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

Abstract

Myosin VI (MYO6) is an actin-based motor that has been implicated in a wide range of cellular processes, including endocytosis and the regulation of actin dynamics. MYO6 is crucial for actin/membrane remodeling during the final step of Drosophila spermatogenesis, and MYO6-deficient males are sterile. This protein also localizes to actin-rich structures involved in mouse spermiogenesis. Although loss of MYO6 in Snell’s waltzer knock-out (KO) mice causes several defects and shows reduced male fertility, no studies have been published to address the role of MYO6 in sperm development in mouse. Here we demonstrate that MYO6 and some of its binding partners are present at highly specialized actin-based structures, the apical tubulobulbar complexes (TBCs), which mediate endocytosis of the intercellular junctions at the Sertoli cell-spermatid interface, an essential process for sperm release. Using electron and light microscopy and biochemical approaches, we show that MYO6, GIPC1 and TOM1/L2 form a complex in testis and localize predominantly to an early endocytic APPL1-positive compartment of the TBCs that is distinct from EEA1-positive early endosomes. These proteins also associate with the TBC actin-free bulbular region. Finally, our studies using testis from Snell’s waltzer males show that loss of MYO6 causes disruption of the actin cytoskeleton and disorganization of the TBCs and leads to defects in the distribution of the MYO6-positive early APPL1-endosomes. Taken together, we report here for the first time that lack of MYO6 in mouse testis reduces male fertility and disrupts spatial organization of the TBC-related endocytic compartment during the late phase of spermiogenesis.

Loss of myosin VI expression affects acrosome/acroplaxome complex morphology during mouse spermiogenesis†

“…Acrosomes were stained either with Coomassie Brilliant Blue (Sigma-Aldrich) or lectin PNA from peanut Alexa Fluor 488 conjugate (Invitrogen). Normal rabbit IgG (Sigma-Aldrich) was used as a negative control during immunofluorescence and immunogold experiments [ 26 ].…”

“…Moreover, the sv/sv mice display several other defects such as changes in Golgi morphology, reduced secretion, and defective endocytosis and autophagy [ 22–25 ]. Moreover, data from our lab shows that male sv/sv mice have significantly reduced fertility and exhibit disruption of the spatial organization of the apical tubulobulbar complexes during the late stage of spermiogenesis [ 26 ]. Here, we show, by comparing the morphology of developing spermatids of control and MYO6-deficient male mice, that this myosin is present in actin-rich structures during acrosome biogenesis and supports the integrity of these testis-specific structures at this stage of mouse sperm development.…”

Formation of Aberrant Myotubes by Myoblasts Lacking Myosin VI Is Associated with Alterations in the Cytoskeleton Organization, Myoblast Adhesion and Fusion

“…Numerous studies, including ours, showed that either a depletion or lack of MVI affects the organization of the actin cytoskeleton in numerous cell types, including myoblasts [ 13 , 52 ]. Furthermore, MVI was shown to be involved in the differentiation of several tissues, including spermiogenesis [ 21 , 22 ] and neuritogenesis [ 53 , 54 ]. All the authors postulated mechanisms associated with the regulation of the cytoskeleton organization [ 21 , 22 , 53 , 54 ].…”

“…Furthermore, MVI was shown to be involved in the differentiation of several tissues, including spermiogenesis [ 21 , 22 ] and neuritogenesis [ 53 , 54 ]. All the authors postulated mechanisms associated with the regulation of the cytoskeleton organization [ 21 , 22 , 53 , 54 ]. Here, we observed changes in the levels of proteins important for cytoskeleton organization and contractile functions, including α-actinin, desmin and γ-actin, as well as fast and slow myosin heavy chains.…”

“…Mutations within MYO6 lead to hearing impairment in mammals, due to the disintegration of the inner ear hair cell stereocilia [ 18 ]. Snell’s waltzer mice ( SV ) lacking functional MVI also have several other defects, such as cardiac hypertrophy, as well as brain, kidney and spermiogenesis dysfunctions [ 11 , 12 , 19 , 20 , 21 , 22 ]. MVI is involved in endocytosis and the intracellular transport of vesicles and organelles, cell migration, the maintenance of Golgi apparatus, actin cytoskeleton organization, and possibly in gene transcription [ 23 , 24 , 25 , 26 , 27 , 28 ].…”