mayday: Biological Overview | References
Gene name - mayday
Cytological map position - 91F11-91F11
Function - signaling
Keywords - calcium ion binding - maintenance of synapse structure - has a positive effect on retrograde trans-synaptic BMP signaling - active in trans-Golgi network - orthologous to human SDF4 (stromal cell derived factor 4).
Symbol - myd
FlyBase ID: FBgn0051475
Genetic map position - chr3R:19,180,659-19,201,096
Classification - EF-hand, calcium binding motif, found in CREC-EF hand family
Cellular location - cytoplasmic
Maintaining synaptic structure and function over time is vital for overall nervous system function and survival. The processes that underly synaptic development are well understood. However, the mechanisms responsible for sustaining synapses throughout the lifespan of an organism are poorly understood. This study demonstrates that a previously uncharacterized gene, CG31475, regulates synaptic maintenance in adult Drosophila NMJs. We named CG31475 (mayday( due to the progressive loss of flight ability and synapse architecture with age. Mayday is functionally homologous to the human protein Cab45 (SDF4 - stromal cell derived factor 4), which sorts secretory cargo from the Trans Golgi Network (TGN). Mayday was found to be required to maintain trans-synaptic BMP signaling at adult NMJs in order to sustain proper synaptic structure and function. Finally, mutations in mayday were shown to result in the loss of both presynaptic motor neurons as well as postsynaptic muscles, highlighting the importance of maintaining synaptic integrity for cell viability (Sidisky, 2021).
Among the most prominent neuromuscular synapses in adult Drosophila are those of the indirect flight muscles. One set of IFMs, the Dorsal Longitudinal Muscles (DLMs), are composed of six large muscle fibers innervated by five motor neurons on each side of the thorax. Once the DLM NMJs are established, these stable structures are present throughout the lifespan of the organism. These NMJs are part of the Giant Fiber (GF) pathway that propels flight behavior. Thus, the activity of DLMs can be monitored by assaying flight behavior as a readout of synaptic integrity. Additionally, the DLM NMJs form a tripartite synapse composed of a presynaptic motor neuron, postsynaptic muscle cell, and associated glial cell, that provide the ability to understand synaptic function at the cellular and molecular level. This model also allows for expression of transgenes in non-essential tissue, particularly the DLM motor neurons that are easily accessible. Together, it is possible assess the morphological and functional properties of adult DLM NMJs to elucidate the mechanisms responsible for sustaining synapses in aging adults, then apply this to understand how synapses deteriorate in neurodegenerative diseases (Sidisky, 2021).
Although the processes involved in maintaining synaptic structure and function may not be understood, there are a few key pathways that are crucial for regulating synaptic growth, organization and stability during synaptic development. Specifically, in Drosophila one key signaling cascade that involves coordination between the presynaptic motor neurons and postsynaptic muscle cells is the bone morphogenic protein (BMP) signaling cascade. The morphogen glass bottom boat (Gbb), the Drosophila ortholog to mammalian BMP7, is secreted in a retrograde manner from the postsynaptic muscle cell to the presynaptic motor neuron. Currently, it is not understood how this pathway could function past development. This suggests that this signaling cascade could play a role in maintaining synaptic integrity (Sidisky, 2021).
Gaining a better understanding of synaptic dysfunction should help to identify strategies involved in maintaining synaptic integrity with age. This study identified Mayday, a resident Golgi protein that is required to maintain trans-synaptic signaling across adult NMJs. Mutations in mayday impair retrograde BMP signaling, resulting in degradation of synaptic structure and function. Finally, this study demonstrates that this sustained trans-synaptic signaling is required to maintain the viability of both presynaptic motor neurons and postsynaptic muscles (Sidisky, 2021).
The current study describes mayday (myd), a previously uncharacterized gene that plays a role in maintaining synaptic integrity with age by promoting trans-synaptic signaling. We found that myd3PM71 mutants have structural and functional deficits in adult DLM NMJs. Through tissue-specific RNAi and rescue experiments, it was determined that Myd is necessary in both postsynaptic muscle tissue and presynaptic motor neurons to maintain synaptic integrity. Myd localizes to the TGN and shares functional homology with human Cab45. Myd sustains retrograde BMP signaling in adult DLM NMJs through genetic interactions with gbb, tkv, wit, and mad mutants and staining of Gbb and pMad markers. Finally, myd sustains the viability of presynaptic motor neurons and postsynaptic muscles (Sidisky, 2021).
From developmental studies, it was learned that Gbb is a morphogen secreted in a retrograde manner trans-synaptically from postsynaptic muscle tissue to the presynaptic motor neuron in larval NMJs to promote synaptic growth. However, relatively little is known regarding the roles of this pathway in fully developed organisms. Recent evidence demonstrates that sustained BMP signaling is required to maintain FMRFamide expression in a subset of neurons in the Drosophila brain. The current results here further demonstrate that retrograde BMP signaling that regulates NMJ development is required in adult NMJs to sustain synaptic integrity with age. It is also possible that several other signaling pathways crucial for organism development may be required throughout the life of the organism (Sidisky, 2021).
Knockdown and rescue experiments using myd demonstrate that it maintains synaptic integrity through roles in both pre- and postsynaptic tissue. While most studies involving BMP in synaptic growth report a retrograde signaling mechanism, recent evidence suggests that this pathway could also signal in an anterograde fashion. While genetic studies provide support for retrograde BMP signaling, it cannot be ruled out that anterograde BMP signaling also plays an important role in maintaining synaptic integrity. In particular, the levels of pMAD that were observed were present within both presynaptic motor neuron terminals as well as postsynaptic muscles. Further studies aimed at characterizing BMP signal activation within muscle cells should help with our understanding of the mechanisms responsible for synaptic maintenance (Sidisky, 2021).
While trans-synaptic BMP signaling plays a clear role in maintaining synapses, myd mutations likely impair other pathways associated with cargo trafficking. In addition to secretory cargo, Cab45 also has a role in trafficking lysosomal proteases. Given the functional homology shared between Cab45 and Myd, it is possible that the trafficking of these lysosomal hydrolases needed for autophagy could be disrupted. Defects in autophagy have been strongly linked with neurodegenerative diseases. Therefore, it is possible that myd mutants have disruptions in autophagy that lead to the loss of synaptic integrity. It will be interesting to investigate how Myd impacts these other processes that are associated with neuronal dysfunction (Sidisky, 2021).
This assessment of synaptic dysfunction in the current study includes flight performance as a readout of functional integrity, as well as morphological measurements of branch length, branch number, and bouton number using a presynaptic membrane marker. In future studies, it will be helpful to further evaluate synaptic integrity in mayday mutants. Additional functional assays may include electrophysiological measurements of activity across these NMJs, and more structural data could be obtained through the use of a wide array of synaptic markers, as well as ultrastructural analysis using Transmission Electron Microscopy. Together, these types of studies should allow for an even greater understanding of synaptic dysfunction and the mechanisms required to maintain these critical structures (Sidisky, 2021).
The trans-Golgi Network (TGN) sorts molecular "addresses" and sends newly synthesized proteins to their destination via vesicular transport carriers. Despite the functional significance of packaging processes at the TGN, the sorting of soluble proteins remains poorly understood. Recent research has shown that the Golgi resident protein Cab45 is a significant regulator of secretory cargo sorting at the TGN. Cab45 (SDF4 - stromal cell derived factor 4) oligomerizes upon transient Ca(2+) influx, recruits soluble cargo molecules (clients), and packs them in sphingomyelin-rich transport carriers. However, the identity of client molecules packed into Cab45 vesicles is scarce. Therefore, this study used a precise and highly efficient secretome analysis technology called hiSPECs. Intriguingly, it was observed that Cab45 deficient cells manifest hypersecretion of lysosomal hydrolases. Specifically, Cab45 deficient cells secrete the unprocessed precursors of prosaposin (PSAP) and progranulin (PGRN). In addition, lysosomes in these cells show an aberrant perinuclear accumulation suggesting a new role of Cab45 in lysosomal positioning. This work uncovers a yet unknown function of Cab45 in regulating lysosomal function (Tran, 2023).
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. This study shows that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. It was further demonstrated that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, these data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi (Lebreton, 2021).
The TGN is a key compartment for the sorting and secretion of newly synthesized proteins. At the TGN, soluble proteins are sorted based on the instructions carried in their oligosaccharide backbones or by a Ca2+-mediated process that involves the cargo-sorting protein Cab45. This study shows that Cab45 is phosphorylated by the Golgi-specific protein kinase Fam20C. Mimicking of phosphorylation translocates Cab45 into TGN-derived vesicles, which goes along with an increased export of LyzC, a Cab45 client. These findings demonstrate that Fam20C plays a key role in the export of Cab45 clients by fine-tuning Cab45 oligomerization and thus impacts Cab45 retention in the TGN (Hecht, 2020).
Sorting of soluble proteins for transport to intracellular compartments and for secretion from cells is essential for cell and tissue homeostasis. The trans-Golgi network (TGN) is a major sorting station that sorts secretory proteins into specific carriers to transport them to their final destinations. The sorting of lysosomal hydrolases at the TGN by the mannose 6-phosphate receptor is well understood. The recent discovery of a Ca(2+)-based sorting of secretory cargo at the TGN is beginning to uncover the mechanism by which cells sort secretory cargoes from Golgi residents and cargoes destined to the other cellular compartments. This Ca(2+)-based sorting involves the cytoplasmic actin cytoskeleton, which through membrane anchored Ca(2+) ATPase SPCA1 and the luminal Ca(2+) binding protein Cab45 sorts of a subset of secretory proteins at the TGN. This study presents this discovery and highlight important challenges that remain unaddressed in the overall pathway of cargo sorting at the TGN (Pakdel, 2018).
Sorting and export of transmembrane cargoes and lysosomal hydrolases at the trans-Golgi network (TGN) are well understood. However, elucidation of the mechanism by which secretory cargoes are segregated for their release into the extracellular space remains a challenge. Previous work has demonstrated that, in a reaction that requires Ca(2+), the soluble TGN-resident protein Cab45 is necessary for the sorting of secretory cargoes at the TGN. This study reports that Cab45 reversibly assembles into oligomers in the presence of Ca(2+) These Cab45 oligomers specifically bind secretory proteins, such as COMP and LyzC, in a Ca(2+)-dependent manner in vitro. In intact cells, mutation of the Ca(2+)-binding sites in Cab45 impairs oligomerization, as well as COMP and LyzC sorting. Superresolution microscopy revealed that Cab45 colocalizes with secretory proteins and the TGN Ca(2+) pump (SPCA1) in specific TGN microdomains. These findings reveal that Ca(2+)-dependent changes in Cab45 mediate sorting of specific cargo molecules at the TGN (Crevenna, 20166).
Ca(2+) import into the lumen of the trans-Golgi network (TGN) by the secretory pathway calcium ATPase1 (SPCA1) is required for the sorting of secretory cargo. How is Ca(2+) retained in the lumen of the Golgi, and what is its role in cargo sorting? This study shows that a soluble, lumenal Golgi resident protein, Cab45, is required for SPCA1-dependent Ca(2+) import into the TGN; it binds secretory cargo in a Ca(2+)-dependent reaction and is required for its sorting at the TGN (von Blume, 2012).
This study identified in a yeast two-hybrid screen the EF-hand Ca(2+)-binding protein Cab45 as an interaction partner of Munc18b. Although the full-length Cab45 resides in Golgi lumen, this study characterized a cytosolic splice variant, Cab45b, expressed in pancreatic acini. Cab45b is shown to bind (45)Ca(2+), and, of its three EF-hand motifs, EF-hand 2 is demonstrated to be crucial for the ion binding. Cab45b is shown to interact with Munc18b in an in vitro assay, and this interaction is enhanced in the presence of Ca(2+). In this assay, Cab45b also binds the Munc18a isoform in a Ca(2+)-dependent manner. The endogenous Cab45b in rat acini coimmunoprecipitates with Munc18b, syntaxin 2, and syntaxin 3, soluble N-ethylmaleimide-sensitive factor attachment protein receptors with key roles in the Ca(2+)-triggered zymogen secretion. Furthermore, it was shown that Munc18b bound to syntaxin 3 recruits Cab45b onto the plasma membrane. Importantly, antibodies against Cab45b are shown to inhibit in a specific and dose-dependent manner the Ca(2+)-induced amylase release from streptolysin-O-permeabilized acini. The present study identifies Cab45b as a novel protein factor involved in the exocytosis of zymogens by pancreatic acini (Lam, 2007).
Search PubMed for articles about Drosophila Mayday
Crevenna, A. H., Blank, B., Maiser, A., Emin, D., Prescher, J., Beck, G., Kienzle, C., Bartnik, K., Habermann, B., Pakdel, M., Leonhardt, H., Lamb, D. C. and von Blume, J. (2016). Secretory cargo sorting by Ca2+-dependent Cab45 oligomerization at the trans-Golgi network. J Cell Biol 213(3): 305-314. PubMed ID: 27138253
Hecht, T. K., Blank, B., Steger, M., Lopez, V., Beck, G., Ramazanov, B., Mann, M., Tagliabracci, V. and von Blume, J. (2020). Fam20C regulates protein secretion by Cab45 phosphorylation. J Cell Biol 219(6). PubMed ID: 32422653
Lam, P. P., Hyvarinen, K., Kauppi, M., Cosen-Binker, L., Laitinen, S., Keranen, S., Gaisano, H. Y. and Olkkonen, V. M. (2007). A cytosolic splice variant of Cab45 interacts with Munc18b and impacts on amylase secretion by pancreatic acini. Mol Biol Cell 18(7): 2473-2480. PubMed ID: 17442889
Lebreton, S., Paladino, S., Liu, D., Nitti, M., von Blume, J., Pinton, P. and Zurzolo, C. (2021). Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells. Proc Natl Acad Sci U S A 118(33). PubMed ID: 34389665
Pakdel, M. and von Blume, J. (2018). Exploring new routes for secretory protein export from the trans-Golgi network. Mol Biol Cell 29(3): 235-240. PubMed ID: 29382805
Sidisky, J. M., Weaver, D., Hussain, S., Okumus, M., Caratenuto, R. and Babcock, D. (2021). Mayday sustains trans-synaptic BMP signaling required for synaptic maintenance with age. Elife 10. PubMed ID: 33667157
Tran, M. L., Tushaus, J., Kim, Y., Ramazanov, B. R., Devireddy, S., Lichtenthaler, S. F., Ferguson, S. M. and von Blume, J. (2023). Cab45 deficiency leads to the mistargeting of progranulin and prosaposin and aberrant lysosomal positioning. Traffic 24(1): 4-19. PubMed ID: 36398980
date revised: 17 April 2023
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