InteractiveFly: GeneBrief

MICAL: Biological Overview | References


Gene name - Molecule interacting with CasL

Synonyms - Mical

Cytological map position- 85F5-85F7

Function - enzyme

Keywords - mesoderm, CNS, brain, synaptogenesis, effector of PlexA signaling

Symbol - Mical

FlyBase ID: FBgn0053208

Genetic map position - 3R: 5,828,033..5,868,543 [-]

Classification - Calponin homology domain, Dehydrogenases

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

The overall size and structure of a synaptic terminal is an important determinant of its function. In a large-scale mutagenesis screen, designed to identify Drosophila mutants with abnormally structured neuromuscular junctions (NMJs), mutations were discovered in Drosophila mical, a conserved gene encoding a multi-domain protein with a N-terminal monooxygenase domain. In mical mutants, synaptic boutons do not sprout normally over the muscle surface and tend to form clusters along synaptic branches and at nerve entry sites. Consistent with high expression of MICAL in somatic muscles, immunohistochemical stainings reveal that the subcellular localization and architecture of contractile muscle filaments are dramatically disturbed in mical mutants. Instead of being integrated into a regular sarcomeric pattern, actin and myosin filaments are disorganized and accumulate beneath the plasmamembrane. Whereas contractile elements are strongly deranged, the proposed organizer of sarcomeric structure, D-Titin, is much less affected. Transgenic expression of interfering RNA molecules demonstrates that MICAL is required in muscles for the higher order arrangement of myofilaments. Ultrastructural analysis confirms that myosin-rich thick filaments enter submembranous regions and interfere with synaptic development, indicating that the disorganized myofilaments may cause the synaptic growth phenotype. As a model, it is suggested that the filamentous network around synaptic boutons restrains the spreading of synaptic branches (Beuchle, 2007).

Members of the semaphorin family of secreted and transmembrane proteins utilize plexins as neuronal receptors to signal repulsive axon guidance. It remains unknown how plexin proteins are directly linked to the regulation of cytoskeletal dynamics. Drosophila MICAL, a large, multidomain, cytosolic protein expressed in axons, interacts with the neuronal plexin A (PlexA) receptor and is required for Semaphorin 1a (Sema-1a)-PlexA-mediated repulsive axon guidance. In addition to containing several domains known to interact with cytoskeletal components, MICAL has a flavoprotein monooxygenase domain, the integrity of which is required for Sema-1a-PlexA repulsive axon guidance. Vertebrate orthologs of Drosophila MICAL are neuronally expressed and also interact with vertebrate plexins, and monooxygenase inhibitors abrogate semaphorin-mediated axonal repulsion. These results suggest a novel role for oxidoreductases in repulsive neuronal guidance (Terman, 2002).

To identify mediators of semaphorin-dependent repulsive axonal guidance, the terminal highly conserved 'C2' portion of the PlexA cytoplasmic domain was used to search for interacting proteins encoded by a Drosophila embryonic (0-24 hr) yeast two-hybrid cDNA library. The strongest interactor has been called MICAL, covers >41 kb of genomic sequence and has at least 25 exons. Based on analysis of isolated cDNAs and Western analysis, there are at least three MICAL isoforms ('long,' 'medium,' and 'short' variants) (Beuchle, 2007).

Drosophila MICAL is named for its recently characterized vertebrate ortholog, MICAL-1 (for molecule interacting with CasL), which has been shown to associate with CasL (see Drosophila p130CAS) and vimentin in nonneuronal cells (Suzuki, 2002). Within the plexin-interacting region in the C terminus identified in the screen described in this paper, there is a predicted heptad-repeat, coiled-coil structure. Interestingly, this region of MICAL shares amino acid similarity with several other coiled-coil domain-containing proteins, including a portion of the alpha domain found in the Ezrin, Radixin, and Moesin (ERM) proteins (~22% identity). N-terminal to this domain there is a region rich in prolines, and the last four amino acids of MICAL (ESII) are a PDZ protein binding motif. There are two regions of varying length, with no significant similarity to other proteins, which appear to determine the size of the different MICAL proteins. MICAL has a single LIM domain, a protein-protein interaction module found in a variety of proteins involved in signal transduction cascades and in cytoskeletal organization, and also a single calponin homology (CH) domain, a domain also found in cytoskeletal and signal transduction proteins and known to be involved in actin filament binding. The MICAL N-terminal ~500 aa is highly conserved among MICAL-related proteins but is unique over its entire length in comparison to other proteins (Terman, 2002).

In situ hybridization analysis using RNA probes corresponding to the N or C terminus of MICAL shows that MICAL and PlexA have similar patterns of embryonic mRNA expression. During early Drosophila development (stages 7-8), both are expressed in the ventral neurogenic region and in many nonneuronal tissues (including developing mesoderm, cells surrounding the cephalic furrow and amnioproctodeal invagination, and in gut primordia). This nonneuronal expression is also seen later in embryonic development (stages 11-17), where both are present within the anterior and posterior midgut primordia, the visceral musculature, and weakly in somatic musculature. During axonal pathfinding (stage 13 onward), both are expressed within the developing brain and ventral nerve cord in most, if not all, CNS neurons, but MICAL, like Sema1a and PlexA, is not highly expressed in peripheral sensory neurons (Terman, 2002).

Western blot analysis using a polyclonal antibody directed against the MICAL C terminus (MICAL-CT) has revealed prominent bands at 530 kDa, 330 kDa, 300 kDa, 200 kDa, and 125 kDa in lysates from wild-type embryos that increase in intensity in lysates from embryos harboring a chromosomal duplication that includes the MICAL locus. The three largest protein bands correspond to the predicted molecular weights of the three MICAL cDNAs (Terman, 2002).

MICAL protein is present in neuronal cell bodies, along axons, and in growth cones. MICAL immunostaining first appears in the nervous system at stage 13 and labels motor and CNS projections, and at later embryonic stages, it is present on axons that make up all motor axon pathways: the intersegmental nerve (ISN), the intersegmental nerves b and d (ISNb and ISNd), and the segmental nerves a and c (SNa and SNc). MICAL immunostaining is also present in segment boundaries at the position of muscle attachment sites and at low levels in the lateral cluster of chordotonal organs (Terman, 2002).

MICAL proteins have conserved protein domains with identical organization in all family members and a high degree of amino acid identity among these domains in different MICALs. There is one MICAL in Drosophila and three mammalian MICALs. The MICALs appear unique with respect to containing both calponin homology (CH) and LIM domains, in addition to their conserved N- and C-terminal regions. There is a family of MICAL-like (MICAL-L) proteins, members of which have a similar organization to MICALs but lack the region N-terminal to the CH domain. There is one MICAL-L protein in Drosophila (MICAL-like) and at least two family members in humans. D-MICAL-L cDNA and genomic DNA sequence information suggest that D-MICAL-L begins just N-terminal to the CH domain. Analysis of publicly available mammalian cDNA and genomic sequences suggests that human MICAL-L1 and MICAL-L2 are similar in overall domain organization to D-MICAL-L and do not contain the highly conserved ~500 aa MICAL N-terminal domain (Terman, 2002).

The high degree of conservation of the MICAL N terminus among family members (up to 62% identical between flies and humans) suggests that this domain is functionally important. Upon closer examination of the 500 aa conserved N-terminal region, a consensus dinucleotide binding sequence, GXGXXG was found, which is distinct from the sequence present in classical mononucleotide binding motifs. Further, this region contains three separate motifs found in flavoprotein monooxygenases (also called hydroxylases), a subclass of oxidoreductases. The amino acid sequence surrounding the GXGXXG motif matches perfectly the consensus sequence for the ADP binding region of flavin adenine dinucleotide (FAD) binding proteins (Rossmann fold or FAD fingerprint 1), and distinguishes this region from consensus NAD, or NADP binding folds. MICALs also have a well-conserved GD motif (FAD fingerprint 2) C-terminal to the FAD fingerprint 1 region, which is important for binding the ribose moiety of FAD. Finally, MICALs have the conserved DG motif between the FAD fingerprint 1 and 2 motifs that has been reported to be involved in binding the pyrophosphate moiety of FAD. Proteins with these consensus FAD binding regions use FAD in the catalysis of oxidation-reduction reactions. Flavoprotein monooxygenases are oxidoreductases (enzymes that catalyze oxidation and reduction reactions) that catalyze the insertion of one atom of molecular oxygen into their substrate using nucleotides as electron donors. These monooxygenases are also defined by their use of FAD as a coenzyme. Apart from these three consensus regions, monooxygenases vary significantly, reflecting the wide range of enzymes in this family and their variable substrate binding pockets also encompassed within this domain. However, MICALs and other monooxygenases show significant similarity within these three FAD binding regions and also similar spacing of these regions within the monooxygenase domain (Terman, 2002).

Biochemical and genetic analyses strongly suggest that MICALs contain functional FAD binding monooxygenase domains required for mediating plexin signaling. In support of this idea, inhibition of flavoprotein monooxygenase enzymatic activity dramatically attenuates semaphorin-mediated axon repulsion and growth cone collapse. However, though the inhibitor EGCG has a high degree of selectivity for flavoprotein monooxygenases, similar concentrations of EGCG inhibit other enzymes including steroid 5alpha-reductase, NADPH-cytochrome P450 reductase, telomerase, matrix metalloproteinases MMP-2 and MMP-9, and phenol sulfotransferase. Although most of these enzymes are unlikely to be expressed in the growth cones of DRG axons, potential nonspecific effects of these inhibitors cannot be ruled out despite their demonstrated selectivity for monooxygenases. Taken together with the in vivo Drosophila experiments showing a requirement for the MICAL FAD binding region in Sema-1a-mediated axon repulsion, these data suggest redox signaling plays an important role in vertebrate semaphorin-mediated axonal repulsion (Terman, 2002).

Flavoprotein monooxygenases specifically catalyze the oxidation of a number of substrates, and in some contexts they can function as oxidases and generate reactive oxygen species. These results suggest that MICALs are flavoproteins most similar to the flavoprotein monooxygenase family of oxidoreductases, but a complete understanding of the chemical nature of the reactions catalyzed by MICALs awaits future study and identification of substrates. The redox regulation of amino acid residues within signaling proteins (including kinases, phosphatases, small GTPases, guanylate cyclases, and adaptor proteins) and cytoskeletal proteins (including actin, actin binding proteins, intermediate filament proteins, and GAP-43) has been shown to modulate their function. In addition, oxidation of actin leads to disassembly of actin filaments, instability and collapse of actin networks, reduced ability of actin to interact with actin crosslinking proteins, and a decrease in the ability of actin monomers to form polymers. Finally, it is also interesting that MICALs have a putative actin filament binding domain (CH domain) and that MICAL-1 interacts with vimentin, an intermediate filament protein (Terman, 2002).

The proline-rich region of vertebrate MICAL-1 interacts with the SH3 domain of the adaptor protein CasL (HEF1) in nonneuronal cells. CasL, along with the related proteins p130Cas (see CAS/CSE1 segregation protein) and Efs (Sin), make up the Cas family of proteins that assembles and transduces intracellular signals that stimulate cell migration and axon outgrowth. These proteins have numerous protein-protein interaction domains, including a Src-homology 3 (SH3) domain, multiple SH2 binding sites in their substrate domain, several proline-rich motifs, and a C-terminal dimerization module. This structure suggests a role for Cas family proteins as docking molecules, and numerous interacting proteins have been identified, including kinases (e.g., FAK, Src, and Abl), phosphatases (e.g., PTP-1B and SHP2), GEFs (e.g., C3G), and adaptor proteins (e.g., Nck, Crk, Grb2, and 14-3-3). Studies indicate that Cas proteins localize mainly to focal adhesions and stress fibers and that they are required in integrin-dependent cell migration and actin filament assembly. Cas proteins, therefore, may play an important role in plexin-mediated repulsive and attractive guidance events (Terman, 2002).

F-actin dismantling through a redox-driven synergy between Mical and cofilin

Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF (actin-depolymerizing factor)/cofilins (encoded by the twinstar gene in Drosophila), sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. This study uncovered a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared with either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodelling, axon guidance and Semaphorin-Plexin repulsion. Mical and cofilin, therefore, form a redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties (Grintsevich, 2016).

This study found that Mical and cofilin function as a pair, synergizing in a Redox-dependent post-translational manner to disassemble F-actin and to control different cellular behaviors. Specifically, cofilin is a well-established actin regulatory protein and a relatively weak severer of F-actin. In contrast, Mical family Redox enzymes have only recently emerged downstream of Semaphorin-Plexin repellents as actin disassembly factors acting via the direct post-translational oxidation of actin. Previous work has also revealed that Mical, whose C-terminus associates with the intracellular portion of the Semaphorin transmembrane receptor plexin, binds with its N-terminal NADPH-dependent Redox domain to F-actin and selectively oxidizes actin's methionine-44 and 47 residues. It is proposed that Mical oxidation-induced changes in filament structure and/or dynamics improve cofilin's binding to actin filaments. This study also found that Mical-oxidized actin copolymers have different properties than unoxidized actin filaments. It is also known that the severing of actin filaments by cofilin is related to the mechanical properties of F-actin. The results support the idea that Mical uses oxidation to weaken the inter-actin (inter-protomer) contacts within filaments and these alterations dramatically speed up cofilin's ability to break/dismantle filaments. These results, therefore, uncover a previously unknown pathway of cellular F-actin disassembly and also present an unusual type of biological synergistic interaction -- one involving two different types of proteins (Mical and cofilin) and the Redox-dependent post-translational modification of a third protein (polymerized actin) (Grintsevich, 2016).

The results also shed new light on the mechanisms of action of both Mical and cofilin. They support a model that Mical and cofilin have been evolutionarily selected to work in tandem to ensure that even a low level of Mical activity in the presence of cofilin would facilitate F-actin disassembly, and vice versa. Moreover, unlike F-actin disassembly by cofilin, which promotes actin turnover by recirculation of monomers for polymerization, Mical post-translationally modifies actin, decreasing its capacity for re-polymerization until the oxidation is reversed. Thus, the Redox-driven synergy between Mical and cofilin not only rapidly disassembles F-actin but also generates post-translationally modified actin that re-assembles abnormally with a net effect of promoting F-actin instability. These results, therefore, provide important insights into how actin-based structures are rapidly and specifically dismantled in cells. Given their widespread overlapping expression patterns and diverse effects on cellular behaviors, this synergistic interaction between Mical and cofilin provides the molecular framework to rapidly dismantle multiple actin-based cellular structures (Grintsevich, 2016).

Retrograde semaphorin-plexin signalling drives homeostatic synaptic plasticity

Homeostatic signalling systems ensure stable but flexible neural activity and animal behaviour. Presynaptic homeostatic plasticity is a conserved form of neuronal homeostatic signalling that is observed in organisms ranging from Drosophila to human. Defining the underlying molecular mechanisms of neuronal homeostatic signalling will be essential in order to establish clear connections to the causes and progression of neurological disease. During neural development, semaphorin-plexin signalling instructs axon guidance and neuronal morphogenesis. However, semaphorins and plexins are also expressed in the adult brain. This study shows that semaphorin 2b (Sema2b) is a target-derived signal that acts upon presynaptic plexin B (PlexB) receptors to mediate the retrograde, homeostatic control of presynaptic neurotransmitter release at the neuromuscular junction in Drosophila. Further, Sema2b-PlexB signalling regulates presynaptic homeostatic plasticity through the cytoplasmic protein Mical and the oxoreductase-dependent control of presynaptic actin. It is proposed that semaphorin-plexin signalling is an essential platform for the stabilization of synaptic transmission throughout the developing and mature nervous system. These findings may be relevant to the aetiology and treatment of diverse neurological and psychiatric diseases that are characterized by altered or inappropriate neural function and behaviour (Orr, 2017).

Semaphorins are a large family of secreted or membrane-associated signalling proteins and plexins serve as signal-transducing semaphorin receptors. Semaphorin-plexin signalling was initially described as mediating growth cone collapse. But, semaphorin-plexin signalling is far more diverse. Notably, semaphorins and plexins continue to be expressed in the mature brain, where their function remains mostly unknown. Semaphorins have been shown to be synaptic signalling proteins, but the activity of semaphorins has been limited to the control of neuroanatomical synapse formation and elimination. This study demonstrates that semaphorin-plexin signalling achieves retrograde, trans-synaptic control of presynaptic neurotransmitter release and homeostatic plasticity (Orr, 2017).

A well-documented assay was used to induce presynaptic homeostatic plasticity (PHP), applying a sub-blocking concentration of the glutamate-receptor antagonist philanthotoxin-433 (PhTx; 15 μM) to significantly decrease the amplitude of average miniature excitatory postsynaptic potentials (mEPSPs; 0.3 μM [Ca2+]e) or miniature excitatory postsynaptic currents (mEPSCs; 1.5 μM [Ca2+]e). This postsynaptic perturbation induces a significant increase in presynaptic neurotransmitter release (the quantal content) that offsets the postsynaptic perturbation and restores normal muscle excitation. This offsetting increase in presynaptic neurotransmitter release is characteristic of PHP1. When this assay was used in larvae containing a null mutation in either the sema2b gene (sema2bC4) or the PlexB gene (PlexBKG0088), PHP was blocked. Consistent with this being a loss-of-function phenotype, heterozygous mutations (either sema2b/+ or PlexB/+) have normal PHP. Remarkably, a double-heterozygous mutant combination of sema2b/+ and PlexB/+ blocks PHP, consistent with both genes acting in concert to drive the expression of PHP (Orr, 2017).

The long-term maintenance of PHP was investigated and the involvement of other semaphorin or Plexin gene family members. Deletion of a non-essential glutamate-receptor subunit (GluRIIA) induces a long-lasting form of PHP1. Long-term PHP is blocked in a sema2b;GluRIIA double mutant as well as in GluRIIA larvae expressing transgenic RNA interference (RNAi) to knockdown PlexB selectively in motor neurons. Next, the effect of mutations was separately tested in all of the remaining semaphorin and Plexin genes encoded in the Drosophila genomet. The sema2b and PlexB mutants are the only mutants that show disruption of PHP (Orr, 2017).

Tissue-specific RNAi and transgenic rescue experiments were performed. Expression of UAS-Sema2b-RNAi in motor neurons (OK371-Gal4) had no effect on PHP, whereas expression in muscle (BG57-Gal4) blocked PHP. In addition, expression of UAS-sema2b in muscle rescues PHP in the sema2b-mutant background. Consistent with these data, sema2b was found to be expressed in muscle and Sema2b protein, expressed under endogenous promoter sequences, concentrates at postsynaptic membranes. Next, it was shown that motor neuron-specific expression of UAS-PlexB-RNAi blocks PHP, whereas muscle-specific expression does not. Motor neuron-specific expression of a previously characterized UAS-PlexBDN dominant-negative transgene, lacking the intracellular signalling domain, blocks PHP. RNA-sequencing analysis of purified motor neurons demonstrates PlexB expression in motor neurons. Finally, motor neuron-specific expression of a PlexB-myc transgene shows that PlexB traffics to the presynaptic nerve terminal. Taken together, these data indicate that Sema2b is a ligand originating in the muscle that acts via presynaptic PlexB to drive expression of PHP (Orr, 2017).

If Sema2b is a retrograde signal that acts upon the presynaptic PlexB receptor, then it should be possible to reconstitute this retrograde signalling by acute application of Sema2b protein. Purified Sema2b protein was acutely applied to the neuromuscular junction (NMJ) of sema2b mutants following PhTx treatment to induce PHP. Sema2b protein (100 nM) was found to completely restores PHP in the sema2b mutant, but fails to restore PHP in the PlexB mutant. In addition, application of Sema2b protein is sufficient to potentiate baseline release, and this effect is also dependent upon PlexB. Finally, a membrane-tethered UAS-sema2b transgene, expressed in muscle, fails to rescue PHP, even though it is concentrated on the postsynaptic membranes. Together, these results indicate that Sema2b is a secreted, postsynaptic ligand that acts upon presynaptic PlexB to enable the expression of PHP. The possibility is acknowledged that PlexB could require a presynaptic co-receptor of, as yet, unknown identity (Orr, 2017).

Given that acute application of Sema2b protein rescues PHP in the sema2b mutant, the failure of PHP in sema2b-mutant larvae cannot be a secondary consequence of altered NMJ development. Nonetheless, Sema2b-PlexB signalling is required for normal NMJ growth. Axon-targeting errors are rare at muscles 6/7, analysed at the third instar larval stage. This study demonstrated that the NMJs in sema2b and PlexB mutants are composed of fewer, larger synaptic boutons with no change in total NMJ area. The abundance of the active-zone-associated protein Bruchpilot (Brp) is unaltered in the sema2b mutant and the sema2b/+;;PlexB/+ double-heterozygous larvae, both of which block PHP. There is a significant decrease in total Brp staining in the PlexB mutant, an effect of unknown consequence. Qualitatively, the ring-like organization of Brp staining was similar across all genotypes, indicative of normal active-zone organization. Finally, there is no consistent difference in synapse ultrastructure across genotypes. Therefore, the Sema2b-PlexB-dependent control of bouton size may be a separate function of Sema2b-PlexB signalling, analogous to anatomical regulation by semaphorins in mammalian systems (Orr, 2017).

PHP occurs through the potentiation of the readily releasable pool (RRP) of synaptic vesicles. Application of PhTx induces a doubling of the apparent RRP in wild-type larvae, an effect that is disrupted in both sema2b and PlexB mutants. Failure to potentiate the RRP is also shown as a failure to maintain the cumulative EPSC amplitude after PhTx application. A strong genetic interaction was subsequently shown with a mutation in the presynaptic scaffolding gene rab3-interacting molecule (rim), a PHP gene. Heterozygous mutations in rim, or in sema2b or PlexB have no effect on PHP. However, double-heterozygous combinations of rim/+ with either sema2b/+ or PlexB/+ strongly impaired the expression of PHP (sema2b/+,rim/+) or abolished PHP (rim/+;;PlexB/+). These data do not, however, reflect direct signalling between PlexB and Rim (Orr, 2017).

To define how PlexB could modulate the RRP, known downstream signalling elements were tested. Mical is necessary for PHP. In Drosophila a single mical gene encodes a highly conserved multi-domain cytoplasmic protein that mediates actin depolymerization, achieved through redox modification of a specific methionine residue (Met44) in actin. Notably, prior genetic evidence has placed Mical downstream of both PlexA and PlexB signalling during axon guidance (Orr, 2017).

An analysis of multiple mical mutations in larvae as well as transgenic rescue animals demonstrates that mical is necessary presynaptically for PHP. Mical protein is present presynaptically and presynaptic expression of a Mical-resistant UAS-Actin5C transgene, which interferes with Mical-mediated actin depolymerization, blocks PHP. This transgenic protein also concentrates within presynaptic boutons. Additional experiments reveal that the homeostatic expansion of RRP is blocked in mical mutants and when Mical-resistant UAS-Act5 is expressed presynaptically. Strong genetic interactions were found between mical and both the PlexB and rim mutants. Finally, anatomical experiments demonstrate that active zones are normal in the mical mutant, including in both light and electron microscopy experiments. It is proposed that Mical enables PlexB-mediated control of the RRP through the regulation of presynaptic actin (Orr, 2017).

For half a century, evidence has underscored the importance of target-derived, retrograde signalling that controls presynaptic neurotransmitter release1. Gene discovery, based on forward genetics, indicates that PHP is controlled by the coordinated action of at least three parallel signalling systems. These data regarding Sema2b, PlexB and Mical can be generalized, then semaphorin-plexin signalling could represent a platform for retrograde, trans-synaptic, homeostatic control of presynaptic release, thereby stabilizing synaptic transmission and information transfer throughout the nervous systems of organisms ranging from Drosophila to humans (Orr, 2017).

Drosophila MICAL regulates myofilament organization and synaptic structure

In a postembryonic screen designed to identify mutations that affect the structure, maintenance and remodeling of neuromuscular synapses, a complementation group was isolated that harbors point mutations in Drosophila mical. Synaptic boutons in mical mutant NMJs have the tendency to cluster around initial nerve-muscle contact sites and along synaptic branches and therefore fail to spread properly along the muscle fiber. The dense bouton accumulations in mical mutants develop postembryonically, starting in first instar larvae. Embryonic NMJs were not visibly altered suggesting that mical mutations do not affect synapse formation per se but synaptic remodeling during the period of rapid larval growth (Beuchle, 2007).

Human MICAL-1 was first discovered by Far Western screening using the SH3-domain of human CasL to probe an expression library. Cas family proteins generally serve as docking molecules that assemble intracellular complexes to transduce extracellular signals, especially at focal adhesions where they regulate the anchorage of integrins to actin-rich stress fibers. MICAL-1 was therefore implicated in the regulation of the cytoskeleton (Suzuki, 2002). Human MICAL-1 has also been shown to interact with Rab1, a small GTPase that plays a role in vesicle trafficking (Weide, 2003), and to assemble into a filamentous network when transfected into tissue culture cells (Suzuki, 2002; Fischer, 2005). In Drosophila, MICAL has been shown to regulate embryonic motor axon guidance by binding to Plexin A, a co-receptor for Semaphorin 1A (Terman, 2002). Consequently, MICAL was suggested to control cytoskeletal changes in migrating axons in response to repulsive environmental cues. Embryonic motor axon guidance errors can persist into larval stages, with NMJs forming at unusual positions or being entirely absent at a high percentage of larval muscles. In mical mutant alleles, however, NMJs generally formed at their wild-type positions, apart from occasional errors on muscles innervated by the SNa pathway. Therefore motor axon trajectories were examined in mutant embryos; relatively mild but statistically significant projection errors were observed. When the original mical deficiency allele was examined, however, motor axon guidance and innervation defects were more pronounced. A possible explanation for this discrepancy could be that Df(3R)swp2mical deletes 12 annotated genes, whereas the currently isolated alleles carry single point mutations (Beuchle, 2007).

Deleterious point mutations were identified in the mical gene in all seven EMS-induced alleles, the most interesting of which are single, non-truncating missense mutations in micalI1367 and micalG158. The mutation in micalI1367 (G134R) alters a highly conserved glycine residue in the monooxygenase domain that is necessary for binding the co-factor FAD (Nadella, 2005; Siebold, 2005). Loss of the ability to interact with FAD likely disrupts the function of the monooxygenase. In fact, point mutations in this motif have been shown to abolish enzymatic activity in related monooxygenases. FAD and the monooxygenase domain are therefore critically required for MICAL function. The mutation in micalG158 (G433S) disrupts a glycine residue located in FAD fingerprint 2 that interacts with the ribose moiety of FAD. This rather subtle change alters the subcellular localization of MICAL in muscles and neurons. MICAL accumulates in the cytoplasm of these cells and is not transported to peripheral locations, e.g. along axons. In addition to binding to FAD, this motif may therefore serve as a checkpoint to control for fully folded and functional MICAL proteins. The remaining mutations are stop codons that truncate the protein prior to the LIM domain, suggesting that the LIM-domain and/or other sequences further downstream are equally important for MICAL function, and that the monooxygenase domain by itself is not sufficient. Due to the nature of its protein–protein interaction domains, MICAL has been suggested to act as a molecular platform that interacts with substrate proteins to bring them in close proximity of its monooxygenase domain. The enzymatic modification of specific substrates may therefore be crucial for MICAL function (Beuchle, 2007).

During embryogenesis MICAL is broadly and dynamically expressed in a variety of tissues. With respect to neuromuscular development, MICAL is initially expressed in muscles and, starting with stage 15, in neurons of the central nervous system (CNS). MICAL accumulates preferentially in axons of the CNS and in peripheral regions of muscle fibers, including muscle attachment sites and the cleft between neighboring muscles. MICAL is present in muscles at least until the end of embryogenesis but is not markedly detected in muscles of third instar larvae. This is also consistent with punctate MICAL accumulations in embryonic but not in larval muscles of micalG158 mutants. At NMJs of third instar larvae, MICAL seems therefore to be expressed predominantly at presynaptic sites. This conclusion is additionally supported by the results of the immunostainings, which detected MICAL predominantly in presynaptic terminals. However, the possibility cannot be excluded that presynaptic MICAL obscures postsynaptically localized proteins. If MICAL has a slow turnover rate, for example, it is quite possible that low levels of embryonic proteins remain stable at postsynaptic sides of NMJs or in muscles until late larval stages. To find further evidence on which side of the NMJ MICAL is required and to determine if the short isoform of MICAL is able to rescue the mutant phenotype, it was expressed in neurons or muscles in heteroallelic mical mutant animals. Neither pre- nor postsynaptic expression could substantially rescue the synaptic or flightless phenotype. One reason could be that the expression level of exogenous MICAL was quite low and higher protein levels would have been required to improve the rescue capability. Since at least three isoforms of MICAL are expressed in embryos, it is also possible that one or both of the other isoforms are functionally required. Indeed, mutations in micalI666 and micalG56 are located in exon 9 and 10, respectively, which should be expressed only in the middle and long isoform, indicating that the short isoform cannot compensate for all MICAL functions. As a third possibility, MICAL function may be required simultaneously on both sides of NMJs to coordinate neuromuscular development (Beuchle, 2007).

While MICAL was identified initially through a neuromuscular phenotype, and taking into account the relatively mild guidance defects, the strongest and most prevailing defects were observed in striation and sarcomeric patterning of muscle fibers. MICAL’s primary role may therefore reside in the spatial organization of myofilaments, or more specifically, in the assembly of myofilaments into a sarcomeric pattern. First, the earliest detectable abnormal filaments in mutant first instar larvae resemble embryonic filaments at stage 15–16, which form long needle-like structures prior to the formation of sarcomeres. Second, known mutations in muscle-specific isoforms of Myosin, Actin or Troponin-T that affect sarcomere assembly, lead to loose and scattered myofilaments in the sarcoplasm of indirect flight muscles. Third, MICAL is expressed in muscles at the time of sarcomere assembly but it could not be detected in considerable amounts in muscles of third instar larvae when it should be expressed if it has a function in the maintenance of sarcomeres. With respect to its subcellular localization in muscles, it is worth noting that MICAL is not homogenously distributed in the sarcoplasm but preferentially accumulates at the plasmamembrane and muscle attachment sites. It has been proposed that myofibrillogenesis starts beneath the plasmamembrane. MICAL would therefore be localized correctly to be involved in myofibril assembly. For these reasons, it is more likely that MICAL is required for the proper assembly of sarcomeres rather than for their maintenance or remodeling. However, since the filament phenotype worsens over time, the possibility cannot be excluded that MICAL plays a role in the maintenance of sarcomeres or in the proteolytic destruction of non-assembled muscle filaments. It is interesting to emphasize that non-functional MICAL affects various sarcomeric proteins differently. While myosin and actin filaments were strongly disorganized, the sarcomeric protein D-Titin showed an almost wild-type distribution. Titins are enormous proteins that span the entire half-sarcomere, with their N-terminus being inserted in Z-discs and their C-terminus in M-lines. Titins may therefore function as a molecular scaffold that directs sarcomere assembly. Since the Titin-based sarcomeric backbone seems to be relatively intact in mical mutants, mutations in mical may uncouple sequential steps in the assembly process of sarcomeres (Beuchle, 2007).

For myofilament organization, MICAL appears to be required in muscles only. Expression of mical double-stranded RNA in specific tissues to downregulate endogenous MICAL revealed that only muscle-specific knock-down could mimic the myofilament phenotype and flight defects of mical mutants. It was not possible, however, to reproduce the neuromuscular phenotype in these experiments, regardless of whether MICAL expression was inhibited pre- or postsynaptically. Possible explanations for this observation could be that the myofilament defects were too weak to interfere with synaptic growth or that the inhibition of MICAL expression was incomplete in neurons despite the use of two different neuronal Gal4-lines. Variability in the efficiency of MICAL downregulation was also observed with muscle-specific Gal4-lines. Whereas Mef2-Gal4 reproducibly disrupted muscle striation, the activity of 24B-Gal4 was much less potent. A requirement of MICAL in muscles is further supported by the notion that the assembly of myofilaments is generally considered to be a cell-autonomous process and independent of motoneuronal input, since it occurs in isolated myocytes, cardiomyocytes and myoblast cell lines that are cultured in the absence of any neurons. Furthermore, if MICAL would be required presynaptically for myofilament organization, muscles that lack NMJs should display myofilament defects. In Drosophila sidestep mutants, a high percentage of ventral muscles permanently lack NMJs due to embryonic motor axon bypass phenotypes. However, sarcomeric organization and muscle striation was normal in non-innervated muscles of sidestep mutants, providing further evidence that MICAL is required in muscles to regulate the higher order assembly of myofilaments. The disturbances in the architecture of contractile filaments in mical mutants do not inhibit muscle contraction completely. They seem to interfere, however, with the speed and vigor of contraction cycles, which may be less problematic for larval muscles but may drastically affect metabolically active muscles, such as the highly structured indirect flight muscles, which would explain why mical mutant adults are unable to fly (Beuchle, 2007).

In wild-type larvae, synaptic boutons are located on the surface of the muscle fiber and are fully wrapped by an insulating layer of subsynaptic reticulum (SSR). The SSR begins to form in first instar larvae. Initial membrane invaginations of the postsynaptic membrane develop into a multiply folded stack of membrane cisternae during larval life. In mical mutants, the SSR appears almost completely absent. The remaining invaginations are flattened to sac-like cavities and widely dispersed in the postsynaptic cytoplasm. The SSR has no assigned function but apart from an exchange of metabolites between the extracellular space and the muscle fiber it may represent a specialized region that facilitates synaptic growth. Repetitive imaging of the same set of NMJs during consecutive stages of development has revealed that sprouting buds of dividing boutons grow out and into the surrounding SSR. Newly forming boutons therefore have to displace the membrane stacks of the SSR in order to gain new synaptic territory. Remnants of the SSR will eventually be adopted by the new bouton. In this view, and due to its flexible and dilatable structure, one of the functions of the SSR could be to facilitate bouton outgrowth. Bouton division and outgrowth seems therefore to be facilitated by two factors: superficial location and SSR envelopment. In mical mutants, synaptic boutons are not superficially located and not wrapped by a soft layer of SSR. They are firmly embedded in the muscle tissue. Actin and myosin filaments accumulate in postsynaptic regions and beneath the sarcolemma. As one possibility, it is proposed that the detached filaments corrupt the development of the SSR. The network of interdigitated cytoskeletal elements around synaptic boutons would then make it difficult for budding boutons to push out into the muscle tissue and would thus hinder the spreading of synaptic boutons across the muscle surface during larval growth. Although this model favors postsynaptic functions of MICAL, at present, the possibility that MICAL has also presynaptic functions, which control synaptic structure, cannot be excluded. Under this scenario, the myofilament phenotype is likely to be independent of the synaptic phenotype. Further studies including high efficient tissue-specific inhibition of MICAL expression, will hopefully allow determination of which of these possibilities is correct. Regardless of whether MICAL functions pre- or postsynaptically, the data presented here show that MICAL is a critical regulator of myofilament architecture and synaptic structure during postembryonic development (Beuchle, 2007).

Drosophila Valosin-Containing Protein is required for dendrite pruning through a regulatory role in mRNA metabolism

The dendritic arbors of the larval Drosophila peripheral class IV dendritic arborization neurons degenerate during metamorphosis in an ecdysone-dependent manner. This process-also known as dendrite pruning-depends on the ubiquitin-proteasome system (UPS), but the specific processes regulated by the UPS during pruning have been largely elusive. This study shows that mutation or inhibition of Valosin-Containing Protein (VCP; termed TER94 by FlyBase), a ubiquitin-dependent ATPase whose human homolog is linked to neurodegenerative disease, leads to specific defects in mRNA metabolism and that this role of VCP is linked to dendrite pruning. Specifically, it was found that VCP inhibition causes an altered splicing pattern of the large pruning gene Molecule interacting with CasL and mislocalization of the Drosophila homolog of the human RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). These data suggest that VCP inactivation might lead to specific gain-of-function of TDP-43 and other RNA-binding proteins. A similar combination of defects is also seen in a mutant in the ubiquitin-conjugating enzyme ubcD1 (Effete) and a mutant in the 19S regulatory particle of the proteasome, but not in a 20S proteasome mutant. Thus, these results highlight a proteolysis-independent function of the UPS during class IV dendritic arborization neuron dendrite pruning and link the UPS to the control of mRNA metabolism (Rumpf, 2014).

To achieve specific connections during development, neurons need to refine their axonal and dendritic arbors. This often involves the elimination of neuronal processes by regulated retraction or degeneration, processes known collectively as pruning. In the Drosophila, large-scale neuronal remodeling and pruning occur during metamorphosis. For example, the peripheral class IV dendritic arborization (da) neurons specifically prune their extensive larval dendritic arbors, whereas another class of da neurons, the class III da neurons, undergo ecdysone- and caspase-dependent cell death. Class IV da neuron dendrite pruning requires the steroid hormone ecdysone and its target gene SOX14, encoding an HMG box transcription factor. Class IV da neuron dendrites are first severed proximally from the soma by the action of enzymes like Katanin-p60L and Mical that sever microtubules and actin cables, respectively. Later, caspases are required for the fragmentation and phagocytic engulfment of the severed dendrite remnants . Another signaling cascade known to be required for pruning is the ubiquitin-proteasome system (UPS). Covalent modification with the small protein ubiquitin occurs by a thioester cascade involving the ubiquitin-activating enzyme Uba1 (E1), and subsequent transfer to ubiquitin-conjugating enzymes (E2s) and the specificity-determining E3 enzymes. Ubiquitylation of a protein usually leads to the degradation of the modified protein by the proteasome, a large cylindrical protease that consists of two large subunits, the 19S regulatory particle and the proteolytic 20S core particle. Several basal components of the ubiquitylation cascade—Uba1 and the E2 enzyme ubcD1—as well as several components of the 19S subunit of the proteasome have been shown to be required for pruning, as well as the ATPase associated with diverse cellular activities (AAA) ATPase Valosin-Containing Protein (VCP) (CDC48 in yeast, p97 in vertebrates, also known as TER94 in Drosophila), which acts as a chaperone for ubiquitylated proteins. Interestingly, autosomal dominant mutations in the human VCP gene cause hereditary forms of ubiquitin-positive frontotemporal dementia (FTLD-U) and amyotrophic lateral sclerosis (ALS). A hallmark of these diseases is the occurrence of both cytosolic and nuclear ubiquitin-positive neuronal aggregates that often contain the RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). It has been proposed that ubcD1 and VCP promote the activation of caspases during dendrite pruning via degradation of the caspase inhibitor DIAP1. However, mutation of ubcD1 or VCP inhibit the severing of class IV da neuron dendrites from the cell body, whereas in caspase mutants, dendrites are still severed from the cell body, but clearance of the severed fragments is affected. This indicates that the UPS must have additional, as yet unidentified, functions during pruning (Rumpf, 2014).

This study further investigated the role of UPS mutants in dendrite pruning. vcp mutation was shown to lead to a specific defect in ecdysone-dependent gene expression, as VCP is required for the functional expression and splicing of the large ecdysone target gene molecule interacting with CasL (MICAL). Concomitantly, mislocalization of Drosophila TDP-43 and up-regulation of other RNA-binding proteins were observed, and genetic evidence suggests that these alterations contribute to the observed pruning defects in VCP mutants. Defects in MICAL expression and TDP-43 localization are also induced by mutations in ubcD1 and in the 19S regulatory particle of the proteasome, but not by a mutation in the 20S core particle, despite the fact that proteasomal proteolysis is required for dendrite pruning, indicating the requirement for multiple UPS pathways during class IV da neuron dendrite pruning (Rumpf, 2014).

Class IV da neurons have long and branched dendrites at the third instar larval stage. In wild-type animals, these dendrites are completely pruned at 16-18 h after puparium formation (h APF). VCP mutant class IV da neurons were generated by the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique for clonal analysis. Mutant vcp26-8 class IV da neurons displayed strong pruning defects and retained long dendrites at 16 h APF. Expression of an ATPase-deficient dominant-negative VCP protein (VCP QQ) under the class IV da neuron-specific driver ppk-GAL4 recapitulated the pruning phenotype and also led to the retention of long and branched dendrites at 16 h APF. VCP inhibition also causes defects in class III da neuron apoptosis. This combination of defects in both pruning and apoptosis is reminiscent of the phenotypes caused by defects in ecdysone-dependent gene expression. Indeed, overexpression of the transcription factor Sox14, which induces pruning genes, led to a nearly complete suppression of the pruning phenotype caused by VCP QQ. This genetic interaction suggested that VCP might be required for the expression of one or several ecdysone target genes during pruning (Rumpf, 2014).

How could VCP be linked to Sox14? The suppression of the vcp mutant phenotype by Sox14 overexpression could be achieved in one of several ways. Sox14 could be epistatic to VCP -- that is, VCP could be required for functional Sox14 expression -- and this effect would be mitigated by Sox14 overexpression. However, VCP could also be required for the expression of one or several Sox14 target genes, and enhanced Sox14 expression could overcome this requirement either via enhanced induction of one or several particular targets or via enhanced induction of other pruning genes, in which case Sox14 would be a bypass suppressor of VCP QQ. To distinguish between these possibilities, the effects were assessed of VCP inhibition on the expression of known genes in the ecdysone cascade required for pruning in class IV da neurons. Class IV da neuron pruning is governed by the Ecdysone Receptor B1 (EcR-B1) isoform, which in turn directly activates the transcription of Sox14 and Headcase (Hdc), a pruning factor of unknown function. Sox14, on the other hand, activates the transcription of the MICAL gene encoding an actin-severing enzyme. In immunostaining experiments, VCP QQ did not affect the expression of EcR-B1, Sox14, or Hdc at the onset of the pupal phase. However, the expression of Mical was selectively abrogated in class IV da neurons expressing VCP QQ, or in vcp26-8 class IV da neuron MARCM clones . These data indicated that VCP might affect dendrite pruning by regulating the expression of the Sox14 target gene Mical, indicating that Sox14 might act as a bypass suppressor of VCP QQ (Rumpf, 2014).

How could VCP inhibition suppress Mical expression? To answer this question, whether Mical mRNA could still be detected in class IV da neurons expressing VCP QQ was assessed. To this end, enzymatic tissue digestion and FACS sorting were used to isolate class IV da neurons from early pupae (1-5 h APF). Total RNA was then extracted from the isolated neurons, and the presence of Mical mRNA expression was assessed by RT-PCR, using control samples or samples from animals expressing VCP QQ under ppk-GAL4. The Mical gene is large (~40 kb) and spans multiple exons that are transcribed to yield a ~15 kb mRNA. To detect Mical cDNA, primer pairs spanning several exons were used for two different regions of Mical mRNA, exons 14-16 and exons 8-12. [MICAL is on the (-) strand, but the exon numbering denoted by Flybase follows the direction of the (+) strand. Therefore, exons 14-16 are upstream of exons 8-12, and the latter are closer to the 3' end of MICAL mRNA.] MICAL mRNA was detectable upon VCP inhibition in these extracts with a primer pair spanning exons 14-16. The second primer pair spanning exons 8-12 also detected MICAL mRNA in both samples, but the RT-PCR product from the VCP QQ-expressing class IV da neurons had a larger molecular weight. Sequencing of the PCR products indicated that MICAL mRNA from VCP QQ-expressing class IV da neurons contained exon 11, which was not present in Mical mRNA from the control sample. Exon 11 is absent from all predicted MICAL splice isoforms except for a weakly supported isoform designated 'Mical-RM'. It introduces a stop codon into MICAL mRNA that would lead to the truncation of the C-terminal 1,611 amino acids from Mical protein. This portion of Mical protein contains several predicted protein interaction domains such as a proline-rich region, a coiled-coil region with similarity to Ezrin/Radixin/Moesin (ERM) domains, and a C-terminal PDZ-binding motif, and is required for the interaction between Mical and PlexinA. In addition, the truncated region contains the epitope for the antibody used in the immunofluorescence experiments, thus explaining the observed lack of Mical expression upon VCP inhibition. Given that a mutant of Mical with a smaller C-terminal truncation (compared with the one induced by VCP inhibition) was not sufficient to rescue the class IV da neuron dendrite pruning defect of mical mutants, disruption of VCP function likely results in expression of a truncated Mical protein without pruning activity. Taken together, these data suggest that the observed defect in MICAL mRNA splicing contributes significantly to the pruning defects of VCP mutants (Rumpf, 2014).

How is VCP linked to alternative splicing of MICAL mRNA? A plausible mechanism for the control of an alternative splicing event would be the modulation of specific (pre)mRNA-binding proteins. VCP has recently been linked to several RNA-binding proteins: human autosomal dominant VCP mutations cause frontotemporal dementia or ALS with inclusion bodies that contain aggregated human TDP-43; a genetic screen in Drosophila identified the RNA-binding proteins Drosophila TDP-43, HRP48, and x16 as weak genetic interactors of the dominant effects of VCP disease mutants; and HuR (a human homolog of the neuronal Drosophila RNA-binding protein elav) was recently shown to bind human VCP. Of these, TDP-43 and also elav have been linked to alternative splicing in various model systems, including Drosophila. Therefore this study used available specific antibodies to assess the levels and distribution of Drosophila TDP-43 (hereafter referred to as TDP-43) and elav. TDP-43 has previously been shown to localize to the nucleus in Drosophila motoneurons and mushroom body Kenyon cells. Surprisingly, TDP-43 was largely localized to the cytoplasm in class IV da neurons, where it was enriched in a punctate pattern around the nucleus, with only a small fraction also detectable in the nucleus, a localization pattern that could be reproduced with transgenic N-terminally HA-tagged TDP-43. Elav is a known nuclear marker for Drosophila neurons; in class IV da neurons, it was somewhat enriched in nuclear punctae. The effects of VCP inhibition on these two RNA-binding proteins was assessed. Elav localization did not change notably upon VCP QQ expression. Strikingly, TDP-43 became depleted from the cytoplasm of class IV da neurons and relocalized to the nucleus upon VCP QQ expression. Closer inspection revealed that TDP-43 in VCP-inhibited neurons was now enriched in nuclear dots that often also exhibited increased elav staining. The relocalization of TDP-43 from the cytoplasm to the nucleus was also observed in vcp26-8 mutant class IV da neuron MARCM clones. Importantly, quantification and normalization of TDP-43 levels showed that VCP inhibition did not alter the absolute levels of TDP-43, suggesting that the observed effects were not a consequence of a defect in TDP-43 degradation. In fact, the only manipulation that resulted in a mild but significant increase in TDP-43 levels -- but without a change in localization -- was the expression of an RNAi directed against the autophagy factor ATG7, perhaps reflecting the degradation of cytoplasmic RNA granules through the autophagy pathway (Rumpf, 2014).

It was next asked whether manipulation of TDP-43 would affect class IV da neuron dendrite pruning. A previously characterized TDP-43 mutant, TDP-43 Q367X (28-128">28), did not display pruning defects, but overexpression of TDP-43 led to strong dendrite pruning defects at 16 h APF. In support of the hypothesis that TDP-43 acts in the same or a similar pathway as VCP during dendrite pruning, it was also found that a more weakly expressed TDP-43 transgene (UAS-TDP-43weak) and VCP A229E, a weakly dominant-active VCP allele corresponding to a human VCP disease mutation, exhibited a synergistic inhibition of pruning when coexpressed. Interestingly, manipulation of elav gave very similar results as with TDP-43: elav down-regulation by RNAi did not affect pruning, but elav overexpression led to highly penetrant pruning defects (Rumpf, 2014).

To exclude the possibility that the pruning defects induced by TDP-43 or elav overexpression were due to long-term expression and aggregation of RNA-binding proteins, TDP-43 and elav overexpression was also induced acutely (24 h before the onset of pupariation). Pruning was still inhibited in these cases. Also, overexpression of several other RNA-binding proteins did not cause pruning defects, with two exceptions: a UAS-carrying P-element in the promotor of the adjacent x16 and HRP48 genes caused a strong pruning defect when expression was induced in class IV da neurons, and levels of a GFP protein trap insertion into the x16 gene were also markedly increased in class IV da neurons expressing VCP QQ, possibly indicating a role for VCP in x16 degradation. In further support of an involvement of VCP with RNA-binding proteins during neuronal pruning processes, it was also found that VCP is required for mushroom body γ neuron axon pruning and induces the accumulation of Boule, an RNA-binding protein that had previously been shown to inhibit γ neuron axon pruning when overexpressed. Thus, the data suggest that VCP regulates a specific subset of RNA-binding proteins and that this regulatory role of VCP is associated with its role in pruning (Rumpf, 2014).

As VCP is an integral component of the UPS, it was next asked whether the role of VCP in MICAL regulation and TDP-43 localization was also dependent on ubiquitylation and/or the proteasome. To address this question, Mical levels and TDP-43 distribution was assessed in UPS mutants with known pruning defects. An ubiquitylation enzyme known to be required for pruning is the E2 enzyme ubcD1. When TDP-43 localization was assessed in larval ubcD1D73 mutant class IV da neurons, TDP-43 was again localized to the nucleus in these cells. Furthermore, a pronounced reduction of Mical expression in ubcD1D73 mutant class IV da neurons was noted during the early pupal stage, indicating that ubiquitylation through ubcD1 is involved in the regulation of TDP-43 localization and Mical expression (Rumpf, 2014).

TDP-43 localization and Mical expression were assessed in proteasome mutants. A previously characterized mutant in the Mov34 gene encoding the 19S subunit Rpn8 was used. TDP-43 was again relocalized to the nucleus in Mov34 mutant class IV da neurons, and Mical expression was absent from Mov34 mutant class IV da neurons at 2 h APF. To rigorously address whether proteasomal proteolysis was also required for TDP-43 localization and Mical expression, the effect was assessed of Pros261, a previously characterized mutation in the 20S core particle subunit Prosβ6. In contrast to Mov34 mutant class IV da neurons, Pros261 mutant class IV da neurons displayed cytoplasmic TDP-43 localization, and robust Mical expression was detected in these neurons at 2 h APF. Thus, although ubiquitylation and the 19S proteasome are both required for Mical expression and normal TDP-43 localization, proteolysis through the 20S core particle of the proteasome is not. Importantly, Pros261 MARCM class IV da neurons showed strong dendrite pruning defects at 16 h APF, as did expression of RNAi constructs directed against subunits of the 20S core particle (Rumpf, 2014).

These data indicate that there must be several ubiquitin- and proteasome-dependent pathways that are required for dendrite pruning: one pathway requires ubcD1, VCP, and the 19S regulatory particle of the proteasome, but not the 20S core particle. This pathway regulates MICAL expression. A second UPS pruning pathway does depend on proteolysis through the 20S core. In an E3 ubiquitin ligase candidate screen, cul-1/lin19 was identified as a pruning mutant. Cul-1 encodes cullin-1, a core component of a class of multisubunit ubiquitin ligases known as SCF (for Skp1/Cullin/F-box) ligases. Class IV da neurons mutant for cul-1 or class IV da neurons expressing an RNAi construct directed against cul-1 had not pruned their dendrites at 16 h APF. However, unlike with VCP, ubcD1, and Mov34, cul-1 mutation did not affect Mical expression at 2 h APF, indicating that cullin-1 is not a component of the VCP-dependent UPS pathway involved in splicing and might thus be a component of a proteolytic UPS pathway. In support of this notion, a recent report independently identified cul-1 as a pruning mutant and associated it with protein degradation (Rumpf, 2014).

It has been proposed that the E2 enzyme ubcD1 and VCP would act to activate caspases during pruning. However, the dendrite pruning defects caused by those UPS mutants are much stronger than the phenotypes caused by caspase inactivation, which mostly causes a delay in the phagocytic uptake of severed dendrites by the epidermis. Although it cannot be excluded that ubcD1 and VCP contribute to caspase activation during pruning, the new mechanism proposed in this study -- control of RNA-binding proteins and MICAL expression -- likely makes a much stronger contribution to the drastic pruning phenotypes of UPS mutants (Rumpf, 2014).

How precisely do VCP, ubcD1, and the 19S proteasome contribute to MICAL expression? The data indicate that VCP inhibition causes missplicing of MICAL mRNA that likely leads to the expression of an inactive Mical protein variant. At the same time, VCP inhibition leads to the mislocalization of TDP-43, and possibly the dysregulation of a number of other RNA-binding proteins. The fact that these phenotypes correlate in the vcp, ubcD1, and Mov34 mutants gives a strong indication that they are related. TDP-43 had previously been identified as a suppressor of the toxicity induced by a weak VCP disease allele in the Drosophila eye. In class IV da neurons, reducing the amounts of TDP-43 (with a deficiency) or elav (by RNAi) did not ameliorate the pruning defect induced by VCP inhibition. Therefore, the possibility cannot be excluded that the two proteins act in parallel rather than in an epistatic fashion. As VCP has been shown to remodel protein complexes that contain ubiquitylated proteins and is structurally similar to the 19S cap, it is interesting to speculate that VCP and the 19S cap might alter the subunit composition of ubiquitylated TDP-43-containing complexes of RNA-binding proteins, and that this activity—rather than a direct action on TDP-43 (or maybe also elav) alone—might lead to both MICAL missplicing and TDP-43 mislocalization (Rumpf, 2014).

Interestingly, autosomal dominant mutations in human VCP cause frontotemporal dementia and ALS, a hallmark of which is the formation of aggregates that contain TDP-43. Most of these aggregates are cytoplasmic (and contain TDP-43 that has relocalized from the nucleus to the cytoplasm), but VCP mutations also induce TDP-43 aggregation in the nucleus, a situation that might be similar to the situation caused by VCP inhibition in class IV da neurons. Although human VCP disease mutations have been proposed to act as dominant-active versions of VCP with enhanced ATPase activity, both the disease allele and the dominant-negative ATPase-dead VCP QQ mutant cause class IV da neuron pruning defects and TDP-43 relocalization to the nucleus of class IV da neurons and therefore act in the same direction. It is thought that VCP can only bind substrates when bound to ATP, and will release bound substrates upon ATP hydrolysis. Thus, it is conceivable that the phenotypic outcome of inhibiting the ATPase (no substrate release) should be similar to that of ATPase overactivation (reduced substrate binding or premature substrate release): in both cases, a substrate protein complex would not be properly remodeled (Rumpf, 2014).

Taken together, these results indicate the existence of a nonproteolytic function of VCP and the UPS in RNA metabolism and highlight its importance during neuronal development (Rumpf, 2014).


REFERENCES

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Siebold, C., et al. (2005). High-resolution structure of the catalytic region of MICAL (molecule interacting with CasL), a multidomain flavoenzyme-signaling molecule, Proc. Natl. Acad. Sci. 102: 16836-16841. PubMed ID: 16275925

Suzuki, T., Nakamoto, T., Ogawa, S., Seo, S., Matsumura, T., Tachibana, K., Morimoto, C. and Hirai, H. (2002). MICAL, a novel CasL interacting molecule, associates with vimentin. J. Biol. Chem. 277: 14933-14941. PubMed ID: 11827972

Terman, J. R., et al. (2002). MICALs, a family of conserved flavoprotein oxidoreductases, function in Plexin-mediated axonal repulsion. Cell 109: 887-900. PubMed ID: 12110185

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date revised: 23 December 2017

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