Abstract
BackgroundSpermatogenesis is comprised of a series of highly regulated developmental changes that transform the precursor germ cell into a highly specialized spermatozoon. The last phase of spermatogenesis, termed spermiogenesis, involves dramatic morphological change including formation of the acrosome, elongation and condensation of the nucleus, formation of the flagella, and disposal of unnecessary cytoplasm. A prominent cytoskeletal component of the developing spermatid is the manchette, a unique microtubular structure that surrounds the nucleus of the developing spermatid and is thought to assist in both the reshaping of the nucleus and redistribution of spermatid cytoplasm. Although the molecular motor KIFC1 has been shown to associate with the manchette, its precise role in function of the manchette and the identity of its testis specific protein partners are unknown. The purpose of this study was to identify proteins in the testis that interact with KIFC1 using a yeast 2 hybrid screen of a testis cDNA library.ResultsThirty percent of the interacting clones identified in our screen contain an identical cDNA encoding a 40 kD protein. This interacting protein has 4 leucine-rich repeats in its amino terminal half and is expressed primarily in the testis; therefore we have named this protein testis leucine-rich repeat protein or TLRR. TLRR was also found to associate tightly with the KIFC1 targeting domain using affinity chromatography. In addition to the leucine-rich repeats, TLRR contains a consensus-binding site for protein phosphatase-1 (PP1). Immunocytochemistry using a TLRR specific antibody demonstrates that this protein is found near the manchette of developing spermatids.ConclusionWe have identified a previously uncharacterized leucine-rich repeat protein that is expressed abundantly in the testis and associates with the manchette of developing spermatids, possibly through its interaction with the KIFC1 molecular motor. TLRR is homologous to a class of regulatory subunits for PP1, a central phosphatase in the reversible phosphorylation of proteins that is key to modulation of many intracellular processes. TLRR may serve to target this important signaling molecule near the nucleus of developing spermatids in order to control the cellular rearrangements of spermiogenesis.
Highlights
Spermatogenesis is comprised of a series of highly regulated developmental changes that transform the precursor germ cell into a highly specialized spermatozoon
A leucine-rich repeat protein interacts with the KIFC1 targeting sequence In an effort to identify proteins associated with the KIFC1 targeting domain, we used the 19 amino acid targeting sequence as bait to screen a mouse testis cDNA library using a yeast 2-hybrid approach [9]
The work presented here supports the idea that the LRRs indicated with overlines (TLRR) is a testis specific regulatory subunit of PP1 that might serve to target PP1 and perhaps other regulatory molecules to the nucleus of developing spermatids
Summary
Spermatogenesis is comprised of a series of highly regulated developmental changes that transform the precursor germ cell into a highly specialized spermatozoon. The final phase requires quite striking cellular reorganization to produce functional sperm including biogenesis of spermatid specific organelles and structures such as the acrosome and microtubule manchette, removal of excess cytoplasm, and streamlining of the spermatid nucleus into its final shape [1]. These unique forms of intracellular motility are expected to require specific adaptations of the spermatid cytoskeleton and associated molecular motor proteins [2]. KIFC1 locates near the spermatid manchette, a spermatidspecific microtubule structure thought to be important for spermatid nuclear shaping [5] and redistribution of cytoplasm (reviewed in [6])
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