Generation of a TMEM43 knockout human induced pluripotent stem cell line (HDZi003-A-1) using CRISPR/Cas9.

TMEM43 (LUMA) is a ubiquitously expressed protein with unknown function. The protein is phylogenetically highly conserved and also found in Drosophila melanogaster (Klinke et al., 2022). TMEM43-p.S358L is a rare, fully penetrant mutation that leads to arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5). To understand the function of the ARVC5-associated mutation it is first important to understand the function of the TMEM43 protein. Therefore, a TMEM43 knockout induced pluripotent stem cell (iPSC) line was generated using the CRISPR/Cas9 genome editing system. The resulting cell line had a deficiency of TMEM43 and showed normal morphology and a stable karyotype. The colonies were positive for pluripotency markers and could be differentiated into the three germ layers.

Regulatory sites in the Mon1-Ccz1 complex control Rab5 to Rab7 transition and endosome maturation.

Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-GTPase exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here, we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro and alterations in early endosomal structures in Drosophila nephrocytes. A shift from Rab5 to more Rab7-positive structures in yeast suggests faster endosomal maturation. Using modeling, we further identify a conserved Rab5-binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Ras-related in brain (Rab) conversion and organelle maturation along the endomembrane system.

Differentiation and function of cardiac valves in the adult Drosophila heart.

Drosophila, like all insects, has an open circulatory system for the distribution of haemolymph and its components. The circulation of the haemolymph is essentially driven by the pumping activity of the linear heart. The heart is constructed as a tube into which the haemolymph is sucked and pumped forward by rhythmic contractions running from the posterior to the anterior, where it leaves the heart tube. The heart harbours cardiac valves to regulate flow directionality, with a single heart valve differentiating during larval development to separate the heart tube into two chambers. During metamorphosis, the heart is partially restructured, with the linear heart tube with one terminal wide-lumen heart chamber being converted into a linear four-chambered heart tube with three valves. As in all metazoan circulatory systems, the cardiac valves play an essential role in regulating the direction of blood flow. We provide evidence that the valves in adult flies arise via transdifferentiation, converting lumen-forming contractile cardiomyocytes into differently structured valve cells. Interestingly, adult cardiac valves exhibit a similar morphology to their larval counterparts, but act differently upon heart beating. Applying calcium imaging in living specimens to analyse activity in valve cells, we show that adult cardiac valves operate owing to muscle contraction. However, valve cell shape dynamics are altered compared with larval valves, which led us to propose our current model of the opening and closing mechanisms in the fly heart.

Drosophila collagens in specialised extracellular matrices.

The basement membrane (BM) constitutes a specialised form of the extracellular matrix (ECM) and plays important roles in many biological processes, such as cell migration, organ and tissue integrity, cell polarity, and the formation of metastases. In metazoans, a canonical BM is formed by only a few conserved structural core proteins: Laminin, Collagen IV, Nidogen and Perlecan. Depending on the tissue's function and mechanical load, additional matrix proteins interact with, or are incorporated into the BM, resulting in tissue-specific mechanical properties, such as higher stiffness or elasticity, or special resistance to mechanical stress or harmful environmental conditions. In flies, the collagen IV-like protein Pericardin forms an integral constituent of matrices around the heart and tension sensors (chordotonal organs) of the peripheral nervous system. The function and integrity of both organ systems strongly relies on the appropriate establishment of a Pericardin (Prc) matrix and the function of its adapter protein-Lonely heart (Loh). In this review, we provide an overview of the four collagens present in flies, and will discuss our recent work on the formation and function of Pericardin-containing matrices, the role of the adapter protein Lonely heart and the necessity of specialised ECM molecules in tissue architecture and function.

Tailup expression in Drosophila larval and adult cardiac valve cells.

In Drosophila larvae, the direction of blood flow within the heart tube, as well as the diastolic filling of the posterior heart chamber, is regulated by a single cardiac valve. This valve is sufficient to close the heart tube at the junction of the ventricle and the aorta and is formed by only two cells; both are integral parts of the heart tube. The valve cells regulate hemolymph flow by oscillating between a spherical and a flattened cell shape during heartbeats. At the spherical stage, the opposing valve cells close the heart lumen. The dynamic cell shape changes of valve cells are supported by a dense, criss-cross orientation of myofibrils and the presence of the valvosomal compartment, a large intracellular cavity. Both structures are essential for the valve cells' function. In a screen for factors specifically expressed in cardiac valve cells, we identified the transcription factor Tailup. Knockdown of tailup causes abnormal orientation and differentiation of cardiac muscle fibers in the larval aorta and inhibits the formation of the ventral longitudinal muscle layer located underneath the heart tube in the adult fly and affects myofibrillar orientation of valve cells. Furthermore, we have identified regulatory sequences of tup that control the expression of tailup in the larval and adult valve cells.

Formation and function of a highly specialised type of organelle in cardiac valve cells.

Within a cell, vesicles play a crucial role in the transport of membrane material and proteins to a given target membrane, and thus regulate a variety of cellular functions. Vesicular transport occurs by means of, among others, endocytosis, where cargoes are taken up by the cell and are processed further upon vesicular trafficking, i.e. transported back to the plasma membrane via recycling endosomes or the degraded by fusion of the vesicles with lysosomes. During evolution, a variety of vesicles with individual functions arose, with some of them building up highly specialised subcellular compartments. In this study, we have analysed the biosynthesis of a new vesicular compartment present in the valve cells of Drosophila melanogaster. We show that the compartment is formed by invaginations of the plasma membrane and grows via re-routing of the recycling endosomal pathway. This is achieved by inactivation of other membrane-consuming pathways and a plasma membrane-like molecular signature of the compartment in these highly specialised heart cells.

Preparation of Drosophila Tissues and Organs for Transmission Electron Microscopy.

Transmission electron microscopy (TEM) is the method of choice to image the ultrastructure of cells or tissues. TEM allows the visualization of molecular complexes up to an atomic resolution. Thus, TEM data have led to important conclusions about cellular processes and supported findings obtained by functional analyses. In this chapter, we describe the preparation of Drosophila tissues for TEM and provide reliable step-by-step protocols for applying classical chemical fixation or high-pressure freezing-freeze substitution (HPF-FS) to preserve cellular structures.

The ER protein Creld regulates ER-mitochondria contact dynamics and respiratory complex 1 activity.

Dynamic contacts are formed between endoplasmic reticulum (ER) and mitochondria that enable the exchange of calcium and phospholipids. Disturbed contacts between ER and mitochondria impair mitochondrial dynamics and are a molecular hallmark of Parkinson's disease, which is also characterized by impaired complex I activity and dopaminergic neuron degeneration. Here, we analyzed the role of cysteine-rich with EGF-like domain (Creld), a poorly characterized risk gene for Parkinson's disease, in the regulation of mitochondrial dynamics and function. We found that loss of Creld leads to mitochondrial hyperfusion and reduced ROS signaling in Drosophila melanogaster, Xenopus tropicalis, and human cells. Creld fly mutants show differences in ER-mitochondria contacts and reduced respiratory complex I activity. The resulting low-hydrogen peroxide levels are linked to disturbed neuronal activity and lead to impaired locomotion, but not neurodegeneration, in Creld mutants. We conclude that Creld regulates ER-mitochondria communication and thereby hydrogen peroxide formation, which is required for normal neuron function.

Neprilysins regulate muscle contraction and heart function via cleavage of SERCA-inhibitory micropeptides.

Muscle contraction depends on strictly controlled Ca2+ transients within myocytes. A major player maintaining these transients is the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, SERCA. Activity of SERCA is regulated by binding of micropeptides and impaired expression or function of these peptides results in cardiomyopathy. To date, it is not known how homeostasis or turnover of the micropeptides is regulated. Herein, we find that the Drosophila endopeptidase Neprilysin 4 hydrolyzes SERCA-inhibitory Sarcolamban peptides in membranes of the sarcoplasmic reticulum, thereby ensuring proper regulation of SERCA. Cleavage is necessary and sufficient to maintain homeostasis and function of the micropeptides. Analyses on human Neprilysin, sarcolipin, and ventricular cardiomyocytes indicates that the regulatory mechanism is evolutionarily conserved. By identifying a neprilysin as essential regulator of SERCA activity and Ca2+ homeostasis in cardiomyocytes, these data contribute to a more comprehensive understanding of the complex mechanisms that control muscle contraction and heart function in health and disease.

A Drosophila melanogaster model for TMEM43-related arrhythmogenic right ventricular cardiomyopathy type 5.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a severe cardiac disease that leads to heart failure or sudden cardiac death (SCD). For the pathogenesis of ARVC, various mutations in at least eight different genes have been identified. A rare form of ARVC is associated with the mutation TMEM43 p.S358L, which is a fully penetrant variant in male carriers. TMEM43 p.S358 is homologous to CG8111 p.S333 in Drosophila melanogaster. We established CRISPR/Cas9-mediated CG8111 knock-out mutants in Drosophila, as well as transgenic fly lines carrying an overexpression construct of the CG8111 p.S333L substitution. Knock-out flies developed normally, whereas the overexpression of CG8111 p.S333L caused growth defects, loss of body weight, cardiac arrhythmias, and premature death. An evaluation of a series of model mutants that replaced S333 by selected amino acids proved that the conserved serine is critical for the physiological function of CG8111. Metabolomic and proteomic analyses revealed that the S333 in CG8111 is essential to proper energy homeostasis and lipid metabolism in the fly. Of note, metabolic impairments were also found in the murine Tmem43 disease model, and fibrofatty replacement is a hallmark of human ARVC5. These findings contribute to a more comprehensive understanding of the molecular functions of CG8111 in Drosophila, and can represent a valuable basis to assess the aetiology of the human TMEM43 p.S358L variant in more detail.

Interplay between SERCA, 4E-BP, and eIF4E in the Drosophila heart.

Appropriate cardiac performance depends on a tightly controlled handling of Ca2+ in a broad range of species, from invertebrates to mammals. The role of the Ca2+ ATPase, SERCA, in Ca2+ handling is pivotal, and its activity is regulated, inter alia, by interacting with distinct proteins. Herein, we give evidence that 4E binding protein (4E-BP) is a novel regulator of SERCA activity in Drosophila melanogaster during cardiac function. Flies over-expressing 4E-BP showed improved cardiac performance in young individuals associated with incremented SERCA activity. Moreover, we demonstrate that SERCA interacts with translation initiation factors eIF4E-1, eIF4E-2 and eIF4E-4 in a yeast two-hybrid assay. The specific identification of eIF4E-4 in cardiac tissue leads us to propose that the interaction of elF4E-4 with SERCA may be the basis of the cardiac effects observed in 4E-BP over-expressing flies associated with incremented SERCA activity.

Wing hearts in four-winged Ultrabithorax-mutant flies-the role of Hox genes in wing heart specification.

Wings are probably the most advanced evolutionary novelty in insects. In the fruit fly Drosophila melanogaster, proper development of wings requires the activity of so-called wing hearts located in the scutellum of the thorax. Immediately after the imaginal ecdysis, these accessory circulatory organs remove hemolymph and apoptotic epidermal cells from the premature wings through their pumping action. This clearing process is essential for the formation of functional wing blades. Mutant flies that lack intact wing hearts are flightless and display malformed wings. The embryonic wing heart progenitors originate from two adjacent parasegments corresponding to the later second and third thoracic segments. However, adult dipterian flies harbor only one pair of wings and only one pair of associated wing hearts in the second thoracic segment. Here we show that the specification of WHPs depends on the regulatory activity of the Hox gene Ultrabithorax. Furthermore, we analyzed the development of wing hearts in the famous four-winged Ultrabithorax (Ubx) mutant, which was first discovered by Ed Lewis in the 1970s. In these flies, the third thoracic segment is homeotically transformed into a second thoracic segment resulting in a second pair of wings instead of the club-shaped halteres. We show that a second pair of functional wing hearts is formed in the transformed third thoracic segment and that all wing hearts originate from the wild-type population of wing heart progenitor cells.

The small GTPase KlRho5 responds to oxidative stress and affects cytokinesis.

Rho5 is the yeast homolog of the human small GTPase Rac1. We characterized the genes encoding Rho5 and the subunits of its dimeric activating guanine-nucleotide-exchange factor (GEF), Dck1 and Lmo1, in the yeast Kluyveromyces lactis. Rapid translocation of the three GFP-tagged components to mitochondria upon oxidative stress and carbon starvation indicate a similar function of KlRho5 in energy metabolism and mitochondrial dynamics as described for its Saccharomyces cerevisiae homolog. Accordingly, Klrho5 deletion mutants are hyper-resistant towards hydrogen peroxide. Moreover, synthetic lethalities of rho5 deletions with key components in nutrient sensing, such as sch9 and gpr1, are not conserved in K. lactis. Instead, Klrho5 deletion mutants display morphological defects with strengthened lateral cell walls and protruding bud scars. The latter result from aberrant cytokinesis, as observed by following the budding process in vivo and by transmission electron microscopy of the bud neck region. This phenotype can be suppressed by KlCDC42G12V, which encodes a hyper-active variant. Data from live-cell fluorescence microscopy support the notion that KlRho5 interferes with the actin moiety of the contractile actomyosin ring, with consequences different from those previously reported for mutants lacking myosin.

A trimeric metazoan Rab7 GEF complex is crucial for endocytosis and scavenger function.

Endosome biogenesis in eukaryotic cells is critical for nutrient uptake and plasma membrane integrity. Early endosomes initially contain Rab5, which is replaced by Rab7 on late endosomes prior to their fusion with lysosomes. Recruitment of Rab7 to endosomes requires the Mon1-Ccz1 guanine-nucleotide-exchange factor (GEF). Here, we show that full function of the Drosophila Mon1-Ccz1 complex requires a third stoichiometric subunit, termed Bulli (encoded by CG8270). Bulli localises to Rab7-positive endosomes, in agreement with its function in the GEF complex. Using Drosophila nephrocytes as a model system, we observe that absence of Bulli results in (i) reduced endocytosis, (ii) Rab5 accumulation within non-acidified enlarged endosomes, (iii) defective Rab7 localisation and (iv) impaired endosomal maturation. Moreover, longevity of animals lacking bulli is affected. Both the Mon1-Ccz1 dimer and a Bulli-containing trimer display Rab7 GEF activity. In summary, this suggests a key role for Bulli in the Rab5 to Rab7 transition during endosomal maturation rather than a direct influence on the GEF activity of Mon1-Ccz1.

The septate junction protein Tetraspanin 2A is critical to the structure and function of Malpighian tubules in Drosophila melanogaster.

Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian tubules, we used the c42-GAL4/UAS system to selectively knock down Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh, and Discs large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh, and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic tubules. Elevated protein levels together with diminished V-type H+-ATPase activity in Tsp2A knockdown tubules are consistent with cell proliferation and reduced transport activity. Indeed, Malpighian tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifests an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian tubules in the late embryo and progresses upstream to distal tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport.

Alary muscles and thoracic alary-related muscles are atypical striated muscles involved in maintaining the position of internal organs.

Alary muscles (AMs) have been described as a component of the cardiac system in various arthropods. Lineage-related thoracic muscles (TARMs), linking the exoskeleton to specific gut regions, have recently been discovered in Drosophila Asymmetrical attachments of AMs and TARMs, to the exoskeleton on one side and internal organs on the other, suggested an architectural function in moving larvae. Here, we analysed the shape and sarcomeric organisation of AMs and TARMs, and imaged their atypical deformability in crawling larvae. We then selectively eliminated AMs and TARMs by targeted apoptosis. Elimination of AMs revealed that AMs are required for suspending the heart in proper intra-haemocelic position and for opening of the heart lumen, and that AMs constrain the curvature of the respiratory tracheal system during crawling; TARMs are required for proper positioning of visceral organs and efficient food transit. AM/TARM cardiac versus visceral attachment depends on Hox control, with visceral attachment being the ground state. TARMs and AMs are the first example of multinucleate striated muscles connecting the skeleton to the cardiac and visceral systems in bilaterians, with multiple physiological functions.

The septate junction protein Mesh is required for epithelial morphogenesis, ion transport, and paracellular permeability in the Drosophila Malpighian tubule.

Septate junctions (SJs) are occluding cell-cell junctions that have roles in paracellular permeability and barrier function in the epithelia of invertebrates. Arthropods have two types of SJs, pleated SJs and smooth SJs (sSJs). In Drosophila melanogaster, sSJs are found in the midgut and Malpighian tubules, but the functions of sSJs and their protein components in the tubule epithelium are unknown. Here we examined the role of the previously identified integral sSJ component, Mesh, in the Malpighian tubule. We genetically manipulated mesh specifically in the principal cells of the tubule at different life stages. Tubules of flies with developmental mesh knockdown revealed defects in epithelial architecture, sSJ molecular and structural organization, and lack of urine production in basal and kinin-stimulated conditions, resulting in edema and early adult lethality. Knockdown of mesh during adulthood did not disrupt tubule epithelial and sSJ integrity but decreased the transepithelial potential, diminished transepithelial fluid and ion transport, and decreased paracellular permeability to 4-kDa dextran. Drosophila kinin decreased transepithelial potential and increased chloride permeability, and it stimulated fluid secretion in both control and adult mesh knockdown tubules but had no effect on 4-kDa dextran flux. Together, these data indicate roles for Mesh in the developmental maturation of the Drosophila Malpighian tubule and in ion and macromolecular transport in the adult tubule.

Identification and In Vivo Characterisation of Cardioactive Peptides in Drosophila melanogaster.

Neuropeptides and peptide hormones serve as critical regulators of numerous biological processes, including development, growth, reproduction, physiology, and behaviour. In mammals, peptidergic regulatory systems are complex and often involve multiple peptides that act at different levels and relay to different receptors. To improve the mechanistic understanding of such complex systems, invertebrate models in which evolutionarily conserved peptides and receptors regulate similar biological processes but in a less complex manner have emerged as highly valuable. Drosophila melanogaster represents a favoured model for the characterisation of novel peptidergic signalling events and for evaluating the relevance of those events in vivo. In the present study, we analysed a set of neuropeptides and peptide hormones for their ability to modulate cardiac function in semi-intact larval Drosophila melanogaster. We identified numerous peptides that significantly affected heart parameters such as heart rate, systolic and diastolic interval, rhythmicity, and contractility. Thus, peptidergic regulation of the Drosophila heart is not restricted to chronotropic adaptation but also includes inotropic modulation. By specifically interfering with the expression of corresponding peptides in transgenic animals, we assessed the in vivo relevance of the respective peptidergic regulation. Based on the functional conservation of certain peptides throughout the animal kingdom, the identified cardiomodulatory activities may be relevant not only to proper heart function in Drosophila, but also to corresponding processes in vertebrates, including humans.

SERCA is critical to control the Bowditch effect in the heart.

The Bowditch effect or staircase phenomenon is the increment or reduction of contractile force when heart rate increases, defined as either a positive or negative staircase. The healthy and failing human heart both show positive or negative staircase, respectively, but the causes of these distinct cardiac responses are unclear. Different experimental approaches indicate that while the level of Ca2+ in the sarcoplasmic reticulum is critical, the molecular mechanisms are unclear. Here, we demonstrate that Drosophila melanogaster shows a negative staircase which is associated to a slight but significant frequency-dependent acceleration of relaxation (FDAR) at the highest stimulation frequencies tested. We further showed that the type of staircase is oppositely modified by two distinct SERCA mutations. The dominant conditional mutation SERCAA617T induced positive staircase and arrhythmia, while SERCAE442K accentuated the negative staircase of wild type. At the stimulation frequencies tested, no significant FDAR could be appreciated in mutant flies. The present results provide evidence that two individual mutations directly modify the type of staircase occurring within the heart and suggest an important role of SERCA in regulating the Bowditch effect.

APC/CFzr regulates cardiac and myoblast cell numbers, and plays a crucial role during myoblast fusion.

Somatic muscles are formed by the iterative fusion of myoblasts into muscle fibres. This process is driven by the recurrent recruitment of proteins to the cell membrane to induce F-actin nucleation at the fusion site. Although several proteins involved in myoblast fusion have been identified, knowledge about their subcellular regulation is rather elusive. We identified the anaphase-promoting complex (APC/C) adaptor Fizzy related (Fzr) as an essential regulator of heart and muscle development. We show that APC/CFzr regulates the fusion of myoblasts as well as the mitotic exit of pericardial cells, cardioblasts and myoblasts. Surprisingly, overproliferation is not causative for the observed fusion defects. Instead, fzr mutants exhibit smaller F-actin foci at the fusion site and display reduced membrane breakdown between adjacent myoblasts. We show that lack of APC/CFzr causes accumulation and mislocalisation of Rols and Duf, two proteins involved in the fusion process. Duf seems to serve as direct substrate of the APC/CFzr and its destruction depends on the presence of distinct degron sequences. These novel findings indicate that protein destruction and turnover constitute major events during myoblast fusion.