mTOR is required for asymmetric division through small GTPases in mouse oocytes.

Mammalian target of rapamycin (mTOR) is central to the control of cell proliferation, growth, and survival in mammalian cells. Prolonged treatment with rapamycin inhibits mTOR complex 2 (mTORC2) activity, and both the mTORC1-mediated S6K1 and 4E-BP1/eIF4E pathways are essential for TORC2-mediated RhoA, Cdc42, and Rac1 expression during cell motility and F-actin reorganization. The functions of mTOR in the mouse oocyte remain unclear, however. The present study shows that rapamycin affects mTOR expression and cytoskeleton reorganization during meiotic maturation of mouse oocytes. mTOR mRNA was expressed in germinal vesicles (GV) until metaphase I (MI), and increased during metaphase II (MII). Immunostaining showed that mTOR localized around the spindle and in the cytoplasm of oocytes. Treatment of oocytes with rapamycin decreased mTOR at the RNA and protein level, and altered asymmetric division. Formation of the actin cap and the cortical granule-free domain were also disrupted after rapamycin treatment, indicating the failure of spindle migration. Injection of an anti-mTOR antibody yielded results consistent with those obtained for rapamycin treatment, further confirming the involvement of mTOR in oocyte polarity. Furthermore, rapamycin treatment reduced the mRNA expression of small GTPases (RhoA, Cdc42, and Rac1), which are crucial regulatory factors for cytoskeleton reorganization. Taken together, these results suggest that rapamycin inhibits spindle migration and asymmetric division during mouse oocyte maturation via mTOR-mediated small GTPase signaling pathways.

Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: eukaryotic clones containing two and three ORF multi-gene cassettes expressed from a single promoter.

Two types of eukaryotic operon-type Expression clones were constructed using the Multisite Gateway system employing six types of att signals. These clones harbored a DNA cassette containing two heterologous ORFs (cDNAs) or three heterologous ORFs in tandem downstream of a single promoter. The most promoter-proximal ORF was translated via a Kozak signal and the downstream one or two ORF(s) were translated as directed by internal ribosome entry site(s) (IRES). These clones were observed to produce two or three different proteins at levels that depended on the activities of the translational initiation signals used. With the intention of modulating the expression level of the first ORF, the translational initiation signals including a Kozak sequence and 11 different IRESs were investigated for their efficiency using a single ORF. The translational activity of these signals varied within a 10-fold magnitude. Using these results, expression at pre-described relative levels was achieved from the optional IRES of the respective ORFs in the cassette. Controllable expression at desired levels of two different ORFs directed by optional IRESs on a bicistronic construct, transcribed from a single promoter, was demonstrated.

[Primary study on proteomics about Ganoderma lucidium spores promoting survival and axon regeneration of injured spinal motor neurons in rats].

OBJECTIVE: To detect some proteins associated with the effect of ganoderma lucidium spores (GASP) on promoting the survival and axon regeneration of injured spinal motor neurons in rats. METHODS: The rats were divided into normal control group, untreated group and GASP-treated group, and the rats in the last two groups received ventral root avulsion. GASP preparation was fed to the rats in the GASP-treated group for 14 days. The gray matter tissues of the lumbar spinal were sampled from rats in each group after 14 days following ventral root avulsion, and the extracted proteins from these tissues were detected by using 2-dimensional electrophoresis. Matrix assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) was utilized to identify the differentially expressed proteins among these three groups. RESULTS: There were six kinds of proteins differentially expressed among the three groups, which were collapsin response mediator protein 2 (CRMP-2), F-actin capping protein beta subunit (FCP-beta), isocitrate dehydrogenase [NAD] subunit beta (IDH-beta), ATPase, glutamate oxaloacetate transaminase-1 (GOT1) and M2 pyruvate kinase (M2-PK). The expression levels of CRMP-2, IDH-beta, ATPase and GOT1 were higher in the GASP-treated group than those in the untreated group, while the expression levels of FCP-beta and M2-PK were lower than those in the untreated group. CONCLUSION: GASP maybe promotes the survival and axon regeneration of injured spinal motor neurons in rats by virtue of up- or down-regulating the expression levels of the proteins mentioned above.

The availability of filament ends modulates actin stochastic dynamics in live plant cells.

A network of individual filaments that undergoes incessant remodeling through a process known as stochastic dynamics comprises the cortical actin cytoskeleton in plant epidermal cells. From images at high spatial and temporal resolution, it has been inferred that the regulation of filament barbed ends plays a central role in choreographing actin organization and turnover. How this occurs at a molecular level, whether different populations of ends exist in the array, and how individual filament behavior correlates with the overall architecture of the array are unknown. Here we develop an experimental system to modulate the levels of heterodimeric capping protein (CP) and examine the consequences for actin dynamics, architecture, and cell expansion. Significantly, we find that all phenotypes are the opposite for CP-overexpression (OX) cells compared with a previously characterized cp-knockdown line. Specifically, CP OX lines have fewer filament-filament annealing events, as well as reduced filament lengths and lifetimes. Further, cp-knockdown and OX lines demonstrate the existence of a subpopulation of filament ends sensitive to CP concentration. Finally, CP levels correlate with the biological process of axial cell expansion; for example, epidermal cells from hypocotyls with reduced CP are longer than wild-type cells, whereas CP OX lines have shorter cells. On the basis of these and other genetic studies in this model system, we hypothesize that filament length and lifetime positively correlate with the extent of axial cell expansion in dark-grown hypocotyls.

Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes.

Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.