ESCRT-III-associated proteins and spastin inhibit protrudin-dependent polarised membrane traffic.

Mutations in the gene encoding the microtubule severing ATPase spastin are the most frequent cause of hereditary spastic paraplegia, a genetic condition characterised by length-dependent axonal degeneration. Here, we show that HeLa cells lacking spastin and embryonic fibroblasts from a spastin knock-in mouse model become highly polarised and develop cellular protrusions. In HeLa cells, this phenotype was rescued by wild-type spastin, but not by forms unable to sever microtubules or interact with endosomal ESCRT-III proteins. Cells lacking the spastin-interacting ESCRT-III-associated proteins IST1 or CHMP1B also developed protrusions. The protrusion phenotype required protrudin, a RAB-interacting protein that interacts with spastin and localises to ER-endosome contact sites, where it promotes KIF5-dependent endosomal motility to protrusions. Consistent with this, the protrusion phenotype in cells lacking spastin also required KIF5. Lack or mutation of spastin resulted in functional consequences for receptor traffic of a pathway implicated in HSP, as Bone Morphogenetic Protein receptor distribution became polarised. Our results, therefore, identify a novel role for ESCRT-III proteins and spastin in regulating polarised membrane traffic.

The AAA ATPase spastin links microtubule severing to membrane modelling.

In 1999, mutations in the gene encoding the microtubule severing AAA ATPase spastin were identified as a major cause of a genetic neurodegenerative condition termed hereditary spastic paraplegia (HSP). This finding stimulated intense study of the spastin protein and over the last decade, a combination of cell biological, in vivo, in vitro and structural studies have provided important mechanistic insights into the cellular functions of the protein, as well as elucidating cell biological pathways that might be involved in axonal maintenance and degeneration. Roles for spastin have emerged in shaping the endoplasmic reticulum and the abscission stage of cytokinesis, in which spastin appears to couple membrane modelling to microtubule regulation by severing.

Spastin couples microtubule severing to membrane traffic in completion of cytokinesis and secretion.

Mutations in the gene encoding the microtubule (MT)-severing protein spastin are the most common cause of hereditary spastic paraplegia, a genetic condition in which axons of the corticospinal tracts degenerate. We show that not only does endogenous spastin colocalize with MTs, but that it is also located on the early secretory pathway, can be recruited to endosomes and is present in the cytokinetic midbody. Spastin has two main isoforms, a 68 kD full-length isoform and a 60 kD short form. These two isoforms preferentially localize to different membrane traffic pathways with 68 kD spastin being principally located at the early secretory pathway, where it regulates endoplasmic reticulum-to-Golgi traffic. Sixty kiloDalton spastin is the major form recruited to endosomes and is also present in the midbody, where its localization requires the endosomal sorting complex required for transport-III-interacting MIT domain. Loss of midbody MTs accompanies the abscission stage of cytokinesis. In cells lacking spastin, a MT disruption event that normally accompanies abscission does not occur and abscission fails. We suggest that this event represents spastin-mediated MT severing. Our results support a model in which membrane traffic and MT regulation are coupled through spastin. This model is relevant in the axon, where there also is co-ordinated MT regulation and membrane traffic.

The hereditary spastic paraplegia proteins NIPA1, spastin and spartin are inhibitors of mammalian BMP signalling.

The hereditary spastic paraplegias (HSPs) are genetic conditions characterized by distal axonopathy of the longest corticospinal tract axons, and so their study provides an important opportunity to understand mechanisms involved in axonal maintenance and degeneration. A group of HSP genes encode proteins that localize to endosomes. One of these is NIPA1 (non-imprinted in Prader-Willi/Angelman syndrome 1) and we have shown recently that its Drosophila homologue spichthyin inhibits bone morphogenic protein (BMP) signalling, although the relevance of this finding to the mammalian protein was not known. We show here that mammalian NIPA1 is also an inhibitor of BMP signalling. NIPA1 physically interacts with the type II BMP receptor (BMPRII) and we demonstrate that this interaction does not require the cytoplasmic tail of BMPRII. We show that the mechanism by which NIPA1 inhibits BMP signalling involves downregulation of BMP receptors by promoting their endocytosis and lysosomal degradation. Disease-associated mutant versions of NIPA1 alter the trafficking of BMPRII and are less efficient at promoting BMPRII degradation than wild-type NIPA1. In addition, we demonstrate that two other members of the endosomal group of HSP proteins, spastin and spartin, are inhibitors of BMP signalling. Since BMP signalling is important for distal axonal function, we propose that dysregulation of BMP signalling could be a unifying pathological component in this endosomal group of HSPs, and perhaps of importance in other conditions in which distal axonal degeneration is found.

Endogenous spartin (SPG20) is recruited to endosomes and lipid droplets and interacts with the ubiquitin E3 ligases AIP4 and AIP5.

The HSPs (hereditary spastic paraplegias) are genetic conditions in which there is distal degeneration of the longest axons of the corticospinal tract, resulting in spastic paralysis of the legs. The gene encoding spartin is mutated in Troyer syndrome, an HSP in which paralysis is accompanied by additional clinical features. There has been controversy over the subcellular distribution of spartin. We show here that, at steady state, endogenous spartin exists in a cytosolic pool that can be recruited to endosomes and to lipid droplets. Cytosolic endogenous spartin is mono-ubiquitinated and we demonstrate that it interacts via a PPXY motif with the ubiquitin E3 ligases AIP4 [atrophin-interacting protein 4; ITCH (itchy E3 ubiquitin protein ligase homologue] [corrected] and AIP5 (WWP1). Surprisingly, the PPXY motif, AIP4 and AIP5 are not required for spartin's ubiquitination, and so we propose that spartin acts as an adaptor for these proteins. Our results suggest that spartin is involved in diverse cellular functions, which may be of relevance to the complex phenotype seen in Troyer syndrome.

Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia.

Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER-endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell-derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER-endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.

Quantitative Gait Analysis Using a Motorized Treadmill System Sensitively Detects Motor Abnormalities in Mice Expressing ATPase Defective Spastin.

The hereditary spastic paraplegias (HSPs) are genetic conditions in which there is progressive axonal degeneration in the corticospinal tract. Autosomal dominant mutations, including nonsense, frameshift and missense changes, in the gene encoding the microtubule severing ATPase spastin are the most common cause of HSP in North America and northern Europe. In this study we report quantitative gait analysis using a motorized treadmill system, carried out on mice knocked-in for a disease-associated mutation affecting a critical residue in the Walker A motif of the spastin ATPase domain. At 4 months and at one year of age homozygous mutant mice had a number of abnormal gait parameters, including in stride length and stride duration, compared to heterozygous and wild-type littermates. Gait parameters in heterozygous animals did not differ from wild-type littermates. We conclude that quantitative gait analysis using the DigiGait system sensitively detects motor abnormalities in a hereditary spastic paraplegia model, and would be a useful method for analyzing the effects of pharmacological treatments for HSP.

An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome.

Mechanisms coordinating endosomal degradation and recycling are poorly understood, as are the cellular roles of microtubule (MT) severing. We show that cells lacking the MT-severing protein spastin had increased tubulation of and defective receptor sorting through endosomal tubular recycling compartments. Spastin required the ability to sever MTs and to interact with ESCRT-III (a complex controlling cargo degradation) proteins to regulate endosomal tubulation. Cells lacking IST1 (increased sodium tolerance 1), an endosomal sorting complex required for transport (ESCRT) component to which spastin binds, also had increased endosomal tubulation. Our results suggest that inclusion of IST1 into the ESCRT complex allows recruitment of spastin to promote fission of recycling tubules from the endosome. Thus, we reveal a novel cellular role for MT severing and identify a mechanism by which endosomal recycling can be coordinated with the degradative machinery. Spastin is mutated in the axonopathy hereditary spastic paraplegia. Zebrafish spinal motor axons depleted of spastin or IST1 also had abnormal endosomal tubulation, so we propose this phenotype is important for axonal degeneration.

A systematic analysis of human CHMP protein interactions: additional MIT domain-containing proteins bind to multiple components of the human ESCRT III complex.

In Saccharomyces cerevisiae 6 closely related proteins (Did2p, Vps2p, Vps24p, Vps32p, Vps60p, Vps20p) form part of the extended ESCRT III complex. This complex is required for the formation of multivesicular bodies and the degradation of internalized transmembrane receptor proteins. In contrast the human genome encodes 10 homologous proteins (CHMP1A (approved gene symbol PCOLN3), 1B, 2A, 2B, 3 (approved gene symbol VPS24), 4A, 4B, 4C, 5, and 6). In this study we have performed a series of protein interaction experiments to generate a more comprehensive picture of the human CHMP protein-interaction network. Our results describe novel interactions between known components of the human ESCRT III complex and identify a range of putative binding partners, which may indicate new ways in which the function of human CHMP proteins may be regulated. In particular, we show that two further MIT domain-containing proteins (AMSH/STAMBP and LOC129531) interact with multiple components of the human ESCRT III complex.

Spastin and atlastin, two proteins mutated in autosomal-dominant hereditary spastic paraplegia, are binding partners.

The pure hereditary spastic paraplegias (HSPs) are a group of conditions in which there is a progressive length-dependent degeneration of the distal ends of the corticospinal tract axons, resulting in spastic paralysis of the legs. Pure HSPs are most frequently inherited in an autosomal-dominant pattern and are commonly caused by mutations either in the SPG4 gene spastin or in the SPG3A gene atlastin. To identify binding partners for spastin, we carried out a yeast two-hybrid screen on a brain cDNA library, using spastin as bait. Remarkably, nearly all of the positive interacting prey clones coded for atlastin. We have verified the physiological relevance of this interaction using co-immunoprecipitation, glutathione S-transferase pull-down and intracellular co-localization experiments. We show that the spastin domain required for binding to atlastin lies within the N-terminal 80 residues of the protein, a region that is only present in the predominantly cytoplasmic, full-length spastin isoform. These data suggest that spastin and atlastin function in the same biochemical pathway and that it is the cytoplasmic function of spastin which is important for the pathogenesis of HSP. They also provide further evidence for a physiological and pathological role of spastin in membrane dynamics.