The vertebrate primary cilium in development, homeostasis, and disease.

Cilia are complex structures that have garnered interest because of their roles in vertebrate development and their involvement in human genetic disorders. In contrast to multicellular invertebrates in which cilia are restricted to specific cell types, these organelles are found almost ubiquitously in vertebrate cells, where they serve a diverse set of signaling functions. Here, we highlight properties of vertebrate cilia, with particular emphasis on their relationship with other subcellular structures, and explore the physiological consequences of ciliary dysfunction.

Metabolic regulation through the endosomal system.

The endosomal system plays an essential role in cell homeostasis by controlling cellular signaling, nutrient sensing, cell polarity and cell migration. However, its place in the regulation of tissue, organ and whole body physiology is less well understood. Recent studies have revealed an important role for the endosomal system in regulating glucose and lipid homeostasis, with implications for metabolic disorders such as type 2 diabetes, hypercholesterolemia and non-alcoholic fatty liver disease. By taking insights from in vitro studies of endocytosis and exploring their effects on metabolism, we can begin to connect the fields of endosomal transport and metabolic homeostasis. In this review, we explore current understanding of how the endosomal system influences the systemic regulation of glucose and lipid metabolism in mice and humans. We highlight exciting new insights that help translate findings from single cells to a wider physiological level and open up new directions for endosomal research.

Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet-Biedl syndrome.

Technological advances hold the promise of rapidly catalyzing the discovery of pathogenic variants for genetic disease. However, this possibility is tempered by limitations in interpreting the functional consequences of genetic variation at candidate loci. Here, we present a systematic approach, grounded on physiologically relevant assays, to evaluate the mutational content (125 alleles) of the 14 genes associated with Bardet-Biedl syndrome (BBS). A combination of in vivo assays with subsequent in vitro validation suggests that a significant fraction of BBS-associated mutations have a dominant-negative mode of action. Moreover, we find that a subset of common alleles, previously considered to be benign, are, in fact, detrimental to protein function and can interact with strong rare alleles to modulate disease presentation. These data represent a comprehensive evaluation of genetic load in a multilocus disease. Importantly, superimposition of these results to human genetics data suggests a previously underappreciated complexity in disease architecture that might be shared among diverse clinical phenotypes.

Adipocyte-derived extracellular vesicles increase insulin secretion through transport of insulinotropic protein cargo.

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic ß-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic ß-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand.

Bardet-Biedl syndrome-associated small GTPase ARL6 (BBS3) functions at or near the ciliary gate and modulates Wnt signaling.

The expansive family of metazoan ADP-ribosylation factor and ADP-ribosylation factor-like small GTPases is known to play essential roles in modulating membrane trafficking and cytoskeletal functions. Here, we present the crystal structure of ARL6, mutations in which cause Bardet-Biedl syndrome (BBS3), and reveal its unique ring-like localization at the distal end of basal bodies, in proximity to the so-called ciliary gate where vesicles carrying ciliary cargo fuse with the membrane. Overproduction of GDP- or GTP-locked variants of ARL6/BBS3 in vivo influences primary cilium length and abundance. ARL6/BBS3 also modulates Wnt signaling, a signal transduction pathway whose association with cilia in vertebrates is just emerging. Importantly, this signaling function is lost in ARL6 variants containing BBS-associated point mutations. By determining the structure of GTP-bound ARL6/BBS3, coupled with functional assays, we provide a mechanistic explanation for such pathogenic alterations, namely altered nucleotide binding. Our findings therefore establish a previously unknown role for ARL6/BBS3 in mammalian ciliary (dis)assembly and Wnt signaling and provide the first structural information for a BBS protein.

Glucose homeostasis is regulated by pancreatic ß-cell cilia via endosomal EphA-processing.

Diabetes mellitus affects one in eleven adults worldwide. Most suffer from Type 2 Diabetes which features elevated blood glucose levels and an inability to adequately secrete or respond to insulin. Insulin producing ß-cells have primary cilia which are implicated in the regulation of glucose metabolism, insulin signaling and secretion. To better understand how ß-cell cilia affect glucose handling, we ablate cilia from mature ß-cells by deleting key cilia component Ift88. Here we report that glucose homeostasis and insulin secretion deteriorate over 12 weeks post-induction. Cilia/basal body components are required to suppress spontaneous auto-activation of EphA3 and hyper-phosphorylation of EphA receptors inhibits insulin secretion. In ß-cells, loss of cilia/basal body function leads to polarity defects and epithelial-to-mesenchymal transition. Defective insulin secretion from IFT88-depleted human islets and elevated pEPHA3 in islets from diabetic donors both point to a role for cilia/basal body proteins in human glucose homeostasis.

The role of primary cilia in obesity and diabetes.

One in 12 people worldwide suffers from diabetes and more than 90% of affected adult individuals are diagnosed with type 2 diabetes mellitus (T2DM). Obesity adds to the personal risk to develop T2DM, and both metabolic diseases are rampantly increasing worldwide. Over recent years, primary cilia have moved into the focus of basic and clinical research, after several human diseases have been identified as ciliopathies (i.e., they are linked to ciliary dysfunction). A subset of ciliopathies presents with obesity and diabetes, either as core symptoms or major complications. Several studies have shown a role for ciliary signaling in the satiety signaling centers of the hypothalamus and in other metabolically active tissues, such as pancreatic islets. Here, we discuss recent advances and perspectives in ciliary metabolic research.

Short and prolonged exposure to hyperglycaemia in human fibroblasts and endothelial cells: metabolic and osmotic effects.

High blood glucose levels are the main feature of diabetes. However, the underlying mechanism linking high glucose concentration to diabetic complications is still not fully elucidated, particularly with regard to human physiology. Excess of glucose is likely to trigger a metabolic response depending on the cell features, activating deleterious pathways involved in the complications of diabetes. In this study, we aim to elucidate how acute and prolonged hyperglycaemia alters the biology and metabolism in human fibroblasts and endothelial cells. We found that hyperglycaemia triggers a metabolic switch from oxidative phosphorylation to glycolysis that is maintained over prolonged time. Moreover, osmotic pressure is a major factor in the early metabolic response, decreasing both mitochondrial transmembrane potential and cellular proliferation. After prolonged exposure to hyperglycaemia we observed decreased mitochondrial steady-state and uncoupled respiration, together with a reduced ATP/ADP ratio. At the same time, we could not detect major changes in mitochondrial transmembrane potential and reactive oxygen species. We suggest that the physiological and metabolic alterations observed in healthy human primary fibroblasts and endothelial cells are an adaptive response to hyperglycaemia. The severity of metabolic and bioenergetics impairment associated with diabetic complications may occur after longer glucose exposure or due to interactions with cell types more sensitive to hyperglycaemia.

Ciliary dysfunction impairs beta-cell insulin secretion and promotes development of type 2 diabetes in rodents.

Type 2 diabetes mellitus is affecting more than 382 million people worldwide. Although much progress has been made, a comprehensive understanding of the underlying disease mechanism is still lacking. Here we report a role for the ß-cell primary cilium in type 2 diabetes susceptibility. We find impaired glucose handling in young Bbs4(-/-) mice before the onset of obesity. Basal body/ciliary perturbation in murine pancreatic islets leads to impaired first phase insulin release ex and in vivo. Insulin receptor is recruited to the cilium of stimulated ß-cells and ciliary/basal body integrity is required for activation of downstream targets of insulin signalling. We also observe a reduction in the number of ciliated ß-cells along with misregulated ciliary/basal body gene expression in pancreatic islets in a diabetic rat model. We suggest that ciliary function is implicated in insulin secretion and insulin signalling in the ß-cell and that ciliary dysfunction could contribute to type 2 diabetes susceptibility.

Ciliary function and Wnt signal modulation.

With the increase in complexity of morphogenetic signaling cascades over the course of evolution and the emergence of broadly ciliated organisms, the cilium seems to have acquired a role as regulator of paracrine signal transduction. Recently, several lines of evidence have provided a link between basal body and ciliary proteins and Wnt signaling. In this chapter, we will evaluate the evidence linking the basal body and cilium with the regulation of beta-catenin-dependent (canonical) and beta-catenin-independent (noncanonical) signaling processes as well as which role(s) Wnt signaling might play in ciliogenesis. In addition, we will discuss aberrant Wnt signaling could contribute to phenotypes common to most ciliopathies and why these phenotypes might be driven by loss of noncanonical rather than gain of noncanonical Wnt signaling.

Disruption of the basal body compromises proteasomal function and perturbs intracellular Wnt response.

Primary cilia and basal bodies are evolutionarily conserved organelles that mediate communication between the intracellular and extracellular environments. Here we show that bbs1, bbs4 and mkks (also known as bbs6), which encode basal body proteins, are required for convergence and extension in zebrafish and interact with wnt11 and wnt5b. Suppression of bbs1, bbs4 and mkks transcripts results in stabilization of beta-catenin with concomitant upregulation of T-cell factor (TCF)-dependent transcription in both zebrafish embryos and mammalian ciliated cells, a defect phenocopied by the silencing of the axonemal kinesin subunit KIF3A but not by chemical disruption of the cytoplasmic microtubule network. These observations are attributable partly to defective degradation by the proteasome; suppression of BBS4 leads to perturbed proteasomal targeting and concomitant accumulation of cytoplasmic beta-catenin. Cumulatively, our data indicate that the basal body is an important regulator of Wnt signal interpretation through selective proteolysis and suggest that defects in this system may contribute to phenotypes pathognomonic of human ciliopathies.

Small molecule intervention in microtubule-associated human disease.

Microtubules are essential for a number of cellular processes that include the transport of intracellular cargo or organelles across long distances and the assembly of the mitotic spindle. The identification of numerous microtubule-associated proteins and the progressive elucidation of the mechanisms of microtubule assembly and transport are beginning to have a profound impact on the study and treatment of human genetic disease. A number of seemingly unrelated phenotypes have now been linked to microtubular dysfunction, especially in systems dependent heavily on microtubule-based transport, such as neurons and ciliated cells. In parallel, the association of microtubule transport defects with human genetic disease has led to the realization that targeting various aspects of microtubular biology with small molecules might offer new therapeutic paradigms, including the development of new therapeutic utility for seemingly old drugs. In this review, we discuss the use of small molecules in the investigation of microtubule-associated processes and particularly the screens of chemical compound libraries for the identification of lead compounds with potential utility in microtubule-associated disease processes.

Direct mass spectrometric monitoring of solid phase organic syntheses.

Direct on-bead monitoring of solid-phase reactions is possible with soft laser desorption time-of-flight mass spectrometry (SLD-TOF MS) without prior cleavage from the resin if photocleavable phenacyl ester or o-nitroveratryl linker groups are employed.

Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene.

Cilia and flagella are microtubule-based structures nucleated by modified centrioles termed basal bodies. These biochemically complex organelles have more than 250 and 150 polypeptides, respectively. To identify the proteins involved in ciliary and basal body biogenesis and function, we undertook a comparative genomics approach that subtracted the nonflagellated proteome of Arabidopsis from the shared proteome of the ciliated/flagellated organisms Chlamydomonas and human. We identified 688 genes that are present exclusively in organisms with flagella and basal bodies and validated these data through a series of in silico, in vitro, and in vivo studies. We then applied this resource to the study of human ciliation disorders and have identified BBS5, a novel gene for Bardet-Biedl syndrome. We show that this novel protein localizes to basal bodies in mouse and C. elegans, is under the regulatory control of daf-19, and is necessary for the generation of both cilia and flagella.