A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion

  1. Katarina Valoskova
  2. Julia Biebl
  3. Marko Roblek
  4. Shamsi Emtenani
  5. Attila Gyoergy
  6. Michaela Misova
  7. Aparna Ratheesh
  8. Patricia Reis-Rodrigues
  9. Kateryna Shkarina
  10. Ida Signe Bohse Larsen
  11. Sergey Y Vakhrushev
  12. Henrik Clausen
  13. Daria E Siekhaus  Is a corresponding author
  1. Institute of Science and Technology Austria, Austria
  2. University of Warwick, United Kingdom
  3. University of Copenhagen, Denmark
6 figures, 1 table and 4 additional files

Figures

Figure 1 with 1 supplement
T antigen is enriched on Drosophila macrophages prior to and during their invasion of the extended germband.

(A) Schematic of T antigen modification of serine (S) and threonine (T) on proteins within the Golgi lumen, through successive addition of GalNAc (yellow) by GalNAcTs and Gal (blue) by C1GalTs. …

https://doi.org/10.7554/eLife.41801.003
Figure 1—source data 1

Source data on the quantification of T antigen levels shown in Figure 1E and Figure 1I, the number of macrophages in the germband shown in Figure 1K–M, and the number on the yolk shown in Figure 1—figure supplement 1N–O,Q and on the vnc (Figure 1—figure supplement 1P).

https://doi.org/10.7554/eLife.41801.005
Figure 1—figure supplement 1
Lectin screen reveals enriched staining for PNA and UEA-1 on macrophages.

(A–L) Confocal images of fixed late Stage 11/early Stage 12 wild type embryos (schematic above) stained with different lectins (visualized in green) indicated in green type in the lower left corner. …

https://doi.org/10.7554/eLife.41801.004
Figure 2 with 1 supplement
An atypical MFS family member, CG8602, located in the Golgi and endosomes, is required for T antigen enrichment on invading macrophages.

(A,B) qPCR quantification (2-ΔCt) of RNA levels in mCherry+ macrophages FACS sorted from srpHemo-3xmCherry wild type embryos reveals no significant change in the expression of (A) the C1GalTA …

https://doi.org/10.7554/eLife.41801.006
Figure 2—source data 1

Source data on the quantification of C1GalTA, Ugalt, and CG8602 expression in macrophages by qPCR (shown in Figure 2A–B,D–E,G) and the Pearson’s coefficient for CG8602 colocalization with different markers (Figure 2H).

https://doi.org/10.7554/eLife.41801.008
Figure 2—figure supplement 1
CG8602 expression and localization.

(A) Schematic depicting the CG8602 gene and the insertion site of the EP3102 P element and the Δ33 excision mutant induced by P element mobilization which removes 914 bp of the ORF. (B–J) Confocal …

https://doi.org/10.7554/eLife.41801.007
Figure 3 with 4 supplements
CG8602, which we name Minerva, is required in macrophages for their efficient invasion of the germband.

(A–C) Representative confocal images of early Stage 12 embryos from (A) control, (B) P{EP}CG8602EP3102 = minerva (mrva)3102 mutant, and (C) mrva3102 mutants with macrophage expression of the gene …

https://doi.org/10.7554/eLife.41801.009
Figure 3—source data 1

Source data on the quantification of macrophages in the germband shown in Figure 3D–E,G and Figure 3—figure supplement 1A, on the yolk (Figure 3F) on the vnc (Figure 3H, Figure 3—figure supplement 1C) and in the whole embryo (Figure 3—figure supplement 1D–E).

Source data on the xyz position of macrophages from the tracks that form the basis of the analysis shown in Figure 3J–K,M and Figure 3—figure supplement 1F,G. Source data on the mismigration of germ cells, the levels of mrva expression in the ovary, and the migration of border cells (Figure 3—figure supplement 1I,J and L, respectively). Source data on the xyz position of macrophages in the movies of the mrva3102 mutant and the control underlying the analysis shown in Figure 3J-K,M and Figure 3—figure supplement 1F-G.

https://doi.org/10.7554/eLife.41801.011
Figure 3—figure supplement 1
CG8602 (Minerva) affects macrophage migration into the germband but not along the vnc and does not alter border cell or germ cell migration.

(A) Quantification of the number of macrophages in the germband (dotted circle in schematic) in embryos from control, mrva23102, and mrva3102 srpHemo(macro)-mrva::HA showing Mrva is required in …

https://doi.org/10.7554/eLife.41801.010
Figure 3—video 1
Representative movie of macrophage migration into the germband in the control.

Macrophages (red) are labeled with srpHemo-H2A::3xmCherry. The time interval between each acquisition is 40 s and the display rate is 15 frames/sec. Scale bar represents 30 μm.

https://doi.org/10.7554/eLife.41801.012
Figure 3—video 2
Representative movie of macrophage migration into the germband in the mrva3102 mutant.

Macrophages (red) are labeled with srpHemo-H2A::3xmCherry. The time interval between each acquisition is 40 s and the display rate is 15 frames/sec. Scale bar represents 30 μm.

https://doi.org/10.7554/eLife.41801.013
Figure 3—video 3
Representative movies of macrophage migration on the vnc in the control and mrva3102 mutant.

Macrophages (red) are labeled with srpHemo-H2A::3xmCherry. The time interval between each acquisition is 40 s and the display rate is 15 frames/sec. Scale bar represents 30 μm.

https://doi.org/10.7554/eLife.41801.014
Figure 4 with 1 supplement
Glycoproteomic analysis reveals Minerva is required for higher levels of T-antigen on a subset of proteins.

(A) Representative Western blot of protein extracts from Stage 11/12 control and mrva3102 mutant embryos probed with T antigen antibody. Arrows indicate decreased/missing bands in the mutant …

https://doi.org/10.7554/eLife.41801.016
Figure 4—figure supplement 1
Related to Supplementary file 1: Further information on the mass spectrometry results.

(A) Work flow for mass spectrometry analysis of T and Tn antigen modifications on proteins in Stage 11/12 control and mrva3102 mutant embryos. (B, C) Annotated ETD MS2 spectra of the VHQPSATPASK …

https://doi.org/10.7554/eLife.41801.017
Figure 5 with 2 supplements
Qsox1 is required for macrophage dissemination and entry into the germband tissue.

(A) Representative confocal images of early Stage 12 embryos from control and P{SUPor-P}Qsox1KG04615 = qsox1KG04615. (B–C) Quantification in early Stage 12 embryos showing a significant reduction in …

https://doi.org/10.7554/eLife.41801.018
Figure 5—source data 1

Source data on the quantification of macrophages in the germband shown in Figure 5B-C, on the yolk shown in Figure 5—figure supplement 1A,1D, on the vnc shown in Figure 5—figure supplement 1B,1E, and in the whole embryo shown in Figure 5—figure supplement 1C.

Source data on the xyz position of macrophages from the tracks that form the basis of the analysis shown in Figure 5E-G. Source data on the quantification of the Pearson's coefficient for Qsox1 colocalization with different markers shown in Figure 5H and the quantification of LanA intensity shown in Figure 5J and Figure 5—figure supplement 1L-N. Source data on the xyz position of macrophages in the movies of the qsox1KG04615 mutant underlying the analysis shown in Figure 5E-G and Figure 5—figure supplement 1F.

https://doi.org/10.7554/eLife.41801.020
Figure 5—figure supplement 1
Qsox1 affects germband entry and Laminin A.

(A–B) Quantification of macrophages in fixed mid-Stage 12 embryos in control and qsox1KG04615 mutants reveals that in the mutant there are (A) increased numbers of macrophages on the yolk …

https://doi.org/10.7554/eLife.41801.019
Figure 5—video 1
Representative movie of macrophage migration into the germband in the qsox1KG04615 mutant.

Macrophages (red) are labeled with srpHemo-H2A::3xmCherry. The time interval between each acquisition is 40 s and the display rate is 15 frames/sec. Scale bar represents 30 μm.

https://doi.org/10.7554/eLife.41801.021
Figure 6 with 1 supplement
Minerva’s murine ortholog, MFSD1, can substitute for Minerva’s functions in migration and T-antigen glycosylation.

(A) Topology prediction of mouse MFSD1 (NP_080089.1) using the online tools TMPred (Hofman and Stoffel, 1993) and Protter (Omasits et al., 2014). 50% of amino acids are identical between the M. …

https://doi.org/10.7554/eLife.41801.022
Figure 6—source data 1

Source data on the quantification of Pearson’s coefficient for MFSD1 colocalization with different markers (Figure 6C), the number of macrophages in the germband (Figure 6E) and the level of T antigen in macrophages (Figure 6F).

https://doi.org/10.7554/eLife.41801.024
Figure 6—figure supplement 1
MFSD1-eGFP localization in colon, breast, lung and skin cancer cells.

(A) Alignment of Minerva and mmMFSD1 by BLAST. The first row shows the Minerva sequence in blue type, the second identical (one letter symbol) or similar (+) amino acids in black, and the third the …

https://doi.org/10.7554/eLife.41801.023

Tables

Key resources table
DesignationSource or referenceIdentifiersAdditional
information
mrvaNAFlyBase:FBgn0035763
qsox1NAFlyBase: FBgn0033814
C1GalTANAFlyBase: FBgn0032078
srp-Gal4PMID: 15239955
srp-3xmCherryPMID: 29321168RRID:BDSC_78358 and 78359
srp-H2A::3xmCherryPMID: 29321168RRID:BDSC_78360 and 78361
UAS-CG8602::FLAG::HAPMID: 22036573
mrva3102Bloomington Drosophila Stock Center (BDSC), RRID:SCR_006457RRID:BDSC_17262
Df(3L)BSC117BDSC, RRID:SCR_006457RRID:BDSC_8976
UAS-mCherry.NLSBDSC, RRID:SCR_006457RRID:BDSC_38425
C1GalTA2.1BDSC, RRID:SCR_006457RRID:BDSC_28834
C1GalTA RNAi 1Vienna Drosophila Resource Centre (VDRC), RRID:SCR_013805VDRC: 2826
C1GalTA RNAi 2VDRC, RRID:SCR_013805VDRC: 110406
CG8602 RNAiVDRC, RRID:SCR_013805VDRC: 101575
qsox1RNAiVDRC, RRID:SCR_013805VDRC: 108288
qsox1 KG04615BDSC, RRID:SCR_006457RRID:BDSC_13824
MC-38OtherGift from Borsig lab, Univ of Zurich (UZH)
4T1OtherATCC Cat# CRL-2539, RRID:CVCL_0125Gift from Borsig lab, UZH
LLC1OtherATCC Cat# CRL-1642, RRID:CVCL_4358Gift from Borsig lab, UZH
B16-BL6OtherNCI-DTP Cat# B16BL-6, RRID:CVCL_0157Gift from Borsig lab, UZH
S2R+OtherGift from Frederico Mauri of the Knoblich lab at IMBA, Vienna
srpHemo-CG8602::3xmCherrythis paperCG8602 amplified from genome cloned into DSPL172 (PMID: 29321168)
MT-CG8602::FLAG::HADrosophila Genomic Resource Center (DGRC), RRID:SCR_002845DGRC: FMO06045
MT-Qsox1::FLAG::HADGRC, RRID:SCR_002845DGRC: FMO06379
PTS1-GFPOtherGift from Dr. McNew
MFSD1-eGFPthis paperMFSD1 amplified from dendritic cell cDNA library, inserted into Doxycycline inducible
expression vector pInducer20
Anti-GFP clone 2B6OtherGift from Ogris lab, MFPL Vienna; (1:100) for WB
Anti-GFP clone 5G4OtherGift from Ogris lab, MFPL Vienna; (1:50) for immunochemistry
Anti-T-antigen (mouse monoclonal)PMID: 23584533(1:5 for
immunochemistry; 1:10 for WB)
Anti-profilin (mouse monoclonal)Developmental Studies
Hybridoma Bank (DSHB), RRID:SCR_013527
DSHB Cat# chi 1J, RRID:AB_528439(1:50)
Anti-GAPDH (rabbit monoclonal)Abcam, RRID:SCR_012931Abcam Cat# ab181603, RRID:AB_2687666(1:10000) for WB
Anti-GRASP65 (rabbit polyclonal)Thermo Fisher Scientific, RRID:SCR_008452ThermoFischer Cat# PA3-910, RRID:AB_2113207(1:200) for immunochemistry
Anti-Rab5 (rabbit monoclonal)Cell Signaling Technology (CST), RRID:SCR_004431, Clone C8B1CST Cat# 3547, RRID:AB_2300649(1:200) for immunochemistry
Anti-Rab7 (rabbit monoclonal)CST, RRID:SCR_004431, Clone D95F2CST Cat# 9367, RRID:AB_1904103(1:200) for immunochemistry
Anti-LAMP1 (rabbit polyclonal)Abcam, RRID:SCR_012931Abcam Cat# ab24170, RRID:AB_775978(1:200) for immunochemistry
Anti- Cnx99a (mouse monoclonal)DSHB, RRID:SCR_013527DSHB Cat# Cnx99A 6-2-1, RRID:AB_2722011(1:5)
Anti- Hrs 27.4 (mouse monoclonal)DSHB, RRID:SCR_013527DSHB Cat# Hrs 27–4, RRID:AB_2618261(1:5)
Anti- Golgin 84
(mouse monoclonal)
DSHB, RRID:SCR_013527DSHB Cat# Golgin8412–1, RRID:AB_2722113(1:5)
Anti Rab7 (mouse monoclonal)DSHB, RRID:SCR_013527DSHB Cat# Rab7, RRID:AB_2722471(1:5)
Anti-GMAP (goat polyclonal)DSHB, RRID:SCR_013527DSHB Cat# GMAP, RRID:AB_2618259(1:50)
Anti- Golgin 245 (goat polyclonal)DSHB, RRID:SCR_013527DSHB Cat# Golgin245, RRID:AB_2618260(1:50)
Anti- HA (rat monoclonal)Roche, RRID:SCR_001326Roche Cat# 3F10, RRID:AB_2314622(1:50)
Anti-LanA (rabbit polyclonal)PMID:9257722gift from Stefan Baumgartner
(1:500)
Anti-Vasa (rat monoclonal)DSHB, RRID:SCR_013527DSHB Cat# anti-vasa, RRID:AB_760351(1:25)
Alexa 488- or 557- or 633- secondariesThermo Fisher Scientific, RRID:SCR_008452(1:500 for 488 and 557; 1:100 for 633)
Goat-anti-rabbit IgG (H + L)-HRPBioRadBio-Rad Cat# 170–6515, RRID:AB_2617112(1:10000)
Goat-anti-mouse IgG (H/L):HRPBioRadBio-Rad Cat# 170–6516, RRID:AB_11125547(1:10000)
LysoTracker Green DND-26Invitrogen, RRID:SCR_008410L752675 nM
Alexa Fluor 488 PhalloidinInvitrogen, RRID:SCR_008410A12379(1:500)
Vectashield mounting mediumVector Laboratories, RRID:SCR_000821VectorLabs: H-1000
Vectashield mounting medium with DAPIVector Laboratories, RRID:SCR_000821VectorLabs: H-1200
Halocarbon Oil 27Sigma-Aldrich, RRID:SCR_008988Sigma Aldrich: Cat# H8773
srpHemo-mrvathis paperCG8602 amplified from genome cloned into srpHemo plasmid
srpHemo-MFSD1this papermmMFSD1 amplified from dendritic cell cDNA library cloned into srpHemo plasmid
Mrva fwFly Primer BankqPCR; 5'TGTGCTTCGTGGGAGGTTTC
Mrva rvFly Primer BankqPCR; 5'GCAGGCAAAGATCAACTGACC
C1GalTA fwFly Primer BankqPCR; 5'TGCCAACAGTCTGCTAGGAAG
C1GalTA rvFly Primer BankqPCR: 5'CTGTGATGTGCATCGTTCACG
Ugalt fwFly Primer BankqPCR; 5'GCAAGGATGCCCAGAAGTTTG
Ugalt rvFly Primer BankqPCR; 5'GATATAGACCAGCGAGGGGAC
RpL32 fwFly Primer BankqPCR; 5'AGCATACAGGCCCAAGATCG
RpL32 rvFly Primer BankqPCR; 5'TGTTGTCGATACCCTTGGGC
Lectin staining kit #2EY LaboratoriesEYLabs:FLK-002
FIJIhttp://fiji.sc/
RRID:SCR_002285)
Imarishttp://www.bitplane.com/imaris/imaris, RRID:SCR_007370
Matlabhttps://www.mathworks.com/products/matlab.html,
RRID:SCR_001622
FlowJohttps://www.flowjo.com/RRID:SCR_008520
LaVision ImSpectorhttp://www.lavisionbiotec.com/,
RRID:SCR_015249
Proteome Discoverer 1.4https://www.thermofisher.com/order/
catalog/product/OPTON-30795, RRID:SCR_014477
LightCycler 480 softwarehttps://lifescience.roche.com/en_at/products/lightcycler14301-480
-software-version-15.html
GraphPad Prismhttps://www.graphpad.com/scientific-software/prism/
RRID:SCR_002798

Additional files

Supplementary file 1

Mass spectrometric analysis of the T and Tn antigen containing O-glycoproteome from wild type and mrva3102 mutant Stage 11–12 Drosophila melanogaster embryos.

Each row lists an individually identified tryptically processed peptide. The 2nd–4th columns describe the analyzed peptide. The 5th, 6th, 7th and 12th are the names and accessions to Uniprot. The 8th indicates the position of the modified amino acid. The 9th indicates the number and 10th the type of glycosylation. The 11th lists the exact position and the 13th the exact description of glycosylation. The 14th is the ratio of the amount of the particular glycopeptide in the control samples (medium) over the amount in the mrva3102 (light). The 15th is the number of missed cleavages after the tryptic digest. The 16th is the measured intensity. The 17th column shows the mass to charge ratio.

https://doi.org/10.7554/eLife.41801.025
Supplementary file 2

All candidate proteins from the O-glycoproteome with at least 3-fold changes in T and Tn antigen in the mrva3102 mutant.

Columns list the gene name, the predicted or known function of the gene, if other T or Tn glycosites on the protein are unchanged or changed in the opposite direction, any known human ortholog (identified by BLAST), references for links to cancer and cancer invasion for the mammalian orthologs, the precise site altered, the T and Tn antigen changes observed at a particular glycosylation site, the number of glycosites on the peptide, the peptide sequence and if the glycosylation site is conserved. The site is considered conserved if the human ortholog has a serine or threonine ±5 amino acids from the Drosophila glycosite. References: 1 (Göhrig et al., 2014); 2. (Fan et al., 2018); 3. (Webb et al., 1999); 4. (C.-C. Chiu et al., 2011); 5. (Huang et al., 2016); 6. (Matos et al., 2015); 7. (Cawthorn et al., 2012); 8. (Cao et al., 2015) 9. (Walls et al., 2017); 10.(Zhou et al., 2017); 11. (Linton et al., 2008); 12. (Bian et al., 2016) 13. (Zhang et al., 2016); 14. (Gonias et al., 2017); 15. (Katchman et al., 2013; Katchman et al., 2011); 16. (Stojadinovic et al., 2007); 17. (Zhou et al., 2016); 18. (Hu et al., 2018); 19. (Li et al., 2008); 20. (Senanayake et al., 2012); 21. (Sheu et al., 2014); 22. (Mao et al., 2018); 23.(Yokdang et al., 2016).

https://doi.org/10.7554/eLife.41801.026
Supplementary file 3

T or Tn antigen modified receptors from the wild-type St 11–12 Drosophila melanogaster embryo O-glycoproteome.

Columns list the gene name for the receptor, its reported function, what kind of glycosylation we identified to be present on the receptors in the wild type sample, and what kind of glycosylation change we observed in the mrva3102 mutant.

https://doi.org/10.7554/eLife.41801.027
Transparent reporting form
https://doi.org/10.7554/eLife.41801.028

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