Endosomal vacuoles of the prepupal salivary glands of Drosophila play an essential role in the metabolic reallocation of iron

Abstract

In the recent past, we demonstrated that a great deal is going on in the salivary glands of Drosophila in the interval after they release their glycoprotein-rich secretory glue during pupariation. The early-to-mid prepupal salivary glands undergo extensive endocytosis with widespread vacuolation of the cytoplasm followed by massive apocrine secretion. Here, we describe additional novel properties of these endosomes. The use of vital pH-sensitive probes provided confirmatory evidence that these endosomes have acidic contents and that there are two types of endocytosis seen in the prepupal glands. The salivary glands simultaneously generate mildly acidic, small, basally-derived endosomes and strongly acidic, large and apical endosomes. Staining of the large vacuoles with vital acidic probes is possible only after there is ambipolar fusion of both basal and apical endosomes, since only basally-derived endosomes can bring fluorescent probes into the vesicular system. We obtained multiple lines of evidence that the small basally-derived endosomes are chiefly involved in the uptake of dietary Fe iron. The fusion of basal endosomes with the larger and strongly acidic apical endosomes appears to facilitate optimal conditions for ferrireductase activity inside the vacuoles to release metabolic Fe iron. While iron was not detectable directly due to limited staining sensitivity, we found increasing fluorescence of the glutathione-sensitive probe CellTracker Blue CMAC in large vacuoles, which appeared to depend on the amount of iron released by ferrireductase. Moreover, heterologous fluorescently-labeled mammalian iron-bound transferrin is actively taken up, providing direct evidence for active iron uptake by basal endocytosis. In addition, we serendipitously found that small (basal) endosomes were uniquely recognized by PNA lectin, whereas large (apical) vacuoles bound DBA lectin.

Transferrin 1 Functions in Iron Trafficking and Genetically Interacts with Ferritin in Drosophila melanogaster

“…Farka s et al (2018) have reported that a key function of the basally derived endosomes in the Drosophila prepupal (PP) salivary glands (SGs) is to support iron import via basal endocytosis. This is followed by strong vacuole acidification to increase the activity of ferrireductase, which is required to release bivalent cationic Fe 2+ for its metabolic utilization in the cytosol (Farka s et al, 2018). These authors provided strong supportive evidence for the involvement of the iron-transferrin system in this process (Farka s et al, 2018), which is similar to the known iron-importing endosomal systems in other model organisms (Lambe et al, 2009;Knutson, 2007;McKie, 2005).…”

“…This is followed by strong vacuole acidification to increase the activity of ferrireductase, which is required to release bivalent cationic Fe 2+ for its metabolic utilization in the cytosol (Farka s et al, 2018). These authors provided strong supportive evidence for the involvement of the iron-transferrin system in this process (Farka s et al, 2018), which is similar to the known iron-importing endosomal systems in other model organisms (Lambe et al, 2009;Knutson, 2007;McKie, 2005). D. melanogaster MCO3 was reported to possess multicopper ferroxidase activity (as does hephaestin in mammals) and to possibly be involved in iron homeostasis (Bettedi et al, 2011;Lang et al, 2012;Wang et al, 2018).…”

Functional disruption of transferrin expression alters reproductive physiology in Anopheles culicifacies

“…However, due to the lack of vertebrate transferrin receptor homolog in insects/mosquitoes, it remains unresolved how loading and unloading of iron are achieved at midgut and ovary surfaces, respectively [8,52]. A recent study by Farkas et.al 2018 suggests the involvement of basally derived endosomes in iron import to the target cells is important in the insect D. melanogaster and proposes that basal endocytosis is followed by vacuole acidification, a process leading to the activation of ferrireductase for iron release and metabolic utilization [53]. Several studies highlight that transferrin plays an antibacterial immune role against microbial infection in insects [54][55][56].…”

Fine structure of Drosophila larval salivary gland ducts as revealed by laser confocal microscopy and SEM

“…To detect chitin (a major component of taenidia) in fi xed tissue, SGs (or tracheae) were stained with Calcofl uor White M2R (also known as Fluorescence Brightener 28, FB28; Sigma, # F-3543) at pH 9 in 0.2 M Tris buffer containing 0.1% Triton X-100 (Serva) for 2-3 h at room temperature. Whenever necessary, samples were also counterstained with Alexa Fluor 488 or Alexa Fluor 546 phalloidin (Molecular Probes/Invitrogen) to detect fi lamentous actin, as previously described (Farkaš et al, 2015(Farkaš et al, , 2018.…”