Synaptotagmin 1 (Syt1) is an abundant and important presynaptic vesicle protein that binds Ca2+ for the regulation of synaptic vesicle exocytosis. Our previous study reported its localization and function on spindle assembly in mouse oocyte meiotic maturation. The present study was designed to investigate the function of Syt1 during mouse oocyte activation and subsequent cortical granule exocytosis (CGE) using confocal microscopy, morpholinol-based knockdown and time-lapse live cell imaging. By employing live cell imaging, we first studied the dynamic process of CGE and calculated the time interval between [Ca2+]i rise and CGE after oocyte activation. We further showed that Syt1 was co-localized to cortical granules (CGs) at the oocyte cortex. After oocyte activation with SrCl2, the Syt1 distribution pattern was altered significantly, similar to the changes seen for the CGs. Knockdown of Syt1 inhibited [Ca2+]i oscillations, disrupted the F-actin distribution pattern and delayed the time of cortical reaction. In summary, as a synaptic vesicle protein and calcium sensor for exocytosis, Syt1 acts as an essential regulator in mouse oocyte activation events including the generation of Ca2+ signals and CGE.

Clathrin heavy chain 1 (CLTC) has been considered a “moonlighting protein” which acts in membrane trafficking during interphase and in stabilizing spindle fibers during mitosis. However, its roles in meiosis, especially in mammalian oocyte maturation, remain unclear. This study investigated CLTC expression and function in spindle formation and chromosome congression during mouse oocyte meiotic maturation. Our results showed that the expression level of CLTC increased after germinal vesicle breakdown (GVBD) and peaked in the M phase. Immunostaining results showed CLTC distribution throughout the cytoplasm in a cell cycle-dependent manner. Appearance and disappearance of CLTC along with β-tubulin (TUBB) could be observed during spindle dynamic changes. To explore the relationship between CLTC and microtubule dynamics, oocytes at metaphase were treated with taxol or nocodazole. CLTC colocalized with TUBB at the enlarged spindle and with cytoplasmic asters after taxol treatment; it disassembled and distributed into the cytoplasm along with TUBB after nocodazole treatment. Disruption of CLTC function using stealth siRNA caused a decreased first polar body extrusion rate and extensive spindle formation and chromosome congression defects. Taken together, these results show that CLTC plays an important role in spindle assembly and chromosome congression through a microtubule correlation mechanism during mouse oocyte maturation.

The prospect of utilizing stem cells for clinical applications has generated an enormous amount of enthusiasm in the stem cell research community and has led to a wealth of new data that offer the possibility for practical applications into clinical translation. Recent advances in stem cell imaging has contributed greatly to the ultimate goal to identify and culture specific cell types for regeneration of tissue that had been affected by disease such as Parkinson's, Alzheimer's, heart disease, muscle diseases, and others.

The role of centrosomes in stem cell division has recently been highlighted and further ascribes important functions to centrosomes in stem cell maintenance, cellular differentiation, and development. Advanced cell and molecular studies coupled with immunofluorescence, electron microscopy, and live cell imaging of specific centrosome proteins have contributed greatly to our knowledge of centrosome composition, structure, and dynamics and have uncovered new insights into mechanisms of centrosome functions in asymmetric cell division. The establishment of asymmetry and differential positioning of mother and daughter centrosomes during stem cell mitosis is important for allowing one cell to maintain stem cell characteristics while the sibling cell undergoes differentiation. Another key role for centrosomes has been revealed in primary cilia of embryonic stem cells that play significant roles in cellular signaling and are therefore critically important for stem cell decisions. Studies of signaling through primary cilia may contribute important information that may aid in the production of specific cells that are suitable for tissue repair and regeneration in the field of regenerative medicine.

Two-photon excitation microscopy (also referred to as multiphoton laser scanning microscopy) has gained increasing popularity during the past few years because of the distinct advantages over single-photon microscopy, which includes increased penetration depth and low out-of-focus photobleaching and photodamage. It allows superior imaging of thick specimen compared to single-photon microscopy, and it excels at imaging live cells either single or within intact tissue. This highly valuable tool has been used with great success to gain important new insights into brain tissue, embryos, whole organs, entire animals, and it has been most useful in numerous other applications.

We previously reported that translocation of mitochondria from the oocyte cortex to the perinuclear area indicates positive developmental potential that was reduced in porcine somatic cell nuclear transfer (SCNT) embryos compared to in vitro–fertilized (IVF) embryos (Katayama, M., Zhong, Z.-S., Lai, L., Sutovsky, P., Prather, R.S. & Schatten, H. (2006). Dev Biol299, 206–220.). The present study is focused on distribution of donor cell mitochondria in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using either pig fibroblasts with mitochondria-stained MitoTracker CMXRos or YFP-mitochondria 3T3 cells (pPhi-Yellow-mito) as donor cells. Transmission electron microscopy was employed for ultrastructural analysis of pig oocyte and donor cell mitochondria. Our results revealed donor cell mitochondrial clusters around the donor nucleus that gradually dispersed into the ooplasm at 3 h after SCNT. Donor-derived mitochondria distributed into daughter blastomeres equally (82.8%) or unequally (17.2%) at first cleavage. Mitochondrial morphology was clearly different between donor cells and oocytes in which various complex shapes and configurations were seen. These data indicate that (1) unequal donor cell mitochondria distribution is observed in 17.2% of embryos, which may negatively influence development; and (2) complex mitochondrial morphologies are observed in IVF and SCNT embryos, which may influence mitochondrial translocation and affect development.

Spindle movement, including spindle migration during first meiosis and spindle rotation during second meiosis, is essential for asymmetric divisions in mouse oocytes. Previous studies by others and us have shown that microfilaments are required for both spindle migration and rotation. In the present study, we aimed to further investigate the mechanism controlling spindle movement during mouse oocyte meiosis. By employing drug treatment and immunofluorescence microscopy, we showed that dynamic microtubule assembly was involved in both spindle migration and rotation. Furthermore, we found that the calcium/CaM/CaMKII pathway was important for regulating spindle rotation.

Increasingly, genetically modified Salmonella are being explored as a novel treatment for cancer because Salmonella preferentially replicate within tumors and destroy cancer cells without causing the septic shock that is typically associated with wild-type S. typhimurium infections. However, the mechanisms by which genetically modified Salmonella strains preferentially invade cancer cells have not yet been addressed in cellular detail. Here we present data that show S. typhimurium strains VNP20009, LT2, and CRC1674 invasion of PC-3M prostate cancer cells. S. typhimurium-infected PC-3M human prostate cancer cells were analyzed with immunofluorescence microscopy and transmission electron microscopy (TEM) at various times after inoculation. We analyzed microfilaments, microtubules, and DNA with fluorescence and immunofluorescence microscopy. 3T3 Phi-Yellow-mitochondria mouse 3T3 cells were used to study the effects of Salmonella infestation on mitochondria distribution in live cells. Our TEM results show gradual destruction of mitochondria within the PC-3M prostate cancer cells with complete loss of cristae at 8 h after inoculation. The fluorescence intensity in YFP-mitochondria-transfected mouse 3T3 cells decreased, which indicates loss of mitochondria structure. Interestingly, the nucleus does not appear affected by Salmonella within 8 h. Our data demonstrate that genetically modified S. typhimurium destroy PC-3M prostate cancer cells, perhaps by preferential destruction of mitochondria.