Pt-LIS1 participates nuclear deformation and acrosome formation via regulating Dynein-1 during spermatogenesis in Portunus trituberculatus

Chang, Le; Xiang, Qiu Meng; Liao, Zai-Tian; Zhu, Jun-Quan; Mu, Chang-Kao; Wang, Chun-Lin; Hou, Cong-Cong

doi:10.1038/s41598-024-83566-2

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Published: 24 February 2025

Pt-LIS1 participates nuclear deformation and acrosome formation via regulating Dynein-1 during spermatogenesis in Portunus trituberculatus

Le Chang¹,
Qiu Meng Xiang¹,
Zai-Tian Liao¹,
Jun-Quan Zhu¹,
Chang-Kao Mu¹,
Chun-Lin Wang¹ &
…
Cong-Cong Hou¹

Scientific Reports volume 15, Article number: 6632 (2025) Cite this article

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Abstract

Spermatogenesis involves complex dynamic mechanisms. Dynein-1 is a key carrier in cellular cargo transport, participating in nuclear deformation and acrosome formation during spermatogenesis. However, the regulatory mechanisms of Dynein-1 during cargo transport remain unknown. In this study, we explored the role of Lissencephaly 1 (LIS1) in spermatogenesis and its impact on Dynein-1 cargo transport in Portunus trituberculatus. LIS1 was known as a Dynein-1 regulator, which is a causative gene of anencephaly syndrome. Pt-Lis1 was cloned from the crab testis, and its highly expression was observed in the testis. Pt-LIS1 dynamically localized around the nucleus and acrosome during spermatogenesis, colocalizing with Dynein-1 subunits, microtubules, mitochondrial markers (PHB), and Acrosin. RNA interference reduced Pt-Lis1 expression, leading to decreased expression of Pt-dhc and Pt-dic. During spermatogenesis, the signals of Pt-LIS1, Pt-DHC, Pt-DIC, and α-Tubulin were weakened and showed disorganized distribution. The colocalization of Pt-LIS1 with Pt-DHC and Pt-DIC decreased, while abnormal colocalization significantly increased. In addition, caspase-3 and p53 expression significantly increased after Pt-Lis1 silencing, indicating association with apoptosis in spermatogenic cells. All these results suggest that LIS1 played a crucial role in crustacean spermatogenesis by regulating nuclear deformation and acrosome formation through modulating Dynein-1 transport cargoes along microtubules.

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Introduction

Spermatogenesis is a highly complex physiological process that is coordinated in a well-regulated stepwise manner by various cell factors and signaling pathways and is involved in mitosis, meiosis and spermiogenesis^1,2. In mammals, spermatids undergo cytoplasmic discarding, nuclear deformation, acrosome formation and tail formation, which eventually develop into mature sperm with athletic ability^3,4. However, in crustaceans, mature sperm have a unique structure, showing irregular morphology, usually without a tail, and are nonmotile sperm without flagella^5,6. During spermatogenesis, the spermatid nucleus undergoes compression and deformation, ultimately forming a highly condensed sperm nucleus. This process is a crucial step in sperm development, enabling the sperm to reduce in volume and adapt to its specialized function and environment. The sperm nucleus morphology of crustaceans also varies according to different groups. In the Brachyura suborder, the nucleus of mature sperm is cup-shaped and wrapped around the acrosomal periphery⁷. In the swimming suborder, most of the sperm nucleus of Macrobrachium are disc-shaped, while the sperm nucleus of Penaeus are spherical^8,9. In Brachyura suborder, the sperm nucleus is cup-shaped and wrapped around the acrosome¹⁰. In early spermatids, the acrosome particles produced by rough endoplasmic reticulum were dispersed in the cytoplasm. In the middle stage of spermatids, they gradually aggregated to form large acrosomal vesicles localized on one side of the cell, and in late spermatids, they gradually specialized into an acrosomal structure consisting of an acrosomal cap, an acrosomal tube, an intermediate layer, a filamentous layer, and a lamellipodium, which involves complex dynamic mechanisms. The mature sperm of P. trituberculatus have the typical structure of the Brachyura suborder.

Motor proteins are the main driving force of intracellular transportation and cell deformation and use the cytoskeleton as a motor track to transport cellular cargo to specific sites for physiological functions in a precise and efficient manner¹¹. Cytoplasmic Dynein-1 (Dynein-1), a large molecular complex of motor proteins, consists of four types of subunits: heavy chain, intermediate chain, light chain and light intermediate chain^12,13. Dynein-1 is mainly responsible for the transport of various cargoes in the cytoplasm, including membranous organelles (lysosomes, Golgi bodies, mitochondria, etc.), mRNA, viruses, protein complexes (such as mitotic checkpoint proteins), etc.¹⁴. Dynein-1 plays an important role in spermatogenesis in Rattus norvegicus. Disruption of dynein function by either RNA interference (RNAi) or Ciliobrevin D inhibitors affected the formation of the blood-testis barrier and disrupt spermatogenesis, leading to an increase in abnormal spermatids¹⁵. In P. trituberculatus, Dynein-1 is involved in nuclear deformation and acrosome formation in spermatogenesis^16,17. At present, how Dynein-1 is regulated during cellular cargo transport in spermatogenesis remains unknown. However, studies have shown that in mammalian neuronal cells, Dynein-1 collaborates with its regulatory protein LIS1 to accomplish complex cellular activities¹⁸.

LIS1, as one of the important regulators of Dynein-1, binds to Dynein-1 through its C-terminal structural domain and changes Dynein-1 from an autoinhibitory state (Phi conformation) to an activated state¹⁹, thus participating in Dynein-1-mediated intracellular transport, nuclear migration, and localization of the spindle and organelles²⁰. At present, researches on the function of LIS1 mainly focus on the mechanism of the migration, survival, and proliferation of vertebrate nerve cells^21–23. A few studies have also shown that LIS1 is indispensable for sperm acrosome formation in mouse spermatogenesis and that Lis1 deficiency results in blocked acrosome formation, irregular nuclear morphology, abnormal apoptosis of spermatogenic cells, and even embryo death^18,24. In Drosophila spermatogenesis, LIS1 is closely associated with spermatid nucleus deformation, localization and acrosome formation by regulating Dynein-1-mediated centrosome localization²⁵. To date, the role and specific mechanism of LIS1 in the spermatogenesis of P. trituberculatus remains unknown.

The purpose of our research is to investigate the role of Pt-LIS1 in the spermatogenesis of P. trituberculatus and identify how Pt-LIS1 is involved in the regulation of cargo transport by Dynein-1. In the present study, we firstly cloned the full-length cDNA of the Pt-Lis1. Then, we utilized qPCR, fluorescence in situ hybridization (FISH), immunofluorescence (IF), Hematoxylin and eosin staining (HE), Western blotting (WB), and RNAi to investigate the expression, distribution and function of Pt-LIS1 during spermatogenesis, including spermatogonial mitosis, spermatocyte meiosis, acrosome formation, nuclear shaping and the maintenance of testicular homeostasis.

Results

Identification and characterization of the Pt-Lis1 gene in P. trituberculatus

The full length of Pt-Lis1 (GenBank accession no. OL841528) cDNA is 1844 bp, with a 1230 bp open reading frame (ORF), a 146 bp 5′ untranslated region (UTR), and a 468 bp 3′UTR, encoding 410 amino acids (aa) (Fig. 1A). The molecular weight of Pt-LIS1 was 46 kDa, and the isoelectric point was 4.99. The similarities of the LIS1 protein sequence with its homologous sequences in Homo sapiens (GenBank accession no. NM_000430.4), Mus musculus (GenBank accession no. NM_013625.4), Xenopus laevis (GenBank accession no. NM_205830.1), Larimichthys crocea (GenBank accession no. NM_027273497.1), Drosophila melanogaster (GenBank accession no. NM_001259432.2), Penaeus monodon (GenBank accession no. NM_037922710.1), Penaeus vannamei (GenBank accession no. NM_027355967.1) and Penaeus japonicus (GenBank accession no. NM_043022649.1) were 84.2, 84.2, 84.0, 83.5, 86.4, 96.8, 96.8 and 97.1%, respectively (Fig. 1B).

We predicted the structural domain of Pt-LIS1 using the online tools SMART and CD-Search. Pt-LIS1 includes the LISH domain at the N-terminus (9–41 aa) and the β-propeller domain at the C-terminus, consisting of seven WD40 structural domains (97–136 aa, 139–178 aa, 181–220 aa, 223–262 aa, 265–324 aa, 327–366 aa, and 369–408 aa) (Fig. 1C). The phylogenetic analysis revealed that Pt-LIS1 clusters with homologs from crustaceans such as Homarus americanus and P. japonicus, showing a closer evolutionary relationship with insects, while exhibiting a more distant connection to vertebrates (Fig. 1D).

The highly expression and distribution of Pt-Lis1 in the testis of P. trituberculatus

The semiquantitative PCR results revealed ubiquitous expression of Pt-Lis1 mRNA across all tissues examined in P. trituberculatus. However, its expression was notably high in the testis (Fig. 2A, B). The distribution of Pt-Lis1 mRNA in the testis of P. trituberculatus during spermiogenesis through FISH experiments. In early spermatids, Pt-Lis1 mRNA exhibited uniform distribution in the cytoplasm around the nucleus (Fig. 2CA1-A3). In the middle stage of spermatids, Pt-Lis1 mRNA was found in the cytoplasm on the opposite side of the nucleus (Fig. 2CB1-B3). In late spermatids, as the nucleus gradually compressed into a cup shape, Pt-Lis1 mRNA predominantly localized on the inner side of the cup-shaped nucleus, with a small amount also present in the acrosomal tube (Fig. 2CC1-C3). The distribution of Pt-Lis1 continuously changes with nuclear deformation, suggesting its potential involvement in this process. Finally, in mature sperm, Pt-Lis1 mRNA was primarily concentrated in the acrosome tube and acrosome cap (Fig. 2CD1-D3). The negative control did not detect signals other than DAPI (Fig. 2CA5-D7), demonstrating the specificity of the Pt-Lis1 mRNA probe. The localization pattern of the Pt-Lis1 mRNA signal in different stages of spermiogenesis was plotted according to the localization results (Fig. 2CA4-D4).

Distribution of Pt-LIS1 protein in the testis of P. trituberculatus

To examine the expression pattern of Pt-LIS1 in the testis of P. trituberculatus, antibodies against Pt-LIS1 were developed. Initially, peptide fragments were expressed in Transetta (DE3) competent cells and then injected into SD rats and New Zealand rabbits. Antibodies were subsequently obtained from the serum of rats and rabbits, revealing specific bands corresponding to Pt-LIS1 at 46 kDa (Fig. 3A-C).

The distribution of Pt-LIS1 in the testis of P. trituberculatus during spermatogenesis was determined through IF experiments. The different types of spermatogenic cells were distinct by HE staining (Fig. 3DA4-G4). In spermatogonia, spermatocytes, and early spermatids, the nuclei are round in shape (Fig. 3DA4-D4). In mid-stage spermatids, the nuclei migrate to one side of the cell (Fig. 3DE4), while in late-stage spermatids, the nuclei gradually compress into a cup-shaped structure (Fig. 3DF4). In mature sperm, the spermatid nucleus undergoes further invagination, forming a cup-shaped nuclear cap (Fig. 3DG4). In spermatogonia, primary spermatocytes in the metaphase of meiosis I and secondary spermatocytes stage, Pt-LIS1 displayed uniform distribution in the perinuclear cytoplasm (Fig. 3DA1-C3). During early and mid-stage spermatids, Pt-LIS1 was observed in the perinuclear cytoplasm and around the proacrosomal vesicle (Fig. 3DD1-E3). In late spermatids, as the nucleus formed a cup shape, Pt-LIS1 signals concentrated in the subacrosomal space and acrosomal vesicles (Fig. 3DF1-F3). In mature sperm, Pt-LIS1 signals were also concentrated in the subacrosomal space and acrosomal tube (Fig. 3DG1-G3). The distribution of Pt-LIS1 continuously changes with nuclear deformation and acrosome formation, suggesting its potential involvement in these processes.

Colocalization of Pt-LIS1 and Pt-DHC, Pt-DIC, α-Tubulin, PHB and Acrosin during spermatogenesis in P. trituberculatus

To further investigate the function of the Pt-LIS1 protein in spermatogenesis in P. trituberculatus, we employed IF combined with laser confocal microscopy to explore the relationship between Pt-LIS1 and Pt-DHC, Pt-DIC, α-Tubulin, PHB and Acrosin.

LIS1 is an important regulator of Dynein-1, while Pt-DHC and Pt-DIC are important subunits of Dynein-1. Therefore, we detected the relationship between Pt-LIS1 and Pt-DHC(/Pt-DIC). The results demonstrated consistent colocalization of Pt-DHC and Pt-DIC with Pt-LIS1 during the spermatogenesis of P. trituberculatus (Figs. 4, 5). From spermatogonia to the early spermatid stage, Pt-DHC and Pt-DIC were distributed in the cytoplasm around the nucleus and were highly colocalized with Pt-LIS1 (Fig. 4A1-D5, Fig. 5A1-C5). In the middle stage of spermatids, Pt-LIS1, Pt-DHC and Pt-DIC signals colocalized in the perinuclear cytoplasm, showing varying intensities between nucleus sides (Fig. 4E1-E5, Fig. 5D1-D5). Late spermatids exhibited strong Pt-LIS1 and Pt-DHC colocalization in the acrosomal cap and tube, with some signal in the subacrosomal space (Fig. 4F1-F5), while Pt-DIC signal was in the subacrosomal space and acrosomal tube (Fig. 5E1-E5). In mature sperm, Pt-DHC and Pt-DIC signals mainly concentrated in the inner nuclear cup and acrosomal tube, highly colocalized with Pt-LIS1 (Fig. 4G1-G5, Fig. 5F1-F5).

As Dynein-1 is a motor protein that moves along microtubules and Pt-LIS1 is an important regulator of Dynein-1, we examined the colocalization of Pt-LIS1 with α-Tubulin. Pt-LIS1 and α-Tubulin exhibited high colocalization during spermatogenesis (Fig. 6). In spermatogonia and spermatocytes, signals were uniformly distributed around the nucleus, with increased α-Tubulin intensity in spermatocytes (Fig. 6A1-B5). In early and middle stages of spermatids, Pt-LIS1 and α-Tubulin signals were colocalized in the perinuclear cytoplasm (Fig. 6C1-D5). In late spermatids, both signals were colocalized in the acrosomal tube (Fig. 6E1-E5). In mature sperm, they were mainly clustered in the acrosomal tube and cap (Fig. 6F1-F5).

As Dynein-1 is involved in mitochondrial transport, we investigated the relationship between Pt-LIS1 and mitochondrial marker PHB. IF results revealed colocalization of Pt-LIS1 and PHB signals throughout spermatogenesis, evenly distributed around the nuclear in spermatogonia, spermatocytes, and early- and middle-stage of spermatids (Fig. 7A1-A5, B1-B5, C1-D5). In late spermatids, colocalization occurred in the acrosomal ducts (Fig. 7E1-E5), and in mature sperm, signals clustered in the acrosomal tubes and caps (Fig. 7F1-F5).

Acrosin, a crucial enzyme in crustacean sperm acrosomes, is transported along the microtubules by motor proteins. We investigated the relationship between Pt-LIS1 and Acrosin, finding colocalization in the cytoplasm around the nucleus in spermatogonia, early and middle stage of spermatids (Fig. 8A1-C5), and in mature sperm, colocalization occurred in the acrosomal cap and tube, with some Acrosin signals around the nucleus (Fig. 8D1-D5).

Detection of Pt-Lis1 RNAi efficiency

To further explore the functions of Pt-LIS1 in the testis of P. trituberculatus, we performed an RNAi experiment by using dsRNA1 (with higher interference efficiency: 70%) (Fig. 9A, B). The mRNA and protein expression levels of Pt-LIS1 were significantly decreased in the Pt-Lis1-dsRNA group when compared to the control group, suggesting that Pt-LIS1 was silenced successfully (Fig. 9C-H).

Deficiency of Pt-Lis1 in vivo affects both the expression level and distribution of Pt-DHC and Pt-DIC

Furthermore, we detected the expression of essential Dynein-1 subunits, finding a significant reduction in Pt-dhc and Pt-dic levels in the Pt-Lis1-dsRNA group (Fig. 10A-D). IF analysis revealed that the signals of Pt-DHC and Pt-DIC exhibited abnormal diffuse distributions in the cytoplasm in the Pt-Lis1-dsRNA group from spermatogonia to early spermatids (Fig. 10EA1-D5, FA1-C5). In the middle stage of spermatids, the signals were confined to one side of the nucleus in the Pt-Lis1-dsRNA group, whereas they evenly distributed around the entire nucleus in the control group (Fig. 10EE1-E5, FD1-D5). In late spermatids, the signals of Pt-DHC and Pt-DIC in the Pt-Lis1-dsRNA group were only weakly distributed within the acrosomal cap, exhibiting a decreased distribution within the subacrosomal space and acrosomal tube to that in the control group (Fig. 10EF1-F5, FE1-E5). In Pt-Lis1-dsRNA group, Pt-DHC and Pt-DIC signals were nearly undetectable compared to mature spermatids in the control group (Fig. 10EG1-G5, FF1-F5). Overall, these results indicate that Pt-Lis1 knockdown disrupts Dynein-1 expression and distribution in P. trituberculatus testes. Statistical analysis further revealed a significant reduction in the number of spermatids with normal Pt-LIS1 and Pt-DHC (or Pt-DIC) signal distribution at each stage (Fig. 10G-N).

Deficiency of Pt-Lis1 in vivo influences the expression level of related genes (Pt-Nude, phb, p53 and caspase-3) in testes

The relative expression levels of related genes in the testis were analyzed by using β-actin and gapdh as double reference genes. In the Pt-Lis1-dsRNA group, the relative expression of phb was not significant changed (Fig. 11A, B), and the relative expression of Pt-Nude (another regulator of Dynein-1) in the testis was not significant changed (Fig. 11C, D). The expression of apoptosis genes (p53 and caspase-3) in the testis was significantly increased (Fig. 11E-H).

Knockdown of Pt-Lis1 leads to abnormal colocalization of Pt-LIS1 and α-Tubulin in the testis of P. trituberculatus

We further explored the relationship between Pt-LIS1 and α-Tubulin after Pt-Lis1 silencing. Compared to the control group, colocalization signals of Pt-LIS1 and α-Tubulin were significantly weakened at all spermatogenesis stages (Fig. 12). From spermatogonia to early spermatids, α-Tubulin signal diffusely distributed in the perinuclear cytoplasm (Fig. 12A1-C5); in the middle stage of spermatids, α-Tubulin signaling was weakly distributed on one side of the nucleus (Fig. 12D1-D5); in late spermatids, α-Tubulin signaling was weakly distributed in acrosome (Fig. 12E1-E5); and in mature sperm, α-Tubulin signaling was observed only on the nucleus side (Fig. 12F1-F5).

Discussion

Structure Analysis of LIS1

Analyzing the structure of LIS1 is crucial for unraveling its functional roles and understanding its contributions to various cellular processes. In this study, we conducted a comprehensive structural analysis of LIS1, shedding light on its intricate composition and potential implications for cellular dynamics. LIS1 serves as a crucial regulator in Dynein-1-mediated cell development across different species^20,25–27. Pt-LIS1 exhibits conserved domains, including the LISH domain at the N-terminus and a β-propeller domain at the C-terminus. The LISH domain, present in various proteins across the eukaryotic genome and associated with genetic syndromes²⁸, plays an essential role in LIS1 dimerization²⁹. The β-propeller domain of LIS1 consists of multiple WD40 structural domains, which are characterized by highly conserved repeating units, and is implicated in assisting Dynein-1 in the transport of cellular cargoes along microtubules and the regulation of cell division³⁰. The presence of these domains is consistent with previous findings in other species such as Homo sapiens, Mus musculus, and Drosophila melanogaster, and aligns with LIS1’s known roles in cellular processes such as microtubule dynamics, intracellular transport, and cell division^31–33.

Pt-LIS1 was necessary for spermatogenesis in P. trituberculatus

Spermatogenesis is an indispensable part of sexual reproduction in animals, and spermatogonia undergo proliferation, differentiation and metamorphosis to form mature sperm³⁴. Compared to other species, the mature sperm of crustaceans have a more specialized morphology, with nonmotile tailless sperm^5,6. Understanding the pivotal role of Pt-LIS1 in spermatogenesis is crucial for unraveling the intricate process of sperm development in P. trituberculatus. Our investigation examining the pattern of Pt-Lis1 mRNA and protein throughout the various stages of spermatogenesis firstly. We observe robust expression of Pt-Lis1 in multiple tissues, with the highest levels found in the testis, indicating its significant involvement in sperm development. This finding indicates that the regulatory factor Pt-Lis1 plays an equally crucial role in sperm development and spermatogenesis as Dynein-1 in P. trituberculatus, a viewpoint consistent with Mesngon et al.²³.

Our analysis demonstrates that Pt-LIS1 exhibits dynamic distribution patterns throughout spermatogenesis, with particularly notable changes during the stages of nuclear deformation and acrosome formation. Pt-LIS1 is uniformly distributed in the perinuclear cytoplasm of spermatogonia and spermatocytes, suggesting its potential involvement in both mitosis of spermatogonia and meiosis of spermatocyte. This aligns with previous studies in Drosophila embryos³⁵ and HeLa cells³⁶, indicating the essential role of LIS1 in mitosis and meiosis regulation. During the transition from early to late spermatids, the Pt-LIS1 mRNA and protein signal exhibited dynamic changes in response to nuclear morphology alterations, hinting at its potential involvement in nuclear deformation. Additionally, as spermatids matured into sperm, Pt-Lis1 signaling gradually concentrated in the subacrosomal space, acrosomal tube and cap, suggesting its association with acrosome formation. These observations align with previous research by Nayernia et al.²², which highlighted the crucial role of LIS1 in acrosome formation and its structural integrity during mouse spermatogenesis.

Pt-LIS1 regulates Dynein-1 transport along microtubule and affects microtubule assembly

Dynein-1 plays a crucial role in regulating diverse cellular processes, including spermatogenesis and oogenesis³⁷, organelle translocation³⁸, spindle assembly^39,40, and nuclear translocation⁴¹, and relies on microtubules to facilitate the transport of cellular cargo to its designated location for proper functioning. Notably, Dynein-1 has been shown only dependent on microtubules⁴². Microtubules, integral components of the cytoskeleton found in nearly all eukaryotes, consist of heterodimers of α-Tubulin and β-Tubulin⁴³. Our colocalization analysis demonstrates the extensive overlap between Pt-LIS1 and α-Tubulin throughout spermatogenesis, supporting Pt-LIS1’s role as a regulator of Dynein-1 in transporting cellular cargo along microtubules, consistent with previous findings²⁶. Furthermore, the signals of Pt-LIS1 and α-Tubulin exhibit a diffuse distribution, with weakened colocalization observed following Pt-Lis1 knockdown (Fig. 12). Considering Dynein-1's role in microtubule formation¹⁵, the knockdown of Pt-Lis1 seems to result in the diffused distribution of α-Tubulin, indicating that Pt-LIS1 affect microtubule assembly.

The transport of cellular cargoes along microtubules is a fundamental process essential for various cellular functions⁴⁴. PHB (prohibitin) is a vital inner mitochondrial membrane protein, and recognized as a marker for mitochondria ^45–47. Studies have highlighted PHB’s expression and functional significance in animal spermatogenesis^48,49. In P. trituberculatus spermatogenesis, PHB colocalizes with Pt-DHC, a Dynein-1 subunit involved in mitochondrial transport¹⁶. Investigating the Pt-LIS1 and mitochondria relationship, we focus on Pt-LIS1 colocalization with PHB. The observed colocalization throughout spermatogenesis suggests LIS1’s involvement in regulating Dynein-1-mediated mitochondrial transport. Additionally, Pt-LIS1 may contribute to Dynein-1-mediated enzymatic transport in spermatogenesis. Acrosin, a trypsin-like serine protease specific to the sperm acrosome, is crucial for the acrosome reaction, penetration of the zona pellucida during sperm-egg binding, and release of other enzymes enhancing sperm motility^50,51, and a previous study showed that motor proteins transport Acrosin along the acrosome⁹. Colocalization of Pt-LIS1 and Acrosin in the acrosome tube and acrosome cap suggesting that Pt-LIS1 may be involved in enzyme transport during spermatogenesis.

Pt-LIS1 plays an important role in the expression of the Dynein −1 subunit

Dynein-1 primarily binds to microtubules via the heavy chain Pt-DHC and attaches to cargo primarily through the intermediate chain Pt-DIC. Consequently, Pt-DHC and Pt-DIC were chosen as the target proteins for colocalization analysis⁵². Our findings indicate that Pt-LIS1 knockdown significantly reduces the expression levels of Pt-dhc and Pt-dic in the testis, key components of the Dynein-1 complex. This suggests that that Pt-LIS1 regulates Dynein-1 subunits at the transcriptional level, impacting the function of Dynein-1. Our immunofluorescence and co-localization analyses reveal that Pt-Lis1 knockdown leads to a reduction in the co-localization signal between Pt-LIS1 and Pt-DHC, as well as between Pt-LIS1 and Pt-DIC. Additionally, an increased number of spermatids exhibit abnormal distribution of colocalization signals (Fig. 10G-N), implying that Pt-Lis1 absence may influence nuclear deformation and acrosome formation in P. trituberculatus. This observation aligns with the studies on mouse testes, where Lis1 deletion resulted in spermatogenesis failure²². The pivotal role of Pt-LIS1 in modulating Dynein-1 expression and function underscores its significance in cellular dynamics and highlights its potential as a target for further investigation into the regulatory mechanisms governing cellular transport and organization.

Pt-LIS1 as a key regulator of Dynein-1 in spermatogenesis of P. trituberculatus

Dynein-1 plays an important role in reproductive development in males^53–55. Previous studies have demonstrated the necessity of motor proteins like Kinesin and Dynein-1 in sperm metamorphosis and acrosome formation during crustacean spermatogenesis^16,56. Furthermore, the indispensable role of Dynein-1 has been firmly established in various cellular processes, including spermatogenesis and acrosome formation in P. trituberculatus^16,17. Through colocalization analysis using IF and laser confocal microscopy techniques, we observed significant colocalization of Pt-LIS1 with Pt-DHC and Pt-DIC throughout different stages of spermatogenesis, including spermatogonia, spermatocytes, spermatids, and mature sperm. These results imply a close functional relationship between Pt-LIS1 and Dynein-1 in coordinating cellular processes during spermatid development. The intricate interplay between Pt-LIS1 and Dynein-1 underscores the complexity of microtubule dynamics and intracellular transport mechanisms. While our study focuses on the interaction between Pt-LIS1 and Dynein-1, further research is needed to elucidate the precise regulatory mechanisms governing their collaboration and its implications for spermatogenesis.

The intricate interplay between different regulatory factors within the Dynein complex highlights the complexity of cellular processes involving microtubule dynamics and intracellular transport^15,22. The relationship between Lis1 and Nude has been previously explored in various biological contexts, and studies have reported that Lis1 can interact with Nude to regulate Dynein function⁵⁷. Our findings indicate that Pt-Nude may not be directly influenced by Pt-Lis1 expression levels or the associated knockdown. The lack of significant alteration in Pt-Nude expression post Pt-Lis1 knockdown suggests a potential independence in the regulatory mechanisms governing these two Dynein-associated factors. However, there is no clear studies on the combination of the two and the regulatory mechanisms.

Moreover, our study provides insights into the potential involvement of Pt-LIS1 in mitochondrial function, apoptosis regulation, and maintenance of spermatogenic cell viability. The expression of phb did not show significant changes. However, further investigation is needed to determine whether Pt-Lis1 affects mitochondrial morphology and function. P53, as a critical tumor suppressor, is activated in response to various stress signals, thereby orchestrating various cellular processes such as cellular senescence, metabolism, apoptosis, and DNA repair⁵⁸. The expression level of p53 can serve as an indicator of the degree of cellular stress⁵⁹. Caspase-3 a pivotal gene in apoptosis, is implicated in the pathogenesis of neurodegenerative diseases, and its overactivation has emerged as a potential therapeutic target for apoptosis-related⁶⁰. Nayernia et al. (2003) reported that reduced Pt-LIS1 expression led to impaired spermatogenesis in mice, while Sitaram et al. (2012) demonstrated that Pt-Lis1 deletion in Drosophila resulted in abnormal spermatogonial division and compromised male spermatogenesis. The observed upregulation of p53 and caspase-3 expression subsequent to Pt-Lis1 knockdown indicates a role for Pt-LIS1 in modulating spermatogenic cell apoptosis. Spermiogenesis, as the final stage of spermatogenesis, is crucial for proper sperm formation. Reduced expression of Lis1 during this stage may lead to excessive apoptosis of spermatogenic cells, which could significantly impair sperm maturation. Therefore, we speculate that Pt-LIS1 may play a crucial role in maintaining the survival of spermatogenic cells and ensuring the quantity of both spermatogenic cells and sperm. But we need to conduct further discussions regarding the extent of apoptosis in spermatogenic cells.

Conclusions

In summary, our study revealed that Pt-Lis1 exhibited high expression levels in the testis, predominantly localized around the nucleus and in the developing acrosome during spermatogenesis (Fig. 13). The localization suggests potential involvement of Pt-LIS1 in mitosis and meiosis, as well as participation in nuclear deformation and acrosome formation processes. After Pt-Lis1 knockdown, we observed decreased expression of Pt-dhc and Pt-dic, accompanied by aberrant colocalization patterns between Pt-LIS1 and Pt-DHC, Pt-DIC, α-Tubulin, PHB, and Acrosin. These findings imply that Pt-LIS1 acts as a crucial regulator ensuring proper Dynein-1-mediated cargo transport along microtubules. Moreover, the upregulation of apoptosis-related genes, p53 and caspase-3, following Pt-Lis1 knockdown suggests its potential involvement in regulating spermatogenic cell apoptosis in P. trituberculatus, suggesting that Pt-LIS1 plays an important role in maintaining the survival of spermatogenic cells and ensuring the number of spermatogenic cells.

Materials and methods

Experimental animals

The study was conducted on 70 mature male and active P. trituberculatus crabs sampled from the Xianxiang Jiandao aquaculture farm (Ningbo, China). All crabs had free access to food with 12:12 h of light/dark cycle and constant environmental conditions (24–26 °C, 25‰ salinity in water) before experimentation and thereafter. Two male New Zealand white rabbits were purchased from Jianfei Experimental Rabbit Farm, Simen Town, Yuyao, Ningbo (SCXK (Zhejiang) 20,220,002) and fed in separate cages at the Experimental Animal Center of Ningbo University (SYXK (Zhejiang) 2019–0005). Four male SD rats were purchased from Zhejiang Viton Lihua Laboratory Animal Technology Co., Ltd. (SCXK (Zhejiang) 2024–0001), and fed in separate cages at the Ningbo University Laboratory Animal Center (SYXK (Zhejiang) 2019–0005). All rabbits and rats had free access to food and water with 12:12 h of light/dark cycle and constant environmental conditions before experimentation and thereafter.

Ethical statement.

All experiments were pursued by the standards of the requirements of the governing regulation for the use of experimental animals in Zhejiang Province (Zhejiang provincial government order No. 263, released on17 August 2009, effective from 1 October 2010) and were performed after the approval of the Animal Care and Use Committee of Ningbo University and the Animal Ethics and Welfare Committee of Ningbo University (approval number NBU20220155). All procedures were designed following the ARRIVE guidelines (PLoS Bio 8(6), e1000412, 2010) and conducted in accordance with the eighth edition (2011) of the rules for the care and use of laboratory animals.

Synthesis of dsRNA

Primers carrying the T7 promoter sequence were designed by Primer Premier 5.0 software (Supplementary Table 1). Fragments were cloned by PCR using the following procedure: 1 cycle at 94 °C for 5 min, 8 cycles at 94 °C for 30 s, 62 °C for 30 s (decrease rate 0.5 °C/cycle), and 72 °C for 30 s, 27 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, and then a final extension at 72 °C for 10 min. The PCR product was used as the template to synthesize dsRNA according to the TranscriptAid T7 High Yield Transcription Kit instructions (Thermo Scientific, Shanghai, China).

Experimental design

Crabs were classified into the following groups: Control group: consisted of 25 healthy male crabs that had not received any type of treatment, PBS group: consisted of 15 male crabs which were injected with 2 μL/g RNase-free PBS (injected once every 48 h into the fifth pleopod, for a total of 5 injections), Pt-Lis1-dsRNA1 group: consisted of 15 male crabs which were injected with 2 μg/g Pt-Lis1-dsRNA1 dilution liquid (injected once every 48 h into the fifth pleopod, for a total of 5 injections), Pt-Lis1-dsRNA2 group: consisted of 6 male crabs which were injected with 2 μg/g Pt-Lis1-dsRNA2 dilution liquid (injected once every 48 h into the fifth pleopod, for a total of 5 injections), egfp-dsRNA group: consisted of 9 male crabs which were injected with 2 μg/g egfp-dsRNA dilution liquid (injected once every 48 h into the fifth pleopod, for a total of 5 injections). All crabs followed a regimen of moderate exercise that reproduced an active lifestyle.

Collection of samples

Crabs were dissected on ice, and five tissues (testis, muscle, gill, hepatopancreas and vas deferens) were detached, immediately frozen in liquid nitrogen, and stored at −80 °C for total RNA and protein extraction. Additionally, some testis sections were fixed in 4% paraformaldehyde in 0.1 M PBS for FISH and IF experiments.

Antigen immunization injections

According to the amino acid sequence of Pt-LIS1 and the antigen site online prediction website, the specific region was selected as the antigen fragment (encoding 212 amino acids, approximately 28.87 kDa), and then the primers were designed (BamH1 and Xho1 cut sites were added at the two ends of the Pt-LIS1 fragment, respectively, YH-LIS1-F/R). The cloned correct DNA fragment was subjected to a double digestion reaction with plasmid pET28-a ( +) (provided by the sperm laboratory of Zhejiang University) and then ligated with T4 ligase (Takara, Beijing, China). Through transformation and sequencing, the correct plasmid was extracted using the Plasmid Extraction Mini Kit (OMEGA, Guangzhou, China). The correct recombinant plasmid was transferred into DE3 competent cells and inoculated into 10 mL Kana⁺ liquid medium to expand the culture (37 °C, 220 g). When the OD value reached 0.4–0.6, 1 mM IPTG solution was added to induce recombinant protein expression (37 °C, 220 g, 6 h). The bacterial solution was resuspended after sedimentation, ultrasonically crushed, and centrifuged (10,000 g, 4 °C, 15 min). Purification of inclusion body proteins from precipitates was performed using a His-tagged protein purification kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. After identification by SDS‒PAGE, the purified recombinant protein was loaded into a dialysis bag for renaturation with a linear 6, 4, 2, 1, and 0 M urea gradient in the dialysate for 8 h. Finally, the protein concentration was detected by using the Super-Bradford Protein Quantification Kit (Beyotime, Shanghai, China).

The purified recombinant protein was fully mixed with Freund’s complete adjuvant at the first immunization injection. Then, each SD rat was injected subcutaneously with 400 μg recombination protein, and each New Zealand rabbit was injected with 600 μg recombination protein. At the 2nd-5th immunization injection (once every other week), the purified Pt-LIS1 recombinant protein was fully mixed with Freund’s incomplete adjuvant, each SD rat was injected with 200 μg recombination protein, and each New Zealand rabbit was injected with 300 μg recombination protein. All rats were overnight fasting, and blood samples were collected from the retro-orbital vein under deep anesthesia using isoflurane (inhalation 100%), the rats were then euthanized via cervical dislocation. All rabbits were anesthetized using isoflurane, and blood samples were collected from the ear vein. The rabbits were then euthanized via auricular vein air injection.

Full-length cDNA cloning of Pt-Lis1

Total RNA was extracted from testes by using TRIzol Reagent (Invitrogen, USA). According to the manufacturer’ instructions, the obtained RNA was used for normal reverse transcription using the Prime-Script® RT Reagent Kit (Takara, Dalia, China). A SMARTer RACE 5′/3′ Kit (Takara, Dalian, China) was used to synthesize 3′ and 5′ terminal cDNA from the testis of P. trituberculatus. The reverse transcribed products (cDNA) were stored at −20 °C for subsequent PCR.

We obtained the Pt-Lis1 cDNA sequence of Drosophila melanogaster (GenBank accession no. NM_001259432.2) from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/, NCBI) database and compared it with the genome of P. trituberculatus, subsequently obtaining the specific fragment. The sequence was imported into Primer Premier 5 software (Premier Biosoft International, Palo Alto, CA, USA) for the design of degenerate primers and sent to Youkang Biotechnology Co., Ltd. (Zhejiang, Hangzhou, China) for synthesis. The testis cDNA of P. trituberculatus was used as a template, and the Touchdown PCR (TD-PCR) program was used to amplify the intermediate fragments of Pt-Lis1. TD-PCR was conducted as follows: 94 °C for 5 min; 8 cycles of 94 °C for 30 s, 55 °C for 30 s (decreased by 0.5 °C/cycle) and 72 °C for 45 s; 27 cycles of 94 °C for 30 s, 53 °C for 30 s, 72 °C for 45 s; and 72 °C for 10 min for the final extension. Then, we designed specific primers by using Primer Premier 5.0 software for RACE. The touch-down PCR used for 3′/5′ RACE was conducted as follows: 94 °C for 5 min; 8 cycles of 94 °C for 30 s, 72/71 °C for 30 s (decreased by 0.5 °C/cycle) and 72 °C for 1 min; 27 cycles of 94 °C for 30 s, 68/67 °C for 30 s and 72 °C for 1 min; and then a final extension at 72 °C for 10 min. These PCR products were separated, identified, and sequenced based on the method described by Chang et al.⁹. We assembled the intermediate segment and the clonal 3′ and 5′ cDNA segment sequences and obtained the full-length cDNA of Pt-Lis1. All primers used in the study are presented in Supplementary Table 2.

Multiple sequence alignment, phylogenetic evolutionary tree analysis and protein structure prediction

The amino acid sequence of Pt-LIS1 was obtained by the Sequence Manipulation Suite (SMS) (http://www.bio-soft.net/sms/); the molecular weight and isoelectric point of Pt-LIS1 were predicted by using the ExPASy-ProtParam tool (https://web.expasy.org/protparam/); the secondary structure and conserved structural domains of Pt-LIS1 were predicted by the SMART (http://smart.embl-heidelberg.de/) software and the CD-Search tool from the NCBI website (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The protein 3D structure of Pt-LIS1 was predicted by the online software I-TASSER (https://zhanggroup.org/I-TASSER/). Sequence alignment and phylogenetic analyses were performed with the software Vector NTI10 (Invitrogen, California, USA) and Mega 5.1 (Informar Technologies, USA), respectively. The amino acid (aa) sequences of LIS1 proteins were downloaded from the National Center of Biotechnology Information (https://www.ncbi.nlm.nih.gov/), and their GenBank accession numbers are listed in Supplementary Table 3.

Expression analysis of Pt-Lis1 mRNA in different tissues of P. trituberculatus

Semiquantitative RT‒PCR was conducted to distinguish the Pt-Lis1 mRNA expression of different tissues of P. trituberculatus. We used the reversed cDNA of the testis, muscle, gill, hepatopancreas and vas deferens for semiquantitative RT‒PCR. We designed different pairs of primers for Pt-Lis1 and the control, β-actin. PCR programs were set as follows: 94 °C for 5 min; 32 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 20 s; 72 °C for 10 min for the final extension. The products were examined by electrophoresis on a 1% agarose gel. The bands were revealed by DNA gel green and analyzed by ImageJ software. One-way ANOVA was performed on the data using Tukey’s HSD and Tukey s-b post hoc tests in IBM SPSS Statistics 25.0 software, and graphs were made using GraphPad Prism 8.0.2 software.

Fluorescence in situ hybridization

The frozen testis sections were fixed in 4% paraformaldehyde in PBS at 4 °C for 18 h, washed twice with 1 × PBS, immersed in 0.5 M sucrose-PBS solution (pH = 7.4) overnight at 4 °C, placed in O.C.T. compound, and frozen at −80 °C. The embedded blocks were cut into slices (5 μm) using a sliding microtome and subsequently adhered to RNase-free poly-lysine-coated slides, which were stored at −80 °C until further use. We designed the reverse primer in the non-conserved sequence region of Pt-Lis1 cDNA, BLASTed it specifically in the genome of P. trituberculatus, and then sent it to Jinweizhi Biotechnology Co. to synthesize the 5ˊ-FAM-modified probe. At the same time, the complementary sequences of the reverse primer were used as the negative control (LIS1-C-probe). The frozen sections were dried and permeabilized and then incubated with the probe at room temperature for 2 h. After incubation, the sections were washed with PBS, stained with DAPI, sealed with a drop of anti-fluorescent bursting agent, and finally observed with a Zeiss laser scanning confocal microscope (LSM880, Carl Zeiss, Germany).

Western blot analysis

The specificity of the rat/rabbit anti-Pt-LIS1 antibody was checked by Western blot based on our previous method⁹, and only one protein band of approximately 46 kDa was detected, coincident with the predicted molecular weight of Pt-LIS1 (Fig. 3). Therefore, IF could be carried out using the rat/rabbit anti-Pt-LIS1 antibody. In addition, we purchased a rabbit anti-β-actin antibody (Cat. No. AF5003), rabbit anti-α-Tubulin antibody (Cat. No. AF0001), HRP-conjugated goat anti-rat IgG (H + L) (Cat. No. A0192), HRP-conjugated goat anti-rabbit IgG (H + L) (Cat. No. A0208), Cy3-labeled goat anti-rat IgG (H + L) (Cat. No. A0507), Alexa Fluor 555-labeled donkey anti-mouse IgG (H + L) (Cat. No. A0460) and Alexa Fluor 488-labeled goat anti-rabbit IgG (H + L) (Cat. No. A0423) from Beyotime (Shanghai, China). In addition, the rabbit anti-Pt-DHC antibody, rat anti-Pt-DIC antibody, rabbit-anti-PHB antibody and rabbit-anti-acrosin antibody were made in our laboratory, and their specificity has been verified⁵² (Supplementary information file).

Immunofluorescence

The experiments were implemented as described in our previous report⁹. The frozen sections were removed from storage at −80 °C and dried for approximately 15 min at room temperature. The dried frozen sections were permeabilized with 0.3% PBST (0.3% Triton X-100 in PBS buffer) for 15 min and then soaked in 5% BSA-PBS (bovine serum albumin in PBS buffer) for 2 h, followed by incubation with primary antibodies (rat/rabbit anti-Pt-LIS1 antibody, rabbit anti-Pt-DHC antibody, rat anti-Pt-DIC antibody, mouse anti-α-Tubulin antibody, rabbit-anti-PHB antibody and rabbit-anti-Acrosin antibody, 1:50 dilution) overnight at 4 °C. Subsequently, the sections were washed with 0.1% PBST three times for 45 min. The washed sections were incubated with secondary antibodies (Cy3-labeled goat anti-rat IgG (H + L), Alexa Fluor 488-labeled goat anti-rabbit IgG (H + L) and Alexa Fluor 555-labeled donkey anti-mouse IgG (H + L), 1:500 dilution) for 2 h at 37 °C. After washing six times with 0.1% PBST for 90 min, the sections were stained with DAPI (Beyotime, Shanghai, China) for 5 min and mounted. Finally, a Zeiss laser scanning confocal microscope (LSM880, Carl Zeiss, Jena, Germany) was used to observe the fluorescence signal. The control groups were incubated with antibody diluent without the primary antibodies.

Hematoxylin and eosin staining

Testes of P. trituberculatus were fixed in 4% paraformaldehyde, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin for 5 min, differentiated in 1% acid alcohol, blued under running water, and counterstained with eosin for 3 min. After dehydration and clearing, sections were mounted with neutral balsam. Stained sections were observed under a light microscope (Olympus BX53, Tokyo, Japan) to analyze the histological features and the distribution of spermatogenic cells at different developmental stages.

Effect of Pt-Lis1 knockdown on the mRNA expression of Pt-dhc, Pt-dic, Pt-Nude, phb, caspase-3 and p53

RT-PCR was used to detect the effect of Pt-Lis1 knockdown on the mRNA expression of Pt-dhc, Pt-dic, Pt-Nude, phb, caspase-3 and p53. β-actin and gapdh were used as the reference genes. The comparative ∆Ct method was used to analyze gene expression. The primers are shown in Supplementary Table 4.

Effect of Pt-Lis1 knockdown on the spatial expression distribution of Pt-LIS1 with Pt-DHC, Pt-DIC and microtubules

IF was used to detect the changes in the colocalization distribution of Pt-LIS1 with Pt-DHC, Pt-DIC, and microtubule (α-Tubulin) in the spermatogenesis of P. trituberculatus after Pt-Lis1 silencing (n = 5).

Statistical analysis

We used GraphPad Prism 8 (GraphPad software, California, USA) for data analysis. All the results are presented as the means ± standard errors of the means. Significance tests using an unpaired t test were performed between the control group and the experimental group. All experiments were repeated three times using different samples. P < 0.05 was considered to indicate a significant difference between the control group and experimental group.

Additional information

Data availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files).

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Acknowledgements

The authors would like to thank the schoolmates in the Laboratory of Reproduction and Development of Ningbo University for their help in this project.

Funding

This research was supported by the NSFC-Ningbo Joint Fund for Regional Innovation Development (Project ID: U23A20248), the Key Project of the Ningbo Natural Science Foundation (Project ID: 2022J071), the National Natural Science Foundation of China (Project ID: 31602140), the National Natural Science Foundation of China (Project ID: 42076087), the Earmarked Fund for CARS-48 (Project ID: CARS-48), the K.C.Wong Magna Fund at Ningbo University, and the Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture.

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Authors and Affiliations

Key Laboratory of Applied Marine Biotechnology By the Ministry of Education and Key Laboratory of Marine Biotechnology of Zhejiang Province, School of Marine Sciences, Ningbo University, Ningbo, 315832, China
Le Chang, Qiu Meng Xiang, Zai-Tian Liao, Jun-Quan Zhu, Chang-Kao Mu, Chun-Lin Wang & Cong-Cong Hou

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Le Chang
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Qiu Meng Xiang
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Zai-Tian Liao
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Jun-Quan Zhu
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Chang-Kao Mu
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Chun-Lin Wang
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Cong-Cong Hou
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Contributions

C.C.H. and L.C. co-designed the study, performed the experiments, the statistical analysis and wrote the paper. Q.M.X. and Z.T.L. implemented the investigation and the experimental animals breeding. L.C. and Q.M.X. performed the validation of data. C.C.H. and J.Q.Z. supervised the work and commented on the manuscript. Acquisition of financial resources occurred through C.C.H., C.K.M. and C.L.W. All authors read and approved the final version of manuscript.

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Correspondence to Cong-Cong Hou.

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Chang, L., Xiang, Q.M., Liao, ZT. et al. Pt-LIS1 participates nuclear deformation and acrosome formation via regulating Dynein-1 during spermatogenesis in Portunus trituberculatus. Sci Rep 15, 6632 (2025). https://doi.org/10.1038/s41598-024-83566-2

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Received: 03 June 2024
Accepted: 16 December 2024
Published: 24 February 2025
Version of record: 24 February 2025
DOI: https://doi.org/10.1038/s41598-024-83566-2

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Abstract

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Lis1 relieves cytoplasmic dynein-1 autoinhibition by acting as a molecular wedge

Microtubule-binding-induced allostery triggers LIS1 dissociation from dynein prior to cargo transport

Lis1 slows force-induced detachment of cytoplasmic dynein from microtubules

Introduction

Results

Identification and characterization of the Pt-Lis1 gene in P. trituberculatus

The highly expression and distribution of Pt-Lis1 in the testis of P. trituberculatus

Distribution of Pt-LIS1 protein in the testis of P. trituberculatus

Colocalization of Pt-LIS1 and Pt-DHC, Pt-DIC, α-Tubulin, PHB and Acrosin during spermatogenesis in P. trituberculatus

Detection of Pt-Lis1 RNAi efficiency

Deficiency of Pt-Lis1 in vivo affects both the expression level and distribution of Pt-DHC and Pt-DIC

Deficiency of Pt-Lis1 in vivo influences the expression level of related genes (Pt-Nude, phb, p53 and caspase-3) in testes

Knockdown of Pt-Lis1 leads to abnormal colocalization of Pt-LIS1 and α-Tubulin in the testis of P. trituberculatus

Discussion

Structure Analysis of LIS1

Pt-LIS1 was necessary for spermatogenesis in P. trituberculatus

Pt-LIS1 regulates Dynein-1 transport along microtubule and affects microtubule assembly

Pt-LIS1 plays an important role in the expression of the Dynein −1 subunit

Pt-LIS1 as a key regulator of Dynein-1 in spermatogenesis of P. trituberculatus

Conclusions

Materials and methods

Experimental animals

Synthesis of dsRNA

Experimental design

Collection of samples

Antigen immunization injections

Full-length cDNA cloning of Pt-Lis1

Multiple sequence alignment, phylogenetic evolutionary tree analysis and protein structure prediction

Expression analysis of Pt-Lis1 mRNA in different tissues of P. trituberculatus

Fluorescence in situ hybridization

Western blot analysis

Immunofluorescence

Hematoxylin and eosin staining

Effect of Pt-Lis1 knockdown on the mRNA expression of Pt-dhc, Pt-dic, Pt-Nude, phb, caspase-3 and p53

Effect of Pt-Lis1 knockdown on the spatial expression distribution of Pt-LIS1 with Pt-DHC, Pt-DIC and microtubules

Statistical analysis

Additional information

Data availability

References

Acknowledgements

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Supplementary Information

Supplementary Information 1.

Supplementary Information 2.

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