The Functions of Auxilin and Rab11 in Drosophila Suggest That the Fundamental Role of Ligand Endocytosis in Notch Signaling Cells Is Not Recycling

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling

The Functions of Auxilin and Rab11 in Drosophila Suggest That the Fundamental Role of Ligand Endocytosis in Notch Signaling Cells Is Not Recycling

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling

Determining the role of a small GTPase, Ral, and an endocytic factor, epsin, in Drosophila Notch signaling

textCell-cell communication events are crucial to determine the fate of each cell during development. Notch signaling is involved in many different contexts in determining cell fate by mediating cell-cell communication. Furthermore, regulation of the Notch transduction pathway is critical for normal cellular function, which is implicated in various diseases, including cancers. At a certain developmental time point, intrinsic or extrinsic developmental cues induce biases in ligands and Notch receptors between neighboring cells. These initial biases are further amplified by various cellular factors which eventually dictate cell fates. In Drosophila, two Notch ligands, Delta and Serrate, trigger Notch receptor activation in nearby cells by virtue of numerous regulating factors. One important question in this area is how cells become Notch signal sending or receiving cells for cell fate decisions. I show evidence about a distinct mechanism for biasing the direction of Notch signaling that depends on a small GTPase, Ral, during Drosophila photoreceptor cell development. Investigations described here indicate that Fz signaling up-regulates Ral transcription in a signal sending fate cell, the R3 precursor, and Ral represses ligand-independent activation of Notch in the R3 precursor. This event ensures R3 to become a signaler and contributes to asymmetric Notch activation in the neighboring cell, R4. Ral is a small Ras-like GTPase that regulates membrane trafficking and signaling. Here, possible Ral effector pathways that are important for Notch regulation will be proposed. To trigger Notch activation in adjacent cells, Notch ligand endocytosis by the signaling cells is necessary. Recently, it was suggested that control of membrane trafficking is important not only for ligand signaling, but also for Notch receptor activation. Furthermore, Notch receptor trafficking regulates critical cellular functions, including proliferation, which is implicated in tumors. Therefore, another important question in Notch signaling is about the role of membrane trafficking in regulation of the Notch transduction pathway. Drosophila endocytic epsin, Liquid facets [Lqf], is a key component necessary for ligand endocytosis, thereby triggering Notch activation in adjacent cells. However, its function in signal receiving cells for Notch activation has not been studied. In this dissertation, I provide evidence that epsin is also required in signal receiving cells for Notch activation in developmental contexts. Furthermore, genetic and molecular evidence suggests that epsin regulates Notch receptor trafficking via Rab5-mediated endosomal sorting pathway for Notch activation. These studies support the idea that Notch activation at the plasma membrane is not the only way to transduce Notch signaling, but the Notch receptor must enter through an epsin-mediated endocytic pathway into subcellular compartments to be activated, at least in some contexts.Cellular and Molecular Biolog

Clustering and Negative Feedback by Endocytosis in Planar Cell Polarity Signaling Is Modulated by Ubiquitinylation of Prickle

<div><p>The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.</p></div

Amplification of asymmetry in membrane clusters and requirement of Pk for excluding Vang.

<p>(A-B”) Clone interface between twin spots expressing either a distal PCP protein fused to ECFP (Fz in A, Dgo in B) or Stbm::EYFP (A’,B’). Note that proximal and distal PCP proteins colocalize in puncta at the clone interface, suggesting that puncta connect PCP complexes of adjacent cells with each other. (C-C”) Clone interface between twin spots expressing either Stbm::ECFP or Stbm::EYFP. (D, E) Quantification of intensity (arbitrary units) along the cell boundaries indicated in A and B, and (F, J) quantification of normalized intensity in C. For F and J, intensity levels of cell boundaries along the clone interface were normalized to the average boundary intensity within the respective twin spot. Clones overexpressing Vang were created adjacent to both wild-type and <i>pk</i> mutant cells by reverse MARCM (see Supporting Information for genotype) (G-I, K-M). (G) Cartoon showing Fz dependent exclusion of Vang-Fmi complexes. (H) Schematic of reverse MARCM clones; red dots indicate <i>vang</i> overexpressing cells (in K-M, red mCherry expressing cells) and yellow dots indicate <i>pk</i> mutant clonal cells facing <i>vang</i> overespressing cells (H, K). (I) Schematic of MARCM experiment. (K) Vang overexpression excludes Vang::YFP from the membrane in neighboring wild-type cells (green arrows), but fails to exclude Vang::YFP in <i>pk</i> mutant cells (yellow dots; yellow arrows indicate membranous Vang::YFP facing <i>vang</i> overexpressing cells in <i>pk</i> mutant cells). (A-C) 16hr APF and (K-M) 26hr APF wing tissues. Scale bars: 5 μm for A-C; 10μm for K-M. Genotypes are (A) <i>y</i>, <i>w</i>, <i>hsflp/+(Y);; ubiP-fz</i>::<i>ECFP</i>, <i>FRT80 / ubiP-vang</i>::<i>EYFP</i>, <i>FRT80</i>, (B) <i>y</i>, <i>w</i>, <i>hsflp/+(Y);; ubiP-ECFP</i>::<i>dgo</i>, <i>FRT80 / ubiP-vang</i>::<i>EYFP</i>, <i>FRT80</i>, (C) <i>y</i>, <i>w</i>, <i>hsflp/+(Y);; ubiP-vang</i>::<i>ECFP</i>, <i>FRT80 / ubiP-vang</i>::<i>EYFP</i>, <i>FRT80</i>, (K-M) <i>y</i>, <i>w</i>, <i>hsflp/D174GAL4; FRT42D</i>, <i>armP-LacZ /FRT42D</i>, <i>pk</i>^{<i>pk-sple13</i>}, <i>actP-vang</i>::<i>YFP</i>, <i>tubP-GAL80; UAS-mCherry/UAS-vang</i>.</p

Models: Cell polarity establishment and the involvement of Pk-mediated endocytosis.

<p>(A-C) Schematics of positive (green arrows) and negative (red arrows) feedback in polarization of single cells (A), and coupled cells (B, C). Two possible modes of positive feedback are shown in (C). (D) Model of mutual exclusion in which competitive interactions between peripheral membrane associated core factors results in endocytosis of either Vang-Fmi or Fmi-Fz complexes.</p

Pk-mediated apical clustering of core proteins requires Vang.

<p>Apical clustering of Fmi (red) is seen in cells clonally overexpressing GFP::Pk (green, A) and Pk (green, D) in wild-type (A) and <i>fz</i> mutant (<i>fz</i>^<i>R52</i><i>/fz</i>^<i>R52</i>, D) but not in <i>vang</i> mutant (<i>vang</i>^<i>A3</i><i>/vang</i>^<i>stbm6</i>, C) wing tissues (26hr APF). Apical clustering of Vang::YFP (green, B) with overexpression of Pk (red, B) is shown. Majority of GFP::Pk, or all of Pk, positive puncta are also positive for Fmi, or Vang::YFP, respectively (A, B). Scale bars: 10μm. Genotypes are (A) <i>y</i>, <i>w</i>, <i>hsflp/+; +/+; actP>CD2>GAL4</i>, <i>UAS-RFP/UAS-GFP</i>::<i>pk</i>, (B) <i>y</i>, <i>w</i>, <i>hsflp/+; UAS-pk/; actP>CD2>GAL4</i>, <i>UAS-RFP/actP-vang</i>::<i>YFP</i>, (C) <i>y</i>, <i>w</i>, <i>hsflp/+; vang</i>^<i>A3</i><i>/vang</i>^<i>stbm6</i><i>; actP>CD2>GAL4</i>, <i>UAS-RFP/UAS-GFP</i>::<i>pk</i>, (D) <i>y</i>, <i>w</i>, <i>hsflp/+; UAS-pk/actP>CD2>GAL4</i>, <i>UAS-GFP; fz</i>^<i>R52</i><i>/fz</i>^<i>R52</i>.</p

Pk protein level is regulated by Cul1-mediated ubiquitinylation and proteasomal degradation.

<p>Levels of endogenous Pk (A) and Myc::Pk expressed with the actin promoter (B) were examined by western blot. Knocking-down <i>cul1</i> using a <i>D174GAL4</i> driven <i>cul1</i> RNAi construct increases the amount of Pk or Myc::Pk in third instar wing discs (lane 3 in A and B). Overexpression of <i>UAS-prosbeta2’</i> induces modification of Myc::Pk (labeled by a red line in C) that is <i>cul1</i> dependent (C; compare lane1 with lane 2). Note that the protein level of unmodified form of Myc::Pk is higher with <i>cul1i</i> in the blot with short exposure time. γ-Tubulin was probed as loading controls (A-C). (D) Myc::Pk from wing discs with genotypes as in (C) was immunoprecipitated and detected with anti-Ub, anti-Myc and anti-Pk antibodies. More Ub signal was detected in the absence as compared to the presence of <i>cul1</i> knock-down. Genotypes are (A) Lane 1: <i>D174GAL4/+(Y); pk</i>^{<i>pk-sple13</i>}<i>/ pk</i>^{<i>pk-sple14</i>}, Lane 2: <i>D174GAL4/+(Y)</i>, Lane 3: <i>D174GAL4/+(Y); UAS-cul1</i>^{<i>IR108558</i>}<i>/+</i>, (B) Lane 1: <i>D174GAL4/+(Y)</i>, Lane 2: <i>D174GAL4/+(Y); +/+; actP-6XMyc</i>::<i>pk/+</i>, Lane 3: <i>D174GAL4/+(Y); UAS-cul1</i>^{<i>IR108558</i>}<i>/+; actP-6XMyc</i>::<i>pk/+</i>, (C) Lane 1: <i>D174GAL4/UAS-lacZi; +/+; actP-6XMyc</i>::<i>pk/UAS-prosbeta2’</i>; Lane 2: <i>D174GAL4/+(Y); UAS-cul1</i>^{<i>IR108558</i>}<i>/+; actP-6XMyc</i>::<i>pk/UAS-prosbeta2’</i>, (D) same as (C).</p

Endocytosis of membranous Pk and Vang.

<p>Windows were created over the wing of live 24-26h APF pupae by opening the pupal case and cuticle, and then incubated in FM4-64 solution. Apical or sub-apical puncta for GFP::Pk (green in A) and Vang::YFP (green in B) were then visualized. (A) Many GFP::Pk positive cytosolic puncta (GFP::Pk driven by <i>ptc-GAL4</i>) co-labeled with FM4-64. (B) In <i>pk</i> overexpressing clones (outlined), Vang::YFP frequently co-labeled with FM4-64. Double positive puncta for GFP::Pk, or Vang::YFP, and FM4-64 are indicated with arrowheads (see the manuscript for quantification). Scale bars: 10μm. Genotypes are (A) <i>ptc-GAL4/+; UAS-gfp</i>::<i>pk</i>, (B) <i>actP>CD2>GAL4/y</i>, <i>w</i>, <i>hsflp; UAS-pk/+; actP-vang</i>::<i>YFP</i>.</p

The Cul1 complex is required for hair and trichome polarity in the wing.

<p>Multiple hairs and swirling of hairs in adult wings (A, C, E) bearing <i>cul1</i> (A) and <i>skpA</i> (C) knock-down, and <i>cul1</i> null (<i>cul1</i>^<i>EX</i>) mutant (E) clones. Growth of pre-hairs in knock-down clones (labeled by GFP) of <i>cul1</i> (B), <i>skpA</i> (D), and <i>slimb</i> (G), and in <i>cul1</i> null mutant clones (F, no RFP expression), is delayed (absent or smaller trichomes). Polarity of pre-hairs in neighboring wild type cells is also affected (white arrow for normal pre-hair direction and yellow arrows for abnormal pre-hair direction in B and G. Yellow dots in D and F indicate wild-type cells with defects in pre-hair polarity). 32~34hr APF pupal wings were stained with phalloidin to label Actin-rich trichomes (red in B, D, and G; green in F). Scale bars: 10μm. Genotypes are (A, B) <i>y</i>, <i>w</i>, <i>hsflp/+(Y); UAS-cul1</i>^{<i>IR108558</i>}<i>/+; actP>CD2>GAL4</i>, <i>UAS-GFP/+</i>, (C, D) <i>y</i>, <i>w</i>, <i>hsflp/+(Y); UAS-skpA</i>^{<i>IR32789</i>}<i>/+; actP>CD2>GAL4</i>, <i>UAS-GFP/+</i>, (E, F) <i>y</i>, <i>w</i>, <i>hsflp/+(Y); FRT42D</i>, <i>cul1</i>^<i>EX</i><i>/FRT42D</i>, <i>ubiP-NLS</i>::<i>mRFP</i>, (G) <i>y</i>, <i>w</i>, <i>hsflp/+(Y); +/+; actP>CD2>GAL4</i>, <i>UAS-GFP/UAS-slimb</i>^{<i>IRFBst0033898</i>}.</p