Coding variants identified in patients with diabetes alter PICK1 BAR domain function in insulin granule biogenesis.

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that mediate curvature of negatively charged lipid membranes during remodeling processes. The BAR domain proteins PICK1, ICA69, and the arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify 4 coding variants in the PICK1 gene from a whole-exome screening of Danish patients with diabetes that each involve a change in positively charged residues in the PICK1 BAR domain. All 4 coding variants failed to rescue insulin content in INS-1E cells upon knock down of endogenous PICK1. Moreover, 2 variants showed dominant-negative properties. In vitro assays addressing BAR domain function suggested that the coding variants compromised BAR domain function but increased the capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists in or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin, from ISGs during the maturation process; and that the coding variants may cause premature budding, possibly explaining their dominant-negative function.

Identifying dominant-negative actions of a dopamine transporter variant in patients with parkinsonism and neuropsychiatric disease.

Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels, but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here, we use clinical information, neuroimaging, and large-scale exome-sequencing data to uncover the occurrence and phenotypic spectrum of a DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PSD-95/Discs-large/ZO-1 homology-binding motif of human DAT (hDAT). We identified the rare but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset neurodegenerative parkinsonism and comorbid neuropsychiatric disease. In cell cultures, hDAT-K619N displayed reduced uptake capacity, decreased surface expression, and accelerated turnover. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDAT-WT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila led to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.

An Amphipathic Helix Directs Cellular Membrane Curvature Sensing and Function of the BAR Domain Protein PICK1.

BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila, disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis.

Mactosylceramide prevents glial cell overgrowth by inhibiting insulin and fibroblast growth factor receptor signaling.

Receptor tyrosine kinase (RTK) signaling controls key aspects of cellular differentiation, proliferation, survival, metabolism, and migration. Deregulated RTK signaling also underlies many cancers. Glycosphingolipids (GSL) are essential elements of the plasma membrane. By affecting clustering and activity of membrane receptors, GSL modulate signal transduction, including that mediated by the RTK. GSL are abundant in the nervous system, and glial development in Drosophila is emerging as a useful model for studying how GSL modulate RTK signaling. Drosophila has a simple GSL biosynthetic pathway, in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3-kinase (PI3K). However, to what extent this effect involves changes in upstream signaling events is unresolved. We show here that glial overgrowth in egh is strongly linked to increased activation of Insulin and fibroblast growth factor receptors (FGFR). Glial hypertrophy is phenocopied when overexpressing gain-of-function mutants of the Drosophila insulin receptor (InR) and the FGFR homolog Heartless (Htl) in wild type SPG, and is suppressed by inhibiting Htl and InR activity in egh. Knockdown of GlcCer synthase in the SPG fails to suppress glial overgrowth in egh nerves, and slightly promotes overgrowth in wild type, suggesting that RTK hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. We conclude that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of insulin and fibroblast growth factor receptors in Drosophila glia.

PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance.

Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine cells and support an important role of PICK1/ICA69 in maintenance of metabolic homeostasis.

Regulation of Toll and Toll-like receptor signaling by the endocytic pathway.

The Toll/TLR receptor family plays a central role in both vertebrate and insect immunity, driving the activation of humoral immunity in response to pathogens. In Drosophila, Toll is also responsible for directing the formation of the Dorsal/NFkappaB gradient specifying dorsoventral patterning of the embryo. Two recent studies have revealed that endocytosis and elements of the molecular machinery governing endosomal progression are required for Drosophila Toll signaling in development and immunity. We demonstrated that Toll is not only present at the plasma membrane but also in a Rab5(+) early endosomal compartment in the embryo and that the distribution of constitutively active Toll(10B) is shifted towards endosomes. Localized inhibition of Rab5 function on the ventral side leads to a reduction of nuclear Dorsal levels, while locally increasing Rab5 function leads to potentiation of signaling. Independently, another laboratory identified the endosomal protein Mop as a potentiator of Toll signaling in Drosophila cell culture and fat-body tissue. Mop functions together with the ESCRT 0 component, Hrs, previously reported to stimulate endosomal progression and the signaling ability of internalized EGFR. We discuss these studies and briefly summarize the most significant findings concerning the role of intracellular localization and trafficking in mammalian TLR function.

Endocytosis is required for Toll signaling and shaping of the Dorsal/NF-kappaB morphogen gradient during Drosophila embryogenesis.

Dorsoventral cell fate in the Drosophila embryo is specified by activation of the Toll receptor, leading to a ventral-to-dorsal gradient across nuclei of the NF-κB transcription factor Dorsal. Toll receptor has been investigated genetically, molecularly, and immunohistologically, but much less is known about its dynamics in living embryos. Using live imaging of fluorescent protein chimeras, we find that Toll is recruited from the plasma membrane to Rab5(+) early endosomes. The distribution of a constitutively active form of Toll, Toll(10b), is shifted from the plasma membrane to early endosomes. Inhibition of endocytosis on the ventral side of the embryo attenuates Toll signaling ventrally and causes Dorsal to accumulate on the dorsal side of the embryo, essentially inverting the dorsal/ventral axis. Conversely, enhancing endocytosis laterally greatly potentiates Toll signaling locally, altering the shape of the Dorsal gradient. Photoactivation and fluorescence recovery after photobleaching studies reveal that Toll exhibits extremely limited lateral diffusion within the plasma membrane, whereas Toll is highly compartmentalized in endosomes. When endocytosis is blocked ventrally, creating an ectopic dorsal signaling center, Toll is preferentially endocytosed at the ectopic signaling center. We propose that Toll signals from an endocytic compartment rather than the plasma membrane. Our studies reveal that endocytosis plays a pivotal role in the spatial regulation of Toll receptor activation and signaling and in the correct shaping of the nuclear Dorsal concentration gradient.

Dynamics of the Dorsal morphogen gradient.

The dorsoventral (DV) patterning of the Drosophila embryo depends on the nuclear localization gradient of Dorsal (Dl), a protein related to the mammalian NF-kappaB transcription factors. Current understanding of how the Dl gradient works has been derived from studies of its transcriptional interpretation, but the gradient itself has not been quantified. In particular, it is not known whether the Dl gradient is stable or dynamic during the DV patterning of the embryo. To address this question, we developed a mathematical model of the Dl gradient and constrained its parameters by experimental data. Based on our computational analysis, we predict that the Dl gradient is dynamic and, to a first approximation, can be described as a concentration profile with increasing amplitude and constant shape. These time-dependent properties of the Dl gradient are different from those of the Bicoid and MAPK phosphorylation gradients, which pattern the anterior and terminal regions of the embryo. Specifically, the gradient of the nuclear levels of Bicoid is stable, whereas the pattern of MAPK phosphorylation changes in both shape and amplitude. We attribute these striking differences in the dynamics of maternal morphogen gradients to the differences in the initial conditions and chemistries of the anterior, DV, and terminal systems.