Niche cells spatially restrict stem-cell self-renewal signaling via receptor-ligand degradation

Abstract Stem-cell niche signaling is short-range in nature, such that only stem cells and not their differentiating progeny experience self-renewing signals1. At the apical tip of the Drosophila testes, 8 to 10 germline stem cells (GSCs) surround the hub, the niche signaling center. Microtubule-based nanotubes (MT-nanotubes) formed by GSCs project into the hub cells, serves as the platform for niche signal reception. Here we show that the receptor for Decapentaplegic (Dpp) accumulated on MT-nanotubes is internalized into hub cells together with the Dpp ligand, and both are degraded in the hub cell lysosomes. Perturbation of hub lysosomal function or MT-nanotube formation lead to excess receptor retention within GSCs as well as excess Dpp ligand that diffuses out of the hub. Our results indicate that degradation of the self-renewal ligand/receptor by niche cells specially restrict the niche signal range, and that might be a general feature of stem-cell niche signaling.

3 arrowheads in Figure 2A, B, E and D). However, CQ treatment of Tkv-YFP protein trap testis showed clear Tkv accumulation in the lysosomes in the hub and later stage spermatocytes ( Figure   S2B, cells distal from hub, B'), indicating that Tkv degradation in GSCs and their immediate progeny are minimum level. Therefore, we conclude that majority of Tkv protein in GSC is degraded in hub cells.
To examine the consequence of defective Tkv degradation on downstream signaling, testes from CQ-fed males were labelled for pMad, an indicator of Dpp signal activation ( Figure 2G, H and I).
CQ-mediated lysosomal inhibition increased pMad levels in GSCs. Overall, these data suggest that Tkv's transport to the hub lysosomes is mediated by MT-nanotubes, and further indicate that Tkv degradation in the hub lysosome is essential for reducing the niche signal.
To determine whether the lysosomes originate in the hub or germ cells, genes involved in lysosomal-dependent degradation were knocked down using hub-and/or germ cell-specific GAL4 drivers. Spinster (Spin) is a lysosomal H + -carbohydrate transporter and a known regulator of lysosomal biogenesis, as well as a regulator of Dpp signaling 15,16 . Lamp1 is an abundant protein in the lysosomal membrane that is required for lysosomes to fuse with endosomes/autophagosomes 17 .
Germ cell-specific knockdown of these genes did not alter pMad level ( Figure  Ubiquitination of membrane proteins is required for recognition by the endosomal sorting complexes required for transport, and thence endocytosis, lysosomal fusion and degradation 18 . SMAD ubiquitination regulatory factor (Smurf)-mediated Tkv ubiquitination is necessary for GSC differentiation both in testicular and ovarian GSCs 19,20 . Smurf is a HECT (Homologous to the E6-AP Carboxyl Terminus) domain containing protein with E3 ubiquitin ligase activity, and disruption of Smurf function enhances Dpp-Tkv signal activation 20 . It has been reported that Tkv Ser238 residue phosphorylation is required for Smurf dependent ubiquitination 19 . To determine if ubiquitinationdefective Tkv no longer localizes to hub cells we mutated the Ser 238 phosphorylation site to alanine.
Unlike wild-type Tkv-GFP, which localized to the hub lysosomes, Tkv-S238A-GFP was predominantly localized to the GSC cell body along plasma membrane. Although Tkv-S238A-GFP All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint illuminates MT-nanotubes (thread-like pattern) in the hub, typical lysosome localization pattern (round punctae-like pattern) dominated in control testis was barely observed, indicating that ubiquitination is critical for Tkv's translocation into hub lysosomes ( Figure 3O and P, Table1).
Expression of Tkv-S238A-GFP in the GSC resulted in elevated signal activation as indicated by higher pMad levels ( Figure 3Q, R and S). Thus, Tkv protein is the target of lysosomal degradation in hub cells, allowing hub lysosomes to negatively regulate Dpp signaling.
Since Dpp ligand also localizes to hub lysosomes ( Figure 1G), we hypothesized that Dpp may be also degraded there. To test this idea, we expressed Dpp-mCherry fusion protein specifically in hub cells using the hub specific GAL4 driver. mCherry signal was only observed within the hub, and no signal was detected outside the hub ( Figure 4A). CQ treatment of the testis to impair lysosome function increased the size of Dpp-mCherry positive punctae within hub cells as seen for Tkv ( Figure 4B, C). We also detected abundant Dpp-mCherry signal outside the hub after a 2 hourtreatment ( Figure 4B, D). Similar results were obtained by using a Dpp-GFP line carrying a fosmid genomic construct in which Dpp has been fused to GFP 21 ( Figure S4). These data support the idea that Dpp is also degraded in hub lysosomes. Furthermore, after 4 hour-CQ treatment, we also detected TIPF (a reporter of ligand-bound Tkv) in broad area outside of the hub, consistent with the idea of Dpp diffusion ( Figure 4E, F).
To determine if Dpp-mCherry detected outside the hub is due to its free diffusion into the extracellular space, we used fluorescence recovery after photobleaching (FRAP) analysis. After photobleaching, the recovery was rapid with an average time to reach to 50% of the original intensity, 8.2 ± 2.2 seconds (n=6) ( Figure 4G, H, movie3, 4). Signal did not fully recover indicating the possible existence of bleached background caused by autofluorescence or the possibility that some Dpp protein might be trapped likely binding to the extracellular matrix components in photobleached field as reported in Drosophila embryo 22 . The Dpp-mCherry signal in lysosomes within the hub did not recover quickly after photobleaching (movie5, no recovery was seen up to 30 min monitoring, n=10 experiments) as seen in the areas outside of the hub when bleached. These data suggest that in the absence of proteolysis Dpp ligand can diffuse from the hub, and that MTnanotubes and lysosome activity prevent it from doing so.

Discussion
We have shown previously that niche cells and stem cells interact in a contact-dependent manner, with GSCs and the hub cells engaging in productive signaling via MT-nanotubes, enabling All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint highly specific cell-cell interactions and excluding non-stem cells from receiving the stem cell signals. Here, we demonstrate that lysosomes in the niche cells, also through MT-nanotube interactions, degrade both the ligand from the niche, its receptor from the stem cell, thus dampening stem-cell renewal signaling ( Figure 4I). This ensures the removal of signaling molecules and further prevents "contact-independent" ligand-receptor interactions outside of the niche. Indeed, lysosomal localization-defective Tkv (S238A), and MT-nanotube loss, both cause elevated Tkv levels within GSCs, and MT-nanotube loss also induces Dpp ligand diffusion in the Drosophila testis. Proteolysis manipulation in our study changed the localization of Tkv from one place to the other (in the Hub lysosomes or GSC plasma membrane, Table1), suggesting that there may be two steps of this regulation, first, Tkv trafficking into hub cells, second; Tkv degradation in hub lysosomes. We examined here Spin and Lamp1, likely regulating the terminal step within lysosomes. Identification of more regulators will help to understand entire molecular mechanism. Cytonemes, another type of actin-dependent signaling protrusion 23 24 , also transfer ligand and receptor, allowing the interaction between cells at a distance. Ligand-producing and receptor-producing cells both form cytonemes and both cells have been observed to take up signaling proteins: receptor into the ligand-producing cells and ligand into the receptor-producing cells 24 , indicating the universality of such transfer in general contact-dependent signaling. It remains to be investigated whether lysosomal proteolysis of ligand and receptor, as demonstrated by our study, might also regulate signaling by cytonemes. editing; this work was supported by an NIH grant 1R35GM128678-01 and start-up funds from UConn Health (to M.I.).

Author Contributions
M.I, Conception and design, acquisition of data, analysis and interpretation of data, drafting or revising the article; S.L, M.A, T.S, Acquisition of data, analysis and interpretation of data, drafting or revising the manuscript. All rights reserved. No reuse allowed without permission.

Declaration of Interests
The authors declare no competing interests.

2.
Kawase   The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint overexpression of Dpp-mCherry, updGal4 ts driver, combination of updGal4 and tubGal80 ts 26 was used to avoid lethality. Temperature shift crosses were performed by culturing flies at 18°C to avoid lethality during development and shifted to 29°C upon eclosion for 4 days before analysis. Control crosses for RNAi screening were designed with matching gal4 and UAS copy number using TRiP control stock were used. Relative quantification was performed using the comparative CT method (ABI manual).
BglII GFP F 5'-ACAGATCTATGGTGAGCAAGGGCGAGGAGCTGTTCA-3' AscI GFP R 5'-TAGGCGCGCCTTACTTGTACAGCTCGTCCATGCCGAGA-3' then digested with BglII and AscI. NotI BglII sites (underlined) were attached to gBlock TkvS238A fragment (Integrated DNA Technologies, sequence as follows). The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint Synthesized fragments were annealed and digested by NotI and BglII. Resultant two inserts (TkvS238A and GFP) were ligated to modified pPGW vector using NotI and AscI sites in the multiple cloning site.
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Images were taken using a Zeiss LSM800 confocal microscope with a 63 ×oil immersion objective (NA=1.4) and processed using Image J and Adobe Photoshop software. Three-dimensional rendering was performed by Imaris software.

In situ hybridization
In situ hybridization on adult testes was performed as described previously 30 .
Briefly, testes were dissected in 1XPBS and then fixed in 4% formaldehyde/PBS for 45 min. After rinsed 2 times with 1XPBS, then resuspended in 70% EtOH, left overnight at 4°C. The next day, testes were washed briefly in the wash buffer containing 2XSSC and 10% deionized formamide, then

Chloroquine or Lysotracker/LysoSensor treatment
Testes from newly eclosed flies were dissected into Schneider's Drosophila medium containing 10% fetal bovine serum. Then testes were incubated at room temperature with or without 100μM Chloroquine (Sigma) in 1mL media for 4 hours or 2 hours prior to imaging. For the lysosome staining, testes were incubated with 50nM of LysoTracker Deep Red (ThermoFisher L12492) or 100nM of LysoSensor Green DND-189 (ThermoFisher L7535) probes in 1mL media for 10 min at room temperature then briefly rinsed with 1mL of media for 3 times prior to imaging. For Tkv-mCherry clonal expression, hs-cre, nos-loxP-stop-loxP-Gal4 with UAS-Tkv-mCherry, UAS-GFP-αTubulin flies were heat-shocked at 37°C. for 15 min. Testes were dissected 24 hour-after the heat shock.
These testes were placed onto Gold Seal™ Rite-On™ Micro Slides two etched rings with media, then covered with coverslips. An inverted Zeiss LSM800 confocal microscope with a 63 ×oil immersion objective (NA=1.4) was used for imaging.
For the Chloroquine feeding, newly eclosed flies were starved for overnight then transferred to food containing 3 mg/ml chloroquine (Sigma) for 3 days. All rights reserved. No reuse allowed without permission.

Quantification of pMad intensities.
Integrated intensity within the GSC nuclear region was measured for anti-pMad staining and divided by the area. To normalize the staining condition, data were further normalized by the average intensities of pMad from randomly picked three cyst cells in the same testes, and the ratios of relative intensities were calculated as each GSC per average cyst cell.

Quantification of Dpp (Dpp-mCherry or Dpp-GFP) intensities.
From a single stack at the level of hub centre, 3 randomly selected squares (5μmX5μm) within 10μm wide region outside of the hub (located next to the hub edge) were measured and background levels were subtracted and normalized by divisions by the mean intensity from 3 randomly selected lysosomal Dpp signal in the same image. Note; Although lysosomal Dpp signal increases size and number in the hub after CQ treatment, but intensity/area did not change. FRAP analysis.
Fluorescence recovery after photo-bleaching (FRAP) of Dpp-mCherry signal was undertaken using a Zeiss LSM800 confocal laser scanning microscope with 63X/1.4 NA oil objective. Zen black software was used for programming of each experiment. A small region (<10μm diameter) of interest was photobleached using laser powers to achieve an approximately 50%-70% bleach using the combination of 405, 480, 555, 640 nm laser. Fluorescence recovery was monitored every second for up to 10 minutes.

Statistical analysis and graphing.
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint Figure 1 All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint Figure 2 All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint Figure 3 All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint Figure 4 All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint   The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint ) or with (CQ+) Chloroquine. I, Quantitation of pMad intensity in the GSCs (relative to somatic cyst cells; CCs, see J and method). Indicated numbers of GSCs from two independent experiments were scored for each data point. J, A representative image of pMad labelling (red) in the wild type (yw: yellow white) testis. Red arrows indicate the pMad signal in CCs which were used as an internal control for pMad staining. G-J White lines divide GSCs attached to the hub and their immediate progeny.
Scale bars, 10μm. Asterisks indicate the hub. The P values were calculated by a student t-test P values. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint The P value was calculated by a student t-test. Scale bars, 10mm. The hub is encircled by blue dotted line.
All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint Figure S3 A-G, Representative images of in situ hybridization using Stellaris FISH probe against dpp mRNA (red) in the testis of indicated genotypes. B, Dpp RNAi (negative control) testis shows almost no detectable signal in the hub, suggesting the specificity of the probe. Hub is encircled by a white dotted line. DAPI (blue). Scale bars, 10 μm. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/414078 doi: bioRxiv preprint Figure S4 A, B Representative images of the testis tips from flies with Dpp-GFP fosmid genomic construct under the after 2-hour culture without (E) or with (F) CQ. C, Diameters of Dpp punctae in the hub after incubation with or without CQ. For the measurement, only well isolated dot was picked to avoid measuring overlapped 2 or more lysosomes. To select single, well-separated lysosomes, lower laser exposure was used. D, Average intensities of Dpp-GFP signal in randomly selected 5μmX5μm square areas next to the hub (see method for measurement). In C, largest diameter chosen from 0.5 μm interval z-stacks for each dot. The indicated numbers (n) of dots from two independent experiments were scored for each data point. Box plot shows 25-75% (box), median (band inside) and minima to maxima (whiskers). The indicated numbers (n) of testes from two independent experiments were scored for each data point. The indicated numbers (n) of testes from two independent experiments were scored for each data point. The P value was calculated by a student ttest. Scale bars, 10mm. The hub is encircled by blue dotted line.  Tkv localization/distribution pattern after inhibition of hub degradation using indicated treatment/genotypes. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. Center.
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