Tektin 4 is located on outer dense fibers, not associated with axonemal tubulins of flagella in rodent spermatozoa

Abstract

Tektins, which are thought to be the constitutive proteins of microtubules in cilia, flagella, basal bodies, and centrioles, have been reported to be involved in the stability and structural complexity of axonemal microtubules. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin 2 and Tektin 4, have been verified to be present in sperm flagella. To elucidate the molecular localization of Tektin 4 in flagella of rodent spermatozoa, we performed immunocytochemistry, fractionation study followed by immunoblot analysis, and immunogold electron microscopy. Confocal laser scanning microscopy and immunogold electron microscopy indicated that Tektin 4 was associated with outer dense fibers (ODFs) in both the middle and principal piece of flagella in rat and mouse spermatozoa. Tektin 4 in rat spermatozoa is completely released by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Pre-embedding immunoelectron microscopy demonstrated that Tektin 4 located on the abaxial (convex) surface of ODFs in flagella, not associate with axonemal microtubules. Our data strongly suggested that Tektin 4 is not associated with axonemal tubulins but an ODFs-affiliated molecule in rodent spermatozoa.

A heat‐shock protein 40, DNAJB13, is an axoneme‐associated component in mouse spermatozoa

“…We then performed indirect immunofluorescence on epididymal sperm and SDS-insoluble tail structures with antibodies against a tubulin, tektin 4 and DNAJB13, respectively. It has been demonstrated that tektin4 is located on the outer dense fibers (Iida et al, 2006) and not associated with axoneme (Cao et al, 2006), while the most abundant axonemal protein, tubulin, is unable to be detected in the SDS-resistant flagellar structures (Cao et al, 2006). We show here that tektin 4 is localized to sperm (Fig.…”

Enriched variations in TEKT4 and breast cancer resistance to paclitaxel

“…In doublet microtubules, tektins also help to organize the longitudinal spacing of accessory structures, such as groups of inner dynein arms and radial spokes 21 . As a microtubule-associated protein, tektin4 colocalizes with the microtubule network and helps to stabilize these structures 31 . TEKT4 variations might induce an abnormal structure of tektin4 and alter the interaction between tektin4 and microtubules, resulting in reduced microtubule stability.…”

Germ cell-less like-2 protein is a new component of outer dense fibers in rat sperm flagella

“…In addition, we have recently reported that Tektin4 and Tektin5, new members of the TEKTIN family, are associated with the cortex of ODF and the inner surface of mitochondria sheath respectively (Iida et al 2006b, Murayama et al 2007. It is therefore probable that Tektin4, Tektin5, and rgcl-2 interact either with each other or with some molecules in SMR to tether the axoneme-ODF complex to the mitochondrial sheath by which physiological connection between flagellar components is maintained.…”

“…Samples for immunoelectron microscopy were prepared by the method described previously (Iida et al 2006b). In brief, purified rat spermatozoa were exposed at 4 8C for 60 min to 50 mmol/l sodium borate buffer (pH 8.0) containing 1% Triton X-100 to remove the plasma membrane.…”