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

During spermiogenesis in mammals, actin filaments and a variety of actin-binding proteins are involved in the formation and function of highly specialized testis-specific structures. Actin-based motor proteins, such as myosin Va and VIIa, play a key role in this complex process of spermatid transformation into mature sperm. We have previously demonstrated that myosin VI (MYO6) is also expressed in mouse testes. It is present in actin-rich structures important for spermatid development, including one of the earliest events in spermiogenesis-acrosome formation. Here, we demonstrate using immunofluorescence, cytochemical, and ultrastructural approaches that MYO6 is involved in maintaining the structural integrity of these specialized actin-rich structures during acrosome biogenesis in mouse. We show that MYO6 together with its binding partner TOM1/L2 is present at/around the spermatid Golgi complex and the nascent acrosome. Depletion of MYO6 in Snell's waltzer mice causes structural disruptions of the Golgi complex and affects the acrosomal granule positioning within the developing acrosome. In summary, our results suggest that MYO6 plays an anchoring role during the acrosome biogenesis mainly by tethering of different cargo/membranes to highly specialized actin-related structures

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

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 show 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.This project was supported by PRELUDIUM grant from National Science Centre (Poland), the grant number 2017/25/N/NZ3/00487 (to P.Z.); ETIUDA doctoral scholarship from National Science Centre (Poland), the grant number 2018/28/T/NZ3/00002 (to P.Z.); a travelling fellowship funded by The Company of Biologists, the grant number JCSTF-171105 (to P.Z.), and a Medical Research Council grant, grant number MR/K000888/1 (to F.B.

A Kinase Anchoring Protein 9 Is a Novel Myosin VI Binding Partner That Links Myosin VI with the PKA Pathway in Myogenic Cells

Myosin VI (MVI) is a unique motor protein moving towards the minus end of actin filaments unlike other known myosins. Its important role has recently been postulated for striated muscle and myogenic cells. Since MVI functions through interactions of C-terminal globular tail (GT) domain with tissue specific partners, we performed a search for MVI partners in myoblasts and myotubes using affinity chromatography with GST-tagged MVI-GT domain as a bait. A kinase anchoring protein 9 (AKAP9), a regulator of PKA activity, was identified by means of mass spectrometry as a possible MVI interacting partner both in undifferentiated and differentiating myoblasts and in myotubes. Coimmunoprecipitation and proximity ligation assay confirmed that both proteins could interact. MVI and AKAP9 colocalized at Rab5 containing early endosomes. Similarly to MVI, the amount of AKAP9 decreased during myoblast differentiation. However, in MVI-depleted cells, both cAMP and PKA levels were increased and a change in the MVI motor-dependent AKAP9 distribution was observed. Moreover, we found that PKA phosphorylated MVI-GT domain, thus implying functional relevance of MVI-AKAP9 interaction. We postulate that this novel interaction linking MVI with the PKA pathway could be important for targeting AKAP9-PKA complex within cells and/or providing PKA to phosphorylate MVI tail domain

Myosin VI in the nucleolus of neurosecretory PC12 cells: its involvement in the maintenance of nucleolar structure and ribosome organization

We have previously shown that unconventional myosin VI (MVI), a unique actin-based motor protein, shuttles between the cytoplasm and nucleus in neurosecretory PC12 cells in a stimulation-dependent manner and interacts with numerous proteins involved in nuclear processes. Among the identified potential MVI partners was nucleolin, a major nucleolar protein implicated in rRNA processing and ribosome assembly. Several other nucleolar proteins such as fibrillarin, UBF (upstream binding factor), and B23 (also termed nucleophosmin) have been shown to interact with MVI. A bioinformatics tool predicted the presence of the nucleolar localization signal (NoLS) within the MVI globular tail domain, and immunostaining confirmed the presence of MVI within the nucleolus. Depletion of MVI, previously shown to impair PC12 cell proliferation and motility, caused disorganization of the nucleolus and rough endoplasmic reticulum (rER). However, lack of MVI does not affect nucleolar transcription. In light of these data, we propose that MVI is important for nucleolar and ribosome maintenance but not for RNA polymerase 1-related transcription

Myosin VI in PC12 cells plays important roles in cell migration and proliferation but not in catecholamine secretion

Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments and is believed to play distinct role(s) than other myosins. We addressed a role of this unique motor in secretory PC12 cells, derived from rat adrenal medulla pheochromocytoma using cell lines with reduced MVI synthesis (produced by means of siRNA). Decrease of MVI expression caused severe changes in cell size and morphology, and profound defects in actin cytoskeleton organization and Golgi structure. Also, significant inhibition of cell migration as well as cell proliferation was observed. Flow cytometric analysis revealed that MVI-deficient cells were arrested in G0/G1 phase of the cell cycle but did not undergo increased senescence as compared with control cells. Also, neither polyploidy nor aneuploidy were detected. Surprisingly, no significant effect on noradrenaline secretion was observed. These data indicate that in PC12 cells MVI is involved in cell migration and proliferation but is not crucial for stimulation-dependent catecholamine release

Myosins and pathology: genetics and biology.

This article summarizes current knowledge on the genetics and possible molecular mechanisms of human pathologies resulted from mutations within the genes encoding several myosin isoforms. Mutations within the genes encoding some myosin isoforms have been found to be responsible for blindness (myosins III and VIIA), deafness (myosins I, IIA, IIIA, VI, VIIA and XV) and familial hypertrophic cardiomyopathy (β cardiac myosin heavy chain and both the regulatory and essential light chains). Myosin III localizes predominantly to photoreceptor cells and is proved to be engaged in the vision process in Drosophila. In the inner ear, myosin I is postulated to play a role as an adaptive motor in the tip links of stereocilia of hair cells, myosin IIA seems to be responsible for stabilizing the contacts between adjacent inner ear hair cells, myosin VI plays a role as an intracellular motor transporting membrane structures within the hair cells while myosin VIIA most probably participates in forming links between neighbouring stereocilia and myosin XV probably stabilizes the stereocilia structure. About 30% of patients with familial hypertrophic cardiomyopathy have mutations within the genes encoding the β cardiac myosin heavy chain and both light chains that are grouped within the regions of myosin head crucial for its functions. The alterations lead to the destabilization of sarcomeres and to a decrease of the myosin ATPase activity and its ability to move actin filaments

Posttranslational modifications of actin

Aktyna, komponent cytoszkieletu komórek eukariotycznych, to jedno z białek najistotniejszych dla funkcjonowania organizmów i najlepiej zachowanych w toku ewolucji. Ta globularna cząsteczka o masie cząsteczkowej około 42,3 kDa występuje zarówno w formie monomerycznej, jak i spolimeryzowanej (filamenty), a zdolność do dynamicznej reorganizacji aktyny jest niezbędna dla życia komórki. Przejście pomiędzy obiema formami jest możliwe dzięki precyzyjnej w czasie i przestrzeni, dynamicznej regulacji organizacji aktyny przez szereg białek wiążących się zarówno z monomerami, jak i filamentami aktyny. Istotnym czynnikiem wpływającym na stopień spolimeryzowania aktyny są także liczne modyfikacje potranslacyjne tego białka. Niniejszy artykuł przeglądowy jest poświęcony omówieniu tego obszernego i wciąż mało poznanego zagadnienia, a w szczególności opisowi jakim modyfikacjom ulega aktyna i w jaki sposób modyfikacje te wpływają na strukturę i funkcje tego wyjątkowego białka.Actin, a constituent of the cytoskeleton of eukaryotic cells, is one of the most important as well as best evolutionary conserved proteins. This globular protein with molecular mass of ~42.3 kDa exists in the cell both in the monomeric and filamentous form, and ability to undergo dynamic reorganization of these two forms is absolutely crucial for cell survival. The monomer-filament transition, precisely controlled in time and space, is possible due to interaction of actin with a panoply of proteins binding to either monomeric or filamentous actin. Yet another factor is affecting actin organization, namely numerous posttranslational modifications. This review article is devoted to presentation of this broad and still unrecognized topic with emphasis on description of the type of actin modifications and how they affect actin structure and function

Studies on the cytoskeleton, muscle contraction and cell motility at the Nencki Institutue: historical view

W 2018 roku Instytut Biologii Doświadczalnej im. M. Nenckiego w Warszawie obchodzi 100. rocznicę istnienia, a artykuł ten jest częścią specjalnego numeru poświęconego wiekowi badań przeprowadzonych w Instytucie w zakresie cytoszkieletu, ruchliwości komórek i skurczu mięśni. Artykuł opisuje historię badań oraz grupy zajmujące się tymi zagadnieniami przez dziesięciolecia. Ponadto, prezentuje wieloletni wkład naukowców z Instytutu Nenckiego w środowisko naukowe w Polsce i za granicą.In 2018 the Nencki Institute of Experimental Biology in Warsaw, Poland, celebrates the 100th Aniversary of its setting-up and this article is a part of the special issue devoted to the century of a research conducted in the Institute related to the cytoskeleton, cell motility and muscle contraction. The article describes the history of the research, and groups working on the motile and contractile processes through the decades. Also, it presents a long-standing contribution of the Nencki Institute researchers to the scientific community in Poland and abroad