abolishes spindle asymmetry. In Patronin-knockdown cells, both sides exhibit low microtubule densities characteristic of pIIa, consistent with Patronin pIIb enrichment in wild type and its activity against depolymerization. Conversely, upon knockdown of Klp10A, both sides exhibit high microtubule densities resembling pIIb (Derivery, 2015).

The parallelism between central spindle asymmetry and asymmetric endosome motility suggests that spindle asymmetry causes biased motility. Indeed, endosome motility at the central spindle and, therefore, segregation become symmetric in Klp10A- and Patronin-knockdowns, while early central spindle targeting is normal. This uncovers a quantitative correlation between spindle and endosomal asymmetry (Derivery, 2015).

Together, a plus-end motor and microtubule plus-ends facing the centre explain why a higher pIIb microtubule density (~20% enrichment) targets endosomes to pIIa (~80% pIIa, that is, >300% enrichment). In other words, endosomes move away from higher microtubule densities in pIIb (Derivery, 2015).

Based on a theoretical model of plus-end endosomal motility on an antiparallel, asymmetric microtubule overlap, the steady-state endosome distribution is

Where PpIIa, PpIIb, the probabilities for an endosome to be on either side of the antiparallel overlap; ρa, ρb, microtubule densities in pIIa/pIIb, respectively; kon, koff, microtubule association/dissociation constants of the motor, respectively; v, the endosome motor-driven velocity; D, the diffusion coefficient of endosomes detached from microtubules; and l, the antiparallel overlap length (Derivery, 2015).

To generate this inverted spindle, 'nanobody assay' was established based on GFP-binding-peptide (GBP)-Pon, a nanobody fused to the Pon localization domain. GBP-Pon traps GFP-Patronin away from the spindle at the pIIb cortex thereby reducing, specifically in pIIb, Patronin-dependent protection against central spindle depolymerization. This inverts spindle asymmetry, which consequently inverts endosomal asymmetry. SiR-Tubulin and endogenous acetyl-tubulin stainings confirmed this spindle inversion (Derivery, 2015).

Interestingly, this assay generates a phenotypic series of different levels of spindle reversal and their corresponding endosomal reversals. These data fall on the theoretical curve obtained with independently measured parameters. Therefore these results uncover the quantitative dependence of asymmetric endosome targeting on spindle asymmetry (Derivery, 2015).

This study has identified Klp10A/Patronin as the machinery generating spindle asymmetry, which is read out by Klp98A to achieve asymmetric targeting of signalling endosomes. Asymmetric endosomal targeting contributes in turn to asymmetric cell fate assignation, confirming previous reports in flies and fish. These data thus uncover a mechanism by which intracellular cargoes in general, and signalling endosomes in particular, can be targeted to one of the daughter cells during asymmetric division (Derivery, 2015).

How could then other cargoes segregate symmetrically, if the spindle is asymmetric? Asymmetric targeting would only be efficient if kon, koff and v are optimized to amplify the mild asymmetry of the spindle, otherwise concealed by noise sources in the cell. More generally, plus- and minus-end motors are present simultaneously in the same vesicle and thereby may counteract each other to achieve symmetrical dispatch (a sort of 'tug of war'). Therefore, the precise landscape of microtubule polarity trails combined with the right cocktail of motors in vesicles provides the plasticity required to generate the plethora of molecular spatial patterns observed in polarized cells (Derivery, 2015).

Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division

Cell fate decision during asymmetric division is mediated by the biased partition of cell fate determinants during mitosis. In the case of the asymmetric division of the fly sensory organ precursor cells, directed Notch signaling from pIIb to the pIIa daughter endows pIIa with its distinct fate. Previous studies have shown that Notch/Delta molecules internalized in the mother cell traffic through Smad anchor for receptor activation (Sara) endosomes and are directed to the pIIa daughter. This study shows that the receptor Notch itself is required during the asymmetric targeting of the Sara endosomes to pIIa. Notch binds Uninflatable, and both traffic together through Sara endosomes, which is essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation. The data uncover a part of the core machinery required for the asymmetric motility of a vesicular structure that is essential for the directed dispatch of Notch signaling molecules during asymmetric mitosis (Loubery, 2014).

The Notch signaling pathway plays multiple roles in organisms ranging from flies and worms to mammals. A powerful model system to elucidate the cell biology of Notch signaling is the Drosophila sensory organs. Each sensory organ precursor (SOP) cell divides asymmetrically to produce a pIIa cell and a pIIb daughter cell, which perform directed Notch signaling: pIIb signals to pIIa. Four independent endocytic mechanisms control asymmetric signaling in the SOP. These include asymmetric endocytic events mediated by the E3 ubiquitin ligase Neuralized, recycling endosomes, and the endocytic adaptors α- and γ-adaptin together with Numb (Loubery, 2014).

During SOP cytokinesis, a fourth mechanism involves a population of endosomes marked by the adaptor protein Sara. Sara endosomes contain as cargo a pool of endocytosed Notch and Delta molecules. Notch and Delta reach the Sara endosome 20 min after their endocytosis in the SOP; this pool is dispatched into pIIa during cytokinesis. In contrast, the pools of Notch in endosomal populations upstream (Rab5 early endosomes) or downstream (Rab7 late endosomes) of Sara endosomes are segregated symmetrically. The specific pool of Notch in Sara endosomes is relevant for signaling: it is cleaved in a ligand- and gamma-secretase-dependent manner to release the transcriptionally active Notch intracellular domain (NICD) in pIIa (Loubery, 2014).

A key question is what machineries control the asymmetric targeting of these endosomes. Is the cargo (the ligand Delta or its receptor Notch) playing a role on the specific targeting of these endosomes? To unravel the machinery regulating the behavior of Sara endosomes during SOP mitosis, candidate factors from previously reported proteomics approaches or genetic screens were tested for Notch signaling. Thus, Uninflatable was identified as a factor involved in the asymmetric dynamics of Sara endosomes (Loubery, 2014).

MARCM homozygous mutant clones were generated for a null allele of Uninflatable (Uif2B7) an the trafficking of Delta, Notch, and the Notch effector Sanpodo through Sara endosomes was monitored. To look at the motility of the endogenous population of Sara endosomes, the cohort of internalized Delta molecules 20 min after its endocytosis was followed in the SOP by means of a pulse-chase antibody uptake assay. Delta, Notch, and Sanpodo traffic normally through Sara endosomes in the absence of Uif, and these endosomes are targeted to the cleavage plane (the central spindle) in cytokinesis (Loubery, 2014).

In Uif mutants or RNAi knockdown conditions, iDl20'/Sara endosomes fail to be asymmetrically dispatched to pIIa after their targeting to the central spindle. These results indicate that Uif is not required to bring Notch to the Sara endosomes or to target the endosomes to the central spindle. However, once in the spindle, Uif is essential for the specific dispatch of Sara endosomes from the spindle into the pIIa cell (Loubery, 2014).

This function of Uninflatable is specific to the asymmetric segregation of Sara endosomes. To gain mechanistic insights into the mechanism of action of Uif, this study has analyzed the density of microtubules in the central spindle and has shown that Uninflatable does not regulate the organization of the microtubular cytoskeleton. In contrast, it was found that Uif controls the residence time of Sara endosomes on the central spindle: in control SOPs, Sara endosomes depart from the central spindle with a decay time of 103 ± 21 s, whereas upon Uif downregulation this decay time goes up to 175 ± 42 s. These data indicate that Uif is not involved in the organization of the spindle, but rather in the motility properties of the endosomes, particularly their last step of departing from the central spindle and end up in pIIa (Loubery, 2014).

Consistent with the role of Uif in the asymmetric targeting of Sara endosomes, Uif contributes to Notch-dependent cell fate assignation in the SOP lineage. To address this, the composition of SOP lineages was examined in homozygous Uif2B7 MARCM clones or upon Uif RNAi. In wild-type animals, the SOP lineage consists of four different cells: two external cells (the shaft and the socket) originating from pIIa and two internal cells (the sheath and the neuron) from pIIb, which can be identified by immunostaining. In Uif mutant clones, instead of a sheath and a neuron per SOP lineage, two sheath cells can be frequently observed in the notum, indicating a symmetric division in the pIIb lineage. Similarly, upon Uif downregulation in the postorbital SOPs, duplications of sockets were observed, which is diagnostic of symmetric divisions in the pIIa lineage. These data uncover a role for Uninflatable in Notch-dependent asymmetric cell fate assignation that is mediated by the asymmetric dispatch of the Sara endosomes (Loubery, 2014).

The Uif phenotype during asymmetric endosomal targeting and cell fate assignation prompted us to look whether Uif is a cargo of Sara endosomes. To detect the endogenous protein, anti-Uif antibodies were generated. To look at Uif trafficking in vivo, transgenic flies were generated expressing a Uif-GFP protein, which can provide activity to rescue the lethality of a Uif lethal mutation at least partly (Loubery, 2014).

Uif-GFP is strongly colocalized with both Sara-GFP and iDelta20'. Since a cargo of Sara endosomes is Notch itself (73% ± 2.7% of the vesicular population of Notch molecules is in Sara endosomes), the presence of Notch cargo was examined in Uif vesicles: 44% ± 4.7% of Uif-positive vesicular structures contain Notch. Therefore, a population of Uninflatable and Notch traffics through Sara endosomes during SOP asymmetric mitosis (Loubery, 2014).

The fact that Uninflatable controls the asymmetric dispatch of the Sara endosomes, which contain internalized Notch and Uninflatable, prompted a look at a possible molecular interaction between Uninflatable and Notch. Uif- and Notch-expressing plasmids were cotransfected in S2 cells and immunoprecipitation experiments were performed by using anti-Uif-coupled beads, followed by immunoblotting with a clean anti-Notch antibody that was purified from a hybridoma cell line (DSHB #C17.9C6). Uif was shown to immunoprecipitate Notch. This coimmunoprecipitation can be reproduced from lysates of S2 cells expressing Notch and Uif tagged with the PC peptide tag and anti-PC-coupled beads; as a control, other transmembrane proteins such as Tkv-GFP are not coimmunoprecipitated with Uif-PC. Together, these results indicate a specific molecular interaction between Notch and Uif (Loubery, 2014).

Uninflatable is a transmembrane protein that, like Notch, contains an array of epidermal growth factor (EGF) repeats. It has been shown that Notch is engaged in protein-protein interactions through its EGF repeats with other factors containing EGF repeats. These include its ligand Delta, but also a number of noncanonical Notch ligands, secreted or membrane proteins lacking the DSL domain characteristic of canonical Notch ligands (Dlk-1, Dlk-2, DNER, Trombospondin, LRP1, EGFL7, and Weary). Consistently, it has recently been reported that a synergistic genetic interaction between Uif and Notch depends on Notch EGF repeats. Therefore, studies were performed to discover which EGF repeats of Uif could be involved in the molecular interaction with Notch. A coimmunoprecipitation experiment was performed in S2 cells coexpressing Notch and an N-terminal, truncated form of Uif tagged with PC (UifΔCter-PC) that lacks the four EGF domains flanking the transmembrane domain but still contains the other 17 EGF repeats and other extracellular domains. While full-length Uif-PC coimmunoprecipitates Notch, UifΔCter-PC does not. This indicates that the interaction between Uif and Notch may be mediated by the four EGF domains of Uif flanking its transmembrane domain (Loubery, 2014).

Although Uif binds and colocalizes with Notch, it does not play a role in core Notch signaling: embryos deprived of maternal and zygotic Uif in germline clones do not show a Notch signaling phenotype, whereas they display loss of inflation of the trachea as previously reported. Consistently, loss of Uif in wing mosaics does not cause a defect of Notch-dependent expression of Wingless at the wing margin. This indicates that Uninflatable interaction with Notch is not essential during core Notch signaling, but rather during the asymmetric dispatch of Notch-containing Sara endosomes during asymmetric cell division. This prompted the possibility that Notch itself is required for the asymmetric motility of the endosomes (Loubery, 2014).

To study whether Notch plays a role during the asymmetric dispatch of Sara endosomes, the trafficking was studied of a Notch-GFP fusion expressed at endogenous levels. The idea was to confirm previous observations using a Notch antibody uptake assay to follow Notch expressed at endogenous levels. To achieve this, a reporter transgenic fly strain was set up in which Notch-GFP fusion is driven by the Notch endogenous promoter and is expressed at endogenous levels. In this fusion, GFP is inserted in the middle of the Notch-intra domain. Since in protein fusions GFP is frequently cleaved out, whether the fusion protein is intact was examined. This would be particularly important in this case, since a cleavage event would lead to a truncated Notch-intra peptide (Loubery, 2014).

In these transgenic Notch-GFP flies, GFP is very efficiently cleaved out (74% of total GFP is cleaved, leading to truncated Notch-intra peptides that can only partially support Notch function and thereby cause a highly penetrant mutant phenotype. This precludes the usage of this reagent as a bona fide marker for Notch. In particular, the cytosolic GFP signal cannot be used as a readout of signaling as previously reported: a nuclear accumulation of the GFP signal in these flies does not solely reflect the accumulation of Notch-intra-GFP, but rather the overall accumulation of different GFP-containing fragments (Loubery, 2014).

Whether, in these conditions, the pool of membrane associated GFP-Notch traffics through Sara endosomes and is asymmetrically dispatched to the pIIa cell was studied. Only 11% ± 1.3% of the total GFP signal in these flies is membrane associated (plasma membrane and intracellular vesicular structures). The rest, representing the vast majority (89%), corresponds to cytosolic and nuclear cleaved GFP (Loubery, 2014).

In Notch-GFP flies, 3.1% of the total GFP signal is associated with intracellular vesicular structures. These correspond to various intracellular vesicular compartments, including Notch in the secretory pathway, as well as in early endosomes, Sara endosomes, recycling endosomes, and late endosomes. To measure the size of the specific pool of Notch in Sara endosomes, a Notch antibody internalization assay was performed, and internalized Notch was chased 20 min after its endocytosis (iNotch20'). As previously established, 73% ± 2.7% of Notch-GFP vesicles are positive for iNotch20'. Of this iNotch20'-positive pool, 79% would be targeted to pIIa . This is consistent with only 65% ± 3.1% of the total pool of Notch-GFP being dispatched to pIIa (Loubery, 2014).

Whether Notch itself plays a role on the asymmetric targeting of Sara endosomes was addressed. Notch was depleated in the SOP by expressing a previously validated Notch dsRNA, and the behavior of Sara endosomes was examined. Upon Notch knockdown in the SOP, iDl20'/Sara endosomes are still targeted to the central spindle, but the subsequent directed dispatch to pIIa is defective. This indicates that Notch itself contributes to the endosomal recruitment of the machinery that endows the Sara endosomes with their asymmetric behavior (Loubery, 2014).

It has been shown that the targeting of Notch to Sara endosomes does not depend on Uninflatable; it was then determined whether the recruitment of Uninflatable on Sara endosomes depended on Notch. Interestingly, it was found that, conversely, the targeting of Uif to Sara endosomes is not controlled by Notch. This implies that these two molecules use different machineries to get to the endosome, where they can interact and are both required for the asymmetric motility of the endosome (Loubery, 2014).

Since the Notch receptor itself is required for the asymmetric targeting of Sara endosomes, it was asked whether Notch signaling plays a role in the process. Notch signaling was blocked by inactivating the ligand Delta through overexpression of Tom in the SOP cell; Tom overexpression leads to inactivation of the Ubiquitin ligase Neuralized and thereby blocks endocytosis-dependent activation of Delta. In the absence of Notch signaling, targeting of Sara endosomes to the central spindle and their asymmetric dispatch to the pIIa cell remains intact. This indicates that although the Notch receptor is essential for the asymmetric targeting of Sara endosomes, Notch signaling is not (Loubery, 2014).

This report has started to unravel the machinery that mediates asymmetric endosome motility during asymmetric cell division. Both Notch and Uninflatable were shown to play a key role in the last step of the asymmetric motility of endosomes: the final, specific stride of the Sara endosomes from the central spindle into the anterior pIIa cell. This is based on the following four key sets of observations (Loubery, 2014).

First, it was confirmed that a functional Notch-GFP fusion expressed at endogenous level does traffic through Sara endosomes, which are indeed dispatched asymmetrically during SOP mitosis. Second, Notch binds Uninflatable, and both colocalize in Sara endosomes. Third, neither Notch nor Uninflatable is essential for the targeting of Notch/Delta/Uif to the Sara endosomes or the targeting of those endosomes to the central spindle, but they are essential for the final dispatch from the central spindle into the pIIa cell. Although Notch is necessary for this process, Notch signaling is not. Fourth, Uninflatable is not an integral component of the Notch signaling pathway, but it plays a role during asymmetric Notch signaling in the SOP, and therefore mutant Uif conditions lead to a lineage identity phenotype. It remains to be elucidated what machineries downstream of Notch/Uninflatable implement the control of the final step toward pIIa and what is asymmetrical in the cytoskeleton so that this final step occurs toward pIIa and not pIIb (Loubery, 2014).

Sara endosomes and the asymmetric division of intestinal stem cells

Tissue homeostasis is maintained by adult stem cells, which self-renew and give rise to differentiating cells. The generation of daughter cells with different fates is mediated by signalling molecules coming from an external niche or being asymmetrically dispatched between the two daughters upon stem cell mitosis. In the adult Drosophila midgut, the intestinal stem cell (ISC) divides to generate a new ISC and an enteroblast (EB) differentiating daughter. Notch signalling activity restricted to the EB regulates intestinal cell fate decision. This study shows that ISCs divide asymmetrically, and Sara endosomes in ISCs are specifically dispatched to the presumptive EB. During ISC mitosis, Notch and Delta traffic through Sara endosomes, thereby contributing to Notch signalling bias, as revealed in Sara mutants: Sara itself contributes to the control of the ISC asymmetric division. These data uncover an intrinsic endosomal mechanism during ISC mitosis, which participates in the maintenance of the adult intestinal lineage (Montagne, 2014).

Midgut stem cell self-renewal is essential to maintain a pool of dividing cells, which compensates the death of differentiated cells. This report showed that ISCs can divide asymmetrically to generate one daughter, which remains a stem cell, whereas the other differentiates into an enterocyte or enteroendocrine cell. During ISC mitosis, Notch/Delta contained within Sara endosomes are directionally dispatched to be inherited by the newly formed EB. These Sara endosomes and their cargo contribute themselves to the generation of asymmetric lineages within the midgut posterior-most region. This conclusion is based on the following five key observations: (1) the asymmetric cortical localization of Pon during ISC mitosis, which is inherited by the newly formed ISC daughter; (2) the asymmetric segregation of Sara endosomes away from the Pon marker in cytokinesis, thereby accumulating into the presumptive EB; (3) the trafficking of both Notch and Delta through Sara endosomes and their asymmetric partitioning during ISC mitosis; (4) the expansion of the ISC compartment in Sara mutants; and (5) the increase of symmetric lineages composed of two Delta-positive cells, both in Sara mutant and Sara overexpression clones, which is similar to Notch loss-of-function phenotype (Montagne, 2014).

The results are therefore consistent with a scenario where Notch/Delta molecules that are endocytosed in the ISC traffic through Sara endosomes. These Sara endosomes go to the central spindle and are finally targeted to the presumptive EB. Carrying Notch and Delta, the EB initiates its fate decision with the activation of Notch signalling and thereby becomes the signal-receiving cell. This mechanism might be conserved in vertebrates. Indeed, it has recently been shown that the mammalian homologue of Delta, Dll1, is asymmetrically inherited during mitosis of mouse neural stem cells, in a mechanism that has been implicated in maintaining stem cell quiescence (Montagne, 2014).

Live imaging of Sara endosomes showed frequent asymmetric inheritance of these endosomes during ISC divisions: indeed, on average, 75% of the Sara endosomes are asymmetrically targeted into one of the two daughter cells. This is consistent withclonal analysis of the midgut lineages and similar previous reports showing that, although symmetric division can also take place in the midgut, the asymmetric lineages are the most frequent ones (Montagne, 2014).

It has recently been shown that homeostasis in the mammalian intestine can also be achieved by a mechanism implicating ISC symmetric division and neutral clonal competition in the ISC compartment. A similar mechanism of self-renewal has been proposed in the Drosophila adult midgut. In this scenario, dividing ISC cells can give rise to two ISCs, two EBs, or one ISC and one EB. It was proposed that ISCs divide symmetrically, and the fate of the progeny is resolved through lateral inhibition mediated by Dl/N signalling. It was speculated that asymmetric cell fate determination occurs by lateral inhibition within the lineage and therefore only one daughter cell is selected for the ISC fate. Conversely, symmetric cell fate determination would happen only when competition happens between two different lineages, so that one sibling pair become ISCs and the other sibling pair become EBs. Alternatively, the data cannot exclude the possibility that an intrinsic mechanism operating in a single lineage determines stochastically whether the division is symmetric or asymmetric. This report show that segregation of Sara/Delta endosomes is consistent with such alternative intrinsic mechanism, whereby asymmetry of Sara would bias divisions to be asymmetric, while more symmetric segregation of these endosomes could allow divisions to be symmetric. (Montagne, 2014).

Sara mutants produce more Delta-positive cells, but the proliferation rate of these cells is affected. Moreover, mutation in Sara causes symmetric ISC divisions [two Delta-positive cells are generated, but this does not lead to an increase in clone size. As their proliferative capability is impaired, it might be more precise to call these cells 'ISC-like' cells. By contrast, in Sara overexpression conditions, the proliferative capability of the supernumerary ISCs are intact, leading to an increased of clone size (Montagne, 2014).

Nevertheless, both Sara mutation and overexpression lead to symmetric generation of two Delta-positive cells. These results imply that it is the comparative levels of Notch signals (the cargo of Sara endosomes) between the two daughters that is instructive to the binary fate choice between EB and ISC. Such a process is consistent with the notion that Notch signalling in this cell pair is mediated by ‘mutual inhibition’ (each of the cells tries to inhibit its neighbour) akin to the mechanism in place during the singling out of SOPs from proneural clusters (Montagne, 2014).

Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division

Endocytosis has a crucial role during Notch signalling after the asymmetric division of fly sensory organ precursors (SOPs): directional signalling is mediated by differential endocytosis of the ligand Delta and the Notch effector Sanpodo in one of the SOP daughters, pIIb. This study shows a new mechanism of directional signalling on the basis of the trafficking of Delta and Notch molecules already internalized in the SOP and subsequently targeted to the other daughter cell, pIIa. Internalized Delta and Notch traffic to an endosome marked by the protein Sara. During SOP mitosis, Sara endosomes containing Notch and Delta move to the central spindle and then to pIIa. Subsequently, in pIIa (but not in pIIb) Notch appears cleaved in Sara endosomes in a gamma-secretase- and Delta internalization-dependent manner, indicating that the release of the intracellular Notch tail to activate Notch target genes has occurred. This study thus uncovers a new mechanism to bias signalling even before asymmetric endocytosis of Sanpodo and Delta takes place in the daughter cells: already during SOP mitosis, asymmetric targeting of Delta and Notch-containing Sara endosomes will increase Notch signalling in pIIa and decrease it in pIIb (Coumailleau, 2009).

Sara endosomes and the maintenance of Dpp signaling levels across mitosis

During development, cells acquire positional information by reading the concentration of morphogens. In the developing fly wing, a gradient of the transforming growth factor-beta (TGF-beta)-type morphogen decapentaplegic (Dpp) is transduced into a gradient of concentration of the phosphorylated form of the R-Smad transcription factor Mad. The endosomal protein Sara (Smad anchor for receptor activation) recruits R-Smads for phosphorylation by the type I TGF-beta receptor. This study that Sara, Dpp, and its type I receptor Thickveins are targeted to a subpopulation of apical endosomes in the developing wing epithelial cells. During mitosis, the Sara endosomes and the receptors therein associated with the spindle machinery to segregate into the two daughter cells. Daughter cells thereby inherited equal amounts of signaling molecules and thus retained the Dpp signaling levels of the mother cell (Bokel, 2006).

PP1 binds Sara and negatively regulates Dpp signaling in Drosophila melanogaster

In signaling involving the transforming growth factor-beta (TGF-beta) superfamily of proteins, ligand binding brings the constitutively active type II receptor kinase into close proximity to its substrate, the type I receptor kinase, which it then activates by phosphorylation. The type I receptor kinase in turn phosphorylates one of the Smad family of transcription factors, which translocates to the nucleus and regulates gene expression. Smads are recruited to the receptor complex by an anchor protein, SARA (Smad anchor for receptor activation). Although several protein kinases in this pathway were known, including the receptors themselves, the relevant phosphatases had not previously been identified. This study reports the isolation of a Drosophila melanogaster homolog of SARA (Sara) in a screen for proteins that bind the catalytic subunit of type 1 serine/threonine protein phosphatase (PP1c). A PP1c-binding motif was identified in Sara, disruption of which reduced the ability of Sara to bind PP1c. Expression of this non-PP1c-binding mutant resulted in hyperphosphorylation of the type I receptor and stimulated expression of a target of TGF-beta signaling. Reducing PP1c activity enhanced the increase in the basal level of expression of genes responsive to Dpp (Decapentaplegic) caused by ectopic expression of the type II receptor Punt. Together these data suggest that PP1c is targeted to Dpp receptor complexes by Sara, where it acts as a negative regulator of Dpp signaling by affecting the phosphorylation state of the type I receptor (Bennett, 2002).

Functions of Sara orthologs in other species

Smad anchor for receptor activation contributes to seizures in temporal lobe epilepsy

Smad anchor for receptor activation (SARA) is an important regulator of transforming growth factor beta (TGF-beta) signaling by recruiting Smad2/3 to TGF-beta receptors. Although TGF-beta signaling is critically involved in epileptogenesis, whether SARA activation is sufficient to facilitate TGF-beta pathway to regulate epilepsy remains unknown. The expression of SARA and downstream Phospho-Smad3 (p-Smad3) was examined in rats with pilocarpine induced epilepsy. Additionally, knockdown of SARA was performed via recombinant lentiviral vector in the pilocarpine-induced rats. This study shows that expressions of SARA and p-Smad3 are increased in the hippocampus as rats subjected to pilocarpine-induced status epilepticus (SE). Both SARA and p-Smad3 are also upregulated in the temporal cortex of epileptic rats. Furthermore, SARA mRNA levels reach peak as early as 6 hr following SE onset and remain elevated in the chronic phase. Transfection of recombinant lentiviral shRNA targeting SARA ktnocks down SARA expression, attenuates TGF-beta/p-Smad3 signaling in the hippocampus, and postpones the SE onset. These results demonstrate that SARA/Smad3 pathway contributes to mechanism of seizure and SARA in TGF-beta signaling may be a potential therapeutic target for epilepsy (Yu, 2017).

The FYVE domain of Smad Anchor for Receptor Activation (SARA) is required to prevent skin carcinogenesis, but not in mouse development

Smad Anchor for Receptor Activation (SARA) has been reported as a critical role in TGF-beta signal transduction by recruiting non-activated Smad2/3 to the TGF-beta receptor and ensuring appropriate subcellular localization of the activated receptor-bound complex. However, controversies still exist in previous reports. This study describes the expression of two SARA isoforms, SARA1 and SARA2, in mice and report the generation and characterization of SARA mutant mice with FYVE domain deletion. SARA mutant mice developed normally and showed no gross abnormalities. Further examination showed that the TGF-beta signaling pathway was indeed altered in SARA mutant mice, with the downregulation of Smad2 protein expression. The decreasing expression of Smad2 was caused by enhancing Smurf2-mediated proteasome degradation pathway. However, the internalization of TGF-beta receptors into the early endosome was not affected in SARA mutant mouse embryonic fibroblasts (MEFs). Moreover, the downregulation of Smad2 in SARA mutant MEFs was not sufficient to disrupt the diverse cellular biological functions of TGF-beta signaling, including growth inhibition, apoptosis, senescence, and the epithelial-to-mesenchymal transition. These results indicate that SARA is not involved in the activation process of TGF-beta signal transduction. Using a two-stage skin chemical carcinogenesis assay, it was found that the loss of SARA promoted skin tumor formation and malignant progression. The data suggest a protective role of SARA in skin carcinogenesis (Chang, 2014).

Phosphatidylinositol 3-kinase and Rab5 GTPase inversely regulate the Smad anchor for receptor activation (SARA) protein independently of transforming growth factor-beta1

SARA has been shown to be a regulator of epithelial cell phenotype, with reduced expression during TGF-beta1-mediated epithelial-to-mesenchymal transition. Examination of the pathways that might play a role in regulating SARA expression identified phosphatidylinositol 3-kinase (PI3K) pathway inhibition as sufficient to reduce SARA expression. The mechanism of PI3K inhibition-mediated SARA down-regulation differs from that induced by TGF-beta1 in that, unlike TGF-beta1, PI3K-dependent depletion of SARA was apparent within 6 h and did not occur at the mRNA or promoter level but was blocked by inhibition of proteasome-mediated degradation. This effect was independent of Akt activity because neither reducing nor enhancing Akt activity modulated the expression of SARA. Therefore, this is likely a direct effect of p85alpha action, and co-immunoprecipitation of SARA and p85alpha confirmed that these proteins interact. Both SARA and PI3K have been shown to be associated with endosomes, and either LY294002 or p85alpha knockdown enlarged SARA-containing endocytic vesicles. Inhibition of clathrin-mediated endocytosis blocked SARA down-regulation, and a localization-deficient mutant SARA was protected against down-regulation. As inhibiting PI3K can activate the endosomal fusion-regulatory small GTPase Rab5, GTPase-deficient Rab5 was expressed and endosomal enlargement and reduced SARA protein expression were observed, similar to that seen with PI3K inhibition. Importantly, either interference with PI3K via LY294002 or p85alpha knockdown, or constitutive activity of the Rab5 pathway, enhanced the expression of smooth muscle alpha-actin. Together, these data suggest that although TGF-beta1 can induce epithelial-to-mesenchymal transition through reduction in SARA expression, SARA is also basally regulated by its interaction with PI3K (Runyan, 2012).

Role of SARA (SMAD anchor for receptor activation) in maintenance of epithelial cell phenotype

By inducing epithelial-to-mesenchymal transition (EMT), transforming growth factor-beta (TGF-beta) promotes cancer progression and fibrosis. This study shows that expression of the TGF-beta receptor-associated protein, SARA (Smad anchor for receptor activation), decreases within 72 h of exposure to TGF-beta and that this decline is both required and sufficient for the induction of several markers of EMT. It has been suggested recently that expression of the TGF-beta signaling mediators, Smad2 and Smad3, may have different functional effects, with Smad2 loss being more permissive for EMT progression. This study found that the loss of SARA expression leads to a concomitant decrease in Smad2 expression and a disruption of Smad2-specific transcriptional activity, with no effect on Smad3 signaling or expression. Further, the effects of inducing the loss of Smad2 mimic those of the loss of SARA, enhancing expression of the EMT marker, smooth muscle alpha-actin. Smad2 mRNA levels are not affected by the loss of SARA. However, the ubiquitination of Smad2 is increased in SARA-deficient cells. Therefore this study examined the E3 ubiquitin ligase Smurf2 and found that although Smurf2 expression was unaltered in SARA-deficient cells, the interaction of Smad2 and Smurf2 was enhanced. These results describe a significant role for SARA in regulating cell phenotype and suggest that its effects are mediated through modification of the balance between Smad2 and Smad3 signaling. In part, this is achieved by enhancing the association of Smad2 with Smurf2, leading to Smad2 degradation (Runyan, 2009).

The role of internalization in transforming growth factor beta1-induced Smad2 association with Smad anchor for receptor activation (SARA) and Smad2-dependent signaling in human mesangial cells

Recent data investigating the role of the Smad anchor for receptor activation (SARA) in TGF-beta signaling have suggested that it has a crucial function in both aiding the recruitment of Smad to the TGF-beta receptor, and ensuring appropriate subcellular localization of the activated receptor-bound complex. The FYVE domain in SARA directs its localization to early endosomal compartments where it can interact with both the TGF-beta receptors and Smads. However, the necessity of endocytosis in the TGF-beta response remains controversial. This study sought to examine the role of internalization in TGF-beta/Smad signaling in human kidney mesangial cells. Using co-immunoprecipitation studies, this study showed that endogenous Smad2 interacts with SARA after TGF-beta1 stimulation. Inhibition of clathrin-mediated internalization only slightly affects TGF-beta1-stimulated association between SARA and Smad2, Smad2 phosphorylation, or Smad2 interaction with Smad4. However, endocytosis inhibition decreases TGF-beta1-induced Smad2 nuclear translocation and thus abrogates Smad2-dependent transcriptional responses. The TGF-beta1-stimulated association between SARA and Smad2 peaks at 30 min followed by separation of the complex components. However, under conditions of inhibited endocytosis, Smad2 remains bound to SARA for at least 6 h without a significant decline in associated levels. This lack of complex dissociation correlates with a lack of Smad2 nuclear accumulation and reduction of Smad2-dependent ARE-Luc reporter activity. The data therefore suggest that endocytosis plays a critical role in TGF-beta signaling in mesangial cells, and that internalization enhances the dissociation of Smad2 from the TGF-beta receptor-SARA complex, allowing Smad2 to accumulate in the nucleus and modulate target gene transcription (Runyan, 2005).

The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-beta/Smad signalling

Transforming growth factor-beta (TGF-beta) initiates intracellular signalling by inducing the formation of a heteromeric complex between TGF-beta type I (TbetaR-I) and TGF-beta type II serine/threonine kinase receptors (TbetaR-II). After the activation of TbetaR-I kinase by TbetaR-II kinase, specific receptor-regulated Smads (R-Smads) are phosphorylated by TbetaR-I kinase. Smad anchor for receptor activation (SARA), which contains a FYVE finger domain, regulates the subcellular localization of R-Smads and presents them to TbetaR-I. However, it is unclear where SARA is localized in the cell and which phospholipid(s) interacts with its FYVE domain. Wild-type SARA and the FYVE domain of SARA (FYVE(SARA)) reveal a punctate staining pattern and co-localize with the early endosomal markers, early endosomal antigen-1 (EEA1) and hepatic growth factor-regulated tyrosine kinase substrate (Hrs). The ectopic expression of dominant negative rab5, a critical regulatory molecule in endosome function, redistributes SARA from punctate to a diffuse cytosolic staining pattern. A lipid binding assay demonstrated that the recombinant FYVE domain from SARA predominantly interacts with phosphatidylinositol 3-phosphate (PtdIns(3)P). Consistent with this result, wortmannin, a PI3 kinase inhibitor, resulted in both a redistribution of SARA from the endosomal compartment to the cytosol and the attenuation of both TGF-beta-induced R-Smad phosphorylation and transcriptional activation. Ectopic expression of the FYVE domain of SARA also induced the redistribution of wild-type SARA and inhibited TGF-beta as well as BMP/Smad-induced transcriptional responses. It is concluded that the FYVE domain is sufficient and necessary for the early endosomal localization of SARA, probably through its interaction with PtdIns(3)P. Moreover, the localization of SARA in early endosomes is required for efficient TGF-beta/Smad signalling (Itoh, 2002).

Structural basis of Smad2 recognition by the Smad anchor for receptor activation

The Smad proteins mediate transforming growth factor-beta (TGFbeta) signaling from the transmembrane serine-threonine receptor kinases to the nucleus. The Smad anchor for receptor activation (SARA) recruits Smad2 to the TGFbeta receptors for phosphorylation. The crystal structure of a Smad2 MH2 domain in complex with the Smad-binding domain (SBD) of SARA has been determined at 2.2 angstrom resolution. SARA SBD, in an extended conformation comprising a rigid coil, an alpha helix, and a beta strand, interacts with the beta sheet and the three-helix bundle of Smad2. Recognition between the SARA rigid coil and the Smad2 beta sheet is essential for specificity, whereas interactions between the SARA beta strand and the Smad2 three-helix bundle contribute significantly to binding affinity. Comparison of the structures between Smad2 and a comediator Smad suggests a model for how receptor-regulated Smads are recognized by the type I receptors (Wu, 2000).


Search PubMed for articles about Drosophila Sara

Bennett, D. and Alphey, L. (2002). PP1 binds Sara and negatively regulates Dpp signaling in Drosophila melanogaster. Nat Genet 31(4): 419-423. PubMed ID: 12134149

Bokel, C., Schwabedissen, A., Entchev, E., Renaud, O. and Gonzalez-Gaitan, M. (2006). Sara endosomes and the maintenance of Dpp signaling levels across mitosis. Science 314(5802): 1135-1139. PubMed ID: 17110576

Chang, H. M., Lin, Y. Y., Tsai, P. C., Liang, C. T. and Yan, Y. T. (2014). The FYVE domain of Smad Anchor for Receptor Activation (SARA) is required to prevent skin carcinogenesis, but not in mouse development. PLoS One 9(8): e105299. PubMed ID: 25170969

Coumailleau, F., Furthauer, M., Knoblich, J. A. and Gonzalez-Gaitan, M. (2009). Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division. Nature 458(7241): 1051-1055. PubMed ID: 19295516

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Biological Overview

date revised: 30 September 2018

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