C-Terminal Kinesin Motor KIFC1 Participates in Acrosome Biogenesis and Vesicle Transport

BIOLOGY OF REPRODUCTION 69, 1719–1729 (2003) Published online before print 25 June 2003. DOI 10.1095/biolreprod.102.014878 C-Terminal Kinesin Motor KIFC1 Participates in Acrosome Biogenesis and Vesicle Transport1 Wan-Xi Yang and Ann O. Sperry2 Department of Anatomy and Cell Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina 27858 ABSTRACT spermatid, spermatogenesis, testis INTRODUCTION The successful production of mammalian sperm capable of fertilization is essential for survival of the species. Spermatogenesis involves intricate cellular change in order to transform the precursor germ cell, the spermatogonium, into a mature spermatozoon. These changes include significant remodeling of the germ cell cytoskeleton, including formation of mitotic and meiotic spindles, and creation of two microtubule structures unique to spermatogenesis: the manchette and the sperm flagella. In addition to changes in the cytoskeleton, formation and redistribution of membrane domains occur during spermiogenesis. Due to the dramatic nature of these cellular rearrangements, mammalian spermatogenesis is an extremely useful model for understanding the role of molecular motor proteins in cellular change. The acrosome is the most visible organelle formed during spermatogenesis, first appearing early in spermatid deThis work was supported by grant GM60628 from the National Institutes of Health (to A.O.S.). 2 Correspondence: Ann O. Sperry, Department of Anatomy and Cell Biology, Brody School of Medicine at East Carolina University, 600 Moye Blvd., Greenville, NC 27858. FAX: 252 816 2850; e-mail: 1 Received: 19 December 2002. First decision: 31 December 2002. Accepted: 17 June 2003. Q 2003 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org 1719 We have identified a possible role for the KIFC1 motor protein in formation of the acrosome, an organelle unique to spermatogenesis. KIFC1, a C-terminal kinesin motor, first appears on membrane-bounded organelles (MBOs) in the medulla of early spermatids followed by localization to the acrosomal vesicle. KIFC1 continues to be present on the acrosome of elongating spermatids as it flattens on the spermatid nucleus; however, increasing amounts of KIFC1 are found at the caudal aspect of the spermatid head and in distal cytoplasm. The KIFC1 motor is also found in the nucleus of very immature round spermatids just prior to its appearance on the acrosome. In some cases, KIFC1 appears localized just below the nuclear membrane adjacent to the subacrosomal membrane. We demonstrate that KIFC1 is associated with importin b and colocalizes with this nuclear transport factor on curvilinear structures associated with the spermatid nuclei. These data support a model in which KIFC1, perhaps in association with nuclear factors, assists in the formation and/or elongation of the spermatid acrosome. This article represents the first demonstration of a direct association of a molecular motor with the spermatid acrosome, the formation of which is essential for fertilization. velopment in the phase termed spermiogenesis. The acrosome begins its life as an acrosomal vesicle adhered to one pole of the nucleus gradually expanding to cover the majority of the spermatid nucleus by the conclusion of spermiogenesis. During the expansion of the acrosome, the underlying nucleus undergoes extensive chromatin condensation and elongation. It is known that the Golgi apparatus supplies the majority of the acrosomal membrane; however, the mechanism by which the acrosome achieves its final extended form and how this process is coordinated with nuclear changes are poorly understood. The Golgi apparatus is the primary, but not exclusive, source of membrane and protein for the developing acrosome [1–3]. This organelle is oriented proximal to the developing acrosome early in spermiogenesis. Acrosome biogenesis begins with the formation of proacrosomal vesicles that are derived from the Golgi and contain dark acrosomal granules. After these vesicles fuse, the resultant acrosomal vesicle adheres to the nucleus, flattening upon contact. During spermatid maturation, the acrosome grows to cover most of the elongated spermatid nucleus due primarily to protein and membrane input from the Golgi [1, 2]. It has been proposed, however, that the differential delivery of proteins to the acrosome might require multiple transport pathways from the Golgi and/or endoplasmic reticulum (ER) [4]. Microtubules play a vital role in the multiple morphogenic processes of spermatogenesis, as demonstrated by multiple studies over the past 2–3 decades [5–9]. The formation and motility of the manchette, the growth of the acrosome, and the condensation and elongation of the spermatid nucleus are tightly coordinated events during spermiogenesis. In fact, the manchette has been proposed to function directly in nuclear condensation and shaping [10]. Recently, specific molecular motor proteins have been demonstrated to play important roles in these movements, including spermatid traffic within the epithelium, formation of the spindle and the manchette, and formation of the acrosome [11–14]. Due to the complexity of the cellular changes required for the formation of viable sperm, it seems likely that additional motor proteins will be identified with crucial roles in various steps in this intricate process. This article describes an analysis of the role of the molecular motor KIFC1 in spermiogenesis. KIFC1 is a member of a highly related group of C-terminal motor proteins that are very similar to one another, diverging only in their tail domains [13]. Our initial hypothesis was that their divergent structure would result in a difference in function for these motors. In order to further examine the role of KIFC1 in the developmental program of spermatogenesis, we have localized this motor to the acrosome of developing spermatids. Prior to its localization on the acrosome, KIFC1 1720 YANG AND SPERRY FIG. 1. KIFC1 is developmentally expressed in rat testis during acquisition of sexual maturity. A) A schematic of the KIFC1 protein showing the sequence unique to KIFC1 (zigzag). The hatched box indicates the motor domain and the white boxes indicate the sequences shared with motor subfamily members. B) KIFC1 protein was detected in extracts prepared from testis obtained from animals at 7, 14, 20, 30, and approximately 90 days (adult) after birth using the KIFC1 antipeptide antibody as described in Materials and Methods. The migration of molecular weight markers (31023) is shown on the right while equal protein loading is demonstrated by ponseau staining of the blot (below). was found on punctate structures between the Golgi and the spermatid nucleus. In addition to its location on the acrosome, this motor is also present in the nucleus during early stages of spermatid development and is associated with the nuclear transport factor importin b. T (...truncated)