BIOLOGICAL OVERVIEW

Phosphoinositide 3-kinases (PI3Ks) have been identified in an evolutionarily diverse range of organisms, including mammals, Drosophila, yeast, plants and Dictyostelium. They are activated by a multitude of extracellular signals and are implicated in mitogenesis, differentiation and cell survival, as well as in the control of the cytoskeleton and cell shape. Inositol is a sugar moiety that is found attached to cell membrane lipids. One type of PI3K, exemplified by Phosphotidylinositol 3 kinase 92E, the protein described in this essay, phosphorylates inositol bearing lipids (phosphatidylinositols or PtdIns) at the D3 position of the inositol ring. Substrates of PI3Ks include PtdIns, PtdIns (4)P, and PtdIns (4,5)P₂: these are phosphatidylinositols with phosphates attached to different hydroxide residues of the inositol ring, generating (respectively) PtdIns (3P), PtdIns (3,4)P₂, and PtdIns (3.4,5)P₃. These phosphoinositide residues created by PI3K phosphorylation have been proposed to act as second messengers, functioning to recruit regulatory proteins to the plasma membrane. The recuited regulatory proteins act in turn to trigger downstream signaling events in multiple regulatory pathways.

Drosophila has three genes coding for PI3Ks. The class I gene, Phosphotidylinositol 3 kinase 92E, informally termed Drosophila p110 or Dp110, the subject of this overview, codes for a protein that phosphorylates PtdIns, PtdIns (4)P, and PtdIns (4,5)P₂ in vitro, though the major substrates of these PI3Ks are though to be PtdIns (4)P and PtdIns (4,5)P₂. This class includes enzymes that mose closely resemble the prototypical p110 catalytic subunit and which associate with a regulatory subunit [e.g. p85 alpha, p85 beta and p55 PIK]. The regulatory subunits contain Sh2 domains that bind to specific phosphotyrosine residues and recruit p110/p85 heterodimers to activated receptor tyrosine kinases, thereby facilitating PI3K activation.

Interest in the Drosophila PI3K pathway has recently be rekindled by the cloning of chico, the Drosophila homolog of vertebrate insulin receptor substrates (IRSs). Many aspects of the insulin system appear to be conserved in flies and mammals. In mammalian cells, activation of the insulin or IGF1 (insulin-like growth factor 1) receptors by insulin and IGF1, respectively, results in the recruitment of IRS1 or IRS2 to the receptor via interaction of the IRS phosphotyrosine-binding domains with a phosphotyrosine motif (NPXY) in the juxtamembrane region of the receptors. Phosphorylation of multiple tyrosine residues of IRS1 triggers the activation of various signaling pathways, including the RAS/MAP kinase pathway via the SH2/SH3 adaptor GRB2 and the PI3K pathway via the p85 SH2 adaptor subunit of p110 PI3K (Yenush, 1997). The Drosophila Insulin-like receptor (InR) shares many structural features with its human homologs, including its heterotetrameric structure and a conserved PTB consensus binding site in the juxtamembrane region. However, Drosophila InR contains a 400-amino acid C-terminal extension not found in any of the vertebrate receptors. This C-terminal tail contains three YXXM consensus binding sites for the SH2 domain of the p60 subunit of PI3K and four additional NPXY consensus PTB-binding sites. The C-terminal domain is functional, since expression of a chimeric receptor consisting of the extracellular domain of the human INR and the intracellular domain of the Drosophila InR in murine 32D cells lacking endogenous IRS1 can partially activate mammalian PI3K and S6K. In contrast, the ability of the human INR to activate PI3K in this system is strictly dependent on the coexpression of IRS1 (Yenush, 1996). These findings and the identification of Chico suggest that in Drosophila, InR couples to the downstream effector PI3K in two different ways, one using docking sites in the InR C-terminal tail and the other connecting through docking sites in Chico (Böhni, 1999).

The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth

Ectopic expression of Dp110 affects wing growth. The adult wing consists of two layers of ectodermal cells (dorsal and ventral) that secrete cuticular structures (wing hairs and veins) and contact sensory organs (campaniform sensilla on the wing surface and sensory bristles along the anterior wing margin). The effect of ectopic Dp110 expression on development during larval stages was examined. At this time the wing imaginal disc is a monolayer epithelium divided by anterior/posterior (A/P) an dorsal/ventral (D/V) compartment boundaries. The expression of WT-Dp110 or Dp110-CAAX (membrane targeted Dp110) in different regions of the wing imaginal disc results in expansion of the corresponding regions in the adult wing blade. Conversely, Dp110D954A (Dp110 with the mutation in the putative ATP binding site) expression reduces the size of these regions. For example, ectopic expression of WT-Dp110 or Dp110-CAAX in the prospective dorsal surface of the wing blade results in wings that are bigger than wild-type and curve downwards. In contrast Dp110D954A wings are smaller and curve upwards. Similarly when Dp110 is expressed along, and immediately anterior to, the A/P boundary of the wing disc, the corresponding region of the adult wing (visualized by assessing the distance between longitudinal veins III and IV) expand or contract. Interestingly, the proximal to distal alignment of the non-sensory wing hairs that are present on the surface of the wing blade, is often disrupted by the expression of Wt-Dp110 or Dp110-CAAX (Leevers, 1996).

To further examine the effect of Dp110 on wing growth, wing discs from third instar larvae were examined in which GAL4 was used to drive the co-expression of nuclear beta-galactosidase and different forms of Dp110 at high levels in the dorsal wing pouch and at lower levels in the ventral wing pouch. The region of Gal4 expression was revealed by immunostaining for beta-galactosidase expression, and ectopic Dp110 expression was detected by a monoclonal antibody directed against a myc peptide epitope engineered into the N-terminus of the Dp110 expression constructs. Consistent with the effects observed in adult wings, the Gal4 expression domain is expanded by WT-Dp110 and Dp110-CAAX, and contracted by Dp110D954A. Similar differences are observed in discs expressing Dp110 along the A/P boundary, even in regions that give rise to non-wing blade adult structures, such as the notum and hinge. Significantly, the same effects are also observed in leg and haltere discs expressing Dp110 along the A/P boundary, indicating that the effect of Dp110 on the growth of imaginal discs is not specific to wing discs (Leevers, 1996).

An examination was made of whether the observed differences in wing size result from alterations in cell size or cell number. The number of cells per unit area was assessed by counting wing hairs, single apical extensions found on the surface of each wing blade cell. Wings generated by transgene expression along the A/P boundary were analyzed by examining an area of fixed size on the wing blade, and by looking at the wing margin between veins III and IV. Surprisingly, it was found that Dp110 and Dp110-CAAX expression increases both the overall number of cells and their size. In contrast, Dp110D954A decreases both cell size and cell number (Leevers, 1996).

To further analyze the role that Dp110 plays in the growth of imaginal discs, effects of the ectopic expression during eye development were examined. The Drosophila compound eye is a repetitive and highly organized structure generated by the stepwise recruitment of cells to ommatidial clusters behind an indentation in the eye imaginal disc known as the morphogenetic furrow. These clusters grow and differentiate during larval and pupal development and ultimately give rise to the adult retina. The Dp110 transgenes are expressed in cells posterior to the morphogenetic furrow using a GMR-Gal4 line (Leevers, 1996).

The expression of wild type or membrane targeted Dp110 generates enlarged and bulging, roughened eyes with fused ommatidia and misplaced or duplicated bristles, whereas Dp110D954A eyes are smaller than normal and flatter. The hexagonal lenses or facets that form the surface of each ommatidium are larger (ectopic WT-Dp110) or smaller (Dp110D954A). The small eyes contain the wild-type number of facets, whereas the enlarged eyes actually contained fewer ommatidia. To investigate the internal changes resulting in these differences, radial and tangential sections through the eyes were examined. The radial sections reflect differences seen from the outside: the adult retina is either increased or decreased, in both size and thickness. In addition, in eyes expressing ectopic WT-Dp110, an apical region above the photoreceptor rhabdomeres and immediately behind the lens was filled with cells. This is particularly evident in apical tangential section, where the space occupied by the secreted pseudocone in control eyes has been found to contain many swollen cell bodies in WT-Dp110 eyes. In more basal transverse sections the normally regular array of photoreceptors is disrupted by WT-Dp110 expression. The rhabdomeres appeared twisted and are disorganized with respect to one another and neighboring ommatidia: the number of photoreceptor rhabdomeres is often reduced. In Dp110D954A eyes the lattice is also disrupted though to a lesser degree, and the orientation of photoreceptors relative to those in neighboring ommatidia is at least partially maintained (Leevers, 1996).

These adult eye phenotypes indicate that Dp110 transgenes can also modulate growth during eye development. Therefore, differences in cell size and/or cell number were sought by examining confocal images in fluorescently labeled larval and pupal discs. The process of photoreceptor determination, as judged by the pattern of expression of the neuronal marker Elav is unaffected by ectopic WT-Dp110 or Dp110D954A expression. Interestingly though, the nuclei of WT-Dp110 discs are more widely spaced. Similarly, the distance between the centers of the developing ommatidial clusters revealed by immunostaining larval and pupal discs with anti-Armadillo, is affected by Dp110 transgene expression. Armadillo, the Drosophila homolog of the vertebrate beta-catenin, localizes to the adherens junctions just below the apical membranes of the developing photoreceptors, where the cells are constricted in close contact. The apical clustering of the photoreceptor membranes is disrupted in Dp110D954A pupal discs, where a 'ring' of Armadillo staining can be seen that persists more basally than in control discs (Leevers, 1996).

Immunostaining of the membranes of pupal cells with anti-alpha-Spectrin indicates that the Dp110 transgenes affect the size and not the number of cells in the eye disc. The pigment, cone, and photoreceptor cell bodies are swollen in ectopic WT-Dp110 discs and smaller than normal in the Dp110D954A discs. Notably, and in spite of these differences in cell size, the specific arrangement of the different cell types within each ommatidial cluster remains undisturbed. Consistent with this, cell proliferation and apoptosis are not detectably affected in the developing eye discs. Presumably the roughness and degeneration of the ommatidial pattern seen in adult WT-Dp110 eyes must arise during the late pupal stages of eye development (Leevers, 1996).

These results implicate Dp110 in the control imaginal disc growth, since the Dp110 transgenes affect both the size of individual cells and (in the wing disc) the overall number of cells. Currently, it cannot be determined how closely linked these two effects are, though it is conceivable that the differences seen in cell number might arise as a direct consequence of the effects on cell size. An alternative possibility is that the differences in the wing cell number result from a more direct effect on the rate of cell division or cell death in the developing discs. Studies in a mammalian system have implicated PI3K in the control of both processes. Interestingly, the simultaneous disruption of two of the three genes encoding class I PI3Ks in Dictyostelium also affects cell growth; double knock-out strains grow slowly and produce cells of reduced size (Leevers, 1996 and references).

Since the effect of the Dp110 transgenes on cell size might arise from alterations in biosynthesis or cytoskeletal architecture, it is noteworthy that likely downstream targets of class I PI3Ks identified in mammalian cells include P70S6K and the small GTP binding protein Rac. P70S6K is a serine/threonine kinase implicated in the up-regulation of translation and cell growth. Furthermore, studies in the class of Drosophila mutants termed 'Minutes' have indicated that protein synthesis is the rate limiting step in growth and development. Heterozygote Minute mutations, many of which correspond to or have been genetically mapped to the vicinity of ribosomal genes, delay development and often result in flies with reduced body size. Studies in mammalian cells have also implied a role for class I PI3Ks in the organization of actin cytoskeleton and membrane ruffling via Rac. Thus it is possible that effects on the architecture of the cytoskeleton or membrane composition and organization contribute to the differences in cell size that are observed. Furthermore, the disrupted adherens junctions in Dp110D954A pupal eye discs, the degeneration seen in adult eyes, and the loss of wing hair polarity observed in the wing blade might all be mediated via effects on actin organization (Leevers, 1996 and references).

The phenotypes generated by ectopic Dp110 expression suggest a possible role downstream of the Drosophila homolog of the EGF receptor or the insulin receptor. One of the multiple phenotypes generated by mutation in Egfr and downstream components of the Ras/MAP kinase pathway is a decrease in wing cell size. Thus, both the Ras/MAP kinase pathway and a signal mediated by Dp110 might cooperate in the control of wing cell size. Although Egfr contains no YXXM motifs (the motif to which the PI3K regulatory subunit binds), the receptor tyrosine kinase substrate Dos, which is also required for correct wing cell size, contains a YXXM motif. Another possible upstream regulator of Dp110 is the Insulin receptor and the Drosophila Insulin receptor substrate protein Chico (Bohni, 1999). Not only does the Drosophila Insulin receptor contain three pYXXM motifs and interact in vitro with the SH2 domains of mammalian p85 (the PI3K regulatory subunit), but flies heteroallelic for mutations in inr, which codes for the Drosophila Insulin receptor, grow slowly and are significantly reduced in size (Fernandez, 1995 and Chen, 1996). Furthermore, these growth defects arise as a result of prolonged larval instars during which the imaginal discs fail to grow to their wild type size. Flies defective in chico exhibit a similar phenotype (Bohni, 1999). Mice deficient in IRS1 are also retarded in growth and have a reduced final body size (Araki, 1994; Tamemoto, 1994 and Leevers, 1996).

Although this study implies a general role for Dp110 in the control of cell growth, many aspects of endogenous Dp110 function may not have been revealed by ectopic expression. Thus it will be informative in the future to examine phenotypes generated by mutations in the gene encoding Dp110. It is of note that when similar experiments were performed to express the Drosophila class II and class III PI3Ks, no general effects on growth are observed. PI3K_59F (class III) expression gives no detectable phenotype, whereas PI3K_68D (class II) expression produces phenotypes that imply a role in differentiation as opposed to growth. It is hoped that further analyses utilizing Drosophila as a model system will enable the characterization of different functions for different PI3Ks during Drosophila development and the performance of genetic screens to identify key downstream targets. The identification of such targets should help to further elucidate the mode of activation of class I PI3Ks in both Drosophila and mammals (Leevers, 1996).

The cytohesin Steppke is essential for insulin signalling in Drosophila

In metazoans, the insulin signalling pathway has a key function in regulating energy metabolism and organismal growth. Its activation stimulates a highly conserved downstream kinase cascade that includes phosphatidylinositol-3-OH kinase (PI(3)K) and the serine-threonine protein kinase Akt. This study identifies a new component of insulin signalling in Drosophila, the steppke gene (step). step encodes a member of the cytohesin family of guanine nucleotide exchange factors (GEFs), which have been characterized as activators for ADP-ribosylation factor (ARF) GTPases. In step mutant animals both cell size and cell number are reduced, resulting in decreased body size and body weight in larvae, pupae and adults. step acts upstream of PI(3)K and is required for the proper regulation of Akt and the transcription factor FOXO. Temporally controlled interference with the GEF activity of the Step protein by feeding the chemical inhibitor SecinH3 causes a block of insulin signalling and a phenocopy of the step mutant growth defect. Step represses its own expression and the synthesis of growth inhibitors such as the translational repressor 4E-BP. These findings indicate a crucial role of an ARF-GEF in insulin signalling that has implications for understanding insulin-related disorders, such as diabetes and obesity (Fuss, 2006).

All animals coordinate growth to reach their final size and shape. The insulin–insulin-like growth factor signalling pathway, which is genetically conserved from flies to humans, has been identified as a key regulator of cell growth in response to extrinsic signals such as growth factors and nutrient availability. In mammals, loss of the ability to respond to insulin, a phenomenon known as insulin resistance, is associated with pathological manifestations such as type 2 diabetes. In Drosophila, activation of a unique insulin-like receptor (InR) stimulates a conserved downstream cascade that includes PI(3)K and Akt. This signalling cascade controls organismal growth directly by regulating cell size and cell number (Fuss, 2006).

In a search for genes controlling larval growth in Drosophila, a genetic locus was identified that was named steppke (step). Molecular analysis and genetic rescue experiments show that the lethality of the P element alleles is linked to the step gene function. The step gene encodes a protein that belongs to the highly conserved cytohesin protein family of GEFs that consists of four family members in humans and one family member in invertebrates such as the nematode, mosquito and fly. GEFs mediate the exchange of GDP for GTP on the ARFs, which belong to the Ras superfamily of small GTPases. Like other Ras-related GTP-binding proteins, the ARF proteins cycle between their active GTP-bound and inactive GDP-bound conformations. In concert with ARFs, cytohesin proteins regulate vesicle trafficking, cell adhesion, migration and structural organization at the cell surface (Fuss, 2006).

Cytohesin proteins contain two characteristic motifs: a Sec7 domain responsible for the GEF activity, and a pleckstrin homology domain (PH) required for plasma membrane recruitment as a result of specific binding to phosphatidylinositol-3,4,5-trisphosphate, the second messenger generated by class I PI(3)Ks. The Sec7 and PH domains of Step are highly conserved compared with the corresponding protein domains of mammalian cytohesins (Fuss, 2006).

Phenotypic analysis of homozygous step^k08110 and step^SH0323 mutants and transheterozygous allelic combinations indicate an essential role of step in regulating growth and body size at all stages of the Drosophila life cycle. Both males and females of step^k08110/step^SH0323 transheterozygous adults are significantly smaller than control animals; however, the body proportions of these animals are not changed. Consistently, larval and pupal development are also slowed down in step mutants and body size is reduced. The observed growth defects mimic a starvation phenotype that is not caused by a failure of food intake, as verified by feeding coloured yeast and by the analysis of a metabolic marker gene (Fuss, 2006).

It is known that larval growth is largely based on an increase in cell size in all terminally differentiated tissues that is accomplished by endoreplication, a modified cell cycle, consisting of successive rounds of DNA synthesis without intervening mitoses. To examine the cause for the growth defects of step mutant larvae, cell cycle activity was investigated in the midgut and the salivary glands, which are representative endoreplicating tissues in the larval stage. A general decrease in endoreplication activity was found, indicated by a slowing down of the S phase of the cell cycle. Both the size and the total number of salivary gland cells are decreased, resulting in a smaller organ (Fuss, 2006).

Because embryonic lethality was observed in a small proportion of the homozygous step^k08110 mutants, it was important to exclude the possibility that the growth defects observed in the mutant larvae derive from a defect laid down during embryogenesis. For this purpose an assay was established to analyse step function exclusively in the larval stage, in which the growth rate is maximal. Use was made of the small molecule SecinH3, which was recently identified as an inhibitor of the Drosophila Step protein and the vertebrate cytohesin family members. SecinH3 binds to the Sec7 domain of Step, thereby inhibiting the guanine nucleotide exchange of interacting ARF proteins. Feeding SecinH3 to wild-type larvae induced a phenocopy of the growth defects observed in step mutants and led to a marked decrease in body size. It is concluded from the phenotypic analysis of step mutants and from the experiments inhibiting Step protein function directly by using the chemical inhibitor SecinH3 that Step is essential for organismal growth of Drosophila larvae, pupae and adults (Fuss, 2006).

In step mutants, organismal growth is strongly reduced and development is delayed, which is also a hallmark of mutants affecting the insulin signalling pathway. To investigate whether step has a function in insulin signalling, the expression of two known target genes of the pathway was analysed in step mutants, namely 4E-BP, encoding a translational repressor, and InR, encoding the insulin receptor, by using quantitative reverse-transcriptase-mediated polymerase chain reaction (RT–PCR); both 4E-BP and InR transcription are upregulated in response to repressed insulin signalling. Lipase3 (Lip3) expression was used as a starvation marker in these experiments. In step mutant larvae and also in wild-type larvae treated with the Step inhibitor SecinH3, 4E-BP and InR transcription is activated, whereas Lip3 expression is unaffected. This indicates that the growth phenotype observed in step mutant larvae is not caused by a complete block of nutrition but is associated with a specific downregulation of insulin signalling activity. Similarly, interfering with Step function by feeding SecinH3 to transheterozygous step mutant flies or applying SecinH3 in S2 tissue culture cells also results in an activation of 4E-BP and InR transcription (Fuss, 2006).

It has been shown previously that 4E-BP and InR are target genes of the transcription factor FOXO (forkhead box, sub-group ‘O’). In Drosophila cells, insulin receptor signalling results in a high activity of PI(3)K and phosphorylation of Akt. Akt phosphorylates FOXO and causes cytoplasmic retention of FOXO, whereas low activities of PI(3)K and Akt allow FOXO to enter the nucleus, where it promotes the expression of factors such as 4E-BP that retard cell growth and proliferation. In step mutant larvae or in S2 tissue culture cells in which Step protein function is inhibited with SecinH3, a nuclear localization of FOXO was found, indicating that step is required for insulin-signalling-dependent cytoplasmic localization of FOXO. Because this is regulated by phosphorylation by means of Akt, whether step is necessary for Akt phosphorylation was tested, and it was found that under conditions in which the step function is affected, the amount of phosphorylated Akt protein is significantly decreased (Fuss, 2006).

It has been shown that activation of Akt during growth in Drosophila is regulated by the class I PI(3)K Dp110. Overexpression of Dp110-CAAX, a constitutively active form of PI(3)K, in wing or eye imaginal discs enhances cellular growth, resulting in enlarged cells and organs, whereas mutations in Dp110 are lethal and result in a larval growth arrest in the third instar. It has been shown previously that Dp110 interacts with key components of the insulin signalling pathway including Chico, PTEN and Akt to control insulin-signalling-dependent cell and organ growth in Drosophila. To test whether step acts together with PI(3)K in a common pathway involved in Akt and FOXO regulation and, if so, to address whether step is genetically upstream or downstream of PI(3)K in the insulin pathway, Dp110-CAAX was expressed in heterozygous and transheterozygous step mutant animals (Fuss, 2006).

step mutant adults are greatly decreased in size and weight in comparison with wild-type animals. In control flies in which Dp110-CAAX has been overexpressed, body size and weight are greatly increased in comparison with wild-type flies. If step were positioned downstream of PI(3)K, the oversize phenotype induced by the expression of Dp110-CAAX should be suppressed or at least strongly reduced, whereas if step were positioned upstream of PI(3)K, Dp110-CAAX expression would rescue the growth phenotype of step mutants. The latter was found, providing in vivo evidence that the cytohesin family member step is upstream of PI(3)K (Fuss, 2006).

Tight regulation of insulin signalling activity has been shown to be crucial for cell and organ growth in Drosophila and for numerous growth-related and homeostasis-related diseases such as cancer and type 2 diabetes in humans. It is known from recent studies in Drosophila that InR represses its own synthesis by a feedback mechanism directed by the transcription factor FOXO. To test whether step is also part of a negative feedback control mechanism, step transcription was analysed at different levels of insulin signalling activity in vivo by using quantitative RT–PCR experiments. Similarly to the 4E-BP and InR genes, step transcription was found to be upregulated under conditions promoting FOXO activity such as starvation or in mutants of the insulin signalling pathway, such as chico mutants. Consistently, step transcription is induced 24-fold in response to a brief pulse of ectopic FOXO expression during larval development. These results indicate a FOXO-dependent transcription of step, which may be direct, presumably through several FOXO consensus binding motifs present in the step promoter, or indirect (Fuss, 2006).

It is therefore proposed that Step is a previously unrecognized and essential component of the insulin signalling cascade in Drosophila that regulates organismal growth. These results are consistent with the findings of a parallel study on the role of mammalian cytohesins. Both papers provide independent evidence for the central involvement of cytohesins in the insulin pathway upstream of PI(3)K and show a functional conservation of these proteins for at least 900 million years (Fuss, 2006).

ER-mitochondrial contact protein Miga regulates autophagy through Atg14 and Uvrag

Mitochondrial malfunction and autophagy defects are often concurrent phenomena associated with neurodegeneration. This study shows that Miga, a mitochondrial outer-membrane protein that regulates endoplasmic reticulum-mitochondrial contact sites (ERMCSs), is required for autophagy. Loss of Miga results in an accumulation of autophagy markers and substrates, whereas PI3P and Syx17 levels are reduced. Further experiments indicated that the fusion between autophagosomes and lysosomes is defective in Miga mutants. Miga binds to Atg14 and Uvrag; concordantly, Miga overexpression results in Atg14 and Uvrag recruitment to mitochondria. The heightened PI3K activity induced by Miga requires Uvrag, whereas Miga-mediated stabilization of Syx17 is dependent on Atg14. Miga-regulated ERMCSs are critical for PI3P formation but are not essential for the stabilization of Syx17. In summary, this study identified a mitochondrial protein that regulates autophagy by recruiting two alternative components of the PI3K complex present at the ERMCSs (Xu, 2022).

Eukaryotic cells are compartmentalized into different organelles that execute distinct functions and communicate with each other through indirect signal transduction or direct organelle-organelle contacts. Mitochondria and the adjacent endoplasmic reticulum (ER) form contacts, which are characterized by a 10-30 nm distance between the two organelles. These contacts mediate lipid exchange and calcium flux between the ER and mitochondria. It has been reported that ER-mitochondrial contact sites (ERMCSs) are important platforms for regulating macroautophagy (hereafter referred to as autophagy) and mitophagy (Xu, 2022).

Autophagosome formation at the ERMCSs in mammalian cells has been reported. Upon starvation, the ER-resident SNARE protein syntaxin 17 (STX17 in mammals; Syx17 in flies) recruits the PI3K complex subunit Atg14 to the ERMCSs and triggers autophagosome formation. However, Syx17 was not required for autophagosome formation in flies , and the major role of Syx17 in both mammals and flies is to mediate the fusion between autophagosome and lysosome. In addition, VAPB and PTPIP51, a pair of ERMCS tethers, also regulate autophagy. Increased ERMCS formation facilitated by VAPB or PTPIP51 overexpression inhibits autophagy; conversely, the weakening of contact by knockdown of these tethers stimulates autophagosome formation. Recent studies have shown that autophagy occurs at ERMCSs to supply free fatty acids for mitochondrial energy metabolism, while mitochondrial respiratory chain activity supports autophagy through the regulation of ERMCS formation. In addition to regulating autophagy at the initiation stage, in a previous study, it was determined that mitochondria play a crucial role in the late stage of autophagy. The loss of Tom40, a key subunit of the mitochondrial protein import channel, results in blockage of autophagosome and lysosome fusion. It was also found that defects in several general mitochondrial metabolic processes, such as ATP production, mitochondrial protein synthesis, or the citrate cycle, do not cause the autophagy defects observed in Tom40-depleted tissues. This implied that the autophagy defects caused by blocking mitochondrial protein import are rather specific. It is therefore hypothesized that certain mitochondrial proteins regulate autophagy directly (Xu, 2022).

In the present study, it was demonstrated that Miga, a mitochondrial outer-membrane protein, is required for autophagy. Loss of Miga led to defects in autophagosome-lysosome fusion. Miga is an evolutionarily conserved protein, with orthologs from worms to humans. In a previous study, it was found to be localized on the mitochondrial outer membrane to regulate mitochondrial fusion by stabilizing MitoPLD. Miga interacts with the ER-localized VAP protein to establish ERMCSs. The interactions between Miga and VAP proteins are regulated by the phosphorylation of the FFAT motif in Miga. A recent study also reported that MIGA2 (the human ortholog of Miga) regulates ERMCSs and contacts between mitochondria and lipid droplets (LDs) . Loss of Miga led to the degeneration of photoreceptor cells in flies. Overexpression of Miga in fly eyes resulted in increased ERMCSs and severe eye degeneration. In mice, loss of MIGA2 led to anxiety-like behavior. This study found that Miga interacts with Atg14 and Uvrag to regulate PI3K activity and Syx17 stability, thereby modulating autophagy (Xu, 2022).

Defects in both mitochondria and autophagy are hallmarks of several types of neurodegenerative diseases. This study found that Miga establishes a direct link between mitochondria and autophagy to maintain cellular homeostasis (Xu, 2022).

It is striking that a mitochondrial protein directly regulates autophagy by interacting with the core components of the autophagy machinery. In the present study, it was found that the mitochondrial protein Miga forms complexes with Uvrag and Atg14 to regulate PI3P production and to stabilize Syx17 during autophagy (Xu, 2022).

Miga interacts with Vap33 to mediate formation of ERMCSs. Overexpression of wild-type Miga, but not MigaFM, led to increased PI3P levels, implying that Miga-induced ERMCSs are required for regulating PI3P formation. However, the ERMCS tether function of Miga is neither required for recruiting Uvrag nor for binding to Atg14 and Syx17 stabilization. It has been shown previously that Atg14 and other components of the PI3K complex, such as Atg16 and Vps34, are enriched in ERMCSs upon starvation. The question remains as to why the PI3K complex needs to be present. Phosphatidylinositol (PI) is a substrate required for the PI3K complex to produce PI3P. PI is synthesized on the ER, and ERMCSs are the sites for the transfer of PI between the ER and mitochondria. During autophagy, the PI3K complex promotes PI3P formation to facilitate autophagic processes, and ERMCSs represent platforms to access PI. It is believed that the enrichment of the PI3K complex at ERMCSs is needed to assess the supply of PI. The present study found that MigaFM failed to promote PI3P formation, although it was still able to recruit key PI3K components, such as Uvrag or Atg14. This implied that PI3P formation during autophagy not only requires the activity of the PI3K complex but also PI supplied from ERMCSs (Xu, 2022).

Previous studies reported that ERMCSs are required for the initiation of autophagy. In the current study, it was found that in Miga mutants, autophagic processes were blocked at the autophagosome-lysosome fusion stage, while autophagosome formation was largely unaffected. Lack of Miga led to a reduction in PI3P and Syx17 levels. Previous studies have demonstrated that PI3K is not only essential for autophagy initiation but is also recruited to the autophagosome together with the HOPS complex to facilitate autophagosome and lysosome fusion in mammalian cells. The remaining PI3P in Miga mutants is probably sufficient for autophagosome formation but not enough for the autophagosome-lysosome fusion process. This study found that the loss of Miga reduced co-localization of FYVE-GFP and Atg8a but not co-localization of FYVE-GFP and CathL. This suggests that the reduction of PI3P in autophagosomes, but not lysosomes, might contribute to fusion defects (Xu, 2022).

The fusion defects observed in Miga mutants were not identical to those found in mutants without Syx17 or HOPS components. The puncta of autophagosome markers are larger in Miga mutants than those in mutants without Syx17 or HOPS components, possibly due to the combined effects of reduction of PI3P and Syx17. In worms and mammalian cells, the lack of EPG5 prevents autophagosome maturation and induces the ectopic fusion of autophagosomes with various endocytic vesicles. The enlarged Atg8a-positive structures in Miga mutants might also be a result of the ectopic fusion of autophagosomes with other vesicles (Xu, 2022).

In mammals, both UVRAG and ATG14 are required for autophagy. In flies, Uvrag regulates PI3P formation under fed conditions, and Atg14 is required for PI3P-positive autophagosome formation. This study found that Miga overexpression induces PI3P formation; additionally, Uvrag, but not Atg14, is required during this process. It was also found that Miga overexpression leads to an upregulation of numerous autophagy markers, such as Atg9, Syx17, Atg18a, Rab7, and LAMP, among others. However, the expression levels or patterns of p62 and Atg8a did not change significantly upon Miga overexpression. This implied that Miga overexpression is not sufficient to fully activate autophagy (Xu, 2022).

STX17, the mammalian ortholog of Syx17, is an autophagosome-localized Q-SNARE that mediates autophagosome and lysosome fusion through interactions with SNAP29 and VAMP8/Vamp7. STX17 contains two tandem transmembrane domains that have low hydrophobicity but are required for autophagosome localization. In fed mammalian cells, STX17 reportedly localizes to the ER, mitochondria, and cytosol. STX17 was enriched in ERMCSs upon autophagy stimulation and was present on completely closed autophagosomes. The detailed translocation mechanism remains unclear. In flies, Syx17 shows diffusely dispersed patterns, and there is no mitochondria-specific localization under normal fed conditions. Syx17 forms puncta and co-localizes with Atg8-positive autophagosomes upon starvation. It was found that Miga is required for the stabilization of Syx17. Miga does not bind to Syx17 but stabilizes it through Atg14. It is puzzling why a mitochondrial protein would be required for the stabilization of a protein that functions in autophagosome maturation. It has been reported that there are three-way contacts among the ER, mitochondria, and late endosomes. It is possible that Miga, Vap33, and Atg14 mediate the contact between the ER, mitochondria, and autophagosomes. Autophagosome-associated Atg14 further stabilizes Syx17 to mediate the fusion between autophagosomes and lysosomes. GFP-Atg14 formed large puncta instead of decreasing in the Miga mutant clones. One possible explanation for this is that the overexpression of GFP-Atg14 overrides the requirement of Miga to stabilize it, but the overexpression of GFP-Atg14 per se is not sufficient to fully rescue the autophagy defects in the Miga mutant. Therefore, similar to other autophagy markers, GFP-Atg14 puncta accumulated in the Miga mutant clones (Xu, 2022).

In summary, this study identified a mitochondrial protein, Miga, that regulates autophagic processes by interacting with Atg14 and Uvrag. This delineates a link between mitochondria and macroautophagy. However, this study did not solve how Miga stabilizes Atg14 and Syx17. It is possible that Miga mediates the three-way contact between the ER, mitochondria, and autophagosomes. Miga interacts with Atg14 and stabilizes Atg14. Furthermore, Atg14 interacts with Syx17 to stabilize it. It is not clear how the relay is carried out during autophagy (Xu, 2022).

Both Atg14 and Uvrag interact with MigaN (1-252 aa), but there is no evident competition between Atg14 and Uvrag. The exact regions of Miga that bind to each protein were not identified in this study (Xu, 2022).

PROTEIN STRUCTURE

Amino Acids - 1088

Structural Domains

A full-length Dp110 cDNA was isolated and found to encode a protein homologous throughout its length to the class I mammalian PI3Ks p110alpha and p110beta. Dp110 can be divided into domains of different predicted function. The most conserved regions, found at the C-terminus, are the catalytic core (HR1) and PIK domain (HR2), a domain that is also found in PI4-kinases. The N-terminus contains regions known to be both necessary and sufficient for mammalian p110alpha to associate with the regulatory subunit p85. The central portion of the protein contains two further PI3K homology regions: HR3, which is of unknown function, and a stretch of basic amino acids followed by a short leucine zipper, reminiscent of the dimerization domains of bZIP transcription factors and which therefore might play a role in intra- or inter-molecular interactions (Leevers, 1996).

date revised: 10 June 2023

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