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.

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

Actins and myosins in the nucleus

Aktyna i miozyna to białka kojarzone przede wszystkim z ich kluczową rolą w generacji skurczu mięśni. Natomiast poza izoformami charakterystycznymi dla mięśni są również izoformy aktyny i miozyny, które występują we wszystkich typach komórek i tkanek (patrz artykuł Suszek i współaut. w tym zeszycie KOSMOSU). Badania prowadzone w ostatnich dwóch dekadach wykazały niezbicie, że zarówno aktyna (i szereg białek wiążących aktynę) oraz liczne miozyny (przedstawiciele rodzin I, II, V, VI, XVI i XVIII) lokalizują się w jądrze komórkowym gdzie są zaangażowane w procesy transkrypcji i naprawy DNA, transport w nukleoplazmie oraz import i eksport jądrowy, a także w utrzymywanie architektury jądra. Niniejszy artykuł opisuje dotychczasowy stan wiedzy o roli układu akto-miozynowego w jądrze komórkowym.Actin and myosins are the proteins mainly known from their key roles in muscle contraction. However, besides typical muscle isoforms there are actins and myosins that are present in all cell and tissue types. Studies performed within the last two decades have irrefutably shown that both the cytoplasmic actin isoforms (along with numerous actin-binding proteins) as well as many myosins (representing class I, II, V, VI, XVI and XVIII) are present within the nucleus. They play important roles in nuclear processes as they are involved in transcription and DNA repair, intranuclear transport as well as nuclear import and export, and in maintenance of nuclear architecture. This article describes the current knowledge on the acto-myosin system in this biggest cellular compartment

Unconventional myosins and their functions in striated muscles and myogenic cells

Miozyny to oddziałujące z aktyną białka motoryczne, zaangażowane w skurcz mięśni, migrację komórek i transport wewnątrzkomórkowy. Występują one we wszystkich organizmach eukariotycznych, w tym w pierwotniakach i roślinach. Miozyny zbudowane są z jednego lub dwóch łańcuchów ciężkich oraz kilku łańcuchów lekkich (1-7 na łańcuch ciężki). Zidentyfikowano kilka tysięcy sekwencji łańcuchów ciężkich miozyn występujących w kilkuset gatunkach. W łańcuchach ciężkich miozyn wyróżniono główkę obejmującą domenę motoryczną (miejsce oddziaływania z aktyną i wiązania ATP) i szyjkę z motywami IQ (miejsce niekowalencyjnego wiązania się z łańcuchami lekkimi) oraz ogonek (zawierający domeny warunkujące specyficzne funkcje poszczególnych izoform miozyny). Na podstawie różnic w sekwencji aminokwasowej domeny motorycznej wyróżniono ponad trzydzieści rodzin w nadrodzinie miozyn, z czego 12, reprezentowanych przez 40 izoform, występuje u człowieka. Miozyny mięśniowe (tworzące rodzinę II) zwane są konwencjonalnymi, a pozostałe - niekonwencjonalnymi. Niniejszy artykuł opisuje nadrodzinę miozyn, a w szczególności budowę i funkcje tych miozyn niekonwencjonalnych, które są obecne w komórkach miogennych i mięśniach poprzecznie-prążkowanych.Myosins, actin-dependent molecular motors, are engaged in muscle contraction, cell migration and intracellular transport. They are present in all eukaryotic organisms including protists and plants. They are composed of one or two heavy chains, and a number of light chains (1-7 per a heavy chain). Several thousands of myosin heavy chains have been sequenced in hundreds of species. The heavy chain is composed of a motor domain (with actin and ATP binding sites), a neck with IQ motifs (where light chains bind to) and a tail (with domains determining specific functions of a given myosin). A myosin superfamily is divided into over 30 families based on differences in the motor domain primary sequence. Twelve families represented by 40 isoforms are expressed in humans. Well known muscle myosins forming a family II are termed as conventional while all others are termed as unconventional. The article describes the myosin superfamily with emphasis on structure and function of unconventional myosins present in myogenic cells and striated muscles

Polyphenolic Compounds of Crataegus Berry, Leaf, and Flower Extracts Affect Viability and Invasive Potential of Human Glioblastoma Cells

Crataegus contains numerous health-promoting compounds that are also proposed to have anti-cancer properties. Herein, we aimed at a contemporaneous evaluation of the effects of polyphenol-rich extracts of berries, leaves, and flowers of six Crataegus species on the viability and invasive potential on the highly aggressive human glioblastoma U87MG cell line. The treatment with the extracts evoked cytotoxic effects, with the strongest in the berry extracts. All extracts not only promoted the apoptosis-related cleavage of poly (ADP-ribose) polymerase 1 (PARP1) but also substantially inhibited the activity of pro-survival kinases, focal adhesion kinase (FAK), and protein kinase B (PKB; also known as Akt), thus indicating the suppression of proliferative and invasive potentials of the examined glioblastoma cells. The qualitative and quantitative characterization of the extracts’ content was also performed and revealed that amongst 37 polyphenolic compounds identified in the examined Crataegus extracts, the majority (29) was detected in berries; the leaf and flower extracts, exerting milder cytotoxic effects, contained only 14 and 13 compounds, respectively. The highest polyphenol content was found in the berries of C. laevigata x rhipidophylla x monogyna, in which flavan-3-ols and phenolic acids predominated. Our results demonstrated that a high content of polyphenolic compounds correlated with the extract cytotoxicity, and especially berries were a valuable source of compounds with anti-cancer potential. This might be a promising option for the development of an effective therapeutic strategy against highly malignant glioblastomas in the future