Thursday, November 21, 2024
11/21/2024
PD/PI: Takemaru, Ken-Ichi 7. PROJECT SUMMARY Cilia are evolutionarily conserved microtubule-based structures that protrude from the apical cell surface to perform important biological functions, ranging from signal transduction to fluid movement. The sperm flagellum is a modified motile cilium that is essential for locomotion and fertilization of the egg. As such, abnormal sperm flagella are one of the leading causes of male infertility. Although not completely equivalent, analogous mechanisms are thought to operate during flagellogenesis and ciliogenesis. Defective ciliogenesis is associated with human disorders, known as ciliopathies, such as primary ciliary dyskinesia, and many of the male patients also show fertility issues. Despite worldwide research efforts to elucidate the role of ciliary proteins in these diseases, their physiological roles in male fertility remain poorly understood. We previously demonstrated that mice lacking the key ciliary protein Chibby1 (Cby1) exhibit ciliopathy phenotypes. Molecularly, Cby1 interacts with membrane-binding BAR domain proteins, called Cby1-interacting BAR domain-containing 1 and 2 (ciBAR1 and 2, formerly known as FAM92A and B), to facilitate ciliogenesis. More recently, we found that a Cby family member, Cby1-Like (Cby1L, also known as Cby3), is expressed abundantly and exclusively in the testis. Interestingly, Cby1L localizes to the annulus, a septin-based ring structure at the midpiece/principal piece (MP/PP) junction in the sperm tail, in the testis. To gain insight into the in vivo function of Cby1L during spermatogenesis, we created Cby1L-/- mice and found that male mice are completely infertile with the majority of sperm showing kinked tails around the MP/PP junction. Curiously, in the sperm from Cby1L-/- mice, the annulus is present but fails to stop at the MP/PP junction and progresses into the PP. Although the annulus is tightly bound to membranes during its migration, in Cby1L-/- testis, the annulus is partially detached from the flagellar membrane, suggesting that Cby1L is involved in anchoring the annulus to the membrane. Consistent with this hypothesis, we found that Cby1L physically interacts with ciBAR1 as well as the annulus septin SEPT4. Furthermore, we found that ciBAR1 localizes to the annulus, and ciBAR1-/- male mice are infertile, producing sperm with kinked tails. Collectively, our preliminary data suggest an exciting working model in which Cby1L in a complex with ciBAR1 stably anchors the annulus to flagellar membranes during its caudal migration and precisely positions it at the MP/PP junction likely via membrane shaping and constriction. The overall goal of this application is to understand the molecular roles of Cby1L and its binding proteins in spermatogenesis and male fertility. To achieve this goal, we propose the following Specific Aims: 1) Investigate the role of Cby1L in flagellogenesis using a Cby1L-/- mouse model; 2) Investigate the involvement of ciBAR1 in flagellogenesis and male fertility; and 3) Identify novel Cby1L-binding proteins and characterize their functions during flagellogenesis. We anticipate that our proposed research will unravel a novel mechanism underlying the proper compartmentalization of the sperm tail by ciliary proteins.
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