A defined tubby domain β-barrel surface region of TULP3 mediates ciliary trafficking of diverse cargoes.

The primary cilium is a paradigmatic subcellular compartment at the nexus of numerous cellular and morphogenetic pathways. The tubby family protein TULP3 acts as an adapter of the intraflagellar transport complex A in transporting integral membrane and membrane-associated lipidated proteins into cilia. However, the mechanisms by which TULP3 coordinates ciliary transport of diverse cargoes is not well understood.

Nitric oxide delivery and heme-assisted S-nitrosation by the bedbug nitrophorin.

Nitrophorins are heme proteins used by blood feeding insects to deliver nitric oxide (NO) to a victim, leading to vasodilation and antiplatelet activity. Cimex lectularius (bedbug) nitrophorin (cNP) accomplishes this with a cysteine ligated ferric (Fe(III)) heme. In the acidic environment of the insect's salivary glands, NO binds tightly to cNP.

Ankmy2 Prevents Smoothened-Independent Hyperactivation of the Hedgehog Pathway via Cilia-Regulated Adenylyl Cyclase Signaling.

The mechanisms underlying subcellular targeting of cAMP-generating adenylyl cyclases and processes regulated by their compartmentalization are poorly understood. Here, we identify Ankmy2 as a repressor of the Hedgehog pathway via adenylyl cyclase targeting. Ankmy2 binds to multiple adenylyl cyclases, determining their maturation and trafficking to primary cilia.

Tulp3 Regulates Renal Cystogenesis by Trafficking of Cystoproteins to Cilia.

Polycystic kidney disease proteins, polycystin-1 and polycystin-2, localize to primary cilia. Polycystin knockouts have severe cystogenesis compared to ciliary disruption, whereas simultaneous ciliary loss suppresses excessive cyst growth. These data suggest the presence of a cystogenic activator that is inhibited by polycystins and an independent but relatively minor cystogenic inhibitor, either of which are cilia dependent.

Using Primary Neurosphere Cultures to Study Primary Cilia.

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function.

Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins.

The primary cilium is a paradigmatic organelle for studying compartmentalized signaling; however, unlike soluble protein trafficking, processes targeting integral membrane proteins to cilia are poorly understood. In this study, we determine that the tubby family protein TULP3 functions as a general adapter for ciliary trafficking of structurally diverse integral membrane cargo, including multiple reported and novel rhodopsin family G protein-coupled receptors (GPCRs) and the polycystic kidney disease-causing polycystin 1/2 complex. The founding tubby family member TUB also localizes to cilia similar to TULP3 and determines trafficking of a subset of these GPCRs to neuronal cilia.

Trafficking to the primary cilium membrane.

The primary cilium has been found to be associated with a number of cellular signaling pathways, such as vertebrate hedgehog signaling, and implicated in the pathogenesis of diseases affecting multiple organs, including the neural tube, kidney, and brain. The primary cilium is the site where a subset of the cell's membrane proteins is enriched. However, pathways that target and concentrate membrane proteins in cilia are not well understood.

Smoothened determines β-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium.

Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein-coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia.

A Mechanical Switch Couples T Cell Receptor Triggering to the Cytoplasmic Juxtamembrane Regions of CD3ζζ.

The eight-subunit T cell receptor (TCR)-CD3 complex is the primary determinant for T cell fate decisions. Yet how it relays ligand-specific information across the cell membrane for conversion to chemical signals remains unresolved. We hypothesized that TCR engagement triggers a change in the spatial relationship between the associated CD3ζζ subunits at the junction where they emerge from the membrane into the cytoplasm.

Studying G protein-coupled receptors: immunoblotting, immunoprecipitation, phosphorylation, surface labeling, and cross-linking protocols.

Primary cilia are signaling organelles that have been shown to coordinate cellular responses to extracellular cues during physiological processes ranging from organ patterning to cell cycle regulation. A variety of receptors, including G protein-coupled receptors (GPCRs), downstream effectors (adenylyl cyclases), and second messengers, such as calcium, accumulate in the ciliary compartment. Isolation of GPCRs is essential for studying posttranslational modifications, intracellular trafficking, and protein-protein interactions that are important in downstream signaling.

Increased Akt signaling in the mosquito fat body increases adult survivorship.

Akt signaling regulates diverse physiologies in a wide range of organisms. We examine the impact of increased Akt signaling in the fat body of 2 mosquito species, the Asian malaria mosquito Anopheles stephensi and the yellow fever mosquito Aedes aegypti. Overexpression of a myristoylated and active form of A.

Piecing together the family portrait of TCR-CD3 complexes.

The pre-T-cell receptor (TCR)-, αβTCR-, and γδTCR-CD3 complexes are members of a family of modular biosensors that are responsible for driving T-cell development, activation, and effector functions. They inform essential checkpoint decisions by relaying key information from their ligand-binding modules (TCRs) to their signaling modules (CD3γε + CD3δε and CD3ζζ) and on to the intracellular signaling apparatus. Their actions shape the T-cell repertoire, as well as T-cell-mediated immunity; yet, the mechanisms that underlie their activity remain an enigma.

Defects in coatomer protein I (COPI) transport cause blood feeding-induced mortality in Yellow Fever mosquitoes.

Blood feeding by vector mosquitoes provides the entry point for disease pathogens and presents an acute metabolic challenge that must be overcome to complete the gonotrophic cycle. Based on recent data showing that coatomer protein I (COPI) vesicle transport is involved in cellular processes beyond Golgi-endoplasmic reticulum retrograde protein trafficking, we disrupted COPI functions in the Yellow Fever mosquito Aedes aegypti to interfere with blood meal digestion. Surprisingly, we found that decreased expression of the γCOPI coatomer protein led to 89% mortality in blood-fed mosquitoes by 72 h postfeeding compared with 0% mortality in control dsRNA-injected blood-fed mosquitoes and 3% mortality in γCOPI dsRNA-injected sugar-fed mosquitoes.