Thesis
Reference
Sara-binding proteins and their role during asymmetric cell division
MORALEDA MERLO, Ana Belen
Abstract
During TGF-ß signaling, Sara acts as a scaffold protein recruiting the transcription factor to the ligand receptor. Sara is a member of the TGF-ß super family, which associates to endosomes by binding PtdIns(3)P. Sara endosomes are involved in the symmetric partitioning of TGF-ß/BMP signaling molecules during wing development and in the asymmetric distribution of Delta and Notch during SOP asymmetric cell division. We still do not know what mechanisms are involved in Sara endosomal dynamics and trafficking. During my PhD I focused on finding the molecules, which drive the targeted motility of Sara endosomes during SOP asymmetric cell division. Other key questions were i) how Notch and Delta are targeted into Sara endosomes during SOP division,? and ii) what are the Activin target genes during Sara-mediated TGF-ß Signaling? In order to answer these questions, the main goal of my PhD has been to unravel the Sara machinery.
MORALEDA MERLO, Ana Belen. Sara-binding proteins and their role during asymmetric cell division. Thèse de doctorat : Univ. Genève, 2011, no. Sc. 4322
URN : urn:nbn:ch:unige-166986
DOI : 10.13097/archive-ouverte/unige:16698
Available at:
http://archive-ouverte.unige.ch/unige:16698
UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Section de chimie et biochimie
Département de biochemie Professeur Marcos González-Gaitán
Sara-binding proteins and their role during asymmetric cell division
THÈSE
présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biochimique
par
Ana Belén MORALEDA-MERLO de
Valdepeñas (Espagne)
Thèse No 4322
GENÈVE
Atelier Repromail
2011
TABLE OF CONTENTS
TABLE OF CONTENTS……….3
SUMMARY………...6
RÉSUMÉ...9
INTRODUCTION………..……….12
THE DROSOPHILA SOP ASYMMETRIC CELL DIVISION………12
FORMATION OF THE PRONEURAL CLUSTER……….14
SOP SPECIFICATION………...….16
SOP PLANAR CELL POLARITY AND ASYMMETRIC SEGREGATION OF CELL FATE DETERMINANTS………18
ENDOCYTIC REGULATION OF NOTCH SIGNALING DURING SOP ASYMMETRIC CELL DIVISION………..……21
SARA, A TGF- ß PATHWAY COMPONENT INVOLVED IN ASYMMETRIC AND SYMMETRIC CELL DIVISION………...…24
THE ROLE OF BINDING SARA PHOSPHATASES ……….28
RESULTS……….31
A PROTEOMICS APROACH TO UNRAVEL “ THE SARA MACHINERIES”………...…… 32
CONDITION FOR SARA IMMUNOPRECIPITATION………...……….33
DISECTING DOMAINS OF THE SARA PROTEIN……….….34
MASS SPECTROMETRY DATA……….……36
STRUCTURE FUNCTION ANALYSIS OF SARA……….41
A MINIMUM SARA FRAGMENT TARGETED TO ASYMMETRIC ENDOSOMES……….44
ROLE OF THE PP1 BINDING DOMAIN DURING ASYMMETRIC MOTILITY OF ENDOSOMES………...46
TABLE OF CONTENTS
STUDY OF SARA PHOSPHORYLATION………..48
UNINFLATABLE BINDS SARA………...55
UNINFLATABLE LOCALIZATION………57
ROLE OF UIF DURING ASYMMETRIC MOTILITY OF SARA ENDOSOMES……….59
UIF IS REQUIRED DURING DIRECTIONAL NOTCH SIGNALING IN THE SOP…………61
ACTIVIN TARGET GENES SCREENING………..63
KLP98A IMMUNOPRECIPITATION………..67
DISCUSSION………...…70
A MOLECULAR COMPLEX FOR THE ASYMMETRIC DISTRIBUTION OF SARA ENDOSOMES………..…………71
FINDING PARTNERS OF SARA……….71
MAPPING SARA DOMAINS………71
DROSOPHILA SARA PHOSPHORYLATION………72
UNINFLATABLE, A NEW PARTNER OF SARA INVOLVED IN ASYMMETRIC PARTIONING OF SARA ENDOSOMES……….73
MATHERIALS AND METHODS……….76
IMMUNOPRECIPITATION………..………76
IMMNOBLOTTING………77
PHOSPHORYLATION ASSAY……….78
MASS SPECTROMETRY………..…78
FLY GENOTYPES………...………78
CLONING……….79
TABLE OF CONTENTS
PRIMERS AND PCR CONDITIONS………79
FLY INJECTIONS………...81
IN SITU HYBRIDAZATION………..81
SENSORY ORGAN PRECURSOR DIVISION……….…...82
DELTA UPTAKES………...…82
IMAGE PROCESSING AND QUANTIFICATION……….…83
NOTUM IMMUNOSTAINING……….….83
IMAGINAL DISC IMMUNOSTAINING………..84
BRAIN IMMUNOSTAINING……….84
BIBLIOGRAPHY………85
APENDIX……….96
ACKNOWLEDGEMENTS………..……120
STATEMENT………....122
SUMMARY
SUMMARY
During development and adults stem cell proliferation, one mechanism to generate daughter cells with distinct fates upon mitosis relies on the unequal segregation of molecules that regulate the fate of the daughter cells. This mode of cell division is named asymmetric cell division. Asymmetric cell division is a fundamental and evolutionarily conserved process for generating cell diversity. The Drosophila neuroblast in the central nervous system and sensory organs of the peripheral nervous system develop from cell lineages are excellent models to examine the gene expression program and the machinery underliying asymmetric distribution of cell fate determinants. Reviewed in (Bardin et al., 2004)
To divide asymmetrically, cells establish an axis of polarity, orient the mitotic spindle along this axis and localize cell fate determinants to one side of the cell.
During cytokinesis, determinants are then segregated into one of the two daughter cells where they are utilized to direct the different cell fate.
The external sensory organs of the adult Drosophila follow a series of asymmetric divisions to generate the adult bristles which act as mechanoreceptors. Each mechanoreceptor comprises four specialized cells derived from a single sensory organ precursor (SOP). In the notum, SOPs divide asymmetrically along the A-P axis of the body to generate an anterior pIIb cell and a posterior pIIa cell. The pIIa or pIIb fate decision depends on the unequal segregation of regulators of Delta/Notch signaling pathway, which localize at the anterior cortex of dividing SOPs. Reviewed in (Betschinger and Knoblich, 2004)
Endocytic trafficking modulates cell fate during asymmetric cell division in Drosophila. The endocytosis of Notch and its ligands is a key mechanism by which Notch mediated cell-cell signaling is regulated. Endocytosis may serve to bring ligand-bound receptors to signal-transducing machinery or may regulate the transport of active ligands from cell to cell (Le Borgne et al., 2005).
SUMMARY
Following the asymmetric division of SOPs, Delta ligands and Notch receptors traffic differently in the two daughter cells, leading to directional signal activation (Coumailleau et al., 2009). This new mechanism of directional signaling is based on 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. Asymmetric targeting of Delta and Notch-containing Sara endosomes will increase Notch signaling in pIIa and decrease it in pIIb (unpublished work in our lab shows that).
Sara endosomes move on microtubules to reach one of the two daughters cells.
To achieve these movements, the Drosophila Kinesin motor KLP98A binds to Phosphatidylinsositol 3 Phosphate PtdIns(3)P vesicles (Sato et al., 2001) and transports endosomes along microtubules.
Sara (Smad anchor for the receptor activation) is a FYVE domain protein, which associates with (PtdIns(3)P) in endosomes. During TGF-ß signaling, Sara acts as a scaffold protein recruiting the transcription factor to the ligand receptor complex (Tsukazaki et al., 1998). Sara is present in a multivesicular population of early endosomes that are located apically in polarized epithelial cells of the wing imaginal disc (Bokel et al., 2006). Sara endosomes are involved in the symmetric partitioning of TGF-ß/BMP signaling molecules among daughter cells during Drosophila wing development (Bokel et al., 2006).
During my PhD, I focused my project on unraveling and dissecting the Sara machinery, which controls the processes, described above.
I have performed a proteomics approach based on immunoprecipitation of Drosophila Sara complexes and Mass Spectrometry (MS) analysis in order to
SUMMARY
identify Sara binding partners that have an important function on the mechanism of directional trafficking of Notch/Delta.
To study the role of Sara during TGF-ß /Activin signaling, I have carried out a candidate gene screening to find Activin targets in the wing imaginal disc.
Finally, in order to understand how asymmetric endosomes motility is achieved I have performed the same type of IP/MS approach to find binding partners of the kinesin involved in asymmetric motility of endosomes.
RÉSUMÉ
RÉSUMÉ
Translated by Dr Sylvain Loubery.
Au cours du développement, et aussi lors de la prolifération des cellules souches adultes, un mécanisme permettant de produire, suite à une mitose, deux cellules aux destinées différentes consiste à répartir de facon inégale entre les cellules-filles des molécules qui contrôlent leurs destinées. Ce mode de division est nommé division asymétrique ; c’est un processus évolutionnairement conservé et fondamental pour la génération de types cellulaires variés. Les neuroblastes du système nerveux central de la drosophile ainsi que les organes sensoriels du système nerveux périphérique consistuent des lignages cellulaires qui sont d’excellents modèles pour l’étude des programmes génétiques et de la machinerie sous-jacente à la distribution asymétrique de déterminants de destinée cellulaire (voir pour revue Allison et al., 2004).
Pour se diviser asymétriquement les cellules établissent un axe de polarité, orientent leur fuseau mitotique selon cet axe et positionnent leurs déterminants de destinée cellulaire d’un côté de la cellule. Lors de la cytocinèse les déterminants sont alors ségrégés dans une des cellules-filles, dans laquelle ils vont être utilisés pour entraîner l’acquisition d’une destinée particulière.
Les organes sensoriels externes de la drosophile subissent une série de divisions asymétriques aboutissant à la génération de l’organe adulte, qui est un mécanorécepteur. Chaque organe comprend quatre cellules spécialisées provenant d’une cellule précurseur unique, la SOP (Sensory Organ Precursor).
Les SOPs se divisent de façon asymétrique selon l’axe antéropostérieur de l’animal pour générer une cellule antérieure nommée pIIb et une cellule postérieure nommée pIIa. Le choix de la destinée pIIa ou pIIb dépend de la ségrégation biaisée de régulateurs de la voie de signalisation Delta/Notch qui sont localisés au cortex antérieur de la SOP en division (voir pour revue Jorg
RÉSUMÉ
and Knoblich, 2004).
Le trafic des compartiments d’endocytose module le choix de destinée cellulaire lors de la division asymétrique chez la drosophile. L’endocytose de Notch et de ses ligands est un mécanisme clef pour la régulation de la signalisation intercellulaire médiée par la voie Notch. L’endocytose pourrait servir à mettre en contact les récepteurs liés aux ligands avec une machinerie de transduction du signal, comme elle pourrait aussi réguler le transport de ligands activés de cellule à cellule (Roland et al., 2005).
Suite à la division asymétrique des SOPs, les ligands Delta et les récepteurs Notch circulent de façon différente dans les deux cellules-filles, induisant une activation du signal directionnelle (Coumailleau, Furthauer et al., 2009). Ce nouveau mécanisme de signalisation directionnelle est basé sur la circulation de molécules de Delta et Notch déjà internalisées dans la SOP, qui sont ensuite ciblées vers la cellule-fille pIIa. Delta et Notch internalisés sont dirigés vers un endosome marqué par la protéine Sara. Lors de la mitose de la SOP, les endosomes Sara contenant Notch et Delta se déplacent jusqu’au fuseau mitotique central, puis dans la cellule-fille pIIa. Le ciblage d’endosomes Sara contenant Notch et Delta accroît la signalisation Notch dans la cellule-fille pIIa et le diminue dans la pIIb (résultats non publiés de notre laboratoire).
Les endosomes Sara se déplacent sur les microtubules pour atteindre une des cellules-filles. A cette fin, la kinésine de drosophile KLP98A se lie à des vésicules de phosphatidylinositol-3-phosphate (PI(3)P) (Sato et al., 2001) et transporte les endosomes le long des microtubules.
Sara (Smad Anchor for Receptor Activation) est une protéine à domaine FYVE qui s’associe au PI(3)P des endosomes. Lors de la signalisation de la voie du TGF-ß, Sara agit comme protéine d’échafaudage en recrutant le facteur de transcription au complexe récepteur-ligand (Tsukazaki et al., 1998). Sara est
RÉSUMÉ
présente sur une population d’endosomes précoces multi-vésiculaires qui, dans les cellules épithéliales polarisées du disque imaginal d’aile, est localisée au niveau apical (Boekel et al., 2006). Les endosomes Sara sont impliqués dans la répartition symétrique des molécules de TGF-ß/BMP entre les cellules-filles lors du développement de l’aile de la drosophile (Boekel et al., 2006).
Pendant ma thèse, j’ai concentré mon projet sur l’élucidation et la dissection de la machinerie Sara contrôlant les mécanismes décrits ci-dessus.
J’ai entrepris une approche de protéomique basée sur l’immunoprécipitation de complexes Sara de drosophile suivie de spectrométrie de masse (IP/MS) afin d’identifier des partenaires de Sara jouant un rôle dans le mouvement directionnel de Notch et Delta.
Pour l’étude du rôle de Sara dans la signalisation TGF-ß /Activine, j’ai entrepris un crible de gènes candidats afin de trouver des cibles de l’Activine dans le disque imaginal d’aile.
Enfin, pour comprendre comment est contrôlé le mouvement asymétrique des endosomes, j’ai entrepris le même type d’approche de protéomique IP/MS pour trouver des partenaires de la kinésine impliquée dans ce mouvement asymétrique.
INTRODUCTION
INTRODUCTION
Animals are made up of a large number of different cell types. During development these are generated from a single cell which generates sister cells with distinct identities through a mitotic cell division. To achieve this process, they can segregate cell fate determinants into only one of the two daughter cells resulting in an asymmetric cell division. Therefore, asymmetric divisions contribute to this expansion in cell type diversity by making two types of cells from one. These distinct mechanisms underlying asymmetric cell division have been elucidated mostly in Drosophila melanogaster but many of the molecules involved are highly conserved in vertebrates.
This introduction is mainly focused on the mechanisms by which cell fates determinants are asymmetrically acquired within the SOP lineage of the peripheral nervous system (PNS) in Drosophila. It also presents the SARA protein involved in asymmetric cell division and as member of the TGF-ß pathway. Finally, the PP1C and presumptive PP2A Sara binding proteins and their phosphatase functions in TGF-ß pathway and in asymmetric cell division are described.
THE DROSOPHILA SOP ASYMMETRIC CELL DIVISION
The sensory organ precursor (SOP) of the fruit fly together with the neuroblast lineage of the central nervous system have been very important models for understanding the mechanisms, which control asymmetric cell division.
After a series of asymmetric cell division, the SOP gives rise to the adult bristle; this external sensory organ is constituted by 4 different types of cells (Fig. 1).
INTRODUCTION
Figure 1. Development of Drosophila mechanosensory bristles. A) The four cells are formed after a series of asymmetric cell divisions from a sensory organ precursor (pI) cell. PI cell will divide into pIIa and pIIb. PIIb cell produces first a glia (which undergoes apoptosis soon afterwards) and pIII. A) and B) each organ is composed of two external (shaft and socket, white arrowheads in B) and two internal (neuron and sheath) cells. SEM image courtesy of Sylvain Loubery.
The bristles are divided into macro and microchaetes depending on their size and location in the fly head and body. These two types of mechanoreceptors have some morphological and functional differences but the same develop- mental patterns and genetic control (Usui et al., 2004) and (Simpson and Marcellini, 2006). There is a relative fixed number of sensory organs in the larval PNS (Dambly‐Chaudiere, 1986) and many of the sensory bristles (macrochaetes) of the adult PNS can be identified individually and occupy fixed positions (Simpson, 1990).
The sensory organ is formed in three phases (Fig. 2). At the first stage, the so- called proneural clusters, groups of 20-30 cells, segregate from the epithelium of the wing imaginal disc. At the second stage, the SOP cell is determined and its position in the proneural cluster is specified. At the final stage, the SOP cell divides and the daughter cells differentiate into the components of mechanoreceptor. Each stage has its own genetic control.
INTRODUCTION
Figure 2. The Drosophila bristles are formed in three stages. 1) Proneural cluster in the wing imaginal disc. 2) SOP specification (in the center darkest cell) in the proneural cluster. 3) Asymmetric cell division of the SOP.
FORMATION OF THE PRONEURAL CLUSTER
The cells of a proneural cluster are differentiated from the surrounding ectodermal cells and the proneural genes play the key role in this process. It is the expression of these genes that renders the cells of this cluster the capability to become SOP. Inactivation of proneural genes causes disappearance of some or all macrochaetes. An ectopic expression of these genes results in development of additional bristles at ectopic positions.
All of the cells in an equivalence group initially express proneural genes of the achaete–scute (AS-C) complex (Campuzano and Modolell, 1992). AS-C genes encode the bHLH family transcription factors, which bind to the specific sites in the regulatory regions of the genes they control, E boxes. Along with the proneural genes, Delta (Dl), Scabrous (SCA), Enhancer of split E(spl)-C, Senseless (SENS), etc., belong to such target genes(Powell et al., 2004). AS-C occupies approximately 90 kpb in the genome and contains nine transcription units separated by untranscribed regions. Each transcript has its own time and spatial distribution profiles. The specificity and expression patterns of the AS- C genes are determined by enhancers located at a distance of up to 100 kpb from this complex (Gomez‐Skarmeta et al., 1995). One type of enhancers initiates the expression of AS-C genes in the cells of individual proneural cluster, and enhancers of the second type trigger this process in the SOP cell (Escudero et al., 2005).
As AS-C proteins are transcription factors, they are able to regulate
INTRODUCTION
transcription, including transcription of the genes that code for them. These factors acquire a regulatory activity within heterodimers with certain other proteins. Depending on the composition, such complexes are either positive or negative regulators of AS-C gene expression. The heterodimers of AC and SC with the protein DA, the product of gene Daughterless (Da), also a bHLH protein, are positive regulators of AS-C gene transcription. The transcription is activated through the binding of such heterodimers to the three E boxes in AS- C regulatory region (Cabrera and Alonso, 1991). The heterodimers of the proteins AS-C and EMC, the product of gene Extramacrochaete, are negative regulators of the AS-C expression, as EMC is an HLH protein, deprived of the DNA-binding basic domain. The complexes formed by proneural proteins and EMC are incapable of binding to DNA. Competing with DA for binding to AS- C proteins, EMC decreases the concentration of functional heterodimers, thereby decreasing the transcription level of AS-C genes. (Van Doren et al., 1992) , (Vaessin et al., 1994) and (Cabrera et al., 1994).
The activity of the proneural genes is regulated not only by the heterodimers containing AC and SC, but also by other factors. The transcription factor Senseless (SENS) plays a dual role in the activity regulation of proneural genes. SENS is an activator or a repressor of the proneural gene transcription depending on its content in the cell. At a low concentration, it acts as a repressor of the proneural gene activity, directly binding to DNA at the corresponding sites of AS-C enhancer regions; at a high concentration, it forms complexes with proneural proteins and DA, acting as a coactivator of the proneural gene expression (Nolo et al., 2000) and (Jafar‐Nejad et al., 2003).
Along with a direct intracellular regulation of the proneural gene activity, the EGFR signaling pathway plays an important role in AS-C genes regulation.
The EGFR transmembrane receptor DER can bind either Spizt (SPI) or Argos (AOS) ligands. An intra cellular signal transduction is activated or blocked depending on the bound ligand (Freeman, 1998), (Livneh et al., 1985), and (Shilo, 2003).
INTRODUCTION
The signal transduction commences from the SPI binding to the DER extracellular domain. Then the intracellular domain of the receptor in the recipient cell is phosphorylated and the EGRF cascade is activated. The intracellular signal transduction from the cell membrane to the nucleus initiates the transcription of gene Pointed and the subsequent synthesis of two isoforms of the protein Pointed, Pnt-P1 and Pnt-P2 (Gabay et al., 1997) and(Kumar et al., 2003). Both isoforms can play the role of transcription factors for proneural genes.
The secreted ligand Argos is a repressor of the EGFR signaling pathway. The gene AOS is activated simultaneously with the activation of proneural genes, and its expression is observed exclusively in the proneural cluster cells.
Secretion of the ligand and its binding to the receptor blocks the EGFR signal transduction into the cells neighboring the cells actively expressing AS-C proteins (Culi and Modolell, 1998) and (Klein et al., 2004). Thus, the local differential expression of AS-C genes and the EGFR signaling pathway determine a precise location of the proneural cluster and provide accumulation of proneural proteins in the cells of the cluster.
SOP SPECIFICATION
The obligatory condition for SOP cell determination is that the concentration of As-C proteins reaches the maximal value as compared with the neighboring cells. The cells with insufficient concentration of proneural proteins remain epidermal.
The conserved Notch signaling pathway restricts proneural gene expression to a single neural progenitor cell through a process of lateral inhibition, a molecular regulatory loop between the neighboring cells (Fig. 3) (Artavanis‐
Tsakonas et al., 1995) and (Heitzler and Simpson, 1991).
INTRODUCTION
Figure 3. Receptor activation during Delta/Notch signaling and SOP specification by lateral inhibition . A) Activation of Notch by its ligand Delta triggers two proteolytic cleavages of Notch (S2 and S3). S3 cleavage releases the Notch intracellular domain, which translocates to the nucleus. In the absence of nuclear Notch intra, CSL associates with a co- repressor complex (R red icon), which actively represses the transcription of Notch target genes. The CSL co-repressor complex is displaced by a co-activator complex containing Notch intra (A blue icon), which mediates Notch target gene activation (Fortini and Artavanis- Tsakonas, 1993) and (Le Borgne et al., 2005). B) A simple scheme of lateral inhibition involving Notch, Delta and the proneural genes. Activation of the Notch signaling cascade by the Delta ligand leads to repression of proneural gene expression in the presumptive non SOP cell. Transcription factors encoded by the proneural genes positively regulate the expression of the transmembrane ligand Delta, which binds to the Notch receptor on adjacent cells causing Notch to be cleaved and resulting in the nuclear localization of the Notch intracellular domain.
Since Delta expression is regulated by proneural transcription factors, down regulation of proneural genes leads to a reduction in Notch activation in the neighboring cell. In this way, the cell that initially has higher levels of proneural gene or Delta expression (or perhaps lower levels of Notch) will acquire the SOP fate, while extinguishing proneural gene expression in the surrounding cells.
The lateral inhibition is efficient for the cells directly adjacent to the presumptive SOP cell. However, the neurogenic gene Scabrous (SCA) acquires an exclusive role in determining the fate of more remote cells of the proneural cluster; expression of this gene is activated by the heterodimers of AS-C and DA proteins (Mlodzik et al., 1990), (Renaud and Simpson, 2001). In its absence,
INTRODUCTION
the neural pathway is not blocked in the cells that do not directly contact the future SOP cell. On the other hand, SCA is not necessary for the lateral inhibition of the cells contacting the SOP cell, as Dl is sufficient for this process. Since SCA distribution gradient is observed within the proneural cluster, it is assumed that this is the particular factor that determined the size of the region where the inhibiting signal is spread. However, the precise mechanism of SCA action in the lateral inhibition process is still vague.
Presumably, this protein is necessary for stabilization of the N–Dl complex.
The lateral inhibition ends by determination of the single proneural cluster cell as a SOP cell. Then the determined cell undergoes successive asymmetric divisions.
One key mechanism by which a cell can produce two different daughter cells is by partitioning cell-fate determinants unequally between the daughter cells. A number of polarity cues have been identified that establish the anterior- posterior (A-P) polarity of SOP and enable the asymmetric segregation of cell fate determinants to one of the daughter cell.
SOP PLANAR CELL POLARITY AND ASYMMETRIC SEGREGATION OF CELL FATE DETERMINANTS
The A-P orientation of the SOP division depends on the Planar Cell Polarization (PCP) of the single-layered notum epithelium (Gho and Schweisguth, 1998), (Lu et al., 1999b) and (Bellaiche et al., 2001). A small number of evolutionarily conserved proteins act to locally coordinate the polarization of epithelial cells perpendicular to their apical-basal axis within the plane of the tissue (Adler, 2002) and (Klein and Mlodzik, 2005). These PCP proteins include the receptor of Wingless (Wnt)/PCP pathway protein Frizzled (Fz) (Vinson et al., 1989), the transmembrane protein Van Gogh (Vang) also known as Strabismus (Adler et al., 2000) and the atypical cadherin Flamingo (Fmi) (Usui et al., 1999). Mutations in the Fz, Vang and Fmi genes randomize the orientation of the SOP division relative to the body axis (Gomes et al.,
INTRODUCTION
Once the polarization of the epithelium is established the SOPs divides asymmetrically within the plane of the epithelium along the anterior-posterior axis of the body to generate an anterior pIIb cell and a posterior pIIa cell (Fig.
4).
Figure 4. Asymmetric division of the sense organ precursor pI. The pI cell asymmetrically divides in the plane of the epidermal epithelium producing the pIIa and pIIb daughter cells. The mitotic spindle aligns with the anterior - posterior axis of the animal (arrows show the movements of centrosomes).
The pIIa or pIIb fate decision relies on the unequal segregation of regulators of Delta/Notch signaling (Numb and Pon) that localize at the anterior cortex of dividing SOPs. The polar localization of these regulators is controlled by the atypical Protein Kinase C (aPKC), Bazooka (Baz) and Par6 complex that localizes at the opposite posterior pole ((Gonczy, 2008), (Knoblich, 2008) and (Wirtz‐Peitz et al., 2008). To ensure the proper mitotic spindle orientation, the complex Pins, Gαi and Discs-Large (Dlg) are localized at the anterior cortex of the pI cells. This complex responds to Fz signaling. Pins associates with another protein Mud, which captures astral microtubules from one of the spindle poles and orients the mitotic spindle (Bellaiche et al., 2001) and (Bowman et al., 2006).
The aPKC protein is activated when Par-6 is phosphorylated by Aurora A (Aur-A) (Wirtz‐Peitz et al., 2008) this allows aPKC to phosphorylate Lethal (2) giant larvae (Lgl) (Betschinger et al., 2003). Lgl is a cytoskeleton protein that is required for the anterior localization of Numb (Plant et al., 2003) and (Yamanaka et al., 2003). Phosphorylated Lgl is released from aPKC and thereby allows protein Baz to enter the complex and this changes the substrate specificity of aPKC, which now can phosphorylate Numb (Wirtz‐Peitz et al.,
INTRODUCTION
2008). Phosphorylated Numb is released from the posterior part of the cell to the anterior part thanks to its adaptor Pon and cytoskeleton components (Lu et al., 1999a) (Fig. 5).
Figure 5. Intrinsically asymmetric division in Drosophila SOP. Epithelial posterior-anterior polarity is used to establish the asymmetric accumulation of Par proteins (Par-3, Par-6 and aPKC, red) to the posterior cortex. Upon entry into mitosis and the activation of Aurora-A kinase, the mitotic spindle is oriented by the microtubule binding protein Mud, which is recruited by Pins, Dlg and Gαi (yellow). The asymmetric localization of cell fate determinants (Numb and Pon pink and violet) to the cortex opposite Par-3/6 and aPKC requires the phosphorylation of Lgl (blue) by aPKC.
After cytokinesis the cell fate determinant Numb is segregated into the pIIb (Rhyu et al., 1994) daughter cell with the help of its adaptor protein Pon. In this cell, Numb represses Notch signaling, while in the pIIa cell, which does not acquire Numb, Notch signaling is active.
The absence of the Dlg partner Inscuteable (Insc) in the SOP generates a symmetric mitotic spindle. The complexes Gαi/Pins/Dlg and Par3/Par6/aPKC are localized to the opposite sides of the cell and Insc misexpression in SOP leads to co-localization of the two complexes in one pole (Bellaiche et al., 2001).
The development of the Drosophila bristles concludes with the asymmetric cell division of the pIIa and pIIb (previous formation of pIIIb) cells. The pIIa cell divides A–P, forming the posterior socket cell and the anterior shaft cell, which inherits Numb. The divisions of the pIIb lineage are reminiscent of the embryonic neuroblast lineage with respect to their division orientation, and the fates of their daughter cells.
Most Drosophila neuroblasts divide asymmetrically by self-renewing and
INTRODUCTION
Baz/Par6/aPKC complex is apical localized and binds through Baz to Inscuteable (Insc). Insc, in turn, recruits Partner of Inscuteable (Pins) and interact with Gαi (Kraut et al., 1996), (Parmentier et al., 2000) and (Yu et al., 2000). Neuroblasts divide in the apical-basal orientation and require Insc to reorientate the spindle along the apical basal axis. In addition, neuroblasts express Miranda, a linker protein that is asymmetrically localized to the basal cortex and acts positioning the homeodomain protein Prospero (Pros) (Doe et al., 1991), (Vaessin et al., 1991) and (Matsuzaki et al., 1992). Prospero and protein Numb (Uemura et al., 1989) are localized as basal crescents and segregate to the basal GMC (Rhyu et al., 1994), (Hirata et al., 1995) (Knoblich et al., 1995),(Spana and Doe, 1995), (Li et al., 1997) and (Broadus et al., 1998). The pIIb cell divides along its A-B axis and expresses Pros, Miranda and Insc, (Carmena et al., 1998) and (Gho et al., 1999). PIIb asymmetric cell division gives rise to a glia cell, which disappears afterwards by apoptosis and the pIIIb cell. In the final division, pIIIb divides apical–basal to form the apical sheath cell and the basal neuron, which inherits Numb.
After the asymmetric division of the SOP, endocytosis plays a crucial role during Notch signaling. There are four different mechanisms by which endocytic trafficking modulates cell fate during asymmetric cell division in Drosophila.
ENDOCYTIC REGULATION OF NOTCH SIGNALING DURING SOP ASYMMETRIC CELL DIVISION
Although both Notch and Delta are expressed in all cells of the SOP lineage, Notch is activated only in daughter cells that do not inherit Numb. Several experiments have suggested that this is because Numb might induce the endocytosis of the four-pass transmembrane protein Sanpodo ((O'Connor‐Giles and Skeath, 2003). Sanpodo endocytosis requires α-Adaptin, a Numb-binding partner involved in Clathrin-mediated (Hutterer and Knoblich, 2005). Numb binds the endocytic protein α-Adaptin and regulates its preferential segregation
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with Numb into pIIb (Berdnik et al., 2002). Numb was also shown to perform a complex with the protein Sanpodo (O'Connor‐Giles and Skeath, 2003). Sanpodo is localized at the plasma membrane in pIIa and is required for Notch activation while in the pIIb cell, Sanpodo is removed from the plasma membrane by Numb and α-Adaptin dependent endocytosis and therefore this cell is unable to activate Notch signaling (Fig. 6).
PIIa Notch activation also depends of the Delta (Dl) endocytosis in the pIIb.
Endocytosis defective Dl proteins have reduced signaling capacity (Parks et al., 2000). Neuralized (Neur) is a cell fate determinant, which is unequally segregated during SOP division and inherited together with Numb by the pIIb cell (Le Borgne and Schweisguth, 2003). Neur is an E3-Ubiquitin ligase that interacts with mono-ubiquitinated Dl. Delta ubiquitination promotes its endocytosis and potentiates its ability to activate Notch in the neighboring pIIa cell (Deblandre et al., 2001), (Itoh et al., 2003), (Lai et al., 2001)(Pavlopoulos et al., 2001) and (Yeh et al., 2001) (Fig. 6).
Figure 6. Cell fate specification after SOP-cell division. During SOP division Numb, α - Adaptin (α-Ada) and Neur segregate asymmetrically into the pIIb cell, where they inhibit Notch signaling. Numb is a binding partner α-Ada and both bind to Notch and Sanpodo inhibiting Notch signaling in the pIIb. Neur ubiquitinates Dl in pIIb promoting Notch signaling in the pIIa cell.
The mechanism by which Neur regulates Notch signaling remains unclear. A model proposes that endocytosis might direct Delta to an Epsin-dependent recycling pathway (Wang and Struhl, 2004). There is another endocytic regulation of Notch signaling in pIIb that could support this model. During
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around the centrosome in the pIIb cell (Emery et al., 2005). Delta traffics through the recycling endosomes, which is marked by Rab11. In pIIa, however, the recycling endosomes does not form because the centrosome fails to recruit Nuclear fallout (Nuf), a Rab11 binding partner that is essential for recycling endosome formation (Rothwell et al., 1998) (Fig. 7). Rab11 plays an important role controlling vesicular protein transport through recycling endosomes to the plasma membrane (Zerial and McBride, 2001) therefore Delta passage through recycling endosomes is very important because when Dl is at the plasma membrane it can activate pIIa Notch signaling.
Figure 7. Asymmetric Rab11 endosomes regulate Notch singnaling in the pIIa. During mitosis Rab11-positives endosomes (yellow dots) are equally partitioned among daughter cells. Shortly after division, a transient Rab11-positive recycling compartment is established in the centrosomal region of the anterior pIIb cell. This enhances Delta recycling (broken arrows in d) from early endosomes (red dots) to the plasma membrane in pIIb.
Directional Delta/Notch signaling following SOP division is achieved through asymmetric segregation of cell fate determinants that regulate trafficking of ligand and receptor in the two daughter cells after division. Recent results show that there is a new mechanism of directional signaling on the basis of trafficking of Delta and Notch molecules already internalized in the SOP and subsequently targeted to the pIIa cell (Coumailleau et al., 2009). Internalized Delta and Notch traffic to a subpopulation of Rab5 endosomes marked by the protein Sara (Smad anchor for the receptor activation).
SARA, A TGF-ß PATHWAY COMPONENT INVOLVED IN ASYMMETRIC AND SYMMETRIC CELL DIVISION
INTRODUCTION
During mitosis, Sara endosomes containing Notch and Delta, concentrate at the acting ring at the cytokinesis cleavage plane and from there they move to the central spindle. Finally they are asymmetrically segregated to the pIIa cell (Fig. 8).
Figure 8. Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division. a) During Metaphase, the majority of Sara endosomes are targeted to the acting contracting ring at the cleavage plane. b) In late anaphase the Sara endosomes move to the central spindle and finally c) they are segregated to the pIIa cell during cytokenesis.
Coumailleau and Furthauer observed that the vast majority of Sara endosomes are localized at the cortex in the actin ring. In anaphase, the Sara endosomes go to the central spindle and in this phase these endosomes jump from actin to microtubules to finally use these microtubules and move along them in other to reach the pIIa.
Sylvain Loubery, a post-doc in Marcos Gonzalez lab is addressing the possibility of some cytoskeleton components being involved in asymmetric distribution of Sara endosomes. Sara endosomes move on microtubules to reach one of the two daughters cells. Sylvain is investigating klp98A protein as a microtubule motor that could be driving this movement toward the pIIa cell.
Very little is known about Klp98A a kinesin motor domain that may transport Phosphatidylinsositol 3 Phosphate PtdIns(3)P enriched cargo vesicles and organelles along microtubules. KLP98A contains the 120-amino acid Phox homology (PX) domain that targetsproteins to organelle membranes through interactions between two conserved basic motifs within the PX domain and specific PtdIns(3)P (Sato et al., 2001). Its human homologous KIF 16B has been shown to be involved in the transport of early endosomes to the plus end
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of microtubules (Hoepfner et al., 2005). Sylvain Loubery has observed that in klp98A mutant flies, during SOP cytokinesis, Sara endosomes fail to use microtubules and the asymmetric distribution of the Sara endosomes is impaired (data unpublished).
Sara itself is dispensable for asymmetric Delta and Notch segregation and for the sensory organ development. Although Sara is not essential, its over expression causes early endosomes, which normally segregate fairly symmetrically, to be targeted to pIIa, although a redundant factor can cover this role in its absence. High levels of Sara over expression (NeurGal4-Sara) saturate the system, leading to remnants of Sara endosomes in pIIb and a lineage phenotype indicative of ectopic Notch signaling (Fig. 9).
Figure 9. SARA over expression elicits ectopic Notch signaling. A) Wild-type bristles comprise two external cells, the socket (red arrowhead) and the shaft (white arrowhead). B) Upon Sara over expression, sensory organs comprise two sockets (red arrowheads) but no shaft, a phenotype indicative of symmetric Notch activation in the SOP lineage. SEM images courtesy of Sylvain Loubery
Sara is involved in Notch signaling activation during SOP asymmetric cell division, but it has been previously shown that Sara is also present in a multivesicular population of early endosomes that are located apically in polarized epithelial cells of the wing imaginal disc. Sara endosomes are involved in the symmetric partitioning of TGF-ß/BMP signaling molecules among daughter cells during Drosophila wing development (Bokel et al., 2006). These endosomes contain the BMP ligand and receptor complex, Decapentaplegic (Dpp) and Thick veins (Tkv) respectively. During division, the Sara endosomes are targeted to the mitotic spindle and after cytokinesis;
they are symmetrically distributed into the two daughter cells dispatching
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equally the TGF-ß/ BMP signaling molecules. In the absence of Sara, one of the daughter cells inherits unequal TGF-ß/BMP signaling. The sara mutant flies exhibit defects in wing vein differentiation, a process that requires BMP signaling (Fig. 10.)
Figure 10. BMP/Dpp Signal distribution during wing epithelial Drosophila cell mitosis.
A) Upon mitosis, Sara endosomes localize at the central spindle. B) After cytokinesis Sara endosomes are symmetrically distributed and the BMP/Dpp signaling is equally activated in the two daughter cells giving rise to normal pattering in the Drosophila wing. C) When Sara endosomes are unequally distributed, BMP/Dpp signaling is unequal on the two daughter cells and the normal wing development is impaired giving rise to defects in the venation pattern. Pictures taken from Boeckel et al., 2006.
Sara is a component of the TGF-β super-family signaling pathway. TGF-β transduction components are found to play important roles in many cellular processes, such as proliferation, differentiation, apoptosis, and cancer. TGF-β factors can be grouped into two major families, the BMP/Dpp and Activin pathways.
In Drosophila, seven TGF-β pathways ligands have been identified from genomic sequence (Adams et al., 2000), (Raftery and Sutherland, 1999) and (Jensen and Bachtrog, 2010). These ligands act through a receptor complex comprised of heterodimeric combinations of type I and type II receptors. Three type I receptors (Thickvein (Tkv), Saxophone (Sax), Baboon (Babo)) and two type II receptors (Punt and wishful thinking (wit)) interact with either of two R-Smads, mothers against Dpp (Mad) or dSmad2 (Smox) (Das et al., 1999) and (Marques et al., 2002). R-Smad molecules are recruited by Sara (Tsukazaki et
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al., 1998). The type I TGF- β receptor phosphorylates R-Smad molecules. Sara is a FYVE domain protein, which associates with phosphatidyl-inositol-3- phosphate (PtdIns(3)P) in endosomes and acts as a scaffold protein binding to R-Smads molecules and to the type receptor I. This binding is mediated by its Smad binding domain (SBD) and by the C-terminus domain of Sara, respectively. Upon phosphorylation, R-Smad dissociates from Sara and binds to Co-Smad (Medea in Drosophila). This active complex rapidly accumulate in the nucleus, where they are directly involved in transcriptional regulation (Lagna et al., 1996) and (Wisotzkey et al., 1998) (Fig. 11).
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Figure 11. Drosophila BMP and Activin TGF-β pathways and Sara scaffold function. A) The BMP ligands Dpp, Gbb and Scw, act through the type I receptor Tkv or Sax, resulting in phosphorylation of Mad, its association with the co-Smad Medea, tranlocation of the complex into the nucleus, and regulation of target gene expression. The type II receptors Put and Wit display dual specificity and function in both the BMP and Activin pathways. An Activin pathway involves the Type I receptor Babo and Smox. Alp23B and β-Activin ligands signal through Babo. B) Sara presents the R-Smad to the receptor I for phosphorylation. Active R- Smad dissociates from Sara and Receptor and forms a complex with Co-Smad. The complex translocate to the nucleus to activate target genes.
Sara is a key regulator of TGF- β signaling. Sara can act as an adaptor by recruiting R-Smad molecules thereby contributing to TGF- β signaling and Sara can also down regulate Dpp signaling binding PP1c a Drosophila phosphatase (Bennett and Alphey, 2002).
THE ROLE OF BINDING SARA PHOSPHATASES
It has been shown that the type I receptor is a relevant substrate of PP1c and controls its phosphorylation state. The type II receptor is a constitutively active kinase, which phosphorylates type I receptor (Ventura et al., 1994). In PP1c mutants the effect of ectopic Punt is dominantly enhanced and leads to Drosophila wing disc overgrowth, which is very similar to ectopic Dpp phenotype. The role of PP1c in the receptor complex is to antagonize the type II receptor by the dephosphorylation of Type I receptor.
Drosophila has four PP1c genes encoding closely related proteins (>85%
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identical), which are named by their chromosome location and subtype (three PP1α (96A, 87B, 13C) and one PP1β (Flw or 9C) (Dombradi et al., 1990). Sara has a canonical PP1c-binding motif (Egloff et al., 1997) and binds to all four isoforms. A mutation in the Sara PP1c binding domain consisting in a replacement of Phenylalanine 678 by Alanine (SaraF678A), strongly reduces the ability of Sara to bind PP1c isoforms. In SaraF678A a threefold increase in the phosphorylation of the type I receptor was found, therefore Sara binds PP1c and negatively regulates Dpp signaling.
The in vitro biochemical activities of the PP1c isoforms are very similar, however, genetic analysis provides a powerful approach to analyze the specific, no redundant functions of each isoform. Of these, PP187B contributes 80% of the total PP1 activity but the phenotypes of PP187B loss of function mutants (Axton et al., 1990),(Dombradi et al., 1990) and (Baksa et al., 1993) may be due to a loss of overall PP1 activity, rather than identifying specific functions unique to the PP187B protein.
It has been shown previously that the Drosophila PP1c catalytic subunit gene PP1 9C corresponds to Flapwing (Flw) or PP1β, (Raghavan et al., 2000). The semilethality of a strong allele, flw6, demonstrated that PP1β is essential in flies. flw6 larval body wall muscles appeared to form normally, but then they appear detached and degenerated leading to a semiparalyzed larva that could not feed properly (Raghavan et al., 2000). The essential role of PP1β in flies is to regulate nonmuscle actomyosin (Vereshchagina et al., 2004). Loss of PP1β leads to increased levels of phosphorylated nonmuscle myosin regulatory light chain (MRLC) or Spaghetti Squash (Sqh) and actin disorganization.
Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) binds specifically to PP1β, and activates PP1 Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo. The similarity between fly and human PP1 β and MYPT genes suggests this role may be conserved.
Similarly, though PP1 is often assumed to be the major nonmuscle MRLC
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phosphatase, PP2A has also been implicated (Holst et al., 2002). Inhibition of PP2A, reduce the cytoskeletal rearrangements, increase the phosphorylation of the myosin heavy and light chains at protein kinase C-specific sites. These findings indicate that a dynamicactomyosin cytoskeleton is partially regulated by both PP1 and PP2A.
PP2A and PP1c have antagonist functions in TGF- β signaling. Contrary to PP1c; PP2A performs a positive regulation of BMP/Dpp pathway. The PP2A catalytic subunit interacts with BMP receptor complexes and dephosphorylates R-Smad molecule in the linker region increasing the nuclear translocation of Smads and amplifying the BMP signaling (Bengtsson et al., 2009). In Drosophila, this PP2A catalytic subunit is known as PP2A-29B.
Immunoprecipitation and Mass Spectrometry analysis show the PP2A-29B as a presumptive binding protein of Drosophila Sara (see later in results).
Recent studies have revealed Drosophila PP2A as a very important asymmetric cell division regulator. The PP2A heterotrimeric complex, composed of the catalytic subunit (Mts), scaffold subunit (PP2A-29B) and a B-regulatory subunit (Tws), is required for the asymmetric cell division of Drosophila neuroblasts (Wang et al., 2009) and (Ogawa et al., 2009). PP2A negatively regulates aPKC signaling by promoting Par-6 dephosphorylation in neuroblast.
Mutation in Tws, Mts and PP2A-29B exhibits neuroblast overgrowth, high phosphorylation levels of Par6 and low phosphorylation levels of Numb.
Therefore, PP2A should act suppressing neuroblast over proliferation and promoting neuronal differentiation inducing asymmetric distribution of Numb which activation depends on Par6-aPKC activated complex.
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Sara is an endosomal protein that has been shown to play a role during signaling mediated by TGF-beta, Dpp and Notch signaling (Tsukazaki et al., 1998), (Bokel et al., 2006) and (Coumailleau et al., 2009). During development has been show to act during symmetric cell division which happens during the interpretation of the Dpp morphogen gradients and during asymmetric cell division of the sensory organ precursors which perform directional Notch signaling by targetting the motility of Sara signaling endosomes to one of the daughter cells. During my PhD, I aimed at contributing to the understading of the role of Sara during these events. In particular, I set up to establish assays and conditions to answer the following questions:
1. What are the molecular machineries in which Sara is implicated to mediate asymmetric signaling? For this I have carried out a proteomic approach based on immunoprecipitation of Sara complexes and Mass Spectrometry (MS) analysis.
2. What other machineries are involved in a asymmetric motility? Following the same immunoprecipitation/MS, I found binding partners to the key PtdIns(3)P binding kinesin responsible for the asymmetric motility of Sara endosomes.
3. What is the role of Sara during Activin signaling? The study of Sara during Activin signaling has been hampered by the fact that no bona fide Activin transcriptional target was known for the wing. I have performed a candidate gene screening to find Activin targets in the wing.
A PROTEOMICS APROACH TO UNRAVEL “THE SARA MACHINERIES”
The development of Drosophila mecanosensory bristles is a powerful model to study Delta/Notch signaling. During the asymmetric cell division of the fly sensory organ precursor (SOP) endocytosis plays a crucial role by regulating Notch signaling (Berdnik et al., 2002) (Hutterer and Knoblich, 2005), (Hutterer
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and Knoblich, 2005), (Le Borgne et al., 2005) and (Emery et al., 2005). Recent data suggest that a subpopulation of multivesicular endosomes marked by the protein Sara is involved in the directional transport of Delta/Notch during asymmetric cell division (Coumailleau et al., 2009).
During SOP mitosis, Notch and Delta are internalized in Sara endosomes and subsenquently targeted to the PIIA daugther cell in which Notch signaling will be activated. Sara is localized at the limiting membrane of these endosomes and contributes to their asymmetric behavior, but is not essential to endow Sara endosomes its asymmetric distribution; the mechanism by which these endosomes are asymmetrically distributed remains unknown.
I have used a proteomic approach based on immunoprecipitation and MS analysis to determine the composition of Sara complexes. The final goal is to explore the possibility that these Sara binding partners have and important function on the mechanism of directional trafficking of Notch/Delta.
CONDITIONS FOR SARA IMMUNOPRECIPITATION
Sara was immunoprecipitated from two different Drosophila melanogaster systems: i) S2 cells, representing the symmetric cell division scenario and ii) the third instar larval brains and imaginal discs, which contain both a high number of asymmetrically dividing cells (the neuroblast stem cells) and symmetrically dividing cells (imaginal disc cells; Fig. 12). The Sara protein was tagged with GFP and over-expressed under the control of either a Metallothionein promoter (Met) for copper-inducible expression in S2 cells or the UAS promoter activated by the Hedgehog-Gal4-Gal80ts driver (hh-Gal4- Gal80ts) in larvae. As a negative control, non-tagged Sara was overexpressed and submitted to the same immunoprecipitation/MS protocol.
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Figure 12. Sara immunoprecipitation. A) Commassie staining from immunoprecipitation (IP) of S2 cells expressing either Sara GFP (Met-Sara GFP) or GFP (Met-GFP) as control. B) Commassie Staining form IP of larval brains and imaginal discs expressing either Sara GFP (hh-Gal4-Gal80ts-Sara-GFP) or untagged Sara (hh-Gal4-Gal80ts-Sara) as control. C) GFP Western blot (WB) from larval IP hh-Gal4-Gal80ts-Sara GFP and hh-Gal4-Gal80ts-Sara and Sara Western blot from larval inputs hh-Gal4-Gal80ts-Sara GFP and hhGal4-Gal80ts-Sara.
Arrowheads correspond to the sara band. IP: immunoprecipitation; WB: Western blot.
DISECTING DOMAINS OF THE SARA PROTEIN
My ultimate goal is to identify the cellular machineries that control the different behaviors of the Sara endosomes, including the targeting of endosomes to the spindle, the asymmetric motility of the Sara endosomes and transduction of the Notch signal from endosomes. To achieve this, my strategy has been to perform IP using GFP combined with MS, not only for GFP-tagged full length Sara as a bait, but also for defined pieces of the Sara protein (Fig.
13, 14). In addition, IP/MS was performed on negative controls where GFP is not fused to Sara or over expressed Sara is untagged. These pieces are
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designed to contain well established domains in the Sara protein: the FYVE domain, the Smad binding domain and the PP1 binding domain (Fig. 13).
Figure 13. Schematic representation of the Sara pieces considered in this work. Sara was dissected in eight different domains encompassing the full length of the protein. Every domain was GFP tagged at its N-terminus. Abreviations of the domains are indicated as in the Introduction.
The logic is that by performing IPs using these distinct fragments, I can uncover the identity of specific partners to better find putative candidates.
Indeed a bona fide binding candidate should not appear in the MS data after IP in negative controls. Furthermore, it might not appear in all the immunoprecipitates, but only in a subset of the fragments considered, corresponding to those in which the binding domain of a particular candidate is present. Indeed, the specificity of the Sara partner candidates is reinforced by comparing the results from different fragments (see MS data).
This approach serves three additional objectives: to map the binding domains, to give hints on the function of the domain and to provide preliminary information on the function of the binding partner. Indeed, using different fragments provides hints on the identity of the binding domain in the Sara
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sequence that could be responsible for the molecular interaction between the candidate and Sara. This medium through put approach is merely used, as an initial step of domain mapping and the definite identity of the fragment will be characterized by further pulldown and immunoprecipitation analysis.
Furthermore, by performing a structure/function analysis of the Sara fragments studied, we can gain insights on the possible molecular/cellular role of the candidate during the biology of the Sara endosomes. Indeed, by studying the behavior of endosomes carrying the mutant fragments tagged by GFP, we can have an initial idea of the role of the partners bound to this fragment during the asymmetric motility of endosomes, Notch signaling, etc. Moreover, when over- expressed, some of the fragments cause a phenotype during Notch signaling, the bristle lineage or growth of imaginal discs (tumors) or have a Dominant negative effect. Again, this gives us preliminary information on the role of those fragments (and their binding partners) during signaling and development.
Fig. 14 shows the IP of the fragments considered in this study.
Figure 14. Verification of the Sara domains. The size of the bands in Commassie staining or GFP Wester blot analysis match the expected molecular weight of the different domains, arrowheads correspond to the Sara bands.
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MASS SPECTROMETRY DATA
In order to identify factors, which are engaged in Sara complexes, each of the immunoprecipitations, were concentrated in one single band (Fig. 15) and subsequently submitted MS analysis.
Figure 15. Concentration of the Sara complex. Proteins were eluted from beads with 30µl of Laemmli buffer, then charged in 10% precast gel and finally ran during 5 minutes. The bands were visualized by Commassie staining and cut out. In order to analyze the Sara complexes composition, MS of the digested bands was performed in the Proteomics Core Facility of Geneva using the NanoLC- ESI-MS/Ms technique.
MS was carried out from immunoprecipitates from full length of Sara extracted either from S2 cells or larvae. In addition, the different Sara domains from larvae were submitted to IP and MS. The first piece of Sara (N-terminus) was not analyzed. As negative controls IP/MS was also performed on blank S2 cells and larvae (Tab. 1)
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Continuation Tab. 1
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Continuation Tab. 1
Table 1. Identification of Sara binding proteins by MS analysis. Eleven partners of Sara are represented in this table from the total Sara binding proteins found in the MS. The different criteria to select these eleven proteins follow: 1) they must have an homologous in Humans; 2) some being already known Sara binding proteins; 3) their posible relevancy in Sara endosomes distribution during SOP asymmetric cell division. In the first column the Drosophila Identification numbers of the eleven different partners are indicated. The second column shows the name of the partners and its function. The third column contains the name of the human homologous of these partners. The rest of the columns have the different domains compending Sara protein. The purple color indicates which Immunoprecipitated Sara piece analyzed (including the cell and larval full length of Sara) is binding to the newly found partner of Sara.
All the molecules considered as a real binding protein of Sara must meet the minimun requirement of two MS analsyis established parameters. The first parameter to be considered is the number of recognized peptides for each binding protein. When the protein complex is digested from the band of the gel different peptide fragments are produced. After MS analysis these peptides will provide an aminoacid sequence containing the information to corresponding proteins. The identification of this sequence along the aminoacids backbone of a protein will provide the identity of the molecule to which the peptide belongs.
Only proteins with at least two aligned peptides within their sequences from the digested complex are taken into account as a part of the analyzed complex.
The second parameter is the probability of the peptide sequence being a certain
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protein. The protein probability calculation is based on the total number of peptide identified for the same molecule. The more peptides fragments we find for the same protein the higher the probability is for a molecule to be identified in the complex. To calculate this probability I have used the Scaffold software, which utilizes proven statistical algorithms to calculate the probability that proteins are actually in the biological samples. Scaffold results help to detect false positives displaying a high confident identification performance. I have defined up to 95% the protein probability identification for a molecule of being a member of the Sara complex. After these filters, I have proceeded to analyze the MS data.
First, the MS data confirmed previous reports, which identified PP1c isoforms (Bennett and Alphey, 2002) as well as the Activin-type R-Smad, Smad2 (Smox) (Tsukazaki et al., 1998) as Sara binding partners. It is worth noting that we could not find the BMP R-Smad Smad1 (Mad) in our immunoprecipitates, confirming the mammalian data showing that Sara only binds Smad2 and Smad3. A report in Drosophila however has suggested that Mad also binds Sara (Bennett and Alphey, 2002), a result that has not been confirmed. In summary, this data suggests that our assay to find Sara partners is efficient at uncovering the machinery that, together with Sara, determines the properties of motility and signaling of the Sara endosomes.
I found in addition 40 bona fide Sara binding candidates. I have focused on those, which show a homology in vertebrate systems, are known partners of Sara and could play an important role in SOP asymmetric cell division. These represent 11 proteins. A few of these interactions are worth noting here, although no further work has been performed to pursuit the significance of all of them.
I found two endocytic proteins, Clathrin heavy chain (Chc) and α-adaptin as binding interactors found in immunnoprecipitates from the minimal asymmetric Sara fragment, which contains the FYVE, SBD ad PP1 domain
