- Email: [email protected]
Macromolecular uptake in Drosophila pericardial cells requires rudhira function
E XP E RI ME N TA L CE LL RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Macromolecular uptake in Drosophila pericardial cells requires rudhira function Debjani Das, Rajaguru Aradhya, D. Ashoka, Maneesha Inamdar⁎ Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bangalore 560064, India
ARTICLE INFORMATION
ABS T R AC T
Article Chronology:
The vertebrate reticuloendothelial system (RES) functions to remove potentially damaging
Received 29 October 2007
macromolecules, such as excess hormones, immune-peptides and -complexes, bacterial-
Revised version received
endotoxins, microorganisms and tumor cells. Insect hemocytes and nephrocytes – which
13 February 2008
include pericardial cells (PCs) and garland cells – are thought to be functionally equivalent to
Accepted 19 February 2008
the RES. Although the ability of both vertebrate scavenger endothelial cells (SECs) and PCs to
Available online 29 February 2008
sequester colloidal and soluble macromolecules has been demonstrated the molecular mechanism of this function remains to be investigated. We report here the functional
Keywords:
characterization of Drosophila larval PCs with important insights into their cellular uptake
Reticuloendothelial System
pathways. We demonstrate the nephrocyte function of PCs in live animals. We also develop
Scavenger endothelial cell
and use live-cell assays to show that PCs take up soluble macromolecules in a Dynamin-
Nephrocyte
dependent manner and colloids by a Dynamin-independent pathway. We had earlier
Microphagocytosis
identified Drosophila rudhira (Drudh) as a specific marker for PCs. Using RNAi mediated knock-
Coomassie Brilliant Blue
down we show that Drudh regulates macropinocytic uptake in PCs. Our study establishes
Rudhira
important functions for Drosophila PCs, describes methods to identify and study them, provides a genetic handle for further investigation of their role in maintaining homeostasis and demonstrates that they perform key subsets of the roles played by the vertebrate RES. © 2008 Elsevier Inc. All rights reserved.
Introduction The molecular mechanisms by which absorption of iron, excretion of toxic wastes and regulation of hormone levels is attuned to the physiological demands of the body, have been of great interest in mammals. Comprised mainly of “professional phagocytes” (macrophages) and “professional pinocytes” (scavenger endothelial cells, SECs), the vertebrate reticuloendothelial system (RES) plays a major role in these processes [1,2]. SECs are found in the liver of all terrestrial vertebrates, gills of cartilaginous fishes and kidney or heart of bony fishes [3]. In invertebrates, equivalent functions are thought to be carried out by the hemocytes, PCs and garland cells (GCs) [4,5].
While hemocytes are the primary phagocytes, PCs located in the heart are considered equivalent to the vertebrate SECs. The phagocytic function of Drosophila hemocytes and vertebrate macrophages has been studied in detail in connection to infection and immunity [6–10]. But mechanisms of macromolecular-uptake and processing by PCs and SECs have not been investigated. Identifying the molecules required for the uptake pathways that eliminate a wide variety of wastes is fundamental to the elucidation of their physiological role and has important implication in viral infections and drug-delivery strategies. These cells also provide an ideal system for investigating endocytic processes in detail. Molecular mechanisms of several aspects of vertebrate development and function are
⁎ Corresponding author. Maneesha Inamdar JNCASR, Jakkur P.O., Bangalore 560064, India. Fax: +91 80 2208 2766. E-mail address: [email protected] (M. Inamdar). 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.02.009
E XP E RI ME N TA L CE L L RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
conserved in Drosophila [11–13]. In addition the wide range of genetic tools and manipulation techniques available has made Drosophila an attractive model for investigating cellular functions. We report here the functional characterization of Drosophila larval PCs with important insights into their cellular uptake pathways. Reports as early as the 1890s suggest that PCs segregate and store waste products [14]. The equivalence of PCs with the vertebrate reticuloendothelial system was first proposed by Max Poll (1934) [15] and supported by Grasse and Lesperon (1937) [14,16,17]. Uptake of injected dyes revealed that PCs sequester molecules in a restricted size range (16–20 Ǻ). Owing to their restricted phagocytic ability PCs are referred to as being “microphagocytic” [18]. They can also sequester small electronegative colloids [4]. These experiments also detected other cells with similar function. Such cells with the ability to sequester macromolecules are termed nephrocytes. Just as macrophages and SECs of the RES eliminate different classes of wastes [3], similar division of labor is also seen among insect nephrocytes— hemocytes remove large particulate matter and PCs microphagocytose colloidal particles [18]. Ultrastructural studies on PCs have demonstrated a cellular architecture with a network of “labyrinthine channels” separated by “pedicles” of cytoplasm interconnected by “junctional complexes”, all of which are typical of cells engaged in ultrafiltration [4,19]. Accordingly PCs were thought to be analogous to podocytes of the vertebrate kidney [4] but this has not been functionally demonstrated. A large body of literature describes the ultrastructural details of PCs as well as their RES-like functional characteristics [4,16,18–20]. However the molecular basis or the mechanistic details of this function have not been amenable to analysis thus far. Hence we chose to analyze PC function in Drosophila, as this would allow a comparative analysis of insect PC and vertebrate SEC function. PC function has not been systematically investigated in Drosophila due to technical difficulties and also due to a lack of suitable genetic markers. We have analyzed Drosophila PC function and its regulation using the tools of microscopy and live imaging techniques available in Drosophila. We investigated PC function at the organism and cellular level by establishing several assays that allow analysis in a more physiologically relevant scenario than injection of dyes. Using these assays we demonstrate in live animals the ability of Drosophila PCs to sequester macromolecules from the hemolymph and hence function as nephrocytes. To understand the mechanism of this sequestration function we analyzed the uptake properties of larval PCs. We show that Drosophila PCs are microphagocytic and take up soluble and colloidal macromolecules but not particulate matter. Use of soluble macromolecules as probes reveals a Dynamin-dependent uptake pathway. We also describe the use of aqueous preparation of Coomassie Brilliant Blue (CBB) as a colloidal probe and show that it is taken up by a Dynaminindependent macropinocytic pathway. To identify molecules that may control the uptake function of PCs we tested whether DRudh, a specific marker for postembryonic PCs is required for PC function. We investigated the role of DRudh in PC function by examining both loss- and gainof-function phenotypes and show that DRudh regulates macropinocytic uptake in these cells. Our results establish a role for DRudh in macromolecular uptake, a key function of PCs.
1805
This is the first report on genetic regulation of PC function and will allow comparative studies on the role of insect PCs and vertebrate SECs in maintaining tissue homeostasis.
Materials and methods Functional assays Cy3 labeled-maleylated Bovine Serum Albumin (C-mBSA) and FITC-labeled dextran (F-dex) uptake assays were performed as described in Supplementary information. For CBB uptake larval preparations were incubated with 0.3% colloidal preparation of CBB in PBS for 1 min, washed, mounted in 70% glycerol and imaged after 30, 60 or 120 min as indicated. Images were obtained with a Zeiss LSM510-Meta confocal microscope using an argon laser line for excitation (488 nm excitation maximum) and detected using a long pass filter at 505 nm (modified from [21]). Images were analyzed using the software LSM Image Examiner (Carl Zeiss, Inc.). Inhibitors used were all obtained from SIGMA Chemical Co. (USA) and working solution made in PBS to obtain: Monodansylcadaverine (0.5 mM), Chlorpromazine (10 μm), Ethylisopropylamiloride (200 μM) and Wortmannin (200 nM). Hemidissected preparations were incubated with the inhibitors for 30 min prior to incubation with probes.
Quantitation for CBB uptake assay Quantitation was done on the confocal images obtained after CBB uptake using Zeiss LSM 510 Meta software. Parameters for quantitation were set on images of wild type cells. Maximum fluorescent intensity obtained from the cuticle was determined using the Profile tool. This value was set as the threshold intensity. Using the Histo tool the total number of pixels with intensity above this threshold value was determined for each cell. The same threshold value was applied to all cells and N30 cells/genotype were quantified in this way. The values obtained for each genotype were statistically analyzed using GraphPad Prism software and the Mean and Standard Error of the Mean values were graphically represented. Student's t test was used to test the significance.
Results and discussion Pericardial cell function: sequestering of hemolymph macromolecules We demonstrate nephrocyte function of Drosophila PCs by tracking movement of molecules from hemolymph into PCs. Ferrandon et al [22] had generated transgenic animals expressing a secreted (s) and a non-secreted (ns) form of GFP driven by the same promoter. They reported no GFP fluorescence in PCs of animals expressing nsGFP indicating that GFP is not produced in PCs in this experimental system (Fig. S1A, C in supplementary material). However GFP was seen in PCs of third instar larvae (L3) expressing sGFP (Fig. S1B, D in supplementary material). This indicates that PCs can take up sGFP from the hemolymph.
1806
E XP E RI ME N TA L CE LL RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
Fig. 1 – Drosophila PCs sequester macromolecules from hemolymph. (A, B) Bright field image showing dissected third instar larva that was fed with (A) normal or (B) silver nitrate-containing food. Arrowheads in (B) indicate PCs showing pigmentation. tr: trachea. Dorsal view, anterior to the left. Scale Bar = 50 μm.
To test the ability of PCs to take up macromolecules released into the hemolymph from the gut following ingestion, we monitored ingested Silver nitrate. As reported [4,23] larvae fed with food containing 0.5 mg/ml Silver nitrate from mid-second larval instar (L2) showed yellowish orange pigmentation in PCs (Fig. 1B), GCs and malphigian tubules (data not shown) by late L3. Age-matched animals not fed with Silver nitrate do not show similar pigmentation (Fig. 1A). Thus Drosophila PCs function as classical nephrocytes sequestering both endogenous (expressed in vivo) and exogenous (acquired) macromolecules from the hemolymph. In their description of Drosophila PCs Mills and King [16] had stressed on the possible “toxic” nature of materials sequestered by PCs. This view was further expanded by Wigglesworth [14] to include elimination of not only toxic but mostly “useless” material by PCs. Our results on uptake of both sGFP and Silver nitrate by PCs provide experimental verification to these speculations. The exact mechanism of Silver nitrate uptake by PCs however remains to be elucidated. Uptake of toxic macro-
molecules from hemolymph also has relevance to pesticide delivery in insects. Ingested macromolecular drugs would presumably get sequestered by PCs thus preventing drug concentration from reaching desired levels. Failure of baculovirus-mediated expression of Juvenile Hormone Esterase in increasing insecticidal activity of the virus is attributed to sequestration by the PCs [20]. Similar challenges posed by vertebrate SECs for drug targeting to non-hepatic tissues are well documented [2]. In this regard mutants with reduced uptake described in this study (see below) would be an extremely valuable model for screening drugs and their vehicle.
Dynamin-dependent microphagocytosis and scavenging as one mechanism of pericardial cell function To understand the uptake properties of PCs, we characterized endocytic pathways operating in PCs. FITC-labeled dextran (F-dex) has been used to study fluid-phase endocytic uptake in Drosophila hemocytes [6]. Using this probe we show that PCs
Fig. 2 – PCs are microphagocytic in both Dynamin-dependent and -independent manner. Wild type (+/+) and shits2 PCs shown as indicated, were incubated with (A–B, E–F, I–J) F-dex (green) or (C–D, G–H, K–L) C-mBSA (red) or (M–S) CBB (gray) at 25 °C, 19 °C or 30 °C as indicated. (B, D) are single optical sections through the cell shown in the panel to their immediate left (i.e. A and C respectively). (M–O) show stacked image of the same cell imaged after 30 min, 1 h or 2 h as indicated. Scale bar = 10 μm.
E XP E RI ME N TA L CE L L RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
actively take-up F-dex (Fig. 2A, B and Fig. S2A, B in supplementary material). However it is not clear if this represents a fluid-phase pathway similar to that in hemocytes (our unpublished data). Earlier experiments suggest that insect PCs exclude particles based on size and electropositive charge [4]. Wigglesworth (1972) called this limited phagocytic ability of pericardial cells as microphagocytosis. But it is not clear how these cells sequester some molecules while rejecting others of the same size and charge. Molecular recognition is extremely important for these cells especially since they sequester denatured or exogenous molecules [4]. We investigated the scavenging function of PCs by testing for uptake of Cy3 labeled-maleylated Bovine Serum Albumin (C-mBSA), a ligand for Anion Ligand Binding Receptor (ALBR), which has a binding specificity similar to the scavenger receptors (dSR) [6, 7]. In live PCs (Fig. 2 C and D) but not in paraformaldehyde (PF) fixed PCs (Fig. S2C and D in supplementary material), after 5 min pulse of C-mBSA, fluorescent vesicles were detected in the cortical cytoplasm. These results indicate that a functional ALBR is likely to be expressed in PCs and could be involved in the scavenging function. Macromolecular uptake in many contexts is known to be regulated by Dynamin [24]. Drosophila Dynamin (dDyn) is encoded by the gene shibire (shi) and null mutations in this gene are lethal [25]. Electron microscopic studies on the larval garland cells of shits mutants suggested that all pathways of endocytosis require dDyn [26]. shits1 and shits2 alleles are temperature-sensitive missense mutations [25], which we used to investigate the requirement of Dynamin in endocytic uptake by PCs. Uptake of both soluble probes (F-dex and C-mBSA) is completely abolished at the restrictive temperature in shits2 PCs indicating that this uptake is Dynamin-dependent (Fig. 2I–L, Fig. S2I–L in supplementary material and Fig. 5; similar results were obtained for both mutants but data shown only for shits2). Like PCs, hemocytes also have an ALBR scavenger receptor [7]. However, these two components of the insect scavenging system differ not only in the size of macromolecules they engulf but also in the pathways recruited for these processes.
1807
In hemocytes, fluid-phase uptake is Dynamin-independent [6] while in PCs we find that it is Dynamin-dependent.
Uptake of insoluble macromolecules is Dynamin-independent Colloid uptake is a prominent feature of insect PCs. We used colloidal preparation of Coomassie Brilliant Blue (referred to as CBB) to investigate colloidal uptake in live Drosophila PCs as it was reported to become autofluorescent following uptake by PCs [21]. PCs imaged 30 min (Fig. 2M), 1 h (Fig. 2N) and 2 h (Fig. 2O) after a 1 min exposure to CBB show increase in both intensity and size of the fluorescent granules suggesting active processing of CBB by the PCs. This processing of CBB is specific to PCs since other tissues, like the cardioblasts lying immediately adjacent to the PCs in the same preparation show no such autofluorescence (see Fig. 4 arrowhead). CBB autofluorescence was completely abolished when PCs were pre-treated with 4% PF (Fig. S2N in supplementary material) providing further evidence of active uptake and processing by PCs. At the restrictive temperature of 30 °C, shits mutant PCs tested for CBB uptake show fluorescent signals (Fig. 2P–S) indicating that shits mutant PCs can take up CBB (Fig. 5). Hence CBB uptake occurs even in absence of Dynamin function.
CBB is taken up by macropinocytosis in PCs To investigate whether CBB uptake occurs through any of the known endocytic pathways we treated the cells with pathwayspecific inhibitors before assaying for uptake. We first used Wortmannin (Wm), a Phosphoinositide-3-kinase inhibitor that affects all types of uptake [27]. 200 nM Wm significantly reduced both C-mBSA (Fig. 3B) and CBB uptake (Fig. 3G) suggesting that CBB could be taken up by one of the known pathways. Monodansylcadaverine (MDc) and Chlorpromazine (Cpz) are inhibitors of clathrin function, and hence of soluble uptake pathways. MDc is a transglutaminase inhibitor which prevents clustering and internalization of the ligand-receptor complexes into clathrin-coated vesicles [28] and Cpz acts by
Fig. 3 – CBB uptake in PCs is by macropinocytosis. C-mBSA (A–E) or CBB (F–J) uptake assays on wild type (+/+) PCs following 30 min treatment with inhibitors as indicated. Outline of PC indicated in B, C, D by white line. PBS: phosphate buffered saline; Wm: Wortmanin; MDc: Monodansylcadaverine; Cpz: Chlorpromazine; EIPA: Ethylisopropylamiloride. Scale bar = 10 μm.
1808
E XP E RI ME N TA L CE LL RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
accumulating clathrin and AP-2 in endosomal compartments, thus preventing the formation of clathrin-coated pits at the plasma membrane [29]. 0.5 mM MDc or 10 μm Cpz completely abolished C-mBSA uptake (which is internalized through a clathrin–dynamin pathway) (Fig. 3C and D) but had no effect on CBB (Fig. 3H and I). Ethylisopropylamiloride (EIPA) is an inhibitor of the Na+/H+exchanger and affects macropinocytosis but neither clathrin- nor raft-mediated endocytosis [27]. 200 μM EIPA did not affect C-mBSA uptake (Fig. 3E), but significantly reduced CBB uptake (Fig. 3J). These data indicate that CBB uptake by PCs occurs by macropinocytosis, a process that does not require Dynamin function (Fig. 5).
DRudh affects Dynamin-independent uptake in PCs We had earlier identified a novel cytoplasmic WD40 domain protein, Rudhira (Flybase name CG32663, NCBI accession # NM_167309) by sequence homology with a mouse protein expressed during primitive erythropoiesis and neo-vascularization [30]. Drosophila Rudh (DRudh) expresses in high levels in PCs (undetected in other tissues) and is temporally regulated [31]. Hence we investigated if it is required for PC function. Drudh
loss-of-function mutants are not available. Hence we reduced DRudh expression in PCs by double-stranded RNA interference (RNAi) of Drudh mRNA using the GAL4-UAS system (See Methods in supplementary material). In absence of an exclusive PCspecific driver we used dorothy-GAL4 (dot-GAL4) driver expressed in the hematopoietic system and PCs [32,33]. We generated dotGAL4 UAS-Drudh-RNAi larvae and achieved a 30% decrease in DRudh expression (Fig. S3A–C in supplementary material) which resulted in a significant (P < 0.01) decrease in CBB uptake by PCs (Fig. 4A, B and F) with no effect on Dynamin-dependent uptake (data shown for C-mBSA uptake Fig. S4 in supplementary material). Thus knock-down of DRudh expression results in impaired Dynamin-independent uptake (Fig. 5). We did not observe a complete loss of CBB uptake in silenced animals, which could be due to incomplete knock down of DRudh expression, or because of redundancy in DRudh function. We next tested if over-expression of DRudh in PCs enhances CBB uptake in these cells. Transgenic UAS-Drudh flies (see Methods in supplementary material) were used to overexpress Drudh in the presence of a GAL4 activator. DRudh overexpression in PCs using dot-GAL4 did not result in increased CBB uptake (Fig. 4C, D and G) although a 60% increase in protein
Fig. 4 – Changing levels of DRudh expression affect CBB uptake. CBB assay on (A) UAS-Drudh-RNAi (B) dot-GAL4; UAS-Drudh-RNAi (C) UAS-Drudh (D) dot-GAL4/UAS-Drudh (E) arm-GAL4/UAS-Drudh PCs of third instar larvae. Arrowhead indicates cardiac tube showing no autofluorescence following CBB treatment. (F, G) Graph showing average pixels/cell observed in the CBB assays for the genotypes indicated (n = 30). Error bars represent Standard Error of the Mean (±s.e.m). Asterisk indicates P < 0.01. Scale Bar = 10 μm.
E XP E RI ME N TA L CE L L RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
1809
Rudhira: a link between PCs and vertebrate SECs Based on their microphagocytic scavenging function, PCs are considered similar to the vertebrate SECs [3]. It is interesting to note in this context that while Drosophila Drudh is expressed in PCs, its murine ortholog is expressed in endothelial precursors during vasculogenesis and angiogenesis [30]. These observations raise interesting questions about evolution of PCs. Are PCs specialized endothelial cells (such as those found in vertebrate liver) strategically placed juxtaposed to the heart to exploit their scavenging function? Attempts to address these intriguing issues regarding PCs will gain significantly from the information about PC function and the reagents generated in this study. Further explorations in PC biology will aid in the comparative functional analysis of vertebrate and invertebrate “professional pinocytes”.
Acknowledgments Fig. 5 – A comparison of macro- and micropinocytic modes of uptake in pericardial cells. A simplistic schematic representation of macro- and micropinocytosis, also showing requirement of Dynamin, DRudhira function and effect of pathway-specific endocytic inhibitors. Wm: Wortmanin; MDc: Monodansylcadaverine; Cpz: Chlorpromazine; EIPA: Ethylisopropylamiloride.
level could be detected by immunostaining in the third larval instar (Fig. S3C–E in supplementary material). DRudh may regulate colloid uptake by interacting with other proteins probably by virtue of its WD40 domains [34]. Hence overexpression of DRudh alone may be ineffective whereas reduction of Rudh levels results in an uptake phenotype. Similar results have been reported in many systems for Dynamin where overexpression of the dominant-negative construct produces a strong inhibitory phenotype but that of the wild type allele has no phenotype owing to the clathrin-mediated regulation of Dynamin's GTPase activity [35,36]. Although dot-GAL4 is expressed in embryonic PCs, it is absent or undetectable in PCs after hatching till late third larval instar. One reason, therefore, for the absence of a gainof-function phenotype could be that over-expression later in development was insufficient to produce an effect. In the absence of any early PC-specific driver we used a ubiquitously expressed driver, armadillo-GAL4 (arm-GAL4), to drive expression of the same UAS-Drudh construct. This combination showed a significant (P < 0.01) enhancement in CBB uptake (Fig. 4C, E and G). This increase in Dynamin-independent uptake is specific since control animals expressing UAS-lacZ instead of UAS-Drudh driven by the same arm-GAL4 driver do not exhibit such enhancement in CBB uptake in PCs (data not shown). The difference in temporal activity of the GAL4 drivers suggests that not just levels but timing of DRudh expression is also important for PC function. However since the arm-GAL4 driver is not specific to PCs, the possibility of ubiquitous Drudh overexpression non-autonomously affecting PC function remains. In either case our results indicate that DRudh regulates macromolecular uptake function in PCs.
We thank Katherine Anderson, Dominique Ferrandon, L. S. Sashidhara, Volker Hartenstein, D. K. Hoshizaki, Kryztof Jagla, Michael Semeriva, Helen Skaer, V. Sriram, and K. VijayRaghavan for fly stocks, reagents and valuable comments. We thank Suma B.S. for help with confocal microscopy. DD is supported by a CSIR-SPM fellowship. This work was funded by the JNCASR, the Council of Scientific and Industrial Research, and Department of Biotechnology, Government of India.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2008.02.009.
REFERENCES
[1] M. Knutson, M. Wessling-Resnick, Iron metabolism in the reticuloendothelial system, Crit. Rev. Biochem. Mol. Biol. 38 (2003) 61–88. [2] B. Smedsrod, Clearance function of scavenger endothelial cells, Comp. Hepatol. 3 (Suppl 1) (2004) S22. [3] T. Seternes, K. Sorensen, B. Smedsrod, Scavenger endothelial cells of vertebrates: a nonperipheral leukocyte system for high-capacity elimination of waste macromolecules, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 7594–7597. [4] A. Crossley, Nephrocytes and pericardial cells, in: G.L. Kerkut, G.A. (Eds.), Comprehensive insect physiology, biochemistry and pharmacology, vol. 3, Pergamon Press, Oxford, 1985, pp. 487–515. [5] E.J. Ward, D.E. Coulter, Odd-skipped is expressed in multiple tissues during Drosophila embryogenesis, Mech. Dev. 96 (2000) 233–236. [6] A. Guha, V. Sriram, K.S. Krishnan, S. Mayor, Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes, J. Cell Sci. 116 (2003) 3373–3386. [7] V. Sriram, K.S. Krishnan, S. Mayor, Deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster, J. Cell Biol. 161 (2003) 593–607.
1810
E XP E RI ME N TA L CE LL RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
[8] M. Elrod-Erickson, S. Mishra, D. Schneider, Interactions between the cellular and humoral immune responses in Drosophila, Curr. Biol. 10 (2000) 781–784. [9] B. Lemaitre, J. Hoffmann, The host defense of Drosophila melanogaster, Annu. Rev. Immunol. 25 (2007) 697–743. [10] D. Hultmark, K. Borge-Renberg, Drosophila immunity: is antigen processing the first step? Curr. Biol. 17 (2007) R22–R24. [11] S. Zaffran, M. Frasch, Early signals in cardiac development, Circ. Res. 91 (2002) 457–469. [12] L. Mandal, U. Banerjee, V. Hartenstein, Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta–gonadal–mesonephros mesoderm, Nat. Genet. 36 (2004) 1019–1023. [13] E. Bier, R. Bodmer, Drosophila, an emerging model for cardiac disease, Gene 342 (2004) 1–11. [14] V.B. Wigglesworth, The pericardial cells of insects:analogue of reticuloendothelail system, J. Reticuloendothel. Soc. 7 (1970) 208–216. [15] M. Poll, Rescheres histophysiologiques sur les tubes de malpighi du Tenebrio molitor, L Rec. Inst. Zool. Torley-Rousseau 5 (1934) 78–126. [16] R.P. Mills, R.C. King, The pericardial cells of Drosophila melanogaster, Q. J. Microsc. Sci. 106 (1965) 261–268. [17] P.P. Grasse, L. Lesperon, Accumulation de colorants acides chez le ver a soie par des tissue differents selon la voie d'acces, Compt. Rend. Acad. Sci. 201 (1935). [18] V.B. Wigglesworth, The circulatory system and associated tissues, the principles of insect physiology, Chapman Hall (1972) 411–473. [19] U. Tepass, V. Hartenstein, The development of cellular junctions in the Drosophila embryo, Dev. Biol. 161 (1994) 563–596. [20] B.C. Bonning, T.F. Booth, B.D. Hammock, Mechanistic studies of the degradation of juvenile hormone esterase in Manduca sexta, Arch. Insect Biochem. Physiol. 34 (1997) 275–286. [21] D. Dulcis, R.B. Levine, Innervation of the heart of the adult fruit fly, Drosophila melanogaster, J. Comp. Neurol. 465 (2003) 560–578. [22] D. Ferrandon, A.C. Jung, M. Criqui, B. Lemaitre, S. Uttenweiler-Joseph, L. Michaut, J. Reichhart, J.A. Hoffmann, A drosomycin-GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway, EMBO J. 17 (1998) 1217–1227.
[23] H.S. Distefano, Effects of silver nitrate on the pigmentation of Drosophila, Am. Nat. 77 (1943) 94–96. [24] G.J. Praefcke, H.T. McMahon, The dynamin superfamily: universal membrane tubulation and fission molecules? Nat. Rev. Mol. Cell Biol. 5 (2004) 133–147. [25] A.M. van der Bliek, E.M. Meyerowitz, Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic, Nature 351 (1991) 411–414. [26] T. Kosaka, K. Ikeda, Reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of Drosophila melanogaster, shibirets1, J. Cell Biol. 97 (1983) 499–507. [27] S. Falcone, E. Cocucci, P. Podini, T. Kirchhausen, E. Clementi, J. Meldolesi, Macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events, J. Cell Sci. 119 (2006) 4758–4769. [28] Y. Rikihisa, Y. Zhang, J. Park, Inhibition of infection of macrophages with Ehrlichia risticii by cytochalasins, monodansylcadaverine, and taxol, Infect. Immun. 62 (1994) 5126–5132. [29] J.J. Chu, M.L. Ng, Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway, J. Virol. 78 (2004) 10543–10555. [30] K. Siva, M.S. Inamdar, Rudhira is a cytoplasmic WD40 protein expressed in mouse embryonic stem cells and during embryonic erythropoiesis, Gene Expr. Patterns 6 (2006) 225–234. [31] D. Das, D. Ashoka, R. Aradhya, M. Inamdar, Gene expression analysis in post-embryonic pericardial cells of Drosophila, Gene Expr. Patterns 8 (2008) 199–205. [32] C.H. Deborah, A. Kimbrell, Clare Bolduc, Kurt Kleinhesselink, Kathy Beckingham, The Dorothy enhancer has Tinman binding sites and drives hopscotch-induced tumor formation, Genesis 34 (2002) 23–28. [33] P. Yi, Z. Han, X. Li, E.N. Olson, The mevalonate pathway controls heart formation in Drosophila by isoprenylation of Ggamma1, Science 313 (2006) 1301–1303. [34] D. Li, R. Roberts, WD-repeat proteins: structure characteristics, biological function, and their involvement in human diseases, Cell Mol. Life Sci. 58 (2001) 2085–2097. [35] H.C. Chang, M. Hull, I. Mellman, The J-domain protein Rme-8 interacts with Hsc70 to control clathrin-dependent endocytosis in Drosophila, J. Cell Biol. 164 (2004) 1055–1064. [36] G. Di Paolo, P. De Camilli, Does clathrin pull the fission trigger? Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 4981–4983.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Macromolecular uptake in Drosophila pericardial cells requires rudhira function Debjani Das, Rajaguru Aradhya, D. Ashoka, Maneesha Inamdar⁎ Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bangalore 560064, India
ARTICLE INFORMATION
ABS T R AC T
Article Chronology:
The vertebrate reticuloendothelial system (RES) functions to remove potentially damaging
Received 29 October 2007
macromolecules, such as excess hormones, immune-peptides and -complexes, bacterial-
Revised version received
endotoxins, microorganisms and tumor cells. Insect hemocytes and nephrocytes – which
13 February 2008
include pericardial cells (PCs) and garland cells – are thought to be functionally equivalent to
Accepted 19 February 2008
the RES. Although the ability of both vertebrate scavenger endothelial cells (SECs) and PCs to
Available online 29 February 2008
sequester colloidal and soluble macromolecules has been demonstrated the molecular mechanism of this function remains to be investigated. We report here the functional
Keywords:
characterization of Drosophila larval PCs with important insights into their cellular uptake
Reticuloendothelial System
pathways. We demonstrate the nephrocyte function of PCs in live animals. We also develop
Scavenger endothelial cell
and use live-cell assays to show that PCs take up soluble macromolecules in a Dynamin-
Nephrocyte
dependent manner and colloids by a Dynamin-independent pathway. We had earlier
Microphagocytosis
identified Drosophila rudhira (Drudh) as a specific marker for PCs. Using RNAi mediated knock-
Coomassie Brilliant Blue
down we show that Drudh regulates macropinocytic uptake in PCs. Our study establishes
Rudhira
important functions for Drosophila PCs, describes methods to identify and study them, provides a genetic handle for further investigation of their role in maintaining homeostasis and demonstrates that they perform key subsets of the roles played by the vertebrate RES. © 2008 Elsevier Inc. All rights reserved.
Introduction The molecular mechanisms by which absorption of iron, excretion of toxic wastes and regulation of hormone levels is attuned to the physiological demands of the body, have been of great interest in mammals. Comprised mainly of “professional phagocytes” (macrophages) and “professional pinocytes” (scavenger endothelial cells, SECs), the vertebrate reticuloendothelial system (RES) plays a major role in these processes [1,2]. SECs are found in the liver of all terrestrial vertebrates, gills of cartilaginous fishes and kidney or heart of bony fishes [3]. In invertebrates, equivalent functions are thought to be carried out by the hemocytes, PCs and garland cells (GCs) [4,5].
While hemocytes are the primary phagocytes, PCs located in the heart are considered equivalent to the vertebrate SECs. The phagocytic function of Drosophila hemocytes and vertebrate macrophages has been studied in detail in connection to infection and immunity [6–10]. But mechanisms of macromolecular-uptake and processing by PCs and SECs have not been investigated. Identifying the molecules required for the uptake pathways that eliminate a wide variety of wastes is fundamental to the elucidation of their physiological role and has important implication in viral infections and drug-delivery strategies. These cells also provide an ideal system for investigating endocytic processes in detail. Molecular mechanisms of several aspects of vertebrate development and function are
⁎ Corresponding author. Maneesha Inamdar JNCASR, Jakkur P.O., Bangalore 560064, India. Fax: +91 80 2208 2766. E-mail address: [email protected] (M. Inamdar). 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.02.009
E XP E RI ME N TA L CE L L RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
conserved in Drosophila [11–13]. In addition the wide range of genetic tools and manipulation techniques available has made Drosophila an attractive model for investigating cellular functions. We report here the functional characterization of Drosophila larval PCs with important insights into their cellular uptake pathways. Reports as early as the 1890s suggest that PCs segregate and store waste products [14]. The equivalence of PCs with the vertebrate reticuloendothelial system was first proposed by Max Poll (1934) [15] and supported by Grasse and Lesperon (1937) [14,16,17]. Uptake of injected dyes revealed that PCs sequester molecules in a restricted size range (16–20 Ǻ). Owing to their restricted phagocytic ability PCs are referred to as being “microphagocytic” [18]. They can also sequester small electronegative colloids [4]. These experiments also detected other cells with similar function. Such cells with the ability to sequester macromolecules are termed nephrocytes. Just as macrophages and SECs of the RES eliminate different classes of wastes [3], similar division of labor is also seen among insect nephrocytes— hemocytes remove large particulate matter and PCs microphagocytose colloidal particles [18]. Ultrastructural studies on PCs have demonstrated a cellular architecture with a network of “labyrinthine channels” separated by “pedicles” of cytoplasm interconnected by “junctional complexes”, all of which are typical of cells engaged in ultrafiltration [4,19]. Accordingly PCs were thought to be analogous to podocytes of the vertebrate kidney [4] but this has not been functionally demonstrated. A large body of literature describes the ultrastructural details of PCs as well as their RES-like functional characteristics [4,16,18–20]. However the molecular basis or the mechanistic details of this function have not been amenable to analysis thus far. Hence we chose to analyze PC function in Drosophila, as this would allow a comparative analysis of insect PC and vertebrate SEC function. PC function has not been systematically investigated in Drosophila due to technical difficulties and also due to a lack of suitable genetic markers. We have analyzed Drosophila PC function and its regulation using the tools of microscopy and live imaging techniques available in Drosophila. We investigated PC function at the organism and cellular level by establishing several assays that allow analysis in a more physiologically relevant scenario than injection of dyes. Using these assays we demonstrate in live animals the ability of Drosophila PCs to sequester macromolecules from the hemolymph and hence function as nephrocytes. To understand the mechanism of this sequestration function we analyzed the uptake properties of larval PCs. We show that Drosophila PCs are microphagocytic and take up soluble and colloidal macromolecules but not particulate matter. Use of soluble macromolecules as probes reveals a Dynamin-dependent uptake pathway. We also describe the use of aqueous preparation of Coomassie Brilliant Blue (CBB) as a colloidal probe and show that it is taken up by a Dynaminindependent macropinocytic pathway. To identify molecules that may control the uptake function of PCs we tested whether DRudh, a specific marker for postembryonic PCs is required for PC function. We investigated the role of DRudh in PC function by examining both loss- and gainof-function phenotypes and show that DRudh regulates macropinocytic uptake in these cells. Our results establish a role for DRudh in macromolecular uptake, a key function of PCs.
1805
This is the first report on genetic regulation of PC function and will allow comparative studies on the role of insect PCs and vertebrate SECs in maintaining tissue homeostasis.
Materials and methods Functional assays Cy3 labeled-maleylated Bovine Serum Albumin (C-mBSA) and FITC-labeled dextran (F-dex) uptake assays were performed as described in Supplementary information. For CBB uptake larval preparations were incubated with 0.3% colloidal preparation of CBB in PBS for 1 min, washed, mounted in 70% glycerol and imaged after 30, 60 or 120 min as indicated. Images were obtained with a Zeiss LSM510-Meta confocal microscope using an argon laser line for excitation (488 nm excitation maximum) and detected using a long pass filter at 505 nm (modified from [21]). Images were analyzed using the software LSM Image Examiner (Carl Zeiss, Inc.). Inhibitors used were all obtained from SIGMA Chemical Co. (USA) and working solution made in PBS to obtain: Monodansylcadaverine (0.5 mM), Chlorpromazine (10 μm), Ethylisopropylamiloride (200 μM) and Wortmannin (200 nM). Hemidissected preparations were incubated with the inhibitors for 30 min prior to incubation with probes.
Quantitation for CBB uptake assay Quantitation was done on the confocal images obtained after CBB uptake using Zeiss LSM 510 Meta software. Parameters for quantitation were set on images of wild type cells. Maximum fluorescent intensity obtained from the cuticle was determined using the Profile tool. This value was set as the threshold intensity. Using the Histo tool the total number of pixels with intensity above this threshold value was determined for each cell. The same threshold value was applied to all cells and N30 cells/genotype were quantified in this way. The values obtained for each genotype were statistically analyzed using GraphPad Prism software and the Mean and Standard Error of the Mean values were graphically represented. Student's t test was used to test the significance.
Results and discussion Pericardial cell function: sequestering of hemolymph macromolecules We demonstrate nephrocyte function of Drosophila PCs by tracking movement of molecules from hemolymph into PCs. Ferrandon et al [22] had generated transgenic animals expressing a secreted (s) and a non-secreted (ns) form of GFP driven by the same promoter. They reported no GFP fluorescence in PCs of animals expressing nsGFP indicating that GFP is not produced in PCs in this experimental system (Fig. S1A, C in supplementary material). However GFP was seen in PCs of third instar larvae (L3) expressing sGFP (Fig. S1B, D in supplementary material). This indicates that PCs can take up sGFP from the hemolymph.
1806
E XP E RI ME N TA L CE LL RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
Fig. 1 – Drosophila PCs sequester macromolecules from hemolymph. (A, B) Bright field image showing dissected third instar larva that was fed with (A) normal or (B) silver nitrate-containing food. Arrowheads in (B) indicate PCs showing pigmentation. tr: trachea. Dorsal view, anterior to the left. Scale Bar = 50 μm.
To test the ability of PCs to take up macromolecules released into the hemolymph from the gut following ingestion, we monitored ingested Silver nitrate. As reported [4,23] larvae fed with food containing 0.5 mg/ml Silver nitrate from mid-second larval instar (L2) showed yellowish orange pigmentation in PCs (Fig. 1B), GCs and malphigian tubules (data not shown) by late L3. Age-matched animals not fed with Silver nitrate do not show similar pigmentation (Fig. 1A). Thus Drosophila PCs function as classical nephrocytes sequestering both endogenous (expressed in vivo) and exogenous (acquired) macromolecules from the hemolymph. In their description of Drosophila PCs Mills and King [16] had stressed on the possible “toxic” nature of materials sequestered by PCs. This view was further expanded by Wigglesworth [14] to include elimination of not only toxic but mostly “useless” material by PCs. Our results on uptake of both sGFP and Silver nitrate by PCs provide experimental verification to these speculations. The exact mechanism of Silver nitrate uptake by PCs however remains to be elucidated. Uptake of toxic macro-
molecules from hemolymph also has relevance to pesticide delivery in insects. Ingested macromolecular drugs would presumably get sequestered by PCs thus preventing drug concentration from reaching desired levels. Failure of baculovirus-mediated expression of Juvenile Hormone Esterase in increasing insecticidal activity of the virus is attributed to sequestration by the PCs [20]. Similar challenges posed by vertebrate SECs for drug targeting to non-hepatic tissues are well documented [2]. In this regard mutants with reduced uptake described in this study (see below) would be an extremely valuable model for screening drugs and their vehicle.
Dynamin-dependent microphagocytosis and scavenging as one mechanism of pericardial cell function To understand the uptake properties of PCs, we characterized endocytic pathways operating in PCs. FITC-labeled dextran (F-dex) has been used to study fluid-phase endocytic uptake in Drosophila hemocytes [6]. Using this probe we show that PCs
Fig. 2 – PCs are microphagocytic in both Dynamin-dependent and -independent manner. Wild type (+/+) and shits2 PCs shown as indicated, were incubated with (A–B, E–F, I–J) F-dex (green) or (C–D, G–H, K–L) C-mBSA (red) or (M–S) CBB (gray) at 25 °C, 19 °C or 30 °C as indicated. (B, D) are single optical sections through the cell shown in the panel to their immediate left (i.e. A and C respectively). (M–O) show stacked image of the same cell imaged after 30 min, 1 h or 2 h as indicated. Scale bar = 10 μm.
E XP E RI ME N TA L CE L L RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
actively take-up F-dex (Fig. 2A, B and Fig. S2A, B in supplementary material). However it is not clear if this represents a fluid-phase pathway similar to that in hemocytes (our unpublished data). Earlier experiments suggest that insect PCs exclude particles based on size and electropositive charge [4]. Wigglesworth (1972) called this limited phagocytic ability of pericardial cells as microphagocytosis. But it is not clear how these cells sequester some molecules while rejecting others of the same size and charge. Molecular recognition is extremely important for these cells especially since they sequester denatured or exogenous molecules [4]. We investigated the scavenging function of PCs by testing for uptake of Cy3 labeled-maleylated Bovine Serum Albumin (C-mBSA), a ligand for Anion Ligand Binding Receptor (ALBR), which has a binding specificity similar to the scavenger receptors (dSR) [6, 7]. In live PCs (Fig. 2 C and D) but not in paraformaldehyde (PF) fixed PCs (Fig. S2C and D in supplementary material), after 5 min pulse of C-mBSA, fluorescent vesicles were detected in the cortical cytoplasm. These results indicate that a functional ALBR is likely to be expressed in PCs and could be involved in the scavenging function. Macromolecular uptake in many contexts is known to be regulated by Dynamin [24]. Drosophila Dynamin (dDyn) is encoded by the gene shibire (shi) and null mutations in this gene are lethal [25]. Electron microscopic studies on the larval garland cells of shits mutants suggested that all pathways of endocytosis require dDyn [26]. shits1 and shits2 alleles are temperature-sensitive missense mutations [25], which we used to investigate the requirement of Dynamin in endocytic uptake by PCs. Uptake of both soluble probes (F-dex and C-mBSA) is completely abolished at the restrictive temperature in shits2 PCs indicating that this uptake is Dynamin-dependent (Fig. 2I–L, Fig. S2I–L in supplementary material and Fig. 5; similar results were obtained for both mutants but data shown only for shits2). Like PCs, hemocytes also have an ALBR scavenger receptor [7]. However, these two components of the insect scavenging system differ not only in the size of macromolecules they engulf but also in the pathways recruited for these processes.
1807
In hemocytes, fluid-phase uptake is Dynamin-independent [6] while in PCs we find that it is Dynamin-dependent.
Uptake of insoluble macromolecules is Dynamin-independent Colloid uptake is a prominent feature of insect PCs. We used colloidal preparation of Coomassie Brilliant Blue (referred to as CBB) to investigate colloidal uptake in live Drosophila PCs as it was reported to become autofluorescent following uptake by PCs [21]. PCs imaged 30 min (Fig. 2M), 1 h (Fig. 2N) and 2 h (Fig. 2O) after a 1 min exposure to CBB show increase in both intensity and size of the fluorescent granules suggesting active processing of CBB by the PCs. This processing of CBB is specific to PCs since other tissues, like the cardioblasts lying immediately adjacent to the PCs in the same preparation show no such autofluorescence (see Fig. 4 arrowhead). CBB autofluorescence was completely abolished when PCs were pre-treated with 4% PF (Fig. S2N in supplementary material) providing further evidence of active uptake and processing by PCs. At the restrictive temperature of 30 °C, shits mutant PCs tested for CBB uptake show fluorescent signals (Fig. 2P–S) indicating that shits mutant PCs can take up CBB (Fig. 5). Hence CBB uptake occurs even in absence of Dynamin function.
CBB is taken up by macropinocytosis in PCs To investigate whether CBB uptake occurs through any of the known endocytic pathways we treated the cells with pathwayspecific inhibitors before assaying for uptake. We first used Wortmannin (Wm), a Phosphoinositide-3-kinase inhibitor that affects all types of uptake [27]. 200 nM Wm significantly reduced both C-mBSA (Fig. 3B) and CBB uptake (Fig. 3G) suggesting that CBB could be taken up by one of the known pathways. Monodansylcadaverine (MDc) and Chlorpromazine (Cpz) are inhibitors of clathrin function, and hence of soluble uptake pathways. MDc is a transglutaminase inhibitor which prevents clustering and internalization of the ligand-receptor complexes into clathrin-coated vesicles [28] and Cpz acts by
Fig. 3 – CBB uptake in PCs is by macropinocytosis. C-mBSA (A–E) or CBB (F–J) uptake assays on wild type (+/+) PCs following 30 min treatment with inhibitors as indicated. Outline of PC indicated in B, C, D by white line. PBS: phosphate buffered saline; Wm: Wortmanin; MDc: Monodansylcadaverine; Cpz: Chlorpromazine; EIPA: Ethylisopropylamiloride. Scale bar = 10 μm.
1808
E XP E RI ME N TA L CE LL RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
accumulating clathrin and AP-2 in endosomal compartments, thus preventing the formation of clathrin-coated pits at the plasma membrane [29]. 0.5 mM MDc or 10 μm Cpz completely abolished C-mBSA uptake (which is internalized through a clathrin–dynamin pathway) (Fig. 3C and D) but had no effect on CBB (Fig. 3H and I). Ethylisopropylamiloride (EIPA) is an inhibitor of the Na+/H+exchanger and affects macropinocytosis but neither clathrin- nor raft-mediated endocytosis [27]. 200 μM EIPA did not affect C-mBSA uptake (Fig. 3E), but significantly reduced CBB uptake (Fig. 3J). These data indicate that CBB uptake by PCs occurs by macropinocytosis, a process that does not require Dynamin function (Fig. 5).
DRudh affects Dynamin-independent uptake in PCs We had earlier identified a novel cytoplasmic WD40 domain protein, Rudhira (Flybase name CG32663, NCBI accession # NM_167309) by sequence homology with a mouse protein expressed during primitive erythropoiesis and neo-vascularization [30]. Drosophila Rudh (DRudh) expresses in high levels in PCs (undetected in other tissues) and is temporally regulated [31]. Hence we investigated if it is required for PC function. Drudh
loss-of-function mutants are not available. Hence we reduced DRudh expression in PCs by double-stranded RNA interference (RNAi) of Drudh mRNA using the GAL4-UAS system (See Methods in supplementary material). In absence of an exclusive PCspecific driver we used dorothy-GAL4 (dot-GAL4) driver expressed in the hematopoietic system and PCs [32,33]. We generated dotGAL4 UAS-Drudh-RNAi larvae and achieved a 30% decrease in DRudh expression (Fig. S3A–C in supplementary material) which resulted in a significant (P < 0.01) decrease in CBB uptake by PCs (Fig. 4A, B and F) with no effect on Dynamin-dependent uptake (data shown for C-mBSA uptake Fig. S4 in supplementary material). Thus knock-down of DRudh expression results in impaired Dynamin-independent uptake (Fig. 5). We did not observe a complete loss of CBB uptake in silenced animals, which could be due to incomplete knock down of DRudh expression, or because of redundancy in DRudh function. We next tested if over-expression of DRudh in PCs enhances CBB uptake in these cells. Transgenic UAS-Drudh flies (see Methods in supplementary material) were used to overexpress Drudh in the presence of a GAL4 activator. DRudh overexpression in PCs using dot-GAL4 did not result in increased CBB uptake (Fig. 4C, D and G) although a 60% increase in protein
Fig. 4 – Changing levels of DRudh expression affect CBB uptake. CBB assay on (A) UAS-Drudh-RNAi (B) dot-GAL4; UAS-Drudh-RNAi (C) UAS-Drudh (D) dot-GAL4/UAS-Drudh (E) arm-GAL4/UAS-Drudh PCs of third instar larvae. Arrowhead indicates cardiac tube showing no autofluorescence following CBB treatment. (F, G) Graph showing average pixels/cell observed in the CBB assays for the genotypes indicated (n = 30). Error bars represent Standard Error of the Mean (±s.e.m). Asterisk indicates P < 0.01. Scale Bar = 10 μm.
E XP E RI ME N TA L CE L L RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
1809
Rudhira: a link between PCs and vertebrate SECs Based on their microphagocytic scavenging function, PCs are considered similar to the vertebrate SECs [3]. It is interesting to note in this context that while Drosophila Drudh is expressed in PCs, its murine ortholog is expressed in endothelial precursors during vasculogenesis and angiogenesis [30]. These observations raise interesting questions about evolution of PCs. Are PCs specialized endothelial cells (such as those found in vertebrate liver) strategically placed juxtaposed to the heart to exploit their scavenging function? Attempts to address these intriguing issues regarding PCs will gain significantly from the information about PC function and the reagents generated in this study. Further explorations in PC biology will aid in the comparative functional analysis of vertebrate and invertebrate “professional pinocytes”.
Acknowledgments Fig. 5 – A comparison of macro- and micropinocytic modes of uptake in pericardial cells. A simplistic schematic representation of macro- and micropinocytosis, also showing requirement of Dynamin, DRudhira function and effect of pathway-specific endocytic inhibitors. Wm: Wortmanin; MDc: Monodansylcadaverine; Cpz: Chlorpromazine; EIPA: Ethylisopropylamiloride.
level could be detected by immunostaining in the third larval instar (Fig. S3C–E in supplementary material). DRudh may regulate colloid uptake by interacting with other proteins probably by virtue of its WD40 domains [34]. Hence overexpression of DRudh alone may be ineffective whereas reduction of Rudh levels results in an uptake phenotype. Similar results have been reported in many systems for Dynamin where overexpression of the dominant-negative construct produces a strong inhibitory phenotype but that of the wild type allele has no phenotype owing to the clathrin-mediated regulation of Dynamin's GTPase activity [35,36]. Although dot-GAL4 is expressed in embryonic PCs, it is absent or undetectable in PCs after hatching till late third larval instar. One reason, therefore, for the absence of a gainof-function phenotype could be that over-expression later in development was insufficient to produce an effect. In the absence of any early PC-specific driver we used a ubiquitously expressed driver, armadillo-GAL4 (arm-GAL4), to drive expression of the same UAS-Drudh construct. This combination showed a significant (P < 0.01) enhancement in CBB uptake (Fig. 4C, E and G). This increase in Dynamin-independent uptake is specific since control animals expressing UAS-lacZ instead of UAS-Drudh driven by the same arm-GAL4 driver do not exhibit such enhancement in CBB uptake in PCs (data not shown). The difference in temporal activity of the GAL4 drivers suggests that not just levels but timing of DRudh expression is also important for PC function. However since the arm-GAL4 driver is not specific to PCs, the possibility of ubiquitous Drudh overexpression non-autonomously affecting PC function remains. In either case our results indicate that DRudh regulates macromolecular uptake function in PCs.
We thank Katherine Anderson, Dominique Ferrandon, L. S. Sashidhara, Volker Hartenstein, D. K. Hoshizaki, Kryztof Jagla, Michael Semeriva, Helen Skaer, V. Sriram, and K. VijayRaghavan for fly stocks, reagents and valuable comments. We thank Suma B.S. for help with confocal microscopy. DD is supported by a CSIR-SPM fellowship. This work was funded by the JNCASR, the Council of Scientific and Industrial Research, and Department of Biotechnology, Government of India.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2008.02.009.
REFERENCES
[1] M. Knutson, M. Wessling-Resnick, Iron metabolism in the reticuloendothelial system, Crit. Rev. Biochem. Mol. Biol. 38 (2003) 61–88. [2] B. Smedsrod, Clearance function of scavenger endothelial cells, Comp. Hepatol. 3 (Suppl 1) (2004) S22. [3] T. Seternes, K. Sorensen, B. Smedsrod, Scavenger endothelial cells of vertebrates: a nonperipheral leukocyte system for high-capacity elimination of waste macromolecules, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 7594–7597. [4] A. Crossley, Nephrocytes and pericardial cells, in: G.L. Kerkut, G.A. (Eds.), Comprehensive insect physiology, biochemistry and pharmacology, vol. 3, Pergamon Press, Oxford, 1985, pp. 487–515. [5] E.J. Ward, D.E. Coulter, Odd-skipped is expressed in multiple tissues during Drosophila embryogenesis, Mech. Dev. 96 (2000) 233–236. [6] A. Guha, V. Sriram, K.S. Krishnan, S. Mayor, Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes, J. Cell Sci. 116 (2003) 3373–3386. [7] V. Sriram, K.S. Krishnan, S. Mayor, Deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster, J. Cell Biol. 161 (2003) 593–607.
1810
E XP E RI ME N TA L CE LL RE S E A RCH 3 1 4 ( 2 00 8 ) 1 8 0 4 –18 1 0
[8] M. Elrod-Erickson, S. Mishra, D. Schneider, Interactions between the cellular and humoral immune responses in Drosophila, Curr. Biol. 10 (2000) 781–784. [9] B. Lemaitre, J. Hoffmann, The host defense of Drosophila melanogaster, Annu. Rev. Immunol. 25 (2007) 697–743. [10] D. Hultmark, K. Borge-Renberg, Drosophila immunity: is antigen processing the first step? Curr. Biol. 17 (2007) R22–R24. [11] S. Zaffran, M. Frasch, Early signals in cardiac development, Circ. Res. 91 (2002) 457–469. [12] L. Mandal, U. Banerjee, V. Hartenstein, Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta–gonadal–mesonephros mesoderm, Nat. Genet. 36 (2004) 1019–1023. [13] E. Bier, R. Bodmer, Drosophila, an emerging model for cardiac disease, Gene 342 (2004) 1–11. [14] V.B. Wigglesworth, The pericardial cells of insects:analogue of reticuloendothelail system, J. Reticuloendothel. Soc. 7 (1970) 208–216. [15] M. Poll, Rescheres histophysiologiques sur les tubes de malpighi du Tenebrio molitor, L Rec. Inst. Zool. Torley-Rousseau 5 (1934) 78–126. [16] R.P. Mills, R.C. King, The pericardial cells of Drosophila melanogaster, Q. J. Microsc. Sci. 106 (1965) 261–268. [17] P.P. Grasse, L. Lesperon, Accumulation de colorants acides chez le ver a soie par des tissue differents selon la voie d'acces, Compt. Rend. Acad. Sci. 201 (1935). [18] V.B. Wigglesworth, The circulatory system and associated tissues, the principles of insect physiology, Chapman Hall (1972) 411–473. [19] U. Tepass, V. Hartenstein, The development of cellular junctions in the Drosophila embryo, Dev. Biol. 161 (1994) 563–596. [20] B.C. Bonning, T.F. Booth, B.D. Hammock, Mechanistic studies of the degradation of juvenile hormone esterase in Manduca sexta, Arch. Insect Biochem. Physiol. 34 (1997) 275–286. [21] D. Dulcis, R.B. Levine, Innervation of the heart of the adult fruit fly, Drosophila melanogaster, J. Comp. Neurol. 465 (2003) 560–578. [22] D. Ferrandon, A.C. Jung, M. Criqui, B. Lemaitre, S. Uttenweiler-Joseph, L. Michaut, J. Reichhart, J.A. Hoffmann, A drosomycin-GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway, EMBO J. 17 (1998) 1217–1227.
[23] H.S. Distefano, Effects of silver nitrate on the pigmentation of Drosophila, Am. Nat. 77 (1943) 94–96. [24] G.J. Praefcke, H.T. McMahon, The dynamin superfamily: universal membrane tubulation and fission molecules? Nat. Rev. Mol. Cell Biol. 5 (2004) 133–147. [25] A.M. van der Bliek, E.M. Meyerowitz, Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic, Nature 351 (1991) 411–414. [26] T. Kosaka, K. Ikeda, Reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of Drosophila melanogaster, shibirets1, J. Cell Biol. 97 (1983) 499–507. [27] S. Falcone, E. Cocucci, P. Podini, T. Kirchhausen, E. Clementi, J. Meldolesi, Macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events, J. Cell Sci. 119 (2006) 4758–4769. [28] Y. Rikihisa, Y. Zhang, J. Park, Inhibition of infection of macrophages with Ehrlichia risticii by cytochalasins, monodansylcadaverine, and taxol, Infect. Immun. 62 (1994) 5126–5132. [29] J.J. Chu, M.L. Ng, Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway, J. Virol. 78 (2004) 10543–10555. [30] K. Siva, M.S. Inamdar, Rudhira is a cytoplasmic WD40 protein expressed in mouse embryonic stem cells and during embryonic erythropoiesis, Gene Expr. Patterns 6 (2006) 225–234. [31] D. Das, D. Ashoka, R. Aradhya, M. Inamdar, Gene expression analysis in post-embryonic pericardial cells of Drosophila, Gene Expr. Patterns 8 (2008) 199–205. [32] C.H. Deborah, A. Kimbrell, Clare Bolduc, Kurt Kleinhesselink, Kathy Beckingham, The Dorothy enhancer has Tinman binding sites and drives hopscotch-induced tumor formation, Genesis 34 (2002) 23–28. [33] P. Yi, Z. Han, X. Li, E.N. Olson, The mevalonate pathway controls heart formation in Drosophila by isoprenylation of Ggamma1, Science 313 (2006) 1301–1303. [34] D. Li, R. Roberts, WD-repeat proteins: structure characteristics, biological function, and their involvement in human diseases, Cell Mol. Life Sci. 58 (2001) 2085–2097. [35] H.C. Chang, M. Hull, I. Mellman, The J-domain protein Rme-8 interacts with Hsc70 to control clathrin-dependent endocytosis in Drosophila, J. Cell Biol. 164 (2004) 1055–1064. [36] G. Di Paolo, P. De Camilli, Does clathrin pull the fission trigger? Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 4981–4983.