Drosophila Tel2 is expressed as a translational fusion with EpsinR and is a regulator of wingless signaling.

Tel2, a protein conserved from yeast to vertebrates, is an essential regulator of diverse cellular processes including telomere maintenance, DNA damage checkpoints, DNA repair, biological clocks, and cell signaling. The Drosophila Tel2 protein is produced as a translational fusion with EpsinR, a Clathrin adapter that facilitates vesicle trafficking between the Golgi and endosomes. EpsinR and Tel2 are encoded by a Drosophila gene called lqfR. lqfR is required for viability, and its specific roles include cell growth, proliferation, and planar cell polarity. We find that all of these functions of lqfR are attributed entirely to Tel2, not EpsinR. In addition, we find that Drosophila LqfR/Tel2 is a component of one or more protein complexes that contain E-cadherin and Armadillo. Moreover, Tel2 modulates E-cadherin and Armadillo cellular dynamics. We propose that at least one of the functions of Drosophila Tel2 is regulation of Wingless signaling.

Ral inhibits ligand-independent Notch signaling in Drosophila.

We discovered recently that the Drosophila Ral GTPase regulates Notch signaling and thereby affects cell patterning in the eye. Although Ral functions in the ligand signaling cells, Ral does not stimulate ligand signaling directly. Rather, in cells that express both Notch receptor and ligand, Ral activity promotes a cell to become the signaler by inhibiting Notch receptor activation in that cell. Moreover, Ral inhibits a particular pathway of Notch activation-receptor activation that occurs independent of ligand binding. In this Commentary, we discuss the phenomenon of ligand-independent Notch receptor activation and how this event might be regulated by Ral.

Drosophila Epsin's role in Notch ligand cells requires three Epsin protein functions: the lipid binding function of the ENTH domain, a single Ubiquitin interaction motif, and a subset of the C-terminal protein binding modules.

Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin's mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, we tested each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand cells. First, we discovered that deletion or mutation of both UIMs destroyed Epsin's function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin's two UIMs was essential. Third, the lipid-binding function of the ENTH domain was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, we found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin's essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. We propose that Epsin's specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin.

Notch ligand ubiquitylation: what is it good for?

In the first volume of Developmental Cell, it was reported that the classic Drosophila neurogenic gene neuralized encodes a ubiquitin ligase that monoubiquitylates the Notch ligand Delta, thus promoting Delta endocytosis. A requirement for ligand internalization by the signal-sending cell, although counterintuitive, remains to date a feature unique to Notch signaling. Ten years and many ubiquitin ligases later, we discuss sequels to these three papers with an eye toward reviewing the development of ideas for how ligand ubiquitylation and endocytosis propel Notch signaling.

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.

Ral GTPase promotes asymmetric Notch activation in the Drosophila eye in response to Frizzled/PCP signaling by repressing ligand-independent receptor activation.

Ral is a small Ras-like GTPase that regulates membrane trafficking and signaling. Here, we show that in response to planar cell polarity (PCP) signals, Ral modulates asymmetric Notch signaling in the Drosophila eye. Specification of the initially equivalent R3/R4 photoreceptor precursor cells in each developing ommatidium occurs in response to a gradient of Frizzled (Fz) signaling. The cell with the most Fz signal (R3) activates the Notch receptor in the adjacent cell (R4) via the ligand Delta, resulting in R3/R4 cell determination and their asymmetric positions within the ommatidium. Two mechanisms have been proposed for ensuring that the cell with the most Fz activation sends the Delta signal: Fz-dependent transcriptional upregulation in R3 of genes that promote Delta signaling, and direct blockage of Notch receptor activation in R3 by localization of an activated Fz/Disheveled protein complex to the side of the plasma membrane adjacent to R4. Here, we discover a distinct mechanism for biasing the direction of Notch signaling that depends on Ral. Using genetic experiments in vivo, we show that, in direct response to Fz signaling, Ral transcription is upregulated in R3, and Ral represses ligand-independent activation of Notch in R3. Thus, prevention of ligand-independent Notch activation is not simply a constitutive process, but is a target for regulation by Ral during cell fate specification and pattern formation.

The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus.

Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface.

Drosophila liquid facets-Related encodes Golgi epsin and is an essential gene required for cell proliferation, growth, and patterning.

Epsin and epsin-Related (epsinR) are multi-modular proteins that stimulate clathrin-coated vesicle formation. Epsin promotes endocytosis at the plasma membrane, and epsinR functions at the Golgi and early endosomes for trans-Golgi network/endosome vesicle trafficking. In Drosophila, endocytic epsin is known as Liquid facets, and it is essential specifically for Notch signaling. Here, by generating and analyzing loss-of-function mutants in the liquid facets-Related (lqfR) gene of Drosophila, we investigated the function of Golgi epsin in a multicellular context. We found that LqfR is indeed a Golgi protein, and that like liquid facets, lqfR is essential for Drosophila viability. In addition, primarily by analyzing mutant eye discs, we found that lqfR is required for cell proliferation, insulin-independent cell growth, and cell patterning, consistent with a role in one or several signaling pathways. Epsins in all organisms share an ENTH (epsin N-terminal homology) domain, which binds phosphoinositides enriched at the plasma membrane or the Golgi membrane. The epsinR ENTH domain is also the recognition element for particular cargos. By generating wild-type and mutant lqfR transgenes, we found that all apparent LqfR functions are independent of its ENTH domain. These results suggest that LqfR transports specific cargo critical to one or more signaling pathways, and lays the foundation for identifying those proteins.

On the roles of the Drosophila KASH domain proteins Msp-300 and Klarsicht.

KASH (Klarsicht, Anc-1, Syne-1 homology) domain-containing proteins anchor the nucleus to the actin cytoskeleton or to microtubules. KASH proteins thus play pivotal roles in a variety of developmental processes where nuclear positioning is critical. Two KASH proteins have been identified in Drosophila: Muscle-specific protein-300 (Msp-300) and Klarsicht (Klar). Msp-300 anchors nuclei to actin, and has been reported to be essential for positioning of nurse cell nuclei during oogenesis, and thus production of mature ooctyes. Klar is required for positioning of photoreceptor and cone cell nuclei in the developing eye, which is critical for proper eye morphology. Here, we asked whether KASH domain-containing forms of Msp-300 are required for nuclear positioning in the eye, and we found that they are not. Moreover, in the course of this work, we discovered that contrary to previous reports, KASH domain-containing forms of Msp-300 are not required for viability, nor for oogenesis. However, we did find that Msp-300 has a function in egg laying, normally redundant with a function of Klar.

A Drosophila model for Angelman syndrome.

Angelman syndrome is a neurological disorder whose symptoms include severe mental retardation, loss of motor coordination, and sleep disturbances. The disease is caused by a loss of function of UBE3A, which encodes a HECT-domain ubiquitin ligase. Here, we generate a Drosophila model for the disease. The results of several experiments show that the functions of human UBE3A and its fly counterpart, dube3a, are similar. First, expression of Dube3a is enriched in the Drosophila nervous system, including mushroom bodies, the seat of learning and memory. Second, we have generated dube3a null mutants, and they appear normal externally, but display abnormal locomotive behavior and circadian rhythms, and defective long-term memory. Third, flies that overexpress Dube3a in the nervous system also display locomotion defects, dependent on the ubiquitin ligase activity. Finally, missense mutations in UBE3A alleles of Angelman syndrome patients alter amino acid residues conserved in the fly protein, and when introduced into dube3a, behave as loss-of-function mutations. The simplest model for Angelman syndrome is that in the absence of UBE3A, particular substrates fail to be ubiquitinated and proteasomally degraded, accumulate in the brain, and interfere with brain function. We have generated flies useful for genetic screens to identify Dube3a substrates. These flies overexpress Dube3a in the eye or wing and display morphological abnormalities, dependent on the critical catalytic cysteine. We conclude that dube3a mutants are a valid model for Angelman syndrome, with great potential for identifying the elusive UBE3A substrates relevant to the disease.

Overexpression of partner of numb induces asymmetric distribution of the PI4P 5-Kinase Skittles in mitotic sensory organ precursor cells in Drosophila.

Unequal segregation of cell fate determinants at mitosis is a conserved mechanism whereby cell fate diversity can be generated during development. In Drosophila, each sensory organ precursor cell (SOP) divides asymmetrically to produce an anterior pIIb and a posterior pIIa cell. The Par6-aPKC complex localizes at the posterior pole of dividing SOPs and directs the actin-dependent localization of the cell fate determinants Numb, Partner of Numb (Pon) and Neuralized at the opposite pole. The plasma membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the plasma membrane localization and activity of various proteins, including several actin regulators, thereby modulating actin-based processes. Here, we have examined the distribution of PIP2 and of the PIP2-producing kinase Skittles (Sktl) in mitotic SOPs. Our analysis indicates that both Sktl and PIP2 reporters are uniformly distributed in mitotic SOPs. In the course of this study, we have observed that overexpression of full-length Pon or its localization domain (LD) fused to the Red Fluorescent Protein (RFP::Pon(LD)) results in asymmetric distribution of Sktl and PIP2 reporters in dividing SOPs. Our observation that Pon overexpression alters polar protein distribution is relevant because RFP::Pon(LD) is often used as a polarity marker in dividing progenitors.

Auxilin is essential for Delta signaling.

Endocytosis regulates Notch signaling in both signaling and receiving cells. A puzzling observation is that endocytosis of transmembrane ligand by the signaling cells is required for Notch activation in adjacent receiving cells. A key to understanding why signaling depends on ligand endocytosis lies in identifying and understanding the functions of crucial endocytic proteins. One such protein is Epsin, an endocytic factor first identified in vertebrate cells. Here, we show in Drosophila that Auxilin, an endocytic factor that regulates Clathrin dynamics, is also essential for Notch signaling. Auxilin, a co-factor for the ATPase Hsc70, brings Hsc70 to Clathrin cages. Hsc70/Auxilin functions in vesicle scission and also in uncoating Clathrin-coated vesicles. We find that like Epsin, Auxilin is required in Notch signaling cells for ligand internalization and signaling. Results of several experiments suggest that the crucial role of Auxilin in signaling is, at least in part, the generation of free Clathrin. We discuss these observations in the light of current models for the role of Epsin in ligand endocytosis and the role of ligand endocytosis in Notch signaling.

Identification of genes that interact with Drosophila liquid facets.

We have performed mutagenesis screens of the Drosophila X chromosome and the autosomes for dominant enhancers of the rough eye resulting from overexpression of liquid facets. The liquid facets gene encodes the homolog of vertebrate endocytic Epsin, an endocytic adapter protein. In Drosophila, Liquid facets is a core component of the Notch signaling pathway required in the signaling cells for ligand endocytosis and signaling. Why ligand internalization by the signaling cells is essential for signaling is a mystery. The requirement for Liquid facets is a hint at the answer, and the genes identified in this screen provide further clues. Mutant alleles of clathrin heavy chain, Rala, split ends, and auxilin were identified as enhancers. We describe the mutant alleles and mutant phenotypes of Rala and aux. We discuss the relevance of all of these genetic interactions to the function of Liquid facets in Notch signaling.

Drosophila klaroid encodes a SUN domain protein required for Klarsicht localization to the nuclear envelope and nuclear migration in the eye.

KASH (Klarsicht/Anc-1/Syne homology) domain proteins are cytoskeleton-associated proteins localized uniquely to the outer nuclear membrane. Klarsicht is a KASH protein required for nuclear migration in differentiating cells of the Drosophila eye. The C-terminal KASH domain of Klarsicht resides in the perinuclear space, and the cytoplasmic moiety connects to the microtubule organizing center. In C. elegans and vertebrate cells, SUN (Sad1/UNC-84) domain proteins reside in the inner nuclear membrane and tether KASH proteins to the outer nuclear membrane. Is there a Drosophila SUN protein that performs a similar function, and if so, is it like Klarsicht, obviously essential for nuclear positioning only in the eye? Here, we identify Drosophila Klaroid, a SUN protein that tethers Klarsicht. klaroid loss-of-function mutants are indistinguishable phenotypically from klarsicht mutants. Remarkably, neither gene is essential for Drosophila viability or fertility, and even in klaroid klorsicht double mutants, the only obvious external morphological defect is rough eyes. In addition, we find that klaroid and klarsicht are required for nuclear migration in differentiating neurons and in non-neural cells. Finally, while perinuclear Klaroid is ubiquitous in the eye, Klarsicht expression is limited to differentiating cells and may be part of the trigger for apical nuclear migration.

Endocytosis, endosome trafficking, and the regulation of Drosophila development.

Endocytosis and endosome trafficking regulate cell signaling in unexpected ways. Here we review the contribution that Drosophila research has made to this exciting field. In addition to attenuating signaling, endocytosis shapes morphogen gradients, activates ligands, and regulates spatially receptor activation within a single cell. Moreover, some receptors signal from within endosomes, and the ability of a specific type of endosome to form controls the ability of cells to signal. Experiments in Drosophila reveal that through regulation of a variety of cell signaling pathways, endocytosis controls cell patterning and cell fate.

KASH 'n Karry: the KASH domain family of cargo-specific cytoskeletal adaptor proteins.

A diverse family of proteins has been discovered with a small C-terminal KASH domain in common. KASH domain proteins are localized uniquely to the outer nuclear envelope, enabling their cytoplasmic extensions to tether the nucleus to actin filaments or microtubules. KASH domains are targeted to the outer nuclear envelope by SUN domains of inner nuclear envelope proteins. Several KASH protein genes were discovered as mutant alleles in model organisms with defects in developmentally regulated nuclear positioning. Recently, KASH-less isoforms have been found that connect the cytoskeleton to organelles other than the nucleus. A widened view of these proteins is now emerging, where KASH proteins and their KASH-less counterparts are cargo-specific adaptors that not only link organelles to the cytoskeleton but also regulate developmentally specific organelle movements.

Drosophila klarsicht has distinct subcellular localization domains for nuclear envelope and microtubule localization in the eye.

The Drosophila klarsicht (klar) gene is required for developmentally regulated migrations of photoreceptor cell nuclei in the eye. klar encodes a large ( approximately 250 kD) protein with only one recognizable amino acid sequence motif, a KASH (Klar, Anc-1, Syne-1 homology) domain, at its C terminus. It has been proposed that Klar facilitates nuclear migration by linking the nucleus to the microtubule organizing center (MTOC). Here we perform genetic and immunohistochemical experiments that provide a critical test of this model. We analyze mutants in the endogenous klar gene and also flies that express deleted forms of Klar protein from transgenes. We find that the KASH domain of Klar is critical for perinuclear localization and for function. In addition, we find that the N-terminal portion of Klar is also important for function and contains a domain that localizes the protein to microtubules apical to the nucleus. These results provide strong support for a model in which Klar links the nucleus to the MTOC.

Fat facets and Liquid facets promote Delta endocytosis and Delta signaling in the signaling cells.

Endocytosis modulates the Notch signaling pathway in both the signaling and receiving cells. One recent hypothesis is that endocytosis of the ligand Delta by the signaling cells is essential for Notch activation in the receiving cells. Here, we present evidence in strong support of this model. We show that in the developing Drosophila eye Fat facets (Faf), a deubiquitinating enzyme, and its substrate Liquid facets (Lqf), an endocytic epsin, promote Delta internalization and Delta signaling in the signaling cells. We demonstrate that while Lqf is necessary for three different Notch/Delta signaling events at the morphogenetic furrow, Faf is essential only for one: Delta signaling by photoreceptor precluster cells, which prevents recruitment of ectopic neurons. In addition, we show that the ubiquitin-ligase Neuralized (Neur), which ubiquitinates Delta, functions in the signaling cells with Faf and Lqf. The results presented bolster one model for Neur function in which Neur enhances Delta signaling by stimulating Delta internalization in the signaling cells. We propose that Faf plays a role similar to that of Neur in the Delta signaling cells. By deubiquitinating Lqf, which enhances the efficiency of Delta internalization, Faf stimulates Delta signaling.

The functions of Klarsicht and nuclear lamin in developmentally regulated nuclear migrations of photoreceptor cells in the Drosophila eye.

Photoreceptor nuclei in the Drosophila eye undergo developmentally regulated migrations. Nuclear migration is known to require the perinuclear protein Klarsicht, but the function of Klarsicht has been obscure. Here, we show that Klarsicht is required for connecting the microtubule organizing center (MTOC) to the nucleus. In addition, in a genetic screen for klarsicht-interacting genes, we identified Lam Dm(0), which encodes nuclear lamin. We find that, like Klarsicht, lamin is required for photoreceptor nuclear migration and for nuclear attachment to the MTOC. Moreover, perinuclear localization of Klarsicht requires lamin. We propose that nuclear migration requires linkage of the MTOC to the nucleus through an interaction between microtubules, Klarsicht, and lamin. The Klarsicht/lamin interaction provides a framework for understanding the mechanistic basis of human laminopathies.

Deubiquitinating enzymes: their roles in development, differentiation, and disease.

The ubiquitin-mediated proteolysis pathway has come a long way in the past decade. At first thought to be an unglamorous garbage dump for damaged proteins, the ubiquitin pathway has been shown to regulate virtually everything that occurs in the cell. Deubiquitinating enzymes, which cleave ubiquitin-protein bonds, are the largest group of enzymes in the pathway, yet they are the least well understood. Deubiquitinating enzymes have two kinds of functions: housekeeping and regulatory. The housekeeping enzymes facilitate the proteolytic pathway. By contrast, the regulatory enzymes control the ubiquitination of specific protein substrates; their relationship to ubiquitination is analgous to that of phosphatases with respect to phosphorylation. Here, I review the current state of knowledge of the deubiquitinating enzymes. I focus particularly on the known regulatory enzymes, and also on the housekeeping enzymes that are implicated in development of disease.