Distribution Of Golgi Apparatus Research Articles

Loss of AMPK activity induces organelle dysfunction and oxidative stress during oocyte aging

BackgroundOocyte quality is critical for the mammalian reproduction due to its necessity on fertilization and early development. During aging, the declined oocytes showing with organelle dysfunction and oxidative stress lead to infertility. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase which is important for energy homeostasis for metabolism. Little is known about the potential relationship between AMPK with oocyte aging.ResultsIn present study we reported that AMPK was related with low quality of oocytes under post ovulatory aging and the potential mechanism. We showed the altered AMPK level during aging and inhibition of AMPK activity induced mouse oocyte maturation defect. Further analysis indicated that similar with its upstream regulator PKD1, AMPK could reduce ROS level to avoid oxidative stress in oocytes, and this might be due to its regulation on mitochondria function, since loss of AMPK activity induced abnormal distribution, reduced ATP production and mtDNA copy number of mitochondria. Besides, we also found that the ER and Golgi apparatus distribution was aberrant after AMPK inhibition, and enhanced lysosome function was also observed.ConclusionsTaken together, these data indicated that AMPK is important for the organelle function to reduce oxidative stress during oocyte meiotic maturation.

The role of cell division cycle 42 in root development of tooth and the involved mechanisms

Objective: To explore the effect of cell division cycle 42 (CDC42) on root development and its regulation on cell proliferation and migration in Hertwig's epithelial root sheath (HERS). Methods: Trace the spatiotemporal expression of CDC42 in root development process [postnatal day 5 (P5), P7, P14] through immunofluorescence staining. Nine eight-week-old C57BL/6J male mice were randomly divided into 3 groups using a simple random sample method (n=3 in each group). P3 tooth germ was cultured in air-liquid system for 1 day and then transplanted to renal capsule each to observe tooth root development. The control group implanted tooth germ only. The phosphate buffered saline (PBS) group implanted tooth germ and gel beads soaked with PBS, while the ML141 group implanted tooth germ and gel beads soaked with CDC42 inhibitor (ML141). Cdc42 in HERS cells was inhibited via lentivirus transfection. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and scratch assay were performed. The distribution of Golgi apparatus (GM130) and cytoskeleton (F-actin) in migrated cells were mapped via immunofluorescence staining. Results: CDC42 was expressed in epithelial cells of HERS, polarized ameloblasts and odontoblasts, as well as adjacent dental papilla and dental follicle cells during tooth root development. The root length of the ML141 group [(0.61±0.09) mm] was substantially shorter than that of control group [(1.03±0.19) mm, P=0.007] and PBS group [(0.98±0.10) mm, P=0.021] according to the data of renal capsule transplantation. After lentiviral transfection, the relative expression of Cdc42 in knockdown group (0.31±0.33) was significantly lower than that in control group (1.05±0.08) (t=15.38, P<0.001), demonstrating the knockdown efficiency closed to 70%. Cell viabilities were significantly inhibited in knockdown group (0.87±0.04, 0.96±0.10, 0.59±0.06, respectively) compared with those in control group (1.09±0.13, 1.55±0.32, 1.10±0.09, respectively) after 3, 4 and 5 days (t=3.16, P=0.016; t=4.23, P=0.002; t=5.08, P<0.01), and the cell proliferation ability in knockdown group [(1.65±0.64)%] also decreased than that in the control group [(4.02±1.12)%](t=5.21, P<0.001). In addition, the cell migration rates after 24 and 48 h [(45.1±4.2)%, (56.4±8.3)%] in knockdown group were obviously lower than those in the control group [(63.8±7.4)%, (80.2±7.8)%] (t=3.78, P=0.019; t=3.62, P=0.023). After Cdc42 was knocked down, Golgi apparatus distributed along the nucleus while behaved oriented in the control group. Conclusions: CDC42 plays an important role in the regulation of root length during root development, which may mediate root elongation by affecting the migration and proliferation of HERS cells.

Glyphosate exposure deteriorates oocyte meiotic maturation via induction of organelle dysfunctions in pigs

BackgroundRecently, defects in mammalian oocytes maturation induced by environmental pollution results in the decreasing animal reproduction. Animal exposed to glyphosate is largely unavoidable because glyphosate is one of the most widely used herbicide worldwide due to its high-efficiency and broad-spectrum effects, which causes glyphosate an environmental contaminant found in soil, water and food. During the last few years, the growing and wider use of glyphosate has raised great concerns about its effects of reproductive toxicity. In this study, using porcine models, we investigated effects of glyphosate on organelle functions during oocyte meiosis.ResultsThe results showed glyphosate exposure disrupted porcine oocyte maturation. Expression levels of cumulus expansion-related genes were interfered, further indicating the meiotic defects. The damaging effects were mediated by destruction of mitochondrial distribution and functions, which induced ROS accumulation and oxidative stress, also indicated by the decreased mRNA expression of related antioxidant enzyme genes. We also found an interference of endoplasmic reticulum (ER) distribution, disturbance of Ca2+ homeostasis, as well as fluctuation of ER stress, showing with the reduced ER stress-related mRNA or protein expression, which could indicate the dysfunction of ER for protein processing and signal transduction in glyphosate-exposed oocytes. Moreover, glyphosate exposure induced the disruption of lysosome function for autophagy, showing with the decrease of LAMP2 expression and autophagy-related genes mRNA expression. Additionally, our data showed the distribution of Golgi apparatus and the functions of ribosome were disturbed after glyphosate exposure, which might affect protein synthesis and transport.ConclusionsCollectively, our study showed that exposed to glyphosate could affect animal reproduction by compromising the quality of oocytes through its wide toxic effects on organelle functions.

Ral GTPase is essential for actin dynamics and Golgi apparatus distribution in mouse oocyte maturation

BackgroundRal family is a member of Ras-like GTPase superfamily, which includes RalA and RalB. RalA/B play important roles in many cell biological functions, including cytoskeleton dynamics, cell division, membrane transport, gene expression and signal transduction. However, whether RalA/B involve into the mammalian oocyte meiosis is still unclear. This study aimed to explore the roles of RalA/B during mouse oocyte maturation.ResultsOur results showed that RalA/B expressed at all stages of oocyte maturation, and they were enriched at the spindle periphery area after meiosis resumption. The injection of RalA/B siRNAs into the oocytes significantly disturbed the polar body extrusion, indicating the essential roles of RalA/B for oocyte maturation. We observed that in the RalA/B knockdown oocytes the actin filament fluorescence intensity was significantly increased at the both cortex and cytoplasm, and the chromosomes were failed to locate near the cortex, indicating that RalA/B regulate actin dynamics for spindle migration in mouse oocytes. Moreover, we also found that the Golgi apparatus distribution at the spindle periphery was disturbed after RalA/B depletion.ConclusionsIn summary, our results indicated that RalA/B affect actin dynamics for chromosome positioning and Golgi apparatus distribution in mouse oocytes.

Podophyllotoxin Exposure Affects Organelle Distribution and Functions in Mouse Oocyte Meiosis.

Podophyllotoxin (POD) is one of the most characterized lignans that is commonly found in podophyllum, and its preparations and derivatives are widely used in clinical treatment due to strong antitumor and antivirus activities. POD has been reported for its neurotoxicity, liver toxicity, and potential reproductive toxicity. In the present study, we investigated the effects of POD on the organelles of mouse oocytes during meiosis. Our results showed that exposure to POD significantly reduced the developmental competence of mouse oocytes. Further analysis revealed that the endoplasmic reticulum (ER) failed to accumulate to the spindle periphery, suggesting that POD exposure might affect protein synthesis during oocyte meiotic maturation. Similarly, abnormal Golgi apparatus distribution was found after POD exposure, which could be confirmed by the aberrant localization of Rab11a-related vesicles, indicating that POD induced vesicle-based protein transport disorder. We also found the aberrant accumulation of lysosomes in the cytoplasm of POD-exposed oocytes, which implied that POD might lead to aberrant protein degradation. Moreover, the perinuclear distribution of mitochondria was also significantly disturbed, indicating the mitochondrial dysfunction after POD exposure. In all, our study illustrated that exposure to POD might disrupt protein synthesis, transport, degradation, and ATP production by its effects on the distribution and functions of organelles during mouse oocyte meiotic maturation.

RAB8A GTPase regulates spindle migration and Golgi apparatus distribution via ROCK-mediated actin assembly in mouse oocyte meiosis†.

Actin filaments are widely involved in multiple cellular processes in oocyte meiosis, such as spindle migration and polar body extrusion. The actin nucleators like Arp2/3 complex and formins are the most recognized molecules for actin assembly in oocytes. In the present study, we report that the vesicle trafficking factor, RAB8A GTPase, is a new regulator critical for actin assembly in meiosis. Our results showed that RAB8A was localized at both the spindle periphery and cortex in mouse oocytes, which was similar to the localization patterns of actin filaments. RAB8A depletion caused spindle migration defects and the failure of polar body extrusion, which could have been due to decreases in both cytoplasmic and cortical actin filaments in oocytes. Based on mass spectrometry analysis, we showed that RAB8A promoted actin assembly through its modulation on the ROCK-LIMK signaling pathway. Moreover, we demonstrated that RAB8A colocalized and interacted with GM130 at the spindle periphery and that RAB8A depletion caused the disruption of GM130-docked Golgi distribution. Taken together, our data indicated that RAB8A was required for Golgi distribution, spindle migration, and polar body extrusion via ROCK-mediated actin assembly in mouse oocyte meiosis.

The role of Golgi reassembly and stacking protein 65 phosphorylation in H2O2-induced cell death and Golgi morphological changes

This study aimed to investigate the effects of H2O2-induced oxidative stress on cell viability and survival, as well as changes in the distribution of Golgi apparatus and in the level of Golgi reassembly and stacking protein 65 (GRASP65). Cell viability of cultured N2a cells treated with H2O2 was measured by the MTT assay. Apoptosis was measured by flow cytometry analyses. Cells labeled by indirect immunofluorescence were observed under confocal microscope to detect any Golgi morphological alterations; electron microscopy of Golgi apparatus was also done. Expression of GRASP65 and phospho-GRASP65 was examined by immunoblotting. H2O2 treatment reduced the cell viability and raised the cell mortality of N2a cells in a time-dependent manner. Notable changes were only observed in the distribution and morphology of Golgi apparatus at 6h after H2O2 treatment. The expression of GRASP65 showed no significant changes at different time points; the phosphorylated GRASP65 level was significantly increased after H2O2 treatment, peaked at 3h, and finally dropped at 6h. Taken together, GRASP65 phosphorylation may have a critical role in inducing cell death at the early stage after H2O2 treatment, while its role in H2O2-induced Golgi morphological changes may be complex.

Maturation of the Golgi apparatus in urothelial cells

The differentiation of urothelial cells is characterized by the synthesis of uroplakins and their assembly into the asymmetric unit membrane. The Golgi apparatus (GA) has been proposed to play a central role in asymmetric unit membrane formation. We have studied the distribution and organization of the GA in normal mouse urothelial cells and in the superficial urothelial cells that undergo differentiation following cyclophosphamide-induced regeneration, in correlation with urothelial cell differentiation. In normal urothelium, immature basal cells have a simple GA, which is small and distributed close to the nucleus. In intermediate cells, the GA starts to expand into the cytoplasm, whereas the GA of terminally differentiated umbrella cells is complex, being large and spread over the whole basal half of the cytoplasm. During early stages of regeneration after cyclophosphamide treatment, the GA of superficial cells is simple and no markers of urothelial differentiation (uroplakins or asymmetric unit membranes, discoidal or fusiform vesicles, apical surface covered with microvilli) are expressed. At a later stage, the GA expands and, in the final stage of regeneration, when cells express all markers of terminal urothelial differentiation, the GA become complex once again. Our results show that: (1) GA distribution and organization in urothelial cells is differentiation-dependent; (2) the GA matures from a simple form in partially differentiated cells to a complex form in terminally differentiated superficial cells; (3) major rearrangements of GA distribution and organization correlate with the beginning of asymmetric unit membrane production. Thus, GA maturation seems to be crucial for asymmetric unit membrane formation.

WAVE1, an A-kinase anchoring protein, during mammalian spermatogenesis.

Proper compartmentalization of signalling cascades is paramount to many intracellular activities during spermatogenesis and sperm function. In the present study we focus on the A-kinase-anchoring protein (AKAP) WAVE1, a member of the Wiskott-Aldrich syndrome (WASP) family of adaptor proteins, to study its localization throughout mammalian spermatogenesis. Using transmission electron microscopy, immunocytochemistry and western blotting, we examined the distribution of WAVE1 and putative partners during mammalian spermatogenesis. The localization and association of PKA RII, the regulatory subunit II of protein kinase A, tyrosine kinase Abl, and small GTPase RAC1 were also explored. WAVE1 localization in spermatocytes and round spermatids coincided with Golgi apparatus distribution, whereas in elongated spermatids and testicular sperm WAVE1 localized to the mitochondrial sheath. Following epididymal passage, WAVE1 was found exclusively on the mitochondrial sheath, suggesting that the protein may function in this region. WAVE1 and PKA RII co-localized along the mitochondrial sheath, PKA RII concentrates in the mid-piece, and RAC1 associated with the post-acrosomal region and the connecting piece. The distribution of WAVE1, PKA RII and RAC1 is conserved in mature mouse, bull, baboon and human sperm. The data support the possibility of a functional signalling unit established by WAVE1 and its associated proteins in the mid-piece of maturing sperm.

Golgi 58K-like protein in pollens and pollen tubes ofLilium davidii

In animal cells, Golgi apparatus is located near the microtubule organizing center (MTOC) and its position is determined partly by 58K protein. By sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting methods, a 58K-like protein has been found in pollen grains and pollen tubes of Lilium davidii. Its molecular weight is very similar to that of the 58K protein of animal cells. By immunofluorescence labeling, under a confocal laser scanning microscope (CLSM), the animal 58K antibody revealed a punctate staining in pollen grains and pollen tubes, which is consistent with the distribution of Golgi apparatus in plant cells. In addition, immuno-gold labeling and transmission electron microscopy showed that the 58K-like protein bound mainly to the membrane of vesicles-like structure near Golgi apparatus. This is the first demonstration of the 58K-like protein in plant cells.

Golgi apparatus in chick skeletal muscle: changes in its distribution during end plate development and after denervation.

In the course of studies about the cellular and molecular mechanisms of motor end plate formation, the distribution of the Golgi apparatus (GA) has been investigated by immunofluorescence methods in chick skeletal muscle in primary culture and in innervated muscles of 15-day-old chicks. By using a monoclonal antibody directed against the GA, we confirmed the known distribution of the GA in myogenic cells: a juxtanuclear polarized organization in myoblasts and a perinuclear nonpolarized distribution in myotubes. In contrast, the innervated anterior latissimus dorsi muscle of "young adult" chicks displayed a focal distribution of GA that appeared restricted to areas located underneath the motor end plates identified by alpha-bungarotoxin fluorescent labeling of the acetylcholine receptor. Five days after denervation of anterior latissimus dorsi muscle, a striking reorganization and expansion of the GA was observed. The GA now showed a perinuclear distribution in close association with every nucleus of the muscle fibers as observed in myotubes. The focal distribution of the GA in innervated muscle fibers and its remodeling upon denervation are interpreted in terms of a model of local synthesis, processing, and routing of acetylcholine receptor to the end plate and of regulation of these processes by functional motor innervation.

The microtubule-organizing complex and the Golgi apparatus are co-localized around the entire nuclear envelope of interphase cardiac myocytes.

In most animal cells, the microtubule-organizing centre (MTOC) and the Golgi apparatus (GA) are co-localized on one side of the nucleus, an arrangement that allows these cells to acquire a functional polarity. An exception has been reported in the skeletal muscle myotube, where the MTOC and GA exhibit a circumnuclear distribution. We wished to determine if this unusual distribution of the MTOC and GA was peculiar to syncytial myotubes or reflected a pattern found in muscle cells generally. Immunofluorescence microscopic studies of cultured chicken skeletal muscle, cardiac muscle and gizzard smooth muscle cells were carried out using preimmune sera that recognized the pericentriolar material, anti-tubulin antibodies to label the MTOC, and fluorescent wheat-germ agglutinin to label the GA. These studies have shown that cardiac myocytes possess a circumnuclear distribution of their MTOC and GA as do skeletal myotubes, but smooth muscle cells exhibit the centrosomal MTOC and GA distribution found in most other cells. The circumnuclear MTOC/GA distribution therefore is associated with striated muscle cells. We also found that as embryonic cardiac myocytes pass through the cell cycle the microtubule-organizing activity in these cells switches from a circumnuclear distribution in interphase to the conventional centrosomal location during mitosis. Thus, cardiac myocytes provide a rare example of mononucleated animal cells that do not display a centrosomal MTOC or a polarized GA, and also reveal a system in which the MTOC structure can be reversibly altered in a cell cycle-dependent manner.