futsch expression was analyzed in mutant backgrounds that specifically alter the 22C10 expression pattern. The Zn finger transcription factor Tramtrack acts as a repressor of 22C10 antigen expression, and in tramtrack mutant embryos, high levels of 22C10 antigen are found in the mesoderm (Giesen, 1997). Consistent with Futsch being the 22C10 antigen, the expression of futsch mRNA is also drastically increased in tramtrack mutant embryos. In a second experiment, a ftz-GAL4 driver line and EP(X)1419 were used to drive ectopic futsch expression in a pair-rule expression pattern. Ectopic expression of the 22C10 antigen correlates with ectopic expression of the deduced futsch transcript, further supporting the possibility that futsch encodes the 22C10 antigen (Hummel, 2000).
Nerfin-1 is a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. The neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development or in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oα47A, Lar, and futsch<, genes whose encoded proteins participate in these early navigational events (Kuzin, 2005).
Given the axon guidance defects in nerfin-1null embryos and the fact that Nerfin-1 is a Zn-finger nuclear protein, it was hypothesized that Nerfin-1 may be required for the correct expression of genes involved in axon guidance. Accordingly, the embryonic expression profiles of over 35 genes that have been shown to play important roles in axon guidance were examined. Included in the candidate screen were genes encoding transcription factors, RNA-binding proteins, cell surface receptor proteins, their ligands, signal transduction proteins, and components of the cytoskeleton. Homozygous nerfin-1null embryos were identified by the absence of Nerfin-1 immunoreactivity. Whole-mount in situ hybridization and/or protein immunostaining for altered spatial or temporal expression in nerfin-1null embryos identified six genes that require nerfin-1 function to achieve full wild-type expression levels (Kuzin, 2005).
Two genes involved in anterior vs. posterior commissure choice, those encoding the receptor tyrosine kinase Derailed, and its ligand Wnt5, both required nerfin-1 for full expression. In the absence of nerfin-1, ventral cord expression levels of Robo and Robo3 were unaffected; however, Robo2 expression levels were significantly reduced. Expression of Slit, the ligand for Robo receptors, and Commissureless, a factor responsible for clearing Robo receptors from commissural axons, was unaffected in nerfin-1null embryos (Kuzin, 2005).
Loss of nerfin-1 function also significantly delayed and/or reduced the early expression of the neuron-specific microtubule-associated MAP1B-like gene futsch. futsch expression is normally activated in newborn neurons starting at stage 11; however, in nerfin-1null embryos expression is first detected only at the stage 13. Not until embryonic stage 15 did the level of futsch expression in mutant embryos approach that of wild type. Reduced mRNA steady state levels for the genes encoding Leukocyte-antigen-related-like (Lar), another receptor tyrosine kinase, and G-oα47A gene, which encodes an alpha subunit of heterotrimeric G proteins, were also detected in nerfin-1null embryos. The reduced level of gene expression in mutant embryos was nervous system specific. For example, G-oα47A gene expression in mesodermal derived tissues was not altered in nerfin-1null embryos (Kuzin, 2005).
Mammalian Fragile X mental retardation gene (FMR1) encodes an RNA binding protein that acts as a negative translational regulator. A Drosophila fragile X syndrome model has been developed using loss-of-function mutants and overexpression of the FMR1 homolog (Fmr1). Drosophila Fmr1 nulls display enlarged synaptic terminals, whereas neuronal overexpression results in fewer and larger synaptic boutons. Synaptic structural defects are accompanied by altered neurotransmission, with synapse type-specific regulation in central and peripheral synapses. These phenotypes mimic those observed in mutants of microtubule-associated Futsch. Immunoprecipitation of Drosophila Fmr1 shows association with Futsch mRNA, and Western analyses demonstrate that Fmr1 inversely regulates Futsch expression. Fmr1;futsch double mutants restore normal synaptic structure and function. It is proposed that Fmr1 acts as a translational repressor of Futsch to regulate microtubule-dependent synaptic growth and function (Zhang, 2001).
Evidence is presented that Fmr1 negatively regulates Futsch expression. (1) Fmr1 associates with Futsch mRNA. This interaction is specific, since Fmr1 fails to bind other targets such as alpha-tubulin mRNAs and the interaction is missing in Fmr1 null mutants. (2) In Fmr1 null mutants, Futsch protein level in the nervous system is increased and Fmr1 neuronal overexpression causes Futsch expression to be reduced. These results show that the level of Futsch in the nervous system is inversely regulated by the level of Fmr1. Taken together, the biochemical association between Fmr1 protein and Futsch mRNA and the inverse regulation of Futsch expression by Fmr1 strongly support a hypothesis that Fmr1 acts as a negative regulator of Futsch translation (Zhang, 2001).
Futsch appears to be the major target for Fmr1 in the regulation of synaptic structure and function. Structurally, futsch hypomorphs display fewer and enlarged NMJ synaptic boutons with dispersed, punctate anti-Futsch immunoreactivity, a phenotype indistinguishable from that caused by overexpression of Fmr in dfxrNOE. However, futschNOE causes synaptic overgrowth, a phenotype similar to Fmr1 null mutants. Functionally, all four genotypes (loss and overexpression of either Fmr1 or Futsch) enhance neurotransmission at the larval NMJ, and all four genotypes impair neurotransmission in the adult eye. Thus, the expression alterations of Futsch are sufficient to explain the synaptic phenotypes of Fmr1 mutants (Zhang, 2001).
The most conclusive experimental result is the suppression of Fmr1 synaptic phenotypes by the Fmr1;futsch double mutants. The double mutant develops normal synaptic architecture, including the normal number of arboreal branches and synaptic boutons. Strikingly, the double mutant reduces NMJ transmission to suppress the peripheral synaptic phenotype, while at the same time it increases photoreceptor transmission to suppress the central synaptic phenotype. Based on these results, it is proposed that the major function of Fmr1 is the negative regulation of Futsch in the nervous system, which in turn regulates microtubule-dependent synaptic structure and function. Of course, it remains probable that Fmr1 is translationally regulating multiple proteins. However, the Futsch misregulation is sufficient to explain the synaptic phenotypes in Fmr1 mutants and, by extrapolation, possibly the mental retardation of FraX patients (Zhang, 2001).
TDP-43 is an evolutionarily conserved RNA binding protein recently associated with the pathogenesis of different neurological diseases. At the moment, neither its physiological role in vivo nor the mechanisms that may lead to neurodegeneration are well known. Previous studies have shown that TDP-43 mutant flies presented locomotive alterations and structural defects at the neuromuscular junctions (Feiguin, 2009). This study investigated the functional mechanism leading to these phenotypes by screening several factors known to be important for synaptic growth or bouton formation. As a result it was found that alterations in the organization of synaptic microtubules correlate with reduced protein levels in the microtubule associated protein futsch/MAP1B. Moreover, TDP-43 was found to physically interact with futsch mRNA and that its RNA binding capacity is required to prevent futsch down-regulation and synaptic defects (Godena, 2011).
TDP-43 is an RNA binding protein of 43 kDa that belongs to the hnRNP family and plays numerous roles in mRNA metabolism such as transcription, pre-mRNA splicing, mRNA stability, microRNA biogenesis, transport and translation. TDP-43 is very well conserved during the evolution, especially with regards to the two RNA-recognition motifs (RRMs), the first (RRM1) being responsible for the binding of TDP-43 with UG rich RNA. In consonance with these described functions, TDP-43 prevalently resides in the cell nucleus where it co-localizes with other members of the RNA processing machinery. Nevertheless, in pathological conditions such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), TDP-43 appears in the form of large insoluble protein aggregates redistributed within the cytoplasm. At the moment, however, it is not clear how these alterations may lead to neurodegeneration. In theory, the cytosolic accumulation of TDP-43 may induce a toxic, gain of function effect on motoneurons while the exclusion of TDP-43 from the cell nucleus may lead to neurodegeneration due to a loss of function mechanism. At present, several lines of evidence mainly from different cellular and animal models support either view suggesting that both models may be acting to lead the disease condition. Recently, to determine the physiological role of TDP-43 in vivo it has been reported that the flies which lack the TDP-43 homologue (TBPH) closely reproduce many of the phenotypes observed in ALS patients, such as progressive defects in the animal locomotion and reduced life span. Moreover, it was observed that loss of TDP-43 function in Drosophila results in reduced number of motoneurons terminal branches and synaptic boutons at neuromuscular junctions (NMJs), indicating TDP-43 may regulate the assembly and organization of these structures. In coincidence with that, it should be noted that overexpression of TDP-43 in Drosophila has been reported to increase dendritic branching (Lu, 2009), lead to motor dysfunction and reduced life span (Hanson, 2010), axon loss and neuronal death (Li, 2010), is generally toxic regardless of inclusion formations (Miguel, 2010), and at least in part is the cause behind the degeneration associated with TER94 mutations which is the Drosophila homologue of the VCP protein (Ritson, 2010). Taken together, and in consideration that Drosophila TDP-43 (TBPH) can functionally substitute for human TDP-43 in functional splicing assays (D'Ambrogio, 2009), these reports confirm that Drosophila may represent a highly suitable animal model to investigate TDP-43 functions both in normal and disease conditions (Godena, 2011).
Drosophila larval NMJ is a well-characterized system to analyze the cellular and molecular events that are involved in synapse development and plasticity. Synaptic growth during larval development is expanded according to muscle size and is accomplished by the addition of new boutons to the existing presynaptic terminals. Typically, defects in synapse formation and synaptic growth are linked to cytoskeleton abnormalities, since the synaptic boutons and the newly formed buds require the underlying presynaptic microtubules to maintain their structural organization and plasticity inside the innervated muscles. Thus, to determine the physiological role of TDP-43 in vivo and the pathological consequences of its altered function, the molecular organization of Drosophila NMJs during larval development was examined in TDP-43 minus flies (Godena, 2011).
This work, shows that in TBPH minus Drosophila model the changes observed at the level of NMJs and synaptic boutons formation can be explained by defects at the cytoskeleton level, which in turn are mediated by a down-regulation of the futsch protein (but not mRNA). These results provide additional insight with regards to potential disease mechanisms mediated by TDP-43 and considerably extend knowledge with regards to defining the basic molecular functions of this protein. Future work will be aimed at better characterizing more in depth the functional mechanism through which TBPH regulates futsch protein levels and how these results can be extended to the human disease model (Godena, 2011).
Mutations in leucine-rich repeat kinase 2 (LRRK2) are linked to familial as well as sporadic forms of Parkinson's disease (PD), a neurodegenerative disease characterized by dysfunction and degeneration of dopaminergic and other types of neurons. The molecular and cellular mechanisms underlying LRRK2 action remain poorly defined. This study shows that LRRK2 controls synaptic morphogenesis at the Drosophila neuromuscular junction. Loss of Drosophila LRRK2 results in synaptic overgrowth, whereas overexpression of Drosophila LRRK or human LRRK2 has opposite effects. Alteration of LRRK2 activity also affects neurotransmission. LRRK2 exerts its effects on synaptic morphology by interacting with distinct downstream effectors at the presynaptic and postsynaptic compartments. At the postsynapse, LRRK2 interacts with the previously characterized substrate 4E-BP (Imai, 2008), an inhibitor of protein synthesis. At the presynapse, LRRK2 phosphorylates and negatively regulates the microtubule (MT)-binding protein Futsch. These results implicate synaptic dysfunction caused by deregulated protein synthesis and aberrant MT dynamics in LRRK2 pathogenesis and offer a new paradigm for understanding and ultimately treating PD (Lee, 2010).
Parkinson's disease (PD) is one of the most common neurodegenerative diseases and is characterized by locomotor abnormalities as a result of the dysfunction and eventual loss of dopaminergic (DA) neurons. Most PD cases are sporadic with no known cause. Recent advances in PD genetics have led to the identification of familial PD (FPD) genes. It is anticipated that understanding the disease mechanisms of the FPD cases will provide insights into PD pathogenesis in general. Despite intensive studies of the FPD gene products at the biochemical and cell biological levels, understanding of their physiological function and the molecular and cellular pathways underlying disease pathogenesis is still fragmentary. Of all FPD genes, leucine-rich repeat kinase 2 (LRRK2) is the most frequently mutated. LRRK2 encodes a large serine/threonine kinase with multiple other domains. Some pathogenic mutations in LRRK2, such as the I2020T and G2019S substitutions in the kinase domain and R1441C substitution in the ROC domain, appear to augment kinase activity. In Drosophila and mouse models, pathogenic human (hLRRK2) or Drosophila (dLRRK) LRRK2 induce parkinsonian phenotypes in an age-dependent manner. A number of LRRK2-interacting proteins and substrates have been identified through in vitro studies, which implicate diverse biological functions for LRRK2 in translational control, vesicular trafficking, and cytoskeletal regulation. The physiological relevance of these interacting proteins and substrates remain to be established (Lee, 2010).
Actin and microtubule (MT) cytoskeleton dynamics play a crucial role in the formation of the nervous system, regulating such fundamental processes as axonal guidance and synaptogenesis. Dynamic modulation of synaptic structure and function is fundamental to neural network formation during development and is the molecular basis of learning and memory. Synaptic dysfunction is tightly linked to the pathogenesis of major neurodegenerative diseases such as Alzheimer's disease, and its role in PD is beginning to be appreciated. In Drosophila, the MT-associated protein 1B (MAP1B) homolog Futsch is required for axonal and dendritic growth during embryogenesis and for synaptic morphogenesis during larval neuromuscular junction (NMJ) development. This study shows that dLRRK phosphorylates and negatively regulates Futsch function at the presynapse. The previously characterized dLRRK substrate 4E-BP functionally interacts with LRRK2 at the postsynapse. These results implicate defects in presynaptic MT cytoskeleton dynamics and postsynaptic protein synthesis in LRRK2 pathogenesis (Lee, 2010).
Genetic mutations in LRRK2 are frequently found in familial and sporadic PD cases. Understanding the physiological function of LRRK2 will therefore offer insights into PD pathogenesis in general. This study reveals a new physiological function of LRRK2 and offers molecular mechanisms underlying such function. The key findings from this study are that LRRK2 plays an important role in regulating synaptic morphogenesis and that it does so through distinct substrate proteins at the presynaptic and postsynaptic compartments. The results also show that the precise level of LRRK2 activity is important for synaptic morphogenesis and neurotransmission, but the regulation of these two synaptic processes likely involve different mechanisms and players. Given the similarity of Drosophila NMJ synapse to mammalian excitatory glutamatergic synapses, it is possible that the findings reported here are relevant to mammalian systems (Lee, 2010).
Synaptic loss is a major neurobiological substrate of cognitive dysfunction in a number of neurological diseases. Extensive studies in patients and animal models have documented that synaptic failure is one of the earliest events in the pathogenesis of Alzheimer's disease. Interestingly, neurotransmission defects have been repeatedly observed in rodent FPD models, including the LRRK2 model, although no obvious signs of neurodegeneration accompany the electrophysiological defects. These results suggest that synaptic dysfunction is a primary effect of FPD gene mutations and that synaptic failure is intimately involved in PD pathogenesis. The molecular mechanisms underlying these synaptic transmission defects, however, remain elusive. This study of the LOF and GOF models of LRRK2 in Drosophila provides mechanistic insights into the possible cause of synaptic dysfunction in LRRK2-associated PD. It was found that LRRK2 regulates synaptic morphogenesis at the presynaptic and postsynaptic compartments through distinct substrates (Lee, 2010).
In the presynaptic side, LRRK2 forms a complex with tubulin and the MT-binding protein Futsch. Furthermore, LRRK2 phosphorylates Futsch and negatively regulates the presynaptic function of Futsch in controlling MT dynamics. MT cytoskeleton is critical for the generation and maintenance of neuronal axons and dendrites, transport of synaptic vesicles and organelles along the processes, and the initiation and maintenance of synaptic transmission. Disrupted MT dynamics in neuronal synapses has been implicated as an underlying cause for several neurological diseases, including hereditary spastic paraplegia and fragile X syndrome. LRRK2-associated PD may share this feature with the aforementioned diseases. Disrupted MT dynamics could be responsible for the presynaptic effects observed in LRRK2 LOF and GOF mutants, such as aberrant mitochondria distribution. The synaptic vesicle transport phenotypes seen in Caenorhabditis elegans LRK-1 mutant could also be attributable to altered MT dynamics. These could all contribute to synaptic dysfunction. Futsch/MAP1B proteins are large multidomain proteins that are phosphorylated by multiple kinases, including Sgg/GSK-3β, PAR-1/MARK, and Cdk5, which also phosphorylate tau and are implicated in tau pathology. Tau-related pathology has been observed in LRRK2 transgenic animals. It would be interesting to test for possible interplay between LRRK2 and these other kinases in regulating MT-binding proteins and MT dynamics. In mammalian hippocampal neurons, overexpression of pathogenic hLRRK2 led to reduced neurite length and branching, whereas deficiency of LRRK2 had opposite effects. Whether MT dynamics regulated by Futsch/MAP1B is contributing to this LRRK2 effect in mammals will await additional investigation (Lee, 2010).
This study also showed that LRRK2 interacts with 4E-BP at the postsynapse. 4E-BP acts as a negative regulator of the translational initiation factor eIF4E through direct binding and sequestration. Phosphorylation of 4E-BP by LRRK2 weakens 4E-BP binding to eIF4E (Imai, 2008), therefore releasing the inhibitory effect of 4E-BP on eIF4E. Previous studies have demonstrated an important postsynaptic role for eIF4E-mediated protein synthesis in activity-dependent synaptic growth at the Drosophila NMJ (Sigrist, 2000). Genetic interaction studies demonstrate a functional role for 4E-BP in mediating the synaptic effects of LRRK2. However, the exact roles of 4E-BP and LRRK2 in this process appear complex. For example, (1) despite the prominent effects of 4E-BP overexpression on NMJ development, its loss of function has no obvious effect. One would expect a gain of eIF4E function in the absence of 4E-BP and therefore a synaptic-overgrowth phenotype. (2) 4E-BP activity is predicted to be high in dLRRK mutant and low in LRRK2 overexpression condition, but this study observed synapse phenotypes opposite of what is predicted based on the presumed roles of eIF4E and 4E-BP on Drosophila NMJ morphogenesis. One possible explanation of these seemingly disparate results is that phospho-4E-BP, the product of LRRK2-mediated phosphorylation of 4E-BP, instead of being inactive and inert, may actually perform some new synaptic function at the NMJ. In this scenario, loss of 4E-BP function in d4E-BP mutant would not show the same phenotype as LRRK2 overexpression, which produces more phospho-4E-BP. Recent studies in Drosophila dopaminergic neurons suggest a functional role for phospho-4E-BP in vivo. Alternately, effectors other than 4E-BP may also mediate the effects of LRRK2 on NMJ development and neurotransmission. Although 4E-BP overexpression might have masked the effects of these other effectors, in dLRRK mutant background, the functional roles of these other effectors might manifest themselves. A similar situation was observed in pumillo mutant, in which a synapse-loss phenotype was observed despite the upregulation of eIF4E activity in this mutant attributable to the derepression of translational inhibition of eIF4E, which would have resulted in a predicted synapse-overgrowth phenotype. Involvement of other effectors in mediating the effects of LRRK2 on NMJ development and neurotransmission, and possibly different effectors for NMJ development versus neurotransmission, could also explain the complex electrophysiological phenotypes of dLRRK mutant and LRRK2 overexpression animals, as well as the uncoupling of the effects on synaptic differentiation and neurotransmission by the various genetic manipulations. Future studies will test these possibilities as well as the relevance of the NMJ studies to dopaminergic neuron synapses (Lee, 2010).
In addition to LRRK2, the TOR pathway also regulates 4E-BP function through phosphorylation. This pathway primarily regulates cell and organism growth through diverse outputs, including protein synthesis, cytoskeletal change, autophagy, and cell survival. This study found that treatment of flies with rapamycin, an inhibitor of TOR, has the same effects as 4E-BP overexpression in wild type as well as LRRK2 overexpression backgrounds. Although rapamycin has been extensively studied in the context of autophagy induction and neurodegeneration models, its effect on Drosophila NMJ development is unlikely attributable to autophagy, because the current results show that presynaptic or postsynaptic induction of autophagy through Atg1 overexpression had no obvious effect on synapse number. The similar effects of rapamycin and 4E-BP overexpression on NMJ development support that rapamycin acts via the 4E-BP translational control pathway to impact NMJ development. A recent report showed that either 4E-BP overexpression or 4E-BP activation by rapamycin could suppress the muscle and dopaminergic neurodegeneration phenotypes seen in Drosophila Pink1 and Parkin models of PD. These results suggest that deregulation of protein synthesis could be generally involved in PD pathogenesis and that rapamycin or its analogs could be developed into effective PD therapeutics (Lee, 2010).
In Drosophila, mAb 22C10 has been widely used to label neuronal cells within the PNS and CNS. In all stages of development, 22C10 antigen expression starts in postmitotic neurons shortly before axonogenesis is initiated. In the embryonic nervous system, first mAb 22C10 reactivity is detected in the MP2 neurons. During subsequent development, several interneurons and motoneurons express the 22C10 antigen. Within the developing PNS, neuronal cells express the 22C10 antigen from early stages of neuronal differentiation onward. It is important to note that the 22C10 epitope is found in the growing axon, the cell body, and the dendrite. At the end of embryogenesis, 22C10 expression can be found in all sensory neurons. During late larval development, expression is found in all photoreceptor neurons. In addition to expression in the nervous system, some 22C10 antigen can be detected in the muscle attachment sites of the ventrolateral musculature and the tip cell of the Malphigian tubules. By using confocal microscopy, the 22C10 epitope has been localized in the central axonal compartment of larval neurons associated with the microtubule cytoskeleton (Hummel, 2000 and references therein).
The Baz/Par-3-Par-6-aPKC complex is an evolutionarily conserved cassette critical for the development of polarity in epithelial cells, neuroblasts, and oocytes. aPKC is also implicated in long-term synaptic plasticity in mammals and the persistence of memory in flies, suggesting a synaptic function for this cassette. At Drosophila glutamatergic synapses, aPKC controls the formation and structure of synapses by regulating microtubule (MT) dynamics. At the presynapse, aPKC regulates the stability of MTs by promoting the association of the MAP1B-related protein Futsch to MTs. At the postsynapse, aPKC regulates the synaptic cytoskeleton by controlling the extent of Actin-rich and MT-rich areas. In addition, Baz and Par-6 are also expressed at synapses and their synaptic localization depends on aPKC activity. These findings establish a novel role for this complex during synapse development and provide a cellular context for understanding the role of aPKC in synaptic plasticity and memory (Ruiz-Canada, 2004).
During expansion of the NMJ, parent boutons located at the distal end of a branch give rise to new synaptic boutons by budding. New buds separate from parent boutons by the formation of a neck, and NMJ branches extend by neck elongation and bouton enlargement. Throughout this process, the postsynaptic membrane and underlying cytoskeleton impose a barrier to presynaptic extension, since synaptic boutons and their buds are completely surrounded by the muscle cell membrane and underlying cytoskeleton. During branch elongation, a presynaptic signal may induce the retraction of the postsynaptic cytoskeleton barrier. It is proposed that changes in both the pre- and postsynaptic cytoskeleton during branch elongation mediate these events and that these processes are regulated by aPKC with the collaboration of Baz and Par-6 in both locales (Ruiz-Canada, 2004).
The localization of aPKC to MT-rich domains at the NMJ and the marked reduction in new synaptic bouton formation observed in dapkc mutants prompted an investigation of the cytoskeletal changes during branch elongation. At the presynaptic arbor of wild-type larvae, NMJ expansion is accompanied by the presence of unbundled MTs at the distal tip of NMJ branches and by little if any colocalization between MTs and the MAP1B-related protein Futsch. In vertebrates, MAP1B protects MTs from depolymerization. Similarly in Drosophila, MT integrity in axons and at the NMJ is preserved by Futsch; severe mutations in futsch result in MT fragmentation. This Futsch-dependent protection of MT integrity at presynaptic arbors has an important role during NMJ expansion, since MT fragmentation in futsch and dapkc mutants results in marked reduction in bouton number (Ruiz-Canada, 2004).
In epithelial cells and growth cones, an increase in aPKC activity enhances MT lifetime, although the involvement of MT-associated proteins in this process has not been investigated. The results suggest that aPKC activity enhances MT stability in a process that depends on Futsch. (1) Coimmunoprecipitation experiments indicate that aPKC, Tubulin, and Futsch exist in the same biochemical complex and that the interaction between aPKC and MTs is likely to be through Futsch. (2) Loss of dapkc function results in fragmented MTs at terminal boutons, and an increase in aPKC activity by pre-PKM (PKM is a persistantly active kinase) results in longer than normal MTs and an increased colocalization between MTs and Futsch. (3) Presynaptic expression of PKM in a futsch mutant background results in MT fragmentation similar to futsch mutants alone, suggesting that PKM acts through Futsch to stabilize MTs (Ruiz-Canada, 2004).
Three futsch mutations lead to a pronounced reduction in the level of 22C10 antigen expression including futschM455, futschP28, and futschN94. The futschM455 allele leads to a uniform reduction in expression, while both futschP28 and futschN94 also lead to an abnormal subcellular distribution of Futsch. In futschP28, a granular expression in the cell body and dendrites is observed. Interestingly, in futschN94, there is diffuse staining within the nerve terminal that is not observed in wild type. This aspect of the phenotype has been analyzed in the visual system. In third instar wild-type photoreceptor cells, distinct 22C10-positive fibers extend from the cell body into the axon and dendrites, but no staining is observed in the nerve terminals. In futschN94, Futsch protein is distributed uniformly in the nerve terminal. The mislocalization of the Futsch protein can be clearly seen in the nerve terminals in the lamina where the photoreceptor axons of cells R1-R6 terminate as well as in the growth cones of photoreceptor cells R7 and R8 in the medulla. In addition, futschN94 mutant larvae show ectopic Futsch expression in the so-called optic lobe pioneer cells (Hummel, 2000).
In two mutations, futschK68 and futschP158, 22C10 antigen expression is eliminated. Homozygous futschK68 flies are viable, and overall neuronal development proceeds normally in the embryo. In the larvae, a reduced number of synaptic boutons visualized by the antibody nc46 is noted. In futschP158, the lethality and the absence of 22C10 expression could not be separated by recombination. In wild-type embryos, highest levels of mAb 22C10 reactivity are seen in all PNS neurons. Futsch is prominently expressed in the dendrites of the five lateral chordotonal organs. To analyze the morphology of the PNS neurons in mutant futschP158 embryos, the GAL4 driver line P0163 was used to express the transmembrane protein CD2 specifically in PNS neurons, thereby visualizing their axonal and dendritic morphology. In mutant futschP158 larvae, about 97% of the PNS neurons fail to develop their normal dendritic morphology. Occasionally, however, dendritic processes are detected. Axonal growth is impaired as well, and few axons grew normally into the CNS. Axonal pathfinding is not affected, and axons appeared to simply stop growing after some distance.
Within the ventral nerve cord, Futsch is expressed in many inter- and motor-neurons that also express the Fasciclin II protein (Fas II). In stage 16 futschP158 mutant embryos, the longitudinal connectives are reduced. Similarly, disruptions are observed in the motoneuronal connections. In particular, SNb nerves fail to reach muscle fibers 12 and 13. Motoneurons stall and fail to set up the correct innervation pattern in 89% of the segments. To determine the null phenotype of futsch, the CNS defects associated with the deficiencies Df(1)A94 and Df(1)S39 that remove the futsch locus were analyzed. In both cases, a nervous system phenotype very similar to the futschP158 mutant phenotype was observed. Thus, P158 appears to be an amorphic mutation, whereas the K68 is a hypomorphic mutation of the futsch gene. Loss of futsch function leads to disruption of dendritic and axonal growth.
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date revised: 15 October 2011
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