The regulation of cell size and branch complexity in the terminal cells of the Drosophila tracheal system.

The terminal cells of the larval Drosophila tracheal system extend dozens of branched cellular processes, most of which become hollow intracellular tubes that support gas exchange with internal tissues. Previously, we undertook a forward genetic mosaic screen to uncover the pathways regulating terminal cell size, morphogenesis, and the generation and maintenance of new intracellular tubes. Our initial work identified several mutations affecting terminal cell size and branch number, and suggested that branch complexity and cell size are typically coupled but could be genetically separated. To deepen our understanding of these processes, we have further characterized and determined the molecular identities of mutations in the genes sprout, denuded and asthmatic, that had been implicated in our initial screen. Here we reveal the molecular identity of these genes and describe their function in the context of the TOR and Hippo pathways, which are widely appreciated to be key regulators of cell and organ size.

GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function.

Microsatellite expansions are the leading cause of numerous neurodegenerative disorders. Here we demonstrate that GGGGCC and CAG microsatellite repeat RNAs associated with C9orf72 in amyotrophic lateral sclerosis/frontotemporal dementia and with polyglutamine diseases, respectively, localize to neuritic granules that undergo active transport into distal neuritic segments. In cultured mammalian spinal cord neurons, the presence of neuritic GGGGCC repeat RNA correlates with neuronal branching defects, and the repeat RNA localizes to granules that label with fragile X mental retardation protein (FMRP), a transport granule component. Using a Drosophila GGGGCC expansion disease model, we characterize dendritic branching defects that are modulated by FMRP and Orb2. The human orthologs of these modifiers are misregulated in induced pluripotent stem cell-differentiated neurons (iPSNs) from GGGGCC expansion carriers. These data suggest that expanded repeat RNAs interact with the messenger RNA transport and translation machinery, causing transport granule dysfunction. This could be a novel mechanism contributing to the neuronal defects associated with C9orf72 and other microsatellite expansion diseases.

Rab and Arl GTPase family members cooperate in the localization of the golgin GCC185.

GCC185 is a large coiled-coil protein at the trans Golgi network that is required for receipt of transport vesicles inbound from late endosomes and for anchoring noncentrosomal microtubules that emanate from the Golgi. Here, we demonstrate that recruitment of GCC185 to the Golgi is mediated by two Golgi-localized small GTPases of the Rab and Arl families. GCC185 binds Rab6, and mutation of residues needed for Rab binding abolishes Golgi localization. The crystal structure of Rab6 bound to the GCC185 Rab-binding domain reveals that Rab6 recognizes a two-fold symmetric surface on a coiled coil immediately adjacent to a C-terminal GRIP domain. Unexpectedly, Rab6 binding promotes association of Arl1 with the GRIP domain. We present a structure-derived model for dual GTPase membrane attachment that highlights the potential ability of Rab GTPases to reach binding partners at a significant distance from the membrane via their unstructured and membrane-anchored, hypervariable domains.

A functional role for the GCC185 golgin in mannose 6-phosphate receptor recycling.

Mannose 6-phosphate receptors (MPRs) deliver newly synthesized lysosomal enzymes to endosomes and then recycle to the Golgi. MPR recycling requires Rab9 GTPase; Rab9 recruits the cytosolic adaptor TIP47 and enhances its ability to bind to MPR cytoplasmic domains during transport vesicle formation. Rab9-bearing vesicles then fuse with the trans-Golgi network (TGN) in living cells, but nothing is known about how these vesicles identify and dock with their target. We show here that GCC185, a member of the Golgin family of putative tethering proteins, is a Rab9 effector that is required for MPR recycling from endosomes to the TGN in living cells, and in vitro. GCC185 does not rely on Rab9 for its TGN localization; depletion of GCC185 slightly alters the Golgi ribbon but does not interfere with Golgi function. Loss of GCC185 triggers enhanced degradation of mannose 6-phosphate receptors and enhanced secretion of hexosaminidase. These data assign a specific pathway to an interesting, TGN-localized protein and suggest that GCC185 may participate in the docking of late endosome-derived, Rab9-bearing transport vesicles at the TGN.

Misincorporation proton-alkyl exchange (MPAX): engineering cysteine probes into proteins.

This unit describes a rapid and efficient method to screen a polypeptide for amino acid residues that contribute to protein-protein interaction interfaces. Cysteine residues are introduced as positional probes in a protein at random by co-expression in bacteria with specific cysteine misincorporator tRNAs. The protein is then purified as an ensemble of polypeptides containing cysteine at low frequency, at different positions in each molecule. The ability of the native protein structure to protect different cysteine residues from chemical modification by iodoacetamide is determined to obtain a protein surface map that reveals candidate surface residues that are likely to be important for protein-protein interaction. Cysteine mutants with altered ligand binding can also be selected simultaneously by affinity chromatography.

Purification and analysis of TIP47 function in Rab9-dependent mannose 6-phosphate receptor trafficking.

TIP47 (tail interacting protein of 47 kDa) is a cytosolic protein that is essential for the transport of mannose 6-phosphate receptors (MPRs) from endosomes to the trans-Golgi. This protein is recruited from the cytosol onto the surface of late endosomes by Rab9 GTPase, which enables TIP47 to bind to MPR cytoplasmic domains with enhanced affinity. A mutation in a deep hydrophobic cleft of TIP47 (F(236)C) confers enhanced affinity binding to MPR cytoplasmic domains and stabilizes MPRs in living cells. We describe the purification of native and recombinant TIP47 proteins and assays that we use to monitor the function of this protein in MPR transport in living cells.

In vitro selection and prediction of TIP47 protein-interaction interfaces.

We present a new method for the rapid identification of amino acid residues that contribute to protein-protein interfaces. Tail-interacting protein of 47 kDa (TIP47) binds Rab9 GTPase and the cytoplasmic domains of mannose 6-phosphate receptors and is required for their transport from endosomes to the Golgi apparatus. Cysteine mutations were incorporated randomly into TIP47 by expression in Escherichia coli cells harboring specific misincorporator tRNAs. We made use of the ability of the native TIP47 protein to protect 48 cysteine probes from chemical modification by iodoacetamide as a means to obtain a surface map of TIP47, revealing the identity of surface-localized, hydrophobic residues that are likely to participate in protein-protein interactions. Direct mutation of predicted interface residues confirmed that the protein had altered binding affinity for the mannose 6-phosphate receptor. TIP47 mutants with enhanced or diminished affinities were also selected by affinity chromatography. These methods were validated in comparison with the protein's crystal structure, and provide a powerful means to predict protein-protein interaction interfaces.

Population depletion activates autonomous CD154-dependent survival in biopsylike Burkitt lymphoma cells.

Population size is governed through cells reacting to a variety of intrinsic and extrinsic cues. Tumors, while liberated from many of the homeostatic constraints placed on physiologic counterparts, can nonetheless remain subject to both social and environmental control. Burkitt lymphoma cells faithful to the biopsy phenotype were used to model the reliance of the colony, if any, on an inbuilt population sensor. Below a normally suicidal threshold number of cells, low picomolar quantities of exogenous CD40 ligand (CD40L/CD154) were found to sustain the clone without the discernible shift in phenotype that accompanies high CD40L encounter. Although CD154 was undetectable in populous cultures, message was induced as numbers became limiting. Correspondingly, attempts to neutralize endogenous CD40L activity failed to perturb cells at optimal densities but resulted in their marked decline as the critical threshold was approached. These data reveal an auto-inducible survival mechanism seemingly regulated through the monitoring of population size, a process somewhat akin to that of "quorum sensing" among gram-negative bacteria in which diffusible molecules provide a means of communication to coordinate gene expression with population density. This process could be activated as cells discern depletions in their community or when deprived of signals otherwise furnished within an appropriate environmental niche.

The myxoid liposarcoma specific TLS-CHOP fusion protein localizes to nuclear structures distinct from PML nuclear bodies.

CHOP in 12q13, also called GADD153 or DDIT3, encodes a transcription factor of the C/EBP type. As a result of t(12;16) translocations, CHOP is rearranged and fused to TLS in 16p11 in about 90% of myxoid liposarcomas/round cell liposarcomas (MLS/RCLS). The TLS-CHOP protein consists of the N-terminal half of TLS juxtaposed to the N-terminal of the entire CHOP. It is capable of forming dimers with the natural dimer partners of CHOP. Here we report that recombinant TLS-CHOP-green fluorescence protein localizes to nuclear structures, similar to, but distinct from, PML nuclear bodies. The TLS-CHOP-green fluorescent protein nuclear structures are resistant to high salt concentration and nuclease treatment. Transfection of TLS-CHOP to normal fibroblasts causes a rapid down regulation and relocation of PML nuclear bodies. An abnormal extra nuclear localization of PML bodies was also found in TLS-CHOP carrying cell lines established from myxoid liposarcomas. Transfection of TLS-CHOP induced a rapid disappearance of PCNA. TLS-CHOP may disturb the nuclear machinery by binding and sequestering important factors from their natural sites.