Atg2, Atg9 and Atg18 in mitochondrial integrity, cardiac function and healthspan in Drosophila.

In yeast, the Atg2-Atg18 complex regulates Atg9 recycling from phagophore assembly site during autophagy; their function in higher eukaryotes remains largely unknown. In a targeted screening in Drosophila melanogaster, we show that Mef2-GAL4-RNAi-mediated knockdown of Atg2, Atg9 or Atg18 in the heart and indirect flight muscles led to shortened healthspan (declined locomotive function) and lifespan. These flies displayed an accelerated age-dependent loss of cardiac function along with cardiac hypertrophy (increased heart tube wall thickness) and structural abnormality (distortion of the lumen surface). Using the Mef2-GAL4-MitoTimer mitochondrial reporter system and transmission electron microscopy, we observed significant elongation of mitochondria and reduced number of lysosome-targeted autophagosomes containing mitochondria in the heart tube but exaggerated mitochondrial fragmentation and reduced mitochondrial density in indirect flight muscles. These findings provide the first direct evidence of the importance of Atg2-Atg18/Atg9 autophagy complex in the maintenance of mitochondrial integrity and, regulation of heart and muscle functions in Drosophila, raising the possibility of augmenting Atg2-Atg18/Atg9 activity in promoting mitochondrial health and, muscle and heart function.

The Gene Expression Program for the Formation of Wing Cuticle in Drosophila.

The cuticular exoskeleton of insects and other arthropods is a remarkably versatile material with a complex multilayer structure. We made use of the ability to isolate cuticle synthesizing cells in relatively pure form by dissecting pupal wings and we used RNAseq to identify genes expressed during the formation of the adult wing cuticle. We observed dramatic changes in gene expression during cuticle deposition, and combined with transmission electron microscopy, we were able to identify candidate genes for the deposition of the different cuticular layers. Among genes of interest that dramatically change their expression during the cuticle deposition program are ones that encode cuticle proteins, ZP domain proteins, cuticle modifying proteins and transcription factors, as well as genes of unknown function. A striking finding is that mutations in a number of genes that are expressed almost exclusively during the deposition of the envelope (the thin outermost layer that is deposited first) result in gross defects in the procuticle (the thick chitinous layer that is deposited last). An attractive hypothesis to explain this is that the deposition of the different cuticle layers is not independent with the envelope instructing the formation of later layers. Alternatively, some of the genes expressed during the deposition of the envelope could form a platform that is essential for the deposition of all cuticle layers.

The Drosophila planar polarity gene multiple wing hairs directly regulates the actin cytoskeleton.

The evolutionarily conserved frizzled/starry night (fz/stan) pathway regulates planar cell polarity (PCP) in vertebrates and invertebrates. This pathway has been extensively studied in the Drosophila wing, where it is manifested by an array of distally pointing cuticular hairs. Using in vivo imaging we found that, early in hair growth, cells have multiple actin bundles and hairs that subsequently fuse into a single growing hair. The downstream PCP gene multiple wing hairs (mwh) plays a key role in this process and acts to antagonize the actin cytoskeleton. In mwh mutants hair initiation is not limited to a small region at the distal edge of pupal wing cells as in wild type, resulting in multiple hairs with aberrant polarity. Extra actin bundles/hairs are formed and do not completely fuse, in contrast to wild type. As development proceeded additional hairs continued to form, further increasing hair number. We identified a fragment of Mwh with in vivo rescue activity and that bound and bundled F-actin filaments and inhibited actin polymerization in in vitro actin assays. The loss of these activities can explain the mwh mutant phenotype. Our data suggest a model whereby, prior to hair initiation, proximally localized Mwh inhibits actin polymerization resulting in polarized activation of the cytoskeleton and hair formation on the distal side of wing cells. During hair growth Mwh is found in growing hairs, where we suggest it functions to promote the fusion of actin bundles and inhibit the formation of additional actin bundles that could lead to extra hairs.

ChtVis-Tomato, a genetic reporter for in vivo visualization of chitin deposition in Drosophila.

Chitin is a polymer of N-acetylglucosamine that is abundant and widely found in the biological world. It is an important constituent of the cuticular exoskeleton that plays a key role in the insect life cycle. To date, the study of chitin deposition during cuticle formation has been limited by the lack of a method to detect it in living organisms. To overcome this limitation, we have developed ChtVis-Tomato, an in vivo reporter for chitin in Drosophila. ChtVis-Tomato encodes a fusion protein that contains an apical secretion signal, a chitin-binding domain (CBD), a fluorescent protein and a cleavage site to release it from the plasma membrane. The chitin reporter allowed us to study chitin deposition in time lapse experiments and by using it we have identified unexpected deposits of chitin fibers in Drosophila pupae. ChtVis-Tomato should facilitate future studies on chitin in Drosophila and other insects.

The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of "upstream" Planar Cell Polarity proteins.

The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.

A novel MitoTimer reporter gene for mitochondrial content, structure, stress, and damage in vivo.

Mitochondrial dysfunction plays important roles in many diseases, but there is no satisfactory method to assess mitochondrial health in vivo. Here, we engineered a MitoTimer reporter gene from the existing Timer reporter gene. MitoTimer encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized. Confocal microscopy confirmed targeting of the MitoTimer protein to mitochondria in cultured cells, Caenorhabditis elegans touch receptor neurons, Drosophila melanogaster heart and indirect flight muscle, and mouse skeletal muscle. A ratiometric algorithm revealed that conditions that cause mitochondrial stress led to a significant shift toward red fluorescence as well as accumulation of pure red fluorescent puncta of damaged mitochondria targeted for mitophagy. Long term voluntary exercise resulted in a significant fluorescence shift toward green, in mice and D. melanogaster, as well as significantly improved structure and increased content in mouse FDB muscle. In contrast, high-fat feeding in mice resulted in a significant shift toward red fluorescence and accumulation of pure red puncta in skeletal muscle, which were completely ameliorated by voluntary wheel running. Hence, MitoTimer allows for robust analysis of multiple parameters of mitochondrial health under both physiological and pathological conditions and will be highly useful for future research of mitochondrial health in multiple disciplines in vivo.

Dusky-like functions as a Rab11 effector for the deposition of cuticle during Drosophila bristle development.

The morphogenesis of Drosophila sensory bristles is dependent on the function of their actin and microtubule cytoskeleton. Actin filaments are important for bristle shape and elongation, while microtubules are thought to mediate protein and membrane trafficking to promote growth. We have identified an essential role for the bristle cuticle in the maintenance of bristle structure and shape at late stages of bristle development. We show that the small GTPase Rab11 mediates the organized deposition of chitin, a major cuticle component in bristles, and disrupting Rab11 function leads to phenotypes that result from bristle collapse rather than a failure to elongate. We further establish that Rab11 is required for the plasma membrane localization of the ZP domain-containing Dusky-like (Dyl) protein and that Dyl is also required for cuticle formation in bristles. Our data argue that Dyl functions as a Rab11 effector for mediating the attachment of the bristle cell membrane to chitin to establish a stable cuticle. Our studies also implicate the exocyst as a Rab11 effector in this process and that Rab11 trafficking along the bristle shaft is mediated by microtubules.

dusky-like is required to maintain the integrity and planar cell polarity of hairs during the development of the Drosophila wing.

The cuticular hairs and sensory bristles that decorate the adult Drosophila epidermis and the denticles found on the embryo have been used in studies on planar cell polarity and as models for the cytoskeletal mediated morphogenesis of cellular extensions. ZP domain proteins have recently been found to be important for the morphogenesis of both denticles and bristles. Here we show that the ZP domain protein Dusky-like is a key player in hair morphogenesis. As is the case in bristles, in hairs dyl mutants display a dramatic phenotype that is the consequence of a failure to maintain the integrity of the extension after outgrowth. Hairs lacking dyl function are split, thinned, multipled and often very short. dyl is required for normal chitin deposition in hairs, but chitin is not required for the normal accumulation of Dyl, hence dyl acts upstream of chitin. A lack of chitin however, does not mimic the dyl hair phenotype, thus Dyl must have other targets in hair morphogenesis. One of these appears to be the actin cytoskeleton. Interestingly, dyl mutants also display a unique planar cell polarity phenotype that is distinct from that seen with mutations in the frizzled/starry night or dachsous/fat pathway genes. Rab11 was previously found to be essential for Dyl plasma membrane localization in bristles. Here we found that the expression of a dominant negative Rab11 can mimic the dyl hair morphology phenotype consistent with Rab11 also being required for Dyl function in hairs. We carried out a small directed screen to identify genes that might function with dyl and identified Chitinase 6 (Cht6) as a strong candidate, as knocking down Cht6 function led to weak versions of all of the dyl hair phenotypes.

Regulation of cell shape, wing hair initiation and the actin cytoskeleton by Trc/Fry and Wts/Mats complexes.

The two NDR kinase family genes in Drosophila are tricornered (trc) and warts (wts). Previous studies on trc have focused on its role in the morphogenesis of extensions of epidermal cells and in dendrite branching and tiling. Studies on wts have focused on its roles as a tumor suppressor, in controlling photoreceptor type and in the maintenance of dendrites. Here we examine and compare the function of these genes in wing cells prior to their terminal differentiation. Mutations in these genes lead to changes in cell shape, cellular levels of F-actin, the timing of differentiation, and the expression of multiple wing hairs and DE-Cadherin. We showed that the effects of wts on all of these processes appear to be mediated by its regulation of the Yorkie transcription factor. We also provide evidence that trc regulates the expression of DE-cadherin and mwh. In addition, we showed that the effects on cell shape and the timing of differentiation appear to be not linked to changes in relative growth rate of cells compared to their neighbors.

Rho1 has multiple functions in Drosophila wing planar polarity.

The frizzled (fz) signaling/signal transduction pathway controls planar cell polarity in both vertebrates and invertebrates. Previous data implicated Rho1 as a component of the fz pathway in Drosophila but it was unclear how it functioned. The existence of a G Protein Binding-Formin Homology 3 (GBD-FH3) domain in Multiple Wing Hairs, a downstream component of the pathway suggested that Rho1 might function by binding to and activating Mwh. We re-examined the role of Rho1 in wing planar polarity and found that it had multiple functions. Aberrant Rho1 activity led to changes in the number of hairs formed, changes in cell shape and F-actin and changes in cellular junctions. Experiments that utilized Rho effector loop mutations argued that these phenotypes were mediated by effects of Rho1 on the cytoskeleton and not by effects on transcription. We found strong positive genetic interactions between Rho1 and mwh, that Rho1 regulated the accumulation of Mwh protein and that these two proteins could be co-immunoprecipitated. The Mwh GBD:FH3 domain was sufficient for co-immunoprecipitation with Rho1, consistent with this domain mediating the interaction. However, further experiments showed that Rho1 function in wing differentiation was not limited to interacting with Mwh. We established by genetic experiments that Rho1 could influence hair morphogenesis in the absence of mwh and that the disruption of Rho1 activity could interfere with the zig zag accumulation pattern of upstream fz pathway proteins. Thus, our results argue that in addition to its interaction with Mwh Rho1 has functions in wing planar polarity that are parallel to and upstream of fz. The upstream function may be an indirect one and associated with the requirement for normal apical basal polarity and adherens junctions for the accumulation of PCP protein complexes.

The Drosophila Fry protein interacts with Trc and is highly mobile in vivo.

BACKGROUND: Cell polarity is a common feature of eukaryotic cells. The NDR kinases have been found to regulate polarized growth in both animal cells and fungi. Drosophila Tricornered is an NDR kinase that is essential for the normal polarized growth of extensions of epidermal cells and for the tiling and branching of dendrites of da sensory neurons. Tricornered function requires interacting with the large Furry protein (3479 amino acid). RESULTS: We constructed a furry (fry) transgene and established that it rescued the lethality of fry null mutations. The encoded protein was tagged at both its amino and carboxy termini and this allowed us to demonstrate that the protein existed as an uncleaved protein in vivo. We used the C terminal GFP tag to follow the protein in vivo and found it to be highly mobile. Interestingly Fry accumulated at the distal tip of growing bristles. We established that Fry and Trc could be co-immunoprecipitated from wing discs. CONCLUSIONS: The mobility of Fry in both bristles and dendrites suggests that it could function in directing/mediating the intracellular transport needed for polarized growth. Our observations that full length Fry and Trc show only partial co-localization in growing bristles while an amino terminal fragment of Fry shows close to complete co-localization with Trc suggests that the interaction between these proteins is transient and regulated.

Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs.

The evagination of Drosophila imaginal discs is a classic system for studying tissue level morphogenesis. Evagination involves a dramatic change in morphology and published data argue that this is mediated by cell shape changes. We have reexamined the evagination of both the leg and wing discs and find that the process involves cell rearrangement and that cell divisions take place during the process. The number of cells across the width of the ptc domain in the wing and the omb domain in the leg decreased as the tissue extended during evagination and we observed cell rearrangement to be common during this period. In addition, almost half of the cells in the region of the leg examined divided between 4 and 8 h after white prepupae formation. Interestingly, these divisions were not typically oriented parallel to the axis of elongation. Our observations show that disc evagination involves multiple cellular behaviors, as is the case for many other morphogenetic processes.

Planar signaling and morphogenesis in Drosophila.

The regulatory mechanisms governing the parallel alignment of hairs, bristles, and ommatidia in Drosophila have all served as model systems for studying planar signaling and tissue level morphogenesis. Polarity in all three systems is mediated by the serpentine receptor Frizzled and a number of additional gene products. The localized accumulation of these proteins within cells plays a key role in the development of planar polarity. A comparison of the function of these gene products in the different cell types suggests cell-specific modifications of the pathway.

The multiple-wing-hairs gene encodes a novel GBD-FH3 domain-containing protein that functions both prior to and after wing hair initiation.

The frizzled signaling/signal transduction pathway controls planar cell polarity (PCP) in both vertebrates and invertebrates. Epistasis experiments argue that in the Drosophila epidermis multiple wing hairs (mwh) acts as a downstream component of the pathway. The PCP proteins accumulate asymmetrically in pupal wing cells where they are thought to form distinct protein complexes. One is located on the distal side of wing cells and a second on the proximal side. This asymmetric protein accumulation is thought to lead to the activation of the cytoskeleton on the distal side, which in turn leads to each cell forming a single distally pointing hair. We identified mwh as CG13913, which encodes a novel G protein binding domain-formin homology 3 (GBD-FH3) domain protein. The Mwh protein accumulated on the proximal side of wing cells prior to hair formation. Unlike planar polarity proteins such as Frizzled or Inturned, Mwh also accumulated in growing hairs. This suggested that mwh had two temporally separate functions in wing development. Evidence for these two functions also came from temperature-shift experiments with a temperature-sensitive allele. Overexpression of Mwh inhibited hair initiation, thus Mwh acts as a negative regulator of the cytoskeleton. Our data argued early proximal Mwh accumulation restricts hair initiation to the distal side of wing cells and the later hair accumulation of Mwh prevents the formation of ectopic secondary hairs. This later function appears to be a feedback mechanism that limits cytoskeleton activation to ensure a single hair is formed.

The flare gene, which encodes the AIP1 protein of Drosophila, functions to regulate F-actin disassembly in pupal epidermal cells.

Adult Drosophila are decorated with several types of polarized cuticular structures, such as hairs and bristles. The morphogenesis of these takes place in pupal cells and is mediated by the actin and microtubule cytoskeletons. Mutations in flare (flr) result in grossly abnormal epidermal hairs. We report here that flr encodes the Drosophila actin interacting protein 1 (AIP1). In other systems this protein has been found to promote cofilin-mediated F-actin disassembly. In Drosophila cofilin is encoded by twinstar (tsr). We show that flr mutations result in increased levels of F-actin accumulation and increased F-actin stability in vivo. Further, flr is essential for cell proliferation and viability and for the function of the frizzled planar cell polarity system. All of these phenotypes are similar to those seen for tsr mutations. This differs from the situation in yeast where cofilin is essential while aip1 mutations result in only subtle defects in the actin cytoskeleton. Surprisingly, we found that mutations in flr and tsr also result in greatly increased tubulin staining, suggesting a tight linkage between the actin and microtubule cytoskeleton in these cells.

The balance between the novel protein target of wingless and the Drosophila Rho-associated kinase pathway regulates planar cell polarity in the Drosophila wing.

Planar cell polarity (PCP) signaling is mediated by the serpentine receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling utilizes Drosophila Frizzled 2 (DFz2) as a receptor and also requires Dsh for transducing signals to regulate cell proliferation and differentiation in many developmental contexts. Distinct pathways are activated downstream of Dsh in Wg- and Fz-signaling pathways. Recently, a number of genes, which have essential roles as downstream components of PCP signaling, have been identified in Drosophila. They include the small GTPase RhoA/Rho1, its downstream effector Drosophila rho-associated kinase (Drok), and a number of genes such as inturned (in) and fuzzy (fy), whose biochemical functions are unclear. RhoA and Drok provide a link from Fz/Dsh signaling to the modulation of actin cytoskeleton. Here we report the identification of the novel gene target of wingless (tow) by enhancer trap screening. tow expression is negatively regulated by Wg signaling in wing imaginal discs, and the balance between tow and the Drok pathway regulates wing-hair morphogenesis. A loss-of-function mutation in tow does not result in a distinct phenotype. Genetic interaction and gain-of-function studies provide evidence that Tow acts downstream of Fz/Dsh and plays a role in restricting the number of hairs that wing cells form.

The tricornered Ser/Thr protein kinase is regulated by phosphorylation and interacts with furry during Drosophila wing hair development.

The Trc/Ndr/Sax1/Cbk1 family of ser/thr kinases plays a key role in the morphogenesis of polarized cell structures in flies, worms, and yeast. Tricornered (Trc), the Drosophila nuclear Dbf2-related (Ndr) serine/threonine protein kinase, is required for the normal morphogenesis of epidermal hairs, bristles, laterals, and dendrites. We obtained in vivo evidence that Trc function was regulated by phosphorylation and that mutations in key regulatory sites resulted in dominant negative alleles. We found that wild-type, but not mutant Trc, is found in growing hairs, and we failed to detect Trc in pupal wing nuclei, implying that in this developmental context Trc functions in the cytoplasm. The furry gene and its homologues in yeast and Caenorhabditis elegans have previously been implicated as being essential for the function of the Ndr kinase family. We found that Drosophila furry (Fry) also is found in growing hairs, that its subcellular localization is dependent on Trc function, and that it can be coimmunoprecipitated with Trc. Our data suggest a feedback mechanism involving Trc activity regulates the accumulation of Fry in developing hairs.

Drosophila Mob family proteins interact with the related tricornered (Trc) and warts (Wts) kinases.

The function of Tricornered (Trc), the Drosophila Ndr (Nuclear Dbf2-related) serine/threonine protein kinase, is required for the normal morphogenesis of a variety of polarized outgrowths including epidermal hairs, bristles, arista laterals, and dendrites. In yeast the Trc homolog Cbk1 needs to bind Mob2 to activate the RAM pathway. In this report, we provide genetic and biochemical data that Drosophila Trc also interacts with and is activated by Drosophila Dmob proteins. In addition, Drosophila Mob proteins appear to interact with the related Warts/Lats kinase, which functions as a tumor suppressor in flies and mammals. Interestingly, the overgrowth tumor phenotype that results from mutations in Dmob1 (mats) was only seen in genetic mosaics and not when the entire animal was mutant. We conclude that unlike in yeast, in Drosophila individual Mob proteins interact with multiple kinases and that individual NDR family kinases interact with multiple Mob proteins. We further provide evidence that Mo25, the Drosophila homolog of the RAM pathway hym1 gene does not function along with Trc.