Alstrom syndrome gene is a stem-cell-specific regulator of centriole duplication in the Drosophila testis

  1. Cuie Chen
  2. Yukiko M Yamashita  Is a corresponding author
  1. Life Science Institute, Department of Cell and Developmental Biology, Michigan Medicine, University of Michigan, United States
  2. Howard Hughes Medical Institute, United States
  3. Whitehead Institute for Biomedical Research, United States
  4. Department of Biology, Massachusetts Institute of Technology, United States

Abstract

Asymmetrically dividing stem cells often show asymmetric behavior of the mother versus daughter centrosomes, whereby the self-renewing stem cell selectively inherits the mother or daughter centrosome. Although the asymmetric centrosome behavior is widely conserved, its biological significance remains largely unclear. Here, we show that Alms1a, a Drosophila homolog of the human ciliopathy gene Alstrom syndrome, is enriched on the mother centrosome in Drosophila male germline stem cells (GSCs). Depletion of alms1a in GSCs, but not in differentiating germ cells, results in rapid loss of centrosomes due to a failure in daughter centriole duplication, suggesting that Alms1a has a stem-cell-specific function in centrosome duplication. Alms1a interacts with Sak/Plk4, a critical regulator of centriole duplication, more strongly at the GSC mother centrosome, further supporting Alms1a’s unique role in GSCs. Our results begin to reveal the unique regulation of stem cell centrosomes that may contribute to asymmetric stem cell divisions.

Introduction

Many stem cells divide asymmetrically to generate daughter cells with distinct fates: one self-renewing stem cell and one differentiating cell. Asymmetrically dividing stem cells in several systems show stereotypical inheritance of the mother vs. daughter centrosomes, where the mother or daughter centrosome is consistently inherited by stem cells (Conduit and Raff, 2010; Habib et al., 2013; Januschke et al., 2011; Wang et al., 2009; Yamashita et al., 2007). This observation has provoked the idea that centrosome asymmetry in stem cells might play a critical role during asymmetric cell divisions and that stem cell centrosomes might be uniquely regulated. However, this question remains largely unanswered due to the lack of known stem-cell-specific centrosomal proteins.

Drosophila male germline stem cells (GSCs) divide asymmetrically by orienting their spindle perpendicular toward the hub cells, the major niche component (Yamashita et al., 2003; Figure 1A). During male GSC divisions, the mother centrosome is always located near the hub-GSC junction, whereas the daughter centrosome migrates to the other side of the cell, leading to spindle orientation perpendicular to the hub and consistent inheritance of the mother centrosome by GSCs (Figure 1A; Yamashita et al., 2007). Previously, we reported that Klp10A, a microtubule-depolymerizing kinesin of kinesin-13 family, is enriched on GSC centrosomes, but not on centrosomes of differentiating germ cells (i.e. gonialblasts (GBs) and spermatogonia (SGs)) (Chen et al., 2016). Klp10A is the first protein reported to exhibit stem-cell-specific centrosome localization. RNAi-mediated depletion of klp10A leads to abnormal elongation of the mother centrosomes in GSCs. The abnormally elongated mother centrosome in klp10A-depleted GSCs result in mitotic spindles with an asymmetric morphology (large vs. small half spindles) and the generation of a large GSC and a small GB that often dies, likely due to an insufficient cell size (Chen et al., 2016). These results suggested that stem cell centrosomes are indeed under unique regulation.

Figure 1 with 3 supplements see all
Identification of Alms1a as a germline stem cell (GSC)-specific Klp10A interactor.

(A) Asymmetric centrosome inheritance in Drosophila male GSCs. (B) Scheme of Klp10A pulldown and mass spectrometry. The Klp10A pull down was conducted using either upd-induced tumor (GSC-enriched) or dpp-induced tumor (SG-enriched) extract, followed by mass spectrometry analysis. (C) An apical tip of the Drosophila testis stained for Alms1a (red), γ-Tub (centrosome/pericentriolar matrix, blue) and Vasa (germ cells, green). Asterisk indicates the hub. GSCs are outlined with yellow dotted lines. Arrowheads (yellow) indicate examples of GSC centrosomes. Arrows (cyan) indicate examples of SG centrosomes. Bar: 5 μm. (D) Co-immunoprecipitation of Klp10A and Alms1a. Control GFP and GFP-Klp10A was immunoprecipitated from GSC-enriched extracts (nos-gal4>UAS-upd) using an anti-GFP antibody and blotted with anti-Alms1a and anti-GFP. (E) Schematic of full-length Alms1a, Klp10A and constructs used for GST-pulldown. (F) Direct interaction of Klp10A and Alms1a C-terminal fragment. GST control and recombinant GST-Alms1a-C were immobilized on glutathione agarose beads, incubated with His-Klp10A E. coli lysate, and blotted with anti-GST and anti-His antibody. (G–H) Bimolecular fluorescence complementation (BiFC) analysis of Alms1a-Klp10A interaction. (G) An example of the apical tip in nos-gal4>UAS-alms1a-VNm9 (control) testis, showing no signal. (H) An example of the apical tip in nos-gal4>UAS-alms1a-VNm9, UAS-VC155-klp10A testis, showing signal specifically at GSC centrosomes. Flies are raised at 18°C to minimize ectopic protein expression. Green: BiFC (Venus YFP fluorescence). Red: γ-Tub. Blue: Vasa. Arrowheads (yellow) indicate examples of GSC centrosomes positive for BiFC. Arrows (cyan) indicate examples of SG centrosomes negative for BiFC. Bar: 5 μm.

To obtain further insights into the mechanism and regulation of GSC centrosome asymmetry, we aimed to identify additional proteins that exhibit GSC-specific centrosome localization by taking advantage of Klp10A’s enrichment on GSC centrosomes. Here, we identify Alms1a, a Drosophila homolog of the causative gene for the human ciliopathy Alstrom syndrome, as a GSC-specific Klp10A-interacting protein. Human ALMS1 is reported to localize to the centrosome/basal body in many cells and function in formation/maintenance of the cilia (Andersen et al., 2003; Hearn et al., 2005). However, the underlying molecular mechanisms of its function remain poorly understood. We found that Drosophila Alms1a exhibits a unique asymmetric localization between mother and daughter centrosomes in GSCs, in which the GSC mother centrosomes have a higher amount of Alms1a than the GSC daughter centrosome. We found that alms1a depletion leads to a failure in centrosome replication, due to a failure to duplicate centrioles (the core of the centrosome), specifically in asymmetrically dividing GSCs. In contrast, alms1a is not required for centriole duplication in symmetrically dividing differentiating cells and GSCs induced to divide symmetrically. We show that Alms1a physically interacts with Sak, a key kinase that promotes centriole duplication, preferentially in GSCs. Taken together, our study begins to reveal the unique molecular composition and regulation of stem cell centrosome, paving the way to understand how stem cell centrosome asymmetry may contribute to asymmetric stem cell divisions.

Results

Identification of Alms1a as a GSC-specific Klp10A interactor

We previously reported that Klp10A, a microtubule-depolymerizing kinesin of kinesin-13 family, is enriched on GSC centrosomes, but not the centrosomes of differentiating germ cells (i.e. GBs and SGs) (Chen et al., 2016). We reasoned that identifying Klp10A-interacting proteins may uncover proteins that regulate the asymmetric behavior of the mother and/or daughter centrosomes in GSCs. As Klp10A localizes to the central spindle of all germ cells in addition to its GSC-specific centrosome localization (Chen et al., 2016; Figure 1B), we compared the results of anti-Klp10A immunopurification-mass spectrometry analysis using GSC-enriched extract vs. SG-enriched extract. We immunopurified Klp10A from testis extract that is enriched for either (1) GSCs due to the overexpression of the self-renewal factor, Upd (Kiger et al., 2001; Tulina and Matunis, 2001) or (2) SGs due to the overexpression of Dpp, a bone morphogenic protein (BMP) (Bunt and Hime, 2004; Kawase et al., 2004; Schulz et al., 2004). We reasoned that GSC centrosome-specific proteins would be enriched in the Klp10A-pulldown from GSC-enriched extracts compared to the Klp10A-pulldown from SG-enriched extracts.

Using this approach, we identified 260 candidates (Supplementary file 1), including two homologs of Alstrom syndrome (Alms) 1, a causal gene for a rare autosomal recessive disorder characterized by childhood obesity and sensory impairment, categorized as multiorgan ciliopathy (Marshall et al., 2011). The mammalian Alms1 protein was previously shown to localize to centrosomes and basal bodies of the primary cilia (Andersen et al., 2003; Hearn et al., 2005). The Drosophila genome encodes two Alms1 homologs, juxtaposed to each other on the X chromosome, which we named alms1a (CG12179) and alms1b (CG12184). The peptide sequences identified by our mass-spectrometry analysis were identical between these two proteins. We raised specific antibodies against Alms1a and Alms1b, and found that Alms1a localized to centrosomes in all germ cells (GSCs to spermatids) (Figure 1C and Figure 1—figure supplement 1), whereas Alms1b was not expressed until a late stage of spermatogenesis and localized to the basal body of elongating spermatids (Figure 1—figure supplement 1D,E). Therefore, Alms1a is likely the relevant Klp10A interactor in GSCs: accordingly, we focused on Alms1a in this study.

Alms1a and Klp10A co-immunoprecipitated in GSC-enriched extract but not in SG-enriched extract, confirming that Alms1a is a GSC-specific Klp10A interactor (Figure 1D). We found that this physical interaction between Alms1a and Klp10A is direct, and mediated via Alms1a’s conserved C-terminal region (‘ALMS motif’), as demonstrated by pulldown of the GST-fusion protein expressed in bacterial cells (Figure 1E–F) as well as by a bacterial two hybrid method (Figure 1—figure supplement 2).

We further assessed the interaction between Alms1a and Klp10A using the bimolecular fluorescence complementation (BiFC) method (Hu et al., 2002), which reveals protein-protein interactions in vivo via reconstitution of a functional Venus YFP from two non-fluorescent fragments (VNm9 and VC155) (Saka et al., 2007). Upon co-expression of alms1a-VNm9 and VC155-klp10A in the germline (nos-gal4>UAS-alms1a-VNm9, UAS-VC155-klp10A), we observed a YFP signal at centrosomes in GSCs, but not in the GB and SGs (Figure 1G,H and Figure 1—figure supplement 3). The lack of BiFC signals in the GB and SG serves as an internal negative control and confirms that the interaction between Alms1a and Klp10A is GSC-specific. These results imply that, although Alms1a localizes to the centrosomes of both GSCs and SGs, Alms1a might have a specific function in GSCs via its interaction with Klp10A.

Alms1a localizes asymmetrically between mother versus daughter centrosomes in male GSCs

To begin to understand the function of Alms1a, we first examined its centrosomal localization in GSCs and SGs in more detail. Each centrosome consists of two centrioles, the mother centriole (‘m’ in Figure 2A) and daughter centriole (‘d’ in Figure 2A). Centrosome duplication generates a mother centrosome that contains the original mother centriole (‘M’ in Figure 2A), and a daughter centrosome that contains the original daughter centriole, now a new mother (‘D’ in Figure 2A).

Figure 2 with 2 supplements see all
Alms1a exhibits asymmetric localization to the mother centrosome in germline stem cells (GSCs).

(A) Schematic of centrosome structure. Each cell contains two centrosomes, mother centrosome (M, pink) and daughter centrosome (D, blue). Each centrosome consists of two centrioles: mother centriole (m, dark purple) and daughter centriole (d, light purple). (B–D) A HyVolution image of wild-type GSC centrosomes (B). (C–D) Magnified images of a mother centrosome (C) and a daughter centrosome (D) from panel (B). Green: Centrobin (Cnb). Red: Alms1a. Blue: Asl. Asterisk indicates the hub. Arrowheads indicate GSC centrosomes. Arrow indicates mother centrosome. Bar: 5 μm. (E–G) A HyVolution image of wild type SG centrosomes (E). (F–G) Magnified images of SG centrosomes from panel (E). (H–J) A HyVolution image of klp10ARNAi GSC centrosomes (H). (I–J) Magnified images of a mother centrosome (I) and a daughter centrosome (J) from panel (H). (K) Ratio of integrated pixel density of Alms1a signal between mother and daughter centrosomes (M/D) in the indicated genotypes. P value was calculated using two-tailed Student’s t-test. Error bars indicate the standard deviation. 14 cells were scored for each data point. (L–O) Lightning images of a GSC mother centrosome (L), a daughter centrosome (M), a GB/SG centrosome (N) and a SC centrosome (O). Green: Cp110-GFP. Red: Alms1a. Blue: Ana1. Bar: 0.5 μm. Cartoon shows the interpretation of protein localization to the mother and daughter centrioles. (P–R) Stimulated emission depletion (STED) super-resolution images of wild-type GSC mother centrosome (O), a GSC daughter centrosome (P), and a SG centrosome (Q). Red: Alms1a. Blue: Asl. Green: Cnb. Bar: 0.5 μm.

We found that, although Alms1a localized to centrosomes in all cell types, it was more enriched on the mother centrosome than the daughter centrosome in GSCs (Figure 2B–D,K). In contrast, the two centrosomes in SGs had an equivalent amount of Alms1a (Figure 2E–G,K). Knockdown of klp10A in the male germline (nos-gal4>UAS-klp10ATRiP.HMS00920UAS-klp10ARNAi hereafter [Chen et al., 2016]) abolished the asymmetric enrichment of Alms1a to the mother centrosome in GSCs (Figure 2H–K), suggesting that Klp10A is necessary to create the asymmetrical distribution of Alms1a to the mother vs. daughter centrosomes in GSCs. klp10ARNAi did not affect Alms1a localization to the SG centrosomes (Figure 2—figure supplement 1A). Germline knockdown of alms1a (nos-gal4>alms1TRiP.HMJ30289, referred as to alms1RNAi hereafter) did not compromise Klp10A localization in GSCs (Figure 2—figure supplement 1B,C).

We further investigated the localization of Alms1a within the centrosome by examining its spatial relationship with known centriolar markers. Alms1a localization to the centrosome mostly did not overlap with Centrobin (Cnb), a daughter centriole marker (Januschke et al., 2013; Figure 2B,D–G), suggesting that Alms1a might localize to the mother centriole (see below). Detailed examination of Alms1a localization using Lightning confocal microscopy (see Materials and methods) confirmed that Alms1a is a mother centriole protein in GSCs and GBs/SGs (Figure 2L–N). Ana1 is a core centriole protein required for centrosome assembly and is known to localize to the mother centriole in early interphase (Fu et al., 2016; Goshima et al., 2007), whereas Cp110 localizes to the distal end of both mother and daughter centrioles, where it ‘caps’ the centriole (Delgehyr et al., 2012; Schmidt et al., 2009). We found that Alms1a localizes to the proximal end of the mother centriole: it localizes to the Ana1-positive centriole (e.g. mother centriole) at the opposite end compared to CP110, which is at the distal end (Figure 2M,N). Its localization to the proximal end was clearly visible in the spermatocytes (SCs), where much longer centrioles allowed easier distinction of distal vs. proximal ends (Figure 2O).

In addition to this localization at the proximal end of the mother centriole, Alms1a appeared to extend toward the proximal end of the daughter centriole specifically in the GSC mother centrosome (Figure 2L). This localization likely accounts for a higher amount of Alms1a at the GSC mother centrosome (Figure 2B–D,K). However, the Lightning microscopy did not resolve whether Alms1a at the mother and daughter centrioles are continuous or distinct entities. To achieve better resolution, we used stimulated emission depletion (STED) super-resolution microscopy: the mother centriole was marked by Asl, which is known to surround the mother centriole in a ring shape (Figure 2—figure supplement 2). The Asl ring observed in GSC centrosomes matches to the dimension of the Asl ring reported in other Drosophila cells (Fu et al., 2016; Galletta et al., 2016). We found that Alms1a staining appears as two spots within the GSC mother centrosome (Figure 2P), but as one spot within the GSC daughter centrosome and GB/SG centrosomes (Figure 2Q–R). Together, these results demonstrate that Alms1a localizes to the mother and daughter centrioles at the GSC mother centrosomes, but only to the mother centrioles elsewhere, generating an asymmetry between mother vs. daughter centrosomes of GSCs.

It should be noted that, although Klp10A was reported to be more concentrated at the distal end of the centriole in Drosophila spermatocytes (Delgehyr et al., 2012; Riparbelli et al., 2018), Klp10A exhibits pan centrosomal (broadly pericentrosomal) localization in GSCs (Chen et al., 2016). We suggest that Klp10A-Alms1a interaction likely occurs at the proximal end of the centrioles, where Alms1a is most concentrated, although other possibilities as to the site of their functional interaction cannot be excluded.

Depletion of alms1a leads to centrosome loss due to defective centriole duplication

To understand the function of alms1a, we examined the phenotypes arising from depletion of alms1a. Depletion of alms1a was confirmed using a specific antibody against Alms1a (Figure 1—figure supplement 1A–C and G). UAS-alms1RNAi is expected to knockdown both alms1a and alms1b, due to the high degree of their sequence similarity: However, because alms1b is not visibly expressed in GSCs/SGs (Figure 1—figure supplement 1D), the phenotypes caused by alms1RNAi in GSCs are likely due to the loss of alms1a (see Materials and methods). Expression of the full-length Alms1a (nos-gal4>alms1a-GFP, made to be insensitive to RNAi, see Materials and methods) rescued the centrosome loss phenotype of alms1RNAi phenotype partially, suggesting that the phenotype is caused, at least in part, by the loss of alms1a (Figure 3—figure supplement 1). The partial rescue may be due to an insufficient level of transgene expression, as nos-gal4 often causes variable expression levels among cells. Alternatively, GFP-tagging of Alms1a in the rescue transgene may partially compromise its functionality, although GFP-tagged Alms1a exhibits identical localization pattern as endogenous Alms1a detected by antibody staining. Also, the possibility of off-target effect of RNAi construct cannot be entirely excluded, although it is unlikely, given that the target sequence of alms1RNAi does not have any other similar sequences within the Drosophila genome.

Knockdown of alms1a (nos-gal4>UAS-alms1RNAi, whereby RNAi is induced starting from embryogenesis) resulted in the complete loss of centrosomes from all germ cells, except for the mother centrosomes in GSCs, suggesting that alms1a is required for generating new centrosomes/centrioles (Figure 3A,B). Wild-type GSCs normally contain mother and daughter centrosomes, each of which contains both mother and daughter centrioles, throughout the cell cycle, due to early duplication of the centrosome right after the previous mitosis (Figure 3C). In contrast, many alms1RNAi GSCs contained only one centrosome (i.e. mother), visualized by Asl as well as γ-Tubulin (Figure 3D). Interestingly, the remaining centrosome in alms1RNAi GSCs gradually elongated (Figure 3D and Figure 3—figure supplement 2), similar to our earlier observation with klp10ARNAi (Chen et al., 2016): in klp10ARNAi GSCs, the mother centrosome gradually elongated, whereas the daughter centrosome was slightly smaller than normal, although it was not lost as is observed in alms1RNAi GSCs. These data suggest that klp10A and alms1a share a similar function, possibly balancing the growth of mother vs. daughter centrosomes (see Discussion). We conclude that alms1a is required for centrosome duplication in GSCs.

Figure 3 with 3 supplements see all
alms1a is required for daughter centriole duplication in germline stem cells (GSCs).

(A–B) Examples of apical tips in control (A) and nos-gal4>UAS-alms1RNAi (B) testes (in this genotype, RNAi is induced since embryogenesis). Green: Vasa. Red: Asl. Blue: DAPI. Asterisk indicates the hub. Arrowheads indicate remaining centrosomes in alms1RNAi GSCs. Bar: 10 μm. (C–D) Examples of centrosomes in control (C) and nos-gal4>UAS-alms1RNAi (D) GSCs. Green: GFP-α-tubulin. Red: Asl. White: γ-Tub. Blue: DAPI. GSCs are outlined with yellow dotted lines. Arrowheads indicate GSC centrosomes. Bar: 5 μm. (E) Quantification of centrosome number in temporarily controlled alms1RNAi GSCs. (0–4 days after RNAi induction, nos-gal4ΔVP16, tub-gal80ts>UAS-alms1RNAi). Centrosome number was quantified by Asl and γ-Tub double staining. Error bars indicate the standard deviation. n = GSC numbers scored for each data point. (F, G) Examples of GSCs in control GSC (F) and alms1RNAi GSC (G, 2 days after RNAi induction, nos-gal4ΔVP16, tub-gal80ts>UAS-alms1RNAi) stained for daughter centriole protein Cnb. Green: Vasa. Red: Cnb. White: γ-Tub. Blue: DAPI. Arrowheads indicate GSC centrosomes. Arrow indicates mother centrosome in alms1RNAi GSC lacking Cnb staining (2 days after RNAi induction). The daughter centrioles are marked by Cnb. Cartoon shows interpretation of centriole composition. The color scheme is the same as Figure 2A. Bar: 5 μm. N = 30 GSCs for each genotype. (H) Quantification of Cnb amount (integrated pixel density) on the mother (M) vs. daughter (D) centrosomes in the indicated genotypes. p-Value was calculated using two-tailed Student’s t-test. Error bars indicate the standard deviation. Twelvecells were scored for each data point. (I, J) Lightning images of GSCs in control (I) and nos-gal4>UAS-alms1RNAi (J) testes. Green: Vasa. Red: Asl. White: Cp110. Asterisk indicates the hub. Bar: 10 μm. (K) Quantification of Cp110 dot number/centrosome in control vs. alms1RNAi GSCs. (L) Model of centrosome loss in alms1RNAi GSCs. Red centriole: the original mother centriole. Green centriole: the original daughter centriole that becomes a mother for the first time after centriole duplication. Blue centriole: the newest daughter centriole generated in the latest cell cycle.

To understand the precise steps of daughter centrosome loss upon depletion of alms1a, we induced alms1RNAi in a temporally controlled manner (nos-gal4ΔVP16, tub-gal80ts>UAS-alms1RNAi): alms1RNAi was induced upon eclosion and the outcome was monitored thereafter. This approach confirmed that GSCs lose their daughter centrosomes as fast as 1–2 days (2–4 GSC cell cycles) upon RNAi-mediated knockdown of alms1a (Figure 3E), and allowed us to examine the process of centrosome loss. In control GSCs, the daughter-centriole-specific protein Cnb always marked both mother and daughter centrosomes, as expected because both centrosomes contain a daughter centriole (Figure 3F,H). However, upon knockdown of alms1a, Cnb is lost or diminished from the mother centrosome prior to centrosome loss: when GSCs still contained two γ-Tubulin-positive centrosomes, the mother centrosome lacked Cnb staining, suggesting that it does not contain the daughter centriole (Figure 3G,H). Based on these results, we infer that centriole duplication is compromised in GSCs upon alms1 depletion: mother centriole (lacking Cnb) and daughter centriole (marked by Cnb) of the GSC mother centrosome likely split from each other, and segregate into GSC and GB, respectively, without duplicating centrioles (Figure 3L). In the following cell cycle, the GSCs that are incapable of duplicating centrioles will continue to divide without passing any new centrosomes to GBs. The inability of alms1RNAi GSCs to duplicate centrioles was further comfirmed by detailed examination of centrosomes upon complete loss of the daughter centrosomes in alms1RNAi GSCs. In contrast to control GSCs, which contain two centrosomes, each of which contains two centrioles marked by Cp110 (Figure 3I,K), alms1RNAi GSCs contained only one centrosome, which contains only one centriole (Figure 3J,K), demonstrating that alms1RNAi compromises centriole duplication. The lack of centriole duplication in GSCs would lead to loss of centrosomes/centrioles in all germ cells, which must inherit the first template for centriole duplication from GSCs (Figure 3L). nos-gal4>UAS-alms1RNAi flies were sterile, likely due to the complete loss of centrosomes, although knockdown of alms1b also contributes to this sterility (Figure 3—figure supplement 3). Taken together, we conclude that alms1a plays a critical role in ensuring centrosome duplication in GSCs, which is essential for the production of centrosomes/centrioles in all downstream germ cells.

alms1a is dispensable for centrosome duplication in differentiating germ cells and GSCs that are induced to divide symmetrically

The above results show that alms1a is required to ensure the duplication of daughter centrioles in GSCs. Considering the unique asymmetric localization of Alms1a only in GSCs but not in differentiating cells, we wondered whether Alms1a is required for centriole duplication in differentiating cells.

To address this question, we depleted alms1a from differentiating cells. The SG-specific driver bam-gal4 was used to deplete alms1a in differentiating germ cells (bam-gal4>UAS-alms1RNAi). We found that SGs, which divide symmetrically, maintained their centrosomes upon depletion of alms1a (Figure 4A). Thus, alms1a is specifically required for centriole duplication in stem cells, but not in symmetrically dividing, differentiating cells. Note that this result was not due to incomplete depletion of alms1a, as Alms1a was undetectable in SGs in the testis from bam-gal4>UAS-alms1RNAi flies (Figure 4A).

alms1a is dispensable for centrosome duplication in symmetrically dividing cells.

(A) An example of Alms1a staining in bam-gal4>UAS-GFP, UAS-alms1RNAi testis. The boundary of bam-gal4-positive vs. -negative SGs is indicated by the yellow line (note that bam expression marked by GFP is initially weak). Green: GFP. White: Alms1a. Red: Asl. Blue: Vasa. Asterisk indicates the hub. Yellow box inset indicates an example of Alms1a staining in germline stem cell (GSC). Cyan box inset indicates an example of Alms1a staining in SG. Bar: 10 μm. (B–C) Examples of upd-induced GSCs without alms1RNAi (B, nos-gal4>UAS-upd) or with alms1RNAi (C, nos-gal4>UAS-upd, UAS-alms1RNAi). Green: Vasa. Red: Asl. White: Alms1a. Blue: DAPI. Inset is magnified image of a single GSC. Arrows indicate centrosomes. Bar: 10 μm. (D) Quantification of centrosome numbers (quantified by Asl and γ-Tub double staining) in the indicated genotypes. Error bars indicate the standard deviation. n = GSC numbers scored.

To test whether the unique requirement of alms1a in GSCs is due to their asymmetric division or to their stem cell identity, we examined whether symmetrically dividing GSCs require alms1a for centriole duplication (Figure 4B–C). We induced symmetric divisions in GSCs by expressing upd together with alms1RNAi (nos-gal4>UAS-upd, UAS-alms1RNAi). Alms1a protein was undetectable under this condition, confirming efficient knockdown (Figure 4C). Importantly, these symmetrically dividing GSCs retained their centrosomes (Figure 4C–D), suggesting that alms1a is required for centriole duplication only in asymmetrically dividing cells.

Alms1a interacts with the core centriole duplication machinery

To further understand how Alms1a regulates centriole duplication in the GSCs, we examined whether Alms1a interacts with key proteins for centriole biogenesis. Sak is a homolog of the Plk4 kinase, a master regulator required for centriole duplication (Bettencourt-Dias et al., 2005; Habedanck et al., 2005), whereas Sas-6 is a component for cartwheel assembly to promote centriole formation (Nakazawa et al., 2007; Rodrigues-Martins et al., 2007a). Alms1a physically interacted with Sak and Sas-6 in testes lysates enriched with GSCs, as indicated by co-immunoprecipitation (Figure 5A–B). Moreover, GST-pulldown experiments using bacterial extracts as well as bacterial two hybrid experiments showed that Sak directly binds to Alms1a via the ALMS motif. In contrast, Sas-6-ALMS motif did not interact in bacterial extract (Figure 5C–E and Figure 5—figure supplement 1), suggesting that the interaction between Alms1a and Sas-6 in testis extract is indirect.

Figure 5 with 1 supplement see all
Alms1a interacts with Sak and Sas-6 in germline stem cells (GSCs).

(A) Co-immunoprecipitation of Sak and Alms1a. Control GFP and Alms1a-GFP were immunoprecipitated from GSC-enriched extracts (nos-gal4>UAS-upd) using an anti-GFP antibody and blotted with anti-Sak and anti-GFP. (B) Co-immunoprecipitation of Sas-6 and Alms1a. Control GFP and Alms1a-GFP were immunoprecipitated from GSC-enriched extracts (nos-gal4>UAS-upd) using an anti-GFP antibody and blotted with anti-Sas-6 and anti-GFP. (C–H) BiFC analysis of Alms1a-Sak interaction. (C) Schematic of Alms1a, Sak and Sas-6 and constructs used for in vitro pull-down experiments. (D–E) GST-pulldown of Sak, Sas-6 and Alms1a C-terminal fragment. GST control and recombinant GST-Alms1a-C was immobilized on glutathione agarose beads and incubated with His-Sak (D) or His-Sas-6 (E) lysate for pull-down assay and blotted with anti-GST and anti-His antibody. (F–G) Examples of apical tips in nos-gal4 >UAS-alms1a-VNm9 (control) and nos-gal4>UAS-alms1a-VNm9, UAS-VC155-sak testes. Flies are raised at 18°C to minimize centrosome over-duplication due to ectopic protein expression. (H–K) Magnified images of a GSC mother centrosome (H), a GSC daughter centrosome (I) and two SG centrosomes (J–K) from panel (G). Green: BiFC. Red: γ-Tub. Blue: Vasa. Asterisk indicates the hub. GSCs are outlined with yellow dotted lines. Arrowheads (yellow) indicate examples of GSC centrosomes. Arrows (cyan) indicate examples of SG centrosomes. Bar: 5 μm. (L) Quantification of BiFC integrated pixel density in the indicated genotypes. P value was calculated using two-tailed Student’s t-test. Error bars indicate the standard deviation. Twenty cells were scored for BiFC signals for each data point.

To further characterize these interactions, we performed the BiFC analysis between Alms1a and Sak. We observed a weak BiFC signal at centrosomes in general, but the BiFC signal was significantly higher at the GSC mother centrosome (Figure 5F–L). Given that Alms1a localizes to the mother centriole in all centrosomes, but to the daughter centriole only in GSCs (Figure 2), we infer that the Alms1a-Sak interaction preferentially occurs at the daughter centriole of the GSC mother centrosome.

Alms1a promotes centriole duplication together with Sak

How does Alms1a regulate centriole duplication, and why it is required only in GSCs? Alms1a’s direct interaction with Sak (Figure 5) indicates that Alms1a might promote centriole duplication together with Sak. Indeed, we found that overexpression of Alms1a dramatically enhanced centrosome overduplication caused by Sak overexpression. Overexpression of alms1a (nos-gal4 >UAS-alms1a-GFP, 25°C) alone did not result in an obvious increase in centrosome number compared to the control (2.09 ± 0.38 centrosomes/cell in alms1a overexpression, vs. 2.00 ± 0.00 centrosomes/cell in control) (Figure 6A,B,E). Sak overexpression increased centrosome numbers slightly (2.79 ± 1.07 centrosomes/cell) (Figure 6C,E), similar to observations in other cell types (Habedanck et al., 2005; Peel et al., 2007; Rodrigues-Martins et al., 2007b). Strikingly, co-expression of alms1a and sak (nos-gal4>UAS-alms1a-GFP, UAS-GFP-sak, 25°C) dramatically enhanced centrosome overduplication (Figure 6D–E and Figure 6—figure supplement 1) (4.61 ± 1.91 centrosomes/cell). These results suggest that Alms1a promotes centrosome duplication together with Sak, possibly by recruiting Sak to the site of centriole duplication in GSCs.

Figure 6 with 1 supplement see all
Alms1a promotes centrosome duplication together with Sak.

(A–E) Alms1a overexpression enhances centrosome overduplication induced by Sak overexpression. Examples of germ cells in control (A), nos-gal4>UAS-alms1a-GFP (B), nos-gal4>UAS-GFP-sak (C) and nos-gal4>UAS-alms1a-GFP, UAS-GFP-sak (D). Flies were raised at 25°C to drive a higher level of protein expression. Green: GFP. Red: Asl. Blue: Vasa. Germ cells are outlined with yellow dotted lines. Bar: 5 μm. (E) Quantification of centrosome number (quantified by Asl and γ-Tub double staining) in GSCs/SGs in indicated genotypes. p-Value was calculated using two-tailed Student’s t-test. Error bars indicate the standard deviation. n = 100 cells were scored for each data point. (F–K) Sak and Sas-6 accumulate on the remaining mother centrioles in alms1RNAi GSCs. (F–G) Examples of GSC centrosomes in control (F) and nos-gal4>UAS-alms1RNAi (G) testes stained for Sak (white), Asl (red), and Vasa (green). Asterisk indicates the hub. GSCs are outlined with yellow dotted lines. Bar: 5 μm. (H) Quantification of Sak integrated pixel density in the indicated genotypes. P value was calculated using two-tailed Student’s t-test. Error bars indicate the standard deviation. n = 20 GSCs were scored for Sak signals for each data point. (I–J) Examples of GSC centrosomes in control (I) and nos-gal4>UAS-alms1RNAi (J) testes stained for Sas-6 (white), Asl (red), and Vasa (green). Bar: 5 μm. (K) Quantification of Sas-6 integrated pixel density in the indicated genotypes. P value was calculated using two-tailed Student’s t-test. Error bars indicate the standard deviation. n = 20 GSCs were scored for Sas-6 signals for each data point.

Why is Alms1a’s function to promote centriole duplication together with Sak only required in GSCs but not in SGs? Interestingly, we found that RNAi-mediated knockdown of alms1a in GSCs resulted in the excessive enrichment of both Sak and Sas-6 at the elongated mother centriole that remains after all other centrosomes are depleted (Figure 6F–K). The excess amount of Sak and Sas-6 on the remaining mother centriole in alms1RNAi GSCs may explain why it continues to elongate. In parallel, the accumulation of Sak and Sas-6 on the mother centriole may deplete Sak and Sas-6 from the site of the daughter centriole production, leading to the failure in daughter centriole duplication. Therefore, the GSC-specific function of Alms1a may be to promote daughter centriole duplication by recruiting Sak to the ‘seeding site’ for daughter centriole duplication (see Discussion).

Discussion

Asymmetric behaviors between mother and daughter centrosomes have been observed in asymmetrically dividing stem cells in several systems (Conduit and Raff, 2010; Habib et al., 2013; Januschke et al., 2011; Wang et al., 2009; Yamashita et al., 2007), provoking the idea that these centrosome asymmetries may contribute to asymmetric cell fates. Here, we identified a centrosomal protein Alms1a, a homolog of a causative gene of human ciliopathy Alstrom syndrome, that exhibits asymmetric localization between mother and daughter centrosomes in Drosophila male GSCs. Alms1a is required for centriole duplication specifically in GSCs, but not in differentiating cells, demonstrating the unique regulation of centrosomes in asymmetrically dividing stem cells.

Although the exact molecular mechanism of Alms1a function remains elusive, a few lines of observations imply its possible function. Alms1a localizes to the mother centriole in all centrosomes, but in GSCs it also localizes to the daughter centriole specifically in the mother centrosome, where it interacts with Sak and Sas-6. Importantly, Alms1a’s function is required in GSCs but not SGs, thus this GSC-specific localization at daughter centriole of the mother centrosome is likely what is required for centriole duplication in GSCs. Therefore, we postulate that Alms1a may promote daughter centriole duplication by recruiting Sak (and Sas-6), to the daughter centriole of the GSC mother centrosome.

Why is Alms1a only required in asymmetrically dividing GSCs for centriole duplication? Given that alms1RNAi leads to excessive overgrowth of mother centriole of the GSC mother centrosome at the expense of the daughter centriole duplication, it is tempting to speculate that Alms1a is required to ‘discourage’ mother centriole’s growth and ‘encourage’ daughter centrioles generation/growth. Indeed, upon alms1a depletion, the mother centriole gradually elongates and accumulates excessive amounts of Sak and Sas-6, while failing at daughter centriole duplication. Perhaps to ensure correct centrosome positioning, the mother centriole of the mother centrosome in GSCs may have a stronger microtubule nucleation activity, which may interfere with production of new centrioles. This imbalance between mother strength and daughter duplication may be achieved by Alms1a. Such a scenario would explain why Alms1a is required specifically in asymmetrically dividing GSCs, although the underlying molecular mechanisms on how Alms1a may achieve this remains elusive.

A recent study identified Ninein as a protein that specifically localizes to the daughter centrosome when overexpressed in Drosophila neuroblasts and male GSCs (Zheng et al., 2016), although no phenotypic consequences were detected upon its depletion, leaving the role of its asymmetric localization unknown. Our Klp10A pull down did not identify Ninein, it will be of interest to study how Klp10A and Alms1a may be functionally related to Ninein.

In summary, our study identified Alms1a as a protein that exhibits asymmetric localization between mother and daughter centrosomes of GSCs. We demonstrate that alms1a is uniquely required for centriole duplication in asymmetrically dividing stem cells. This specific requirement of alms1a only in a subset of cell types (e.g. asymmetrically dividing adult stem cells) may partly explain the reported late-onset nature of Alstrom syndrome, which exhibits symptoms progressively during childhood, as opposed to many other ciliopathies that are associated with disease symptoms at birth (Braun and Hildebrandt, 2017). Our study revealed that GSC centrosomes are indeed under unique regulation, involving GSC centrosome-specific proteins Klp10A and Alms1a. The identification of these proteins (Alms1a, Klp10A [Chen et al., 2016] and Ninein [Zheng et al., 2016]) paves the way to understand how asymmetric behavior of centrosomes contributes to asymmetric stem cell division.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (D. melanogaster)nos-gal4PMID:9501989
Strain, strain background (D. melanogaster)bam-gal4PMID:12571107
Strain, strain background (D. melanogaster)tub-gal4Bloomington
Stock Center
ID_BSC: 5138
Strain, strain background (D. melanogaster)UAS-updPMID:10346822
Strain, strain background (D. melanogaster)tub-gal80tsPMID:14657498
Strain, strain background (D. melanogaster)nos-gal4 without VP16 (nos-gal4ΔVP16)PMID:26131929
Strain, strain background (D. melanogaster)UAS-EGFPBloomington Stock CenterID_BSC: 5430
Strain, strain background (D. melanogaster)UAS-dppBloomington Stock CenterID_BSC: 1486
Strain, strain background (D. melanogaster)ywBloomington Stock CenterID_BSC: 189
Strain, strain background (D. melanogaster)UAS-GFP-alpha-tubulin84BBloomington Stock CenterID_BSC: 7373
Strain, strain background (D. melanogaster)UAS-klp10ARNAiBloomington Stock CenterID_BSC: 33963
Strain, strain background (D. melanogaster)UAS-klp10A-EGFPPMID:26131929
Strain, strain background (D. melanogaster)centrobin-YFPPMID:21407209
Strain, strain background (D. melanogaster)ubi-asl-tdTomatoPMID:21694707
Strain, strain background (D. melanogaster)ubi-ana1-tdTomatoPMID:18854586
Strain, strain background (D. melanogaster)ubi-Cp110-GFPPMID:27185836
Strain, strain background (D. melanogaster)UAS-alms1RNAiBloomington Stock CenterID_BSC: 63721
Strain, strain background (D. melanogaster)UAS-alms1a-EGFPThis study
Strain, strain background (D. melanogaster)UAS-alms1a-EGFP (insensitive to RNAi)This study
Strain, strain background (D. melanogaster)UAS-alms1a-VNm9This study
Strain, strain background (D. melanogaster)UAS-VC155-klp10AThis study
Strain, strain background (D. melanogaster)UAS-VC155-sakThis study
Strain, strain background (D. melanogaster)UAS-EGFP-ALMS motifThis study
Strain, strain background (D. melanogaster)cep135-EGFPBloomington Stock CenterID_BSC: 60183
AntibodyAnti-vasa (Rabbit polyclonal)Santa Cruz BiotechnologyID_SCB: d-26IF: 1:200
AntibodyAnti-alpha-Tubulin (Mouse
monoclonal)
Developmental Studies Hybridoma BankWB: 1:2000
AntibodyAnti-vasa (Rat monoclonal)Developmental Studies Hybridoma BankIF: 1:20
AntibodyAnti-gamma-Tubulin (Mouse monoclonal)Sigma-AldrichID_Sigma:T6557IF: 1:100
AntibodyAnti-Asl (Rabbit polyclonal)A gift from Jordan RaffIF: 1:5000
AntibodyAnti-Sas-6 (Rabbit polyclonal)A gift from Jordan RaffIF: 1:5000
WB: 1:40000
AntibodyAnti-Sak (Rabbit polyclonal)A gift from Monica Bettencourt-DiasIF: 1:400
WB: 1:4000
AntibodyAnti-Spd2 (Rabbit polyclonal)A gift from Maurizio GattiIF: 1:25
AntibodyAnti-GFP (Chick monoclonal)Aves LabsID_aves: GFP-1020WB: 1:6000
AntibodyAnti-GST (Mouse monoclonal)BiolegendWB: 1:4000
AntibodyAnti-His (Mouse monoclonal)Millipore-SigmaWB: 1:4000
AntibodyAnti-Alms1a (Guinea pig polyclonal)This studyIF: 1:4000
WB: 1:40000
AntibodyAnti-Alms1b (Guinea pig polyclonal)This studyIF: 1:4000
AntibodyAnti-Centrobin (Rabbit polyclonal)This studyIF: 1:4000
Recombinant DNA reagentpGEX-4T-LP (plasmid)Addgene
Recombinant DNA reagentpMCSG7 (plasmid)Addgene
Recombinant DNA reagentpGEX-alms1a-C (plasmid)This study
Recombinant DNA reagentpMCSG7-klp10A (plasmid)This study
Recombinant DNA reagentpMCSG7-sak (plasmid)This study
Recombinant DNA reagentpMCSG7-sas-6 (plasmid)This study
Recombinant DNA reagentpKNT-25
(plasmid)
Euromedex
Recombinant
DNA reagent
pUT18C (plasmid)Euromedex
Recombinant DNA reagentpKT25-zip (plasmid)Euromedex
Recombinant DNA reagentpUT18C-zip (plasmid)Euromedex
Recombinant DNA reagentpKNT-alms1a (plasmid)This study
Recombinant DNA reagentpUT-klp10A (plasmid)This study
Recombinant DNA reagentpUT-sak (plasmid)This study
Recombinant DNA reagentpUT-sas-6 (plasmid)This study

Fly husbandry, strains and transgenic flies

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All fly stocks were raised on standard Bloomington medium at 25°C, and young flies (0- to 1-day-old adults) were used for all experiments unless otherwise noted. The following fly stocks were used: nos-gal4 (Van Doren et al., 1998), UAS-upd (Zeidler et al., 1999), UAS-dpp (Stock #1486, Bloomington Drosophila Stock Center (BDSC)) (Staehling-Hampton et al., 1994), tub-gal80ts (Stock # 7017, BDSC) (McGuire et al., 2003), UAS-EGFP (Stock #5430, BDSC), yw (Stock #189, BDSC), cep135-EGFP (Stock#60183, BDSC) bam-gal4 (Chen and McKearin, 2003), tub-gal4 (Stock # 5138, BDSC) (Lee and Luo, 1999), UAS-klp10A-GFP (Inaba et al., 2015), UAS-GFP-α-tubulin84B (Stock #7373, BDSC) (Grieder et al., 2000), UAS-klp10ARNAi (TRiP.HMS00920, BDSC), UAS-alms1RNAi (TRiP.HMJ30289, BDSC), nos-gal4 without VP16 (nos-gal4ΔVP16) (Inaba et al., 2015), centrobin-YFP (Januschke et al., 2011) (a gift from Cayetano Gonzalez), ubi-asl-tdTomato (Gopalakrishnan et al., 2011) and ubi-ana1-tdTomato (Blachon et al., 2008) (gifts from Tomer Avidor-Reiss), ubi-Cp110-GFP (Galletta et al., 2016) (a gift from Nasser Rusan). Note that UAS-alms1RNAi is expected to knockdown both alms1a and alms1b. However, because alms1b is not expressed in GSCs, the phenotypes resulting from expression of this RNAi construct in GSCs are due to the loss of alms1a. Although we attempted to generate alms1a-specific RNAi lines, none of them efficiently knocked down alms1a as evidenced by western blotting.

For the construction of UAS-alms1a-VNm9, the VNm9 fragment was PCR-amplified from a plasmid with pUASP-VNm9 (a gift from Mayu Inaba), and subcloned into NotI/KpnI sites of pUAST-attB. The alms1a fragment was PCR-amplified from a cDNA ((LD15034) Drosophila Genomics Resource center), and subcloned into BglII/NotI sites of pUAST-VNm9-attB. For the construction of UAS-VC155-klp10A and UAS-VC155-sak, the VC155 fragment was PCR-amplified from a plasmid with pUASP-VC155 (a gift from Mayu Inaba), and subcloned into EcoRI/XhoI sites of pUAST-attB. The klp10A fragment was PCR-amplified from a cDNA ((LD29208) Drosophila Genomics Resource center), and subcloned into XhoI/KpnI sites of pUAST-VC155-attB. The sak fragment was PCR-amplified from a cDNA ((RE70136) Drosophila Genomics Resource center), and subcloned into XhoI/KpnI sites of pUAST-VC155-attB. For the construction of UAS-alms1a-GFP, UAS-GFP-ALMS motif and UAS-GFP-sak, the alms1a, ALMS motif and sak fragments were PCR-amplified from cDNAs, and subcloned into BglII/NotI sites of pUAST-GFP-attB (C-terminal GFP tag) or NotI/KpnI sites of pUAST-GFP-attB (N-terminal GFP tag). UAS-alms1a-GFP that is insensitive to RNAi was generated using QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies) from UAS-alms1a-GFP with the following primers:5’-gaattatagtgtattcattcggtttagtctgggtaacctcggatttcggtgatgtgaactgactgatg-3’ and 5’-catcagtcagttcacatcaccgaaatccgaggttacccagactaaaccgaatgaatacactataattc-3’. Transgenic flies were generated using PhiC31 integrase-mediated transgenesis at the P{CarryP}attP40 (FBti0114379) or P{CarryP}attP2 (FBst0008622) integration sites (BestGene).

Recombinant protein expression and GST pull-down assays cDNAs for alms1a, klp10A, sak and sas-6 were purchased from Drosophila Genomics Resource center as described above. Full-length or fragments of these genes were generated by PCR amplification of the cDNAs. For the construction of pGEX-alms1a-C, the alms1a-C fragment was PCR-amplified from cDNA, and subcloned into BamHI/NotI sites of pGEX-4T-LP (Addgene). Glutathione S-transferase (GST)-tagged Alms1a-C (1115-1322aa) or GST alone as a control were expressed in Rosetta (DE3) competent cells (Novagen). For the construction of pMCSG7-klp10A, pMCSG7-sak and pMCSG7-sas-6, full-length genes were amplified from cDNAs, and subcloned into KpnI/EcoRI, KpnI/NotI and KpnI/BamHI sites of pMCSG7 (Addgene), respectively. N-terminal 6xHis-tagged klp10A, sak and sas-6 were expressed in Rosetta competent cells as described above. Cells were grown at 37°C in Terrific Broth (TB) (Millipore) in the presence of ampicillin (100 μg/mL). After optical density (OD) values reaches to 0.6, protein expression was induced by isopropyl β-D-1-thiogalactopyranoside at a final concentration of 0.3 mM for an additional 20 hr at 16°C. The cells were harvested by centrifugation at 5000 rpm for 5 min, and lysed by sonication in lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM EDTA, 1% NP-40 and 5% glycerol) supplemented with complete protease inhibitor cocktail (Roche) and 1 mM phenylmethyl sulphonyl fluoride (PMSF). The immunoprecipitations between Alms1a-C and Klp10A/Sak/Sas-6 were performed by in vitro GST pull-down assay using Pierce GST Protein Interaction Pull-Down kit (Thermo Scientific). The cell lysates were incubated for 30 min at 4°C followed by centrifugation at 14,000 rpm for 10 min at 4°C. GST and GST-Alms1a-C lysates with equal total protein level were incubated with glutathione agarose beads for 1 hr at 4°C, and washed three times in lysis buffer. Then, His-Klp10A, His-Sak and His-Sas-6 lysates with equal total protein level were incubated with washed beads for 2 hr at 4°C, and washed five times in lysis buffer. Before the last wash, the beads were moved to a fresh tube, and eluted with 2xLaemmli buffer (Bio-Rad) containing 5% β-mercaptoethanol, and boiled for 10 min. SDS-PAGE and Western blot were then conducted to examine the protein interactions.

Bacterial two-hybrid analysis

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The bacterial two-hybrid analysis was performed to detect direct protein-protein interactions using bacterial adenylate cyclase-based two-hybrid, BACTH systems (Euromedex). For the construction of pKNT-alms1a, the alms1a full-length was PCR-amplified from cDNA, and subcloned into HindIII/SmaI sites of pKNT25. For the construction of pUT-klp10A, pUT-sak and pUT-sas-6, full-length genes were amplified from cDNAs, and subcloned into KpnI/EcoRI, PstI/KpnI and KpnI/EcoRI sites of pUT18C, respectively. Two recombinant plasmids encoding alms1a-T25 and T18-klp10A/T18-sak/T18-sas-6 are co-transformed into BTH101 competent cells. Transformants are plated on MacConkey selective plate in the presence of maltose, ampicillin (100 μg/mL) and kanamycin (50 μg/mL), and incubated at 30°C for 24 hr. Complementation can be detected as red colonies on the MacConkey-maltose (Sigma) plates, while colonies will be colorless if no interaction occurs. Co-transformation of pKT25-zip and pUT18C-zip with BTH101 competent cells was used as a positive control. Co-transformation of pKNT25 or pUT18C with one of the recombinant plasmids served as negative controls.

Immunofluorescent staining and confocal microscopy

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Drosophila testes were dissected in phosphate-buffered saline (PBS), transferred to 4% formaldehyde in PBS and fixed for 30 min. The testes were then washed in PBST (PBS containing 0.1% Triton X-100) for at least 30 min, followed by incubation with primary antibody in 3% bovine serum albumin (BSA) in PBST at 4°C overnight. Samples were washed for 60 min (three 20 min washes) in PBST, incubated with secondary antibody in 3% BSA in PBST at 4°C overnight, washed as above, and mounted in VECTASHIELD with 4',6-diamidino-2-phenylindole (DAPI; Vector Labs).

The primary antibodies used were as follows: rat anti-Vasa (1:20; developed by A. Spradling and D. Williams, obtained from Developmental Studies Hybridoma Bank (DSHB)); mouse anti-γ-Tubulin (GTU-88; 1:100; Sigma-Aldrich); rabbit anti-Vasa (d-26; 1:200; Santa Cruz Biotechnology); rabbit anti-Asl (1:2000; a gift from Jordan Raff); rabbit anti-Sas-6 (1:2000; a gift from Jordan Raff); rabbit anti-Sak (1:200; a gift from Monica Bettencourt-Dias); rabbit anti-Spd2 (1:25; a gift from M. Gatti); chicken anti-GFP (1:1000, Aves Labs). Anti-Alms1a and Alms1b antibody were generated by injecting peptides (QEMEVEPKKQLEKEQHQNDMQQGEPKGREC) and (CNISQRGNHLEKIE) into guinea pigs (Covance). Affinity purification was used to purify the antibodies. Specificity of the antibodies was validated by the lack of staining in alms1RNAi testis (Figure 1—figure supplement 1). Anti-Centrobin antibody was generated by injecting a peptide (GRPSRELHGMVHSTPKSGSVEPLRHRPLDDNIC) into rabbits (Covance). Alexa Fluor-conjugated secondary antibodies (Life Technologies) were used with a dilution of 1:200. Images were taken using an upright Leica TCS SP8 confocal microscope with a 63 × oil immersion objective (NA = 1.4) and processed using Adobe Photoshop software. As necessary, better resolution was obtained with HyVolution/Lightning on TCS SP8 confocal microscopy or STED imaging.

Co-immunoprecipitation and western blotting

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For immunoprecipitation using Drosophila testis lysate, testes enriched with GSCs due to ectopic Upd expression (100 pairs/sample) were dissected into PBS at room temperature within 30 min. Testes were then homogenized and solubilized with lysis buffer (10 mM Tris-HCl pH 7.5; 150 mM NaCl; 0.5 mM EDTA supplemented with 0.5% NP40 and protease inhibitor cocktail (EDTA-free, Roche)) for 30 min at 4°C. Testes lysates were centrifuged at 13,000 rpm for 15 min at 4°C using a table centrifuge, and the supernatants were incubated with GFP-Trap magnetic agarose beads (ChromoTek) for 4 hr at 4°C. The beads were washed three times with wash buffer (10 mM Tris-HCl pH 7.5; 150 mM NaCl; 0.5 mM EDTA). Bound proteins were resolved in SDS-PAGE and analyzed by western blotting.

For anti-Klp10A pull-down and mass spectrometry assays, testes enriched with GSCs (nos-gal4>UAS-upd) or enriched with SGs (nos-gal4>UAS-dpp) (250 pairs/sample) were dissected into PBS. Testes were then homogenized and solubilized with lysis buffer (10 mM Tris-HCl pH 7.5; 150 mM NaCl; 0.5 mM EDTA, 0.1% NP40) and protease inhibitor cocktail for 30 min at 4°C. After centrifugation at 13,000 rpm for 15 min at 4°C, supernatants were incubated with anti-Klp10A conjugated Dynabeads for 2 hr at 4°C. The beads were washed three times with wash buffer (10 mM Tris-HCl pH 7.5; 150 mM NaCl; 0.5 mM EDTA), followed by boiling in 2xLaemmli buffer and sent for Mass spectrometry analysis (MS Bioworks).

For western blotting, samples subjected to SDS-PAGE (NuPAGE Bis-Tris gels (8%; Invitrogen)) were transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P; Millipore). Membranes were blocked in PBS containing 5% nonfat milk and 0.1% Tween-20, followed by incubation with primary antibodies diluted in PBS containing 5% nonfat milk and 0.1% Tween-20. Membranes were washed with PBS containing 5% nonfat milk and 0.1% Tween-20, followed by incubation with secondary antibody. After washing with PBS, detection was performed using an enhanced chemiluminescence system (Amersham). Primary antibodies used were rabbit anti-GFP (abcam; 1:4000), guinea pig anti-Alms1a (this study; 1:5000), rabbit anti-Sas-6 (1:10000; a gift from Jordan Raff), rabbit anti-Sak (1:2000; a gift from Monica Bettencourt-Dias), mouse anti-GST (Biolegend; 1:1500), and mouse anti-His (Millipore-Sigma; 1:2500). Secondary antibodies used were goat anti-guinea pig IgG, goat anti-rabbit IgG and goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (abcam; 1:5000).

Data analyses

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Statistical analysis was performed using GraphPad Prism seven software. Data are shown as means ± standard deviation. The p-value (two-tailed Student’s t-test) is provided for comparison with the control. Integrated pixel intensity was used to measure the amount of proteins at mother and daughter centrosomes. Regions of interest were manually drawn in ImageJ and background intensity was subtracted from each value.

Fertility assays

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Individual males of the genotypes shown were allowed to mate with two yw virgin female flies for 1 week. The flies were removed after a week, and the numbers of progenies were scored.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

References

    1. Braun DA
    2. Hildebrandt F
    (2017) Ciliopathies
    Cold Spring Harbor Perspectives in Biology 9:a028191.
    https://doi.org/10.1101/cshperspect.a028191
    1. Grieder NC
    2. de Cuevas M
    3. Spradling AC
    (2000)
    The fusome organizes the microtubule network during oocyte differentiation in Drosophila
    Development 127:4253–4264.

Decision letter

  1. Anna Akhmanova
    Senior and Reviewing Editor; Utrecht University, Netherlands
  2. Moe R Mahjoub
    Reviewer; Washington University in St Louis, United States
  3. Jens Januschke
    Reviewer; University of Dundee School of Life Sciences, United Kingdom

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study provides evidence for the role of the Alms1a, a Drosophila homolog of the human ciliopathy gene Alstrom syndrome, as a specific centriole duplication factor in male germline stem cells in flies. The study also describes the interactions of Alms1a with some key components of the centriole duplication pathway, and these data will inform further mechanistic studies on the centriole duplication in stem cells and the function of Alms1a in this process.

Decision letter after peer review:

Thank you for submitting your article "Alstrom syndrome gene is a stem cell-specific regulator of centriole duplication in the Drosophila testis" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Anna Akhmanova as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Moe R Mahjoub (Reviewer #1); Jens Januschke (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

This study investigates the role of Alms1a in regulating the asymmetric behavior of centrosomes in asymmetrically dividing Drosophila stem cells. Using a biochemical approach, they identify Alms1a as an interactor of Klp10A, a protein they previously showed to be enriched on centrosomes of male germline stem cells (GSC). They show that Alms1a exhibits asymmetric localization between mother and daughter centrosomes in GSCs, with enrichment on the mother centrosome compared to the daughter centrosome. Using super-resolution imaging, they show that Alms1a localizes to the proximal region of the mother centriole and extends toward the proximal end of the daughter centriole specifically in the GSC mother centrosome. Depletion of Alms1a in vivo during stem cell proliferation resulted in centriole duplication defects, which was specifically associated with asymmetrically dividing GSCs, resulting in loss of centrosomes over time/cell division. Finally, using a combination of biochemical and fluorescence microscopy methods (e.g. BiFC), they demonstrate that Alms1a interacts with key components of the centriole duplication pathway, including Sak and Sas6, to regulate centriole assembly in these stem cells.

The manuscript contains a lot of interesting data and valid points. The discovery of a potential cell type specific centriole duplication factor is very interesting, of broad interest to the readership of eLife and thus well worth reporting. The story is consistent with prior results from this lab (and others) that highlight asymmetric behaviors between mother and daughter centrosomes in asymmetrically-dividing stem cells across several systems. On the other hand, mechanistic insights and their interpretation as to how Alms1 would execute this function are as is not straightforward. The proposed model that Alsm1a is a male GSC specific factor that specifically regulates daughter centriole duplication by being present on the daughter centriole within the mother centrosome needs additional support and could benefit, if possible, from more super-resolution data.

Essential revisions:

1) The main observation is based on one RNAi line causing the phenotype. It would be important to add additional controls, a rescue or perhaps the use of a different RNAi line, or a mutant. In addition to the rescue with full-length protein, the authors can attempt the rescue experiment using the C-terminal fragment of the gene, since they suggest this is the key motif sufficient for interactions with both Klp10 at GSC centrosome, and for "recruiting" Sak during centriole duplication. Both points could be addressed with this one approach.

2) Furthermore, while the central observation is remarkable, the study is somewhat preliminary in terms of the molecular mechanism. Refining the localization of Alms1a (and its C-terminal fragment) using a quantitative approach (potentially via STED or another superresolution imaging modality) would also strengthen the conclusion about the relative localization at mother vs daughter centrioles. At the same time, they can use this method to check the status of mother vs daughter centrioles in Alms1a-depleted cells.

3) Figure 6, the overexpression of Alms1 and Sak give rise to extra centrosomes even if Sak alone only results in a milder effect. This is very interesting as Sak over-expression is normally sufficient to generate extra centrosomes. However, the centrosomes in the panel Sak, Alms1 (Figure 6D) have different sizes and Asl intensities. Are the authors sure that they all represent extra bona fide centrosomes? Maybe using other centriole markers, such as Sas4, would be useful.

4) Finally, additional quantifications and controls (e.g. for the bipartide system to detect protein interactions) indicated in specific comments of the reviewers below would be important.

We include below specific comments of individual reviewers for more background.

Reviewer 1:

1) The authors say "RNAi-mediated depletion of klp10A leads to abnormal elongation of the mother centrosomes in GSCs". Do they mean mother centriole? Or are both mother and daughter centrioles of the mother centrosome elongated? This also applies to Figure 3C-D, where the authors suggest "elongated mother centrosomes" upon Alms1 depletion. Based on the centriole duplication defect, I imagine they are referring to the existing mother centriole as being elongated.

2) Figure 1E-F: The authors demonstrate interaction between Alms1a C-terminal motif with Klp10a, indicating this domain is sufficient. Did they test whether the N-terminus plays a role in this too? There are a number of centrosomal proteins that can interact via multiple domains.

3) Figure 2: Is the C-terminal domain of Alms1a, if expressed in the GSC, still show asymmetric association between mother and daughter centrosomes?

4) Figure 2—figure supplement 1A: The authors show that klp10A-RNAi did not affect Alms1a localization to the SG centrosomes. Did they quantify the relative signal intensity between mother and daughter (since depletion of Klp10A does not cause mislocalization of Alms1a from centrosomes in GSCs completely either)?

5) Figure 5: Similar to Figure 1, they show interaction between Alms1a C-terminal motif with Sak, but not with Sas6. Did they identify interaction with Sas6 via the N-terminal half of Alms1a?

6) Figure 6: The authors say "These results suggest that Alms1a promotes centrosome duplication together with Sak, possibly by recruiting Sak to the site of centriole duplication in GSCs". Do the authors know whether Alm1a is phosphorylated, and if so, is this Sak-dependent (since Sak's role in centriole duplication is typically mediated via phosphorylation)?

7) Figure 6: Importantly, if Alms1a's role in duplication is merely to recruit Sak, then one experiment to test that would be to express the C-terminal domain of Alms1a (which they show to be sufficient for interaction), to see if it can rescue the centriole duplication defect in Alms1a-depleted cells.

Reviewer 2:

1) In S-phase both the mother and daughter centrosomes of GSCs contain normally both a set of two centrioles. When centriole duplication is triggered again, both centrioles within the mother centrosome should be competent to duplicate. The data supporting that Alms1a regulates duplication of a specific type of centrioles are currently not strong. Cnb depletion in mother centrioles in GSCs is not quantified. Given that cnb mutant germ line cells do have centrioles despite basal body length regulation problems in spermatocytes (Reina, JCB 2018), it is not clear what depleted Cnb in the Alms1 RNAi GSCs means. While a possibility, it does not demonstrate per se that centriole duplication has not occurred. There is further no solid evidence to support that the mother centriole within the mother centrosome keeps duplicating when Alms1a is depleted, while the daughter centriole does not (appears also unlikely given the morphological changes (elongation) detected. The results point out that Alsm1a localizes to the proximal end of the mother centriole and does not overlap with Cnb, yet the discussion summarizes "Importantly, Alms1a's function is required in GSCs but not SGs, thus this GSC-specific localization at daughter centriole of the mother centrosome is likely what is required for centriole duplication." I got confused here and think that the rationale here needs to be better worked out.

2) Within the GSC centrosomes, would two rings of Asl in GSCs be expected normally in S-phase? This is what Figure 2C, D suggest. However, one Asl ring seems to be detected in Figure 2F, G (SG cells) and in Figure 2O-Q (GSCs). It is possible that Asl labels the wall of only one centriole, despite two centrioles being present as at least in embryos Asl is only incorporated in the new daughter later in the centriole duplication cycle (e.g. Raff lab). Thorough quantifications of the localization of Asl signal (one or two centriole walls detected) is missing. This might be of relevance to assign centriole specific roles, especially as statements on the precise point of the cell and the centriole duplication cycle are tough to make in the system and could potentially be a confounding factor. Is there any way to control that rule that out? It is a pity that the STED analysis had not been extended to measure centrioles upon Alms1a depletion. STED on Asl, Alsm1a and Cnb in controls and upon Alms1 depletion in GSCs plus quantification might be desirable to better understand how loss of a specific pool/enrichment of Alms1 might drive a particular function at a given location.

3) Faulty centriole duplication is a likely possibility of centriole loss here, but centrioles could also be destabilized (fragmented and then lost). Can that be excluded? Especially since the gal80ts experiment revealed that after 4 days almost half of the GSCs appear to be centrosome less (Figure 3H model points at loss of centrosomes from GSCs), while in the nosGa4 driven depletion the conclusion is the GSCs specifically retain centrosomes (quantification missing). Or is mother centrosome anchoring depending on Alms1a? In my understanding the mother centrosome is "immortal" when properly anchored in this system.

Reviewer 3:

1) Alms1 RNAi. The authors show indeed a clear reduction by western blot. Did they rescue the depletion? Even if Alms RNAi results in lower levels, they should show that the phenotypes obtained are only due to this depletion. Using a resistant line or a mutation that can be rescued by Alms1 transgene will show this.

2) Figure 2L, not sure I agree with the interpretation of Alms1 being localized to the proximal site of the two centrioles in the mother centrosome. Maybe super resolution will help and drawing line scans to illustrate the fluorescence intensity distribution?

3) Figure 5E, I am not sure I am interpreting this western blot correctly. While I can see in the IP for Sak the pull down of GST- Alms1 (Figure 5D), in the Sas-6 lane, this is not the case.

4) The bipartide system to detect interactions- VNm9. I wonder if all the controls are included in the quantifications. Because in Figure 5L the interaction in mother GSCs is more prominent, but still is not zero in the other centrosomes. Does it mean that they do not interact or that they interact with more molecules in the mother centrosome?

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Alstrom syndrome gene is a stem cell-specific regulator of centriole duplication in the Drosophila testis" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Anna Akhmanova as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Moe R Mahjoub (Reviewer #1); Jens Januschke (Reviewer #2).

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. All reviewers agreed that the paper has been improved and many comments were satisfactorily addressed. However, there are some remaining points that need to be addressed, as outlined below:

Essential revisions:

1) The rescue experiment deserves some additional attention. The experimental setup used here shows that the RNAi line targets Alms1a, but it does not seem to conclusively show that only targets this gene. Alms1a-GFP in the RNAi background only partially restores the normal centrosome number of 2 centrosomes per cell. According to the data included in Figure 1—figure supplement 3, all control cells have 2 centrosomes, and in Alms1a RNAi, the large majority has zero and around 25% contain 1 centrosome. While Alms1a-GFP does not cause a change in centrosome number, in the RNAi background, only 25% of the cells contain 2 centrosomes. It is true that there is a decrease in the percentage of cells showing zero centrosomes, but this could be due to the excess of Alms1a-GFP and therefore reduced effect of the RNAi. Therefore, there was a concern that these data are insufficient to prove specificity of the RNAi used, and that the RNAi line is depleting other genes responsible for the phenotypes. Ideally, one would like to see characterization of another RNAi line or a mutation that can be fully rescued by the GFP line. Alternatively, one could generate a GFP line that it is not targeted by the RNAi. If this is not possible, the reviewers would like to see proof that in the rescue experiments, the depletion of the endogenous Alms1a not affected by the expression of Alms1a-GFP and some detailed discussion about the quality of the rescue experiment, which should get a more prominent place in the paper.

2) The point that Alms1a RNAi leads to the loss of the diplosome configuration of the GSC centrosomes is not demonstrated with sufficient clarity (if one looks closely there is a faint CNB signal at the mother centrosome). Therefore, it appears problematic to obtain sufficiently conclusive data with CNB as a marker. The authors should provide conclusive, quantified experimental evidence about centriole numbers per centrosome in GSCs upon knock down of Alsm1a (as in Figure 3F,G and Figure 2—figure supplement 2). The reviewers suggest using STED looking for cell cycle dependent ASL signal that allows visualizing the diplosome. The authors have now done this in controls (from where it appears that anaphase is suitable), but not in the relevant experimental conditions. These data should be included.

https://doi.org/10.7554/eLife.59368.sa1

Author response

Essential revisions:

1) The main observation is based on one RNAi line causing the phenotype. It would be important to add additional controls, a rescue or perhaps the use of a different RNAi line, or a mutant. In addition to the rescue with full-length protein, the authors can attempt the rescue experiment using the C-terminal fragment of the gene, since they suggest this is the key motif sufficient for interactions with both Klp10 at GSC centrosome, and for "recruiting" Sak during centriole duplication. Both points could be addressed with this one approach.

The rescue experiments have been performed and the results are shown in Figure 3—figure supplement 1. Full-length Alms1a protein is able to rescue the RNAi phenotype partially (possibly because of variable expression levels, whereby the expression level does not reach to a sufficient level in some cells/tissues). ALMS motif alone failed to rescue the RNAi phenotype. Even if this motif may be an essential domain to interact with Klp10A and Sak, it does not mean that this motif would be sufficient for Alms1a function(s) as the remaining region might mediate other essential functions such as linking Klp10A/Sak to something else.

2) Furthermore, while the central observation is remarkable, the study is somewhat preliminary in terms of the molecular mechanism. Refining the localization of Alms1a (and its C-terminal fragment) using a quantitative approach (potentially via STED or another superresolution imaging modality) would also strengthen the conclusion about the relative localization at mother vs daughter centrioles. At the same time, they can use this method to check the status of mother vs daughter centrioles in Alms1a-depleted cells.

Detailed characterization of Alms1a localization together with Cnb and Asl have been performed using STED (Figure 2P-R). Alms1a localizes to both mother and daughter centriole specifically in GSC mother centrosome as we stated. At the daughter centriole of the mother centrosome, it completely colocalizes with Cnb. We also included spermatocytes’ centrosome (Figure 2O) to show that Alms1a indeed localizes to the proximal side of the centriole.

The status of mother vs daughter centrioles in wild type and alms1RNAi GSCs has been examined with centrosome markers Cp110 and Asl using Lightning microscopy (Figure 3—figure supplement 2). Cp110 marks both mother and daughter centrioles in interphase wild type GSCs, consistent with previous studies that in GSCs centriole duplication occurs immediately after centrioles disengage. (Note that, this is in contrast to Drosophila neuroblast, where mother and daughter centrioles each organize centrosome without duplicating centrioles for quite some time during the cell cycle.) In contrast to wild type centrosomes, where two Cp110 dots were always observed, there is only one Cp110 spot on the elongated mother centrosome upon Alms1a depletion, suggesting that elongated centrosome is made of a single, elongated mother centriole, and there is no daughter centriole. Accordingly, many core centriole proteins and PCM proteins including Sak, Sas6, Ana1, Asl and g-Tub observed on “elongated mother centrosome” reflects their localization to the mother centriole (Figure 3 and 6).

3) Figure 6, the overexpression of Alms1 and Sak give rise to extra centrosomes even if Sak alone only results in a milder effect. This is very interesting as Sak over-expression is normally sufficient to generate extra centrosomes. However, the centrosomes in the panel Sak, Alms1 (Figure 6D) have different sizes and Asl intensities. Are the authors sure that they all represent extra bona fide centrosomes? Maybe using other centriole markers, such as Sas4, would be useful.

Additional markers including Sas-6 (centrioles) and Spd2 (both centrioles and PCM) have been used to characterize the centrosomes upon overexpression of Alms1a and Sak (Figure 6—figure supplement 1). Indeed, supernumerary centrosomes observed upon overexpression of Alms1a and Sak contains all centrosomal/centriolar markers, suggesting that these are bona fide centrosomes.

4) Finally, additional quantifications and controls (e.g. for the bipartide system to detect protein interactions) indicated in specific comments of the reviewers below would be important.

Quantifications and controls for BiFC have to added as shown in Figure 1—figure supplement 3 and Figure 5L. For threshold, we subtract the BiFC signal on ROI (centrosomes) to background signal (same size area in background), then use imageJ to quantify the integrated intensity density of IF signals.

We include below specific comments of individual reviewers for more background.

Reviewer 1:

1) The authors say "RNAi-mediated depletion of klp10A leads to abnormal elongation of the mother centrosomes in GSCs". Do they mean mother centriole? Or are both mother and daughter centrioles of the mother centrosome elongated? This also applies to Figure 3C-D, where the authors suggest "elongated mother centrosomes" upon Alms1 depletion. Based on the centriole duplication defect, I imagine they are referring to the existing mother centriole as being elongated.

In nos>klp10ARNAi GSCs, only mother centriole elongates. Daughter centriole exists but not elongates (see Author response image 1, which is reproduced from Figure 2A-B in Chen et al., 2016 (published under the Creative Commons Attribution 4.0 International Public License, (CC BY 4.0, https://creativecommons.org/licenses/by/4.0/))). In nos>alms1RNAi GSCs, only mother centriole exists and elongates (Figure 3—figure supplement 2). Also, Response 2 to Essential revision point 2 hopefully address this point further.

Author response image 1

2) Figure 1E-F: The authors demonstrate interaction between Alms1a C-terminal motif with Klp10a, indicating this domain is sufficient. Did they test whether the N-terminus plays a role in this too? There are a number of centrosomal proteins that can interact via multiple domains.

We haven’t tested the function of Alms1a N-terminal motif yet. The reasons we expressed C-terminal domain only in some experiments reported here were 1) to test the function of ALMS motif (as it is the only conserved domain), and also 2) full length Alms1a does not express well in bacteria. Although it is possible that N-ter domain may also interact with other proteins, we feel that it is beyond the scope of the present study that identifies a protein that exhibits unique asymmetric localization on the GSC centrosomes for the first time.

3) Figure 2: Is the C-terminal domain of Alms1a, if expressed in the GSC, still show asymmetric association between mother and daughter centrosomes?

We did observe some asymmetric localization between mother and daughter centrosome in nos>GFP-ALMS motif GSCs (panel in Author response image 2). But we are not sure about the significance/meaning of this, and we feel that addressing such details is beyond the scope of the present study.

Author response image 2

4) Figure 2—figure supplement1A: The authors show that klp10A-RNAi did not affect Alms1a localization to the SG centrosomes. Did they quantify the relative signal intensity between mother and daughter (since depletion of Klp10A does not cause mislocalization of Alms1a from centrosomes in GSCs completely either)?

Quantification have been added to the Figure 2—figure supplement 1.

5) Figure 5: Similar to Figure 1, they show interaction between Alms1a C-terminal motif with Sak, but not with Sas6. Did they identify interaction with Sas6 via the N-terminal half of Alms1a?

We haven’t done the GSC-pulldown assay to test the interaction of Sas6 with Alms1a N-ter. However, based on Bacterial Two Hybrid experiment (Figure 5—figure supplement 1), where we did not detect direct interaction between Sas6 and full-length Alms1a, there is no reason to believe Alms1a N-ter would interact with Sas-6.

6) Figure 6: The authors say "These results suggest that Alms1a promotes centrosome duplication together with Sak, possibly by recruiting Sak to the site of centriole duplication in GSCs". Do the authors know whether Alm1a is phosphorylated, and if so, is this Sak-dependent (since Sak's role in centriole duplication is typically mediated via phosphorylation)?

Based on the general knowledge of the centrosome field, it would not be surprising if Alms1a is phosphorylated by Sak. But we feel that this level of analysis is beyond the scope of the present study. We hope that the reviewers agree with our notion.

7) Figure 6: Importantly, if Alms1a's role in duplication is merely to recruit Sak, then one experiment to test that would be to express the C-terminal domain of Alms1a (which they show to be sufficient for interaction), to see if it can rescue the centriole duplication defect in Alms1a-depleted cells.

Please see our response to Essential revision 1.

Reviewer 2:

1) In S-phase both the mother and daughter centrosomes of GSCs contain normally both a set of two centrioles. When centriole duplication is triggered again, both centrioles within the mother centrosome should be competent to duplicate. The data supporting that Alms1a regulates duplication of a specific type of centrioles are currently not strong. Cnb depletion in mother centrioles in GSCs is not quantified. Given that cnb mutant germ line cells do have centrioles despite basal body length regulation problems in spermatocytes (Reina, JCB 2018), it is not clear what depleted Cnb in the Alms1 RNAi GSCs means. While a possibility, it does not demonstrate per se that centriole duplication has not occurred. There is further no solid evidence to support that the mother centriole within the mother centrosome keeps duplicating when Alms1a is depleted, while the daughter centriole does not (appears also unlikely given the morphological changes (elongation) detected. The results point out that Alsm1a localizes to the proximal end of the mother centriole and does not overlap with Cnb, yet the discussion summarizes "Importantly, Alms1a's function is required in GSCs but not SGs, thus this GSC-specific localization at daughter centriole of the mother centrosome is likely what is required for centriole duplication." I got confused here and think that the rationale here needs to be better worked out.

We hope that the revision experiments (described above) have already clarified most of this reviewer’s concerns about the mode of centriole duplication in wild type and Alms1a-RNAi GSCs.

Quantification of Cnb staining has been added (new Figure 3H).

We would like to note that, unlike Drosophila neuroblasts, where single centriole (split mother and daughter centrioles) each organizes two centrosomes for quite some time during cell cycle before they eventually duplicate centrioles, male GSCs appear to duplicate centrioles as soon as mother and daughter centrioles split from each other during G1/S transition (Salzmann, 2014 MBoC). Noting this difference likely clarifies some concerns raised by this reviewer.

We believe that the lack of Cnb indicates the lack of daughter centrioles: please note the aforementioned centrosome composition in GSCs during cell cycle. Although this reviewer might have inferred that the lack of Cnb localization means that Cnb disappeared from a centriole, our data rather suggests that the daughter centriole to which Cnb can localize is not present. We hope the loss of daughter centriole is better supported now based on revision experiments (described above). We are not claiming that Alms1a regulates centriole duplication via Cnb—instead, we believe that the lack of Cnb simply demonstrates that there is no daughter centrioles (to which Cnb can localize).

2) Within the GSC centrosomes, would two rings of Asl in GSCs be expected normally in S-phase? This is what Figure 2C, D suggest. However, one Asl ring seems to be detected in Figure 2 F, G (SG cells) and in Figure 2O-Q (GSCs). It is possible that Asl labels the wall of only one centriole, despite two centrioles being present as at least in embryos Asl is only incorporated in the new daughter later in the centriole duplication cycle (e.g. Raff lab). Thorough quantifications of the localization of Asl signal (one or two centriole walls detected) is missing. This might be of relevance to assign centriole specific roles, especially as statements on the precise point of the cell and the centriole duplication cycle are tough to make in the system and could potentially be a confounding factor. Is there any way to control that rule that out? It is a pity that the STED analysis had not been extended to measure centrioles upon Alms1a depletion. STED on Asl, Alsm1a and Cnb in controls and upon Alms1 depletion in GSCs plus quantification might be desirable to better understand how loss of a specific pool/enrichment of Alms1 might drive a particular function at a given location.

In GSC centrosomes, Asl labels only the wall of mother centriole at interphase. Also the above-mentioned centrosome composition/centriole duplication timing in GSCs may now clarify some of confusions, and we hope that most of issues are clarified for this reviewer. In addition, STED analysis of the Asl localization has been performed and the results are shown as Figure 2—figure supplement 2.

3) Faulty centriole duplication is a likely possibility of centriole loss here, but centrioles could also be destabilized (fragmented and then lost). Can that be excluded? Especially since the gal80ts experiment revealed that after 4 days almost half of the GSCs appear to be centrosome less (Figure 3H model points at loss of centrosomes from GSCs), while in the nosGa4 driven depletion the conclusion is the GSCs specifically retain centrosomes (quantification missing). Or is mother centrosome anchoring depending on Alms1a? In my understanding the mother centrosome is "immortal" when properly anchored in this system.

The mother centrosome show ~95% of anchoring near the niche even in wild type, which means that the mother centrosome can be lost to differentiating cells at some frequency (not 100% “immortal”). After 4 days, which is 6-8 cell cycles, it is not surprising that significant population of GSCs have lost the centrosomes. Although the possibility of centriole destabilization may not be entirely excluded, given that the mother centrioles are stable enough (such that they can elongate so long) rather seems to favor the possibility of centriole loss. Also, we hope that the added revision experiments and explanations above clarify this reviewer’s concern on this point.

Reviewer 3:

1) Alms1 RNAi. The authors show indeed a clear reduction by western blot. Did they rescue the depletion? Even if Alms RNAi results in lower levels, they should show that the phenotypes obtained are only due to this depletion. Using a resistant line or a mutation that can be rescued by Alms1 transgene will show this.

This issue was addressed in the Essential revision point 1.

2) Figure 2L, not sure I agree with the interpretation of Alms1 being localized to the proximal site of the two centrioles in the mother centrosome. Maybe super resolution will help and drawing line scans to illustrate the fluorescence intensity distribution?

The added revision experiments hopefully address this concern. Also, we added Alms1a localization in the spermatocytes, where much longer centrioles help visualize proximal vs. distal sides of centrioles (new Figure 2O). This clearly showed that Alms1a localizes at the proximal end of the centrioles.

3) Figure 5E, I am not sure I am interpreting this western blot correctly. While I can see in the IP for Sak the pull down of GST- Alms1 (Figure 5D), in the Sas-6 lane, this is not the case.

Correct. Sas-6 doesn’t interact with Alms1a C-ter, which is our conclusion (Sas-6 and Alms1a interacts in the GSC extract, but not via GST-pulldown using bacterial extract. Based on this, we concluded that Sas-6-Alms1a interaction is indirect).

4) The bipartide system to detect interactions- VNm9. I wonder if all the controls are included in the quantifications. Because in Figure 5 L the interaction in mother GSCs is more prominent, but still is not zero in the other centrosomes. Does it mean that they do not interact or that they interact with more molecules in the mother centrosome?

Quantifications of controls have been added in Figure 5L. There is no detectable BiFC signals in controls (both nos>alms1a-VNm9 and nos>Vc155-sak).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Essential revisions:

1) The rescue experiment deserves some additional attention. The experimental setup used here shows that the RNAi line targets Alms1a, but it does not seem to conclusively show that only targets this gene. Alms1a-GFP in the RNAi background only partially restores the normal centrosome number of 2 centrosomes per cell. According to the data included in Figure 1—figure supplement 3, all control cells have 2 centrosomes, and in Alms1a RNAi, the large majority has zero and around 25% contain 1 centrosome. While Alms1a-GFP does not cause a change in centrosome number, in the RNAi background, only 25% of the cells contain 2 centrosomes. It is true that there is a decrease in the percentage of cells showing zero centrosomes, but this could be due to the excess of Alms1a-GFP and therefore reduced effect of the RNAi. Therefore, there was a concern that these data are insufficient to prove specificity of the RNAi used, and that the RNAi line is depleting other genes responsible for the phenotypes. Ideally, one would like to see characterization of another RNAi line or a mutation that can be fully rescued by the GFP line. Alternatively, one could generate a GFP line that it is not targeted by the RNAi. If this is not possible, the reviewers would like to see proof that in the rescue experiments, the depletion of the endogenous Alms1a not affected by the expression of Alms1a-GFP and some detailed discussion about the quality of the rescue experiment, which should get a more prominent place in the paper.

We apologize for the confusion. We should have been clearer, but as was explained in the Materials and methods section, our rescue transgene is already RNAi-resistant. Therefore, it is highly unlikely that the introduction of transgene is weakening the RNAi effect, as the reviewers were concerned. Also, please note that the RNAi line that we used is from the “TRiP line” series that were designed and generated by Nobert Perrimon’s group, where the target sequence was designed computationally to avoid similarity to any other location of the Drosophila genome. The target sequence for this construct is TCGGAAGTAACTCAGACGAAA and upon BLAST search against Drosophila genome, we do not find any matching sequences except for Alms1a and Alms1b genes. Therefore, it is highly unlikely that the RNAi construct we used have unknown off-target effect (expect for targeting Alms1b additionally, because these two genes are almost identical).

Also, as explained in the manuscript, our attempts to generate any additional RNAi lines (only target Alms1a without targeting Alms1b) have been unsuccessful (we generated 2 lines but none of them efficiently targeted Alms1a).

Based on these information (in particular, the unlikeliness of off-target effects), our best explanation for partial rescue is the expression level of the transgene. It is widely known that nos-gal4 (the driver that we use for transgene expression) varies in expression level among cells. As necessary, we can add additional discussions in the main text to add caveats of our experiments.

Additional Editor comment for Essential revision 1

Rescue: please make sure to explain in the main text how the rescue was done and include all the data pertaining to the rescue in the main figures. Please discuss all the various potential reasons explaining why the rescue is not complete (insufficient expression levels of the rescue construct, problems with the GFP fusion, etc). Since the rescue is incomplete, potential off-target effects of the RNAi remain a distinct possibility and must be included in the discussion - please soften your conclusions accordingly, and make sure that this possibility is very explicitly mentioned and will be obvious to the readers of the manuscript. With other words, you must clearly state in the manuscript that you cannot be sure that the phenotypes described are unambiguously linked to the lack of Alms1a function.

We have added the explanation in the main text in the Result section about these possibilities. With this addition of the text clearly describing caveats etc., we wish to keep the rescue data in the supplementary figures, simply because the data are too bulky to be included in the main figures. With a clear explanation in the text, this information (only partial rescue) is not hidden, and we believe that it is sufficient for transparency. On the contrary, we are concerned that the insertion of the bulky data into the main figure disrupts the flow of logic: there are many published papers whose entire organization had become very confusing and fragmented as a result of many revision processes, preventing the understanding by general readers. We believe that dissemination of scientific results must be mindful of broad scientific community (current and future), not just for authors and reviewers. By “flow” we do not mean the one that is convenient for us/authors, but the one better for the understanding by readers. If the editor/reviewers feel that the text dealing with experimental caveat is insufficient, we will further edit the text, but we hope that they agree that our treatment of this rescue data by the text provides sufficient transparency.

We also think that the question pertaining whether ALMS motif only might rescue the phenotype (which is included in this rescue figure) is rather too specific, and inclusion of such data into main figure does not benefit general readers. We added this experiment per reviewers’ request: if the result were to be positive, i.e. ALMS motif was sufficient to rescue the phenotype, it would have added an important knowledge, so we agreed with the reviewers’ point on the potential value of such an experiment. But now that we obtained the completely negative data, addition of such data in the main figure is not warranted. Again, inclusion of such data rather confuses readers, instead of providing better understanding and transparency.

2) The point that Alms1a RNAi leads to the loss of the diplosome configuration of the GSC centrosomes is not demonstrated with sufficient clarity (if one looks closely there is a faint CNB signal at the mother centrosome). Therefore, it appears problematic to obtain sufficiently conclusive data with CNB as a marker. The authors should provide conclusive, quantified experimental evidence about centriole numbers per centrosome in GSCs upon knock down of Alsm1a (as in Figure 3F,G and Figure 2—figure supplement 2). The reviewers suggest using STED looking for cell cycle dependent ASL signal that allows visualizing the diplosome. The authors have now done this in controls (from where it appears that anaphase is suitable), but not in the relevant experimental conditions. These data should be included.

We understand the potential concerns of reviewers on this point. However, we would like to bring their attention to the terminal phenotype of Alms1a-RNAi, as shown in Figure 3C, D, which is clear loss of daughter centrosomes. At this point, as is shown in Figure 3—figure supplement 2, the already-elongated mother centrosome contains only one Cp110 signal, as opposed to 2 dots/centrosome in the control (if deemed necessary, we can provide the quantification of Cp110 number per centrosome in control vs. Alms1a-RNAi GSCs). This clearly demonstrates that the final phenotype of Alms1a-RNAi is the loss of diplosome configuration. We believe that this is already sufficient to draw our conclusion that Alms1a is required for centriole duplication.

In this regard, Figure 3 F, G, H are only additional confirmation to our conclusion by capturing the moment of centrosome loss: when the mutant GSCs still contain 2 centrosomes = 2 g-tubulin dots (i.e. before GSCs exhibit the terminal phenotype of having elongated mother centrosome only), the mother centrosome already has a diminished amount (almost none) of Cnb---that’s the only point that we’re trying to convey, and these panels are not the only evidence to support our conclusion (because in the end, clearly the elongated mother centrosome does not have the diplosome configuration). We agree that one can see a tiny amount of Cnb still left on the mother centrosome, but this is clearly much less than the control situation (which has equal amount of Cnb on two centrosomes), and the fact that a tiny amount of Cnb is left on the mother centrosome does not negate our conclusion that the Alms1a-RNAi leads to “compromised daughter centriole generation”.

Moreover, as the data shown in Figure 3F-H represent “transitional phenotype” (instead of terminal phenotype), when the GSC is “about to lose the daughter centrosome”, capturing the right moment is extremely challenging. Anaphase cells are quite infrequent: Anaphase is <10% of entire mitotic cells, and GSCs’ overall mitotic index is only 2% (of total GSCs), this means that anaphase GSC is only less than 0.2% of entire GSCs. This is still feasible when we’re imaging wild type (Figure 2—figure supplement 2), but when combined with the need to capture the moment of centriole loss (specific time point when RNAi becomes effective), it becomes essentially impossible. Of course, lack of feasibility is not the reason for not needing the result, if the entire conclusion relies on that specific result, but as we outlined above, we believe that our overall conclusion (centriole duplication defect) is already sufficiently supported by Alms1a RNAi’s terminal phenotype (Figure 3—figure supplement 2).

We can certainly rephrase our main text to convey these points. We sincerely ask reviewers to consider these issues and hope they agree that we do not need additional experiments.

Additional Editor comment for Essential revision 2

Conclusive, quantified experimental evidence about centriole numbers per centrosome in GSCs: indeed, please provide the quantification of Cp110 number per centrosome in control vs. Alms1a-RNAi GSCs. Overall, the reviewers have already raised the question about insufficient quantification in the first round of review and were somewhat disappointed by this aspect of the revised paper. Since CNB marker raises concerns, perhaps quantification would be appropriate as well? Again, please make sure that none of this information is "hidden" in the supplements.

We apologize if the addition of quantification data was not clear enough to reviewers in the first round of the revision. We had added the quantification of Cnb intensity (Figure 3H) per reviewers’ request during the first round of the revision, which shows a very low intensity of Cnb on the mother centrosome in Alms1-RNAi GSCs. In this revised version, we added more emphasis in the text on this aspect. Also Cp110 data was moved to the main Figure as new Figure 3I, J, with additional quantification as new Figure 3K.

In addition, as explained in our response letter that accompanied the first round of the revision, data quantification had been added to multiple places, addressing all of reviewers’ request during the first round of revision (Figure 5L, Figure 1—figure supplement 3, Figure 2—figure supplement 1B). Therefore, we are somewhat confused by the reviewers’ remarks that they were disappointed by the lack of quantification.

https://doi.org/10.7554/eLife.59368.sa2

Article and author information

Author details

  1. Cuie Chen

    Life Science Institute, Department of Cell and Developmental Biology, Michigan Medicine, University of Michigan, Ann Arbor, United States
    Contribution
    Conceptualization, Resources, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5498-9753
  2. Yukiko M Yamashita

    1. Life Science Institute, Department of Cell and Developmental Biology, Michigan Medicine, University of Michigan, Ann Arbor, United States
    2. Howard Hughes Medical Institute, Cambridge, United States
    3. Whitehead Institute for Biomedical Research, Cambridge, United States
    4. Department of Biology, Massachusetts Institute of Technology, Cambridge, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Investigation, Writing - original draft, Writing - review and editing
    For correspondence
    yukikomy@wi.mit.edu
    Competing interests
    Reviewing editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5541-0216

Funding

Howard Hughes Medical Institute

  • Yukiko M Yamashita

National Institute of General Medical Sciences (R01GM118308)

  • Yukiko M Yamashita

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Drs. Cheng-Yu Lee, Jordan Raff, Cayetano Gonzales, Monica Bettencourt-Dias, Nasser Rusan, Tomer Avidor-Reiss, Mayu Inaba, the Bloomington Stock Center, the Vienna Drosophila RNAi Center, the Drosophila Genomics Resource Center and the Developmental Studies Hybridoma Bank for reagents, and the Yamashita laboratory, Clemens Cabernard, Sue Hammoud and Life Science Editors for discussions and comments on the manuscript, MS Bioworks for mass-spectrometry analysis. This work was supported by R01GM118308 (to YMY). The research in the Yamashita laboratory is supported by Howard Hughes Medical Institute.

Senior and Reviewing Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Reviewers

  1. Moe R Mahjoub, Washington University in St Louis, United States
  2. Jens Januschke, University of Dundee School of Life Sciences, United Kingdom

Publication history

  1. Received: May 27, 2020
  2. Accepted: September 22, 2020
  3. Accepted Manuscript published: September 23, 2020 (version 1)
  4. Version of Record published: October 5, 2020 (version 2)

Copyright

© 2020, Chen and Yamashita

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Cuie Chen
  2. Yukiko M Yamashita
(2020)
Alstrom syndrome gene is a stem-cell-specific regulator of centriole duplication in the Drosophila testis
eLife 9:e59368.
https://doi.org/10.7554/eLife.59368

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