Cyclin-G-associated kinase GAK/dAux regulates autophagy initiation via ULK1/Atg1 in glia.

Autophagy is a major means for the elimination of protein inclusions in neurons in neurodegenerative diseases such as Parkinson's disease (PD). Yet, the mechanism of autophagy in the other brain cell type, glia, is less well characterized and remains largely unknown. Here, we present evidence that the PD risk factor, Cyclin-G-associated kinase (GAK)/Drosophila homolog Auxilin (dAux), is a component in glial autophagy. The lack of GAK/dAux increases the autophagosome number and size in adult fly glia and mouse microglia, and generally up-regulates levels of components in the initiation and PI3K class III complexes. GAK/dAux interacts with the master initiation regulator UNC-51like autophagy activating kinase 1/Atg1 via its uncoating domain and regulates the trafficking of Atg1 and Atg9 to autophagosomes, hence controlling the onset of glial autophagy. On the other hand, lack of GAK/dAux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/dAux might play additional roles. Importantly, dAux contributes to PD-like symptoms including dopaminergic neurodegeneration and locomotor function in flies. Our findings identify an autophagy factor in glia; considering the pivotal role of glia under pathological conditions, targeting glial autophagy is potentially a therapeutic strategy for PD.

Dopamine transporter and synaptic vesicle sorting defects underlie auxilin-associated Parkinson's disease.

Auxilin participates in the uncoating of clathrin-coated vesicles (CCVs), thereby facilitating synaptic vesicle (SV) regeneration at presynaptic sites. Auxilin (DNAJC6/PARK19) loss-of-function mutations cause early-onset Parkinson's disease (PD). Here, we utilized auxilin knockout (KO) mice to elucidate the mechanisms through which auxilin deficiency and clathrin-uncoating deficits lead to PD. Auxilin KO mice display cardinal features of PD, including progressive motor deficits, α-synuclein pathology, nigral dopaminergic loss, and neuroinflammation. Significantly, treatment with L-DOPA ameliorated motor deficits. Unbiased proteomic and neurochemical analyses of auxilin KO brains indicated dopamine dyshomeostasis. We validated these findings by demonstrating slower dopamine reuptake kinetics in vivo, an effect associated with dopamine transporter misrouting into axonal membrane deformities in the dorsal striatum. Defective SV protein sorting and elevated synaptic autophagy also contribute to ineffective dopamine sequestration and compartmentalization, ultimately leading to neurodegeneration. This study provides insights into how presynaptic endocytosis deficits lead to dopaminergic vulnerability and pathogenesis of PD.

Auxilin regulates intestinal stem cell proliferation through EGFR.

Adult tissue homeostasis is maintained by residential stem cells. The proliferation and differentiation of adult stem cells must be tightly balanced to avoid excessive proliferation or premature differentiation. However, how stem cell proliferation is properly controlled remains elusive. Here, we find that auxilin (Aux) restricts intestinal stem cell (ISC) proliferation mainly through EGFR signaling. aux depletion leads to excessive ISC proliferation and midgut homeostasis disruption, which is unlikely caused by defective Notch signaling. Aux is expressed in multiple types of intestinal cells. Interestingly, aux depletion causes a dramatic increase in EGFR signaling, with a strong accumulation of EGFR at the plasma membrane and an increased expression of EGFR ligands in response to tissue stress. Furthermore, Aux co-localizes and associates with EGFR. Finally, blocking EGFR signaling completely suppresses the defects caused by aux depletion. Together, these data demonstrate that Aux mainly safeguards EGFR activation to keep a proper ISC proliferation rate to maintain midgut homeostasis.

Epidermal chloroplasts are defense-related motile organelles equipped with plant immune components.

In addition to conspicuous large mesophyll chloroplasts, where most photosynthesis occurs, small epidermal chloroplasts have also been observed in plant leaves. However, the functional significance of this small organelle remains unclear. Here, we present evidence that Arabidopsis epidermal chloroplasts control the entry of fungal pathogens. In entry trials, specialized fungal cells called appressoria triggered dynamic movement of epidermal chloroplasts. This movement is controlled by common regulators of mesophyll chloroplast photorelocation movement, designated as the epidermal chloroplast response (ECR). The ECR occurs when the PEN2 myrosinase-related higher-layer antifungal system becomes ineffective, and blockage of the distinct steps of the ECR commonly decreases preinvasive nonhost resistance against fungi. Furthermore, immune components were preferentially localized to epidermal chloroplasts, contributing to antifungal nonhost resistance in the pen2 background. Our findings reveal that atypical small chloroplasts act as defense-related motile organelles by specifically positioning immune components in the plant epidermis, which is the first site of contact between the plant and pathogens. Thus, this work deepens our understanding of the functions of epidermal chloroplasts.

Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic.

Clathrin-coated vesicles lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 "uncoating ATPase." The J- and PTEN-like domain-containing proteins, auxilin 1 (Aux1) and auxilin 2 (GAK), recruit Hsc70. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin-mediated membrane scission has released the vesicle from the plasma membrane. These bursts contain a very small number of auxilins, and even four to six molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated pit assembly. We previously showed that clathrin-coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. The differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.

Human rhomboid family-1 modulates clathrin coated vesicle-dependent pro-transforming growth factor α membrane trafficking to promote breast cancer progression.

BACKGROUND: Epidermal growth factor receptor (EGFR) signalling is critical in epithelial cancer development. Human rhomboid family-1 (RHBDF1) facilitates the secretion of TGFα, an EGFR ligand, in breast cancer; however, the underlying mechanism remains unclear. We evaluated the role for RHBDF1 in clathrin-coated vesicle (CCV)-dependent pro-TGFα membrane trafficking in breast cancer cells upon stimulation by G-protein coupled receptor (GPCR) agonists. METHODS: RHBDF1 was silenced in various breast cancer cells using shRNA. TGFα levels, subcellular localization, and secretion were evaluated using ELISA, immunofluorescent staining, and coimmunoprecipitation. Phosphorylation and expression of relevant proteins were measured by western blotting. RHBDF1-dependent cell viability and invasion were measured. FINDINGS: RHBDF1 mediates GPCR agonist-induced EGFR phosphorylation by promoting TGFα secretion in various types of breast cancer cells. RHBDF1 not only mediates ADAM17-dependent shedding of TGFα, but is essential in membrane trafficking of pro-TGFα. RHBDF1 silencing results in blocking of clathrin uncoating from CCV, a crucial step for the plasma membrane release of pro-TGFα. Interaction of RHBDF1 with auxilin-2, a CCV protein, determines the recruitment of HSC70 to CCV to facilitate clathrin uncoating. RHBDF1 function is required for the proliferation and mobility of breast cancer cells upon stimulation by Sphingosine 1 Phosphate (S1P), a GPCR agonist. We demonstrate a significant correlation between RHBDF1 overexpression and EGFR activation in breast cancer tissues. INTERPRETATION: RHBDF1 is an indispensable component of the protein trafficking machinery involved in GPCR-mediated EGFR transactivation, and is an attractive therapeutic target for cancer. FUND: National Natural Science Foundation of China (81,672,740 to ZSZ, 81,272,356 and 81,330,029 to LYL).

Cargo regulates clathrin-coated pit invagination via clathrin light chain phosphorylation.

Clathrin light chains (CLCs) control selective uptake of a range of G protein-coupled receptors (GPCRs), although the mechanism by which this occurs has remained elusive thus far. In particular, site-specific phosphorylation of CLCb controls the uptake of the purinergic GPCR P2Y12, but it is dispensable for the constitutive uptake of the transferrin receptor (TfR). We demonstrate that phosphorylation of CLCb is required for the maturation of clathrin-coated pits (CCPs) through the transition of flat lattices into invaginated buds. This transition is dependent on efficient clathrin exchange regulated by CLCb phosphorylation and mediated through auxilin. Strikingly, this rearrangement is required for the uptake of P2Y12 but not TfR. These findings link auxilin-mediated clathrin exchange to early stages of CCP invagination in a cargo-specific manner. This supports a model in which CCPs invaginate with variable modes of curvature depending on the cargo they incorporate.

Reconstitution of Clathrin Coat Disassembly for Fluorescence Microscopy and Single-Molecule Analysis.

The disassembly of the clathrin lattice surrounding coated vesicles is the obligatory last step in their life cycle. It is mediated by the coordinated recruitment of auxilin and Hsc70, an ATP-driven molecular clamp. Here, we describe the preparation of reagents and the single-particle fluorescence microscopy imaging assay in which we visualize directly the Hsc70-driven uncoating of synthetic clathrin coats or clathrin-coated vesicles.

LRRK2 phosphorylation of auxilin mediates synaptic defects in dopaminergic neurons from patients with Parkinson's disease.

Recently identified Parkinson's disease (PD) genes involved in synaptic vesicle endocytosis, such as DNAJC6 (auxilin), have further implicated synaptic dysfunction in PD pathogenesis. However, how synaptic dysfunction contributes to the vulnerability of human dopaminergic neurons has not been previously explored. Here, we demonstrate that commonly mutated, PD-linked leucine-rich repeat kinase 2 (LRRK2) mediates the phosphorylation of auxilin in its clathrin-binding domain at Ser627. Kinase activity-dependent LRRK2 phosphorylation of auxilin led to differential clathrin binding, resulting in disrupted synaptic vesicle endocytosis and decreased synaptic vesicle density in LRRK2 patient-derived dopaminergic neurons. Moreover, impaired synaptic vesicle endocytosis contributed to the accumulation of oxidized dopamine that in turn mediated pathogenic effects such as decreased glucocerebrosidase activity and increased α-synuclein in mutant LRRK2 neurons. Importantly, these pathogenic phenotypes were partially attenuated by restoring auxilin function in mutant LRRK2 dopaminergic neurons. Together, this work suggests that mutant LRRK2 disrupts synaptic vesicle endocytosis, leading to altered dopamine metabolism and dopamine-mediated toxic effects in patient-derived dopaminergic neurons.

Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic.

Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe 'coincidence-detecting' sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.

Steered molecular dynamics simulation of the binding of the bovine auxilin J domain to the Hsc70 nucleotide-binding domain.

The Hsp70 and Hsp40 chaperone machine plays critical roles in protein folding, membrane translocation, and protein degradation by binding and releasing protein substrates in a process that utilizes ATP. The activities of the Hsp70 family of chaperones are recruited and stimulated by the J domains of Hsp40 chaperones. However, structural information on the Hsp40-Hsp70 complex is lacking, and the molecular details of this interaction are yet to be elucidated. Here we used steered molecular dynamics (SMD) simulations to investigate the molecular interactions that occur during the dissociation of the auxilin J domain from the Hsc70 nucleotide-binding domain (NBD). The changes in energy observed during the SMD simulation suggest that electrostatic interactions are the dominant type of interaction. Additionally, we found that Hsp70 mainly interacts with auxilin through the surface residues Tyr866, Arg867, and Lys868 of helix II, His874, Asp876, Lys877, Thr879, and Gln881 of the HPD loop, and Phe891, Asn895, Asp896, and Asn903 of helix III. The conservative residues Tyr866, Arg867, Lys868, His874, Asp876, Lys877, and Phe891 were also found in a previous study to be indispensable to the catalytic activity of the DnaJ J domain and the binding of it with the NBD of DnaK. The in silico identification of the importance of auxilin residues Asn895, Asp896, and Asn903 agrees with previous mutagenesis and NMR data suggesting that helix III of the J domain of the T antigen interacts with Hsp70. Furthermore, our data indicate that Thr879 and Gln881 from the HPD loop are also important as they mediate the interaction between the bovine auxilin J domain and Hsc70.

Prion-specific Hsp40 function: The role of the auxilin homolog Swa2.

Yeast prions are protein-based genetic elements that propagate through cell populations via cytosolic transfer from mother to daughter cell. Molecular chaperone proteins including Hsp70, the Hsp40/J-protein Sis1, and Hsp104 are required for continued prion propagation, however the specific requirements of chaperone proteins differ for various prions. We recently reported that Swa2, the yeast homolog of the mammalian protein auxilin, is specifically required for the propagation of the prion [URE3]. 1 [URE3] propagation requires both a functional J-domain and the tetratricopeptide repeat (TPR) domain of Swa2, but does not require Swa2 clathrin binding. We concluded that the TPR domain determines the specificity of the genetic interaction between Swa2 and [URE3], and that this domain likely interacts with one or more proteins with a C-terminal EEVD motif. Here we extend that analysis to incorporate additional data that supports this hypothesis. We also present new data eliminating Hsp104 as the relevant Swa2 binding partner and discuss our findings in the context of other recent work involving Hsp90. Based on these findings, we propose a new model for Swa2's involvement in [URE3] propagation in which Swa2 and Hsp90 mediate the formation of a multi-protein complex that increases the number of sites available for Hsp104 disaggregation.

Auxilin Underlies Progressive Locomotor Deficits and Dopaminergic Neuron Loss in a Drosophila Model of Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disorder that exhibits motor and non-motor symptoms, as well as pathological hallmarks, including dopaminergic (DA) neuron death and formation of α-synuclein (α-Syn) Lewy bodies. Cyclin-G-associated kinase (GAK), a PD susceptibility gene identified through genome-wide association studies (GWAS), is a ubiquitous serine/threonine kinase involved in clathrin uncoating, though its PD-related function remains elusive. Here, we implicate the Drosophila GAK homolog, auxilin (aux), in a broad spectrum of parkinsonian-like symptoms. Downregulating aux expression leads to progressive loss of climbing ability, decreased lifespan, and age-dependent DA neuron death similar to α-Syn overexpression. Reduced aux expression further enhances and accelerates α-Syn-mediated DA neuron loss. Flies with reduced aux expression are more sensitive to the toxin paraquat, suggesting that genetic and environmental factors intertwine. Taken together, these findings decipher a pivotal role for GAK/aux and suggest mechanisms underlying PD.

DNAJC6 Mutations Associated With Early-Onset Parkinson's Disease.

OBJECTIVE: DNAJC6 mutations were recently described in two families with autosomal recessive juvenile parkinsonism (onset age < 11), prominent atypical signs, poor or absent response to levodopa, and rapid progression (wheelchair-bound within ∼10 years from onset). Here, for the first time, we report DNAJC6 mutations in early-onset Parkinson's disease (PD). METHODS: The DNAJC6 open reading frame was analyzed in 274 patients with early-onset sporadic or familial PD. Selected variants were followed up by cosegregation, homozygosity mapping, linkage analysis, whole-exome sequencing, and protein studies. RESULTS: We identified two families with different novel homozygous DNAJC6 mutations segregating with PD. In each family, the DNAJC6 mutation was flanked by long runs of homozygosity within highest linkage peaks. Exome sequencing did not detect additional pathogenic variants within the linkage regions. In both families, patients showed severely decreased steady-state levels of the auxilin protein in fibroblasts. We also identified a sporadic patient carrying two rare noncoding DNAJC6 variants possibly effecting RNA splicing. All these cases fulfilled the criteria for a clinical diagnosis of early-onset PD, had symptoms onset in the third-to-fifth decade, and slow disease progression. Response to dopaminergic therapies was prominent, but, in some patients, limited by psychiatric side effects. The phenotype overlaps that of other monogenic forms of early-onset PD. INTERPRETATION: Our findings delineate a novel form of hereditary early-onset PD. Screening of DNAJC6 is warranted in all patients with early-onset PD compatible with autosomal recessive inheritance. Our data provide further evidence for the involvement of synaptic vesicles endocytosis and trafficking in PD pathogenesis.

Auxilin facilitates membrane traffic in the early secretory pathway.

Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER-Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.

The clathrin-binding and J-domains of GAK support the uncoating and chaperoning of clathrin by Hsc70 in the brain.

Cyclin-G-associated kinase (GAK), the ubiquitously expressed J-domain protein, is essential for the chaperoning and uncoating of clathrin that is mediated by Hsc70 (also known as HSPA8). Adjacent to the C-terminal J-domain that binds to Hsc70, GAK has a clathrin-binding domain that is linked to an N-terminal kinase domain through a PTEN-like domain. Knocking out GAK in fibroblasts caused inhibition of clathrin-dependent trafficking, which was rescued by expressing a 62-kDa fragment of GAK, comprising just the clathrin-binding and J-domains. Expressing this fragment as a transgene in mice rescued the lethality and the histological defects caused by knocking out GAK in the liver or in the brain. Furthermore, when both GAK and auxilin (also known as DNAJC6), the neuronal-specific homolog of GAK, were knocked out in the brain, mice expressing the 62-kDa GAK fragment were viable, lived a normal life-span and had no major behavior abnormalities. However, these mice were about half the size of wild-type mice. Therefore, the PTEN-like domains of GAK and auxilin are not essential for Hsc70-dependent chaperoning and uncoating of clathrin, but depending on the tissue, these domains appear to increase the efficiency of these co-chaperones.

JACALIN-LECTIN LIKE1 Regulates the Nuclear Accumulation of GLYCINE-RICH RNA-BINDING PROTEIN7, Influencing the RNA Processing of FLOWERING LOCUS C Antisense Transcripts and Flowering Time in Arabidopsis.

Lectins selectively recognize sugars or glycans for defense in living cells, but less is known about their roles in the development process and the functional network with other factors. Here, we show that Arabidopsis (Arabidopsis thaliana) JACALIN-LECTIN LIKE1 (AtJAC1) functions in flowering time control. Loss of function of AtJAC1 leads to precocious flowering, whereas overexpression of AtJAC1 causes delayed flowering. AtJAC1 influences flowering through regulation of the key flowering repressor gene FLOWERING LOCUS C (FLC). Genetic analysis revealed that AtJAC1's function is mostly dependent on GLYCINE-RICH RNA-BINDING PROTEIN7 (GRP7), an upstream regulator of FLC. Biochemical and cell biological data indicated that AtJAC1 interacted physically with GRP7 specifically in the cytoplasm. AtJAC1 influences the nucleocytoplasmic distribution of GRP7, with predominant nuclear localization of GRP7 when AtJAC1 function is lost but retention of GRP7 in the cytoplasm when AtJAC1 is overexpressed. A temporal inducible assay suggested that AtJAC1's regulation of flowering could be compromised by the nuclear accumulation of GRP7. In addition, GRP7 binds to the antisense precursor messenger RNA of FLC through a conserved RNA motif. Loss of GRP7 function leads to the elevation of total FLC antisense transcripts and reduced proximal-distal polyadenylation ratio, as well as histone methylation changes in the FLC gene body region and increased total functional sense FLC transcript. Attenuating the direct binding of GRP7 with competing artificial RNAs leads to changes of FLC antisense precursor messenger RNA processing and flowering transition. Taken together, our study indicates that AtJAC1 coordinates with GRP7 in shaping plant development through the regulation of RNA processing in Arabidopsis.

Interactions of peripheral proteins with model membranes as viewed by molecular dynamics simulations.

Many cellular signalling and related events are triggered by the association of peripheral proteins with anionic lipids in the cell membrane (e.g. phosphatidylinositol phosphates or PIPs). This association frequently occurs via lipid-binding modules, e.g. pleckstrin homology (PH), C2 and four-point-one, ezrin, radixin, moesin (FERM) domains, present in peripheral and cytosolic proteins. Multiscale simulation approaches that combine coarse-grained and atomistic MD simulations may now be applied with confidence to investigate the molecular mechanisms of the association of peripheral proteins with model bilayers. Comparisons with experimental data indicate that such simulations can predict specific peripheral protein-lipid interactions. We discuss the application of multiscale MD simulation and related approaches to investigate the association of peripheral proteins which contain PH, C2 or FERM-binding modules with lipid bilayers of differing phospholipid composition, including bilayers containing multiple PIP molecules.