James E. Wilhelm

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600–900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz

In Drosophila oocytes, precise localization of the posterior determinant, Oskar, is required for posterior patterning. This precision is accomplished by a localization-dependent translational control mechanism that ensures translation of only correctly localized oskar transcripts. Although progress has been made in identifying localization factors and translational repressors of oskar, none of the known components of the oskar complex is required for both processes. Here, we report the identification of Cup as a novel component of the oskar RNP complex. cup is required for oskar mRNA localization and is necessary to recruit the plus end–directed microtubule transport factor Barentsz to the complex. Surprisingly, Cup is also required to repress the translation of oskar. Furthermore, eukaryotic initiation factor 4E (eIF4E) is localized within the oocyte in a cup-dependent manner and binds directly to Cup in vitro. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. We propose that Cup coordinates localization with translation.

Mouse oocytes within germ cell cysts and primordial follicles contain a Balbiani body

The Balbiani body or mitochondrial cloud is a large distinctive organelle aggregate found in developing oocytes of many species, but its presence in the mouse has been controversial. Using confocal and electron microscopy, we report that a Balbiani body does arise in mouse neonatal germline cysts and oocytes of primordial follicles but disperses as follicles begin to grow. The mouse Balbiani body contains a core of Golgi elements surrounded by mitochondria and associated endoplasmic reticulum. Because of their stage specificity and perinuclear rather than spherical distribution, these clustered Balbiani body mitochondria may have been missed previously. The Balbiani body also contains Trailer hitch, a widely conserved member of a protein complex that associates with endoplasmic reticulum/Golgi-like vesicles and transports specific RNAs during Drosophila oogenesis. Our results provide evidence that mouse oocytes develop using molecular and developmental mechanisms widely conserved throughout the animal kingdom.

Identification of novel filament-forming proteins in Saccharomyces cerevisiae and Drosophila melanogaster

A screen for GFP-tagged yeast proteins that can assemble into visible structures reveals four new filamentous structures in the cytoplasm formed by metabolic enzymes and translation factors.

Mechanisms of translational regulation in Drosophila

Translational regulation plays an essential role in many phases of the Drosophila life cycle. During embryogenesis, specification of the developing body pattern requires co‐ordination of the translation of oskar, gurken and nanos mRNAs with their subcellular localization. In addition, dosage compensation is controlled by Sex‐lethal‐mediated translational regulation while dFMR1 (the Drosophila homologue of the fragile X mental retardation protein) controls translation of various mRNAs which function in the nervous system. Here we describe some of the mechanisms that are utilized to regulate these various processes. Our review highlights the complexity that can be involved with multiple factors employing different mechanisms to control the translation of a single mRNA.

A functional genomic screen in planarians identifies novel regulators of germ cell development

Germ cells serve as intriguing examples of differentiated cells that retain the capacity to generate all cell types of an organism. Here we used functional genomic approaches in planarians to identify genes required for proper germ cell development. We conducted microarray analyses and in situ hybridization to discover and validate germ cell-enriched transcripts, and then used RNAi to screen for genes required for discrete stages of germ cell development. The majority of genes we identified encode conserved RNA-binding proteins, several of which have not been implicated previously in germ cell development. We also show that a germ cell-specific subunit of the conserved transcription factor CCAAT-binding protein/nuclear factor-Y is required for maintaining spermatogonial stem cells. Our results demonstrate that conserved transcriptional and post-transcriptional mechanisms regulate germ cell development in planarians. These findings suggest that studies of planarians will inform our understanding of germ cell biology in higher organisms.

The Structure of the Lingo-1 Ectodomain, a Module Implicated in Central Nervous System Repair Inhibition

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200Å2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.

Common regulatory control of CTP synthase enzyme activity and filament formation

The ability of enzymes to assemble into visible supramolecular complexes is a widespread phenomenon. Such complexes have been hypothesized to play a number of roles; however, little is known about how the regulation of enzyme activity is coupled to the assembly/disassembly of these cellular structures. CTP synthase is an ideal model system for addressing this question because its activity is regulated via multiple mechanisms and its filament-forming ability is evolutionarily conserved. Our structure-function studies of CTP synthase in Saccharomyces cerevisiae reveal that destabilization of the active tetrameric form of the enzyme increases filament formation, suggesting that the filaments comprise inactive CTP synthase dimers. Furthermore, the sites responsible for feedback inhibition and allosteric activation control filament length, implying that multiple regions of the enzyme can influence filament structure. In contrast, blocking catalysis without disrupting the regulatory sites of the enzyme does not affect filament formation or length. Together our results argue that the regulatory sites that control CTP synthase function, but not enzymatic activity per se, are critical for controlling filament assembly. We predict that the ability of enzymes to form supramolecular structures in general is closely coupled to the mechanisms that regulate their activity.

Kinesin-73 is a processive motor that localizes to Rab5-containing organelles

Drosophila Kinesin-73 (Khc-73), which plays a role in mitotic spindle polarity in neuroblasts, is a metazoan-specific member of the Kinesin-3 family of motors, which includes mammalian KIF1A and Caenorhabditis elegans Unc-104. The mechanism of Kinesin-3 motors has been controversial because some studies have reported that they transport cargo as monomers whereas other studies have suggested a dimer mechanism. Here, we have performed single-molecule motility and cell biological studies of Khc-73. We find that constructs containing the motor and the conserved short stretches of putative coiled-coil-forming regions are predominantly monomeric in vitro, but that dimerization allows for fast, processive movement and high force production (7 piconewtons). In Drosophila cell lines, we present evidence that Khc-73 can dimerize in vivo. We also show that Khc-73 is recruited specifically to Rab5-containing endosomes through its “tail” domain. Our results suggest that the N-terminal half of Khc-73 can undergo a monomer-dimer transition to produce a fast processive motor and that its C-terminal half possesses a specific Rab5-vesicle binding domain.

A one-hybrid system for detecting RNA-protein interactions.

Background : mRNA translation, stability, and localization are controlled by regulatory proteins that bind to specific RNA motifs. Since biochemical isolation of such proteins has often proven to be difficult, a genetic system for studying RNA–protein interactions would be of great utility in the identification of novel RNA binding proteins and in understanding how these proteins recognize particular RNA sequences.  The bacteriophage lambda gene product N protein is a sequence specific RNA binding protein that when bound to its target sequence allows RNA polymerase to ignore transcription termination signals. The fact that the binding of N protein to RNA is directly coupled to gene expression suggests that N protein could be used to develop a general system for studying RNA–protein interactions.