Parkinson's disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria

Mutations in Pten-induced kinase 1 (PINK1) are linked to early-onset familial Parkinson's disease (FPD). PINK1 has previously been implicated in mitochondrial fission/fusion dynamics, quality control, and electron transport chain function. However, it is not clear how these processes are interconnected and whether they are sufficient to explain all aspects of PINK1 pathogenesis. Here we show that PINK1 also controls mitochondrial motility. In Drosophila, downregulation of dMiro or other components of the mitochondrial transport machinery rescued dPINK1 mutant phenotypes in the muscle and dopaminergic (DA) neurons, whereas dMiro overexpression alone caused DA neuron loss. dMiro protein level was increased in dPINK1 mutant but decreased in dPINK1 or dParkin overexpression conditions. In Drosophila larval motor neurons, overexpression of dPINK1 inhibited axonal mitochondria transport in both anterograde and retrograde directions, whereas dPINK1 knockdown promoted anterograde transport. In HeLa cells, overexpressed hPINK1 worked together with hParkin, another FPD gene, to regulate the ubiquitination and degradation of hMiro1 and hMiro2, apparently in a Ser-156 phosphorylation-independent manner. Also in HeLa cells, loss of hMiro promoted the perinuclear clustering of mitochondria and facilitated autophagy of damaged mitochondria, effects previously associated with activation of the PINK1/Parkin pathway. These newly identified functions of PINK1/Parkin and Miro in mitochondrial transport and mitophagy contribute to our understanding of the complex interplays in mitochondrial quality control that are critically involved in PD pathogenesis, and they may explain the peripheral neuropathy symptoms seen in some PD patients carrying particular PINK1 or Parkin mutations. Moreover, the different effects of loss of PINK1 function on Miro protein level in Drosophila and mouse cells may offer one explanation of the distinct phenotypic manifestations of PINK1 mutants in these two species.     

PINK1 and Parkin flag Miro to direct mitochondrial traffic

The Parkinson's disease proteins PINK1 and Parkin are proposed guardians of mitochondrial fidelity, targeting damaged mitochondria for degradation by mitophagy. In this issue of Cell, Wang et al. (2011) now show that PINK1 and Parkin also regulate mitochondrial trafficking and quarantine damaged mitochondria by severing their connection to the microtubule network.     

Msp1: patrolling mitochondria for lost proteins

Intracellular protein localization is critical for establishing organelle identity, avoiding inappropriate interactions, and maintaining cellular function. An emerging mechanism for ensuring high‐fidelity protein localization is the selective destruction of mislocalized copies. Two new papers find that the mitochondrial outer membrane is kept free of mislocalized proteins by Msp1 (Chen et al, 2014 ; Okreglak & Walter, 2014 ).     

A Highlights from MBoC Selection: APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane

Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations.     

The Miro GTPases: At the heart of the mitochondrial transport machinery

Mitochondria are organelles of elaborate structure that in addition to supplying cellular energy, have significant roles in calcium homeostasis and apoptosis. Failure to maintain mitochondrial dynamics results in neurodegenerative diseases and neuromuscular pathologies. The Miro GTPases, which constitute a unique subgroup of the Ras superfamily, have emerged as essential regulators of mitochondrial morphogenesis and trafficking along microtubules. Miro GTPases function as calcium-dependent sensors in the control of mitochondrial motility. Increased awareness of the biological function of Miro GTPases can contribute to elucidate the molecular mechanisms underlying diseases caused by deregulated mitochondrial dynamics.     

Understanding Miro GTPases: Implications in the Treatment of Neurodegenerative Disorders

The Miro GTPases represent an unusual subgroup of the Ras superfamily and have recently emerged as important mediators of mitochondrial dynamics and for maintaining neuronal health. It is now well-established that these enzymes act as essential components of a Ca²⁺-sensitive motor complex, facilitating the transport of mitochondria along microtubules in several cell types, including dopaminergic neurons. The Miros appear to be critical for both anterograde and retrograde mitochondrial transport in axons and dendrites, both of which are considered essential for neuronal health. Furthermore, the Miros may be significantly involved in the development of several serious pathological processes, including the development of neurodegenerative and psychiatric disorders. In this review, we discuss the molecular structure and known mitochondrial functions of the Miro GTPases in humans and other organisms, in the context of neurodegenerative disease. Finally, we consider the potential human Miros hold as novel therapeutic targets for the treatment of such disease.     

Old dog,new tricks: Arf1 required for mitochondria homeostasis

The small GTPase Arf1 that is classically required for the budding of COPI‐coated vesicles from the Golgi membrane is now proposed to have novel and conserved roles in the morphological and functional maintenance of mitochondria: It functionally localizes to ER/mitochondria contact sites; it allows for the recruitment of a degradation machinery to mitochondria to remove toxic mitofusin/Fzo1 clusters; and it allows the extension of autophagy sequestration membranes needed for mitophagy to clear damaged mitochondria.     

Purification and characterization of a spinach-leaf protein capable of transferring phospholipids from liposomes to mitochondria or chloroplasts

A phospholipid transfer protein has been purified 195-fold from an extract of spinach leaves. This protein is capable of transferring phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol and phosphatidylethanolamine from liposomes to mitochondria. In addition to this protein, a minor part of the total activity was associated with a less purified fraction. The pure protein has an isoelectric point of 9.0 +/- 0.2 determined by a chromatofocusing technique. Electrophoresis on sodium dodecyl sulfate/polyacrylamide gel showed that the protein is homogeneous and has an apparent molecular weight of 9000 +/- 1000, in agreement with the value (8832) calculated from the amino acid composition. This composition is characterized by a high amount of alanine and glycine and by the absence of phenylalanine, whereas arginine, glutamine, histidine and methionine are minor components. The spinach protein is also able to transfer phosphatidylcholine and phosphatidylglycerol from liposomes to intact chloroplasts. This observation reinforces the hypothesis that plastid phospholipids are partly imported from outside the organelle by a transfer process.     

Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

Mutations in PINK1 and Parkin are associated with early-onset Parkinson''s disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser⁶⁵) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser⁶⁵ phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser⁶⁵ by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser⁶⁵ phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin''s ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser⁶⁵ is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson''s disease.     

Inflammasome assembly: The wheels are turning

Inflammasomes control host cell death and inflammation in response to sterile or infectious stimuli. Two recent reports published in Science reveal the structural basis for the assembly of NAIP-NLRC4 inflammasomes.The innate immune response relies on germline-encoded pattern-recognition receptors that monitor the extracellular and intracellular compartments of host cells for the presence of pathogens. Detection of microbe-derived ligands or endogenous danger signals in the host cell cytosol can result in the assembly of large multi-protein complexes termed inflammasomes, which drive the activation of caspase-1, a cysteine protease. Once activated, caspase-1 initiates a pro-inflammatory cell death program called pyroptosis, and promotes the maturation and secretion of cytokines like interleukin (IL)-1β and -18. While inflammasomes play a protective role against microbial infections, uncontrolled inflammasome activation is implicated in chronic inflammatory disorders such as atherosclerosis, gout and diabetes.NLRC4, a member of the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family, assembles an inflammasome in response to bacterial pathogens. Although often referred to as a receptor, NLRC4 does not directly bind microbe-derived ligands, but employs so-called NAIP proteins (NLR family, apoptosis inhibitory protein) as upstream sensors for different ligands^1,2. In mice, NAIP5/6 were shown to recognize bacterial flagellin, while the inner rod and the needle subunit of the Salmonella typhimurium Type 3 Secretion System is recognized by NAIP2 and NAIP1, respectively. Previous work showed that ligand binding induces the interaction of NAIPs with NLRC4 and the formation of disk-shaped oligomeric complexes³, but the underlying mechanism was unknown.Zhang et al.⁴ and Hu et al.⁵ have now used Cryo-electron microcopy (Cryo-EM) to examine the structure of the PrgJ-NAIP2-NLRC4 complex and its assembly mechanism. Both studies found that the complex has a wheel- or disk-like architecture, with 10-12 spokes corresponding to individual protomers. While it was not possible to distinguish NAIP2 and NLRC4 protomers within the complex, probably due to their conserved domain organization, analysis of unfinished NAIP2-NLRC4 disks indicated that the protomer at one end is different from the remaining subunits⁵. Nanogold labeling demonstrated that only one NAIP2 is found per complex, thus supporting a model in which the assembly of the entire NAIP2-NLRC4 inflammasome starts with the ligand-induced activation of one single NAIP molecule. As the authors point out that this differs strikingly from the assembly of the heptameric apoptosome, a caspase-9 activating platform, which requires ligand-mediated activation of every Apaf-1 protomer of the complex^4,5.How does activated NAIP2 induce the progressive oligomerization of NLRC4? Structural comparison of the atomic model of the NAIP2-NLRC4 complex and the previously determined crystal structure of inactive monomeric NLRC4⁶ revealed that a striking ∼90° hinge rotation accompanies NLRC4 activation. This conformational change results in the formation of a new oligomerization surface (referred to as catalytic or nucleating surface) that interacts with the next protomer in the wheel-like structure and thus facilitates progressive oligomerization (Figure 1). A matching receptor surface is preformed in the inactive NLRC4 molecule, and mediates the recruitment of NLRC4 into the growing complex. Intriguingly, missense mutations in NLRC4 that are associated with autoinflammatory conditions map to this important hinge region^7,8,9. Whether such mutations induce the formation of NLRC4 disks in the absence of activated NAIPs remains to be shown, but the new studies provide the structural basis for understanding these gain-of-function mutations.Open in a separate windowFigure 1(A) Domain organization of NAIPs and NLRC4 in mice. BIR, Baculovirus Inhibitor of apoptosis protein Repeat; NOD, nucleotide-binding and oligomerization domain; HD1, helical domain 1; WHD, winged-helical domain; HD2, helical domain 2; LRR, leucine-rich repeats; CARD, caspase-recruitment domain. (B) Assembly mechanism of NAIP-NLRC4 inflammasomes. Binding of their specific ligand activates mouse NAIP proteins. The activated NAIP interacts with an inactive NLRC4 molecule and induces a conformational change that activates NLRC4, resulting in the generation of a new catalytic or nucleating surface. Active NLRC4 molecules recruit and activate NLRC4 molecules in a domino-like reaction. The completed NAIP-NLRC4 inflammasome is a multi-subunit disk-like structure containing 9-11 molecules of NLRC4, but only one NAIP molecule. The NLRC4^CARD (not shown for simplicity reasons) initiates caspase-1 activation either through the adaptor protein ASC or directly. Active caspase-1 promotes inflammation and host defense by inducing pyroptotic cell death and cytokine maturation.Both studies also found that NAIP2 itself was precluded from self-oligomerization and further recruitment into the complex. Structural analysis showed that all NAIP family members feature a catalytic or nucleating surface, which interacts with the receptor surface of inactive NLRC4 and induces the activating conformational changes that mediate progressive oligomerization. However, in contrast to NLRC4, NAIPs lack a receptor surface matching the nucleating surface of their own or of NLRC4. Thus, NAIPs only act as initiators of inflammasome disk formation and only one single NAIP member can be found to be incorporated into a NAIP-NLRC4 complex.Revealing the mechanism of NAIP-NLRC4 inflammasome formation is a major breakthrough, but many mysteries remain. An important next step is to define how the wheel-like complex is connected to the downstream signaling components. NLRC4 features a caspase-recruitment domain (CARD) that can either recruit pro-caspase-1 or ASC, a small adaptor protein featuring a CARD and a Pyrin domain (PYD), through which it oligomerizes to form a larger macromolecular complex known as “ASC speck”. Zhang et al.⁴ and Hu et al.⁵ used CARD-deleted NLRC4 for their studies to avoid unspecific oligomerization of the CARD, therefore the orientation of the NLRC4^CARD within the disk could not be determined. Consistent with the ability of NLRC4 to directly interact with and activate caspase-1 even in the absence of ASC, Zhang et al.⁴ showed that complexes containing full-length NLRC4 induce the oligomerization of the caspase-1^CARD. Yet since ASC is recruited to activated NLRC4 in wild-type cells, it will be also interesting to determine whether and how NAIP-NLRC4 complexes initiate ASC filaments, and how such a macromolecular structure looks like.Another open question is how the ligand PrgJ induces NAIP activation and where it is located within the wheel-like structure. Previous work showed that the NOD domain of NAIPs confers ligand binding and specificity¹⁰, yet according to the new studies the same domain also forms the nucleating surface that initiates complex assembly. Since thus far the structure of inactive or activated NAIPs has not been reported, the structural basis underlying ligand-NOD domain interaction is unknown and the resulting conformational changes remain to determined.Finally, it needs to be investigated whether the mechanism of NAIP-NLRC4 complex formation applies to other types of inflammasomes. The PYHIN family member AIM2, which activates caspase-1 upon recognizing pathogen- or host-derived DNA in the cytosol, was proposed to oligomerize along double-stranded DNA instead of forming wheel-like structures. Nonetheless, other NLR family members like NLRP3 might form disk-shaped inflammasomes in analogy to the NAIP-NLRC4 complex. Indeed, gain-of-function mutations in NLRP3 that are associated with autoinflammatory disorders were shown to result in auto-oligomerization¹¹, but the Cryo-EM structure of such complexes has not yet been solved. Determining the structure of other inflammasomes will thus be an exiting field of research for structural biologist and might reveal important insight into the activation mechanisms of these receptors.     

Functional impairment in RHOT1/Miro1 degradation and mitophagy is a shared feature in familial and sporadic Parkinson disease

Mitophagy is a conserved and highly regulated process of selective degradation crucial in maintaining normal cellular physiology. Genetic defects and cellular aberrations affecting mitophagy have been associated with the development of Parkinson disease. In their recently published article (highlighted in a punctum in this issue of the journal) Hsieh et al. present a putative mitophagy marker, which serves as a mechanistic link between sporadic and familial Parkinson disease.     

Protein conformation wheels: cytochromes and lysozymes

Conformation wheels, directly relating to amino acid sequence to the local torsion angles in a protein molecule, are presented for cytochromes c, c2, c550, and c551 and for lysozymes from hen egg-white and T4 bacteriophage. The circular plots for the cytochrome molecules aid in visualizing the common three-dimensional folding ("cytochrome fold") observed in this family of proteins. Conformation wheels for lysozymes from two different species reveal the characteristic differences in their folding patterns. These novel plots are also useful in storing and comparing the several sets of crystallographic data reported for lysozyme.     

Simple device for automatically transferring broth cultures

A simple device is described for automatically transferring broth cultures from one tube to another by means of a tilting platform.     

植物转基因方法概述

综述了植物基因转化的不同方法。植物基因转化方法主要包括直接基因转移和农杆菌介导的转化,直接基因转移又包括PEG介导、电激介导和基因枪方法。相比之下,农杆菌转化方法具有明显的优势;本文对其方法和机制作了较为全面的介绍。