Macrophage mitochondrial bioenergetics and tissue invasion are boosted by an Atossa‐Porthos axis in Drosophila
2022; Springer Nature; Volume: 41; Issue: 12 Linguagem: Inglês
10.15252/embj.2021109049
ISSN1460-2075
AutoresShamsi Emtenani, Elliot T. Martin, Attila Gyoergy, Julia Bicher, Jakob‐Wendelin Genger, Thomas Köcher, Maria Akhmanova, Mariana Guarda, Marko Roblek, Andreas Bergthaler, Thomas R. Hurd, Prashanth Rangan, Daria E. Siekhaus,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoArticle23 March 2022Open Access Source DataTransparent process Macrophage mitochondrial bioenergetics and tissue invasion are boosted by an Atossa-Porthos axis in Drosophila Shamsi Emtenani Shamsi Emtenani orcid.org/0000-0001-6981-6938 Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft Search for more papers by this author Elliot T Martin Elliot T Martin Department of Biological Sciences, RNA Institute, University at Albany, Albany, NY, USA Contribution: Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Attila Gyoergy Attila Gyoergy orcid.org/0000-0002-1819-198X Institute of Science and Technology Austria, Klosterneuburg, Austria Search for more papers by this author Julia Bicher Julia Bicher Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Investigation Search for more papers by this author Jakob-Wendelin Genger Jakob-Wendelin Genger orcid.org/0000-0003-4502-1094 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Contribution: Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Thomas Köcher Thomas Köcher Vienna BioCenter Core Facilities, Vienna, Austria Search for more papers by this author Maria Akhmanova Maria Akhmanova orcid.org/0000-0003-1522-3162 Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Investigation, Visualization, Writing - review & editing Search for more papers by this author Mariana Guarda Mariana Guarda Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Investigation Search for more papers by this author Marko Roblek Marko Roblek orcid.org/0000-0001-9588-1389 Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Methodology Search for more papers by this author Andreas Bergthaler Andreas Bergthaler orcid.org/0000-0003-0597-1976 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Contribution: Resources, Data curation, Funding acquisition, Writing - review & editing Search for more papers by this author Thomas R Hurd Thomas R Hurd Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Contribution: Conceptualization, Resources, Methodology, Writing - review & editing Search for more papers by this author Prashanth Rangan Prashanth Rangan orcid.org/0000-0002-1452-8119 Department of Biological Sciences, RNA Institute, University at Albany, Albany, NY, USA Contribution: Conceptualization, Resources, Data curation, Funding acquisition, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Daria E Siekhaus Corresponding Author Daria E Siekhaus [email protected] orcid.org/0000-0001-8323-8353 Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Shamsi Emtenani Shamsi Emtenani orcid.org/0000-0001-6981-6938 Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft Search for more papers by this author Elliot T Martin Elliot T Martin Department of Biological Sciences, RNA Institute, University at Albany, Albany, NY, USA Contribution: Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Attila Gyoergy Attila Gyoergy orcid.org/0000-0002-1819-198X Institute of Science and Technology Austria, Klosterneuburg, Austria Search for more papers by this author Julia Bicher Julia Bicher Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Investigation Search for more papers by this author Jakob-Wendelin Genger Jakob-Wendelin Genger orcid.org/0000-0003-4502-1094 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Contribution: Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Thomas Köcher Thomas Köcher Vienna BioCenter Core Facilities, Vienna, Austria Search for more papers by this author Maria Akhmanova Maria Akhmanova orcid.org/0000-0003-1522-3162 Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Investigation, Visualization, Writing - review & editing Search for more papers by this author Mariana Guarda Mariana Guarda Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Investigation Search for more papers by this author Marko Roblek Marko Roblek orcid.org/0000-0001-9588-1389 Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Methodology Search for more papers by this author Andreas Bergthaler Andreas Bergthaler orcid.org/0000-0003-0597-1976 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Contribution: Resources, Data curation, Funding acquisition, Writing - review & editing Search for more papers by this author Thomas R Hurd Thomas R Hurd Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Contribution: Conceptualization, Resources, Methodology, Writing - review & editing Search for more papers by this author Prashanth Rangan Prashanth Rangan orcid.org/0000-0002-1452-8119 Department of Biological Sciences, RNA Institute, University at Albany, Albany, NY, USA Contribution: Conceptualization, Resources, Data curation, Funding acquisition, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Daria E Siekhaus Corresponding Author Daria E Siekhaus [email protected] orcid.org/0000-0001-8323-8353 Institute of Science and Technology Austria, Klosterneuburg, Austria Contribution: Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Shamsi Emtenani1, Elliot T Martin2, Attila Gyoergy1, Julia Bicher1, Jakob-Wendelin Genger3, Thomas Köcher4, Maria Akhmanova1, Mariana Guarda1, Marko Roblek1, Andreas Bergthaler3, Thomas R Hurd5, Prashanth Rangan2 and Daria E Siekhaus *,1 1Institute of Science and Technology Austria, Klosterneuburg, Austria 2Department of Biological Sciences, RNA Institute, University at Albany, Albany, NY, USA 3CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria 4Vienna BioCenter Core Facilities, Vienna, Austria 5Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada *Corresponding author. Tel: +43 2243 9000 5001; E-mail: [email protected] The EMBO Journal (2022)41:e109049https://doi.org/10.15252/embj.2021109049 See also: P Latorre-Muro & P Puigserver (June 2022) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cellular metabolism must adapt to changing demands to enable homeostasis. During immune responses or cancer metastasis, cells leading migration into challenging environments require an energy boost, but what controls this capacity is unclear. Here, we study a previously uncharacterized nuclear protein, Atossa (encoded by CG9005), which supports macrophage invasion into the germband of Drosophila by controlling cellular metabolism. First, nuclear Atossa increases mRNA levels of Porthos, a DEAD-box protein, and of two metabolic enzymes, lysine-α-ketoglutarate reductase (LKR/SDH) and NADPH glyoxylate reductase (GR/HPR), thus enhancing mitochondrial bioenergetics. Then Porthos supports ribosome assembly and thereby raises the translational efficiency of a subset of mRNAs, including those affecting mitochondrial functions, the electron transport chain, and metabolism. Mitochondrial respiration measurements, metabolomics, and live imaging indicate that Atossa and Porthos power up OxPhos and energy production to promote the forging of a path into tissues by leading macrophages. Since many crucial physiological responses require increases in mitochondrial energy output, this previously undescribed genetic program may modulate a wide range of cellular behaviors. Synopsis How immune cells satisfy increased energy demands during tissue infiltration remains unclear. This study combines live imaging, genetics and metabolomic analysis of fly macrophages and identifies a new pathway supporting pioneer cell invasion via metabolic reprogramming. The uncharacterised nuclear protein Atossa (encoded by CG9005) promotes macrophage invasion of the germband in Drosophila embryos. Atossa increases expression of metabolic enzymes NADPH glyoxylate reductase (GR/HPR) and lysine α-ketoglutarate reductase (LKR/SDH), enhancing the mitochondrial Krebs cycle and ATP levels. Atossa targets DEAD-box protein Porthos (Pths), increasing assembled 40S ribosome levels and translation of mitochondrial OXPHOS components. Mammalian orthologs can substitute for Atossa in flies to increase invasion and macrophage bioenergetics. Video Synopsis Macrophage mitochondrial bioenergetics and tissue invasion are boosted by an Atossa-Porthos axis in Drosophila by Shamsi Emtenani, Daria E Siekhaus and colleagues Introduction Charged with protecting the organism against continuously changing threats, the immune system must constantly adapt, altering the location, number, and differentiation status of its different immune cell subtypes (Nicholson, 2016). Such continuous adjustment requires high levels of energy. How immune cells satisfy these increased metabolic requirements is just beginning to be understood (O'Neill et al, 2016; Guak & Krawczyk, 2020). The main energy currency in the cell is ATP, produced from carbohydrates by cytoplasmic glycolysis and the mitochondrial TCA cycle that feeds electron donors into oxidative phosphorylation (OxPhos) complexes I through IV, components in the electron transport chain (ETC). Anaerobic glycolysis is quick, but respiratory OxPhos extracts considerably more ATP from a single molecule of glucose, albeit more slowly (Berg et al, 2002). OxPhos is most directly regulated by the activity and the amount of complexes I through V that carry it out (Hüttemann et al, 2007). Upregulation of OxPhos is known to be required for many important immune cell functions, such as B cell antibody production (Price et al, 2018), pathogenic T-cell differentiation during autoimmunity (Shin et al, 2020), CD8+ memory T-cell development and expansion (van der Windt et al, 2012), T-reg suppressive function (Weinberg et al, 2019), T cell activation by macrophages (Kiritsy et al, 2021), and the maturation of anti-inflammatory macrophages (Vats et al, 2006). However, what changes immune cells initiate to upregulate OxPhos remains unclear and how such shifts in metabolism could influence immune cell migration is unexplored. Immune cells move within the organism to enable distribution and maturation (Kierdorf et al, 2015) and to respond to homeostatic challenges, injuries, tumors, or infections (Luster et al, 2005; Ratheesh et al, 2015). To migrate across unimpeded environments, cells expend energy restructuring their actin cytoskeleton, activating myosin ATPase and reorganizing their cell membrane (Cuvelier et al, 2007). Even greater energy requirements exist when cells must also remodel their surroundings as they move ahead against the resistance of flanking cells or extracellular matrix (Zanotelli et al, 2018, 2019; Kelley et al, 2019). Most in vitro or in vivo studies on the metabolism that enables the migration of diverse immune cell types have highlighted the importance of glycolysis (Semba et al, 2016; Guak et al, 2018; Kishore et al, 2018). To our knowledge, only one study has demonstrated a need for a functional ETC, to speed neutrophil migration in vivo potentially by enabling the polarized secretion of ATP to amplify guidance cues (Zhou et al, 2018). Increases in OxPhos triggered by PGC-1’s transcriptional upregulation of mitochondrial proteins can underlie enhanced invasion and metastasis in some cancer types and suppress it in others (LeBleu et al, 2014; Torrano et al, 2016; Davis et al, 2020). OxPhos has been shown to be particularly required in the first cancer cell leading coordinated chains into challenging environments in vitro (Khalil & Friedl, 2010; Commander et al, 2020); these leader cells have been shown to need higher ATP levels to create a path (Zhang et al, 2019). Although the ability of immune cells to invade tissues or tumors also depends on movement against surrounding resistance, it is not known if immune cells similarly require enhanced levels of OxPhos for such infiltration and if they do how they achieve this energy boost. To identify new mechanisms governing in vivo migration, we study Drosophila macrophages, also called plasmatocytes. Macrophages are the primary innate immune cell in Drosophila and share remarkable similarities with vertebrate macrophages in ontogeny, functions, and migratory behavior (Ratheesh et al, 2015; Wood & Martin, 2017). These macrophages not only resolve infections, but also influence development and homeostasis (Bunt et al, 2010; Buck et al, 2016; Caputa et al, 2019; Riera-Domingo et al, 2020). To reach places where they are needed to enable proper development, some macrophages follow guidance cues and invade the extended germband between the closely apposed ectoderm and mesodermal tissues, moving against the resistance of surrounding tissues (Siekhaus et al, 2010; Ratheesh et al, 2018; Valoskova et al, 2019; Belyaeva et al, 2022). Importantly, the rate-limiting step for this tissue invasion is the infiltration of the pioneer macrophage, a process affected both by the properties of the surrounding tissues (Ratheesh et al, 2018) as well as macrophages themselves (Valoskova et al, 2019; Belyaeva et al, 2022). Here we identify a previously uncharacterized pathway that induces concerted metabolic and mitochondrial reprogramming to support the higher energy levels needed for pioneer cell invasion through changes in translation and metabolic enzyme expression. Our data lay the foundation for mammalian studies on diverse pathological conditions, from autoimmunity to cancer, as well as those independent of migration. Results CG9005 is required in macrophages for their early invasion into the extended germband To find new molecular pathways potentially mediating germband invasion, we examined the BDGP in situ project and identified CG9005 as a previously uncharacterized gene whose mRNA is enriched in macrophages prior to and during germband tissue entry (BDGP in situ of CG9005 mRNA) (Tomancak et al, 2002, 2007). CG9005 mRNA is maternally deposited and expressed in the mesoderm, including the region in which macrophages are specified during Stage 4–6. CG9005 is further upregulated in macrophages starting at Stage 7 while its expression decreases in the remaining mesoderm. CG9005 mRNA remains expressed during Stages 9–12 in macrophages, during their ingression, dissemination, and movement toward and into the germband. After invasion, CG9005 mRNA is downregulated in macrophages to match the lower expression levels found ubiquitously in the embryo. We examined a P-element insertion allele, CG9005BG02278 (CG9005PBG), visualizing macrophages with a nuclear fluorescent marker. Quantification revealed a 36% decrease in macrophages within the germband in CG9005PBG mutant embryos compared to the control (Fig 1A, B and D), similar to CG9005PBG placed over either Df(2R)ED2222 or Df(2R)BSC259 that remove the gene entirely (Fig 1D), demonstrating the allele is a genetic null for invasion. Expressing CG9005 in macrophages in the mutant completely restored their capacity to invade the germband (Fig 1C and D). Driving any of three independent CG9005 RNA interference (RNAi) lines in macrophages decreased macrophages within the germband by 37–40% compared to controls (Fig 1E) and increased macrophages sitting on the yolk near the entry site that have not yet invaded the germband (Fig EV1A) by 24–27%, a shift also seen in CG9005PBG (Fig EV1B). We counted macrophages migrating along the ventral nerve cord (vnc) in late Stage 12 embryos, a route guided by the same factors that lead into the germband (Wood & Martin, 2017) but not requiring tissue invasion (Siekhaus et al, 2010; Ratheesh et al, 2018). There was no significant difference in the CG9005PBG mutant (Fig EV1C) and the CG9005 RNAi-expressing macrophages (Fig EV1D–F) compared to their controls, arguing that basic migratory processes and recognition of chemotactic signals are unperturbed. Moreover, we detected no significant change in the total number of macrophages for these genotypes (Fig EV1G and H). Taken together, these results from fixed embryos indicate that CG9005 is specifically required in macrophages for the early steps of germband invasion. Figure 1. CG9005 acts in macrophages to spur pioneer cell infiltration into the germband tissue A–C. Confocal images of Stage 12 embryos from control, P{GT1}CG9005BG02278 P-element mutant (CG9005PBG), and CG9005PBG with CG9005 expression restored in macrophages. Macrophage: red. Phalloidin to visualize embryo: green. Germband edge: dotted white line. D. Quantification of macrophages that have penetrated the germband from genotypes in (A-C) and from CG9005PBG over two deficiencies (Df) that remove the gene. n = 35, 56, 25, 9, 18 embryos, respectively; P < 0.0001 for control versus CG9005PBG, Df1, or Df2; P = 0.98 for control versus mac>CG9005 rescue; P = 0.91, 0.90 for CG9005PBG versus Df1 or Df2. E. Macrophage-specific knockdown of CG9005 by UAS-RNAi lines. n = 22, 20, 21, 23, 35, 28 embryos. P < 0.0001 for all comparisons. F. Stills from two-photon movies of control and CG9005PBG mutant embryos showing macrophages (nuclei, red) migrating starting at Stage 10 from the head toward the germband and invading the germband tissue. Elapsed time indicated in minutes. Germband edge (white dotted line) detected by yolk autofluorescence. For quantification of migration parameters in movies see (G-L). G, H. Macrophage migration speed (G) in the head or (H) between the yolk sac and the germband edge. For (G): control n = 8 movies, CG9005PBG mutant n = 3; control n = 360 tracks, mutant n = 450, P = 0.65. For (H): control n = 7 movies, mutant n = 3; control n = 46 tracks, mutant n = 19, P = 0.62. I. The time required for the first macrophage nucleus to enter into the extended germband. Control n = 7 movies, mutant n = 5. Time to entry: control = 23 min, CG9005PBG = 38 min, P < 0.0001. J–L. The migration speed of the (J) 1st, (K) 2nd, or (L) 3rd-5th macrophages along the first 25–30 µm into the germband between the mesoderm and ectoderm. In schematics, analyzed macrophages—light blue, other macrophages—red, ectoderm—green, mesoderm—purple, and yolk—beige. For (J): control n = 6 movies, mutant n = 5, P = 0.012. For (K): control n = 5 movies, mutant n = 5, P = 0.03. For (L): control n = 5 movies, mutant n = 4, P = 0.17. Data information: Scale bars: 50 µm (A–C), 30 µm (F). Throughout paper mac> indicates GAL4 driven expression of a UAS construct specifically in macrophages by srpHemo-GAL4. N for movies represents imaging from different embryos. Throughout this work, embryos with stomodeal invagination and germband retraction away from the anterior of < 29% were defined as Stage 10, 29–31% Stage 11, and 35–40% Stage 12. (D) One-way ANOVA with Tukey. (E and G-L) Unpaired t-tests. Graphs show mean ± SEM; ns = P > 0.05, *P < 0.05, ****P < 0.0001. See Source Data 1 and 2 for Fig 1. Source data are available online for this figure. Source Data for Figure 1 [embj2021109049-sup-0008-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. CG9005PBG mutant macrophages migrate normally within the head and along the vnc A, B. Quantification of macrophages in fixed early Stage 12 embryos shows a significant increase on the yolk in (A) lines expressing each of the CG9005 RNAis specifically in macrophages (mac>) and in (B) the P element mutant CG9005PBG compared to the control. For (A) control (n = 43 embryos) versus CG9005PB mutant (n = 50), CG9005PB mutant/Df1 (n = 28) or CG9005PB mutant/Df2 (n = 9), all P < 0.0001. Control versus CG9005PB mutant with mac>CG9005 rescue (n = 20) P = 0.99. CG9005PB mutant alone versus, mutant with mac>CG9005 rescue P = 0.001. For (B) control 1 (n = 21 embryos) versus CG9005 RNAi 1 (n = 20) P = 0.0002; control 2 (n = 25) versus CG9005 RNAi 2 (n = 19) P < 0.0001; control 3 (n = 16) versus CG9005 RNAi 3 (n = 15) P = 0.001. C–F. Macrophage quantification in ventral nerve cord (vnc) segments reveals no significant difference in macrophage migration along the vnc between (C) CG9005PBG mutant and control embryos or (D-F) mac>CG9005 RNAi embryos compared to the controls. For (C) control (n = 7 embryos) versus CG9005PB mutant (n = 15) P > 0.05. For (D) control 1 (n = 8 embryos) versus CG9005 RNAi 1 (n = 13) P = 0.25; for (E) control 2 (n = 8 embryos) versus CG9005 RNAi 2 (n = 16) P = 0.5; for (F) control 3 (n = 8 embryos) versus CG9005 RNAi 3 (n = 16) P > 0.99. G, H. Quantification of the total macrophage number reveals no significant difference between (G) the control and CG9005PBG mutant embryos, or (H) the control and mac>CG9005 RNAi embryos. For (G) control (n = 43 embryos) versus CG9005PBG mutant (n = 50) P = 0.69. For (H) control 1 (n = 12 embryos) versus CG9005 RNAi 1 (n = 17) P = 0.9; control 2 (n = 27) versus CG9005 RNAi 2 (n = 19) P = 0.84; control 3 (n = 23) versus CG9005 RNAi 3 (n = 27) P = 0.16. I. Stills from two-photon movies of control and CG9005PBG mutant embryos, showing macrophages migrating starting at Stage 10 from the head toward the germband. Elapsed time indicated in minutes. The germband edge (white dotted line) was detected by yolk autofluorescence. For quantification of migration parameters from two-photon live imaging of macrophages, see (J-L). J. Macrophages on the yolk sac in the CG9005PBG mutant reach the germband with a similar speed to control macrophages. Speed: control and mutant = 2.2 µm/min, P = 0.78; control n = 8 movies, mutant n = 3; control n = 373 tracks, mutant n = 124. K, L. Macrophage directionality (K) in the head or (L) on the yolk sac shows no change in the CG9005PBG mutant compared to the control. For (K) head directionality: control = 0.39, mutant = 0.37, P = 0.74; control n = 7 movies, mutant n = 3. For (L) yolk sac directionality: control = 0.40, mutant = 0.39, P = 0.86; control n = 7 movies, mutant n = 3. Data information: Macrophages analyzed in (A-L) were labeled with srpHemo-H2A::3xmCherry to visualize nuclei. In schematics, macrophages are shown in red and analyzed macrophages in light blue, the ectoderm in green, the mesoderm in purple, and the yolk in beige. Throughout this work mac> indicates srpHemo-GAL4 driving UAS constructs specifically in macrophages. Mean ± SEM, ns=P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA with Tukey (A) and unpaired t-test (B-H) and (J-L). Scale bar: 30 µm (I). See Source Data 1 and 2 for Fig EV1. Source data are available online for this figure. Download figure Download PowerPoint Atossa (CG9005) promotes efficient invasion of pioneer macrophages into the germband tissue To directly assess CG9005’s role in germband invasion, we conducted two-photon live imaging in control and CG9005PBG embryos, visualizing macrophage nuclei with srpHemo-H2A::3xmCherry (Figs 1F and EV1I, Movies EV1 and EV2). We observed no significant change in CG9005PBG in macrophage speed or directionality during their migration starting at Stage 9 from the head mesoderm up to the yolk neighboring the germband entry point and beyond between the yolk and the ectoderm (Figs 1G and H, and EV1J–L) (Speed in the head and on yolk: 2.2 µm/min for control and CG9005PBG; P = 0.65, P = 0.78, respectively. Directionality: 0.39 in control, 0.37 in mutant in both regions, P = 0.74 for head, P = 0.86 for yolk. Speed along ectoderm control = 2.6, CG9005PBG = 2.5 µm/min, P = 0.62). However, the first macrophage in CG9005PBG required 65% more time than the control to enter into the germband tissue (time to entry: control = 23 min, CG9005PBG = 38 min, P < 0.0001) (Fig 1I). The speed of the first two pioneering macrophages is also significantly slower as they invade along the path between the mesoderm and ectoderm in CG9005PBG mutant embryos compared to the control (Fig 1J and K) (1st cell: control = 2.5, CG9005PBG = 2 µm/min, P = 0.012; 2nd cell: control = 2.9, CG9005PBG = 2.1 µm/min, P = 0.03). However, the speed of the next few cells migrating along this path was not affected (Fig 1L) (3rd–5th cells: control = 2.5, CG9005PBG = 2.4 µm/min, P = 0.17). We conclude that CG9005 specifically regulates initial tissue invasion, facilitating the entry into and subsequent movement within the germband tissue of the first two pioneer macrophages. Since the macrophage stream into the germband becomes much reduced in CG9005PBG, we called the gene atossa (atos), for the powerful Persian queen whose name means trickling. Atossa (CG9005) is a nuclear protein whose conserved motifs and TADs are important for macrophage tissue invasion Atossa (Atos) contains a conserved domain of unknown function (DUF4210) and a chromosome segregation domain (Chr_Seg) (Fig 2A). Atos also displays two trans-activating domains (TADs) common among transcription factors, three nuclear localization signals (NLS), and a nuclear export signal (NES). We found FLAG::HA-tagged Atos mainly in the nucleus in embryonic macrophages in vivo (Fig 2B) and in macrophage-like S2R+ cells where it was also partially in the cytoplasm (Fig EV2A). Atos mutant forms lacking the conserved domains and TADs were similarly present in the nucleus (Fig EV2A) yet were unable to rescue germband invasion (Figs 2C and D, and EV2B and C). Consistent with a germband invasion defect, atosPBG embryos expressing these atos mutants had more macrophages sitting on the yolk at the germband entry site prior to invasion than those expressing wild-type Atos (Fig EV2D). Atos is 40% identical to its uncharacterized murine orthologs, mFAM214A-B, which maintain these domains (Fig 2A). Expression in macrophages of either mFAM214A or B in atosPBG rescued the germband invasion defect as efficiently as the Drosophila protein itself (Fig 2E and F) and restored the normal number of macrophages on the yolk neighboring the extended germband (Fig EV2E). These data clearly show that the conserved domains and TADs are critical for the primarily nuclear protein, Atos, to facilitate macrophage invasion. Figure 2. CG9005/Atossa requires conserved domains linked to transcriptional activation to enhance tissue invasion, a function maintained by its mammalian orthologs A. Deduced protein structure of Drosophila CG9005/Atossa (Atos) and its murine orthologs, mFAM214A-B, highlighting conserved domains. FAM214A-B are 44–45% identical to Atos. B. Macrophages (red) near the germband in Stage 11/12 embryos. Atos tagged at N terminus with HA (HA-antibody, green) and expressed under direct control of macrophage-specific promoter. Nucleus stained by DAPI (blue). C, D. Confocal images or (D) quantification of the macrophages in germband in Stage 12 embryos from the control, atosPBG, and atosPBG expressing Atos itself or variants lacking particular domains. Transgene expression directly from macrophage-specific promoter (mac-). For control (n = 32 embryos) versus atosPBG mutant (n = 56) P < 0.0001; versus rescue with mac-atos (n = 18) P > 0.99; versus rescue with mac-atosDUF- (n = 17) P = 0.0003; versus rescue with mac-atosChrSeg-(n = 21) P = 0.0003; versus rescue with mac-atosDUF-/ChrSeg-(n = 19) P = 0.00014; versus rescue with mac-atosTAD1-/ TAD2- (n = 25) P = 0.0009, atosPBG mutant versus rescue with mac-atos P = 0.0031. E. Confocal images of atosPBG rescued by expressing Atossa’s murine orthologs, mFAM214A or B (mFAMA-B) in macrophages, F. Quantification of macrophages in the germband in Stage 12 embryos from the control, atosPBG, and atosPBG embryos expressing mFAM214A or B specifically in macrophages (mac-). For control (n = 24 embryos) versus atosPBG mutant (n = 56) P < 0.0001; versus mac-atos rescue (n = 18) P = 0.7; versus mac-mFAMA rescue (n = 22) P = 0.6; versus mac-mFAMB rescue (n = 25) P = 0.086. For atosPBG mutant versus mac-atos rescue P = 0.0006; versus mac-mFAMA rescue P = 0.0002; versus mac-mFAMB rescue P = 0.0043. Data information: Germband edge: dotted white line. Mac indicates direct expression from the srpHemo promoter. (C, E) Macrophage nuclei (red), actin by Phalloidin staining (green). (D,F) One-way ANOVA with Tukey. Mean ± SEM, ns=P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars: 5 µm (B), 50 µm (C, E). See Source Data 1 and 2 for Fig 2. Source data are available online for this figure. Source Data for Figure 2 [embj2021109049-sup-0009-SDa
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