Rab4A GTPase catenin interactions are involved in cell junction dynamics in the testis.

A plethora of evidence has recently accumulated to suggest that Rab guanosine triphosphates (GTPases) may have functions other than those originally proposed in vesicle formation, movement, docking, and fusion. Studies have shown, for example, that Rab proteins interact with actin filaments and microtubules, illustrating cross-talk between intracellular transport and cytoskeletal dynamics. In this report, we show that Rab4A associates with adherens junction signaling proteins in the testis. By immunoprecipitation, Rab4A was found to interact with alpha- and beta-catenin as well as with actin, vimentin, alpha- and beta-tubulin, and protein kinase C (PKC)-alpha and -epsilon. Additionally, administration of Adjudin to adult rats up-regulated the Rab4A level, which coincided with the loss of spermatocytes, round and elongating/elongated spermatids from the seminiferous epithelium. More importantly, the ability of Rab4A to associate with alpha- and beta-catenin increased during Adjudin-induced junction restructuring in the testis, illustrating that Rab4A-catenin interactions are likely to be involved in the disassembly of Sertoli-germ cell contacts. Taken collectively, these results suggest that Rab4A participates in adherens junction dynamics.

Crosstalk between Rab GTPases and cell junctions.

For the past several years, studies from other laboratories, as well as ours, have begun to unravel the mechanism of germ cell movement in the testis by using several in vitro and in vivo models of tight and adherens junction assembly and disassembly, two cellular phenomena that confer cell movement. However, for cell movement to be fully appreciated, the importance of "intracellular" cell movements, such as those involving actin and microtubule filaments, must be better understood. Recent research on Rab GTPases has shown that members of this superfamily function in the trafficking of vesicles containing cargo to distinct subcellular sites such as the plasma membrane while utilizing actin and microtubule filaments as tracks. In this mini-review, we provide an overview of Rab GTPase structure, function, and regulation, while placing added emphasis on the role of Rabs in cell junction dynamics in the testis.

Rab8B GTPase and junction dynamics in the testis.

Throughout spermatogenesis, germ cells migrate from the basal to the adluminal compartment while remaining attached to Sertoli cells via actin-based adherens and intermediate filament-based anchoring junctions. However, the events that trigger deadhesion and adhesion remain largely unknown. As part of our continued effort in elucidating the mechanism of germ cell movement, we have examined the role of Rab8B, a GTPase probably participating in intracellular trafficking events at the site of the adherens junction. By RT-PCR Rab8B mRNA was found in the brain, testis, heart, kidney, and spleen. Immunohistochemical studies revealed that Rab8B was concentrated predominantly in the basal compartment, localizing to a similar site at which immunoreactive E-cadherin was found. Additional experiments demonstrated that Rab8B associated with the actin, intermediate filament, and microtubule cytoskeletal networks. When Sertoli cells were cultured at high density or germ cells were cocultured with Sertoli cells, Rab8B increased significantly during junction assembly. Moreover, inclusion of germ cell-conditioned medium in Sertoli cell cultures resulted in stimulation of Rab8B expression. Conversely, treatment of adult rats with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide reduced Rab8B mRNA and protein levels, coinciding with the time of germ cell loss from the epithelium. Taken collectively, these studies suggest that Rab8B participates in adherens junction dynamics in the testis.

Role of tissue inhibitor of metalloproteases-1 in junction dynamics in the testis.

Using multiple high-performance liquid chromatography steps, we have identified and purified a polypeptide to apparent homogeneity from primary Sertoli cell conditioned culture medium that consisted of 2 molecular variants of 31 and 29 kDa when electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel run under reducing conditions. Partial N-terminal amino acid sequence analysis of these 2 proteins revealed a sequence of NH(2)-IKMAKMLKGFDAVGNATG, which is homologous to tissue inhibitor of metalloproteases-1 (TIMP-1). Studies by semiquantitative reverse transcription-polymerase chain reaction using a primer pair specific to rat TIMP-1 demonstrated that both Sertoli and germ cells express TIMP-1. During maturation, the steady-state TIMP-1 mRNA level in the testis increased significantly from 40 to 60 days of age, which suggests its role in the restructuring of the epithelium during spermiation. This increase in testicular TIMP-1 expression was apparently not due to the increase in germ cell number, because TIMP-1 expression decreased approximately fivefold in germ cells isolated from testes of aging rats. Using Sertoli cells cultured at low (0.05 x 10(6) cells/cm(2)) and high (0.5 x 10(6) cells/cm(2)) densities, it was found that TIMP-1 expression increased transiently but significantly during junction assembly. A similar induction of TIMP-1 mRNA was also detected in Sertoli-germ cell cocultures during germ cell adhesion onto Sertoli cells. More important, the inclusion of either alpha(2)-macroglobulin (a protease inhibitor produced by Sertoli cells) or aprotinin (a serine protease inhibitor) into an in vitro germ cell adhesion assay facilitated the attachment of fluorescently labeled germ cells onto the Sertoli cell epithelium when compared to control, which suggests that the assembly of adherens junctions may involve protease inhibitors.

RAB13 participates in ectoplasmic specialization dynamics in the rat testis.

During spermatogenesis, leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) to gain entry into the adluminal compartment for further development. At the same time, these as well as other germ cell types in the epithelium must retain their close association with Sertoli cells via specialized cell junctions. In this study, we demonstrate that RAB13-a guanosine triphosphatase (GTPase) known to participate in tight junction function in other epithelia-also participates in the dynamics of the ectoplasmic specialization, a testis-specific type of anchoring junction. By immunohistochemistry microscopy, RAB13 localized to the ectoplasmic specialization. Moreover, RAB13 was found to associate with vinculin (VCL) and espin (ESPN), two putative ectoplasmic specialization actin (ACT)-binding proteins, by coimmunoprecipitation and immunofluorescence microscopy experiments. To address the role of RAB13 in ectoplasmic specialization dynamics, an in vivo model was used in which administration of Adjudin induced the disassembly of Sertoli-germ cell anchoring junctions. Following administration of this drug, the RAB13 level decreased steadily when the loss in testicular weight was taken into account. Similarly, the association of RAB13 with VCL decreased but was not completely lost during Adjudin-mediated ectoplasmic specialization restructuring. Taken collectively, these results suggest that RAB13 functions in ectoplasmic specialization dynamics in the testis.