Pten regulates neuronal soma size: a mouse model of Lhermitte-Duclos disease.

Somatic inactivation of PTEN occurs in different human tumors including glioblastoma, endometrial carcinoma and prostate carcinoma. Germline mutations in PTEN result in a range of phenotypic abnormalities that occur with variable penetrance, including neurological features such as macrocephaly, seizures, ataxia and Lhermitte-Duclos disease (also described as dysplastic gangliocytoma of the cerebellum). Homozygous deletion of Pten causes embryonic lethality in mice. To investigate function in the brain, we used Cre-loxP technology to selectively inactivate Pten in specific mouse neuronal populations. Loss of Pten resulted in progressive macrocephaly and seizures. Neurons lacking Pten expressed high levels of phosphorylated Akt and showed a progressive increase in soma size without evidence of abnormal proliferation. Cerebellar abnormalities closely resembled the histopathology of human Lhermitte-Duclos disease. These results indicate that Pten regulates neuronal size in vivo in a cell-autonomous manner and provide new insights into the etiology of Lhermitte-Duclos disease.

The SH2 tyrosine phosphatase shp2 is required for mammalian limb development.

The tyrosine phosphatase Shp2 is recruited into tyrosine-kinase signalling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs, concurrent with its re-localization and stimulation of phosphatase activity. Shp2 can potentiate signalling through the MAP-kinase pathway and is required during early mouse development for gastrulation. Chimaeric analysis can identify, by study of phenotypically normal embryos, tissues that tolerate mutant cells (and therefore do not require the mutated gene) or lack mutant cells (and presumably require the mutated gene during their developmental history). We therefore generated chimaeric mouse embryos to explore the cellular requirements for Shp2. This analysis revealed an obligatory role for Shp2 during outgrowth of the limb. Shp2 is specifically required in mesenchyme cells of the progress zone (PZ), directly beneath the distal ectoderm of the limb bud. Comparison of Ptpn11 (encoding Shp2)-mutant and Fgfr1 (encoding fibroblast growth factor receptor-1)-mutant chimaeric limbs indicated that in both cases mutant cells fail to contribute to the PZ of phenotypically normal chimaeras, leading to the hypothesis that a signal transduction pathway, initiated by Fgfr1 and acting through Shp2, is essential within PZ cells. Rather than integrating proliferative signals, Shp2 probably exerts its effects on limb development by influencing cell shape, movement or adhesion. Furthermore, the branchial arches, which also use Fgfs during bud outgrowth, similarly require Shp2. Thus, Shp2 regulates phosphotyrosine-signalling events during the complex ectodermal-mesenchymal interactions that regulate mammalian budding morphogenesis.

Rheb is an essential regulator of S6K in controlling cell growth in Drosophila.

Understanding the mechanisms through which multicellular organisms regulate cell, organ and body growth is of relevance to developmental biology and to research on growth-related diseases such as cancer. Here we describe a new effector in growth control, the small GTPase Rheb (Ras homologue enriched in brain). Mutations in the Drosophila melanogaster Rheb gene were isolated as growth-inhibitors, whereas overexpression of Rheb promoted cell growth. Our genetic and biochemical analyses suggest that Rheb functions downstream of the tumour suppressors Tsc1 (tuberous sclerosis 1)-Tsc2 in the TOR (target of rapamycin) signalling pathway to control growth, and that a major effector of Rheb function is ribosomal S6 kinase (S6K).

Separate but linked functions of conventional myosins modulate adhesion and neurite outgrowth.

The potential functional diversity of closely related myosin isoforms found in eukaryotic cells is not yet understood in detail. We have previously provided evidence from functional knockouts of Neuro-2A neuroblastoma cells that myosin IIB is essential for neurite outgrowth. Here we investigate the role of non-muscle myosin IIA in the same cell line. We show that suppression of myosin IIA transcript and protein expression, brought about through exposure to isoform-specific antisense oligonucleotides, caused a rearrangement of the actin cytoskeleton and loss of cell adhesion. This also led to disruption of focal contacts, as evidenced by coincident reduction in paxillin and vinculin immunofluorescence, but did not diminish transcript expression. All effects were fully reversible. Before myosin IIA antisense-induced detachment, neurite outgrowth remained unaffected. By contrast, antisense oligonucleotides directed against myosin IIB transcripts had no effect on adhesion but severely attenuated neurite outgrowth. We infer that the two main isoforms of neuronal conventional myosin, myosins IIA and IIB, have separate but linked functions during neuronal adhesion and neurite outgrowth.

The cell junction protein VAB-9 regulates adhesion and epidermal morphology in C. elegans.

Epithelial cell junctions are essential for cell polarity, adhesion and morphogenesis. We have analysed VAB-9, a cell junction protein in Caenorhabditis elegans. VAB-9 is a predicted four-pass integral membrane protein that has greatest similarity to BCMP1 (brain cell membrane protein 1, a member of the PMP22/EMP/Claudin family of cell junction proteins) and localizes to the adherens junction domain of C. elegans apical junctions. Here, we show that VAB-9 requires HMR-1/cadherin for localization to the cell membrane, and both HMP-1/alpha-catenin and HMP-2/beta-catenin for maintaining its distribution at the cell junction. In vab-9 mutants, morphological defects correlate with disorganization of F-actin at the adherens junction; however, localization of the cadherin-catenin complex and epithelial polarity is normal. These results suggest that VAB-9 regulates interactions between the cytoskeleton and the adherens junction downstream of or parallel to alpha-catenin and/or beta-catenin. Mutations in vab-9 enhance adhesion defects through functional loss of the cell junction genes apical junction molecule 1 (ajm-1) and discs large 1 (dlg-1), suggesting that VAB-9 is involved in cell adhesion. Thus, VAB-9 represents the first characterized tetraspan adherens junction protein in C. elegans and defines a new family of such proteins in higher eukaryotes.

Dmoesin controls actin-based cell shape and polarity during Drosophila melanogaster oogenesis.

Ezrin, Radixin and Moesin (ERM) proteins are thought to constitute a bridge between the actin cytoskeleton and the plasma membrane (PM). Here we report a genetic analysis of Dmoesin, the sole member of the ERM family in Drosophila. We show that Dmoesin is required during oogenesis for anchoring microfilaments to the oocyte cortex. Alteration of the actin cytoskeleton resulting from Dmoesin mutations impairs the localization of maternal determinants, thus disrupting antero-posterior polarity. This study also demonstrates the requirement of Dmoesin for the specific organization of cortical microfilaments in nurse cells and, consequently, mutations in Dmoesin produce severe defects in cell shape.

Coordination of microtubules and the actin cytoskeleton by the Rho effector mDia1.

Coordination of microtubules and the actin cytoskeleton is important in several types of cell movement. mDia1 is a member of the formin-homology family of proteins and an effector of the small GTPase Rho. It contains the Rho-binding domain in its amino terminus and two distinct regions of formin homology, FH1 in the middle and FH2 in the carboxy terminus. Here we show that expression of mDia1(DeltaN3), an active mDia1 mutant containing the FH1 and FH2 regions without the Rho-binding domain, induces bipolar elongation of HeLa cells and aligns microtubules in parallel to F-actin bundles along the long axis of the cell. The cell elongation and microtubule alignment caused by this mutant is abolished by co-expression of an FH2-region fragment, and expression of mDia1(DeltaN3) containing point mutations in the FH2 region causes an increase in the amount of disorganized F-actin without cell elongation and microtubule alignment. These results indicate that mDia1 may coordinate microtubules and F-actin through its FH2 and FH1 regions, respectively.

Increased adhesion and aggregation of platelets lacking cyclic guanosine 3',5'-monophosphate kinase I.

Atherosclerotic vascular lesions are considered to be a major cause of ischemic diseases, including myocardial infarction and stroke. Platelet adhesion and aggregation during ischemia-reperfusion are thought to be the initial steps leading to remodeling and reocclusion of the postischemic vasculature. Nitric oxide (NO) inhibits platelet aggregation and smooth muscle proliferation. A major downstream target of NO is cyclic guanosine 3', 5'-monophosphate kinase I (cGKI). To test the intravascular significance of the NO/cGKI signaling pathway in vivo, we have studied platelet-endothelial cell and platelet-platelet interactions during ischemia/reperfusion using cGKI-deficient (cGKI-/-) mice. Platelet cGKI but not endothelial or smooth muscle cGKI is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. The defect in platelet cGKI is not compensated by the cAMP/cAMP kinase pathway supporting the essential role of cGKI in prevention of ischemia-induced platelet adhesion and aggregation.

RhoGAP: the next big thing for small mice?

The size of an organism is determined by the number and size of its constituent cells. The insulin/IGF-1 signaling systems have been long recognized to play a critical role in the determination of body size. Now the generation of mice deficient for a RhoGAP suggests that this small G protein might also regulate the growth of animals.

Regulating shapes and sizes.

Mutations at many loci lead to altered shapes and sizes, suggesting complex regulation of the overall morphology of an organism. Two recent studies present data on how orientation of growth axes and perception of maturation signals might regulate growth processes.

NPK1, an MEKK1-like mitogen-activated protein kinase kinase kinase, regulates innate immunity and development in plants.

Mitogen-activated protein kinase (MAPK) cascades are rapidly activated upon plant recognition of invading pathogens. Here, we describe the use of virus-induced gene silencing (VIGS) to study the role of candidate plant MAP kinase kinase kinase (MAPKKK) homologs of human MEKK1 in pathogen-resistance pathways. We demonstrate that silencing expression of a tobacco MAPKKK, Nicotiana Protein Kinase 1 (NPK1), interferes with the function of the disease-resistance genes N, Bs2, and Rx, but does not affect Pto- and Cf4-mediated resistance. Further, NPK1-silenced plants also exhibit reduced cell size, defective cytokinesis, and an overall dwarf phenotype. Our results provide evidence that NPK1 functions in the regulation of N-, Bs2-, and Rx-mediated resistance responses and may play a role in one or more MAPK cascades, regulating multiple cellular processes.

Evidence for tension-based regulation of Drosophila MAL and SRF during invasive cell migration.

Cells migrating through a tissue exert force via their cytoskeleton and are themselves subject to tension, but the effects of physical forces on cell behavior in vivo are poorly understood. Border cell migration during Drosophila oogenesis is a useful model for invasive cell movement. We report that this migration requires the activity of the transcriptional factor serum response factor (SRF) and its cofactor MAL-D and present evidence that nuclear accumulation of MAL-D is induced by cell stretching. Border cells that cannot migrate lack nuclear MAL-D but can accumulate it if they are pulled by other migrating cells. Like mammalian MAL, MAL-D also responds to activated Diaphanous, which affects actin dynamics. MAL-D/SRF activity is required to build a robust actin cytoskeleton in the migrating cells; mutant cells break apart when initiating migration. Thus, tension-induced MAL-D activity may provide a feedback mechanism for enhancing cytoskeletal strength during invasive migration.

Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.

Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while unspread, round cells became adipocytes. Cell shape regulated the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA committed hMSCs to become adipocytes, while constitutively active RhoA caused osteogenesis. However, the RhoA-mediated adipogenesis or osteogenesis was conditional on a round or spread shape, respectively, while constitutive activation of the RhoA effector, ROCK, induced osteogenesis independent of cell shape. This RhoA-ROCK commitment signal required actin-myosin-generated tension. These studies demonstrate that mechanical cues experienced in developmental and adult contexts, embodied by cell shape, cytoskeletal tension, and RhoA signaling, are integral to the commitment of stem cell fate.

Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance.

Insulin signaling in adipose tissue plays an important role in lipid storage and regulation of glucose homeostasis. Using the Cre-loxP system, we created mice with fat-specific disruption of the insulin receptor gene (FIRKO mice). These mice have low fat mass, loss of the normal relationship between plasma leptin and body weight, and are protected against age-related and hypothalamic lesion-induced obesity, and obesity-related glucose intolerance. FIRKO mice also exhibit polarization of adipocytes into populations of large and small cells, which differ in expression of fatty acid synthase, C/EBP alpha, and SREBP-1. Thus, insulin signaling in adipocytes is critical for development of obesity and its associated metabolic abnormalities, and abrogation of insulin signaling in fat unmasks a heterogeneity in adipocyte response in terms of gene expression and triglyceride storage.

Phosphorylation of actopaxin regulates cell spreading and migration.

Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.

SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development.

G protein-coupled receptors trigger the reorganization of the actin cytoskeleton in many cell types, but the steps in this signal transduction cascade are poorly understood. During Dictyostelium development, extracellular cAMP functions as a chemoattractant and morphogenetic signal that is transduced via a family of G protein-coupled receptors, the cARs. In a strain where the cAR2 receptor gene is disrupted by homologous recombination, the developmental program arrests before tip formation. In a genetic screen for suppressors of this phenotype, a gene encoding a protein related to the Wiskott-Aldrich Syndrome protein was discovered. Loss of this protein, which we call SCAR (suppressor of cAR), restores tip formation and most later development to cAR2(-) strains, and causes a multiple-tip phenotype in a cAR2(+) strain as well as leading to the production of extremely small cells in suspension culture. SCAR-cells have reduced levels of F-actin staining during vegetative growth, and abnormal cell morphology and actin distribution during chemotaxis. Uncharacterized homologues of SCAR have also been identified in humans, mouse, Caenorhabditis elegans, and Drosophila. These data suggest that SCAR may be a conserved negative regulator of G protein-coupled signaling, and that it plays an important role in regulating the actin cytoskeleton.

ncl-1 is required for the regulation of cell size and ribosomal RNA synthesis in Caenorhabditis elegans.

Regulation of ribosome synthesis is an essential aspect of growth control. Thus far, little is known about the factors that control and coordinate these processes. We show here that the Caenorhabditis elegans gene ncl-1 encodes a zinc finger protein and may be a repressor of RNA polymerase I and III transcription and an inhibitor of cell growth. Loss of function mutations in ncl-1, previously shown to result in enlarged nucleoli, result in increased rates of rRNA and 5S RNA transcription and enlarged cells. Furthermore, ncl-1 adult worms are larger, have more protein, and have twice as much rRNA as wild-type worms. Localization studies show that the level of NCL-1 protein is independently regulated in different cells of the embryo. In wild-type embryos, cells with the largest nucleoli have the lowest level of NCL-1 protein. Based on these results we propose that ncl-1 is a repressor of ribosome synthesis and cell growth.

Dominant cataracts result from incongruous mixing of wild-type lens connexins.

Gap junctions are composed of proteins called connexins (Cx) and facilitate both ionic and biochemical modes of intercellular communication. In the lens, Cx46 and Cx50 provide the gap junctional coupling needed for homeostasis and growth. In mice, deletion of Cx46 produced severe cataracts, whereas knockout of Cx50 resulted in significantly reduced lens growth and milder cataracts. Genetic replacement of Cx50 with Cx46 by knockin rescued clarity but not growth. By mating knockin and knockout mice, we show that heterozygous replacement of Cx50 with Cx46 rescued growth but produced dominant cataracts that resulted from disruption of lens fiber morphology and crystallin precipitation. Impedance measurements revealed normal levels of ionic gap junctional coupling, whereas the passage of fluorescent dyes that mimic biochemical coupling was altered in heterozygous knockin lenses. In addition, double heterozygous knockout lenses retained normal growth and clarity, whereas knockover lenses, where native Cx46 was deleted and homozygously knocked into the Cx50 locus, displayed significantly deficient growth but maintained clarity. Together, these findings suggest that unique biochemical modes of gap junctional communication influence lens clarity and lens growth, and this biochemical coupling is modulated by the connexin composition of the gap junction channels.

Oriented clonal cell growth in the developing mouse myocardium underlies cardiac morphogenesis.

During heart morphogenesis, cardiac chambers arise by differential expansion of regions of the primitive cardiac tube. This process is under the control of specific transcription factors such as Tbx5 and dHAND. To gain insight into the cellular mechanisms that underlie cardiogenesis, we have used a retrospective clonal approach based on the spontaneous recombination of an nlaacZ reporter gene targeted to the murine alpha-cardiac actin locus. We show that clonal growth of myocardial cells is oriented. At embryonic day (E) 10.5, the shape of clones is characteristic of a given cardiac region and reflects its morphology. This is already detectable in the primitive cardiac tube at E8.5, and is maintained after septation at E14.5 with additional modulations. The clonal analysis reveals new subdivisions of the myocardium, including an interventricular boundary region. Our results show that the myocardium, from the time of its formation, is a polarized and regionalized tissue and point to the role of oriented clonal cell growth in cardiac chamber morphogenesis.

Cell size regulation, a mechanism that controls cellular RNA accumulation: consequences on regulation of the ubiquitous transcription factors Oct1 and NF-Y and the liver-enriched transcription factor DBP.

Cell sizes can differ vastly between cell types in individual metazoan organisms. In rat liver, spleen, and thymus, differences in average cell size roughly reflect differences in RNA:DNA ratios. For example, hepatocytes were found to have a cytoplasmic:nuclear volume ratio and an RNA:DNA ratio which were 34- and 21-fold higher, respectively, than those in thymocytes. RNA synthesis per DNA-equivalent in the hepatocytes was 25-fold greater than that in thymocytes, suggesting that differences in overall transcriptional activity, not differences in overall RNA stability, were primarily responsible for determining cellular RNA:DNA ratios. The mechanisms determining the capacity of large cells to synthesize and accumulate more ubiquitous cytoplasmic macromolecules, such as ribosomes, than smaller cells is entitled "cell size regulation." Cell size regulation may have important consequences on the tissue distribution of transcription factors. Thus, in liver, lung, kidney, spleen, and brain, cellular levels of the mRNA encoding the leucine zipper protein DBP correlate closely to cellular RNA:DNA ratios. Our results suggest that DBP mRNA levels, like rRNA levels, are transcriptionally determined. Thus the dbp gene, like the ribosomal genes, may be subject to cell size regulation. As a consequence, nuclei from liver, a tissue with a very large average cell size, accumulated higher levels of DBP protein than nuclei from small-celled tissues, such as spleen or lung. In contrast to DBP, the ubiquitous transcription factors Oct1 and NF-Y escaped cell size control. Nuclei from most tissues contained similar amounts of these factors irrespective of cell size. Likewise, tissues with large or small average cell sizes contained similar levels of the mRNAs encoding Oct1 or NF-Ya, one of the subunits of the heteromeric CCAAT-binding factor NF-Y, per DNA-equivalent. Interestingly, mRNA encoding NF-Yb, another subunit of NF-Y, was subject to cell size regulation. Our results suggest that NF-Yb protein escapes cell size regulation at a posttranslational level.