DNALI1 interacts with the MEIG1/PACRG complex within the manchette and is required for proper sperm flagellum assembly in mice
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Evaluation Summary:
This paper is of potential interest to a broad audience in the fields of germ cell biology and cytoskeleton, as it implies a microtubule-based motor function in intra-manchette cargo transport in developing sperm tail. However, some conclusions of this paper require stronger experimental support.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii , recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.
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Evaluation Summary:
This paper is of potential interest to a broad audience in the fields of germ cell biology and cytoskeleton, as it implies a microtubule-based motor function in intra-manchette cargo transport in developing sperm tail. However, some conclusions of this paper require stronger experimental support.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
It was previously shown that MEIG1/PACRG form a protein complex in the manchette and participated in sperm flagellum formation. However, how the MEIG1/PACRG complex associates with the axonemal motor system for cargo transport remained unclear. In this study, using Y2H screening, Yap et al., report DNALI1 is associated with MEIG1/PACRG complex and further show this interaction by several heterologous expression systems and co-localization of the PACRG and DNALI1 in elongating spermatids. To further understand the roles of DNAL1 in developing male germ cells, the authors generated a conditional knockout model using Stra8-Cre and DNALI1 flox mouse lines. The conditional males are infertile with dramatically reduced sperm numbers, abnormal sperm morphology, and severe spermiogenesis deficiency. The authors also …
Reviewer #1 (Public Review):
It was previously shown that MEIG1/PACRG form a protein complex in the manchette and participated in sperm flagellum formation. However, how the MEIG1/PACRG complex associates with the axonemal motor system for cargo transport remained unclear. In this study, using Y2H screening, Yap et al., report DNALI1 is associated with MEIG1/PACRG complex and further show this interaction by several heterologous expression systems and co-localization of the PACRG and DNALI1 in elongating spermatids. To further understand the roles of DNAL1 in developing male germ cells, the authors generated a conditional knockout model using Stra8-Cre and DNALI1 flox mouse lines. The conditional males are infertile with dramatically reduced sperm numbers, abnormal sperm morphology, and severe spermiogenesis deficiency. The authors also show the PACRG, MEIG1, and SPAG16L normally expressed in DNALI1 lacking spermatocytes and spermatids, but MEIG1 and SPAG16L are not present in the manchette in the absence of DNALI1. Based on the suggested impaired sperm individualization in Dnali1 mutant mice, which was not observed in the MEIG1 nor PACRG-deficient mice, the author claim that DNALI1 is upstream of MEIG1/PACRG and that has MEIG1/PACRG associated and non-associated functions in mammalian spermatogenesis.
The phenotype of Dnali1 male germ cell conditional knockout is interesting and strong, which demonstrates well that DNALI1 deficiency results in defective flagellar development and organization. Yet, its functional association with PACRG and MEIG in male germ cells is less clear than those from in vitro and heterologous interaction studies. The claimed MEIG/PACRG associated vs. non-associated function of DNALI1 in flagellar development also needs further delineation to support the current title.
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Reviewer #2 (Public Review):
The paper by Li et al reports the interaction of a subunit of inner arm dyneins DNALI1 with MEIG1/PACRG, which are known components associated with the manchette of spermatids. The authors generated a DNALI1 knockout mouse and found that the deficiency of this protein impacts diverse ranges in spermiogenesis. In particular, the authors showed that DNALI1 knockdown affected not only the flagellar structure but also the formation of both sperm nucleus and flagellum. The paper would contribute to the understanding of the molecular mechanism of intra-manchette transport. The major criticism of this paper is that the authors discuss the axonemal inner arm dynein as a binding partner of DNALI1 but any experimental evidence supporting this idea is not presented. Several other possibilities would be taken into …
Reviewer #2 (Public Review):
The paper by Li et al reports the interaction of a subunit of inner arm dyneins DNALI1 with MEIG1/PACRG, which are known components associated with the manchette of spermatids. The authors generated a DNALI1 knockout mouse and found that the deficiency of this protein impacts diverse ranges in spermiogenesis. In particular, the authors showed that DNALI1 knockdown affected not only the flagellar structure but also the formation of both sperm nucleus and flagellum. The paper would contribute to the understanding of the molecular mechanism of intra-manchette transport. The major criticism of this paper is that the authors discuss the axonemal inner arm dynein as a binding partner of DNALI1 but any experimental evidence supporting this idea is not presented. Several other possibilities would be taken into account, including that DNALI1 might have a stand-alone function separated from dynein assembly and that DNALI1 could be bound to cytoplasmic dynein (IFT dynein). In fact, DNALI1 is reported to interact with cytoplasmic dynein (Rashid et al, 2006). The paper would be therefore strengthened with the experimental evidence showing the direct interaction of DNALI1 with any axonemal dyneins.
In Chlamydomonas, the mutants of DNALI1 (p28), ida4, lack subsets of inner arm Dynein. This is primarily expected for mouse DNALI1 mutant. Authors are recommended first to focus more on the structure of inner arm dynein. Nonetheless, it is interesting that the authors' group, in relation to this paper, have found some axonemal proteins, other than those in inner arms, that are localized on manchette, including PACRG and SPAG16L. PACRG or SPAG16L is a protein localized to the inner junction of a doublet microtubule or the central apparatus, respectively. Authors may thus mention that a wide range of axonemal substructures is affected by the deficiency of DNALI1/MEG1/PACRG.
One of the phenotypes in sperm morphology, multiple flagella, would make the readers confused. This appears to be the simple disintegration or the split of a flagellum. Data to show multiple flagella, ie. two closely positioned flagella, or two axonemes (or basal bodies) in a flagellum, could be included in the EM data. In addition, supposing that DNALI1 is involved in a transport system for flagellar formation, I am concerned about how the authors explain the formation of full-length flagella in DNALI1 knockout mice, although the authors discussed this due to incomplete protein loss.
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Reviewer #3 (Public Review):
The authors of the present manuscript had previously shown that a complex of mouse meiosis-expressed gene 1 (MEIG1) and Parkin co-regulated gene (PACRG) are required to deliver essential cargoes to the developing sperm tail through the poorly understood microtubule-based process of intramanchette transport (IMT). How this MEIG1/PACRG complex associates with microtubule-based motors in IMT, however, was unclear. In this manuscript, the authors identified a microtubule motor protein, axonemal dynein light intermediate polypeptide 1 (DNALI1), as a PARG binding partner via a yeast two-hybrid screen. Further, they revealed that DNALI1 stabilises PACRG abundance in vitro, is expressed in the spermatid manchette, and that PACRG localisation to the manchette is dependent on DNALI1 but not vice versa. Utilising a …
Reviewer #3 (Public Review):
The authors of the present manuscript had previously shown that a complex of mouse meiosis-expressed gene 1 (MEIG1) and Parkin co-regulated gene (PACRG) are required to deliver essential cargoes to the developing sperm tail through the poorly understood microtubule-based process of intramanchette transport (IMT). How this MEIG1/PACRG complex associates with microtubule-based motors in IMT, however, was unclear. In this manuscript, the authors identified a microtubule motor protein, axonemal dynein light intermediate polypeptide 1 (DNALI1), as a PARG binding partner via a yeast two-hybrid screen. Further, they revealed that DNALI1 stabilises PACRG abundance in vitro, is expressed in the spermatid manchette, and that PACRG localisation to the manchette is dependent on DNALI1 but not vice versa. Utilising a Dnali1 germ cell-specific knockout mouse they reveal DNALI1 is essential for male fertility, with Dnali1 cKO sperm having a number of abnormalities that could be consistent with abnormal intramanchette transport, in addition to other abnormalities that are consistent with DNALI1 also having roles prior to the manchette appearing and after its dissolution (e.g. double flagella, spermiation failure). This paper is a key conceptual advance in understanding the mechanisms of intramanchette transport, a process that is still very poorly defined and direct evidence for has been largely lacking. Indeed, the strength of this manuscript is the data defining the interaction and the nature of the interaction, between DNALI1 and MEIG1/PACRG. However, in its current form, the characterisation of the role of DNALI1 in spermatogenesis needs to be improved to properly validate some of the data and to clarify the functions of DNALI1 in spermatogenesis. Once this is done the manuscript will be of interest and of use to both the germ cell biology and broader cell and cytoskeletal biology fields.
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