TGFbeta1-induced aortic endothelial morphogenesis requires signaling by small GTPases Rac1 and RhoA

TGFbeta is a potent regulator of cell differentiation in many cell types. On aortic endothelial cells, TGFbeta1 displays angiogenic properties in inducing capillary-like tube formation in collagen I gels, in vitro. We investigated cytoskeletal changes that precede tube formation and related these alterations to the effects of TGFbeta1 on the activation state of members of the RhoGTPase family. TGFbeta1 promotes cell elongation and stress fiber formation in aortic endothelial cells. Using cell lines with inducible expression of Rac1 mutants, we show that these events are mimicked by expression of dominant-negative Rac1 whereas the constitutively active mutant prevents the TGFbeta1-mediated change of phenotype. Although TGFbeta1 induces an initial rise in the Rac1-GTP content, this phase is followed by a prolonged loss of the active form. In contrast, RhoA activity increases progressively and reaches a plateau when Rac1-GTP is no longer detectable. Prolonged inhibition of Rac1 appears necessary and sufficient for the increase in RhoA-GTP. In situ examination of Rho activity in TGFbeta1-treated cells provides evidence that active RhoA relocalizes to the tips of elongated cells. Inhibiting the Rho effector ROCK abrogates tube formation. Thus, Rac1 and RhoA are regulated by TGFbeta1 in the process of endothelial tube formation in collagen I gels.     

Non-visual arrestins regulate the focal adhesion formation via small GTPases RhoA and Rac1 independently of GPCRs

Arrestins recruit a variety of signaling proteins to active phosphorylated G protein-coupled receptors in the plasma membrane and to the cytoskeleton. Loss of arrestins leads to decreased cell migration, altered cell shape, and an increase in focal adhesions. Small GTPases of the Rho family are molecular switches that regulate actin cytoskeleton and affect a variety of dynamic cellular functions including cell migration and cell morphology. Here we show that non-visual arrestins differentially regulate RhoA and Rac1 activity to promote cell spreading via actin reorganization, and focal adhesion formation via two distinct mechanisms. Arrestins regulate these small GTPases independently of G-protein-coupled receptor activation.     

RhoA signaling is required for respiratory syncytial virus-induced syncytium formation and filamentous virion morphology

Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants, the elderly, and immunocompromised adults. RSV infection of HEp-2 cells induces the activation of RhoA, a small GTPase. We therefore asked whether RhoA signaling is important for RSV replication or syncytium formation. The treatment of HEp-2 cells with Clostridium botulinum C3, an enzyme that ADP-ribosylates and specifically inactivates RhoA, inhibited RSV-induced syncytium formation and cell-to-cell fusion, although similar levels of PFU were released into the medium and viral protein expression levels were equivalent. Treatment with another inhibitor of RhoA signaling, the Rho kinase inhibitor Y-27632, yielded similar results. Scanning electron microscopy of C3-treated infected cells showed reduced numbers of single blunted filaments, in contrast to the large clumps of long filaments in untreated infected cells. These data suggest that RhoA signaling is associated with filamentous virus morphology, cell-to-cell fusion, and syncytium formation but is dispensable for the efficient infection and production of infectious virus in vitro. Next, we developed a semiquantitative method to measure spherical and filamentous virus particles by using sucrose gradient velocity sedimentation. Fluorescence and transmission electron microscopy confirmed the separation of spherical and filamentous forms of infectious virus into two identifiable peaks. The C3 treatment of RSV-infected cells resulted in a shift to relatively more spherical virions than those from untreated cells. These data suggest that viral filamentous protuberances characteristic of RSV infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play a role in initiating cell-to-cell fusion.     

Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation.

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.     

Differential regulation of cyclooxygenase 2 expression by small GTPases Ras, Rac1, and RhoA

Cyclooxygenase 2 (COX-2) is an immediate early gene induced by a variety of stimuli and its expression is stimulated by individual activation of Ras or Rho GTPases. Here we investigate the role of coordinate activation of Ras and Rho GTPases in the induction of COX-2. Individual expression of constitutively active Ras, RhoA, or Rac1 was capable of stimulating COX-2 expression in NIH3T3 cells, but co-expression of constitutively active RhoA with either constitutively active Ras or Rac1 was required for full stimulation of COX-2 expression. Serum growth factors differentially activated Ras, RhoA, and Rac1, which correlated with the activation of Raf-1, ERK, and c-Jun as well as with induction of COX-2. Inhibition of Ras significantly blocked the activation of Raf-1, ERK, and c-Jun and the stimulation of COX-2 expression in response to serum. In contrast, inhibition of Rho family GTPases partially blocked serum induction of ERK activation but had little effects on COX-2 expression. Both inhibitors of MEK (PD098059) and JNK (SP600125) inhibited serum induction of COX-2. PD98059 only inhibited constitutively active Ras-induced COX-2 expression, while SP600125 significantly inhibited both constitutively active Ras- and RhoA-induced COX-2 expression. Together, our data suggest that constitutively active oncogenic Ras and Rho coordinately stimulate COX-2 expression whereas transient activation of Ras but not RhoA or Rac1 mediates the induction of COX-2 in response to serum. Furthermore, ERK and JNK activation are both required for serum- and oncogenic Ras-mediated COX-2 expression whereas only JNK activation is required for oncogenic RhoA-mediated stimulation of COX-2 expression.     

Small GTPases Rap1 and RhoA regulate superoxide formation by Rac1 GTPases activation during the phagocytosis of IgG-opsonized zymosans in macrophages

Phagocytic NADPH oxidase plays a critical role in superoxide generation in macrophage cells. Small GTPases, including Rac1 and Rac2, have been implicated in the regulation of NADPH oxidase activity. Rap1, which has no effect in a cell-free system of oxidase activation, recently has been proven to colocalize with cytochrome b(558). In addition, neutrophils from rap1A(-/-) mice reduce fMLP-stimulated superoxide production. Here, we tried to determine whether Rap1 also plays a role in the production of superoxide. IgG-opsonized zymosan (IOZ) particles treatment induced Rap1 activation and superoxide generation. Knock-down of Rap1 by si-Rap1 suppressed IOZ-induced superoxide formation. Sh-RhoA also reduced superoxide levels, but 8CPT-2Me-cAMP, an activator of Epac1 (a guanine nucleotide exchange factor (GEF) of Rap1), could recover the levels to the control value. When cells were stimulated by IOZ, Rap1 and Rac1 were translocated to the membrane, and then interacted with p22(phox). 8CPT-2Me-cAMP rescued sh-RhoA-induced reduction of the interaction between Rac1 and p22(phox), and enhanced lysophosphatidic acid (LPA)-induced increase of their interaction. Moreover, Rac1 activity was increased by both LPA and 8CPT-2Me-cAMP when treated with IOZ particles. Si-Vav2 impaired GTP-Rac1 levels in response to 8CPT-2Me-cAMP/IOZ. Phosphorylation of RhoA activates Rac1 in response to IOZ by the enhanced binding of phospho-RhoA to RhoGDI, leading to the release of Rac1 from the Rac1-RhoGDI complex. In conclusion, IOZ treatment induces Rap1 activation and phosphorylation of RhoA, which in turn cause Rac1 activation and promote Rac1 translocation to the membrane leading to binding with p22(phox) that activates NADPH oxidase and produces superoxide.     

The small GTPases RhoA and Rac1 regulate cerebellar development by controlling cell morphogenesis,migration and foliation

The small GTPases RhoA and Rac1 are key cytoskeletal regulators that function in a mutually antagonistic manner to control the migration and morphogenesis of a broad range of cell types. However, their role in shaping the cerebellum, a unique brain structure composed of an elaborate set of folia separated by fissures of different lengths, remains largely unexplored. Here we show that dysregulation of both RhoA and Rac1 signaling results in abnormal cerebellar ontogenesis. Ablation of RhoA from neuroprogenitor cells drastically alters the timing and placement of fissure formation, the migration and positioning of granule and Purkinje cells, the alignment of Bergmann glia, and the integrity of the basement membrane, primarily in the anterior lobules. Furthermore, in the absence of RhoA, granule cell precursors located at the base of fissures fail to undergo cell shape changes required for fissure initiation. Many of these abnormalities can be recapitulated by deleting RhoA specifically from granule cell precursors but not postnatal glia, indicating that RhoA functions in granule cell precursors to control cerebellar morphogenesis. Notably, mice with elevated Rac1 activity due to loss of the Rac1 inhibitors Bcr and Abr show similar anterior cerebellar deficits, including ectopic neurons and defects in fissure formation, Bergmann glia organization and basement membrane integrity. Together, our results suggest that RhoA and Rac1 play indispensable roles in patterning cerebellar morphology.     

Structural and Functional Regulation of Tight Junctions by RhoA and Rac1 Small GTPases

Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturbed TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy revealed abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appeared normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupted the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane were unaffected. Expression levels and protein complexes of occludin and ZO-1 appeared normal in all mutant cells, although ZO-1 was more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane.     

Palmitoylation,membrane-proximal basic residues,and transmembrane glycine residues in the reovirus p10 protein are essential for syncytium formation

Avian reovirus and Nelson Bay reovirus are two unusual nonenveloped viruses that induce extensive cell-cell fusion via expression of a small nonstructural protein, termed p10. We investigated the importance of the transmembrane domain, a conserved membrane-proximal dicysteine motif, and an endodomain basic region in the membrane fusion activity of p10. We now show that the p10 dicysteine motif is palmitoylated and that loss of palmitoylation correlates with a loss of fusion activity. Mutational and functional analyses also revealed that a triglycine motif within the transmembrane domain and the membrane-proximal basic region were essential for p10-mediated membrane fusion. Mutations in any of these three motifs did not influence events upstream of syncytium formation, such as p10 membrane association, protein topology, or surface expression, suggesting that these motifs are more intimately associated with the membrane fusion reaction. These results suggest that the rudimentary p10 fusion protein has evolved a mechanism of inducing membrane merger that is highly dependent on the specific interaction of several different motifs with donor membranes. In addition, cross-linking, coimmunoprecipitation, and complementation assays provided no evidence for p10 homo- or heteromultimer formation, suggesting that p10 may be the first example of a membrane fusion protein that does not form stable, higher-order multimers.     

RhoA and Rac1 GTPases mediate the dynamic rearrangement of actin in peripheral glia

Peripheral glial cells in both vertebrates and insects are born centrally and travel large distances to ensheathe axons in the periphery. There is very little known about how this migration is carried out. In other cells, it is known that rearrangement of the Actin cytoskeleton is an integral part of cell motility, yet the distribution of Actin in peripheral glial cell migration in vivo has not been previously characterized. To gain an understanding of how glia migrate, we specifically labeled the peripheral glia of Drosophila melanogaster using an Actin-GFP marker and analyzed their development in the embryonic PNS. It was found that Actin cytoskeleton is dynamically rearranged during glial cell migration. The peripheral glia were observed to migrate as a continuous chain of cells, with the leading glial cells appearing to participate to the greatest extent in exploring the extracellular surroundings with filopodia-like Actin containing projections. We hypothesized that the small GTPases Rho, Rac and Cdc42 are involved in Actin cytoskeletal rearrangements that underlie peripheral glial migration and nerve ensheathement. To test this, transgenic forms of the GTPases were ectopically expressed specifically in the peripheral glia during their migration and wrapping phases. The effects on glial Actin-GFP distribution and the overall effects on glial cell migration and morphological development were assessed. We found that RhoA and Rac1 have distinct roles in peripheral glial cell migration and nerve ensheathement; however, Cdc42 does not have a significant role in peripheral glial development. RhoA and Rac1 gain-of-function and loss-of-function mutants had both disruption of glial cell development and secondary effects on sensory axon fasciculation. Together, Actin cytoskeletal dynamics is an integral part of peripheral glial migration and nerve ensheathement, and is mediated by RhoA and Rac1.     

Rac1 and RhoA GTPases have antagonistic functions during N-cadherin-dependent cell-cell contact formation in C2C12 myoblasts

Background information. N‐cadherin, a member of the Ca²⁺‐dependent cell—cell adhesion molecule family, plays an essential role in the induction of the skeletal muscle differentiation programme. However, the molecular mechanisms which govern the formation of N‐cadherin‐dependent cell—cell contacts in myoblasts remain unexplored. Results. In the present study, we show that N‐cadherin‐dependent cell contact formation in myoblasts is defined by two stages. In the first phase, N‐cadherin is highly mobile in the lamellipodia extensions between the contacting cells. The second stage corresponds to the formation of mature N‐cadherin‐dependent cell contacts, characterized by the immobilization of a pool of N‐cadherin which appears to be clustered in the interdigitated membrane structures that are also membrane attachment sites for F‐actin filaments. We also demonstrated that the formation of N‐cadherin‐dependent cell—cell contacts requires a co‐ordinated and sequential activity of Rac1 and RhoA. Rac1 is involved in the first stage and facilitates N‐cadherin‐dependent cell—cell contact formation, but it is not absolutely required. Conversely, RhoA is necessary for N‐cadherin‐dependent cell contact formation, since, via ROCK (Rho‐associated kinase) signalling and myosin 2 activation, it allows the stabilization of N‐cadherin at the cell—cell contact sites. Conclusions. We have shown that Rac1 and RhoA have opposite effects on N‐cadherin‐dependent cell—cell contact formation in C2C12 myoblasts and act sequentially to allow its formation.     

RhoA of the Rho family small GTPases is essential for B lymphocyte development

RhoA is a member of the Rho family small GTPases that are implicated in various cell functions including proliferation and survival. However, the physiological role of RhoA in vivo remains largely unknown. Here, we deleted RhoA in the B cell and hematopoietic stem cell (HSC) populations in RhoA(flox/flox) mice with CD19 and Mx promoter-driven Cre expression, respectively. Deletion of RhoA by CD19(Cre/+) significantly blocked B cell development in spleen, leading to a marked reduction in the number of transitional, marginal zone, and follicular B cells. Surprisingly, neither B cell proliferation in response to either LPS or B cell receptor (BCR) engagement nor B cell survival rate in vivo was affected by RhoA deletion. Furthermore, RhoA(-/-) B cells, like control cells, were rescued from apoptosis by BCR crosslinking in vitro. In contrast, RhoA deficiency led to a defect in B cell activating factor (BAFF)-mediated B cell survival that was associated with a dampened expression of BAFF receptor and a loss of BAFF-mediated Akt activation. Finally, HSC deletion of RhoA by Mx-Cre severely reduced proB/preB and immature B cell populations in bone marrow while common lymphoid progenitors were increased, indicating that RhoA is also required for B cell progenitor/precursor differentiation. Taken together, our results uncover an important role for RhoA at multiple stages of B cell development.     

Spatial and temporal activation of the small GTPases RhoA and Rac1 by the netrin-1 receptor UNC5a during neurite outgrowth

Netrin-1 attracts or repels growing axons during development. The UNC5 receptors mediate the repulsive response, either alone or in complex with DCC receptors. The signaling mechanisms activated by UNC5 are poorly understood. Here, we examined the role of Rho GTPases in UNC5a signaling. We found that UNC5a induced neurite outgrowth in N1E-115 neuroblastoma cells in a netrin-1- and Rac1-dependent manner. UNC5a lacking its cytoplasmic tail also mediated this effect. In fibroblasts, UNC5a was able to activate RhoA and to a lower extent Rac1 and Cdc42 in response to netrin-1. Using Fluorescence Resonance Energy Transfer (FRET) intermolecular probes, we visualized the spatial and temporal activation of Rac1, Cdc42 and RhoA in live N1E-115 cells expressing UNC5a during neurite outgrowth. We found that Rac1 but not Cdc42 was transiently activated at the leading edge of the cell during neurite initiation. However, at later times when well-developed neurites were formed, active RhoA was found in the cell body and at the base of the neuronal leading process in UNC5a-expressing cells. Together, these findings demonstrate that the netrin-1 receptor UNC5a is able to induce neurite outgrowth and to differentially activate RhoA and Rac1 during neurite extension in a spatial and temporal manner.     

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.     

Activation of Six1 target genes is required for sensory placode formation

In vertebrates, cranial placodes form crucial parts of the sensory nervous system in the head. All cranial placodes arise from a common territory, the preplacodal region, and are identified by the expression of Six1/4 and Eya1/2 genes, which control different aspects of sensory development in invertebrates as well as vertebrates. While So and Eya can induce ectopic eyes in Drosophila, the ability of their vertebrate homologues to induce placodes in non-placodal ectoderm has not been explored. Here we show that Six1 and Eya2 are involved in ectodermal patterning and cooperate to induce preplacodal gene expression, while repressing neural plate and neural crest fates. However, they are not sufficient to induce ectopic sensory placodes in future epidermis. Activation of Six1 target genes is required for expression of preplacodal genes, for normal placode morphology and for placode-specific Pax protein expression. These findings suggest that unlike in the fly where the Pax6 homologue Eyeless acts upstream of Six and Eya, the regulatory relationships between these genes are reversed in early vertebrate placode development.     

Activation of type I phosphatidylinositol 4-phosphate 5-kinase isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the formation of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is implicated in many cellular processes. The Rho GTPases, RhoA and Rac1, have been shown previously to activate PIP5K and to bind PIP5K. Three type I PIP5K isoforms (Ialpha,Ibeta, and Igamma) have been identified; however, it is unclear whether these isoforms are differentially or even sequentially regulated by Rho GTPases. Here we show that RhoA and Rac1, as well as Cdc42, but not the Ras-like GTPases, RalA and Rap1A, markedly stimulate PIP(2) synthesis by all three PIP5K isoforms expressed in human embryonic kidney 293 cells, both in vitro and in vivo. RhoA-stimulated PIP(2) synthesis by the PIP5K isoforms was mediated by the RhoA effector, Rho-kinase. Stimulation of PIP5K isoforms by Rac1 and Cdc42 was apparently independent of and additive with RhoA- and Rho-kinase, as shown by studies with C3 transferase and Rho-kinase mutants. RhoA, and to a lesser extent Rac1, but not Cdc42, interacted in a nucleotide-independent form with all three PIP5K isoforms. Binding of PIP5K isoforms to GTP-bound, but not GDP-bound, RhoA could be displaced by Rho-kinase, suggesting a direct and constitutive PIP5K-Rho GTPase binding, which, however, does not trigger PIP5K activation. In summary, our findings indicate that synthesis of PIP(2) by the three PIP5K isoforms is controlled by RhoA, acting via Rho-kinase, as well as Rac1 and Cdc42, implicating that regulation of PIP(2) synthesis has a central position in signaling by these three Rho GTPases.     

RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells

Repair of the airway epithelium after injury is critical for restoring normal lung. The reepithelialization process involves spreading and migration followed later by cell proliferation. Rho-GTPases are key components of the wound healing process in many different types of tissues, but the specific roles for RhoA and Rac1 vary and have not been identified in lung epithelial cells. We investigated whether RhoA and Rac1 regulate wound closure of bronchial epithelial cells. RhoA and Rac1 proteins were efficiently expressed in a cell line of human bronchial epithelial cells (16HBE) by adenovirus-based gene transfer. We found that both constitutively active RhoA and dominant negative RhoA inhibited wound healing, suggesting that both activation and inhibition of RhoA interfere with normal wound healing. Overexpression of wild-type Rac1 induced upregulation of RhoA, disrupted intercellular junctions, and inhibited wound closure. Dominant negative Rac1 also inhibited wound closure. Inhibition of the downstream effector of RhoA, Rho-kinase, with Y-27632 suppressed actin stress fibers and focal adhesion formation, increased Rac1 activity, and stimulated wound closure. The activity of both RhoA and Rac1 are influenced by the polymerization state of microtubules, and cell migration involves coordinated action of actin and microtubules. Microtubule depolymerization upon nocodazole treatment led to an increase in focal adhesions and decreased wound closure. We conclude that coordination of both RhoA and Rac1 activity contributes to bronchial epithelial wound repair mechanisms in vitro, that inhibition of Rho-kinase accelerates wound closure, and that efficient repair involves intact microtubules.     

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.