High Resolution Localization of Rab5, EEA1, and Nectin‐3 to Tubulobulbar Complexes in the Rat Testis

Abstract

Tubulobulbar complexes are clathrin/actin-based structures that internalize intercellular junctions in the testis. They resemble coated pits with extremely long necks that are cuffed by dendritic actin networks. As the structures mature, a swollen region or bulb develops near the end of each complex. The bulbs lack actin cuffs and are closely associated with cisternae of endoplasmic reticulum. The bulbs expand and are internalized and enter endocytic compartments of the Sertoli cell. Previous immunofluorescence studies have demonstrated that markers for early endosomes (Rab5 and EEA1) are associated with tubulobulbar complexes and are localized at or near the ends of the structures. Here we use a pre-embedding immunoelectron microscopic technique to accurately localize these markers to apical tubulobulbar complexes that occur at junctions between Sertoli cells and spermatids. Staining for Rab5 occurs at bulbs, identified by the presence of two plasma membranes and a close association with cisternae of endoplasmic reticulum. EEA1 is associated with large vesicles that lack an association with the endoplasmic reticulum. Labeling for nectin-3, an adhesion junction protein in the spermatid plasma membrane, occurs at junctions, TBC bulbs, and in associated double membrane vesicles. Our results suggest that Rab5 associates with junction protein containing bulbs prior to their internalization and that EEA1 associates with the structures later and after internalization. We conclude that at tubulobulbar complexes in Sertoli cells of the seminiferous epithelium, the identity of 'bulbs' as putative early endosomes begins to be established prior to their undergoing scission or budding from their parent structures. Anat Rec, 300:1160-1170, 2017. © 2017 Wiley Periodicals, Inc.

Myosin VI maintains the actin-dependent organization of the tubulobulbar complexes required for endocytosis during mouse spermiogenesis†‡

“…APPL1 is an effector of Rab5, which is also present at TBC bulbs. APPL1 recruits MYO6 to early endosomes by binding directly to the MYO6 adaptor protein GIPC1 [6,27]. Interestingly, the localization of APPL1 was very similar to the distribution of MYO6; both proteins were concentrated at the focus point of the TBCs (compare Figure 2C.a-a"and D.a-a" ) and sometimes appeared in a vesicular pattern (compare Figure 2C.b-b" and D.b-b").…”

“…In addition to clathrin, two other marker proteins of the endocytic pathway have been localized to the TBCs-Rab5 [4] and its effector, the early endosomal antigen 1 (EEA1) [5]. Whereas Rab5 localizes to the bulbular region, EEA1 is present on large endocytic vesicles [6]. Thus, endocytosis of cell surface receptors for degradation or recycling might occur at the TBCs by the formation of Rab5-positive vesicles at the bulbs, which then fuse and mature into EEA1-positive early endosomes (Figure 1b).…”

Internalization of Intact Intercellular Junctions in the Testis by Clathrin/Actin‐Mediated Endocytic Structures: Tubulobulbar Complexes

“…Results, mainly from immunofluorescence, show that TBCs contain junction proteins, and electron microscopic images demonstrate that “intact” junctions occur in the structures; that is, adjacent membranes do not separate, but remain attached. At apical sites, the structures have been shown to contain the adhesion junction proteins nectin 2 and α6β1 integrin (in the Sertoli cell membrane) and nectin 3 (in the spermatid membrane) (Guttman et al, ; Young et al, , ; Adams and Vogl, ). At basal sites, TBCs have been shown to contain the tight junction protein claudin 11 and the gap junction protein connexin 43 (Du et al, ), and both tight and gap junctions have been morphologically identified in electron micrographs of the structures (Russell, ; Du et al, ).…”

“…At basal sites, TBCs have been shown to contain the tight junction protein claudin 11 and the gap junction protein connexin 43 (Du et al, ), and both tight and gap junctions have been morphologically identified in electron micrographs of the structures (Russell, ; Du et al, ). Importantly, TBCs are associated with the endocytosis markers Rab5, EEA1, and LAMP1 (Guttman et al, ; Young et al, , ; Du et al, ; Adams and Vogl, ). Together these results are consistent with the conclusion that TBCs internalize intercellular junctions.…”

“…The calcium channel IP 3 R is concentrated in the ER at the sites, and at least one TRP channel occurs in the adjacent Sertoli cell plasma membrane (Lyon et al, ). In addition, the bulbs are associated with the endosome marker Rab5 (Young et al, ; Adams and Vogl, ), and then after scission with EEA1 and LAMP1 (Guttman et al, ; Du et al, ; Adams and Vogl, ). Other endosomal proteins reported to be present at TBCs include endophilin, syntaxin‐7, and Vamp‐2, and the lysosomal marker Lamp‐2 (Upadhyay et al, ).…”