InteractiveFly: GeneBrief

ADP ribosylation factor at 79F: Biological Overview | References


Gene name - ADP ribosylation factor at 79F

Synonyms - Arf1

Cytological map position - 80B1-80B1

Function - signaling

Keywords - regulates secretory pathway, the cellular immune response and planar cell polarity - vesicle transportation between the endoplasmic reticulum and the Golgi - required for dendrite pruning of sensory neurons - kills normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway - Arf GAP Asap promotes Arf1 function at the Golgi for cleavage furrow biosynthesis

Symbol - Arf79F

FlyBase ID: FBgn0010348

Genetic map position - chr3L:22,868,508-22,872,231

NCBI classification - ARF-like small GTPases; ARF, ADP-ribosylation factor

Cellular location - cytoplasmic



NCBI link: EntrezGene
BIOLOGICAL OVERVIEW

Pruning, whereby neurons eliminate their exuberant neurites, is central for the maturation of the nervous system. In Drosophila, sensory neurons, ddaCs, selectively prune their larval dendrites without affecting their axons during metamorphosis. However, it is unknown whether the secretory pathway plays a role in dendrite pruning. This study shows that the small GTPase Arf1, an important regulator of secretory pathway, is specifically required for dendrite pruning of ddaC/D/E sensory neurons but dispensable for apoptosis of ddaF neurons. Analyses of the GTP and GDP-locked forms of Arf1 indicate that the cycling of Arf1 between GDP-bound and GTP-bound forms is essential for dendrite pruning. Sec71 was identified as a guanine nucleotide exchange factor for Arf1 that preferentially interacts with its GDP-bound form. Like Arf1, Sec71 is also important for dendrite pruning, but not apoptosis, of sensory neurons. Arf1 and Sec71 are interdependent for their localizations on Golgi. Finally, Sec71/Arf1-mediated trafficking process is a prerequisite for Rab5-dependent endocytosis to facilitate endocytosis and degradation of the cell adhesion molecule Neuroglian (Wang, 2017).

In the developing nervous systems, neurons often extend excessive neurites and form superfluous connections at early stages. Subsequent removal of those exuberant or inappropriate neurites without causing the death of parental neurons, a process known as pruning, is crucial for the refinement of neural circuits at late developmental stages. Neuronal pruning is a conserved process widely occurring in both vertebrates and invertebrates. In vertebrates, many neurons in the neocortex, neuromuscular system and hippocampal dendate gyrus prune their unwanted neurites to control the proper wiring of the nervous systems. In invertebrates, such as Drosophila, the nervous systems undergo drastic remodelling during metamorphosis, a transition stage from a larva to an adult fly. In the central nervous system (CNS), mushroom body (MB) γ neurons prune their dorsal and medial axon branches as well as entire dendrites. In the peripheral nervous system (PNS), some dorsal dendritic arborization (da) neurons, ddaC, ddaD and ddaE, selectively eliminate their larval dendrites without affecting their axons, whereas ddaF neurons are apoptotic during early metamorphosis. The pruning event involves both local degeneration and retraction, resembling neurodegeneration associated with brain injury and neurodegenerative diseases. Thus, a complete understanding of cellular and molecular mechanisms of developmental pruning would shed some light on pathological neurodegeneration following neurological diseases and injury (Wang, 2017).

In Drosophila, ddaC sensory neurons have emerged as an attractive model system to elucidate the molecular and cellular mechanisms of dendrite-specific pruning during early metamorphosis. In response to the steroid-molting hormone 20-hydroxyecdysone (ecdysone) at the late larval stage, ddaC neurons sever the proximal region of their dendrites and subsequently undergo rapid fragmentation of the severed dendrites as well as dendritic clearance via phagocytosis. It has been well documented that the Ecdysone Receptor and its co-receptor Ultraspiracle are required to activate the expression of several downstream targets to initiate dendrite pruning. Endocytic pathways have been identified that are critical for dendrite pruning (Kanamori, 2015; Zhang, 2014). Rab5/Avalanche and ESCRT complexes, the components of the endocytic pathways, are required for downregulation of the L1-type cell adhesion molecule (L1-CAM) Neuroglian (Nrg) (Zhang, 2014). Nrg is drastically redistributed to endosomes and its protein levels are strongly downregulated prior to pruning, suggesting that massive Nrg endocytosis promotes dendrite pruning (Zhang, 2014). It is conceivable that Nrg endocytosis might be triggered by secreted ligands/signals through the secretory pathway. However, it is completely unknown whether the secretory pathway, an opposite route of the endocytic pathway, also plays a role in dendrite pruning of ddaC neurons (Wang, 2017).

The primary sites of the secretory pathway consist of the endoplasmic reticulum (ER), the Golgi apparatus and the trans-Golgi network in eukaryotic cells. Newly synthesized membrane proteins and lipids exit from the ER, pass through the Golgi complexes and are delivered to the plasma membrane via the post-Golgi exocytosis. In developing neurons, the continuous addition of membrane proteins and lipids via the secretory pathway plays a key role in the outgrowth and elongation of dendrites and axons. Disruption of the ER-to-Golgi transport leads to the inhibition of dendritic or axonal growth in Drosophila sensory neurons and rodent hippocampal neurons. In an attempt to isolate novel players of dendrite pruning, a large-scale RNA interference (RNAi) screen was carried out, and ADP-ribosylation factor 1(Arf1), also known as ADP-ribosylation factor at 79F, was identified as an important player for dendrite pruning in ddaC sensory neurons. Arf1 is a small GTPase and belongs to the Class I Arf family. It has been reported that Arf1 can recruit COPI coat proteins on cis-Golgi and clathrin adaptor proteins, such as AP-1, AP-3, and GGAs, on trans-Golgi in a GTP- dependent manner, and thereby facilitate vesicle formation and trafficking. Studies from yeast and mammals indicate that Arf1 is activated by two conserved families of guanine nucleotide exchange factors (GEFs), including the Gea1/GBF1 family on cis-Golgi and Sec7/BIG family on trans-Golgi (Gillingham, 2007). Arf1 cycles between GDP- and GTP-bound forms, and both the GTP- and GDP-locked forms can interfere with its functions and disrupt secretory trafficking. In mammalian hippocampal neurons, overexpression of the GTP-locked form of Arf1 (Arf1Q71L), which abolishes Arf1 activity, inhibits dendrite growth (Horton, 2005). The mammalian Arf1GEF, BIG2, is required for vesicle trafficking and mutations in human BIG2 gene lead to autosomal recessive periventricular heterotopia with microcephaly (ARPHM), a brain disorder characterized by defective neural proliferation and migration. Thus, various studies have documented that the secretory pathway plays a critical role in neurite growth and extension in developing neurons. However, very little is known about its role in regulating neurite pruning, a developmental degenerative process (Wang, 2017).

This study reports the identification of Arf1 as an important player of dendrite pruning in ddaC sensory neurons. The cycling of Arf1 between GDP-bound and GTP-bound forms is essential for dendrite pruning. Sec71 was identified as a GEF for Arf1 in Drosophila. Sec71, like Arf1, is cell-autonomously required for dendrite pruning of ddaC/D/E sensory neurons but not for apoptosis of ddaF neurons during early metamorphosis. Arf1 and Sec71 co-localize on Golgi apparatus and regulate secretory vesicle biogenesis in ddaC neurons. Furthermore, it was shown that Sec71/Arf1-dependent secretory pathway acts upstream of Rab5-dependent endocytosis and facilitates the internalization and downregulation of the cell adhesion molecule Nrg prior to dendrite pruning. Thus, this study demonstrates a novel and important role of Arf1/Sec71-mediated secretory pathway in promoting developmental pruning via the regulation of Nrg endocytosis (Wang, 2017).

The small G protein Arf1 regulates vesicular trafficking in eukaryotes and is activated on cis-Golgi by the Gea1/GBF1 family of Arf1GEF or on trans-Golgi by the Sec7/BIG1 family (Cherfils, 2014). It has been reported that Drosophila Arf1 regulates hematopoietic niche maintenance, blood cell precursor differentiation in vivo, planar cell polarity, and lamellipodium formation in S2 cells (Carvajal-Gonzalez, 2015; Humphreys, 2012; Khadilkar, 2014). Arf1 and other Golgi proteins were reported to exhibit upregulation of their transcripts in axon pruning of MB γ neurons during the larval-pupal transition. This study reports an important role of Arf1 in regulating dendrite pruning of ddaC sensory neurons. Arf1 puncta overlap with the trans- Golgi marker GalT and partially with the cis-Golgi marker GM130, suggesting that Arf1 is primarily localized on trans-Golgi in ddaC sensory neurons. A specific GEF for Arf1 has not been identified in Drosophila. In vivo and in vitro data provide compelling evidence that Sec71 is a GEF for Arf1 in Drosophila. First, Sec71 was co-localized with Arf1 on Golgi and their localizations were inter-dependent in ddaC neurons. Second, Sec71 preferentially bound to the GDP-bound form of Arf1 instead of GTP-bound form. Third, in the GEF assays Sec71 accelerated the release of GDP from Arf1. Fourth, both Arf1 and Sec71 are required for dendrite pruning, as loss of Sec71 or Arf1 led to comparable pruning defects in ddaC sensory neurons. Finally, the expression of Arf1 fully restored WP dendrite morphology and rescued the pruning defects in Sec71 RNAi ddaC neurons. Thus, Sec71 is specifically required for the GDP-to-GTP exchange of Arf1. Structure-function analyses indicate that DCB domain of Sec71 is important for its targeting on Golgi. In contrast, another small GTPase Arl1 was reported to bind to the N-terminal region of Sec71 (DCB and HUS1 domains) and recruit Sec71 on trans-Golgi apparatus in Drosophila S2 cells, and mammalian Arl1 is required for the recruitment of BIG1/2 (mammalian homologues of Sec71) on trans-Golgi (Wang, 2017).

Secretory pathway plays a novel and important role in governing neurite pruning Extensive studies have attempted to understand roles of post-Golgi trafficking in outgrowth and elaboration of dendrites in growing neurons. Post-Golgi trafficking is polarized toward apical dendrites of rodent hippocampal neurons and selectively regulates the growth of dendrites. The dynamics of the Golgi outposts, mediated by the Golgin Lava Lamp, dynein-dynactin complex and Leucine- rich repeat kinase (Lrrk), is important for dendrite growth in Drosophila class IV da neurons. A small GTPase Rab10, which mediates post-Golgi vesicle trafficking, regulates dendrite growth and branching of multi-dendritic sensory neurons in both C. elegans and Drosophila (Wang, 2017).

This study provides compelling evidence to demonstrate that post-Golgi trafficking plays a crucial role in proper dendrite pruning in sensory neurons. First, the key small GTPase Arf1 was identified that is important for post-Golgi trafficking regulates secretory vesicle biogenesis and dendrite pruning in sensory neurons during metamorphosis. Second, a Sec7-domain-containing protein Sec71 acts as a specific GEF for Arf1 and co- localizes with Arf1. Like Arf1, Sec71 is also an essential factor for regulating dendrite pruning. Third, given that both Arf1 and Sec71 also regulate dendrite growth and arborization in ddaC neurons, a critical role of Arf1 and Sec71 in dendrite pruning was further controlled using the Gene-Switch system. Pulse induction of Arf1T31N or Sec71DN at the middle third instar larval stage when the complete larval dendrite arbors form in ddaC neurons consistently caused much more severe dendrite pruning defects. These results highlight that the secretory pathway play separable roles in two distinct processes, namely dendrite growth and dendrite pruning. Arf1 was reported to regulate post-Golgi secretion by recruiting its downstream effectors, including the clathrin adaptors AP-1 and AP-3, and GGA (Cherfils, 2014). Post-Golgi trafficking pathways include the transport from Golgi to plasma membrane (exocyst complex-mediated), from Golgi to early/sorting endosomes (AP-1-meditated), from Golgi to late endosomes (Golgi-localized Gamma-ear containing Arf-binding protein or GGA-mediated) as well as from Golgi to lysosomes (AP-3-mediated). It is conceivable that at least one of these post-Golgi trafficking routes is involved in dendrite pruning of sensory neurons (Wang, 2017).

It has been reported previously that Rab5/ESCRT-dependent endocytic pathways facilitate dendrite pruning by downregulating the L1-CAM Nrg in ddaC neurons during metamorphosis (Zhang, 2014). In MB γ neurons, the JNK pathway promotes axon pruning by downregulating another adhesion molecule Fasciclin II (Bornstein, 2015). These studies suggest a general mechanism whereby cell adhesion molecules are internalized and downregulated to destabilize dendrites and/or axons during neurite pruning. However, the mechanism that triggers Nrg endocytosis is poorly understood. This study demonstrated that Arf1/Sec71-mediated secretory pathway promotes endocytosis and downregulates Nrg prior to dendrite pruning. First, while Nrg levels were strongly reduced prior to dendrite pruning, loss of Arf1 or Sec71 led to elevated levels of Nrg protein in dendrites, axons and soma, comparable to Rab5 mutant neurons. Second, Nrg was no longer redistributed on FYVE-positive endosomes in Arf1 or Sec71 mutant ddaC neurons, suggesting a blockage of Nrg endocytosis. Third, while Rab5 mutant neurons exhibited robust ubiquinated protein aggregates and enlarged endosomes, further removal of either Arf1 or Sec71 suppressed these rab5 mutant phenotypes, suggesting that the secretory pathway acts upstream of Rab5 to positively regulates endocytosis. Finally, knockdown of Nrg significantly suppressed the dendrite pruning defect in Arf1 or Sec71 mutant neurons, supporting the notion that the secretory pathway promotes Nrg endocytosis and downregulation. Thus, the secretory pathway not only secretes the cell adhesion molecules to the dendrite surface and stabilize dendrites (Taylor, 2015; Zou, 2015), but also unexpectedly promotes the internalization and turnover of the adhesion molecules. It is conceivable that in response to ecdysone pulse, the secretory pathway might be required to specifically secrete an as-yet-unidentified ligand to trigger massive endocytosis of the L1-CAM Nrg and thereby leads to degeneration of larval dendrites. Further studies may continue to elucidate what ligand or secreted protein promotes Nrg endocytosis (Wang, 2017).

Differential modulation of the cellular and humoral immune responses in Drosophila is mediated by the endosomal ARF1-Asrij axis

How multicellular organisms maintain immune homeostasis across various organs and cell types is an outstanding question in immune biology and cell signaling. In Drosophila, blood cells (hemocytes) respond to local and systemic cues to mount an immune response. While endosomal regulation of Drosophila hematopoiesis is reported, the role of endosomal proteins in cellular and humoral immunity is not well-studied. This study demonstrated a functional role for endosomal proteins in immune homeostasis. The ubiquitous trafficking protein ADP Ribosylation Factor 1 (ARF1) and the hemocyte-specific endosomal regulator Asrij differentially regulate humoral immunity. Asrij and ARF1 play an important role in regulating the cellular immune response by controlling the crystal cell melanization and phenoloxidase activity. ARF1 and Asrij mutants show reduced survival and lifespan upon infection, indicating perturbed immune homeostasis. The ARF1-Asrij axis suppresses the Toll pathway anti-microbial peptides (AMPs) by regulating ubiquitination of the inhibitor Cactus. The Imd pathway is inversely regulated- while ARF1 suppresses AMPs, Asrij is essential for AMP production. Several immune mutants have reduced Asrij expression, suggesting that Asrij co-ordinates with these pathways to regulate the immune response. This study highlights the role of endosomal proteins in modulating the immune response by maintaining the balance of AMP production. Similar mechanisms can now be tested in mammalian hematopoiesis and immunity (Khadilkar, 2017).

A balanced cellular and humoral immune response is essential to achieve and maintain immune homeostasis. In Drosophila, aberrant hematopoiesis and impaired hemocyte function can both affect the ability to fight infection and maintain immune homeostasis. Endosomal proteins are known to regulate Drosophila hematopoiesis. This study shows an essential function for endosomal proteins in regulating immunity (Khadilkar, 2017).

Altered hemocyte number and distribution as a result of defective hematopoiesis, can also lead to immune phenotypes like increased melanization or phagocytosis. This study shows that perturbation of normal levels of endocytic molecules ARF1 or Asrij leads to aberrant hematopoiesis, affecting the circulating hemocyte number. This in turn leads to an impaired cellular immune response. The aberrant hematopoietic phenotypes with pan-hemocyte tissue-specific depletion of ARF1 using e33cGal4 or HmlGal4 are comparable to the phenotypes observed in the case of asrij null mutant. Hence this study has compared Gal4-mediated ARF1 knockdown to asrij null mutant (Khadilkar, 2017).

In addition, it was also shown that ARF1 and Asrij have a direct role in humoral immunity by regulating AMP gene expression. This is likely to be a contribution from the hemocyte compartment which is primarily affected upon perturbation of Asrij or ARF1. It is well established that hemocytes, apart from acting as the cellular arm of the immune response, also act as sentinels and relay signals to the immune organs that mount the humoral immune response. Hemocytes have been shown to produce ligands like Spaetzle and upd3 that activate immune pathways and induce anti-microbial peptide secretion from the fat body or gut. Asrij or ARF1 could also be affecting the production of such ligand molecules thereby affecting the target immune-activation pathways (Khadilkar, 2017).

Considering the involvement of Asrij and ARF1 in both the arms of immune response, a model is proposed for the role of the ARF1-Asrij axis in maintaining immune homeostasis that can be used for testing additional players in the process (Khadilkar, 2017).

It is known that ARF1 is involved in clathrin coat assembly and endocytosis and has a critical role in membrane bending and scission. In this context it is also intriguing to note that ARF1, like Asrij, does not seem to have an essential role in phagocytosis. This suggests that hemocytes could be involved in additional mechanisms beyond phagocytosis in order to combat an infection (Khadilkar, 2017).

Both ARF1 and Asrij control hemocyte proliferation as their individual depletion leads to an increase in the total and differential hemocyte counts. Also, both mutants have higher crystal cell numbers due to over-activation of Notch as a result of endocytic entrapment. This suggests that increased melanization accompanied by increase in phenoloxidase activity upon ARF1 or Asrij depletion is a consequence of aberrant hematopoiesis and not likely due to a cellular requirement in regulating the melanization response. Constitutive activation of the Toll pathway or impaired Jak/Stat or Imd pathway signaling in various mutants also leads to the formation of melanotic masses. Thus the phenotypes seen on Asrij or ARF1 depletion could either be due to the defective hematopoiesis which directly affects the cellular immune response or leads to a mis-regulation of the immune regulatory pathways (Khadilkar, 2017).

Regulation of many signaling pathways, including the immune regulatory pathways takes place at the endosomes. For example, endocytic proteins Mop and Hrs co-localize with the Toll receptor at endosomes and function upstream of MyD88 and Pelle, thus indicating that Toll signalling is regulated by endocytosis. This study shows that loss of function of the ARF1-Asrij axis leads to an upregulation of some AMP targets of the Toll pathway. Upon depletion of ARF1-Asrij endosomal axis, increased ubiquitination of Cactus, a negative regulator of the Toll pathway, was found in both hemocytes and fat bodies. This suggests non-autonomous regulation of signals by the ARF1-Asrij axis, which is in agreement with an earlier model of signalling through this route (Khadilkar, 2014). Thus the endosomal axis may systemically control the sorting and thereby degradation of Cactus, which in turn promotes the nuclear translocation of Toll effector, Dorsal. This could explain the significant increase in Toll pathway reporter expression such as Drosomycin-GFP. Interestingly the effect of ARF1 depletion on the Toll pathway is more pronounced than that of Asrij depletion. This is not surprising as ARF1 is a ubiquitous and essential trafficking molecule that regulates a variety of signals. This suggests that ARF1 is likely to be involved with additional steps of the Toll pathway and may also interact with multiple regulators of AMP expression (Khadilkar, 2017).

ARF1 and Asrij show complementary effects on IMD pathway target AMPs. While ARF1 suppresses the production of IMD pathway AMPs, Asrij has a discriminatory role. Asrij seems to promote transcription of AttacinA and Drosocin, whereas it represses Cecropin. However in terms of AMP production only Drosocin and Diptericin are affected, but not to the extent of ARF1. In addition, Relish shows marked nuclear localization in fat body cells of hemocyte-specific arf1 knockdown larvae whereas there is no significant difference in the localization in Asrij depleted larval fat bodies. This indicates that ARF1-Asrij axis exerts differential control over the Imd pathway. Thus ARF1 causes strong generic suppression of the Imd pathway while the role of Asrij could be to fine tune this effect. Mass spectrometric analysis of purified protein complexes indicates that ARF1 and Imd interact. Hence it is very likely that ARF1 regulates Imd pathway activation at the endosomes. Whether this interaction involves Asrij or not remains to be tested and will give insight into modes of differential activation of immune pathways (Khadilkar, 2017).

This analysis shows that Asrij is the tuner for endosomal regulation of the humoral immune response by ARF1 and provides specialized tissue- specific and finer control over AMP regulation. This is in agreement with earlier data showing that Asrij acts downstream of ARF127. Since ARF1 is expressed in the fat body it could communicate with the hemocyte- specific molecule, Asrij, to mediate immune cross talk (Khadilkar, 2017).

As reduced Asrij expression is seen in Toll and Jak/Stat pathway mutants such as Rel E20 and Hop Tum1, it is likely that these effectors also regulate Asrij, setting up a feedback mechanism to modulate the immune response. Earlier work has shown that ARF1-Asrij axis modulates different signalling outputs like Notch by endosomal regulation of NICD (Notch Intracellular Domain) transport and activity and JAK/STAT by endosomal activation of Stat92e. Further, ARF1 along with Asrij regulates Pvr signaling in order to maintain HSC's. ARF1 acts downstream of Pvr. Surprisingly, Asrij levels are downregulated in the Pvr mutant. Hence it is likely that the ARF1-Asrij axis regulates trafficking of the Pvr receptor, which then also regulates Asrij levels thus providing feedback regulation. While active modulation of signal activity and outcome at endosomes could be orchestrated by ARF1 and Asrij, their activities in turn need to be modulated. The data suggest that targets of Asrij endosomal regulation may in turn regulate Asrij expression at the transcript level. Further, upon Gram positive infection in wild type flies, asrij transcript levels decrease with a concomitant increase in suppressed AMPs such as Cecropin. This indicates additional regulatory loops such as that mediated by the IMD pathway effector NFκB may regulate asrij transcription. Using bioinformatics tools, presence of binding sites for NFκβ and Rel family of transcription factors are seen in the upstream regulatory sequence (1kb upstream) of asrij and arf1. Hence, feedback regulation is proposed of Asrij and ARF1 by the effectors of the Toll and Imd pathway respectively. This is reflected in the regulation of Asrij expression by these pathways. This also implies multiple modes of regulation of asrij and arf1, which are likely important in its role as a tuner of the generic immune response, thereby allowing it to discriminate between AMPs that were thought to be uniformly regulated, such as those downstream of IMD. Thus this analysis gives insight into additional complex regulation of the Drosophila immune response that can now be investigated further (Khadilkar, 2017).

Asrij and ARF1 being endocytic proteins are likely to interact with a number of molecules that regulate different cell signalling cascades. Due to endosomal localization, molecular interactions may be favored that further translate into signalling output. Hence, it is not surprising that Asrij and ARF1 genetically interact with multiple signalling pathways and can aid crosstalk to regulate important developmental and physiological processes like hematopoiesis or immune response. It is quite likely that Asrij and ARF1 are themselves also part of different feedback loops or feed-forward mechanisms as their levels need to be tightly regulated. Evidence for this is found with respect to the Toll, JAK/STAT and Pvr pathway as described earlier. Hence it is proposed that the Asrij-ARF1 endosomal signalling axis genetically interacts with various signalling components thereby regulating blood cell and immune homeostasis (Khadilkar, 2017).

AMP transcript level changes upon ARF1 or Asrij depletion also correspond to reporter-AMP levels seen after infection. This suggests that although ARF1 is known to have a role in secretion, mutants do not have an AMP secretion defect. Hence aberrant regulation of immune pathways on perturbation of the ARF1-Asrij axis is most likely due to perturbed endosomal regulation (Khadilkar, 2017).

ARF1 has a ubiquitous function in the endosomal machinery and is well-positioned to regulate the interface between metabolism, hematopoiesis and immunity in order to achieve homeostasis. Along with Asrij and other tissue-specific modulators, it can actively modulate the metabolic and immune status in Drosophila. In this context, it is interesting to note that Asrij is a target of MEF253, which is required for the immune-metabolic switch in vivo. Thus Asrij could bring tissue specificity to ARF1 action, for example, by modulating insulin signalling in the hematopoietic system (Khadilkar, 2017).

It is likely that in Asrij or ARF1 mutants, the differentiated hemocytes mount a cellular immune response and perish as in the case of wild type flies where immunosenescence sets in with age and the ability of hemocytes to combat infection declines. Since their hematopoietic stem cell pool is exhausted, they may fail to replenish the blood cell population, thus compromising the ability to combat infections. Alternatively, mechanisms that downregulate the inflammatory responses and prevent sustained activation may be inefficient when the trafficking machinery is perturbed. This could result in constitutive upregulation thus compromising immune homeostasis (Khadilkar, 2017).

In summary, this study shows that in addition to its requirement in hematopoiesis, the ARF1-Asrij axis can differentially regulate humoral immunity in Drosophila, most likely by virtue of its endosomal function. ARF1 and Asrij bring about differential endocytic modulation of immune pathways and their depletion leads to aberrant pathway activity and an immune imbalance. In humans, loss of function mutations in molecules involved in vesicular machinery like Amphyphysin I in which clathrin coated vesicle formation is affected leads to autoimmune disorders like Paraneoplastic stiff-person syndrome. Synaptotagmin, involved in vesicle docking and fusion to the plasma membrane acts as an antigenic protein and its mutation leads to an autoimmune disorder called Lambert-Eaton myasthenic syndrome. Mutations in endosomal molecules like Rab27A, β subunit of AP3, SNARE also lead to immune diseases like Griscelli and Hermansky-Pudlak syndrome. Mutants of both ARF1 and Asrij are likely to have drastic effects on the immune system. Asrij has been associated with inflammatory conditions such as arthritis, thyroiditis, endothelitis and tonsillitis, whereas the ARF family is associated with a wide variety of diseases. ARF1 has been shown to be involved in mast cell degranulation and IgE mediated anaphylaxis response. Generation and analysis of vertebrate models for these genes such as knockout and transgenic mice will provide tools to understand their function in human immunity (Khadilkar, 2017).

The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila

Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. This study investigated stem-cell death in the digestive system of adult Drosophila melanogaster. It was found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively kills normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spares differentiated cells. The dying stem cells are engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduce CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers (Singh, 2016).

To investigate the molecular mechanism behind the resistance of CSCs to therapeutics, the death of stem cells with different degrees of quiescence was studied in the adult Drosophila digestive system, including intestinal stem cells (ISCs), renal and nephric stem cells (RNSCs) and hindgut intestinal stem cells (HISCs). Expression of the proapoptotic genes rpr and p53 effectively ablated differentiated cells but had little effect on stem cells (Singh, 2016).

In mammals, treatment-resistant leukaemic stem cells (LSCs) can be eliminated by a two-step protocol involving initial activation by interferon-α (IFNα) or colony-stimulating factor (G-CSF), followed by targeted chemotherapy. In Drosophila, activation of the hopscotch (also known as JAK)-Stat92E signalling pathway induces hyperplastic stem cells, which are overproliferating, but retain their apico-basal polarity and differentiation ability. This study conducted a slightly different two-step protocol in Drosophila stem cells by overexpressing the JAK-Stat92E pathway ligand unpaired (upd) and rpr together. The induction of upd + rpr using the temperature-sensitive (ts) mutant esg-Gal4 effectively ablated all of the ISCs and RNSCs through apoptosis within four days. Consistent with this result, expressing a gain-of-function Raf mutant (Rafgof) also accelerated apoptotic cell death of hyperplastic ISCs (Singh, 2016).

Expressing a constitutively active form of Ras oncogene at 85D (also known as RasV12) in RNSCs and the knockdown of Notch activity in ISCs can transform these cell types into CSC-like neoplastic stem cells, which were not only overproliferating, but also lost their apico-basal polarity and differentiation ability. Expressing rpr in RasV12-transformed RNSCs (esgts > RasV12 + rpr) or in ISCs expressing a dominant-negative form of Notch (NDN) (esgts > NDN + rpr) caused the ablation of only a proportion of the transformed RNSCs and few transformed ISCs and it did not affect differentiated cells; substantial populations of the neoplastic stem cells remained even seven days after rpr induction (Singh, 2016).

These results suggest that the activation of proliferation can accelerate the apoptotic cell death of hyperplastic stem cells, but that a proportion of actively proliferating neoplastic RNSCs and ISCs are resistant to apoptotic cell death. Neoplastic tumours in Drosophila are more similar to high-grade malignant human tumours than are the hyperplastic Drosophila tumours (Singh, 2016).

Vesicle-mediated COPI and COPII are essential components of the trafficking machinery for vesicle transportation between the endoplasmic reticulum and the Golgi. In addition, the COPI complex regulates the transport of lipolysis enzymes to the surface of lipid droplets for lipid droplet usage. In a previous screen, it was found that knockdown of COPI components (including Arf79F, the Drosophila homologue of ADP-ribosylation factor 1 (Arf1)) rather than COPII components resulted in stem-cell death, suggesting that lipid-droplet usage (lipolysis) rather than the general trafficking machinery between the endoplasmic reticulum and Golgi is important for stem-cell survival.

To further investigate the roles of these genes in stem cells, a recombined double Gal4 line of esg-Gal4 and wg-Gal4 was used to express genes in ISCs, RNSCs, and HISCs (esgts wgts > X). Knockdown of these genes using RNA interference (RNAi) in stem cells ablated most of the stem cells in 1 week. However, expressing Arf79FRNAi in enterocytes (NP1ts > Arf79FRNAi) or in differentiated stellate cells in Malpighian tubules (tshts > Arf79FRNAi) did not cause similar marked ablation. These results suggest that Arf79F knockdown selectively kills stem cells and not differentiated cells (Singh, 2016).

It was also found that expressing Arf79FRNAi (esgts > RasV12 + Arf79FRNAi) or ζ-COPRNAi (esgts wgts >RasV12 + ζ-COPRNAi) in RasV12-transformed RNSCs ablated almost all of the transformed stem cells. Similarly, expressing Arf79FRNAi (esgts > NDN + Arf79FRNAi) or δ-COPRNAi (esgts wgts > NDN + δ-COPRNAi) in NDN-transformed ISCs ablated all of the cells within one week, but restored differentiated cells to close to their normal levels within one week (Singh, 2016).

δ-COP- and γ-COP-mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique and the COPI complex was found to cell-autonomously regulated stem cell survival. In summary, knockdown of the COPI-Arf79F complex effectively ablated normal and transformed stem cells but not differentiated enterocytes or stellate cells (Singh, 2016).

In the RNAi screen acyl-CoA synthetase long-chain (ACSL), an enzyme in the Drosophila lipolysis-β-oxidation pathway, , and bubblegum (bgm), a very long-chain fatty acid-CoA ligase were also identified. RNAi-mediated knockdown of Acsl (esgts wgts > AcslRNAi) effectively killed ISCs and RNSCs, but killed HISCs less effectively. Expressing AcslRNAi in RasV12-transformed RNSCs (esgts wgts > RasV12 + AcslRNAi also ablated almost all of the transformed RNSCs in one week (Singh, 2016).

Brummer (bmm) is a triglyceride lipase, the Drosophila homologue of mammalian ATGL, the first enzyme in the lipolysis pathway. Scully (scu) is the Drosophila orthologue of hydroxy-acyl-CoA dehydrogenase, an enzyme in the β-oxidation pathway. Hepatocyte nuclear factor 4 (Hnf4) regulates the expression of several genes involved in lipid mobilization and β-oxidation. To determine whether the lipolysis-β-oxidation pathway is required for COPI-Arf79F-mediated stem cell survival, upstream activating sequence (UAS)-regulated constructs (UAS-bmm), UAS-Hnf4, and UAS-scu were expressed in stem cells that were depleted of Arf79F, or ζ-COP . Overexpressing either scu or Hnf4 significantly attenuated the stem cell death caused by knockdown of the COPI-Arf79F complex. Expressing UAS-Hnf4 in FRT82B-γ-COP10 MARCM clones also rescued the stem cell death phenotype induced by γ-COP knockdown. However, bmm overexpression did not rescue the stem-cell death induced by Arf79F knockdown. Since there are several other triglyceride lipases in Drosophila in addition to bmm, another lipase may redundantly regulate the lipolysis pathway (Singh, 2016).

To further investigate the function of lipolysis in stem cells, the expression of a lipolysis reporter (GAL4-dHFN4; UAS-nlacZ), which consisted of hsp70-GAL4-dHNF4 combined with a UAS-nlacZ reporter gene, was examined. The flies were either cultured continuously at 29°C or heat-shocked for 30 min at 37°C, 12 h before dissection. Without heat shock, the reporter was expressed only in ISCs and RNSCs of mature adult flies, but not in enteroendocrine cells, enterocytes, quiescent HISCs or quiescent ISCs of freshly emerged young adult flies (less than 3 days old). Expressing δ-COPRNAi (esgts > δ-COPRNAi + GAL4-dHFN4; UAS-nlacZ) almost completely eliminated the reporter expression, suggesting that the reporter was specifically regulated by the COPI complex. After heat shock or when a constitutively active form of JAK (hopTum-l) was expressed, the reporter was strongly expressed in ISCs, RNSCs and HISCs, but not in enteroendocrine cells or enterocytes. These data suggest that COPI-complex-regulated lipolysis was active in stem cells, but not in differentiated cells, and that the absence of the reporter expression in quiescent HISCs at 29°C was probably owing to weak hsp70 promoter activity rather than to low lipolysis in these cells (Singh, 2016).

Lipid storage was further investigated, and it was found that the size and number of lipid droplets were markedly increased in stem cells after knockdown of Arf79F (esgts > Arf79FRNAi) (Singh, 2016).

Arf1 inhibitors (brefeldin A, golgicide A, secin H3, LM11 and LG8) and fatty-acid-oxidation (FAO) inhibitors (triacsin C, mildronate, etomoxir and enoximone) was used andthese inhibitors markedly reduced stem-cell tumours in Drosophila through the lipolysis pathway but had a negligible effect on normal stem cells (Singh, 2016).

These data together suggest that the COPI-Arf1 complex regulates stem-cell survival through the lipolysis-β-oxidation pathway, and that knockdown of these genes blocks lipolysis but promotes lipid storage. Further, the transformed stem cells are more sensitive to Arf1 inhibitors and may be selectively eliminated by controlling the concentration of Arf1 inhibitors (Singh, 2016).

These data suggest that neither caspase-mediated apoptosis nor autophagy-regulated cell death regulates the stem-cell death induced by the knockdown of components of the COPI-Arf79F complex. Therefore, whether necrosis regulates the stem-cell death induced by knockdown of the COPI-Arf79F complex was investigated. Necrosis is characterized by early plasma membrane rupture, reactive oxygen species (ROS) accumulation and intracellular acidification. Propidium iodide detects necrotic cells with compromised membrane integrity, the oxidant-sensitive dye dihydroethidium (DHE) indicates cellular ROS levels and LysoTracker staining detects intracellular acidification. The membrane rupture phenotype was detected only in esgts.wgts > Arf79FRNAi ISCs but not in wild-type ISCs and the propidium iodide signal was observed only in ISCs from flies that had RNAi-induced knockdown of expression of COPI-Arf79F components (esgts wgts > XRNAi), and not in cells from wild-type, scu-rescued Arf79FRNAi (esgts wgts > scu + Arf79FRNAi) or Hnf4-rescued Arf79FRNAi (esgts wgts > Hnf4 + Arf79FRNAi) flies. In the esgts wgts > AcslRNAi flies, all of the ISCs and RNSCs were ablated after four days at 29°C, but a fraction of the HISCs remained, and these were also propidium iodide positive, indicating that the HISCs were dying slowly. This slowness may have been due to either a lower GAL4 (wg-Gal4) activity in these cells compared to ISCs and RNSCs (esg-Gal4) or quiescence of the HISCs. Furthermore, strong propidium iodide signals were detected in transformed ISCs from esgts > NDN + Arf79FRNAi but not esgts > NDN flies, indicating that the transformed stem cells were dying through necrosis (Singh, 2016).

Similarly, DHE or LysoTracker signals were detected only in ISCs from esgts > Arf79FRNAi flies, but not from wild-type flies, indicating that the dying ISCs had accumulated ROS and were intracellularly acidified. Overexpressing catalase (a ROS-chelating enzyme) rescued the stem-cell death specifically induced by the γ-COP mutant clone or by Arf79F knockdown, and the ROS inhibitor NAC blocked the Arf1 inhibitor-induced death of RasV12-induced RNSC tumours. These data together suggest that knockdown of the COPI-Arf1 complex induced the death of stem cells or of transformed stem cells (RasV12-RNSCs, NDN-ISCs) through ROS-induced necrosis. Although ISCs, RNSCs, and HISCs exhibit different degrees of quiescence, they all rely on lipolysis for survival, suggesting that this is a general property of stem cells (Singh, 2016).

Cases were noticed where the GFP-positive material of the dying ISCs was present within neighbouring enterocytes, suggesting that these enterocytes had engulfed dying ISCs (Singh, 2016).

The JNK pathway, autophagy and engulfment genes are involved in the engulfment of dying cells. Therefore whether these genes are required for COPI-Arf79F-regulated ISC death was investigated. It was found that: (1) ISC death activated JNK signalling and autophagy in neighbouring enterocytes; (2) knockdown of these genes in enterocytes but not in ISCs rescued ISC death to different degrees; (3) the drpr-mbc-Rac1-JNK pathway in enterocytes is not only necessary but also sufficient for ISC death; and (4) inhibitors of JNK and Rac1 could block Arf1-inhibitor-induced cell death of the RasV12-induced RNSC tumours. These data together suggest that the drpr-mbc-Rac1-JNK pathway in neighbouring differentiated cells controls the engulfment of dying or transformed stem cells (Singh, 2016).

The finding that the COPI-Arf79F-lipolysis-β-oxidation pathway regulated transformed stem-cell survival in the fly led to an investigation of whether the pathway has a similar role in CSCs. Two Arf1 inhibitors (brefeldin A and golgicide A) and two FAO inhibitors (triascin C and etomoxir) were tested on human cancer cell lines, and it was found that the growth, tumoursphere formation and expression of tumour-initiating cell markers of the four cancer cell lines were significantly suppressed by these inhibitors, suggesting that these inhibitors suppress CSCs. In mouse xenografts of BSY-1 human breast cancer cells, a novel low-cytotoxicity Arf1-ArfGEF inhibitor called AMF-26 was reported to induce complete regression in vivo in five days. Together, this report and the current results suggest that inhibiting Arf1 activity or blocking the lipolysis pathway can kill CSCs and block tumour growth.

Stem cells or CSCs are usually localized to a hypoxic storage niche, surrounded by a dense extracellular matrix, which may make them less accessible to sugar and amino acid nutrition from the body's circulatory system. Most normal cells rely on sugar and amino acids for their energy supply, with lipolysis playing only a minor role in their survival. The results suggest that stem cells and CSCs are metabolically unique; they rely mainly on lipid reserves for their energy supply, and blocking COPI-Arf1-mediated lipolysis can starve them to death. It was further found that transformed stem cells were more sensitive than normal stem cells to Arf1 inhibitors. Thus, selectively blocking lipolysis may kill CSCs without severe side effects. Therefore, targeting the COPI-Arf1 complex or the lipolysis pathway may prove to be a well-tolerated, novel approach for eliminating CSCs (Singh, 2016).

PH Domain-Arf G protein interactions localize the Arf-GEF Steppke for cleavage furrow regulation in Drosophila

The recruitment of GDP/GTP exchange factors (GEFs) to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. This paper reports how the pleckstrin homology (PH) domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. The PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. The Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. Arf1-GFP, Arf6-GFP and Arl4-GFP localized to furrows. However, probably due to redundancies it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, these data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos (Lee, 2015).

The Arf GAP Asap promotes Arf1 function at the Golgi for cleavage furrow biosynthesis in Drosophila

Biosynthetic traffic from the Golgi drives plasma membrane growth. For Drosophila embryo cleavage, this growth is rapid, but regulated, for cycles of furrow ingression and regression. The highly conserved small G protein Arf1 organizes Golgi trafficking. Arf1 is activated by guanine nucleotide exchange factors, but essential roles for Arf1 GTPase activating proteins (GAPs) are less clear. This study reports that the conserved Arf GAP Asap is required for cleavage furrow ingression in the early embryo. Since Asap can affect multiple sub-cellular processes, genetic approaches were used to dissect the primary effect of Asap. The data argue against cytoskeletal or endocytic involvement, and reveal a common role for Asap and Arf1 in Golgi organization. Although Asap lacked Golgi enrichment, it was necessary and sufficient for Arf1 accumulation at the Golgi, and a conserved Arf1-Asap binding site was required for Golgi organization and output. Notably, Asap re-localized to the nuclear region at metaphase, a shift that coincided with subtle Golgi re-organization preceding cleavage furrow regression. It is concluded that Asap is essential for Arf1 to function at the Golgi for cleavage furrow biosynthesis. Asap may recycle Arf1 to the Golgi from post-Golgi membranes, providing optimal Golgi output for specific stages of the cell cycle (Rodrigues, 2016).

Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling

During illumination, the light sensitive plasma membrane (rhabdomere) of Drosophila photoreceptors undergoes turnover with consequent changes in size and composition. However the mechanism by which illumination is coupled to rhabdomere turnover remains unclear. This study found that photoreceptors contain a light-dependent phospholipase D (PLD) activity. During illumination, loss of PLD resulted in an enhanced reduction in rhabdomere size, accumulation of Rab7 positive, rhodopsin1-containing vesicles (RLVs) in the cell body and reduced rhodopsin protein. These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD. In wild type photoreceptors, during illumination, enhanced PLD activity was sufficient to clear RLVs from the cell body by a process dependent on Arf1-GTP levels and retromer complex function. Thus, during illumination, PLD activity couples endocytosis of RLVs with their recycling to the plasma membrane thus maintaining plasma membrane size and composition (Thakur, 2016).

The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo

A key step in generating a planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. This study describes the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo-distal axis, producing severe PCP phenotypes. Using novel tools, a direct and specific Arf1 involvement was detected in Fz trafficking in vivo. Moreover, a conserved Arf1 PCP function was uncovered in vertebrates. These data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo (Carvajal-Gonzalez, 2015).

This study demonstrates that the AP-1 complex and Arf1 are critical for PCP-core protein membrane localization during PCP establishment in multiple Drosophila tissues, and that it likely serves as a conserved function in zebrafish. Detailed analysis in Drosophila wings and zebrafish cells during gastrulation revealed that Arf1 function is required for polarized accumulation of core PCP components during PCP axis establishment. Defects in correct planar-polarized localization resulted in classical PCP defects in both animal models, and thus this study reveals a conserved function of the Arf1 protein network (Carvajal-Gonzalez, 2015).

Arf1 and AP-1 act as the trafficking machinery for the core PCP components in epithelial cells during PCP establishment. A few studies suggested connections between PCP and trafficking before. First Rabenosyn, an endocytic-related protein, was found polarized in a PCP-type fashion following Fmi localization. Rabenosyn mutant cells display defects in cellular packing and accumulation of PCP-core components at the PM50. Second, the GTPase Rab23, which, similar to Rabenosyn, causes packing defects and multiple cellular hairs (mch), interacts with the cytosolic PCP-core protein Pk. And third, Gish/Rab11/nuf were recently shown to affect hair formation by either increasing the numbers of trichomes per cell (mch) or shortening the hairs (stunted hair phenotype). However, none of these proteins affected PCP-core component localization, including Fmi or Fz, or hair orientation in the adult, similar to how the Arf1/AP-1 network does (Carvajal-Gonzalez, 2015).

Arf1 is known to act during trafficking from the Golgi in Drosophila and vertebrate cells. In the TGN, Arf1 aids in sorting of cargo proteins into carrier vesicles, regulating the binding of clathrin adaptor proteins such as GGAs (Golgi-localized, γ-adaptin ear-containing and ARF-binding proteins) or the clathrin adaptor complex AP-1. It was demonstrated that perturbation of Arf1 and AP-1 function reduced polarized accumulation of Fmi, Fz and Vang with minimal effects on non-planar-polarized membrane proteins (for example, DE-cadherin and FasIII). A distinct subpopulation of PCP-core component complexes with different turnover dynamics from unpolarized membrane junctional domains has recently been identified. These complexes are polarized, stable and restricted to large puncta, and also insensitive to manipulations of the endocytic/recycling machinery. Together with established Arf1/AP-1 functions at the Golgi, the current data support the notion that stable PCP complexes originate mainly from newly synthesized PM proteins. A hypothesis that is further supported by using the DmrD4-Fz-GFP fusion and manipulations to synchronize its ER release. Inhibition of Arf1 with BFA treatment prevented the arrival of Fz to the membrane in S2 cells. Importantly, Arf1 inhibition also inhibited Fz delivery to the apical junctional regions in vivo, where it must localize to participate in PCP establishment. A model is proposed whereby Arf1/AP-1 are involved in the specific transport of the PCP-core factor Fz, and most likely also Vang and Fmi, to the PM during PCP establishment, a process required for enrichment of these proteins in polarized complexes. Recently, it was shown in a mammalian non-polarized culture system that Vangl2 trafficking out of the Golgi depends on Arfaptin and the AP-1 complex, which is consistent with the current data and supports the evolutionary conserved task for these proteins in promoting PCP-core component localization (Carvajal-Gonzalez, 2015).

Arf1 can also act through the COPI complex, and, for example, in flies, at the cis-Golgi, Arf1 together with its guanine nucleotide exchange factor/GEF Gartenzwerg regulate COPI retrograde transport. Thus attempts were made to test this possibility. However, the dsRNA-based KD in vivo of all COPI complex components is cell lethal and thus not informative in this context. Similarly, in cell culture COPI KD, depending on the level, was either too toxic or failed to show an effect on Fz trafficking. As such, although it cannot be completely excluded that COPI is also involved in the process, the comparable results of Arf1 and AP-1 indicate that Arf1 acts at least in part on AP-1 in PCP trafficking (Carvajal-Gonzalez, 2015).

Several studies have identified genes that affect localized actin hair formation in Drosophila wing cells. Interestingly, apart from the PCP-core components, these usually fall into two cellular machinery categories: actin polymerization-related proteins and trafficking-related proteins. Importantly, both sets of factors have been linked to each other in diverse cellular environments, including carrier formation at the Golgi, yeast budding, formation of phagocytic cups or lamellipodia formation in migrating cells. Silencing of Arf1, Rab11, PI4KIIIβ and AP-1 leads to defects in actin-based hair formation in wing cells, notably leading to the formation of mch. Using Drosophila S2 cells, Arf1 is found not only in the Golgi but also at actin-rich cell edges, and that it is essential for lamellipodium biogenesis. In vertebrates, Arf1 is also required for actin polymerization, for example, in neuronal tissues during plasticity Arf1 regulates Arp2/3 through PICK1. Based on these data, a direct/parallel involvement of the Arf1 protein network is likely during hair formation, and indeed an enrichment of these proteins was observed in the growing actin hairs. Thus, in summary, the data on Arf1/AP-1 and its network suggest that it functions repeatedly in different PCP establishment processes, ranging from the initial PCP-core component localization to later restricting the foci of actin polymerization (Carvajal-Gonzalez, 2015).

PH Domain-Arf G protein interactions localize the Arf-GEF Steppke for cleavage furrow regulation in Drosophila

The recruitment of GDP/GTP exchange factors (GEFs) to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. This paper reports how the pleckstrin homology (PH) domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. The PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. The Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. Arf1-GFP, Arf6-GFP and Arl4-GFP localized to furrows. However, probably due to redundancies it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, these data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos (Lee, 2015).

ARF1-GTP regulates Asrij to provide endocytic control of Drosophila blood cell homeostasis

Drosophila melanogaster larval hematopoiesis is a well-established model to study mechanisms that regulate hematopoietic niche maintenance and control of blood cell precursor (prohemocyte) differentiation. Molecules that perturb niche function affect the balance between prohemocytes and differentiated hemocytes. The conserved hemocyte-specific endosomal protein Asrij is essential for niche function and prohemocyte maintenance. Elucidating how subcellular trafficking molecules can regulate signaling presents an important challenge. This study shows that Asrij function is mediated by the Ras family GTPase Arf79F, the Drosophila homolog of ADP ribosylation factor 1 (ARF1), essential for clathrin coat assembly, Golgi architecture, and vesicular trafficking. ARF1 is expressed in the larval lymph gland and in circulating hemocytes and interacts with Asrij. ARF1-depleted lymph glands show loss of niche cells and prohemocyte maintenance with increased differentiation. Inhibiting ARF1 activation by knocking down its guanine nucleotide exchange factor (Gartenzwerg) or overexpressing its GTPAse-activating protein showed that ARF1-GTP is essential for regulating niche size and maintaining stemness. Activated ARF1 regulates Asrij levels in blood cells thereby mediating Asrij function. Asrij controls crystal cell differentiation by affecting Notch trafficking. ARF1 perturbation also leads to aberrant Notch trafficking and the Notch intracellular domain is stalled in sorting endosomes. Thus, ARF1 can regulate Drosophila blood cell homeostasis by regulating Asrij endocytic function. ARF1 also regulates signals arising from the niche and differentiated cells by integrating the insulin-mediated and PDGF-VEGF receptor signaling pathways. It is proposed that the conserved ARF1-Asrij endocytic axis modulates signals that govern hematopoietic development. Thus, Asrij affords tissue-specific control of global mechanisms involved in molecular traffic (Khadilkar, 2014).

Functional characterization of protein-sorting machineries at the trans-Golgi network in Drosophila melanogaster

Targeting of proteins to their final destination is a prerequisite for living cells to maintain their homeostasis. Clathrin functions as a coat that forms transport carriers called clathrin-coated vesicles (CCVs) at the plasma membrane and post-Golgi compartments. This study established an experimental system using Schneider S2 cells derived from Drosophila as a model system to study the physiological roles of clathrin adaptors, and to dissect the processes of CCV formation. It was found that a clathrin adaptor Drosophila GGA (Golgi-localizedγ-adaptin ear containing, ARF binding protein or dGGA), a homolog of mammalian GGA proteins, localizes to the trans-Golgi network (TGN) and is capable of recruiting clathrin from the cytosol onto TGN membranes, through direct protein interaction. dGGA itself is recruited from the cytosol to the TGN in an ARF1 small GTPase (dARF79F)-dependent manner. dGGA recognizes the cytoplasmic acidic-cluster-dileucine (ACLL) sorting signal of Lerp (lysosomal enzyme receptor protein), a homolog of mammalian mannose 6-phosphate receptors. Moreover, both dGGA and another type of TGN-localized clathrin adaptor, AP-1 (adaptor protein-1 complex), are shown to be involved in the trafficking of Lerp from the TGN to endosomes and/or lysosomes. Taken together, these findings indicate that the protein-sorting machinery in fly cells is well conserved relative to that in mammals, enabling the use of fly cells to dissect CCV biogenesis and clathrin-dependent protein trafficking at the TGN of higher eukaryotes (Kametaka, 2010).

The trans-Golgi localization of dGGA suggested that this protein plays a role in the formation of clathrin-coated transport intermediates that transport integral membrane cargo proteins from the TGN to endosomes. To assess the involvement of clathrin in this process, an antibody to dCHC was raised. To examine the contribution of dGGA to the recruitment of dCHC onto the trans-Golgi, in vitro pull-down assays were performed to see whether dGGA interacts with dCHC. A series of dGGA recombinant proteins was prepared as GST-fusion proteins. The full-length dGGA, Hinge and GAE region, and Hinge region efficiently pulled-down dCHC from a cytosolic extract from S2 cells, whereas VHS-GAT pulled-down dCHC at levels close to the control. Thus, the binding site maps to the hinge region (amino acid position 292-590 in dGGA). Mutations were introduced in two putative clathrin binding sequences, D331LL to DAA and/or E327LL to EAA , and in a putative ACLL motif, D496VPLL to DVPAA. That mutations in the two putative clathrin-binding boxes were observed to strong affect the interaction with dCHC, whereas the putative ACLL motif was dispensable for this interaction. These results indicate that the D331LL and E327LL sequences in the hinge region of dGGA have crucial roles in interaction with dCHC. Golgi-association of clathrin heavy chain (Kametaka, 2010).

The intracellular localization of dCHC was also examined in S2 cells. The antibody preferentially labeled large perinuclear puncta and smaller dots beneath the cell surface. The dCHC-positive, perinuclear large structures were juxtaposed to p120-positive structures and colocalized with the signal for HA-dGGA, indicating that they correspond to the trans-Golgi compartments. By contrast, the small peripheral dots colocalized with AP50, a mu2 subunit of AP-2, indicating that they represent the plasma-membrane-associated clathrin-coated pits. These results indicated that a significant amount of cellular clathrin localizes to the trans-Golgi, together with dGGA (Kametaka, 2010).

In mammals, expression of the VHS-GAT domains of mammalian GGAs has been previously shown to exert a dominant-effect on clathrin localization and MPR sorting at the TGN (Puertollano, 2001). Thus, to further assess the role of dGGA vis-à-vis clathrin in vivo, the effect of overexpression of dGGA-VHS-GAT on clathrin localization was examined in S2 cells. Overexpression of the truncated dGGA reduced dCHC association with the Golgi compartment. Together with the above GST-pull down experiments, these results strongly suggest that dGGA has a role in clathrin recruitment at the TGN (Kametaka, 2010).

Genome sequencing projects have revealed the conservation of clathrin adaptor genes throughout the eukaryotic kingdom, including well-established model organisms such as Drosophila and C. elegans. Drosophila is a particularly appealing organism for analysis of GGA function because of the existence of a single dGGA, the ease of RNAi approaches and the conservation of Golgi function. Although the morphology of the Drosophila Golgi is different from that of mammals, the basic features of the organelle (e.g., polarity, number of cisternae per stack and function in the secretory pathway) are quite similar (Kondylis, 2003; Kondylis, 2005). The molecular machineries responsible for protein sorting from the Golgi complex to the endosomal system, however, have not been characterized in Drosophila. Dennes (2005) showed that Lerp, a Drosophila homolog of the mammalian CI-MPR, could rescue the defects in the lysosomal cathepsin sorting in MPR-deficient fibroblasts. However, the properties and functions of Lerp and its putative adaptor dGGA in Drosophila cells have remained uncharacterized. In this study Drosophila S2 cells were used to assess the role of dGGA and its regulators in the trafficking of Lerp (Kametaka, 2010).

The results indicate that dGGA meets the requirements to be a clathrin adaptor for membrane cargo molecules such as Lerp. Its ability to interact with GTP-ARF1, the ACLL motif of Lerp and the dCHC all imply that dGGA functions in CCV formation for trafficking of Lerp between the TGN and endosomal compartments. In addition to the molecular interaction between dGGA and Lerp in vitro, it was also observed that mCherry-tagged Lerp and EGFP-dGGA depart together from the Golgi in vesicular structures in living cells. Moreover, a dominant-negative form of dGGA caused redistribution of clathrin from the Golgi complex and also inhibited recruitment of clathrin in vitro. Furthermore, dGGA knockdown caused a decrease in the level of Lerp processing at endosomal and/or lysosomal compartments in vivo. Thus, dGGA is likely to function at the exit step of Lerp from the TGN. These assays, however, do not allow precise determination of dGGA function at a molecular level. More extensive biochemical and in vivo functional analyses will be required (Kametaka, 2010).

Despite the overlapping localization and common biochemical features of GGAs and AP-1, they are thought to function differently in mammalian cells. It has been shown that AP-1 and cargo molecules, but not GGAs, are concentrated in purified CCV fractions. Moreover, knockdown of GGAs or AP-1 in mammalian cells causes only slight missorting of pro-cathepsin D to the extracellular space without detectable perturbation in the localization of its sorting receptors, MPRs. On the basis of these findings, it has been presumed that GGAs and AP-1 function cooperatively, but not at exactly the same step in cargo trafficking. Indeed, Kornfeld's group proposed a 'hand-off' model in which GGAs first prime the cargo molecule and AP-1 is subsequently recruited through direct interaction with GGAs. GGAs are thus replaced by AP-1 to execute clathrin-coated carrier formation (Ghosh, 2003). In this model, GGA is displaced by AP-1 through a phosphorylation state-dependent structural change. Internal ACLL motifs in the hinge region of human GGA1 and GGA3 are thought to mediate this structural change. Through the characterization of dGGA and Drosophila AP-1 (dAP-1) in the current study, it was found that many structural and functional features of these adaptors were strikingly conserved from mammals to fly. Like mammalian AP-1, dAP-1 was shown to localize to the TGN in S2 cells and the Golgi localization was sensitive to BFA. Moreover, knockdown of AP47, which encodes a mu1 subunit of dAP-1, accentuated the defect in processing of Lerp seen in dGGA-depleted cells, even though knockdown of AP47 itself had little effect. These observations support the notion that dGGA and dAP-1 function closely together in Lerp trafficking in S2 cells (Kametaka, 2010).

It was also noticed that dGGA has one putative internal ACLL sequence in the hinge region. Interestingly, this ACLL motif is preceded by a Ser493 residue that could be a target of casein kinase II (CKII). CKII is known to be involved in the phospho-regulation of CI-MPR trafficking in mammals through phosphorylation of the cytoplasmic tail of CI-MPR, human GGA1 and GGA3, and PACS-1. Although this idea needs further assessment in the future, the dGGA signal was often observed as a doublet on immunoblotting, so dGGA might be under phospho-regulation control like the mammalian GGAs. Taken together, these findings strengthen the idea that Drosophila possesses similar mechanisms of CCV formation at the TGN and of regulation of clathrin adaptors (Kametaka, 2010).

This is the first report that the putative lysosomal enzyme receptor Lerp and its sorting proteins dGGA and dCHC localize to trans-Golgi compartments in Drosophila cells, where Lerp is believed to be sorted and packaged into CCV destined for endosomal compartments. It was also found that the entire molecular system responsible for post-Golgi protein trafficking in Drosophila is highly conserved relative to that in mammals. This conservation should enable genome-wide screens for novel factors involved in the complex processes of CCV formation and regulation of protein sorting at the TGN. Recently, it has been shown with isolated CCVs that double knockdown of dGGA and dAP-1 causes significant reduction of Lerp incorporation into the CCVs in Dmel2, one of another Drosophila cell lines. Although the molecular relationship of these clathrin adaptors needs to be assessed more carefully, these results support the current results showing the physiological consequence of dGGA in cargo sorting at the trans-Golgi. Thus, this approach using the insect systems will lead to a better understanding of how those clathrin adaptors are important in the development of multicellular organisms and in the molecular basis for lysosomal diseases in higher organisms (Kametaka, 2010).

The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila

Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. This study investigated stem-cell death in the digestive system of adult Drosophila melanogaster. It was found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively kills normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spares differentiated cells. The dying stem cells are engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduce CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers (Singh, 2016).

To investigate the molecular mechanism behind the resistance of CSCs to therapeutics, the death of stem cells with different degrees of quiescence was studied in the adult Drosophila digestive system, including intestinal stem cells (ISCs). Expression of the proapoptotic genes rpr and p53 effectively ablated differentiated cells but had little effect on stem cells (Singh, 2016).

In mammals, treatment-resistant leukaemic stem cells (LSCs) can be eliminated by a two-step protocol involving initial activation by interferon-α (IFNα) or colony-stimulating factor (G-CSF), followed by targeted chemotherapy. In Drosophila, activation of the hopscotch (also known as JAK)-Stat92E signalling pathway induces hyperplastic stem cells, which are overproliferating, but retain their apico-basal polarity and differentiation ability. A slightly different two-step protocol was conducted in Drosophila stem cells by overexpressing the JAK-Stat92E pathway ligand unpaired (upd) and rpr together. The induction of upd + rpr using the temperature-sensitive (ts) mutant esg-Gal4 (esgts > upd + rpr effectively ablated all of the ISCs and RNSCs through apoptosis within four days. Consistent with this result, expressing a gain-of-function Raf mutant (Rafgof) also accelerated apoptotic cell death of hyperplastic ISCs (Singh, 2016).

Expressing a constitutively active form of Ras oncogene at 85D (also known as RasV12) in RNSCs and the knockdown of Notch activity in ISCs can transform these cell types into CSC-like neoplastic stem cells, which were not only overproliferating, but also lost their apico-basal polarity and differentiation abilit. It ws found that expressing rpr in RasV12-transformed RNSCs or in ISCs expressing a dominant-negative form of Notch (NDN) caused the ablation of only a proportion of the transformed RNSCs and few transformed ISCs and it did not affect differentiated cells; substantial populations of the neoplastic stem cells remained even seven days after rpr induction (Singh, 2016).

These results suggest that the activation of proliferation can accelerate the apoptotic cell death of hyperplastic stem cells, but that a proportion of actively proliferating neoplastic RNSCs and ISCs are resistant to apoptotic cell death. Neoplastic tumours in Drosophila are more similar to high-grade malignant human tumours than are the hyperplastic Drosophila tumours (Singh, 2016).

Vesicle-mediated COPI and COPII are essential components of the trafficking machinery for vesicle transportation between the endoplasmic reticulum and the Golgi. In addition, the COPI complex regulates the transport of lipolysis enzymes to the surface of lipid droplets for lipid droplet usage. In a previous screen, it was found that knockdown of COPI components (including Arf79F, the Drosophila homologue of ADP-ribosylation factor 1 (Arf1)) rather than COPII components resulted in stem-cell death, suggesting that lipid-droplet usage (lipolysis) rather than the general trafficking machinery between the endoplasmic reticulum and Golgi is important for stem-cell survival (Singh, 2016)

To further investigate the roles of these genes in stem cells, a recombined double Gal4 line of esg-Gal4 and wg-Gal4 was used to express genes in ISCs, RNSCs, and HISCs (esgts wgts > X). Knockdown of these genes using RNA interference (RNAi) in stem cells ablated most of the stem cells in 1 week. However, expressing Arf79FRNAi in enterocytes or in differentiated stellate cells in Malpighian tubules did not cause similar marked ablation. These results suggest that Arf79F knockdown selectively kills stem cells and not differentiated cells (Singh, 2016).

It was also found that expressing Arf79FRNAi in RasV12-transformed RNSCs ablated almost all of the transformed stem cells. Similarly, expressing Arf79FRNAi in NDN-transformed ISCs ablated all of the cells within one week, but restored differentiated cells to close to their normal levels within one week (Singh, 2016).

δ-COP- and γ-COP-mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique, and it was found that the COPI complex cell-autonomously regulated stem cell survival. In summary, knockdown of the COPI-Arf79F complex effectively ablated normal and transformed stem cells but not differentiated enterocytes or stellate cells (Singh, 2016)

In the RNAi screen acyl-CoA synthetase long-chain (ACSL), an enzyme in the Drosophila lipolysis-β-oxidation pathway, and bubblegum (bgm), a very long-chain fatty acid-CoA ligase, were also identified. RNAi-mediated knockdown of Acsl and bgm effectively killed ISCs and RNSCs, but killed HISCs less effectively. Expressing AcslRNAi in RasV12-transformed RNSCs also ablated almost all of the transformed RNSCs in one week (Singh, 2016).

Brummer (bmm) is a triglyceride lipase, the Drosophila homologue of mammalian ATGL, the first enzyme in the lipolysis pathway. Scully (scu) is the Drosophila orthologue of hydroxy-acyl-CoA dehydrogenase, an enzyme in the β-oxidation pathway. Hepatocyte nuclear factor 4 (Hnf4) regulates the expression of several genes involved in lipid mobilization and β-oxidation. To determine whether the lipolysis-β-oxidation pathway is required for COPI-Arf79F-mediated stem cell survival, upstream activating sequence (UAS)-regulated constructs (UAS-bmm, UAS-Hnf4, and UAS-scu) were also expressed in stem cells that were depleted of Arf79F, β-COP, or ζ-COP. Overexpressing either scu or Hnf4 significantly attenuated the stem cell death caused by knockdown of the COPI-Arf79F complex. Expressing UAS-Hnf4 MARCM clones also rescued the stem cell death phenotype induced by γ-COP knockdown. However, bmm overexpression did not rescue the stem-cell death induced by Arf79F knockdown. Since there are several other triglyceride lipases in Drosophila in addition to bmm, another lipase may redundantly regulate the lipolysis pathway (Singh, 2016).

To further investigate the function of lipolysis in stem cells, the expression of a lipolysis reporter (GAL4-dHFN4; UAS-nlacZ which consisted of hsp70-GAL4-dHNF4 combined with a UAS-nlacZ reporter gene was investigated. The flies were either cultured continuously at 29°C or heat-shocked for 30 min at 37°C, 12 h before dissection. Without heat shock, the reporter was expressed only in ISCs and RNSCs of mature adult flies, but not in enteroendocrine cells, enterocytes, quiescent HISCs or quiescent ISCs of freshly emerged young adult flies (less than 3 days old. Expressing δ-COPRNAi almost completely eliminated the reporter expression, suggesting that the reporter was specifically regulated by the COPI complex. After heat shock or when a constitutively active form of JAK (hopTum-l) was expressed, the reporter was strongly expressed in ISCs, RNSCs and HISCs, but not in enteroendocrine cells or enterocytes. These data suggest that COPI-complex-regulated lipolysis was active in stem cells, but not in differentiated cells, and that the absence of the reporter expression in quiescent HISCs at 29°C was probably owing to weak hsp70 promoter activity rather than to low lipolysis in these cells (Singh, 2016).

Lipid storage was futher investigated, and it was found that the size and number of lipid droplets were markedly increased in stem cells after knockdown of Arf79F (Singh, 2016).

Arf1 inhibitors (brefeldin A, golgicide A, secin H3, LM11 and LG8) and fatty-acid-oxidation (FAO) inhibitors (triacsin C, mildronate, etomoxir and enoximone) were used, and it was found that these inhibitors markedly reduced stem-cell tumours in Drosophila through the lipolysis pathway but had a negligible effect on normal stem cells (Singh, 2016).

These data together suggest that the COPI-Arf1 complex regulates stem-cell survival through the lipolysis-β-oxidation pathway, and that knockdown of these genes blocks lipolysis but promotes lipid storage. Further, the transformed stem cells are more sensitive to Arf1 inhibitors and may be selectively eliminated by controlling the concentration of Arf1 inhibitors (Singh, 2016).

These data suggest that neither caspase-mediated apoptosis nor autophagy-regulated cell death regulates the stem-cell death induced by the knockdown of components of the COPI-Arf79F complex. Therefore whether necrosis regulates the stem-cell death induced by knockdown of the COPI-Arf79F complex was investigated. Necrosis is characterized by early plasma membrane rupture, reactive oxygen species (ROS) accumulation and intracellular acidification. Propidium iodide detects necrotic cells with compromised membrane integrity, the oxidant-sensitive dye dihydroethidium (DHE) indicates cellular ROS levels and LysoTracker staining detects intracellular acidification. The membrane rupture phenotype was detected only in esg and the propidium iodide signal was observed only in ISCs from flies that had RNAi-induced knockdown of expression of COPI-Arf79F components, and not in cells from wild-type flies. In the esgts wgts > AcslRNAi flies, all of the ISCs and RNSCs were ablated after four days at 29°C, but a fraction of the HISCs remained, and these were also propidium iodide positive, indicating that the HISCs were dying slowly. This slowness may have been due to either a lower GAL4 (wg-Gal4) activity in these cells compared to ISCs and RNSCs (esg-Gal4) or quiescence of the HISCs. Furthermore, strong propidium iodide signals were detected in transformed ISCs from esgts > NDN + Arf79FRNAi but not esgts flies, indicating that the transformed stem cells were dying through necrosis (Singh, 2016).

Similarly, DHE signals were detected only in ISCs from esgts > Arf79FRNAi flies, indicating that the dying ISCs had accumulated ROS and were intracellularly acidified. Overexpressing catalase (a ROS-chelating enzyme) rescued the stem-cell death specifically induced by the γ-COP mutant clone, and the ROS inhibitor NAC blocked the Arf1 inhibitor-induced death of RasV12-induced RNSC tumours. These data together suggest that knockdown of the COPI-Arf1 complex induced the death of stem cells or of transformed stem cells (RasV12-RNSCs, NDN-ISCs) through ROS-induced necrosis. Although ISCs, RNSCs, and HISCs exhibit different degrees of quiescence, they all rely on lipolysis for survival, suggesting that this is a general property of stem cells (Singh, 2016).

Cases were noticed where the GFP-positive material of the dying ISCs was present within neighbouring enterocytes, suggesting that these enterocytes had engulfed dying ISCs (Singh, 2016).

The JNK pathway, autophagy and engulfment genes are involved in the engulfment of dying cells. Therefore, whether these genes are required for COPI-Arf79F-regulated ISC death was investigated. The following was found: (1) ISC death activated JNK signalling and autophagy in neighbouring enterocytes; (2) knockdown of these genes in enterocytes but not in ISCs rescued ISC death to different degrees; (3) the drpr-mbc-Rac1-JNK pathway in enterocytes is not only necessary but also sufficient for ISC death; and (4) inhibitors of JNK and Rac1 could block Arf1-inhibitor-induced cell death of the RasV12-induced RNSC tumours. These data together suggest that the drpr-mbc-Rac1-JNK pathway in neighbouring differentiated cells controls the engulfment of dying or transformed stem cells (Singh, 2016).

The finding that the COPI-Arf79F-lipolysis-β-oxidation pathway regulated transformed stem-cell survival in the fly led to an investigation of whether the pathway has a similar role in CSCs. WTwo Arf1 inhibitors (brefeldin A and golgicide A) and two FAO inhibitors (triascin C and etomoxir) were tested on human cancer cell lines, and it was found that the growth, tumoursphere formation and expression of tumour-initiating cell markers of the four cancer cell lines were significantly suppressed by these inhibitors, suggesting that these inhibitors suppress CSCs. In mouse xenografts of BSY-1 human breast cancer cells, a novel low-cytotoxicity Arf1-ArfGEF inhibitor called AMF-26 was reported to induce complete regression in vivo in five days. Together, this report and the current results suggest that inhibiting Arf1 activity or blocking the lipolysis pathway can kill CSCs and block tumour growth (Singh, 2016).

Stem cells or CSCs are usually localized to a hypoxic storage niche, surrounded by a dense extracellular matrix, which may make them less accessible to sugar and amino acid nutrition from the body's circulatory system. Most normal cells rely on sugar and amino acids for their energy supply, with lipolysis playing only a minor role in their survival. The current results suggest that stem cells and CSCs are metabolically unique; they rely mainly on lipid reserves for their energy supply, and blocking COPI-Arf1-mediated lipolysis can starve them to death. It was further found that transformed stem cells were more sensitive than normal stem cells to Arf1 inhibitors. Thus, selectively blocking lipolysis may kill CSCs without severe side effects. Therefore, targeting the COPI-Arf1 complex or the lipolysis pathway may prove to be a well-tolerated, novel approach for eliminating CSCs (Singh, 2016).

The Drosophila Arf1 homologue Arf79F is essential for lamellipodium formation

The WAVE regulatory complex (WRC) drives the polymerisation of actin filaments located beneath the plasma membrane to generate lamellipodia that are pivotal to cell architecture and movement. By reconstituting WRC-dependent actin assembly at the membrane, it was recently discovered that several classes of Arf family GTPases directly recruit and activate WRC in cell extracts, and that Arf cooperates with Rac1 to trigger actin polymerisation. This study demonstrated that the Class 1 Arf1 homologue Arf79F colocalises with the WRC at dynamic lamellipodia. Arf79F is required for lamellipodium formation in Drosophila S2R+ cells, which only express one Arf isoform for each class. Impeding Arf function either by dominant-negative Arf expression or by Arf double-stranded RNA interference (dsRNAi)-mediated knockdown uncovered that Arf-dependent lamellipodium formation was specific to Arf79F, establishing that Class 1 Arfs, but not Class 2 or Class 3 Arfs, are crucial for lamellipodia. Lamellipodium formation in Arf79F-silenced cells was restored by expressing mammalian Arf1, but not by constitutively active Rac1, showing that Arf79F does not act via Rac1. Abolition of lamellipodium formation in Arf79F-silenced cells was not due to Golgi disruption. Blocking Arf79F activation with guanine nucleotide exchange factor inhibitors impaired WRC localisation to the plasma membrane and concomitant generation of lamellipodia. These data indicate that the Class I Arf GTPase is a central component in WRC-driven lamellipodium formation (Humphreys, 2012).

Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels

Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. This study shows that before a critical time period during rhabdomere biogenesis in Drosophila photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. It is proposed that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis (Raghu, 2009).

During development, eukaryotic cells undergo morphogenetic changes to suit ongoing physiological needs. Effecting cell shape changes involves complex cell biological processes, including changes in both the cell membrane and the cytoskeletal. An essential element of membrane biogenesis is the need to achieve regulated vesicular transport such that membranes can be delivered to the desired domain of the cell. This process is thought to involve a complex interplay of the physical properties of the lipid constituents in membranes as well as the activities of proteins that can affect membrane curvature. Conceptually, the lipid constituents of the cell membranes could be those with essentially structural roles (such as phosphatidylcholine [PC], phosphatidylethanolamine, phosphatidylserine (PS), and cholesterol) and signaling lipids whose levels change in a regulated manner. These signaling lipids include DAG, its phosphorylated derivative phosphatidic acid (PA), and several phosphorylated species of phosphatidylinositol (PI) (Raghu, 2009).

In the simple eukaryote Saccharomyces cerevisiae that recapitulates most basal transport pathways conserved in higher eukaryotes, genetic analysis has implicated several lipids in regulating membrane traffic. Evidence showing that DAG and PA can affect membrane transport comes from yeast through analysis of SEC14, a gene that encodes a PI/PC transfer protein essential for viability and transport from the Golgi. The sec14 phenotype can be suppressed/bypassed by mutants in several genes that control biosynthesis of PI and PC. However, the ability of such mutants to bypass sec14 has an obligate requirement for SPO14 that encodes phospholipase D (PLD), an enzyme that generates PA from PC. Although Spo14p is not required for vegetative growth, it is required to form the prospore membrane and for PA synthesis during sporulation; loss of Spo14p leads to accumulation of undocked prospore membrane precursors vesicles on the spindle pole body. Thus, in yeast, PA generated by Spo14p activity plays a key role in this membrane trafficking event. Although the analysis of spo14 has implicated PA and its downstream lipid metabolites in membrane transport, to date there is little direct evidence to suggest that PA can function as a regulator of membrane traffic in metazoans. The idea that PA can function in a signaling capacity during membrane transport has been fueled by the observations that (1) in vitro ADP ribosylation factor (Arf) proteins, key mediators of membrane transport, can regulate the activity of PLD, (2) overexpression of PLD in several different cell types affects processes likely to require exocytosis, and (3) overexpression of mammalian PLD1 is reported to promote generation of β-amyloid precursor protein-containing vesicles from the TGN. However, the role of PA in regulating secretion in these settings remains unclear, and currently, there is little evidence linking demonstrable changes in PA levels with the molecular machinery that regulates membrane traffic in vivo (Raghu, 2009).

This study used Drosophila melanogaster photoreceptors as a model system to test the effect of altered PA levels on membrane traffic. It was shown that elevated levels of PA, achieved by manipulation of three genes (CDP diglyceride synthetase, Phospholipase D and retinal degeneration A), disrupt membrane transport to the apical domain of photoreceptors with defects in the endomembrane system (Raghu, 2009).

Work in mammalian cell culture models has suggested that the activity of a PI4K enzyme generating a Golgi localized pool of PI(4)P is important for regulating TGN exit. To test whether the activity of a PI(4)P-generating enzyme might be critical for rhabdomere biogenesis, the effect of down-regulating PI4K activity in developing photoreceptors was tested.The effect of down-regulating two genes that could encode PI4K activity: CG2929 (PI4KIIβ) and CG10260 (PI4KIIIα). This analysis revealed that down-regulation of CG2929 using RNAi phenocopied key aspects of the phenotype of Pld overexpression: (1) down-regulation in the levels of Rh1 protein, (2) formation of small and deformed rhabdomere, and (3) accumulation of abnormal endomembranes within the cell body. These findings suggest that the activity of PI4K is important for membrane transport (Raghu, 2009).

This study elevated PA levels using either cds1 and Pld or rdgA overexpression in each of which the only common and immediate biochemical outcome is the accumulation of PA. Using EM to directly visualize photoreceptor membranes, it was demonstrate that all three genetic manipulations cause defects in endomembrane organization characterized by a reduction in the size of the apical rhabdomere membrane and/or the accumulation of expanded membranous structures in the cell body. These observations, which are consistent with a defect in membrane transport to the apical domain, are highly reminiscent of defects seen in photoreceptors from Drosophila p47 and Rab11 mutants. Importantly, it was also demonstrated that in all three genotypes used to modulate PA levels, the abundance of a single molecular species of PA (16:0/18:2) was elevated without changes in the mass of structural lipids such as PC or of signaling lipids such as PI and DAG. Because this species of PA accounts for <10% of the total PA in photoreceptors, it is hypothesized that it represents a quantitatively minor phospholipid that functions in a signaling capacity to modulate membrane transport. The importance of PA for the described phenotypes is supported by the observation that overexpression of a type II PA phosphatase is able to partially revert the defects in rhabdomere biogenesis and endomembrane structure. Together, these findings provide compelling evidence that PA can affect the transport and organization of endomembranes in metazoan cells (Raghu, 2009).

Interestingly, although cds1, Pld, and rdgA overexpression all caused endomembrane defects in photoreceptors, the ultrastructural features of the abnormal transport intermediates were variable. All three genotypes showed variable degrees of defect in rhabdomere biogenesis. In addition, in the case of cds1, the accumulated endomembranes in the cell body resembled ER-like structures; with Pld overexpression, there were concentric and sheetlike tubular membranes, whereas with rdgA overexpression, in addition to tubular membranes, there were several vesicular intermediates that accumulated. It is likely that these differences reflect the distinct subcellular locations at which PA accumulates in each genotype. In cds1, PA probably accumulates in the ER site at which CDP-DAG synthase activity is normally present; PLD localization is limited to a compartment at the base of the rhabdomeres, and when overexpressed, DGK is distributed in punctate fashion throughout the ER. The generation of a suitable probe to visualize PA levels in a spatial dimension will be required to address this issue (Raghu, 2009).

During development, the precursor cells of the Drosophila eye undergo a substantial increase in size with the concomitant requirement for generating new plasma membrane. During the last 30% of pupal development, photoreceptors show an approximately fourfold increase in plasma membrane surface area, a process that requires a massive surge in polarized membrane transport capacity starting at ~70% pupal development. This study has defined a critical time window ~70% pupal development before which elevation of PA levels by overexpressing Pld results in the endomembrane defects. As this window precedes the onset of rapid membrane transport accompanying rhabdomere biogenesis, it is postulated that PA regulates the activity of a component of the molecular machinery that mediates polarized membrane transport during this period. Conceptually, in this respect the current findings are reminiscent of observations in the yeast Spo14 mutant, in which membrane transport defects are evident only during the generation of the prospore membrane. These findings are the first report of regulation of polarized membrane transport by PA in metazoans (Raghu, 2009).

During this study, it was observed that the effects of elevated PA (through both cds1 and Pld overexpression) were sensitive to the activation state of Arf1. In the cds1 mutant, in which PA is likely to be elevated in the ER, overexpression of the Arf1-GEF garz resulted in significantly less developed apical rhabdomere membrane but was not associated with enhanced accumulation of membranes in the cell body, which is consistent with the known effects of expressing constitutively active Arf1 in cells. In contrast, overexpression of dArf1-GAP resulted in an enhancement of defective rhabdomere biogenesis as well as a massive accumulation of ER membrane-like intermediates in the cell body. This observation suggests that the PA accumulating at the ER in cds1 influences the Arf1 cycle in this setting, resulting in the transport defects described. Previous biochemical analysis has shown that the activity of Arf1-GAP proteins can be regulated by at least three different lipids relevant to this study, namely PC, DAG, and PA. In the lipidomic analysis of cds1 retinae, it was found that the levels of 34:2 DAG and 34:2 PC were no different from wild type, whereas levels of 34:2 PA were elevated. On the basis of these findings, it is likely that the 34:2 PA that accumulates in cds1 photoreceptors causes the transport defects that were described by down-regulating the activity of Arf1 via dArf1-GAP (Raghu, 2009).

The development and maintenance of apical membranes in polarized cells requires both sorting at the TGN with exocytic transport as well as endocytosis. Thus, the phenotypes resulting from PLD overexpression could be a result of (1) altered membrane transport along one of the steps in the secretory pathway from the ER to the developing rhabdomere or (2) the consequence of enhanced endocytosis from the rhabdomere into the cell body (Raghu, 2009).

Experimental evidence presented in this study shows that in photoreceptors overexpressing Pld, the defect in rhabdomere biogenesis was dependent on the levels of active Arf1. In contrast, it was found that (1) altering the activity of Arf6, (2) down-regulation of α-adaptin, and (3) a reduction in the function of dynamin (shi) did not suppress the effects of overexpressing Pld. Collectively, these three observations strongly suggest that excessive clathrin-mediated endocytosis of rhabdomeral plasma membrane does not underlie the endomembrane defects resulting from Pld overexpression. A recent study has suggested a role for Arf1 in regulating a dynamin-independent endocytic pathway in Drosophila cells. The role of this pathway in the effects of Pld overexpression remains unknown (Raghu, 2009).

Arf1 also exerts several effects on distinct steps of the exocytic pathway, including bidirectional transport between the ER and Golgi between Golgi cisternae and the regulation of exit from the late Golgi. In photoreceptors overexpressing Pld, the current analysis suggests that ER to trans-Golgi transport was normal, implying that the observed phenotypes are likely to involve a transport step between the TGN and plasma membrane, although observed phenotypes do not phenocopy exocyst loss of function. In Drosophila photoreceptors, PLD localizes to a restricted subcompartment at the base of the rhabdomeres. Although the molecular identity of this compartment has not been established, its subcellular localization is consistent with the ability of PA produced by PLD to regulate transport between the rhabdomeres and cell body. In TEMs of photoreceptors overexpressing Pld, the endomembranes that were observed in the cell body showed a tubulovesicular morphology extending throughout the cytoplasm. These membranes resemble large pleiomorphic carriers, transport intermediates that derive from the TGN destined for acceptor compartments like the plasma membrane. Furthermore, vesicles containing proteins destined for and normally restricted to the apical rhabdomere membrane (such as Rh1) are found in the cell body of photoreceptors overexpressing Pld. These observations are particularly interesting in the light of previous studies suggesting that PA generated by PLD can regulate the release of vesicles from the Golgi in an Arf1-dependent manner. However, in the absence of a clear identification of the accumulated membranes, the precise definition of the affected transport intermediates that were observed remains elusive (Raghu, 2009).

Arf1 can influence several events at the TGN, including the recruitment and activation of phospholipid-metabolizing enzymes. These include the recruitment and activation of PI4KIIIβ, generating PI(4)P, as well a direct role in activating the type I PIPkin on Golgi membranes in vitro. During this study, it was found that (1) down-regulating the levels of a PI4K expressed during photoreceptor development phenocopies key aspects of that seen with Pld overexpression, and (2) a strong hypomorph of the type I PIPkin (sktl) was able to substantially suppress the effects of Pld overexpression on rhabdomere biogenesis. These observations reflect the importance of tightly regulating type I PIPkin activity by PA for normal transport to the apical domain in polarized cells. They suggest that the regulation of PI(4)P levels is critical for rhabdomere biogenesis. In the context of interpreting the effects of Pld overexpression, it is possible that raised PA levels lead to enhanced activity of type I PIPkin consuming PI(4)P at the TGN, resulting in consequent transport defects to the apical membrane. Although it has not been possible to demonstrate reduced PI(4)P or increased PI(4,5)P2 levels at the Golgi in photoreceptors overexpressing Pld, the observation that overexpression of sktl in developing photoreceptors before the critical time window (but not a kinase-dead version) results in a massive defect in rhabdomere biogenesis underscores the importance of tight regulation of type I PIPkin activity during this process. Thus, a tight regulation of the balance of PI(4)P and PI(4,5)P2 levels through Arf1 activity may underlie the effects of PA in this system (Raghu, 2009).

Given the large number of effectors that can be regulated by PA, in the future, it will be important to identify and understand the functions of those that play a role in the biogenesis of rhabdomeres during photoreceptor development (Raghu, 2009).


REFERENCES

Search PubMed for articles about Drosophila Arf1

Bornstein, B., Zahavi, E. E., Gelley, S., Zoosman, M., Yaniv, S. P., Fuchs, O., Porat, Z., Perlson, E. and Schuldiner, O. (2015). Developmental axon pruning requires destabilization of cell adhesion by JNK signaling. Neuron 88(5): 926-940. PubMed ID: 26586184

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Horton, A. C., Racz, B., Monson, E. E., Lin, A. L., Weinberg, R. J. and Ehlers, M. D. (2005). Polarized secretory trafficking directs cargo for asymmetric dendrite growth and morphogenesis. Neuron 48(5): 757-771. PubMed ID: 16337914

Humphreys, D., Liu, T., Davidson, A. C., Hume, P. J. and Koronakis, V. (2012). The Drosophila Arf1 homologue Arf79F is essential for lamellipodium formation. J Cell Sci 125(Pt 23): 5630-5635. PubMed ID: 22992458

Kanamori, T., Yoshino, J., Yasunaga, K., Dairyo, Y. and Emoto, K. (2015). Local endocytosis triggers dendritic thinning and pruning in Drosophila sensory neurons. Nat Commun 6: 6515. PubMed ID: 25761586

Kametaka, S., Sawada, N., Bonifacino, J. S. and Waguri, S. (2010). Functional characterization of protein-sorting machineries at the trans-Golgi network in Drosophila melanogaster. J. Cell Sci. 123: 460-471. PubMed ID: 20067992

Khadilkar, R. J., Rodrigues, D., Mote, R. D., Sinha, A. R., Kulkarni, V., Magadi, S. S. and Inamdar, M. S. (2014). ARF1-GTP regulates Asrij to provide endocytic control of Drosophila blood cell homeostasis. Proc Natl Acad Sci U S A 111(13): 4898-4903. PubMed ID: 24707047

Khadilkar, R. J., Ray, A., Chetan, D. R., Sinha, A. R., Magadi, S. S., Kulkarni, V. and Inamdar, M. S. (2017). Differential modulation of the cellular and humoral immune responses in Drosophila is mediated by the endosomal ARF1-Asrij axis. Sci Rep 7(1): 118. PubMed ID: 28273919

Lee, D. M., Rodrigues, F. F., Yu, C. G., Swan, M. and Harris, T. J. (2015). PH domain-Arf G protein interactions localize the Arf-GEF Steppke for cleavage furrow regulation in Drosophila. PLoS One 10: e0142562. PubMed ID: 26556630

Raghu, P., Coessens, E., Manifava, M., Georgiev, P., Pettitt, T., Wood, E., Garcia-Murillas, I., Okkenhaug, H., Trivedi, D., Zhang, Q., Razzaq, A., Zaid, O., Wakelam, M., O'Kane, C. J. and Ktistakis, N. (2009). Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels. J Cell Biol 185: 129-145. PubMed ID: 19349583

Rodrigues, F. F., Shao, W. and Harris, T. J. (2016). The Arf GAP Asap promotes Arf1 function at the Golgi for cleavage furrow biosynthesis in Drosophila. Mol Biol Cell 27(20):3143-3155. PubMed ID: 27535433

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Biological Overview

date revised: 15 August 2017

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