CRISPR/Cas9-mediated rapid generation of multiple mouse lines identified Ccdc63 as essential for spermiogenesis

Spermatozoa are flagellated cells whose role in fertilization is dependent on their ability to move towards an oocyte. The structure of the sperm flagella is highly conserved across species, and much of what is known about this structure is derived from studies utilizing animal models. One group of proteins essential for the movement of the flagella are the dyneins. Using the advanced technology of CRISPR/Cas9 we have targeted three dynein group members; Dnaic1, Wdr63 and Ccdc63 in mice. All three of these genes are expressed strongly in the testis. We generated mice with amino acid substitutions in Dnaic1 to analyze two specific phosphorylation events at S124 and S127, and generated simple knockouts of Wdr63 and Ccdc63. We found that the targeted phosphorylation sites in Dnaic1 were not essential for male fertility. Similarly, Wdr63 was not essential for male fertility; however, Ccdc63 removal resulted in sterile male mice due to shortened flagella. This study demonstrates the versatility of the CRISPR/Cas9 system to generate animal models of a highly complex system by introducing point mutations and simple knockouts in a fast and efficient manner.Young, S.A.M.; Miyata, H.; Satouh, Y.; Kato, H.; Nozawa, K.; Isotani, A.; Aitken, R.J.; Baker, M.A.; Ikawa, M. CRISPR/Cas9-Mediated Rapid Generation of Multiple Mouse Lines Identified Ccdc63 as Essential for Spermiogenesis. Int. J. Mol. Sci. 2015, 16, 24732-24750. https://doi.org/10.3390/ijms16102473

A Phosphatidylinositol 3-Kinase/Protein Kinase B-independent Activation of Mammalian Target of Rapamycin Signaling Is Sufficient to Induce Skeletal Muscle Hypertrophy

Overexpression of Rheb activates mTOR signaling via a PI3K/PKB-independent mechanism and is sufficient to induce skeletal muscle hypertrophy. The hypertrophic effects of Rheb are driven through a rapamycin-sensitive (RS) mechanism, mTOR is the RS element that confers the hypertrophy and the kinase activity of mTOR is necessary for this event

Canine and human gastrointestinal stromal tumors display similar mutations in c-KIT exon 11

<p>Abstract</p> <p>Background</p> <p>Gastrointestinal stromal tumors (GISTs) are common mesenchymal neoplasms in the gastrointestinal tract of humans and dogs. Little is known about the pathogenesis of these tumors. This study evaluated the role of <it>c-KIT </it>in canine GISTs; specifically, we investigated activating mutations in exons 8, 9, 11, 13, and 17 of <it>c-KIT </it>and exons 12, 14, and 18 of platelet-derived growth factor receptor, alpha polypeptide (<it>PDGFRA</it>), all of which have been implicated in human GISTs.</p> <p>Methods</p> <p>Seventeen canine GISTs all confirmed to be positive for KIT immunostaining were studied. Exons 8, 9, 11, 13 and 17 of <it>c-KIT </it>and exons 12, 14, and 18 of <it>PDGFRA</it>, were amplified from DNA isolated from formalin-fixed paraffin-embedded samples.</p> <p>Results</p> <p>Of these seventeen cases, six amplicons of exon 11 of <it>c-KIT </it>showed aberrant bands on gel electrophoresis. Sequencing of these amplicons revealed heterozygous in-frame deletions in six cases. The mutations include two different but overlapping six base pair deletions. Exons 8, 9, 13, and 17 of <it>c-KIT </it>and exons 12, 14, and 18 of <it>PDGFRA </it>had no abnormalities detected by electrophoresis and sequencing did not reveal any mutations, other than synonymous single nucleotide polymorphisms (SNPs) found in exon 11 of <it>c-KIT </it>and exons 12 and 14 of <it>PDGFRA</it>.</p> <p>Conclusions</p> <p>The deletion mutations detected in canine GISTs are similar to those previously found in the juxtamembrane domain of <it>c-KIT </it>in canine cutaneous mast cell tumors in our laboratory as well as to those reported in human GISTs. Interestingly, none of the other <it>c-KIT </it>or <it>PDGFRA </it>exons showed any abnormalities in our cases. This finding underlines the critical importance of <it>c-KIT </it>in the pathophysiology of canine GISTs. The expression of KIT and the identification of these activating mutations in <it>c-KIT </it>implicate KIT in the pathogenesis of these tumors. Our results indicate that mutations in <it>c-KIT </it>may be of prognostic significance and that targeting KIT may be a rational approach to treatment of these malignant tumors. This study further demonstrates that spontaneously occurring canine GISTs share molecular features with human GISTs and are an appropriate model for human GISTs.</p

Involvement of Girdin in the Determination of Cell Polarity during Cell Migration

Cell migration is a critical cellular process that determines embryonic development and the progression of human diseases. Therefore, cell- or context-specific mechanisms by which multiple promigratory proteins differentially regulate cell migration must be analyzed in detail. Girdin (girders of actin filaments) (also termed GIV, Gα-interacting vesicle associated protein) is an actin-binding protein that regulates migration of various cells such as endothelial cells, smooth muscle cells, neuroblasts, and cancer cells. Here we show that Girdin regulates the establishment of cell polarity, the deregulation of which may result in the disruption of directional cell migration. We found that Girdin interacts with Par-3, a scaffolding protein that is a component of the Par protein complex that has an established role in determining cell polarity. RNA interference-mediated depletion of Girdin leads to impaired polarization of fibroblasts and mammary epithelial cells in a way similar to that observed in Par-3-depleted cells. Accordingly, the expression of Par-3 mutants unable to interact with Girdin abrogates cell polarization in fibroblasts. Further biochemical analysis suggests that Girdin is present in the Par protein complex that includes Par-3, Par-6, and atypical protein kinase C. Considering previous reports showing the role of Girdin in the directional migration of neuroblasts, network formation of endothelial cells, and cancer invasion, these data may provide a specific mechanism by which Girdin regulates cell movement in biological contexts that require directional cell movement

Transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic strategy for female infertility

Ten to 15% of couples are infertile, with the most common causes being linked to the production of few or no oocytes or sperm. Yet, our understanding of human germ cell development is poor, at least in part due to the inaccessibility of early stages to genetic and developmental studies. Embryonic stem cells (ESCs) provide an in vitro system to study oocyte development and potentially treat female infertility. However, most studies of ESC differentiation to oocytes have not documented fundamental properties of endogenous development, making it difficult to determine the physiologic relevance of differentiated germ cells. Here, we sought to establish fundamental parameters of oocyte development during ESC differentiation to explore suitability for basic developmental genetic applications using the mouse as a model prior to translating to the human system. We demonstrate a timeline of definitive germ cell differentiation from ESCs in vitro that initially parallels endogenous oocyte development in vivo by single-cell expression profiling and analysis of functional milestones including responsiveness to defined maturation media, shared genetic requirement of Dazl, and entry into meiosis. However, ESC-derived oocyte maturation ultimately fails in vitro. To overcome this obstacle, we transplant ESC-derived oocytes into an ovarian niche to direct their functional maturation and, thereby, present rigorous evidence of oocyte physiologic relevance and a potential therapeutic strategy for infertility

Basal ganglia dysfunction in OCD: subthalamic neuronal activity correlates with symptoms severity and predicts high-frequency stimulation efficacy

Functional and connectivity changes in corticostriatal systems have been reported in the brains of patients with obsessive–compulsive disorder (OCD); however, the relationship between basal ganglia activity and OCD severity has never been adequately established. We recently showed that deep brain stimulation of the subthalamic nucleus (STN), a central basal ganglia nucleus, improves OCD. Here, single-unit subthalamic neuronal activity was analysed in 12 OCD patients, in relation to the severity of obsessions and compulsions and response to STN stimulation, and compared with that obtained in 12 patients with Parkinson's disease (PD). STN neurons in OCD patients had lower discharge frequency than those in PD patients, with a similar proportion of burst-type activity (69 vs 67%). Oscillatory activity was present in 46 and 68% of neurons in OCD and PD patients, respectively, predominantly in the low-frequency band (1–8 Hz). In OCD patients, the bursty and oscillatory subthalamic neuronal activity was mainly located in the associative–limbic part. Both OCD severity and clinical improvement following STN stimulation were related to the STN neuronal activity. In patients with the most severe OCD, STN neurons exhibited bursts with shorter duration and interburst interval, but higher intraburst frequency, and more oscillations in the low-frequency bands. In patients with best clinical outcome with STN stimulation, STN neurons displayed higher mean discharge, burst and intraburst frequencies, and lower interburst interval. These findings are consistent with the hypothesis of a dysfunction in the associative–limbic subdivision of the basal ganglia circuitry in OCD's pathophysiology

Met-Independent Hepatocyte Growth Factor-mediated regulation of cell adhesion in human prostate cancer cells

BACKGROUND: Prostate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF), was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells. METHODS: We searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF) were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models. RESULTS: We have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM) and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM) or nucleolin (on the cell surfaces) eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of laminin-binding integrins, nor can they be linked to expression of the known HGF receptor Met, as neither LNCaP nor clonally-derived C4-2 sub-line contain any detectable Met protein. Even in the absence of Met, small GTPases are activated, linking HGF stimulation to membrane protrusion and integrin activation. Membrane-localized nucelolin levels increase during cancer progression, as modeled by both the PC3 and LNCaP prostate cancer progression cell lines. CONCLUSION: We propose that cell surface localized nucleolin protein may function in these cells as a novel HGF receptor. Membrane localized nucleolin binds heparin-bound growth factors (including HGF) and appears upregulated during prostate cancer progression. Antibodies against nucleolin are able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. HGF-nucleolin interactions could be partially responsible for the complexity of HGF responses and met expression reported in the literature