BIOLOGICAL OVERVIEW

In the Drosophila embryo, the correct association of muscle cells with their specific ectodermally derived tendon cells, also known as epidermal muscle attachment or EMA cells, is achieved through reciprocal interactions between these two distinct cell types. Vein, a neuregulin-like factor secreted by the approaching myotube, activates the EGF-receptor signaling pathway within the tendon cells to initiate tendon cell differentiation. kakapo, renamed short stop because it is now realized that the gene was first discovered by van Vactor et al. (1993), is expressed in the tendons and is essential for muscle-dependent tendon cell differentiation. Short stop/Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. A major defect in the kakapo mutant tendon cells is the failure of Vein to localize at the muscle-tendon junctional site; instead, Vein is dispersed and its levels are reduced. This may lead to aberrant differentiation of tendon cells and consequently to the kakapo mutant's deranged somatic muscle phenotype (Strumpf, 1998). The flies display wing blisters because the mutant epidermal cells fail to adhere to the opposing layer of wild-type cells in the wing bilayer. This led to the significance of Kakapo’s Maori name, based upon an ineptly flying New Zealand parrot (Gregory, 1998).

To elucidate the function of kak in epidermal muscle attachment (EMA) cell differentiation, an examination was made of the expression of various markers characteristic of tendon cell terminal differentiation, including Stripe, Delilah, and beta1 tubulin mRNA. The expression of the regulatory protein Stripe, a transcription factor of the early growth response (EGR) family, determines the fate of the EMA competent cells at the first phase of tendon cell development. Stripe expression leads to the expression of an array of EMA-specific genes that contribute to the correct guidance of the myotubes. The second phase of tendon cell differentiation depends on inductive interactions between the myotube and the EMA cell. These interactions lead to terminal differentiation of the EMA competent cells into tendon cells, in which high protein levels of Stripe, Groovin (now known as Kakapo), and Alien are maintained, and the transcription of the genes delilah and beta1 tubulin is induced (Strumpf, 1998 and references).

In kak mutants, an excess of EMA cells, marked by the expression of Stripe and Delilah, is observed at a number of sites in the epidermis. This phenotype is particularly notable in domains in which a group of muscles extend together towards neighboring epidermal attachment cells, such as along the ventral segmental border cells, to which the four ventral longitudinal muscles bind. To further study the state of differentiation of the EMA cells in the kak mutant embryos, the expression of the beta1 tubulin gene was examined. In wild-type embryos, the expression of the beta1 tubulin gene is significantly elevated toward the end of tendon cell differentiation. In contrast to the expression of Stripe and Delilah, the mRNA expression of beta1 tubulin in kak mutant embryos is significantly reduced, suggesting that transcription of the latter gene requires different levels of signaling. It is suspected that Vein signaling from mesodermal cells, which is required for terminal differentiation of tendon cells (Yarnitzky, 1997), may be reduced in the mutant embryos; while there is enough signal to trigger Delilah and Stripe expression, the signal is not capable of inducing beta1 tubulin transcription (Strumpf, 1998).

The expression of delilah, stripe, and beta1 tubulin is induced in the epidermal attachment cells as a result of the EGF-receptor pathway activation by the neuregulin-like growth factor, Vein (Yarnitzky, 1997). Vein is secreted by mesodermal cells underlying the EMA cells. Vein protein localization is restricted to the muscle-tendon junctional site in wild-type embryos. However, in kak mutant embryos, Vein protein is not localized and appears rather diffuse. This altered pattern of Vein may explain the multiple number of cells expressing delilah and stripe: since Vein is not strictly localized at a given muscle-tendon junction site, it apparently weakly activates the EGF-receptor pathway in neighboring cells as well. It is presumed that the only cells that can respond to the ectopic Vein protein are the competent population of EMA cells, defined by the early expression of stripe. These cells express stripe during early developmental stages in a muscle-independent manner and normally lose their stripe expression by stage 16 of embryonic development. When these competent EMA cells receive the muscle-derived Vein signal, the expression of stripe and delilah is reactivated. It appears that only this population of cells is capable of responding to Vein, since the pattern of the ectopic Stripe- or Delilah-expressing cells in the kak mutant embryos resembles that of the early population of Stripe-expressing cells. The reduced levels of beta1 tubulin mRNA in the mutant tendon cells may also result from the abnormal pattern of Vein localization, since lower levels of Vein may not be sufficient to induce maximal beta1 tubulin expression. It therefore appears that the primary defect in kak mutant embryos stems from the lack of Vein accumulation at the muscle-tendon junctional site (Strumpf, 1998).

Is the abnormal differentiation of the epidermal muscle attachment (EMA) cells in kak mutant embryos reflected by the pattern of the somatic musculature? kak mutant embryos at stage 16 of embryonic development were labeled with anti-myosin heavy chain antibody to visualize the somatic muscles, and the muscle pattern was compared with that of wild-type embryos. A significant disruption of the somatic muscle pattern is observed in kak mutant embryos. In many cases, individual myotubes are not oriented correctly, and in some cases the myotube rounds up. Since Kak cannot be detected in myotubes using the available antibodies, it is assumed that the somatic muscle derangement is secondary to the abnormal differentiation of the EMA cells. A similar phenotype is also observed in stripe mutant embryos, in which the EMA cells do not differentiate correctly (Frommer, 1996). The similarity between the stripe and kak muscle phenotype and the reduced beta1 tubulin mRNA expression are consistent with the conclusion that EMA cell differentiation is defective in kak mutants. The correct recognition between the muscle and the tendon cell is essential for arresting the extension of the myotube and establishment of the final pattern of somatic musculature (Yarnitzky, 1997). It appears that the muscle development in kak partial loss of function embryos does not represent a complete loss of function phenotype since a more severe muscle defect is observed in kakV104/DfMK1 embryos (Strumpf, 1998).

How could this intracellular protein affect the localization of Vein at the extracellular matrix surrounding the EMA cell? At least two possibilities, which are not mutually exclusive, are considered. The first is the association of Kak with the unique cytoskeletal network of the EMA cell, which is critical for the cell's polarized organization. Tendon cell polarity may be essential for maintaining the characteristic junctional complexes formed between the basal surfaces of the EMA cell and the muscle cells. The space between these junctional complexes contains many extracellular matrix proteins, some of which may possess a Vein binding function. Impaired tendon cell polarity may lead to the loss of the putative Vein-binding component(s). Alternatively, Kak may be associated with a transmembrane protein(s) responsible for Vein localization either by direct binding or by association with additional extracellular matrix components that may directly bind Vein. Immunoprecipitation experiments with anti-Kak antibody indicated that Kakapo forms protein complexes containing the extracellular protein Tiggrin. These results favor the latter possibility that Kak is directly associated with protein complexes that may be important for Vein binding. The reduced amount of electron-dense material observed at the muscle-tendon junction site in the kak mutant embryos described in Prokop, et al. (1998) is in agreement with both mechanisms mentioned above (Strumpf, 1998).

The excess number of Stripe- and Delilah-expressing cells in the kak mutant embryos may be attributed to the dispersed levels of Vein, which could induce partial activation of the EGF-receptor signaling pathway in neighboring cells. An alternative explanation is that muscle-dependent differentiation of tendon cells may be accompanied by lateral inhibition of neighboring cells. The differentiated tendon cell may activate the Notch-signaling pathway in the surrounding cells. Aberrant contacts between tendon cells and their neighboring EMA competent cells in the kak mutant embryos may prevent efficient lateral inhibition, resulting in an excess of Stripe- and Delilah-expressing cells. An observation that supports this possibility is that an excess in beta1 tubulin-expressing cells is detected in Delta mutant embryos. Delta, a well-characterized Notch ligand, mediates lateral inhibition in a large array of tissues during embryonic and adult development. The lack of Delta may prevent lateral inhibition of the competent EMA cells, leading to their differentiation into beta1 tubulin-expressing cells. The impaired integrity of the epidermis described by Gregory (1998) is consistent with this explanation (Strumpf, 1998).

short stop (shot) is required for sensory and motor axons to reach their targets in the Drosophila embryo. Growth cones in shot mutants initiate at the normal times, and they appear normal with respect to overall morphology and their abilities to orient and fasciculate. However, sensory axons are unable to extend beyond a short distance from the cell body, and motor axons are unable to reach target muscles. The shot gene encodes novel actin binding proteins that are related to plakins and dystrophin and expressed in axons during development. The longer isoforms identified are predicted to contain an N-terminal actin binding domain, a long central triple helical coiled-coil domain, and a C-terminal domain that contains two EF-hand Ca2+ binding motifs and a short stretch of homology to the growth arrest-specific 2 protein. Other isoforms lack all or part of the actin binding domains or are truncated and contain a different C-terminal domain. Only the isoforms containing full-length actin binding domains are detectably expressed in the nervous system. shot is allelic to kakapo, a gene that may function in integrin-mediated adhesion in the wing and embryo. It is proposed that Shot's interactions with the actin cytoskeleton allow sensory and motor axons to extend (Lee, 1999).

kakapo mutation affects terminal arborization and central dendritic sprouting of Drosophila motoneurons. Four mutant alleles of kak are described that are embryonic lethals, that fail to complement one another, and that show a paralytic phenotype when homozygous, transheterozygous or hemizygous over deficiencies. Paralysis might be caused by dysfunction or developmental defects in either the nervous system or the musculature. In kakapo mutant embryos, defects are found in both tissues: muscles detach from the epidermis in all alleles, and there is a reduction in the size of motoneuronal terminals on muscles and of neuronal branches in the CNS at late stage 17 (Prokop, 1998).

In wild-type embryos at stage 17, motoneuronal terminals have branches on their target muscles with varicosities (boutons) of up to 1 micro meters in diameter. In kak mutant embryos NMJs in all locations occupy far less surface on their respective muscles; their branches are reduced in length, and boutons appear reduced in number and size. Whereas some allelic combinations exhibit an almost complete absence of NMJs, other combinations show less severe phenotypes, but their phenotype is nevertheless significant. Although NMJs are severely reduced in kak mutant embryos, presynaptic marker expression is mainly restricted to neuromuscular sites and can hardly be found in ectopic locations. This reduced and restricted appearance of synaptic markers in kak mutant embryos hints at a requirement for kak within the presynaptic terminal (Prokop, 1998).

Ultrastructural analyses of kak mutant embryos reveal that presynaptic boutons can form normal cell junctions with the muscle, interspersed by morphologically normal synapses. However, examples are found where synapses are indicated by structured material in the neuromuscular cleft, but typical presynaptic specializations (T-bars) are missing. If T-bars are found, they are restricted to neuromuscular sites, corroborating light microscopic findings. Furthermore, neuromuscular contacts and synapses are found less frequently when compared with controls, which is in agreement with the reduction of NMJs observed at the light microscopic level. To test whether transmission occurs at kak mutant NMJs, patch recordings were carried out on kak mutant muscles. These recordings reveal excitatory junctional currents, clearly indicating that neuromuscular transmission occurs. In four cases the NMJs were stained with antibodies raised against cysteine string protein subsequent to recording and it was confirmed that in all cases the NMJ is clearly misshapen and reduced in size. Occurrence of neuromuscular transmission is furthermore demonstrated by the presence of strong muscle contractions in kak mutant embryos observed under polarized light in vivo. Taken together, ultrastructural, electrophysiological and in vivo observations suggest that NMJs, although abnormal in shape, are functional in kak mutant embryos. This suggests that kak might be required specifically for growth and shaping of branches at motoneuronal terminals. In kak mutant embryos motoneurons appear capable of navigating along correct paths to their target muscles and maintaining these contacts thereafter. This suggests that the kak mutant alleles affect NMJ formation during the differentiation phase, when muscle-attached growth cones reshape into the branches and boutons of mature NMJs (Prokop, 1998).

To investigate whether kak function might be required directly within the nerve terminal, an anti-kak antiserum was used. The staining procedure used fails to detect strong staining at neuro-muscular junctions in wild type or hemizygous embryos, however, the NMJs of kak overexpressing embryos are labeled more reliably and strongly than wild type. Local neuronal growth is not restricted to branch formation at the NMJ but also occurs within the CNS during the development of dendritic branches at stage 16/17. A test was carried out to see if this growth might also be affected in kak mutant embryos. Dendrites were labeled in a retrograde manner by applying DiI to the NMJ of RP3 motoneurons on muscles VL3/4. In wild-type embryos, RP3 sends an axon contralaterally through the dorsally located anterior root of the intersegmental nerve. On the ipsilateral side, a second projection leaves the soma of RP3, projecting along a similar path as the contralateral process, but remaining confined to the neuropile. Both projections have numerous local arborizations. In kak mutant embryos, the ipsilateral local arborizations are almost normal, but the contralateral arborizations are severely reduced and often form swellings or blobs. The failure of RP3 to elaborate its contralateral dendrites is apparent from late stage 16, suggesting kak function is required for the process of outgrowth rather than maintenance of dendrites. Consistent with the findings for the RP3 dendrites, the whole neuropile is reduced in size in kak mutant embryos, as compared with wild type, but appears normal in its organization. Taken together, these findings suggest that neurons project correctly, but fail to elaborate part of their local branches, leading to smaller NMJs in the periphery and smaller dendrites and thus reduced neuropile volume in the central nervous system (Prokop, 1998).

The phenotypes shown so far strongly suggest a specific requirement for kak function in specific local growth events. Below, two further kak mutant phenotypes are described: mislocalization of axonal proteins and disorganization of the cytoskeleton, both of which are potential causes underlying the specific defects in neuronal branch formation. A mislocalization of proteins along neuronal processes has been observed. For example, Fas II, which encodes a transmembrane protein of the immunoglobulin superfamily, is expressed at low levels in the nerve roots and stops at the entry point into the neuropile of stage 16 and 17 control embryos. By stage 17, Fas II expression in all nerve roots is strongly upregulated and the anterior root of the intersegmental nerve extends to the dorsal part of the neuropile. Thus, it appears as if Fas II fails to localize properly along neuronal processes. In contrast, 22C10 immunoreactivity (see Futsch) appears to be distributed normally in kak mutant nerve roots, but a mislocalization phenotype is found in another type of neuron, the dorsal bipolar neuron of the peripheral nervous system. The dorsal bipolar neurons have longitudinal projections that span the entire length of the segment, but only the proximal regions of these processes are labeled by 22C10 antibodies in the wild type. However, in kak mutant embryos the entire length of these lateral bipolar projections is 22C10-positive. Thus, kak function is required for the correct localization of (membrane) proteins within neuronal processes, and the mislocalization of such proteins is a potential cause for defects in local branching in the neuropile or at the NMJ (Prokop, 1998).

Both defects, the localization of axonal proteins and defects in the microtubule organization may be the underlying cause for the observed reduction in local growth of dendrites and at NMJs. Branching of motoneuronal terminals and axonal defasciculation require a reduction of neuronal cell adhesion molecule (N-CAM)-mediated interaxonal adhesion in vertebrates and, in agreement with this, the overexpression of Fas II, the Drosophila homolog of N-CAM, antagonizes nerve branching. Hence, it can be reasoned that the inhibition of dendrite and branch formation might be due to the observed mislocalization of Fas II to axonal areas where dendrites and terminal branches are usually forming. However, combining kak0 with a Fas II null allele does not show any obvious suppression of the neuromuscular phenotype. Thus, mislocalization of Fas II alone does not explain the growth defects, but its involvement might be obscured by mislocalization of other redundant CAMs of similar function. Mislocalization of membrane proteins might be the consequence of their lack of a Kak-mediated linkage to the membrane-associated cytoskeleton. Conversely, loss of such a physical link could cause disruption of growth regulation, since transmembrane proteins have been shown to instruct the assembly of the actin cytoskeleton in neuronal growth cones (Prokop, 1998).

Neuronal growth defects in kak mutant embryos might be caused directly by defects in cytoskeleton assembly. Microtubules are essential for axonal growth and are regulated in a complex way. The assembly of microtubules during growth is preceded by formation of the actin cytoskeleton. The fine regulation of actin could require actin-associated proteins, and Kak might be one of them. This might explain why loss of kak function suppresses only a specific subset of neuronal growth events, i.e., local growth at NMJs and of contralateral RP3 dendrites but not long distance growth or ipsilateral RP3 arbors. The specific growth defects in kak mutant embryos might be due to subcellular-specific compartmentalization of Kak or local posttranslational modifications. Alternatively, unaffected branches may contain redundant cytoskeletal molecules that the affected branches lack. Possible molecular differences might reflect a general difference between affected and unaffected branches. For example, affected branches might represent preferentially presynaptic output branches (certainly true for NMJs) and unaffected branches may represent postsynaptic or input branches. Alternatively, the qualitative differences might consist in the origin of the branches: arborizations derived from an axon (NMJ, contralateral RP3 dendrites) may require Kak function, but not those derived from somatic extensions (ipsilateral RP3 dendrites) (Prokop, 1998).

Loss of the spectraplakin Short stop activates the DLK injury response pathway in Drosophila

The MAPKKK dual leucine zipper-containing kinase (DLK, Wallenda in Drosophila) is an evolutionarily conserved component of the axonal injury response pathway. After nerve injury, DLK promotes degeneration of distal axons and regeneration of proximal axons. This dual role in coordinating degeneration and regeneration suggests that DLK may be a sensor of axon injury, and so understanding how DLK is activated is important. Two mechanisms are known to activate DLK. First, increasing the levels of DLK via overexpression or loss of the PHR ubiquitin ligases that target DLK activate DLK signaling. Second, in Caenorhabditis elegans, a calcium-dependent mechanism, can activate DLK. This study describe as new mechanism that activates DLK in Drosophila: loss of the spectraplakin short stop (shot). In a genetic screen for mutants with defective neuromuscular junction development, this study identified a hypomorphic allele of shot that displays synaptic terminal overgrowth and a precocious regenerative response to nerve injury. Both phenotypes are the result of overactivation of the DLK signaling pathway. It was further shown that, unlike mutations in the PHR ligase Highwire, loss of function of shot activates DLK without a concomitant increase in the levels of DLK. As a spectraplakin, Shot binds to both actin and microtubules and promotes cytoskeletal stability. The DLK pathway is also activated by downregulation of the TCP1 chaperonin complex, whose normal function is to promote cytoskeletal stability. These findings support the model that DLK is activated by cytoskeletal instability, which is a shared feature of both spectraplakin mutants and injured axons (Valakh, 2013).

Spectraplakins are huge, multidomain proteins that bind to both actin and microtubules to regulate cytoskeletal dynamics. The family of spectraplakins consists of mammalian bpag1/dystonin and ACF7/MAC1, Drosophila short stop (shot)/kakapo, and C. elegans vab-10. Spectraplakins function in many cellular processes, including regulating ER-Golgi transport in mammalian sensory neurons (Ryan, 2012a; Ryan, 2012b) and mediating polarized locomotion of skin stem cells upon injury (Wu, 2011). They also play an essential role in axons, as mutations in mammalian spectraplakins lead to peripheral neuropathy in both mice and humans. In Drosophila, the spectraplakin short stop has been extensively studied for its role in embryonic axon outgrowth and its regulation of microtubule dynamics. All prior loss-of-function mutants in shot are embryonic lethal, and these strong alleles have severe impairments of their cytoskeleton and poor axon outgrowth, leading axons to 'stop short' of their targets. This embryonic phenotype is in apparent contradiction to the larval phenotype described for the shot^VV allele, in which the synaptic terminal is overgrown with additional synaptic boutons. However the shot^VV allele is a hypomorph and motor axons successfully navigate to their targets, so this mutant must retain sufficient function to allow for axonal outgrowth. Although shot^VV behaves as a hypomorph, the molecular lesion was not identified, and so it is plausible that this allele is a neomorph and that its regulation of DLK may not reflect the normal function of the protein. However, RNAi knockdown of shot was found to generate the same phentoypes as shot^VV and leads to the same activation of Wallenda/DLK. Hence, both the shot^VV allele and shot RNAi provide evidence for a new function for shot, namely, as a negative regulator of Wallenda/DLK signaling. It will be interesting to determine whether mammalian spectraplakins also restrain DLK signaling and whether dysregulated MAP kinase pathways may mediate some of the phenotypes of spectraplakin mutants, such as peripheral neuropathy (Valakh, 2013).

A series of recent studies highlights the central role of DLK in the developing and injured mammalian nervous system. DLK is required for normal developmental cell death in motor and sensory neurons, for Wallerian degeneration of injured peripheral axons, for cell death and axon degeneration of retinal ganglion cells in models of glaucoma, and for the proregenerative preconditioning response in injured DRG axons. Because DLK appears to be central to the neuronal injury response, there is great interest in understanding its mechanism of activation. In particular, it is important to understand how axon injury leads to the activation of DLK (Valakh, 2013).

To date, methods that increase the levels of DLK are the best understood mechanism for increasing DLK activity. In worms, flies, and mice, loss of the PHR ubiquitin ligase leads to an increase in the levels of DLK; and in worms and flies, there are extensive data demonstrating that this activates the kinase. Similarly, in both worms and flies, the overexpression of DLK is sufficient to activate the DLK signaling pathway. In Drosophila, injury leads to a loss of the PHR ubiquitin ligase Highwire, potentially via autophagosomal degradation, which in turn leads to an increase in DLK and may be a method of injury-induced activation. In mammals, a positive feedback loop between DLK and JNK inhibits Phr1-dependent degradation of DLK, increasing the levels of DLK and activating the pathway (Huntwork-Rodriguez, 2013). In addition, a calcium-dependent activation mechanism was recently demonstrated in C. elegans, which is very exciting because calcium influx is an early step after axon injury. However, the key hexapeptide sequence that mediates this calcium-dependent regulation is absent from both Drosophila and mouse DLK, suggesting that additional activation mechanisms for DLK may exist. Indeed, the current data show that loss of the spectraplakin shot can also activate DLK and leads to the hypothesis that cytoskeletal disruptions may activate DLK. These findings demonstrate that loss of function of shot leads to activation of Wallenda/DLK without a concomitant increase in the levels of Wallenda/DLK. Hence, loss of shot is not acting upstream or in concert with Highwire because loss of Highwire or components of the Highwire ubiquitin ligase complex lead to increased levels of Wallenda/DLK. The shot mutant is the first manipulation that activates Wallenda/DLK signaling in Drosophila without altering the levels of Wallenda/DLK. Hence, loss of shot must activate Wallenda/DLK via a novel mechanism. It is hypothesized that this mechanism is related to the biochemical function of Shot, which is to stabilize the cytoskeleton by simultaneously binding both actin and microtubules. Prior studies demonstrate that shot null mutants have a destabilized microtubule network, and ehis study demonstrates that the microtubule network is more dynamic in shot RNAi knockdown larvae. It is proposed that a destabilized cytoskeleton activates Wallenda/DLK. Consistent with this model, mutations in either of two subunits of the TCP-1 complex, which like Shot regulates both the actin and microtubule cytoskeleton, leads to activation of DLK signaling. By demonstrating this activation of DLK signaling, the current results support and extend the work of Bounoutas (2011) who demonstrated that microtubule disruption leads to DLK-dependent changes in protein levels in C. elegans. Moreover, the findings are consistent with studies in mammalian cell culture demonstrating that drugs that destabilize microtubules can activate MAP kinase signaling, as well as studies in Drosophila showing that genetic disruption of the cytoskeleton can activate JNK signaling (Valakh, 2013).

Axonal injury as a result of trauma or neurotoxic insults, such as chemotherapy drug treatment, is accompanied by a change in microtubule network stability. A model is proposed in which DLK functions as a sensor of microtubule network stability. When the cytoskeleton is destabilized as a result of injury, DLK will be activated. The consequence of that activation will depend on downstream signaling pathways and may differ by cellular compartment. For example, DLK in the distal axon will promote axonal degeneration, whereas DLK activation in proximal axons will facilitate the retrograde transport of injury signals that can activate regenerative and/or apoptotic gene expression programs (Valakh, 2013).

In the mammalian PNS, DLK is required for the preconditioning response that boosts the efficacy of peripheral DRG axon regeneration after a prior nerve injury (Shin, 2012). In both worms and flies, activation of DLK by increasing its abundance improves the regenerative response in the absence of a prior nerve injury. Hence, it is attractive to speculate that activation of DLK in the absence of injury may also improve regeneration in mammalian axons. The findings with shot suggest that relatively mild disruptions to the axonal cytoskeleton can activate DLK and accelerate the regenerative response in Drosophila in the absence of a prior trauma. Future studies will test whether pharmacological agents that disrupt the cytoskeleton can activate DLK in mammalian neurons and whether such activation promotes axon regeneration (Valakh, 2013).

In conclusion, this study demonstrates that, in the absence of Shot, Wallenda/DLK signaling is activated resulting in synaptic terminal overgrowth and more rapid regenerative axonal sprouting. The role of Shot as an actin-microtubule cross-linker suggests that Wallenda/DLK is activated by cytoskeletal disruption and suggests novel approaches for controlling DLK activity in the injured or diseased nervous system (Valakh, 2013).

Tau and spectraplakins promote synapse formation and maintenance through Jun kinase and neuronal trafficking

The mechanisms regulating synapse numbers during development and aging are essential for normal brain function and closely linked to brain disorders including dementias. Using Drosophila, this study demonstrates roles of the microtubule-associated protein Tau in regulating synapse numbers, thus unravelling an important cellular requirement of normal Tau. In this context, it was found that Tau displays a strong functional overlap with microtubule-binding spectraplakins, establishing new links between two different neurodegenerative factors. Tau and the spectraplakin Short Stop act upstream of a three-step regulatory cascade ensuring adequate delivery of synaptic proteins. This cascade involves microtubule stability as the initial trigger, JNK signalling as the central mediator, and kinesin-3 (see Drosophila Unc-103) mediated axonal transport as the key effector. This cascade acts during development (synapse formation) and aging (synapse maintenance) alike. Therefore, these findings suggest novel explanations for intellectual disability in Tau deficient individuals, as well as early synapse loss in dementias including Alzheimer's disease (Voelzmann, 2016).

The correct formation and subsequent maintenance of synapses is a key prerequisite for brain development, function and longevity. Precocious loss of synapses is observed in late onset neurodegenerative diseases including Alzheimer's disease (AD) and Frontotemporal Dementia (FTD), likely contributing to the cognitive decline and neuronal decay observed in patients. Therefore, the characterisation of mechanisms maintaining synapses during ageing would have major implications for understanding of dementias (Voelzmann, 2016).

The development of synapses and their maintenance during ageing is dependent on sustained transport of synaptic proteins from the distant soma, driven by motor proteins which trail along the bundles of microtubules in axons and dendrites. Microtubules are regulated by microtubule binding proteins which are therefore in a key position to regulate synapse formation and maintenance (Voelzmann, 2016).

Tau is a microtubule associated protein (MAP) discovered in the mid-seventies. Reduction in Tau levels has been linked to intellectual disability and a class of brain disorders termed 'dementias which lack distinctive histopathology' (DLDH). Tau detachment from MTs is linked to prominent neurodegenerative diseases such as Alzheimer's disease, Frontotemporal Dementia and some forms of Parkinson's disease. In vitro, Tau has the ability to regulate microtubule properties including stability, cross-linkage and polymerisation. Through such functions, Tau would be expected to regulate multiple aspects of neuronal cell biology, but its physiological roles are still not understood and highly debated. This might partly be due to experimental challenges posed by functional redundancy, where other MAPs are proposed to mask physiological roles of Tau (Voelzmann, 2016).

A good model in which to deal with functional redundancy is the fruit fly Drosophila melanogaster. As is ideal for studies of Tau, Drosophila neurons provide access to powerful genetics, they are readily established for research on the neuronal cytoskeleton, on neuronal transport and on synapses. Importantly, concepts and mechanisms gained from work in flies are often well conserved in higher organisms (Voelzmann, 2016).

Work in Drosophila suggested that the spectraplakin Short Stop (Shot), a large actin-MT linker molecules and potent regulators of microtubules, could display potential functional overlap with Tau during microtubule stabilisation. This hypothesis is attractive because the well-conserved mammalian spectraplakin Dystonin is already linked to a neurodegenerative disease (type VI hereditary sensory autonomic neuropathy; OMIM #614653), and its paralogue ACF7/MACF1 plays important roles during brain development). Since ACF7 continues to be expressed in the brain, it is tempting to speculate that it might be required for neuronal maintenance (Voelzmann, 2016).

This study used Drosophila neurons, in culture and in vivo alike, to demonstrate novel roles of Tau in regulating the formation and maintenance of synapses during ageing, by coordinating the intracellular trafficking of synaptic proteins. Thus, this study shows that the role of Tau in synapse regulation occurs in functional overlap with Shot. The robust shot-tau double-mutant phenotypes enabled study of the mechanistic cascade composed of three steps: microtubule stability as the trigger, the JNK signalling pathway as the mediator and kinesin-3 mediated axonal transport of synaptic proteins as the key effector. It is propose that a new mechanism based on the loss of Tau function which could explain intellectual disability in MAPT (the human tau gene) mutant individuals and precocious synapse loss in tau-related neurodegeneration (Voelzmann, 2016).

The aim of these studies was to understand the role of endogenous Tau in neurons with particular attention to synapses. This effort was essentially aided by the finding that Tau and Shot are functionally redundant, and the subsequent incorporation of Shot into these studies. The robust phenotypes of shot-tau double-mutant neurons enabled this study to demonstrate roles of Shot-Tau during the formation and maintenance of pre-synaptic sites in axons, and unravel the underlying mechanistic cascade which involves three major steps. Firstly, the absence of Shot-Tau causes microtubule destabilisation. Secondly, this cytoskeletal stress causes aberrant JNK activity patterns with upregulation in somata and downregulation at axon tips. Thirdly, aberrant JNK activation leads to a somatic roadblock for kinesin-3 mediated transport, thus inhibiting the delivery of synaptic proteins and eventually causing synapse loss. Depending on whether the functions of Tau and/or Shot are removed during development or ageing, either the formation or the maintenance of synapses are affected, respectively (Voelzmann, 2016).

The model explaining the function of Tau and Shot in synapse establishment and maintenance by regulating intracellular transport, is supported by loss- and gain-of-function experiments, genetic interactions and cross-rescue experiments. The initial finding that shot-tau mutant neurons had reduced branch numbers, could have suggested that defects on synapse numbers is indirect. However, experiments with double knock-down in culture and in the adult brain clearly showed strong synapse reduction whilst maintaining normal branch patterns, and Unc-104 rescued synapse reduction in shot-tau mutant neurons without major increases of the branch pattern in these neurons. These results clearly demonstrate that changes in neuronal morphology are not the cause of changes in synapse number (Voelzmann, 2016).

Notably, the synaptic function of Tau described in this study for Drosophila might be conserved in higher animals or humans, since also aged Tau knock-out mice develop a reduction of synaptic proteins from the hippocampus (Voelzmann, 2016 and references therein).

These findings provide potential new mechanistic explanations for various tau related brain disorders. For example, microdeletions in the region of MAPT (the human tau gene) cause intellectual disability, and Tau's synapse-promoting roles may well contribute to this pathology. Furthermore, various tauopathies are characterised by precocious pathological loss of synapses. The currnet data suggest that loss of tau could lead to defective synapse maintenance and eventually synapse loss. For example, a prominent group of dementias which lacks distinctive histopathology (DLDH) are characterised by the loss of Tau. Further tauopathies including Alzheimer disease, typically involve hyper-phosphorylation and aggregate formation of Tau. In this scenario, there are two parallel, non-exclusive modalities through which Tau can cause pathology. Firstly, detached hyper-phosphorylated tau attains gain-of-function roles in the cytoplasm damaging neurons through a number of mechanisms. Secondly, hyper-phosphorylation of tau causes a loss-of-function condition by depleting Tau from microtubules. However, since Tau knock-out mouse models mostly failed to show significant phenotypes and the neuronal functions of endogenous tau remain little understood, the pathological importance of Tau loss from microtubules has been marginalised. The current results now re-emphasise the notion that loss of Tau from microtubules could contribute to neurodegenerative pathology and deliver mechanistic explanations (Voelzmann, 2016).

To unravel pathomechanisms caused by the loss of Tau, a combined depletion of Shot and Tau gave strong phenotypes, ideal for short-term experimental approaches. However, similar, yet milder phenotypes were found if only Tau was depleted, suggesting that the mechanisms described in this study could well contribute to slow disease progression in tauopathies. The discovery that spectraplakins are MAPs which functionally overlap with Tau, opens up new experimental avenues for Tau studies. So far, spectraplakins have been linked to the degeneration of sensory and autonomous neurons, and it remains to be elucidated whether they may have similar roles also in the brain. These results clearly hint at this possibility (Voelzmann, 2016).

The loss of Tau and/or Shot inhibits kinesin-3 mediated transport leading to accumulation of synaptic proteins in the soma of neurons. A road-block mechanism is proposed suppressing the initiation of axonal transport in somata of Shot-Tau depleted neurons, which is caused indirectly through microtubule stress and mediated by JNK (Voelzmann, 2016).

The involvement of microtubules in causing a transport block is supported by experiments using microtubule stabilising and de-stabilising drugs which rescued or mimicked the shot-tau mutant phenotypes, respectively. Similarly, axonal transport defects and cognitive deficits of PS19Tg mice (expressing the P301S mutant form of human tau) and various other mouse and fly tauopathy models were shown to be rescued by microtubule-stabilising drugs, suggesting that the mechanisms described may be conserved and relevant to disease (Voelzmann, 2016).

The somatic road-block is a novel mechanism through which the loss of Tau can interfere with the transport of synaptic proteins and provides potential explanations also for somatic accumulations of postsynaptic proteins such as PSD-95, AMPA and NMDA receptors observed in mouse tauopathy models. A likely mechanism causing a roadblock in intracellular transport could be the direct inactivation of Unc-104 or its associated adaptor proteins, for example through JNK or other kinases within its pathway. This mode of regulation has a clear precedent in kinesin-1 and its adaptor Jip which are directly phosphorylated by JNK leading to transport inhibition. Unfortunately, extensive attempts to co-immunoprecipitate JNK and Kinesin-3 were unsuccessful, leaving open for now the exact molecular mechanism (Voelzmann, 2016).

It is proposed that aberrant JNK activation downstream of microtubule destabilisation or stress is the ultimate cause for the defective delivery of synaptic proteins in Tau and/or Shot loss of function. Also in mouse, microtubule stress leads to somatic activation of the JNK pathway, suggesting this mechanism is likely to be conserved with vertebrates (Voelzmann, 2016).

The JNK pathway is emerging as a central player in neurodegenerative diseases. Its activation is prompted by various neurodegeneration risk factors including oxidative stress, inflammation, and ageing. Furthermore, JNK is activated in AD patients and in several AD models where it triggers progression of the pathology. The new link between Tau/spectraplakins, JNK and synapses proposed in this study, is therefore likely to provide mechanistic explanations for synaptic pathology observed in AD and other tauopathies (Voelzmann, 2016).

This study has delivered an important conceptual advance by revealing a new mechanistic cascade which can explain synaptic decay as the consequence of Tau loss from microtubules. Furthermore, a previously unknown functional redundancy with spectraplakins was identified as a promising new avenue for research on Tau. These findings emphasize that Tau detachment from microtubules can be an important aspect contributing to the pathology of tauopathies in parallel to roles of hyper-phosphorylated Tau in the cytoplasm. Synaptic decay, axonal transport and alterations in the JNK pathway are emerging as central players in a wider range of adult-onset neurodegenerative diseases, and here this study has aligned these factors into a concrete mechanistic cascade (Voelzmann, 2016).

Shot and Patronin polarise microtubules to direct membrane traffic and biogenesis of microvilli in epithelia

In epithelial tissues, polarisation of microtubules and actin microvilli occurs along the apical-basal axis of each cell, yet how these cytoskeletal polarisation events are coordinated remains unclear. This study examines the hierarchy of events during cytoskeletal polarisation in Drosophila and human epithelia. Core apical-basal polarity determinants polarise the Spectrin cytoskeleton to recruit the microtubule-binding proteins Patronin (CAMSAP1/2/3 in humans) and Shortstop (Shot; MACF1/BPAG1 in humans) to the apical membrane domain. Patronin and Shot then act to polarise microtubules along the apical-basal axis to enable apical transport of Rab11 endosomes by the Nuf-Dynein microtubule motor complex. Finally, Rab11 endosomes are transferred to the MyoV actin motor to deliver the key microvillar determinant Cadherin99C to the apical membrane to organise the biogenesis of actin microvilli (Khanal, 2016).

These results reveal a mechanism linking determinants of cell polarity with stepwise polarisation of the spectrin cytoskeleton, microtubule cytoskeleton and biogenesis of actin microvilli through apical trafficking of Cad99C. The results suggest that polarisation of the apical spectrin βH-Spectrin is dependent on polarity determinants, likely through interactions with the FERM domain proteins and the apical polarity determinant Crb. The spectraplakin Shot is highly similar to βH-Spectrin, and is able to bind to and colocalise with it at the apical domain of epithelial cells, suggesting that the two proteins might have a similar function. βH-Spectrin is linked to microtubules through Patronin, whereas Shot can directly bind microtubules. Consequently, redundancy is anticipated between βH-Spectrin and Shot, or between Patronin and Shot. Accordingly, this study found that mutation of βH-spectrin only had a mild phenotype, whereas mutation of α-spectrin simultaneously disrupted both pairs of proteins in parallel and caused a drastic phenotype, completely disrupting the apical trafficking of Cad99C and microvillar biogenesis. More importantly, double mutants for shot and βH-spectrin had a more severe effect on microtubule and Cad99C localisation than either alone, therefore demonstrating that the two proteins act in a redundant fashion (Khanal, 2016).

Downstream of the spectrin cytoskeleton, Patronin and Shot are required in parallel to drive apical-basal polarisation of microtubules, which is then responsible for orienting the apical transport of Cad99C, within Rab11 endosomes, by the Dynein motor protein. Eliminating microtubules from cells by overexpressing Katanin60 results in loss of Nuf-Dynein-based apical Rab11 endosome transport and failure to efficiently deliver Cad99C to the apical membrane. The effect on Cad99C polarisation is not an indirect effect of loss of polarity due to impaired Rab11 and Dynein function in localising the apical polarity determinant Crumbs to the apical membrane because, firstly, polarity is maintained in cells expressing Rab11 or Dynein RNAi, as indicated by the normal localisation of aPKC and, secondly, loss of Crb does not strongly affect cell polarity in the follicle cell epithelium owing to redundancy with Bazooka. The results indicate that even under conditions with severe depletion of microtubules, the overall shape of the follicle cell epithelium is relatively normal, indicating that polarised microtubules are required to influence formation of apical microvilli, rather than for other functions of the actin cytoskeleton in epithelial cells. Similarly, no strong effects are seen on cell shape upon loss of either Patronin or Shot (or both), raising questions over the claimed requirement for Patronin homologs and microtubules in formation or maintenance of adherens junctions epithelial cells in culture (Khanal, 2016).

The final step in delivery of Cad99C to the apical membrane also requires actin-based transport through the action of Rip11-MyoV complex. Compromising normal MyoV function in Drosophila follicle cells by expressing a dominant-negative version of the protein, results in loss of Rab11 polarisation from the apical membrane and its abnormal accumulation in the sub-apical region. This phenotype in Drosophila shows similarities with the human microvillus inclusion disease, where mutations in the Myo5b gene also cause loss of Rab11 endosomes from the apical membrane (Khanal, 2016).

In summary, these results reveal how the spectrin cytoskeleton acts to polarise microtubules in epithelial cells, and how polarised microtubules then direct trafficking of Rab11 endosomes carrying Cad99C to the apical membrane. This process relies on a hierarchy of events, and disruption at any stage can lead to failure in delivering Cad99C to the apical membrane, resulting in defective biogenesis of microvilli. These findings are directly relevant to human diseases such as Usher's Syndrome Type 1 and microvillus inclusion disease, helping to outline the molecular and cellular basis for these conditions (Khanal, 2016).

Patronin/Shot cortical foci assemble the noncentrosomal microtubule array that specifies the Drosophila anterior-posterior axis

Noncentrosomal microtubules play an important role in polarizing differentiated cells, but little is known about how these microtubules are organized. This study identified the spectraplakin, Short stop (Shot), as the cortical anchor for noncentrosomal microtubule organizing centers (ncMTOCs) in the Drosophila oocyte. Shot interacts with the cortex through its actin-binding domain and recruits the microtubule minus-end-binding protein, Patronin, to form cortical ncMTOCs. Shot/Patronin foci do not co-localize with gamma-tubulin, suggesting that they do not nucleate new microtubules. Instead, they capture and stabilize existing microtubule minus ends, which then template new microtubule growth. Shot/Patronin foci are excluded from the oocyte posterior by the Par-1 polarity kinase to generate the polarized microtubule network that localizes axis determinants. Both proteins also accumulate apically in epithelial cells, where they are required for the formation of apical-basal microtubule arrays. Thus, Shot/Patronin ncMTOCs may provide a general mechanism for organizing noncentrosomal microtubules in differentiated cells (Nashchekin, 2016).

The recent discovery of the Patronin family of MT minus-end-binding proteins, consisting of Patronin in Drosophila, CAMSAP1, 2, and 3 in mammals, and PTRN-1 in worms, has begun to reveal how the minus ends of noncentrosomal MTs are organized and maintained. The Patronins recognize and stabilize free MT minus ends by protecting them from depolymerization. Patronins appear to play a particularly important role in organizing MTs in differentiated cells. CAMSAP3 localizes to the apical domain in epithelial cells, where it is required for the formation of the apical-basal array of MTs. CAMSAP2 stabilizes neuronal MTs in axon and dendrites, and its knockdown leads to defects in axon specification and dendritic branch formation. Similarly, Caenorhabditis elegans PTRN-1 is required for normal neurite morphology and axon regeneration. The function of Drosophila Patronin has only been examined in cultured S2 cells, where its depletion leads to a decrease in MT number and an increase in free moving MTs (Nashchekin, 2016 and references therein).

The polarized arrangement of the MTs in the Drosophila oocyte depends on the posterior crescent of the Par-1 kinase, which excludes MT minus ends from the posterior cortex. This study shows that Par-1 acts by preventing the association of Shot with the posterior actin cortex, thereby restricting the formation of noncentrosomal MTOCs to the anterior and lateral cortex. Computer modeling has shown that this asymmetric localization of MT minus ends is sufficient to explain the formation of the weakly polarized MT network that directs the transport of oskar mRNA to the posterior pole. Thus, the regulation of the interaction of Shot with the cortex by Par-1 transmits cortical PAR polarity into the polarization of the MT cytoskeleton that localizes the axis determinants (Nashchekin, 2016).

The mechanism by which Par-1 excludes Shot is unknown. The interaction of Shot with the cortex depends on its N-terminal calponin homology domains, which bind to F-actin. Thus, Par-1 could phosphorylate Shot to inhibit its binding to the cortex. If this is the case, Par-1 would have to modify the activity or accessibility of the N-terminal ABD of Shot, as this domain recapitulates the posterior exclusion and cortical recruitment of the full-length protein. Phosphorylation of the ABD by Par-1 was not detected in vitro, however, and it seems more likely that Par-1 acts by modifying the cortex to block the binding of Shot (Nashchekin, 2016).

Shot and its vertebrate ortholog, MACF1, have previously been shown to interact with the MT plus-end tracking protein EB1 through their C-terminal SxIP motifs and with the MT lattice through their Gas2 and C-terminal domains (Nashchekin, 2016).

The current results indicate that in addition to binding to MT plus ends and to the MT lattice, Shot also interacts with MT minus ends through its association with the Patronin/Katanin complex. The exact nature of the interaction between Shot and the Patronin complex is unclear, but Shot was found to interact with Katanin 60 in a high-throughput yeast two-hybrid screen. Thus, one possibility is that Katanin acts as a link between Shot and Patronin. Since Shot is exclusively cortical in the oocyte, the protein does not appear to bind to MT plus ends or along the body of MTs in this system. It will therefore be interesting to investigate whether the different modes of MT binding by Shot are mutually exclusive and how this is regulated (Nashchekin, 2016).

Several models have been proposed to explain the formation of noncentrosomal MTs. Upon centrosome inactivation in postmitotic Drosophila tracheal cells and C. elegans intestinal cells, γ-TuRC complexes and other pericentriolar material (PCM) components are released from the centrosome and transported toward the apical membrane, where they nucleate MT. Whole MTs released from the centrosome can also be delivered and anchored to the apical domain or cell junctions by Ninein. Alternatively, new MTs can grow from MT ends generated by severing enzymes, a mechanism that is thought to be important in plant cells and neurons (Nashchekin, 2016).

This study presents evidence that this latter mechanism is responsible for the formation of the MT array that directs Drosophila axis formation. Firstly, Shot/Patronin ncMTOCs contain stable minus ends even after treatment with the MT-depolymerizing drug, colcemid, as shown by the persistent recruitment of Tau-GFP and EB1-GFP to these foci. This is consistent with the ability of Patronin and CAMPSAPs to capture and stabilize minus ends of single MTs in vitro and in cells. Secondly, MTs start to grow out in all directions from the Shot/Patronin foci immediately after colcemid inactivation. Indeed all visible growing MTs emanate from Patronin foci, indicating that they are the principal source of MTs in the oocyte. Thirdly, the foci contain no detectable γ-tubulin and do not co-localize with PCM proteins. This is consistent with observations in Caco-2 cells, which showed that CAMSAP2 and CAMSAP3 do not co-localize with γ-tubulin and in the C. elegans epidermis, where PTRN-1 and γ-tubulin function in parallel pathways to assemble circumferential MTs (Nashchekin, 2016).

Taken together, these results suggest a model in which the Shot/Patronin foci act as ncMTOCs by capturing and stabilizing MT minus-end stumps that then act as templates for new MT growth. One attractive feature of this model is that it uncouples MT organization from MT nucleation in both space and time. The Shot/Patronin complex bypasses the need to continually nucleate new MTs by preventing existing microtubules from completely depolymerizing. Thus, once a cell has nucleated sufficient MTs, it can maintain and reorganize its MT cytoskeleton by stabilizing MT minus-end stumps in appropriate locations and using these, rather than the γ-tubulin ring complex, to provide the seeds from which new MTs grow. The number of MTs can even increase in the absence of new MT nucleation if MT-severing proteins chop up existing MTs to produce new minus ends that can then be captured and stabilized. The presence of the severing protein, Katanin, in the Shot/Patronin foci is intriguing in this context, as it raises the possibility that it severs existing MTs to provide a local source of minus ends for Patronin to capture (Nashchekin, 2016).

Shot and Patronin also co-localize at the apical cortex of the epithelial follicle cells, where they are required for apical-basal MT organization. This consistent with the recent observation that CAMSAP3 is required for the recruitment of MT minus ends to the apical cortex of mammalian intestinal epithelial cells (Toya, 2016). Thus, this function of Patronin has been evolutionarily conserved. Furthermore, the similarities between roles of Shot and Patronin in the oocyte and the follicle cells suggest that the complex may provide a general mechanism for organizing noncentrosomal MTs. The relationship between Shot and Patronin is different in the follicle cells compared with the oocyte, however, as Shot is not required for the apical recruitment of Patronin. Nevertheless, loss of either protein produces a very similar loss of apical MT and a reduction in overall MT density. Although it cannot be ruled out that they act in parallel pathways, this observation suggests that they collaborate to anchor MTs to the apical cortex. The combination of Patronin binding to the MT minus ends and Shot binding to the MT lattice may therefore provide a robust anchor to retain MTs at the apical cortex (Nashchekin, 2016).

A modifier screen identifies regulators of cytoskeletal architecture as mediators of Shroom-dependent changes in tissue morphology

Regulation of cell architecture is critical in the formation of tissues during animal development. The mechanisms that control cell shape must be both dynamic and stable in order to establish and maintain the correct cellular organization. Previous work has identified Shroom family proteins as essential regulators of cell morphology during vertebrate development. Shroom proteins regulate cell architecture by directing the subcellular distribution and activation of Rho-kinase, which results in the localized activation of non-muscle myosin II. Because the Shroom-Rock-myosin II module is conserved in most animal model systems, Drosophila melanogaster was used to further investigate the pathways and components that are required for Shroom to define cell shape and tissue architecture. Using a phenotype-based heterozygous F1 genetic screen for modifiers of Shroom activity, several cytoskeletal and signaling protein were identified that may cooperate with Shroom. Two of these proteins, Enabled and Short stop, are required for ShroomA-induced changes in tissue morphology and are apically enriched in response to Shroom expression. While the recruitment of Ena is necessary, it is not sufficient to redefine cell morphology. Additionally, this requirement for Ena appears to be context dependent, as a variant of Shroom that is apically localized, binds to Rock, but lacks the Ena binding site, is still capable of inducing changes in tissue architecture. These data point to important cellular pathways that may regulate contractility or facilitate Shroom-mediated changes in cell and tissue morphology (Hildebrand, 2021).

Tissue architecture is typically defined during specific stages of embryonic development and errors in these processes can result in human disease. One example is formation of the vertebrate neural tube. The neural tube is formed via the concerted effort of many cellular pathways that functionally convert a plate of neural ectoderm into a closed tube. Errors in this process can result in birth defects such as spina bifida, exencephaly, or craniorachischisis. One cellular pathway that controls this process is regulated by the Shroom3 cytoskeletal adaptor protein. Shroom3 controls neural tube morphogenesis via the formation of apically positioned contractile networks of actomyosin and these networks facilitate neural tube closure by inducing apical constriction and the anisotropic contraction of actin filaments. This is accomplished via the modular nature of Shroom3. Shroom3 localizes to the apical compartment of epithelial adherens junctions via a direct interaction with F-actin. This interaction is mediated by the Shroom Domain (SD) 1, a unique actin-binding motif present in most Shroom proteins characterized to date. Shroom3 function is also dependent on Rho-kinase (Rock), such that Shroom3 directly binds to Rock and regulates both its localization and catalytic activity. The interaction between Shroom and Rock has been elucidated at the molecular level and is mediated by the conserved SD2 region of Shroom and a conserved coiled-coil region of Rock. The interaction between Shroom and Rock results in the localized activation of non-muscle myosin II (myosin II) contractility, which provides the mechanical force needed to facilitate neural tube morphogenesis. The regulation of myosin II activity by Rock and other cellular pathways has been well described. Rock modulates myosin II activity in two ways. First, Rock can directly phosphorylate the associated regulatory light chain (RLC), which modulates the actin-associated ATPase activity and the conformation of myosin II. Secondly, Rock negatively regulates the phosphatase that dephosphorylates the RLC, thus preventing the inactivation of myosin II (Hildebrand, 2021 and references therein).

Shroom proteins are required for numerous biological processes and are associated with several human diseases. In mammals, there are three definitive Shroom proteins, Shroom2, Shroom3, and Shroom4, each of which contains an N-terminal PDZ domain, the centrally located SD1, and the C-terminally located SD2. All three proteins can directly interact with F-actin and regulate cell morphology via Rock. In humans, SHROOM2 has been linked to neural tube morphogenesis, colorectal cancer, and medulloblastoma, while in vitro studies indicate it is important for cell migration, vasculogenesis, metastasis, and melanosome biogenesis. SHROOM3 mutations have been implicated in chronic kidney disease, heart morphogenesis, and neural tube closure in humans. Using model organisms or cell culture, Shroom3 has been shown to control neural tube closure, axon growth, intestine architecture, eye morphogenesis, thyroid budding, and kidney development. Finally, SHROOM4 mutations have been associated with X-linked mental defects (Hildebrand, 2021).

The Shroom gene is conserved in Drosophila and encodes multiple protein isoforms that have different subcellular distributions and activities in vivo. The most highly conserved region of Drosophila Shroom is the SD2, the region that binds to Drosophila Rho-kinase (Rok). Drosophila Shroom also contains a divergent SD1 motif and this appears to mediate localization to adherens junctions in polarized epithelia. Consistent with the known activities of mammalian Shroom3, expression of Drosophila Shroom in epithelial cells induces apical constriction in a Rok and myosin II dependent manner. While Shroom3 is essential for mouse and human development, Shroom is not absolutely essential for Drosophila viability, as Shroom null flies can be recovered, albeit with significantly reduced frequency. In Drosophila embryos, Shroom is planarly distributed and works in a complicated network with RhoA, Rok, and myosin II to control convergent extension movements. These elegant studies showing the role of Shroom in regulating directional contractility are supported by observations that Shroom proteins can be polarly distributed in mammalian tissues and cells (Hildebrand, 2021 and references therein).

To better understand the mechanisms that control Shroom-regulated changes in cell and tissue morphology, this study has established tools to perform genetic screens for modifiers of Shroom activity in Drosophila. Shroom gain-of-function phenotypes in the eye and wing can be suppressed or enhanced by known components of the Shroom pathway. Using a candidate approach, several cytoskeletal regulators were identified, including Short stop and Enabled, as participants in Shroom-mediated changes in cell morphology. Shroom regulates the distribution of Ena and this is likely mediated by conserved proline-rich sequences in Shroom and the EVH1 domain of Ena. This study further shows that while Ena is required for the Shroom gain-of-function phenotypes, apical recruitment of Ena is not sufficient to cause changes in cell morphology. Additionally, by using an isoform of Shroom that does not bind Ena, but still engages Rok, this study showed that apical constriction can be modulated by different cellular pathways depending on the context (Hildebrand, 2021).

This study describes a genetic approach to identify cellular pathways that participate in tissue morphogenesis. This method takes advantage of the observation that ectopic Shroom protein can utilize the endogenous contractile machinery within epithelial cells to induce apical constriction and disrupt normal tissue morphology. While this work focuses on candidate genes that encode known regulators of epithelial and tissue architecture, it is predicted these tools can be used to perform unbiased, genome-wide screens to identify novel participants in Shroom-mediated cellular processes. Two different tissues, eye and wing imaginal discs, were used for these studies, and these screens can identify factors that are used in a wide range of tissues and cells to control cell dynamics. This is based on the observations that ShroomA, the isoform most similar to mammalian Shroom3, induces similar cellular phenotypes in both types of imaginal discs, and the phenotypes can be modified in both tissues. A powerful aspect of this screen is that these processes are functionally conserved in vertebrate cells and tissues. Additionally, the simplified nature of the Drosophila genome makes these screens possible. Due to genetic and functional redundancy, it is predicted that the analysis performed in this study would be more complicated using vertebrate or cell culture model systems. Drosophila have single genes for Shroom, Rok, myosin II, and Ena while mammals possess gene families for these factors. In support of this, previous work has shown that both Rock1 and Rock2 must be inhibited to prevent Shroom3-mediated apical constriction in cell culture. This screening approach should allow for the identification of novel genetic interactions in Drosophila that can be further verified in mammalian model systems to define their potential role in human disease (Hildebrand, 2021).

Most of the modifiers identified in this study participate in defining actin or microtubule architecture. Of these, several regulate actin dynamics at the level of polymerization or stability, including Ena, Diaphanous, Chickadee, and Slingshot. Interestingly, three of these proteins can be linked, directly or indirectly, to neural tube formation in mice. It should be noted that several classes of actin regulators did not appear to modify the Shroom phenotypes, including nucleators, binding proteins, or adaptors, suggesting that specific types of actin organization are required for Shroom-induced perturbation of cell architecture. This is further supported by the observation that Tropomyosin was also identified in the screen. Tropomyosin regulates the structure of actin filaments and the binding of other proteins, including myosin II and cofilin, that in turn modulate cell architecture or behavior. It is particularly intriguing to note that Tropomyosin mutations can suppress phenotypes caused by the loss of Flapwing, presumably caused by increased myosin II activity. In addition to the actin cytoskeleton, these studies also support a role for microtubules in Shroom-induced phenotypes. This is consistent with the role of microtubules in apical constriction in Drosophila. Recent evidence indicates that apical-medial microtubules play an important role in ventral furrow invagination and this is mediated by Patronin, a protein known to interact with Shot. These studies show that microtubules stabilize the connection of contractile networks to cell junctions to facilitate tissue morphogenesis. These studies are consistent with the current results in relation to Shroom function and Shot distribution in the wing epithelium. It will be interesting to determine if the identified proteins act upstream or downstream of Shroom. While the data suggest Ena acts downstream of Shroom, proteins such as Tropomyosin could function upstream by regulating the amount of Shroom that can bind to F-actin or downstream by modulating the amount of myosin II that can be recruited or activated by the Shroom-Rok complex. It was surprising that determinants of cell adhesion or polarity, such as cadherins or Par complex proteins, were not identified in this screen. It is possible that these proteins are present in sufficient quantity and reducing the dosage is unable to modify the Shroom overexpression phenotype and thus other genetic approaches will be needed to assess the role of these pathways (Hildebrand, 2021).

The data show that endogenous Shroom protein is expressed in epithelial cells during wing and eye development, suggesting it functions in these tissues under normal circumstances. Shroom null flies that survive to adults do not exhibit significant defects in the eyes or wings, although null embryos do exhibit defects in convergent extension and perhaps this could contribute to the observed reduction in viability. In embryos, Shroom is important for the polarized distribution of contractile myosin II needed for convergent extension. It is possible that Shroom activity in disc epithelial cells is redundant to other pathways that regulate Rok and myosin II and Shroom normally functions to make these pathways more robust or function with higher fidelity. Uncovering these subtle interactions will require additional genetic approaches. The localization of Shroom in the eye and wing disc appears to be highly regulated and is reminiscent of that exhibited by myosin II and phosphorylated Sqh, particularly in the eye imaginal disc. A dramatic increase was observed in Shroom protein in cells that are exiting the morphogenetic furrow and forming the pre-clusters that will give rise to the ommatidia. As the ommatidia form, Shroom expression becomes restricted to the R3/4 cells and eventually is lost from these cells. This distribution is essentially the inverse to that of E-cadherin, which is highest in the radial junctions and lower in the circumferential junctions. This could reflect differences in adhesive interactions between the ommatidia pre-clusters and the inter-ommatidia cells, which facilitates rotation of the ommatidia. This hypothesis is supported by previous studies demonstrating that differential adhesion generates specific cellular organization and compartmentalization in the developing eye. Interestingly, the PCP protein Flamingo is also expressed in R3 and R4 and previous studies have identified interactions between the Shroom3 and PCP pathways in the neural tube. As eye development continues, this study observed Shroom expression in the pigment cells of the pupal retina. In both the imaginal disc and the retina, Shroom distribution is restricted to specific cell junctions, suggesting there are differential adhesive or contractile forces associated with these membranes (Hildebrand, 2021).

In the wing imaginal disc, expression of Shroom protein was observed in rows of cells that border the anterior half of the wing margin. Consistent with the genetic interactions, a similar expression pattern was observed for both Ena and Shot in these cells. It is currently unclear if the co-expression of Shroom, Ena, and Shot is controlled pre- or post-transcriptionally. It is possible that the expression of Shroom, Ena, and Shot is coordinately regulated in a gene network. Alternatively, the stability or apical localization of these proteins may be interdependent or closely orchestrated. This expression pattern in the anterior wing margin is similar to members of the Irre cell Recognition Module (IRM), including cell surface receptors Roughest, Hibris, and Kirre, which help position the sensory organs. This is particularly interesting in light of the fact that the vertebrate orthologs of these genes, Neph and Nephrin-1, and Shroom3 are all involved in formation of podocytes in the glomerulus of the mammalian kidney. It will be exciting to apply genetic analysis to investigate if these pathways cooperate to regulate tissue morphology (Hildebrand, 2021).

Ena and Shroom show extensive co-expression and colocalization in both the wing and eye imaginal disc, although Ena is more widely expressed than Shroom. In both the wing and eye imaginal disc, Ena is expressed in most cells and is localized primarily in the tricellular junctions with lower expression in the adherens junctions. However, as seen in the wing margin and the morphogenetic furrow, cells that express Shroom protein also exhibit high levels of Ena in the cell junctions. Importantly, reducing the amount of Shroom protein perturbs the localization of Ena in the anterior wing margin. The relationship between Ena, Shroom, Rok, and myosin II in defining cell shape is likely to be complicated. This stems from the observations that these factors could be placed both upstream and downstream of Shroom. For example, it has been previously shown that Shroom distribution to the apical adherens junctions is mediated, at least in part, by direct binding to F-actin. However, it has also been established that RhoA and Rok regulate F-actin architecture to influence Shroom distribution, which then facilitates the polarized distribution of Rok and myosin II. Ena has been shown to have multiple roles in Drosophila development, including axon guidance, collective cell migration, and epithelial morphogenesis. The role Ena plays in Shroom-mediated apical constriction is unclear. The current data suggest that Ena functions downstream of Shroom and is recruited to adherens junctions via an LPPPP-EVH1 interaction. Ena is primarily defined as a modulator of F-actin dynamics that facilitates the formation of long filaments by competing with barbed-end capping and promoting the addition of actin monomers to the barbed end. This activity may be important for providing the substrate for activated myosin II to drive cell contraction. This is consistent with studies in vertebrate cells showing that Diaphanous 1, is also required for contractility in adherens junctions and that this study has also identified Dia as a potential modifier of Shroom activity (Hildebrand, 2021).

Elegant studies from several groups have identified many other signaling pathways that control the distribution of contractile myosin II networks during Drosophila development, including the Fog, PCP, HH, Dpp, EGF, Toll, and integrin signaling pathway. How all these signaling pathways are orchestrated and converge on myosin II at the cellular and tissue level is a fascinating question. It has been shown that the above processes use a variety of methods to regulate the small GTPase RhoA, which activates Rok, including several GTP exchange factors or GTPase Activating Proteins. It should be noted that other GTPases such as Rap1 or CDC42 also regulate apical constriction. This work has shown that Shroom3 may activate Rock independent of RhoA, suggesting that there as mechanisms to bypass small GTPases in the activation of myosin II. It will be informative to utilize this screening approach to further test how these pathways might work with ShroomA to control cell morphology (Hildebrand, 2021).

GENE STRUCTURE

Northern blot analysis using a kak cDNA fragment reveals two transcripts of ~17.6 and ~15.4 kbp (Strumpf, 1998). Two cDNA sequences overlap to give mRNAs of 17,420 nucleotides encoding a 5,497-amino acid protein (form A) and 17,217 nt encoding a 5,385-amino acid protein (Gregory, 1999)

PROTEIN STRUCTURE

Amino Acids - 4,151 (Strumpf, 1998) and 5,497 and 5,338 (Gregory, 1998)

Structural Domains

The primary amino acid sequence of Kak reveals several domains and motifs that show high degrees of similarity to three distinct vertebrate cytoskeletal-related protein families: plakin (Ruhrberg, 1997a); dystrophin (Koenig, 1988), and Gas2/GAR22 (Schneider, 1988; Zucman-Rossi, 1996). The NH2-terminal region of Kak is homologous to the NH2-terminal domain of members of the plakin family of cytoskeletal cross-linker proteins, comprising plectin, BPAG1, and ACF7 (Ruhrberg, 1997). These large proteins link actin microfilaments and intermediate filaments to the plasma membrane at specialized attachment sites, called hemidesmosomes. Abnormal function of various plakin family members leads to skin (e.g., bullous pemphigous) as well as neurological (e.g., dystonia musculorum) disorders (for reviews see Ruhrberg, 1997a; Fuchs, 1998). The region of similarity between Kak and plakin family members includes the actin binding region but does not exhibit similarity to the intermediate filament-associated domain. The area of strongest similarity is with an actin-binding domain originally defined in alpha-actinin, but subsequently found in dystrophins and spectrins as well as the plakin family. Across this 240-amino acid region, Kakapo shares ~65% amino acid identity with plectin and BPAG1. The high level of conservation suggests that this domain in Kak does bind to actin. All proteins so far described in the plakin family have a carboxy-terminal domain that binds intermediate filaments, encoded by a single large final exon (Ruhrberg, 1997a). This does not appear to be the case for the Kak protein, which, after residue 1200, has no further sequence similarity with plakins, and instead becomes similar to dystrophin. The central region of Kak (amino acids 408-3574) consists of 22 repeats, 105-113 amino acids long. A computerized search has indicated that the central region of Kak shares sequence similarity (~20% identity) with spectrin-like repeats present in an array of cytoskeletal-associated proteins, including dystrophin, alpha-actinin, and spectrin. These repeats are predicted to adopt a triple-helical conformation. In dystrophin, the multiple repeat domain functions as a spacer between the NH2-terminal actin-binding domain and the COOH-terminal domain associated with a group of membrane proteins. The consensus sequence deduced from the alignment of the spectrin-like repeats in Kak shares 46% identity (51% similarity) with the human dystrophin repeat consensus, CS1 (Koenig, 1990). This similarity suggests the presence of a similar domain containing multiple spectrin-like, triple-helical repeats in the central region of Kak protein. This region also contains five Leucine-zipper motifs. A somewhat lower degree of similarity between the Kak COOH-terminal domain (sequence 3725-3793) and the region in dystrophin containing the two EF-hand motifs is also observed. The COOH-terminal domain of dystrophin-related proteins is highly conserved and includes a WW domain, implicated in mediating interactions with the transmembrane protein, beta-dystroglycan. This domain also includes two putative Ca2+-binding EF-hands (Kawasaki, 1995) and a region involved in the binding to members of the syntrophin family of PDZ domain-containing proteins. The similarity between Kak and dystrophin in the COOH-terminal domain is detected only along the EF-hand motifs (~30% identity along a sequence of 70 amino acids). This limited similarity may suggest that although Kak does not appear to be a dystrophin family member, these genes may share a common ancestor (Strumpf, 1998; Gregory, 1998, and their references).

The COOH-terminal region of Kak shows sequence conservation with yet another family of cytoskeletal-related proteins represented by the Gas2/GAR22 proteins (Schneider, 1988; Zucman-Rossi, 1996). Mouse Gas2, a member of this protein family, belongs to a set of proteins that have been shown to be selectively expressed in growth-arrested cells in culture (Schneider, 1988). It is a highly regulated protein (Brancolini, 1992; Manzow, 1996) that interacts with the microfilament system (Brancolini, 1992; Brancolini, 1994). Deletion analysis of the Gas2 protein suggests that the region in Gas2 that is homologous to Kak has a significant function in cytoskeletal organization (Brancolini, 1995). During apoptosis, Gas2 is cleaved by ICE proteases, presumably leading to microfilament derangement (Brancolini, 1995). An additional partially cloned cDNA species of unknown function from human brain exhibits a high level of identity to the COOH-terminal domain of Kak protein. This similarity extends beyond the Gas2 homology domain and exhibits 40% overall identity along the entire 1,658-amino acid sequence available in the data base. It is not clear whether this partially cloned cDNA represents a human Kak homolog. In addition, a putative protein from C. elegans shares domain contents with Kak, including plectin, dystrophin, and Gas2/ GAR22-like domains. The function of this putative protein is yet to be elucidated (Strumpf, 1998 and references).

Taken together, the deduced amino acid sequence of Kak predicts a novel large intracellular protein that carries two distinct cytoskeletal-associated domains separated by a spacer consisting of elongated triple-helical spectrin-like repeats. At the NH2-terminal domain, Kak may interact with actin microfilaments, while at its COOH terminus, it may be associated with membrane structures or with additional cytoskeletal components. The similarities between Kak and its C. elegans and putative human homologs suggest that Kak structure is conserved through evolution (Strumpf, 1998 and references).

Two more isoforms of Shot/Kak have been identified. Two shot cDNAs (form C) encode a third unique N-terminal sequence of 210 amino acids followed by half of the actin binding domain. This second half of the actin binding domain is less evolutionarily conserved than the first half of the actin binding domain. The mammalian BPAG1n3 protein also contains a similar half actin binding domain and associates poorly with the actin cytoskeleton in cultured cells. Finally, isoform D lacks an identifiable actin binding domain and contains no N-terminal globular domain. The likely initiator methionine codon for isoform D is located downstream of sequences encoding the actin binding domain, at the start of sequences encoding the central rod domain. Thus, shot encodes various rod-like proteins predicted to differ in their actin binding properties. The genes for other plakins also encode similarly spliced actin binding variants. Four different 5' isoforms are encoded by the BPAG1 gene. Two neuronal isoforms, BPAG1n1 (dystonin-1) and BPAG1n2 (dystonin-2), contain an actin binding domain; a third neuronal isoform, BPAG1n3, contains a half-actin binding domain analogous to that found in Shot isoform C, and the epidermal isoform BPAG1e contains no actin binding domain, as in Shot isoform D. Mouse ACF7 is 71% identical to BPAG1 within the predicted actin binding domain and encodes N-terminal isoforms similar to Shot isoforms A, B, and C. Both isoforms 1 and 2 of mACF7 are predicted to contain a complete actin binding domain; mACF7 isoform 3 lacks the most conserved portion of the actin binding domain and contains the less conserved portion exactly as in Shot isoform C (Lee, 1999 and references therein).

Thus the shot gene may contain as many as four different promoters. The authors investigated the relationship between the P-element insertions in shot and the shot promoter and transcription start sites. A combination of PCR, Southern analysis, and sequencing was used to map the shotP1, shotP2, kakP1, and kakP2 insertion sites and the cDNA sequences onto a 15 kb genomic DNA contig. The kakP1 and kakP2 P-elements are inserted at the same site, in an intron 1917 bp before the first exon common to mRNAs encoding isoforms A and B. The shotP2 insertion is 49 bp upstream of the start of alternative transcript C. The shotP1 insertion is located in an exon common to both isoforms C and D, 131 bp downstream of the alternative splice site that generates isoform C. Although they are inserted at different sites with respect to the shot transcripts, all of the P-element insertions disrupt the protein expression of the long isoforms of Shot and appear to be similar in their axon growth phenotypes (Lee, 1999 and references therein).

Additional isoforms have been identified of Shot/Kak, the longest of which encodes a 5501 amino acid protein that is almost completely identical to the previously reported 5497 amino acid protein (Gregory, 1998; Strumpf, 1998). The central region of this protein is likely to be rod-like and contains 22 triple helical coiled-coil repeats similar to those found in spectrin and dystrophin. The C-terminal globular domain contains two EF-hand motifs. The C-terminal globular domain also contains a short stretch of sequence homology to the mammalian growth arrest-specific 2 (Gas2) protein. Gas2 is a cytoskeletal protein of unknown function that appears to be associated with microfilaments in cultured cells and is highly induced in cultured fibroblasts during serum starvation. The cDNA sequences isolated for the 3' end of the shot gene reveal that the central rod region is alternatively spliced. Seven of eight cDNA clones predict an isoform that shares the same central rod sequence reported previously (Strumpf, 1998). The other clone encodes a 300 amino acid sequence within the central rod region, as previously reported for Shot/Kak isoforms A and B (Gregory, 1998). A cDNA was also isolated in which the sequence for the 300 amino acid region is spliced to a novel sequence that encodes a globular domain of 436 amino acids. This 436 amino acid domain shows low homology to the six tandem repeat domains in the C-terminal globular region of plectin and is unlikely to form an extensive coiled-coil structure. Although it still contains considerable coiled-coil forming sequence, this truncated isoform lacks the 22 triple helical repeats found in the longer isoforms. By comparing the known lengths of plectin and dystrophin proteins, and the relative lengths of coiled-coil forming sequence in Shot, plectin and dystrophin, it is inferred that the truncated isoforms of Shot are ~75 nm long and the long isoforms of Shot are ~200-220 nm long. Thus, shot encodes rod-like proteins of varying length with different C-terminal domains, as well as different predicted actin binding properties (Lee, 1999 and references therein).

The sequence of the C-terminal domain in the longer Shot isoforms matches the C-terminal sequences of several vertebrate proteins, including the full-length sequence of mouse ACF7 recently reported in GenBank. The sequence of the EF-hand and GAS2 domains are particularly well conserved. Sequencing of the shot cDNAs also reveals diversity in this C-terminal domain. The C-terminal sequence after the Gas2 homology domain is alternatively spliced, with the variant reported here being a closer match to the vertebrate proteins. Taken together, overlapping cDNAs have been cloned that predict multiple isoforms of Shot/Kak and greatly expand the potential functional diversity of this gene. The gene encodes rod-like proteins of varying lengths, only some of which contain complete N-terminal actin binding domains. These proteins contain two different classes of globular C-terminal domains, which in plakins mediate protein-protein interactions. By analogy, the different Shot proteins may therefore interact with diverse cytoskeletal targets (Lee, 1999).

date revised: 2 September 2021

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