New insights into FAK function and regulation during spermatogenesis

Abstract

Germ cell transport across the seminiferous epithelium during the epithelial cycle is crucial to spermatogenesis, although molecular mechanism(s) that regulate these events remain unknown. Studies have shown that spatiotemporal expression of crucial regulatory proteins during the epithelial cycle represents an efficient and physiologically important mechanism to regulate spermatogenesis without involving de novo synthesis of proteins and/or expression of genes. Herein, we critically review the role of focal adhesion kinase (FAK) in coordinating the transport of spermatids and preleptotene spermatocytes across the epithelium and the BTB, respectively, along the apical ectoplasmic specialization (ES) - blood-testis barrier - basement membrane (BM) functional axis during spermatogenesis. In the testis, p-FAK-Tyr³⁸⁷ and p-FAK-Tyr⁴⁰⁷ are spatiotemporally expressed during the epithelial cycle at the actin-rich anchoring junction known as ES, regulating cell adhesion at the Sertoli-spermatid (apical ES) and Sertoli cell-cell (basal ES) interface. Phosphorylated forms of FAK exert their effects by regulating the homeostasis of F-actin at the ES, mediated via their effects on actin polymerization so that microfilaments are efficiently re-organized, such as from their "bundled" to "de-bundled/branched" configuration and vice versa during the epithelial cycle to facilitate the transport of: (i) spermatids across the epithelium, and (ii) preleptotene spermatocytes across the BTB. In summary, p-FAK-Tyr⁴⁰⁷ and p-FAK-Tyr³⁸⁷ are important regulators of spermatogenesis which serve as molecular switches that turn "on" and "off" adhesion function at the apical ES and the basal ES/BTB, mediated via their spatiotemporal expression during the epithelial cycle. A hypothetical model depicting the role of these two molecular switches is also proposed.

Fig. 1

A schematic drawing that illustrates different functional domains of focal adhesion kinase (FAK) and its related non-receptor protein kinases in mammalian cells. FAK is the downstream signaling molecule that transduces signals mediated by an integrin-based receptor following its activation by various ligands (e.g., fragments of collagens and/or laminins), which usually takes place via an interaction with an integrin receptor near its N-terminus. FAK is functionally composed of three domains from its N-terminus: the FERM (band 4.1, ezrin, radixin, noesin homology) domain, the catalytic kinase domain and the FAT (focal adhesion targeting) domain near its C-terminus. There are also three proline-rich (PR) domain, PRI inside the FERM domain, and PRII and PRII within the FAT domain. It also has six putative phosphorylation sites at Tyr-397, -407, -576, -577, -861 and -925. FAK also contains the nuclear localization signal (NLS) and nuclear export signal (NES) that allows its translocation to the nucleus. FAK, unlike other non-receptor protein kinases, such as Abl, CSK, FRK, c-Src, and TEC, possess no SH2 or SH3 domain for protein-protein interactions, instead it utilizes its three PR regions to recruit a large number of signaling proteins (e.g., c-Src, PI-3K), and adaptors (e.g., vincluin, talin, p130Cas) to form a giant regulatory protein complex. Abbreviations used: AbI, Abelson leukemia virus tyrosine kinase; Btk motif, Btk-type zinc finger motif; CSK, cellular Src or C-terminal Src kinase; c-Src; a non-receptor protein tyrosine kinase in cells encoded by the Rous sarcoma virus (SRC) gene; FCH, Fes/CIP4 homology domain; FRK, Fyn-related kinase; Grb7, growth factor receptor-bound protein 7; JAK, Janus kinase also known as JAK1; PI-3K, phosphoinositide 3-kinase; p130Cas, Crk-associated substrate; PLCγ, phospholipase C γ; SH2, Src homology 2; SH3, Src homology 3; TEC, tyrosine protein kinase Tec.

Fig. 2

A schematic drawing illustrating the anatomical features of the seminiferous epithelium in adult rat testes. The BTB physically divides the seminiferous epithelium into the basal and the adluminal (apical) compartment in the mammalian testis. This drawing highlights the actin-rich ultrastructure known as ectoplasmic specialization (ES) in the seminiferous epithelium. ES is present at the Sertoli cell-cell interface at the BTB called basal ES, it is also found at the Sertoli-spermatid interface and designated apical ES. ES is typified by the presence of an array of actin filament bundles that line perpendicular to the Sertoli cell plasma membrane. These actin filament bundles are sandwiched in-between cisternae of endoplasmic reticulum and the Sertoli cell plasma membrane, and these bundles of actin filaments thus confer unusual adhesive strength to the basal and the apical ES. These morphological features are virtually identical between the apical and the basal ES. Since the array of actin filament bundles is only found in Sertoli cells, so that a single array of actin filament bundles is present at the apical ES versus two arrays of microfilament bundles at the basal ES, and the constituent proteins of the apical and the basal ES are also somewhat different (Cheng and Mruk, 2002, 2010). During spermiogenesis, apical ES appears in step 8 spermatids, once it forms, the apical ES replaces desmosome and gap junction, and remains to be the only anchoring device until step 19 spermatids prior to spermiation that occurs at stage VIII of the epithelial cycle (see micrograph on the lower right panel). Due to the unique cellular associations between different germ cells, such as developing spermatids, and the Sertoli cell in the seminiferous epithelium, the epithelium can be divided into 14, 12 and 6 stages in the rat, mouse and man, respectively, and known as the epithelial cycle of spermatogenesis. Herein, two stages of the seminiferous tubule at V and VIII of the epithelial cycle are shown. At stage V, elongating spermatids (ES) (step 17) are transported back to the basal compartment with the spermatid head almost touches the Sertoli cell (SC) nucleus. In stage VIII, preleptotene spermatocytes (PreLS) transformed from type B spermatogonia are transported across the BTB which is made possible via BTB restructuring, step 19 ES also line-up near the lumen of the seminiferous tubule so that spermiation, the release of sperm, can take place in late stage VIII of the cycle. Thus, it is conceivable that the basal and the apical ES undergo extensive restructuring in stage VIII tubules. The schematic drawing illustrates features of the apical and basal ES at stage VII of the epithelial cycle when these junctions remain intact. p-FAK-Tyr³⁹⁷ and -Tyr⁴⁰⁷ are highly expressed at the apical ES, but restricted to different sites with p-FAK-Tyr³⁹⁷ (red fluorescence) restricted to the convex (dorsal) side of the spermatid head, while p-FAK-Tyr⁴⁰⁷ (red fluorescence) is most prominent on the concave (ventral) side of the spermatid head. At the BTB, p-FAK-Tyr⁴⁰⁷ (red fluorescence), but not p-FAK-Tyr³⁹⁷, is highly expressed. It is likely that these two phosphorylated forms of FAK regulate the dynamic organization of actin microfilament bundles at the ES via its effects on actin filament bundling proteins Eps8 and palladin versus branched actin polymerization-inducing proteins Arp2/3 complex/N-WASP and filamin A, depending on the stage of the epithelial cycle.

Fig. 3

A hypothetical model illustrating the physiological role of p-FAK-Tyr³⁹⁷ and p-FAK-Tyr⁴⁰⁷ in coordinating the apical and the basal ES disruption to facilitate spermiation and BTB restructuring at stage VIII of the epithelial cycle. At stage VIII of the epithelial cycle, spermiation and BTB restructuring that facilitate the release of sperm and the transport of preleptotene spermatocytes across the BTB take place simultaneously, but at opposite ends of the seminiferous epithelium. As detailed in the text, p-FAK-Tyr³⁹⁷ and -Tyr⁴⁰⁷ play crucial roles in coordinating these cellular events. Shown on the left panel, biologically active fragments of laminin chains released via the action of MMP-2 during the degeneration of apical ES induce BTB restructuring possibly via the action of p-FAK-Tyr⁴⁰⁷, which likely induces an activation and the recruitment of Arp2/3 protein complex to induce branched actin polymerization, effectively converting actin filaments from a “bundled” to a “branched/de-bundled” configuration, this thus enhances endocytic vesicle-mediated protein trafficking including transcytosis and recycling so that a “new” BTB is assembled at the basal region of the preleptotene spermatocyte being transported across the BTB prior to the disassembly of the “old” BTB above the spermatocyte (see bottom right panel). On the other hand, p-FAK-Tyr³⁹⁷ at the apical ES also recruits the Arp2/3 complex but re-localizes actin bundling proteins Eps8 and palladin away from the apical ES, the net results thus favors the “branched/de-bundled” actin filaments at the apical ES, facilitating endocytic vesicle-mediated protein degradation and/or transcytosis and recycling to assemble “new” apical ES for the newly formed step 8 spermatids at stage VIII of the epithelial cycle. This hypothesis is supported by recent findings in the field as discussed in the text.