In a groundbreaking study published in Nature Communications, researchers have unveiled a novel molecular player intimately involved in the regulation of sperm motility—a critical factor underpinning male fertility. Utilizing an advanced technique known as proximity labeling on the axonemal protein CFAP91, the team successfully identified EFCAB5, a calcium-binding protein whose precise role had previously remained elusive. The findings not only deepen our understanding of the molecular architecture and functionality of sperm flagella but also open new avenues for investigating male infertility and potential therapeutic interventions.
Sperm motility, the capacity of sperm cells to navigate through the female reproductive tract towards the oocyte, is fundamental for successful fertilization. Despite decades of research, the intricate protein networks orchestrating this motility remain only partially mapped. The axoneme, a highly conserved microtubule-based structure forming the skeleton of eukaryotic cilia and flagella, houses a myriad of proteins critical to flagellar beating and energy transduction. CFAP91—Cilia and Flagella Associated Protein 91—has been identified previously as a scaffolding component within the axoneme, but its interactome and functional partners in sperm remained largely unknown.
Leveraging a proximity-dependent biotin identification technique—proximity labeling—researchers were able to go beyond traditional protein interaction studies. This method allows for the tagging of proteins that lie in the immediate vicinity of a bait protein under physiological conditions, providing a refined snapshot of protein neighborhoods within complex structures like the axoneme. CFAP91 was used as the bait, and by fusing it with an engineered enzyme that biotinylates nearby proteins, the team successfully captured a dynamic interactome within sperm flagella.
Among the proteins identified, EFCAB5 (EF-hand Calcium Binding domain 5) stood out due to its unique domain architecture and its potential calcium regulation capability. Calcium ions have long been implicated in modulating flagellar beating patterns, yet how specific calcium-binding proteins integrate into the axonemal framework has remained speculative. EFCAB5’s designation suggested a pivotal role in sensing or transducing calcium signals necessary for motility regulation.
Subsequent functional characterization using gene-editing techniques in murine sperm elucidated the essentiality of EFCAB5 for proper flagellar function. Loss of EFCAB5 led to severely impaired sperm motility characterized by erratic and non-progressive movement patterns. High-speed video microscopy and subsequent kinematic analyses revealed that the absence of EFCAB5 disrupted the fine-tuned coordination of axonemal components that produce symmetrical and well-tempered flagellar waves. This deficient motility phenotype closely mirrors clinical characteristics observed in certain cases of idiopathic male infertility.
The mechanistic investigation extended to decipher how EFCAB5 interacts with CFAP91 and other axonemal proteins. Co-immunoprecipitation assays, combined with in situ proximity ligation, confirmed a physical and functional coupling between EFCAB5 and CFAP91 within the radial spoke complex—a critical regulator of flagellar beat frequency and amplitude. This suggests that EFCAB5 functions as a crucial calcium sensor or modulator that influences axonemal mechanics via CFAP91, thus directly impacting the physical forces driving sperm progressivity.
Notably, calcium regulation in flagella is a rapidly reversible and highly localized process, critical for enabling sperm to adjust their motility patterns in response to chemical and mechanical cues encountered during their journey in the female reproductive tract. EFCAB5’s ability to bind calcium via its EF-hand domains likely positions it as a molecular switch modulating conformational changes in the axoneme, thereby translating transient calcium fluxes into mechanical output. Such nuanced regulation extends our conceptual framework of how local signaling cascades integrate with cytoskeletal dynamics to generate motility.
From a methodological perspective, the success of proximity labeling in dissecting the axonemal proteome underscores the power of innovative biochemical tools to map organelle-specific interactomes. Traditional co-immunoprecipitation approaches often fall short due to the insolubility, low abundance, or transient interactions of key axonemal proteins. Proximity labeling overcomes these limitations, facilitating a more comprehensive and physiologically relevant portrait of protein-protein networks, especially in structurally intricate systems like sperm flagella.
This discovery also holds clinical implications. Male infertility affects approximately 7% of men worldwide, and in nearly half of these cases, sperm motility defects are the underlying cause. Identification of EFCAB5 as a crucial regulator invites exploration of its genetic variants among infertile men. Screening for mutations or dysregulation of EFCAB5 could complement current diagnostic paradigms, offering a molecular target for personalized assessment and possibly therapeutic modulation.
Moreover, understanding how EFCAB5 orchestrates calcium-mediated signaling to govern flagellar dynamics may inspire novel fertility treatments aimed at restoring or enhancing sperm motility. Pharmacological agents or gene therapies designed to modulate EFCAB5’s function could become a reality, presenting alternatives to assisted reproductive technologies that bypass—but do not address—the motility deficits.
Expanding the scope, this study also contributes to the broader field of cilia and flagella biology. Given that ciliary dysfunction underpins a host of human diseases collectively termed ciliopathies—with manifestations spanning respiratory, renal, and neurological deficits—the principles learned from EFCAB5’s role in sperm flagella may have wider relevance. Similar calcium-dependent regulatory mechanisms might exist in other motile cilia, revealing broader biological paradigms and therapeutic opportunities.
Structurally, EF-hand motifs exemplify versatile calcium-binding domains found across various proteins involved in signal transduction and cytoskeletal regulation. EFCAB5’s localization within the highly specialized confines of the radial spoke complex suggests a finely adapted function integrating calcium sensing with mechanical modulation. This attests to the evolutionary refinement of motility regulation in cells where precision and responsiveness are paramount.
Future lines of investigation will likely explore the dynamic interplay between EFCAB5 and other calcium-binding proteins within the axoneme, elucidating how combinatorial interactions and post-translational modifications influence sperm motility patterns under physiological and pathological conditions. Integrating high-resolution cryo-electron microscopy with live-cell imaging and proteomics could provide unprecedented insights into these processes.
In conclusion, the identification of EFCAB5 as a key regulatory protein for sperm motility marks a seminal advance in reproductive biology. By harnessing proximity labeling centered on CFAP91, the research team unlocked new dimensions in our understanding of the molecular machinery driving sperm function. Beyond male infertility, these findings ripple across the fields of cell motility, calcium signaling, and organelle biology, reinforcing the centrality of precise protein networks in cellular function.
The impact of this work extends into translational realms, offering hope for novel diagnostics and treatments tailored to combat male infertility. Furthermore, the methodological innovations exemplified herein stand as a beacon for future studies investigating complex protein-interaction landscapes within challenging cellular environments. As we venture deeper into the nanoscale choreography of sperm flagella, insights such as those revealed by EFCAB5 promise to illuminate fundamental principles sculpting life’s earliest moments.
Subject of Research: Sperm motility regulation via axonemal protein interactions and calcium signaling.
Article Title: Proximity labeling of axonemal protein CFAP91 identifies EFCAB5 that regulates sperm motility.
Article References:
Wang, H., Shimada, K., Pham, A.H. et al. Proximity labeling of axonemal protein CFAP91 identifies EFCAB5 that regulates sperm motility. Nat Commun 16, 8238 (2025). https://doi.org/10.1038/s41467-025-63705-7
Image Credits: AI Generated
