BIOLOGICAL OVERVIEW

The human tumor suppressor gene PTEN gets its name from its biochemical function, its domain structure and its chromosomal location: PTEN stands for the combination of phosphatase and tensin homolog on chromosome 10 (J. Li, 1997). The protein is also known as MMAC1 (Steck, 1997) or TEP1 (D. Li, 1997). The protein exhibits phosphatase activity against proteins and lipid phosphate residues. The PTEN protein contains a protein phosphatase domain with similarity to dual specificity phosphatases (D. Li, 1997; Li and Sun, 1997; Steck, 1997). Dual specificity phosphatases act both on serine/threonine and tyrosine phosphotyrosine residues. The phosphatase domain is embedded within a domain with homology to an actin-binding motif in the focal adhesion-associated protein tensin. Immunohistochemical evidence has suggested that like tensin, PTEN is associated with the cytoskeleton (D. Li, 1997).

Now that the domain structure of PTEN has been described, the known and potential functions of the PTEN homolog in Drosophila and mammals is presented. The lipid phosphatase function of PTEN places it in the middle of the insulin pathway, known to involve lipid signaling (Goberdhan, 1999; Huang, 1999). Drosophila Pten modulates cell size, and consequently tissue mass, by acting antagonistically to the lipid modifiying enzyme Phosphotidylinositol 3 kinase 92E, also known as Dp110, and its upstream activator Chico, an insulin receptor binding and signal transduction protein (Goberdhan, 1999). All signals from the Insulin-like receptor can be antagonized by Pten (Huang, 1999). In terms of its protein phosphatase function, mammalian PTEN targets focal adhesion kinase, a major effector of cytoskeletal function. Overexpression of wild-type mammalian PTEN and mutant PTEN that lacks lipid phosphatase activity can reduce levels of focal adhesion kinase (FAK: see Drosophila Focal adhesion kinase-like) phosphorylation and the formation of focal adhesions, thereby inhibiting cell migration and invasiveness (Tamura, 1998). In terms of its cytoskeletal connection, Drosophila Pten appears to regulate the subcellular organization of the actin cytoskeleton in multiple cell types. The bristle, hair, and rhabdomere phenotypes observed in Drosophila Pten mutant tissue have not been reported in flies defective in insulin or Dp110 signaling, indicating that unlike the Pten-linked growth defects, involving insulin signaling, these effects are probably not derived from alterations in lipid signaling but from direct influences on cytoskeletal function (Goberdhan, 1999).

The tumor suppressor gene PTEN is one of the most frequently mutated genes involved in the development of mammalian cancer. PTEN mutations are found in a wide variety of tumors such as glioblastomas, endometrial carcinomas, advanced prostate cancers and melanoma cells. Germ-line mutations in PTEN are linked to three rare autosomal dominant syndromes: Cowden Disease, Bannayan-Zonana syndrome and Lhermitte-Duclose disease. A common feature of these syndromes is a predisposition for the development of hamartomas: benign tumors that have differentiated but disorganized cells. Pten mutant mice exhibit early embryonic lethality (E7.5-E9.5) and the heterozygotes display a predisposition to tumor development. Consistent with the role for PTEN as a lipid phosphatase, Pten mutant cells exhibit increased lipid signaling. The C. elegans gene daf-18 encodes a distant PTEN homolog. In addition to a conserved phosphatase domain, daf-18 encodes a large non-homologous C-terminal region. Daf-18 is associated with the insulin signaling pathway. In C. elegans, daf-18 mutants can suppress the mutant phenotypes of daf-2, the C. elegans insulin-like receptor, and age-1, the C. elegans PI3K homolog (Huang, 1999 and references therein).

Drosophila Pten was identified in two laboratories using two different approaches, one based on homology to mammalian PTEN (Huang, 1999) and the second based on intensive analysis of a chromosomal region containing several other genes coding for signaling proteins (Goberdhan, 1999). This second approach will be examined in detail, since it foreshadows future approaches that will be taken to analyze Drosophila genes based on genomics. Previously, two Drosophila genes had been identified in the vicinity of the gene that was later to be identified as Drosophila Pten: Dror encodes a neural-specific receptor tyrosine kinase, and basket encodes the homolog of c-Jun amino-terminal kinase. These two genes map adjacent to each other at 31B/C on the second chromosome. Several deficiency chromosomes have been characterized that uncover one or both of these genes and also affect the adjacent gene chico , which codes for a homolog of mammalian insulin receptor substrates, IRS1-4 (Böhni, 1999). One of these deficiencies, Df(2L)170B, is of particular interest because it deletes sequences proximal to Dror and produces an overgrowth phenotype in homozygous clones. This effect was not observed with deficiencies [Df(2L)41C and Df(2L)147F] that only delete Dror, DJNK, and chico. Clones generated with these smaller deficiencies contain reduced numbers of small cells attributable to the loss of chico function (Böhni, 1999). During a chemical mutagenesis screen using the Df(2L)170B chromosome, a new lethal complementation group has been identified that maps proximal to Dror and affects tissue growth. Sequence analysis of genomic and cDNA clones reveals a novel gene at this locus encoding the Drosophila PTEN homolog, Pten (Goberdhan, 1999).

Drosophila Pten regulates cell number and size and affects assembly of specific cytoskeleton-dependent structures. Because animals transheterozygous for strong Pten alleles die with no obvious phenotypes, the functions of this gene have been elucidated further by generating homozygous mutant clones in heterozygous animals using the FLP/FRT system. Two Pten alleles, DPTEN¹ and DPTEN³, produce growth phenotypes slightly more severe than those generated by Df(2L)170B, the chromosomal deficiency deleting Pten, Dror, DJNK, and chico. DPTEN¹ and DPTEN³ also behave in a manner similar to the deficiency in combination with a weak, nonlethal Pten mutation; this suggests that they are strong or null Pten alleles (Goberdhan, 1999).

The adult compound eye in Drosophila is composed of ~700 regularly arranged unit eyes or ommatidia, each containing the same complement of photoreceptors and accessory cells. Mutant clones can be generated in the eye and other tissues by somatic recombination of homologous chromosomes in heterozygous animals. If only the nonmutant chromosome and not the mutant chromosome is marked with a copy of the white (w⁺) gene, mutant clones can be distinguished by the absence of pigment. The wild-type twin spot resulting from a recombination event with these chromosomes contains increased levels of pigment relative to the remainder of the eye, because the cells in this region carry two copies of the w⁺ gene. Ommatidia within clones homozygous for DPTEN¹, DPTEN³, or Df(2L)170B are enlarged and bulge from the surface of the eye. Ommatidial facets are occasionally fused, and there frequently are clusters of morphologically abnormal interommatidial bristles or clustered sockets in mutant regions, particularly (but not exclusively) in the dorsal half of the eye. To assess the effect of loss of Pten function on the regulation of cell number, the numbers of ommatidia in multiple mutant clones and their wild-type twin spots were compared. Mutant clones typically contain at least twice as many ommatidia as neighboring twin spots, suggesting that mutant cells overproliferate, a result supported by analysis of clones at larval stages. The largest Pten clones include more than half of the adult eye and form a hyperplastic tumor-like overgrowth. This hyperplastic phenotype, however, is mild compared to the effects of several other tumor suppressor mutations in Drosophila (Goberdhan, 1999).

Sections demonstrate that the number and identities of ommatidial cells are not obviously altered in Pten mutant clones, indicating that this gene does not have a major role in specifying cell fate in the eye. Analysis of nonpigmented mutant clones, however, reveals that the cell bodies of all mutant photoreceptors are greatly enlarged, but not those of their wild-type/heterozygous neighbors. By using a w⁺-marked mutant chromosome to visualize the pigment cell layer in clones, it is possible to show that the volume of this layer is also increased. These cellular growth defects account for the increased size of mutant ommatidia. Furthermore, in mutant photoreceptors, the light-sensing rhabdomeres are elliptical and not circular in cross-section. This phenotype is observed in several mutants that are believed to affect assembly of the actin cytoskeleton. Analysis of ommatidia that contain both wild-type and mutant photoreceptors reveals that both the cell size and rhabdomeric phenotypes are cell-autonomous (Goberdhan, 1999).

Cells in the wing, each of which normally possesses a single wing hair, are also significantly enlarged in mutant Pten clones. Wing hairs are occasionally duplicated, a defect observed under conditions where the cytoskeleton of wing epithelial cells is disrupted. In addition, crossvein formation in the wing, a process involving specialized contacts between wing epithelial cells and the extracellular matrix (ECM), is abnormal in mutant regions. In ~70% of cases, anterior cross-veins are absent or partially absent in mutant clones, whereas the posterior cross-vein is affected in ~35% of cases. Occasionally, extra wing vein material is observed. Longitudinal veins are affected only very rarely (Goberdhan, 1999).

Overexpression of Pten also produces enlargement of wing cells. Wild-type Pten cDNA is overexpressed in particular areas of the wing using the GAL4-UAS system. Initially flies carrying a dpp-GAL4 construct were used. This drives gene expression in cells that will normally populate the region between the third and fourth longitudinal wing veins (LIII and LIV). Overexpression of Pten reduces the size of these regions by nearly 25% compared with wild type. This is not a consequence of a general reduction in wing size in overexpressing flies, since an adjacent area of the wing between LIV and LV is essentially unaffected. The effect on wing area is similar to that produced by overexpression of Dp110^D954A, a dominant-negative, kinase-dead version of Pi3K92E. The reduction is caused by both a decrease in cell size and cell number and is opposite of the effect of overexpressing an activated, membrane-associated form of Pi3K92E, Dp110-CAAX, in the same region. To test whether Pten's growth regulatory functions are primarily mediated by its effects on the insulin receptor-Pi3K92E signaling pathway and not by an independent signaling cascade, the genetic effects of Pten alleles were sought using mutant phenotypes associated with chico and Pi3K92E (Goberdhan, 1999).

Interestingly, both overexpression of a dominant negative form of Phosphotidylinositol 3 kinase 92E (also known as Dp110 or Pi3K92E) and mutations either in the Drosophila insulin receptor or in chico/IRS1-4 also reduce cell size as well as proliferation. Furthermore, overexpression of wild-type and activated forms of Pi3K92E produces similar size and proliferation defects as those seen in Pten mutant cells. These observations are consistent with a model in which growth is regulated in Drosophila by specific phosphoinositides whose levels are controlled by the balance of Pten and Pi3K92E activities. Pten and Pi3K92E. At 25°C, flies do not survive to adult when wild-type Pi3K92E is ectopically expressed by means eyeless-GAL4. Interestingly, this lethality can be rescued by coexpression of Pten. Furthermore, the small eye phenotype of Pten overexpression is suppressed by overexpression of wild-type Pi3K92E and enhanced by overexpression of dominant negative Pi3K92E. These results clearly indicate that Pten and Pi3K92E function antagonistically in Drosophila. The recent characterization of chico, a Drosophila IRS1-4 homolog, has shown that chico, Pi3K92E and Insulin receptor(Inr) act as positive elements in a Drosophila insulin signaling pathway to regulate cell proliferation and cell size (Bohni, 1999). Consistent with the role of Pten as a negative regulator in this insulin pathway, removal of one copy of the chico gene genetically enhances the eye/Pten eye phenotype. Overexpression of Inr (eye/Inr) causes lethality at 25°C. At room temperature, few animals survive with overproliferated eyes. Strikingly, co-overexpression of Pten completely rescues lethality and the overproliferation phenotype. This suggests that all signals from the insulin receptor can be antagonized by Pten function. Together with the previous findings that mammalian and C. elegans PTEN molecules interact with components of the insulin pathway (Furnari, 1998; Maehama, 1998; Myers, 1998; Ogg , 1998; Stambolic, 1998; Sun, 1999), these genetic data argue that PTEN functions as a major conserved negative regulator in the insulin signaling pathway (Huang, 1999).

The importance of Drosophila Pten in negatively regulating the growth-promoting effects of insulin signaling in vivo, however, is best illustrated in homozygous clones mutant for both chico and Pten. In these clones, the reduced growth phenotype normally seen in chico mutant cells is masked completely by the overgrowth phenotype associated with loss of Pten function, suggesting that Pten normally has a critical role downstream of Chico in maintaining growth-promoting signals at nonhyperplastic levels (Goberdhan, 1999).

Pten also affects the organization of the actin cytoskeleton in differentiating adult cells. The bristle, hair, and rhabdomere phenotypes observed in Pten mutant tissue have not been reported in flies defective in insulin or Dp110 signaling, indicating that unlike the Pten-linked growth defects, these effects are probably not caused simply by an increase in PIP3 levels. Pten mutant clones have been analyzed in the larval imaginal discs to determine whether there is an underlying defect in microfilament organization during differentiation that might explain these phenotypes. Consistent with the adult eye phenotype, Pten mutant ommatidial preclusters in the eye imaginal disc were more widely spaced, and specific staining of photoreceptors indicates that these cells are enlarged in mutant clones. Phalloidin staining demonstrates that there are defects in the actin cytoskeletal network in mutant tissue. In particular, analysis of mutant photoreceptors reveals disorganization in the most basal part of the apical cytoskeleton, the scaffold on which apical photoreceptor projections are normally assembled in the disc. An altered distribution of neighboring actin microfilaments is also observed. Because rhabdomere assembly in photoreceptors requires the apical cytoskeleton, this defect may account for the rhabdomeric phenotype seen in adult Pten mutant ommatidia. Analysis of the wing imaginal disc also shows disturbed cytoskeletal organization in mutant tissue. Therefore, although Pten mutant cells can still assemble actin microfilaments, the subcellular regulation of this process appears to be abnormal, potentially accounting for the structural defects in adult bristles, rhabdomeres, and wing hairs. One interesting hypothesis that remains to be tested is that these effects on the cytoskeleton may have an important role in regulating cell growth by controlling spreading and shape change at the cell surface. It is intriguing to speculate that the loss of Pten function might alter communication between the peripheral actin cytoskeleton, the plasma membrane and the extracellular matrix, and therefore affect the invasiveness of cells (Goberdhan, 1999), as has been suggested from studies in mammalian cell culture (Tamura, 1998).

Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam

Both cell-intrinsic and extrinsic pathways govern axon regeneration, but only a limited number of factors have been identified and it is not clear to what extent axon regeneration is evolutionarily conserved. Whether dendrites also regenerate is unknown. This study reports that, like the axons of mammalian sensory neurons, the axons of certain Drosophila dendritic arborization (da) neurons are capable of substantial regeneration in the periphery but not in the CNS, and activating the Akt pathway enhances axon regeneration in the CNS. Moreover, those da neurons capable of axon regeneration also display dendrite regeneration, which is cell type-specific, developmentally regulated, and associated with microtubule polarity reversal. Dendrite regeneration is restrained via inhibition of the Akt pathway in da neurons by the epithelial cell-derived microRNA bantam but is facilitated by cell-autonomous activation of the Akt pathway. This study begins to reveal mechanisms for dendrite regeneration, which depends on both extrinsic and intrinsic factors, including the PTEN-Akt pathway that is also important for axon regeneration. This study has thus established an important new model system -- the fly da neuron regeneration model that resembles the mammalian injury model -- with which to study and gain novel insights into the regeneration machinery (Song, 2012).

The present study shows that Drosophila sensory neuron dendrites and axons are capable of regeneration in a cell type-specific manner. While dendrites and axons share the same cell type specificity in their capacity for regeneration, they differ in their developmental regulation, with axons but not dendrites retaining the regeneration ability throughout larval development. It was further shown that the evolutionarily conserved PTEN-Akt signaling pathway is important for the regeneration of dendrites as well as axons and that both axon regeneration and dendrite regeneration are accompanied by the reversal of microtubule polarity (Song, 2012).

It is known that Drosophila larval axons undergo a scaling process in which axons substantially increase their length in accordance with the growth of the organism. Thus, it raises an important issue of whether larval axons regenerate or simply scale after axotomy. This question may be addressed with the following two considerations. First, larval axons scale while maintaining their neural connections; da neuron axons have already formed synaptic connections with neurons in the VNC, and these axon projections are not significantly altered as larvae grow in size. Therefore, this increase of axon length does not involve bona fide axon pathfinding or synaptogenesis. Thus, axon scaling differs from the developmental axon outgrowth before synapse formation and is different from axon regeneration, which involves reinitiation of the developmental program for severed axons to generate growth cones or growth cone-like structures and pathfind to reach their targets. In the larval injury model, the axon is severed and therefore develops a new growing tip, reroutes following the presumptive trajectory by active or passive cues, and may or may not eventually establish synaptic contacts with their right targets in the CNS. This process resembles the regeneration program rather than axon scaling. Second, all of the da neurons, including class I, class III, and class IV, show similar axon scaling during larval stages. However, class IV but not class I or class III da neurons displayed axon regrowth after axotomy. The fact that only class IV da neurons are capable of regrowth, although all of these different types of da neurons undergo scaling, strongly suggests that class IV da neurons possess a unique regrowth potential that allows their severed axons to reinitiate the developmental program for axon outgrowth. For these reasons, it is believed that a subset of the larval axons can regenerate after injury, although the possibility cannot be excluded that the ability of these axons to scale contributes to their regeneration potential. While this regeneration process may or may not fully recapitulate the regeneration program in adults, understanding how this process takes place in larvae will provide invaluable insights into the axon regeneration machinery (Song, 2012).

The ability of class IV but not class I or class III da neurons to readily regenerate their axons and dendrites could conceivably reflect cell type-specific features, including the transcription programs. One interesting question is whether the same program that governs the cell type morphology may also influence their regeneration capacity. For dendrite regeneration of class IV da neurons, either the regenerated dendrite or the neighboring dendritic branch continues to grow to fill the available space. Thus, they may possess a persistent growing potential that is inhibited by neighboring branches nearby so that the branches might overgrow if those inhibitory signals are removed. Therefore, with some branches removed due to injury, the remaining branches will regrow to take over the vacant space. Since class I and class III da neuron dendrites show very limited space-filling ability, these dendrites may lack the growth potential required for regeneration (Song, 2012).

In response to injury, class IV da neurons regenerate their axons substantially, while class I da neurons partially reverse the microtubule polarity of nearby dendrites and convert one of these dendrites into a pseudo-axon (Stone, 2010). Taken together with the current finding that class IV but not class I or class III da neurons are able to regenerate their dendrites, which are also associated with the reversal of microtubule polarity, these observations raise the question of whether pathways controlling neuronal polarity and/or cytoskeletal rearrangement may influence dendrite and axon regeneration (Song, 2012).

Several lines of evidence suggest that dendrite regeneration depends on a balance of influences. First, there may be competition between de novo dendrite regeneration and invasion of neighboring branches. Successful regeneration prevents invasion and vice versa. Second, there could be a balance of extrinsic inhibitory cues, as in the form of the ban miRNA in epithelial cells, and intrinsic growth-promoting signals, as conveyed by the activation of the Akt pathway (Song, 2012).

Moreover, given that activation of the Akt pathway at later stages of development is not sufficient to elevate the extent of dendrite regeneration to that during early larval development, it seems likely that either factors downstream from Akt are developmentally regulated to turn off the regeneration program at later stages or, alternatively, other pathways may contribute to this inhibition (Song, 2012).

Whereas dendrite and axon regeneration display differences with respect to developmental regulation, the PTEN-Akt pathway is important for regeneration of axons as well as dendrites. This pathway not only regulates the extent of dendrite growth of class IV da neurons during development, but also affects their dendrite regeneration and axon regeneration in the CNS. Together with previous work (Park, 2008; Liu, 2010), these results support the notion that modulating neuronal intrinsic PTEN and Akt activity is a potential therapeutic strategy for promoting axon regeneration and functional repair after CNS trauma (Song, 2012).

This work focuses on class IV da neurons, which behave very differently from class I da neurons in regeneration. In particular, unlike class I da neurons, the class IV da neuron is capable of regenerating its axon in the periphery but not inside the CNS, thereby providing the first example of this phenomenon in invertebrates. A recent study of Caenorhabditis elegans PLM neurons, a type of mechanosensory neurons that consistently regrow their axons upon laser-mediated axotomy, has identified multiple genes important for axon regeneration (Chen, 2011), illustrating the power of the genetic approach. The injury model involving Drosophila class IV da neuron axotomy in the CNS (VNC) in the current study has the additional feature that it resembles the injury model involving mammalian DRG neuron axotomy in the CNS (spinal cord) at the cellular and molecular level: Both da neuron and DRG neuron axons regenerate poorly in the CNS even though they display robust regeneration in the periphery, and in both cases, axon regeneration in the CNS is enhanced by activation of the PTEN-Akt pathway. Importantly, while the PTEN-Akt pathway has been shown to be critical for mammalian axon regeneration in the CNS, this has not been shown in invertebrate models; for example, in C. elegans, PTEN (DAF-18) has no effect on axon regrowth. The current finding underscores the usefulness of the Drosophila system that this study developed as a model to uncover evolutionarily conserved mechanisms for CNS axon regeneration. Moreover, the elaborate and stereotyped dendritic branching pattern of da neurons provides a sensitive assay system to begin studying the injury responses and regeneration of dendrites, which may yield clues to facilitate studies of mammalian neuronal dendrites and identify novel approaches to promote dendrite recovery for the treatment of nervous system trauma (Song, 2012).

Novel functions for integrin-associated proteins revealed by analysis of myofibril attachment in Drosophila

This study used the myotendinous junction of Drosophila flight muscles to explore why many integrin associated proteins (IAPs) are needed and how their function is coordinated. These muscles revealed new functions for IAPs not required for viability: Focal Adhesion Kinase (FAK), RSU1, tensin and vinculin. Genetic interactions demonstrated a balance between positive and negative activities, with vinculin and tensin positively regulating adhesion, while FAK inhibits elevation of integrin activity by tensin, and RSU1 keeps PINCH activity in check. The molecular composition of myofibril termini resolves into 4 distinct layers, one of which is built by a mechanotransduction cascade: vinculin facilitates mechanical opening of filamin, which works with the Arp2/3 activator WASH to build an actin-rich layer positioned between integrins and the first sarcomere. Thus, integration of IAP activity is needed to build the complex architecture of the myotendinous junction, linking the membrane anchor to the sarcomere (Green, 2018).

The adult indirect flight muscles of Drosophila have proved to be an excellent system to identify functions for integrin-associated proteins (IAPs) that are not essential for viability. The mechanical linkage between the last Z-line of each myofibril and the plasma membrane is a well ordered and multi-layered structure, ideal for elucidating the mechanisms by which actin can be organized into different structures at subcellular resolution. In the layer closest to the membrane, the integrin signaling layer, an important counterbalancing is found between IAPs, with FAK inhibiting the activation of integrin by tensin, and RSU1 inhibiting excess PINCH activity. It was discovered that the muscle actin regulatory layer (MARL) has a different composition to the fibroblast ARL, containing a mechanotransduction cascade of vinculin and filamin, which, together with WASH and the Arp2/3 complex, builds an actin-rich zone linking the adhesion machinery at the membrane to the first Z-line (Green, 2018).

The modified terminal Z-lines [MTZ - composed of 4 zones: (1) an integrin signalling layer at the membrane; and then zones containing different actin structures-(2) a force transduction layer (FTL); (3) a muscle actin regulatory layer (MARL); and (4) the first Z-line followed by the first sarcomere] revealed both positive and inhibitory actions of FAK, with the latter consistent with the role of FAK in adhesion disassembly. Both loss of FAK and activated integrin suppressed the phenotypes caused by loss of RSU1 or vinculin, but only activated integrin alleviated the defects caused by the absence of tensin, suggesting that FAK inhibition requires tensin activity, and in turn, tensin elevates integrin activity. This fits with the recent discovery that tensin contributes to the inside-out activation of integrins via talin (Georgiadou, 2017). FAK and tensin thus form a balanced cassette that is thought to respond to upstream signals to regulate integrin activity. Further work is needed to discover how tensin increases integrin activity, how this is inhibited by FAK, and what signals control this regulatory cassette. One model would have tensin activating integrin by direct binding to the β subunit cytoplasmic tail, and FAK inhibition by phosphorylation of tensin, but an alternative is that they have antagonistic roles in integrin recycling (Green, 2018).

RSU1 is part of the complex containing ILK, PINCH and Parvin (IPP complex), and binds the 5th LIM domain of PINCH. Loss of RSU1 causes milder phenotypes than loss of ILK, PINCH or parvin, and these phenotypes have previously been interpreted as a partial loss of IPP activity. The current findings indicate that the phenotypes observed in the absence of RSU1 are due to too much PINCH activity, and therefore the role of RSU1 is to keep PINCH activity in check. This suggests that PINCH is perhaps the key player of the IPP complex, and is recruited to adhesions by integrin via ILK, and kept in check by integrin and RSU1. The importance of regulating active PINCH levels is consistent with the dosage sensitivity of PINCH: reducing PINCH partially rescues the dorsal closure defect in embryos lacking the MAPK Misshapen, and elevating PINCH rescues hypercontraction caused by loss of Myosin II phosphatase. Reducing the interaction of PINCH with ILK had unexpectedly no phenotype, but in combination with the loss of RSU1 becomes lethal; the lethality can now be interpreted as being caused by too much PINCH activity, rather than too little. Excess 'free' PINCH results in elongated membrane interdigitations and elevated paxillin levels. This suggests that PINCH has an important role at the cell cortex, consistent with cortical proteins in the PINCH interactome. Too much parvin activity also causes lethality, which is suppressed by elevating ILK levels. Thus, it is increasingly clear that the functions of IPP components need to be tightly controlled. This study gained some insight into how RSU1 inhibits PINCH activity by demonstrating that ΔLIM4, 5 PINCH still caused longer interdigitations. This rules out RSU1 blocking the binding of another protein from binding LIM5, and suggests instead that RSU1 bound to LIM5 must be inhibiting the activity of LIM1-3 (Green, 2018).

Vinculin has a dual function in the MTZ: its head domain promotes force transduction layer (FTL; containing actin, the C-terminus of talin and vinculin) stability via binding talin, and its tail promotes muscle actin regulatory layer (MARL) formation. This analysis of the vinculin mutant by electron microscopy showed a phenotype within the electron dense layer close to the membrane that is presumed to corresponds to the integrin signalling layer. It suggests that vinculin may mediate interactions between IAPs that aid in keeping this as an even layer. The fact that the disruption to this layer is only evident on the muscle side of the interaction raises the question of how similar the integrin junctions are on the two sides of this cell-cell interaction via an intervening ECM. Many other sites of integrin-mediated adhesion in Drosophila involve integrins on both sides of the interaction and by electron microscopy the electron dense material looks similar on the two sides, and it would be expected that both sides need to resist the same forces. Even with structured illumination microscopy the two sides of the membrane cannot be resolved, but the results show that the C-terminus of talin and vinculin are not pulled away from the membrane in the adult tendon cells. This suggests either that vinculin has a different role in the tendon cell, with a different configuration, as was observed for talin in the pupal wing, or it is absent (Green, 2018).

The vinculin tail function in MARL formation does not require that vinculin is bound to talin, but it is suspected that in the wild type it is talin-binding that converts vinculin into an open conformation, permitting the tail to trigger MARL formation with filamin, as outlined in a working model (see Model of IAP function in the IFM MTZ). A key function of vinculin tail in the MARL is to aid the mechanical opening of the filamin mechanosensitive region. This study presents evidence suggesting this is achieved by the vinculin tail anchoring the C-terminus to actin, but further work is required to determine if there is direct binding between the two proteins. Similarly, the results indicate that the Arp2/3 nucleation promoting factor WASH is part of the same pathway as filamin and acts downstream of it, but the connection between the two has yet to be resolved. This new function for WASH is distinct from its best characterized role regulating actin on intracellular vesicles during endosomal sorting and recycling, but WASH also has additional roles in the nucleus and the oocyte cortex, showing that it is a versatile protein (Green, 2018).

Given the myofibril defects seen with loss of RSU1, tensin, vinculin and filamin it might be expected that mutations in genes encoding these IAPs might be implicated in muscle disease. Indeed, mutations in integrin α7, talin and ILK are associated with muscular myopathies in humans and mice. Mutations in the genes encoding RSU1, tensin and vinculin have not been linked to muscle myopathies, but mutations in filamin are linked to myofibrillar myopathies. However, given the subtlety of these defects in Drosophila, one might predict that mutations in genes encoding these IAPs are associated with subtle defects in humans such as reduced sporting performance or susceptibility to muscle injury. The authors were unaware of any mutations in genes encoding these IAPs being related to athletic performance or injury susceptibility, but these IAPs would be good candidates for further study in this area (Green, 2018).

One way that these IAPs may contribute to athletic performance is by building a muscle shock absorber, the MARL, which protects the myofibrils from contraction-induced damage. The concept of muscle shock absorbers is well established since tendons perform this function. The presence of filamin, Arp3, vinculin and α-actinin in the MARL suggests that the MARL contains branched and bundled actin filaments. Branched actin networks have been shown to be viscoelastic and actin crosslinkers such as filamin have been shown to reduce viscosity and increase elasticity of actin networks. Further study into the functional nature of the MARL should increase understanding of athletic performance and injury susceptibility (Green, 2018).

Roles of PINK1 in regulation of systemic growth inhibition induced by mutations of PTEN in Drosophila

The maintenance of mitochondrial homeostasis requires PTEN-induced kinase 1 (PINK1)-dependent mitophagy, and mutations in PINK1 are associated with Parkinson's disease (PD). PINK1 is also downregulated in tumor cells with PTEN mutations. However, there is limited information concerning the role of PINK1 in tissue growth and tumorigenesis. This study shows that the loss of pink1 caused multiple growth defects independent of its pathological target, Parkin. Moreover, knocking down pink1 in muscle cells induced hyperglycemia and limited systemic organismal growth by the induction of Imaginal morphogenesis protein-Late 2 (ImpL2). Similarly, disrupting PTEN activity in multiple tissues impaired systemic growth by reducing pink1 expression, resembling wasting-like syndrome in cancer patients. Furthermore, the re-expression of PINK1 fully rescued defects in carbohydrate metabolism and systemic growth induced by the tissue-specific pten mutations. These data suggest a function for PINK1 in regulating systemic growth in Drosophila and shed light on its role in wasting in the context of PTEN mutations (Han, 2021).

This study reports that PINK1 regulated systemic growth in a non-autonomous manner. The loss of PINK1 in muscle cells specifically upregulated the insulin/IGF signaling antagonist ImpL2, thus inhibiting organ growth by dysregulating carbohydrate metabolism and insulin signaling. Furthermore, strong genetic evidence is provided that PINK1 activity is dispensable for cell growth but absolutely required for the induction of tissue wasting following the knockdown of the tumor suppressor gene PTEN. This effect is mediated through systemic insulin signaling. In sharp contrast to wild-type cells, PTEN mutant cells retain high levels of membrane PIP3 independent of InR/PI3K, thus maintaining high cellular insulin signaling and a high rate of cell growth despite low circulatory insulin activities. Thus, mutations in PTEN enhance cell growth by maintaining high levels of insulin signaling, but they also inhibit the growth of other tissues in a non-autonomous manner through the transcriptional repression of PINK1 (Han, 2021).

Although induced by the tumor suppressor PTEN, PINK1 does not inhibit cell growth in a number of cancer cell lines. Instead, PINK1 has been shown to promote cell growth and proliferation in various models. This study demonstrated that in the fly fat body, wing, and eye, the loss of PINK1 impaired cell growth. This is the opposite of PTEN, which leads to overgrowth when mutated. Moreover, despite the fact that the expression of PINK1 is reduced by mutations in pten, ectopic expression of PINK1 does not affect the growth of either wild-type or pten mutant cells, indicating that PINK1 may not simply function as a regulator of cellular growth, particularly in pten-associated tumors. Interestingly, knocking down pten or pink1 in specific tissues reduces the insulin signaling and cell growth of peripheral tissue and leads to hyperglycemia and weight loss. Moreover, re-expression of PINK1 completely rescued the insulin resistance and systemic growth inhibition caused by tissue-specific mutations of pten. Supporting these findings, pten mutant clones have been shown to reduce the insulin signaling and growth potential of surrounding tissues. Furthermore, this study found that the secreted insulin/IGF antagonist impl2 is highly expressed upon the loss of both pink1 and pten and is reduced to wild-type levels by the re-expression of PINK1. Importantly, the systemic growth defects resulting from the tissue-specific knockdown of pten or pink1 were completely rescued by reducing impl2 to wild-type levels. These data suggest that reducing PINK1 expression may mediated cachexia and systemic metabolic dysfunction induced by tumors associated with PTEN mutations (Han, 2021).

Much like PTEN mutations, tumors induced in the adult Drosophila midgut by expressing the oncogene Yorkie (Yki) in intestine stem cells, or by transplanting tumors that express RasV12 in cells mutant for scribble, also secrete high levels of ImpL2, thereby reducing systemic insulin/IGF signaling and inducing organ wasting. It was established that the overexpression of ImpL2 in specific tissues in flies led to wasting in surrounding tissues. ImpL2 is a major mediator that is both necessary and sufficient for wasting. Together with the current results, these data suggest that a common mechanism underlying tumor cachexia may be the disruption of systemic insulin/IGF signaling that then results in hyperglycemia, insulin resistance, and organ wasting. There are seven IGF-binding proteins (IGFBPs) in mammals, which are dysregulated in different kinds of tumors. In the context of cancer, analyses of IGFBPs have largely focused on their effects on tumor growth. Recently, IGFBP-3 was found to be upregulated in human pancreatic cancer, supporting a potential role for insulin-binding peptides in the induction of tumor cachexia. Moreover, IGFBP-7 expression is associated with tumor growth and functions as a tumor suppressor in the colon, rectum, breast, and thyroid by either directly inducing apoptosis or inhibiting tumor growth via the deregulation of p16, p21, p53, and ERK signaling. In glioblastoma, however, IGFBP-7 plays an oncogenic role and stimulates tumor cell proliferation. It is possible that besides functions in tumor cells, IGFBP-7 may play a role in tumor cachexia as ImpL2 in flies (Han, 2021).

The InR/PI3K cascades in tumor cells with expression of Yki and RasV12 may also be shut down by reduced circulatory insulin/IGF signaling. This suggests that other mechanisms of organ wasting may exist. It has recently been demonstrated that Yki-associated tumors produce the epidermal growth factor receptor (EGFR) ligand, Vein, to trigger autonomous EGFR signaling and produce the platelet-derived growth factor (PDGF)- and vascular endothelial growth factor (VEGF)-related factor 1 (Pvf1) ligand to non-autonomously activate PVR (PDGF and VEGF receptor-related) signaling and wasting in peripheral cells. For PTEN mutant tumors, PI3K/AKT signals remain high regardless of circulatory ImpL2 level. This is explained by the function of PTEN as a negative regulator of AKT downstream of InR/PI3K. The re-expression of impl2 completely abolished the systemic growth defects without affecting the overgrowth of pten mutant cells. Therefore, it is suggested that the induction of ImpL2 expression by mutations in pten through PINK1 is both necessary and sufficient for peripheral organ wasting. The results, together with previous studies, highlight the importance of dissecting metabolic responses to cancer. The focus of cancer research is gradually expanding, from cancer cells to the tumor microenvironment to the system as a whole. In addition, it will be interesting to test whether some malignant tumors that harbor PTEN and/or PINK1 mutations (e.g., glioblastoma) display the elevated expression of IGFBPs, which alter systemic metabolism in a manner similar to Drosophila. Furthermore, these findings may provide a new strategy for treating such tumors (Han, 2021).

PTEN is endowed with both lipid and protein phosphatase activities. Because PTEN functionally antagonizes the lipid kinase PI3K, the depletion of PTEN results in high levels of the lipid second messenger PIP3, resulting in the increased membrane recruitment and activation of Akt. This leads to enhanced cellular growth, proliferation, and survival. PINK1 promotes cell growth and prevents cell death through its cytosolic downstream targets, such as mTORC2 and p53. However, PINK1 has also been shown to function inside mitochondria to promote the activity of complex I of the mitochondrial respiratory chain, and a recent study using three-dimensional (3D) structured illumination super-resolution microscopy suggested that under physiological conditions, PINK1 localizes to the cristae membrane and intracristae space. Moreover, endogenous PINK1 localizes to both cytosolic and mitochondrial fractions. Consistent with the localization of PINK1 to the IMM, this study found that targeting PINK1 to the IM fully restored complex I activity and rescued the growth defects of pink1 mutants. By contrast, OMM-localized PINK1 did not affect growth associated with pink1 mutation, but completely rescued muscle degeneration phenotypes of pink1 mutants associated with the loss of mitophagy. These data suggest that PINK1 activity is required inside mitochondria but not in the cytosol to promote complex I activity and cell growth. As a protein kinase, PINK1 kinase activity is required to maintain complex I activity in mitochondria, and PINK1 kinase activity in cytosol protects neurons. Inside mitochondria, PINK1 directly phosphorylates the NDUFA10/ND42 subunit to promote complex I activity. However, the overexpression of both wild-type and phosphorylation-mimic ND42 is not able to suppress the defective growth of pink1 mutants, indicating that multiple physiological targets of PINK1 may exist. It has been recently reported that PINK1 could phosphorylate the IMM protein MIC60 to promote complex I assembly and maintain oxidative phosphorylation. Nevertheless, PINK1 promotes growth when inside the mitochondria and protects cells against stress by initiating mitophagy when anchored to the OMM (Han, 2021).

Similar to knocking down pink1 in muscle cells, the disruption of complex I activity by muscle-specific knockdown of NDUFS1/ND75 reduces insulin signaling non-autonomously by inducing ImpL2 expression. Although the mechanisms are unclear, this work, together with the observations of other groups, strongly implies that the knockdown of pink1 disrupts the function of complex I, thus upregulating the expression of ImpL2 and inhibiting cell growth non-autonomously. These findings describe key functional associations between PINK1 and PTEN in tumorgenesis and systemic growth. This study shows that impaired PINK1-associated mitochondrial function plays a key role in wasting-like syndrome and reveal that protecting mitochondrial function in cancer patients may provide tremendous benefit (Han, 2021).

GENE STRUCTURE

There are three alternatively spliced PTEN mRNAs, producing proteins differing by a few amino acids at their carboxyl termini (Smith, 1999 and Goberdhan, 1999)

Bases in 5' UTR - 527

Exons - 12

Bases in 3' UTR - 921

PROTEIN STRUCTURE

Amino Acids - 509

Structural Domains

The predicted Pten protein was aligned and compared with other known PTEN homologs. The amino-terminal half of the Drosophila Pten protein has been particularly highly conserved over evolution, sharing ~65% identity with human PTEN in this region and ~40% and 30% identity, respectively, with the more divergent Caenorhabditis elegans (Ogg. 1998) and yeast (L. Li, 1997) PTEN homologs. Like its counterparts in other organisms, Drosophila Pten contains a putative protein phosphatase motif within a larger region related to an actin binding domain in the cytoskeletal protein tensin. A number of amino acids shared by all members of the PTEN family are not present in other protein phosphatases or tensin family members. These residues may have an important role in the specific functions of PTEN, such as its phosphoinositide 3-phosphatase activity. Sequence comparison of the carboxy-terminal half of Drosophila Pten with human PTEN also highlights a number of conserved regions, which may be involved in the regulation of this molecule (Goberdhan, 1999).

date revised: 5 August 2023

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