Focal adhesion kinase is a regulator of F-actin dynamics: New insights from studies in the testis

Abstract

During spermatogenesis, spermatogonia (2n, diploid) undergo a series of mitotic divisions as well as differentiation to become spermatocytes, which enter meiosis I to be followed by meiosis II to form round spermatids (1n, haploid), and then differentiate into spermatozoa (1n, haploid) via spermiogenesis. These events take place in the epithelium of the seminiferous tubule, involving extensive junction restructuring at the Sertoli-Sertoli and Sertoli-germ cell interface to allow the transport of developing germ cells across the epithelium. Although structural aspects of these cell-cell junctions have been studied, the underlying mechanism(s) that governs these events has yet to be explored. Earlier studies have shown that a non-receptor protein tyrosine kinase known as focal adhesion kinase (FAK) is a likely regulator of these events due to the stage-specific and spatiotemporal expression of its various phosphorylated/activated forms at the testis-specific anchoring junctions in the testis, as well as its association with actin regulatory proteins. Recent studies have shown that FAK, in particular its two activated phosphorylated forms p-FAK-Tyr⁴⁰⁷ and p-FAK-Tyr³⁹⁷, are crucial regulators in modulating junction restructuring at the Sertoli cell-cell interface at the blood-testis barrier (BTB) known as the basal ectoplasmic specialization (basal ES), as well as at the Sertoli-spermatid interface called apical ES during spermiogenesis via its effects on the filamentous (F)-actin organization at the ES. We herein summarize and critically evaluate the current knowledge regarding the physiological significance of FAK in regulating BTB and apical ES dynamics by governing the conversion of actin filaments at the ES from a "bundled" to a "de-bundled/branched" configuration and vice versa. We also provide a molecular model on the role of FAK in regulating these events based on the latest findings in the field.

Keywords: F-actin; ectoplasmic specialization; focal adhesion kinase; seminiferous epithelial cycle; spermatogenesis; testis.

Figure 1. An schematic drawing illustrating the regulatory events mediated by the β1-integrin/FAK signaling cascades during apical ES degeneration at spermiation. This is a schematic drawing of the seminiferous epithelium in the rat testis in which the blood-testis barrier (BTB) physically divides the epithelium into the adluminal (apical) and the basal compartment with the base of the Sertoli cell lies on the basement membrane of the tunica propria. Apical ES first appears at the interface of step 8 spermatids and Sertoli cells at stage VIII of the epithelial cycle, once it forms, it is the only anchoring device in these spermatids, replacing desmosome and gap junction at the Sertoli-spermatid interface, and to confer spermatid polarity. Also, apical ES is found in step 8–19 spermatids, and it begins to undergo degeneration at stage VII of the cycle until it is disintegrated entirely at late stage VIII to allow the release of sperm at spermiation.^,, In stage VII-early stage VIII tubules (left panel) as shown herein, spermatids attach to the Sertoli cell through an adhesion protein complex containing α6β1-integrin and laminin-α3β3γ3 (other adhesion protein complexes at the apical ES are: JAM-C-ZO-1, nectin2/3-afadin, and N-cahderin-β-catenin²¹). This complex is known to associate with a large number of regulatory proteins,^,, which include PKB, DOCK180, PI3K, and most notably FAK, in particular p-FAK-Tyr³⁹⁷, p-FAK-Tyr⁴⁰⁷, and p-FAK-Tyr⁵⁷⁶, which recruit actin cross-linking and bundling proteins, such as Eps8, palladin, and filamin A, to maintain the integrity of the actin filament bundles at the ES. Collectively, these proteins to confer the integrity of the apical ES. At late stage VIII of the epithelial cycle, to prepare for spermiation, the expression of FAK, in particular p-FAK-Tyr³⁹⁷ and -Tyr⁴⁰⁷, at the apical ES is downregulated.^,, This loss of FAK at the apical ES fails to retain the actin filament cross-linking, barded end-capping and bundling proteins (e.g., Eps8, palladin, filamin A) at the apical ES to maintain the integrity of the actin filament bundles, instead, N-WASP activated-Arp2/3 protein complex induces branched actin polymerization, converting the actin filaments from their “unbundled” and their “de-bundled” configuration, destabilizing the apical ES, facilitating endocytic vesicle-mediated protein trafficking to further destabilize apical ES adhesion. Furthermore, apical ES disruption involves also the PI3K/PKB signaling cascades in which ERK1/2, a downstream signaling protein, is activated via phosphorylation (arrow),^,,, which further enhances apical ES disruption and, thus, facilitating the release of sperm at spermiation.

Figure 2. A schematic diagram illustrating the molecular architecture of the BTB and its restructuring events that are mediated by FAK during the seminiferous epithelial cycle. The panel on the left is a schematic drawing that illustrates the relative location of the BTB in the seminiferous epithelium. The BTB is enlarged and shown in the right panel. The upper part of the diagram on the right displays the molecular architecture of an intact BTB. The relatively high expression of p-FAK-Tyr⁴⁰⁷ at the BTB, coupled with the upregulation of Eps8 (an actin barbed end capping and bundling protein), and two actin cross-linking and bundling proteins palladin and filamin A at the BTB thus maintain the integrity of the actin filament bundles at the BTB. Occludin/ZO-1 and other TJ proteins (e.g., JAM-B/ZO-1, JAM-A-ZO-1), together with basal ES proteins (e.g., N-cadherin/β-catenin, nectin-2/afadin), gap junction proteins (e.g., connexin-43, connexin-33), and desmosomal proteins (e.g., desmoglein-2), thus confer Sertoli cell-cell adhesion to constitute the blood-testis barrier (BTB). This thus maintains the BTB integrity, such as at stage VII of the epithelial cycle. At stage VIII of the epithelial cycle, BTB undergoes modifications as shown in the lower part of the diagram on the right panel. This is likely mediated via a downregulation of p-FAK-Tyr⁴⁰⁷, which coupled with an upregulation of the Arp2/3 complex and N-WASP, thereby converting actin filament bundles from their “bundled” to their “unbundled/branched” configuration, destabilizing the BTB to facilitate endocytic vesicle-mediated protein trafficking, facilitating BTB restructuring to allow the transport of preleptotene spermatocytes across the BTB. Other signaling proteins, such as the phosphatase, SHP2 (Src homology domain-containing phosphatase-2) may also take part in this event.

Figure 3. The role of p-FAK-Tyr³⁹⁷ and -Tyr⁴⁰⁷ in coordinating cellular events at the apical ES-BTB functional axis during the epithelial cycle of spermatogenesis. FAK, in particular its two activated/phosphorylated forms of p-FAK-Tyr³⁹⁷ and -Tyr⁴⁰⁷ is tightly involved in coordinating the events of spermiation and BTB restructuring that take place simultaneously across the epithelium at stage VIII of the seminiferous epithelial cycle. p-FAK-Tyr³⁹⁷ that is highly expressed at early stage VIII of the cycle may: (1) involve in the activation and/or upregulation of matrix metalloproteinase 2 (MMP-2) which cleaves laminin-β3 and -γ3 chains. (2) This thus generates biologically active laminin fragments which are released from the apical ES to activate BTB restructuring via an “inside-outside-in” signaling cascade, involving p-FAK-Tyr⁴⁰⁷. (3) It is likely that the biologically active laminin fragments are working in concert with p-FAK-Tyr⁴⁰⁷ to recruit N-WASP-Arp2/3 complex to the site to induce F-actin re-organization, converting actin filaments from their “bundled” to their “unbundled/branched” configuration, thereby destabilizing the BTB. (4) The “unbundled/branched” F-actin network at the BTB thus favors endocytic vesicle-mediated protein trafficking, inducing protein endocytosis, such as TJ protein occludin, leading to BTB restructuring. It is obvious that this model will be rapidly updated when more functional data are available in the near future.