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 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.

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.

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.

The protozoan parasite Toxoplasma gondii is a representative of apicomplexan parasites that invades host cells through an unconventional motility mechanism. During host cell invasion it forms a specialized membrane-surrounded compartment that is called the parasitophorous vacuole. The interactions between the host cell and parasite membranes are complex and recent studies have revealed in more detail that both the host cell and the parasite membrane contribute to the formation of the parasitophorous vacuole. By using our a new specimen preparation technique that allows three-dimensional imaging of thick-sectioned internal cell structures with high-resolution, low-voltage field emission scanning electron microscopy, we were able to visualize continuous structural interactions of the host cell membrane with the parasite within the parasitophorous vacuole. Fibrous and tubular material extends from the host cell membrane and is connected to parasite membrane components. Shorter protrusions are also elaborated from the parasite. Several of these shorter fine protrusions connect to the fibrous material of the host cell membrane. The elaborate network may be used for modifications of the parasitophorous vacuole membrane that will allow utilization of nutrients from the host cell by the parisite while it is being protected from host cell attacks. The structural interactions between parasite and host cells undergo time-dependent changes, and a fission pore is the most prominent structure left connecting the parasite with the host cell. The fission pore is anchored in the host cell by thick structural components of unknown nature. The new information gained with this technique includes structural details of fibrous and tubular material that is continuous between the parasite and host cell and can be imaged in three dimensions. We present this technique as a tool to investigate more fully the complex structural interactions of the host cell and the parasite residing in the parasitophorous vacuole.