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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.

Growth differentiation factor 3 (GDF-3) is structurally a bone morphogenetic protein/growth differentiation factor subfamily member of the TGF-beta superfamily. GDF-3 exhibits highest level of expression in white fat tissue in mice and is greatly induced by high fat diet if fat metabolic pathway is blocked. To identify its biological function, GDF-3 was overexpressed in mice by adenovirus mediated gene transfer. Mice transduced with GDF-3 displayed profound weight gain when fed with high fat diet. The phenotypes included greatly expanded adipose tissue mass, increased body adiposity, highly hypertrophic adipocytes, hepatic steatosis, and elevated plasma leptin. GDF-3 stimulated peroxisome proliferator activated receptor expression in adipocytes, a master nuclear receptor that controls adipogenesis. However, GDF-3 was not involved in blood glucose homeostasis or insulin resistance, a condition associated with obesity. In contrast, similar phenotypes were not observed in GDF-3 mice fed with normal chow, indicating that GDF-3 is only active under high lipid load. Thus, GDF-3 is a new non-diabetic adipogenic factor tightly coupled with fat metabolism.

To test whether reduced immune responsiveness in early life may be related to the immaturity of neonatal antigen-presenting cells, we comparatively assessed the phenotypic and functional characteristics of dendritic epidermal leukocytes (DEL) and epidermal Langerhans cells (LC) in newborn (NB) and adult mice, respectively. We report that purified, 3-day-cultured DEL do not acquire the morphology and phenotype typical of LC and are significantly weaker stimulators of naive, allogeneic CD4+ and CD8+ T cells than LC. Freshly isolated DEL are twice as efficient as LC in the uptake of fluorescein isothiocyanate-conjugated tracers but are not able to present these to antigen-specific T cell hybridomas. To clarify the underlying cause, cytokine expression of NB and adult epidermal cells (EC) was examined. We found that DEL express considerable amounts of interleukin (IL)-10, that IL-10 in NB EC supernatants partially inhibits LC maturation, and that DEL-enriched EC from IL-10-/- mice induce stronger primary T cell responses compared with those from IL-10+/+ mice. We conclude that IL-10 is one of the factors preventing maturation and differentiation of DEL into immunocompetent LC in intrauterine life and is at least partly responsible for the poor immune responsiveness of neonates.

Background: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine., Results: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo., Conclusion: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

In Brn3b(-/-) mice, where 80% of retinal ganglion cells degenerate early in development, the remaining 20% include most or all ganglion cell types. Cells of the same type cover the retinal surface evenly but tile it incompletely, indicating that a regular mosaic and normal dendritic field size can be maintained in the absence of contact among homotypic cells. In Math5(-/-) mice, where only approximately 5% of ganglion cells are formed, the dendritic arbors of at least two types among the residual ganglion cells are indistinguishable from normal in shape and size, even though throughout development they are separated by millimeters from the nearest neighboring ganglion cell of the same type. It appears that the primary phenotype of retinal ganglion cells can develop without homotypic contact; dendritic repulsion may be an end-stage mechanism that fine-tunes the dendritic arbors for more efficient coverage of the retinal surface.

We have established a primary pancreatic duct epithelial cell culture (PDEC) system to investigate the relationship between oncogenic activation of K-ras and pancreatic ductal tumorigenesis. We have found that the acute introduction of physiological levels of oncogenic K-ras (K-rasV12) into quiescent PDECs stimulates S-phase entry and induces a pronounced increase in cell size. Both effects are dependent on the functional integrity of the phosphatidylinositol 3'-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. In addition, K-rasV12 promotes the loss of epithelial E-cadherin and the gain of mesenchymal N-cadherin in PDEC. Our observations indicate that the oncogenic activation of K-ras is sufficient to elicit mitogenic and morphogenic responses in pancreatic ductal cells and hence is likely to play an instructive role in the initiation of pancreatic ductal adenocarcinoma.

The coxsackie and adenovirus receptor (CAR) is a key factor in adenoviral cancer gene therapy. Reduced expression of CAR during progression of prostate and bladder cancer has been reported. In embryonic development and tissue differentiation, CAR is also differentially expressed. This study suggests a role of CAR expression in cell adhesion and cell motility of human cancer cells. Stable CAR-expressing clones from E-cadherin-deficient A2780 ovarian and CaSki cervical cancer cells with originally low and high CAR expression levels, respectively, were established. CAR reexpression in otherwise singularly growing A2780 parental cells resulted in formation of cell-cell contacts and aggregation in cell clusters. CAR overexpression in cell adhesion-forming CaSki cells did not result in morphological changes. Migration of the A2780 CAR clones was strongly reduced as characterized by using spread-off assays. Using migration chambers, formation of satellite colonies was reduced by 97% in CAR-expressing A2780 cell clones and by 23% in CAR-expressing CaSki cell clones. Parental A2780 and CaSki cells selected for high migratory ability by using migration chambers expressed endogenous CAR on lower levels associated with lower adenoviral transduction efficiency. Our data suggest CAR as a new inhibitory factor for cancer cell migration.

Genetic studies of neocortical area patterning are limited, because mice deficient for candidate regulatory genes die before areas emerge and have other complicating issues. To define roles for the homeodomain transcription factor EMX2, we engineered nestin-Emx2 transgenic mice that overexpress Emx2 in cortical progenitors coincident with expression of endogenous Emx2 and survive postnatally. Cortical size, lamination, thalamus, and thalamocortical pathfinding are normal in homozygous nestin-Emx2 mice. However, primary sensory and motor areas are disproportionately altered in size and shift rostrolaterally. Heterozygous transgenics have similar but smaller changes. Opposite changes are found in heterozygous Emx2 knockout mice. Fgf8 expression in the commissural plate of nestin-Emx2 mice is indistinguishable from wild-type, but Pax6 expression is downregulated in rostral cortical progenitors, suggesting that EMX2 repression of PAX6 specification of rostral identities contributes to reduced rostral areas. We conclude that EMX2 levels in cortical progenitors disproportionately specify sizes and positions of primary cortical areas., (Copyright 2004 Cell Press)

Individuals with tuberous sclerosis complex (TSC) develop central nervous system abnormalities that may reflect astrocyte dysfunction. In an effort to model astrocyte dysfunction in TSC, we generated mice lacking Tsc1 (hamartin) expression in astrocytes and demonstrated that Tsc1-null astrocytes exhibit abnormalities in contact inhibition growth arrest. In this study, we demonstrate that hamartin-deficient astrocytes are also defective in cell size regulation. We show that the increase in Tsc1-null astrocyte size is associated with increased activation of the S6-kinase pathway. In keeping with recent reports that the hamartin/tuberin complex may regulate Rheb and downstream S6K activation, we demonstrate that expression of either Rheb or S6K in primary astrocytes results in increased S6 pathway activation, and that inhibition of Rheb activity in Tsc1-deficient astrocytes using either pharmacologic or genetic strategies markedly reduces S6 activation. Collectively, these observations suggest that TSC inactivation in astrocytes results in defective cell size regulation associated with dysregulated Rheb/mTOR/S6K pathway activity., (Copyright 2004 Wiley-Liss, Inc.)

Many cholinergic parasympathetic and enteric neurons require neurturin signaling through GDNF family receptor GFRalpha2 for target innervation. Since a distinct minority of sympathetic neurons are cholinergic, we examined whether GFRalpha2 is important for their development. We detected GFRalpha2 in neonatal sympathetic cholinergic neurons and neurturin mRNA in their target tissues, sweat glands in footpads, and periosteum. Lack of GFRalpha2 in mice did not affect the number of sympathetic cholinergic neurons, but their soma size was decreased in comparison to wild types. In adult and in 3-week-old GFRalpha2 knockout mice, the density of sympathetic cholinergic innervation was reduced by 50-70% in the sweat glands, and was completely absent in the periosteum. Sympathetic noradrenergic innervation of blood vessels in the footpads was unchanged. The density of sympathetic axons in sweat glands was unaffected at postnatal day P4 reflecting successful growth into the target area. Our results indicate that the cholinergic subpopulation of sympathetic neurons requires GFRalpha2 signaling for soma size and for growth or maintenance of target innervation. Thus, neurturin may be a general target-derived innervation factor for postganglionic cholinergic neurons in all parts of the autonomic nervous system., (Copyright 2004 Elsevier Inc.)

In order to furnish a combined model of relevance to human inclusion-body myopathy and Alzheimer's disease, transgenic mice expressing human betaAPP-C99 in skeletal muscle and brain under the control of the cytomegalovirus/beta-actin promoter were produced (Tg13592). These transgenic mice develop Abeta deposits in muscles but not in brain. Cell metabolic activity was analyzed in brain regions and muscle by cytochrome oxidase (CO) histochemistry, the terminal enzyme of the electron transport chain. By comparison to age-matched controls of the C57BL/6 strain, CO activity was selectively increased in dark skeletal muscle fibers of Tg13592 mice. In addition, only increases in CO activity were obtained in those brain regions where a significant difference appeared. The CO activity of Tg13592 mice was elevated in several thalamic nuclei, including laterodorsal, ventromedial, and midline as well as submedial, intralaminar, and reticular. In contrast, the groups did not differ in most cortical regions, except for prefrontal, secondary motor, and auditory cortices, and in most brainstem regions, except for cerebellar (fastigial and interpositus) nuclei and related areas (red and lateral vestibular nuclei). No variation in cell density and surface area appeared in conjunction with these enzymatic alterations. The overproduction of betaAPP-C99 fragments in brain without (amyloidosis did not appear to affect the metabolic activity of structures particularly vulnerable in Alzheimer's disease.

In support of leptin's physiological role as humoral signal of fat mass, we have shown that adipocyte volume is a predominant determinant of leptin mRNA levels in anatomically distinct fat depots in lean young mice in the postabsorptive state. In this report, we investigated how obesity may affect the relationship between leptin mRNA levels and adipocyte volume in anatomically distinct fat depots in mice with genetic (Lep(ob)/Lep(ob) and A(y)/+), diet-induced, and aging-related obesity. In all of the obese mice examined, tissue leptin mRNA levels relative to the average adipocyte volume were lower in the perigonadal and/or retroperitoneal than in the inguinal fat depots and were lower than those of the lean young mice in the perigonadal fat depot. A close, positive correlation between leptin mRNA level and adipocyte volume was present from small to hypertrophic adipocytes within each perigonadal and inguinal fat pad in the obese mice, but the slopes of the regression lines relating leptin mRNA level to adipocyte volume were significantly lower in the perigonadal than in the inguinal fat pads of the same mice. These results suggest that obesity per se is associated with a decreased leptin gene expression per unit of fat mass in mice and that the positive correlation between leptin mRNA level and adipocyte volume is an intrinsic property of adipocytes that is not disrupted by adipocyte hypertrophy in obese mice.

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.

Cyclin-dependent kinase (CDK) activity initiates the eukaryotic cell division cycle by turning on a suite of gene expression in late G1 phase. In metazoans, CDK-dependent phosphorylation of the retinoblastoma tumor suppressor protein (Rb) alleviates repression of E2F and thereby activates G1/S transcription. However, in yeast, an analogous G1 phase target of CDK activity has remained elusive. Here we show that the cell size regulator Whi5 inhibits G1/S transcription and that this inhibition is relieved by CDK-mediated phosphorylation. Deletion of WHI5 bypasses the requirement for upstream activators of the G1/S transcription factors SBF/MBF and thereby accelerates the G1/S transition. Whi5 is recruited to G1/S promoter elements via its interaction with SBF/MBF in vivo and in vitro. In late G1 phase, CDK-dependent phosphorylation dissociates Whi5 from SBF and drives Whi5 out of the nucleus. Elimination of CDK activity at the end of mitosis allows Whi5 to reenter the nucleus to again repress G1/S transcription. These findings harmonize G1/S control in eukaryotes.

Neuronal apoptosis is a key component in the sculpting of tissues during embryonic and postnatal development and is driven largely by the action of caspases. In the mouse olfactory system, caspase-3 and -9 are expressed in olfactory receptor neurons (ORNs) of adult mice, and their selective retrograde activation drives ORN apoptosis following ablation of their target, the olfactory bulb (OB). Here, we show that both of these caspases are expressed at the earliest stages of ORN embryonic development, and their expression is concentrated in outgrowing ORN axons. The retention, in null mice for both caspases, of a population of ORNs that would normally undergo developmental apoptosis beginning at E13 of development, results in a permanently expanded population of ORNs. In turn, in some caspase-3 null mice, the ORN target organ, the OB, also develops abnormally, resulting in the formation of secondary, apparently functional, extracranial ectopic OBs., (Copyright 2004 Wiley-Liss, Inc.)

The FGF receptor Heartless (HTL) is required for mesodermal cell migration in the Drosophila gastrula. We show that mesoderm cells undergo different phases of specific cell shape changes during mesoderm migration. During the migratory phase, the cells adhere to the basal surface of the ectoderm and exhibit extensive protrusive activity. HTL is required for the protrusive activity of the mesoderm cells. Moreover, the early phenotype of htl mutants suggests that HTL is required for the adhesion of mesoderm cells to the ectoderm. In a genetic screen we identified pebble (pbl) as a novel gene required for mesoderm migration. pbl encodes a guanyl nucleotide exchange factor (GEF) for RHO1 and is known as an essential regulator of cytokinesis. We show that the function of PBL in cell migration is independent of the function of PBL in cytokinesis. Although RHO1 acts as a substrate for PBL in cytokinesis, compromising RHO1 function in the mesoderm does not block cell migration. These data suggest that the function of PBL in cell migration might be mediated through a pathway distinct from RHO1. This idea is supported by allele-specific differences in the expressivity of the cytokinesis and cell migration phenotypes of different pbl mutants. We show that PBL is autonomously required in the mesoderm for cell migration. Like HTL, PBL is required for early cell shape changes during mesoderm migration. Expression of a constitutively active form of HTL is unable to rescue the early cellular defects in pbl mutants, suggesting that PBL is required for the ability of HTL to trigger these cell shape changes. These results provide evidence for a novel function of the Rho-GEF PBL in HTL-dependent mesodermal cell migration.

A synRas mouse model was used expressing constitutively activated Ha-Ras (Val12 mutation) in neurons to investigate the role of Ras-MAPkinase signalling for neuronal connectivity in adult brain. Expression of the transgene in the cortex of these mice starts after neuronal differentiation is completed and allows to directly investigate the effects of enhanced Ras activity in differentiated neurons. Activation of Ha-Ras induced an increase in soma size which was sensitive to MEK inhibitor in postnatal organotypic cultures. Adult cortical pyramidal neurons showed complex structural rearrangements associated with an increased size and ramification of dendritic arborization. Dendritic spine density was elevated and correlated with a twofold increase in number of synapses. In acute brain slices of the somatosensory and of the visual cortex, extracellular field potentials were recorded from layer II/III neurons. The input-output relation of synaptically evoked field potentials revealed a significantly higher basal excitability of the transgenic mice cortex compared to wild-type animals. In whole cell patch clamp preparations, the frequency of AMPA receptor-mediated spontaneous excitatory postsynaptic currents was increased while the ratio between NMDA and AMPA-receptor mediated signal amplitude was unchanged. A pronounced depression of paired pulse facilitation indicated that Ras contributes to changes at the presynaptic site. Furthermore, synRas mice showed an increased synaptic long-term potentiation, which was sensitive to blockers of NMDA-receptors and of MEK. We conclude that neuronal Ras is a common switch of plasticity in adult mammalian brain sculpturing neuronal architecture and synaptic connectivity in concert with tuning synaptic efficacy.

The process of vascular smooth muscle cell (vSMC) differentiation is critical to embryonic angiogenesis. However, despite its importance, the vSMC differentiation program remains largely undefined. Murine gene disruption studies have identified several gene products that are necessary for vSMC differentiation, but these methodologies cannot establish whether or not a factor is sufficient to initiate the differentiation program. A gain-of-function system consisting of normal vSMC progenitor cells would serve as a useful complement to whole animal loss-of-function studies. We use such a system here, namely freshly isolated rat neural crest stem cells (NCSCs), to show that activation of the calcineurin signaling pathway is sufficient to drive these cells toward a smooth muscle fate. In addition, we present data suggesting that transforming growth factor (TGF)-beta1, which also causes NCSCs to differentiate into smooth muscle, activates calcineurin signaling in NCSCs, leading to a model in which activation of calcineurin signaling is the mechanism by which TGF-beta1 causes SMC differentiation in these cells.

Immunohistochemistry for parvalbumin, a marker for primary proprioceptors, was performed on the dorsal root ganglion (DRG) of wildtype and knockout mice for Brn-3a at postnatal day 0 and embryonic day 18.5. The DRG contained many parvalbumin-immunoreactive (ir) neurons in wildtype (5.4%) and knockout mice (5.6%). Cell size analysis demonstrated that such neurons were mostly medium-sized to large in these mice. Therefore, it is unlikely that the survival of proprioceptors is dependent upon Brn-3a in the DRG. In the dorsal column and gray matter of the spinal cord of knockout mice, however, parvalbumin-ir nerve fibers were sparse compared to wildtype mice. The number of parvalbumin-ir varicosities around motoneurons decreased in the mutant. Thus, our data suggest that Brn-3a may play an important role in the central projection and terminal formation of DRG proprioceptors in the spinal cord.

B-1 cells, distinguishable from conventional B-2 cells by their cell surface marker, anatomical location, and self-replenishing activity, play an important role in innate immune responses. B-1 cells constitutively express the IL-5R alpha-chain (IL-5Ralpha) and give rise to Ab-producing cells in response to various stimuli, including IL-5 and LPS. Here we report that the IL-5/IL-5R system plays an important role in maintaining the number and the cell size as well as the functions of mature B-1 cells. The administration of anti-IL-5 mAb into wild-type mice, T cell-depleted mice, or mast cell-depleted mice resulted in reduction in the total number and cell size of B-1 cells to an extent similar to that of IL-5Ralpha-deficient (IL-5Ralpha(-/-)) mice. Cell transfer experiments have demonstrated that B-1 cell survival in wild-type mice and homeostatic proliferation in recombination-activating gene 2-deficient mice are impaired in the absence of IL-5Ralpha. IL-5 stimulation of wild-type B-1 cells, but not IL-5Ralpha(-/-) B-1 cells, enhances CD40 expression and augments IgM and IgG production after stimulation with anti-CD40 mAb. Enhanced IgA production in feces induced by the oral administration of LPS was not observed in IL-5Ralpha(-/-) mice. Our results illuminate the role of IL-5 in the homeostatic proliferation and survival of mature B-1 cells and in IgA production in the mucosal tissues.

Lineage-marker depleted (Lin(-)) murine bone marrow cells expressing stem cell antigen 1 (Sca-1) were sorted on the basis of stem cell factor receptor (c-kit) expression to obtain Lin(-)Sca(+)Kit(+) or Lin(-)Sca(+)Kit(-) cells. Lin(-)Sca(+)Kit(-) cells have a markedly greater chemotactic response to stromal derived factor-1 (SDF-1). Using a novel fluorescent stain, we show that both populations generate similar levels of a key messenger, phosphatidylinositol 3,4,5 trisphosphate (PIP(3)), in response to SDF-1. Differences in motile behavior may therefore lie downstream of phosphatidylinositol 3-kinase (PI3-kinase) activation at the level of cytoskeleton regulation. The 2 cell populations were compared using 2-dimensional difference gel electrophoresis (2D-DIGE), with a maleimide CyDye fluorescent protein labeling technique that has enhanced sensitivity for low abundance samples. Comparative proteomic analysis of Cy3- and Cy5-labeled protein samples allows relative quantification of protein spots present in both cell populations; of these, 73% were common. Key protein differences were adseverin and gelsolin, actin micro-filament splicing proteins, regulated by Rac, downstream of PI3-kinase activation. Adseverin was shown to be acetylated, a novel modification for this protein. Differences in major regulators of cell shape and motility between the 2 populations can explain the differential response to SDF-1.

Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo., (Copyright the Rockefeller University Press)

Rat cortical astrocytes regulate their cell volume in response to hypotonic challenge. This regulation is believed to depend largely on the release of chloride or organic osmolytes through anion channels. Using whole-cell recordings, we identified weakly outwardly rectifying chloride currents that could be activated in response to hypotonic challenge. These currents exhibited the following permeability sequence upon replacement of chloride in the bathing solution with various anions: I- > NO3- > Cl- > Gluc- > or = MeS- > Ise-. Interestingly, extracellular I-, albeit showing the greatest permeability, blocked the currents with an IC50 of approximately 50 mM. Currents were almost completely inhibited by 123 microM NPPB and partially inhibited by 200 microM niflumic acid or 200 microM DIDS. Additionally, the total number of Cl- ions effluxed through the hypotonically activated channels was markedly similar to the total solute efflux during volume regulation. We therefore propose the hypotonically activated chloride channel as a major contributor to volume regulation of astrocytes. To examine potential candidate chloride channel genes expressed by astrocytes, we employed RT-PCR to demonstrate the presence of transcripts for ClC-2, 3, 4, 5, and 7, as well as for VDAC and CFTR in cultured astrocytes. Moreover, we performed immunostaining with antibodies against each of these channels and showed the strongest expression of ClC-2 and ClC-3, strong expression of ClC-5 and VDAC, weak expression of ClC-7 and very weak expression of ClC-4 and CFTR. Intriguingly, although we found at least seven Cl- channel proteins from three different gene families in astrocytes, none appeared to be active in resting cells., (Copyright 2004 Wiley-Liss, Inc.)

Down syndrome is the most frequent genetic cause of mental retardation, having an incidence of 1 in 700 live births. In the present study we used a transgenic mouse in vivo library consisting of 4 yeast artificial chromosome (YAC) transgenic mouse lines, each bearing a different fragment of the Down syndrome critical region 1 (DCR-1), implicated in brain abnormalities characterizing this pathology. The 152F7 fragment, in addition to genes also located on the other DCR-1 fragments, bears the DYRK1A gene, encoding for a serine-threonine kinase. The neurobehavioral analysis of these mouse lines showed that DYRK1A overexpressing 152F7 mice but not the other lines display learning impairment and hyperactivity during development. Additionally, 152F7 mice display increased brain weight and neuronal size. At a biochemical level we found DYRK1A overexpression associated with a development-dependent increase in phosphorylation of the transcription factor FKHR and with high levels of cyclin B1, suggesting for the first time in vivo a correlation between DYRK1A overexpression and cell cycle protein alteration. In addition, we found an altered phosphorylation of transcription factors of CREB family. Our findings support a role of DYRK1A overexpression in the neuronal abnormalities seen in Down syndrome and suggest that this pathology is linked to altered levels of proteins involved in the regulation of cell cycle.

Background: Mesoderm migration in the Drosophila gastrula depends on the fibroblast growth factor (FGF) receptor Heartless (Htl). During gastrulation Htl is required for adhesive interactions of the mesoderm with the ectoderm and for the generation of protrusive activity of the mesoderm cells during migration. After gastrulation Htl is essential for the differentiation of dorsal mesodermal derivatives. It is not known how Htl is activated, because its ligand has not yet been identified., Results: We performed a genome-wide genetic screen for early zygotic genes and identified seven genomic regions that are required for normal migration of the mesoderm cells during gastrulation. One of these genomic intervals produces upon its deletion a phenocopy of the htl cell migration phenotype. Here we present the genetic and molecular mapping of this genomic region. We identified two genes, FGF8-like1 and FGF8-like2, that encode novel FGF homologs and were only partially annotated in the Drosophila genome. We show that FGF8-like1 and FGF8-like2 are expressed in the neuroectoderm during gastrulation and present evidence that both act in concert to direct cell shape changes during mesodermal cell migration and are required for the activation of the Htl signaling cascade during gastrulation., Conclusions: We conclude that FGF8-like1 and FGF8-like2 encode two novel Drosophila FGF homologs, which are required for mesodermal cell migration during gastrulation. Our results suggest that FGF8-like1 and FGF8-like2 represent ligands of the Htl FGF receptor.

Semaphorins are known to play an important role in axon guidance of vertebrate olfactory sensory neurons to their targets in specific glomeruli of the olfactory bulb (OB). However, it is not clear how semaphorin-mediated guidance contributes to a systematic hierarchy of cues that govern the organization of this system. Because of the putative role that odorant receptor molecules such as P2 could play in establishing appropriate glomerular destinations for growing olfactory axons, we have also determined the spatial organization of P2 glomeruli in semaphorin 3A (Sema3A) mutant mice. First, in the postnatal OB of control and Sema3A(-/-) mice, we analysed the trajectories of olfactory axons that express the Sema3A receptor, neuropilin-1 (npn-1) and the positions of npn-1(+) glomeruli. Sema3A at the ventral OB midline guides npn-1(+) axons to targets in the lateral and medial OB. Absence of Sema3A permits many npn-1 axons to terminate aberrantly in the rostral and ventral OB. Second, in Sema3A(-/-) mice, many P2 axons are abnormally distributed throughout the ventral OB nerve layer and converge in atypical locations compared with littermate controls where P2 axons converge on stereotypically located lateral and medial glomeruli. In addition to their radically altered spatial distribution, P2 glomeruli in Sema3A(-/-) mice are significantly smaller and more numerous than in heterozygote littermates. These data show that Sema3A is an important repulsive olfactory guidance cue that establishes restricted npn-1(+) subcompartments in the olfactory bulb. Furthermore, Sema3A plays a key role in the convergence of axons expressing the odorant receptor P2 onto their appropriate targets.

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Galpha subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.

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.

Although the mechanisms of cell cycle control are well established, the factors controlling cell growth and target size are still poorly understood. Much evidence suggests that ribosome biogenesis, and in particular the synthesis of the rRNAs, plays a central role not only in permitting growth, but also in regulating it. In the past few years we have begun to penetrate the network linking rRNA gene transcription to growth.

In this paper, we describe the characterization of DEF6, a novel PH-DH-like protein related to SWAP-70 that functions as an upstream activator of Rho GTPases. In NIH 3T3 cells, stimulation of the PI 3-kinase signaling pathway with either H2O2 or platelet-derived growth factor (PDGF) resulted in the translocation of an overexpressed DEF6-GFP fusion protein to the cell membrane and induced the formation of filopodia and lamellipodia. In contrast to full-length DEF6, expression of the DH-like (DHL) domain as a GFP fusion protein potently induced actin polymerization, including stress fiber formation in COS-7 cells, in the absence of PI 3-kinase signaling, indicating that it was constitutively active. The GTP-loading of Cdc42 was strongly enhanced in NIH 3T3 cells expressing the DH domain while filopodia formation, membrane ruffling, and stress fiber formation could be inhibited by the co-expression of the DH domain with dominant negative mutants of either N17Rac1, N17Cdc42, or N19RhoA, respectively. This indicated that DEF6 acts upstream of the Rho GTPases resulting in the activation of the Cdc42, Rac1, and RhoA signaling pathways. In vitro, DEF6 specifically interacted with Rac1, Rac2, Cdc42, and RhoA, suggesting a direct role for DEF6 in the activation of Rho GTPases. The ability of DEF6 to both stimulate actin polymerization and bind to filamentous actin suggests a role for DEF6 in regulating cell shape, polarity, and movement.

To identify new nonessential genes that affect genome integrity, we completed a screening for diploid mutant Saccharomyces cerevisiae strains that are sensitive to ionizing radiation (IR) and found 62 new genes that confer resistance. Along with those previously reported (Bennett et al., Nat. Genet. 29:426-434, 2001), these genes bring to 169 the total number of new IR resistance genes identified. Through the use of existing genetic and proteomic databases, many of these genes were found to interact in a damage response network with the transcription factor Ccr4, a core component of the CCR4-NOT and RNA polymerase-associated factor 1 (PAF1)-CDC73 transcription complexes. Deletions of individual members of these two complexes render cells sensitive to the lethal effects of IR as diploids, but not as haploids, indicating that the diploid G1 cell population is radiosensitive. Consistent with a role in G1, diploid ccr4Delta cells irradiated in G1 show enhanced lethality compared to cells exposed as a synchronous G2 population. In addition, a prolonged RAD9-dependent G1 arrest occurred following IR of ccr4Delta cells and CCR4 is a member of the RAD9 epistasis group, thus confirming a role for CCR4 in checkpoint control. Moreover, ccr4Delta cells that transit S phase in the presence of the replication inhibitor hydroxyurea (HU) undergo prolonged cell cycle arrest at G2 followed by cellular lysis. This S-phase replication defect is separate from that seen for rad52 mutants, since rad52Delta ccr4Delta cells show increased sensitivity to HU compared to rad52Delta or ccr4Delta mutants alone. These results indicate that cell cycle transition through G1 and S phases is CCR4 dependent following radiation or replication stress.

Highwire (Hiw), a putative RING finger E3 ubiquitin ligase, negatively regulates synaptic growth at the neuromuscular junction (NMJ) in Drosophila. hiw mutants have dramatically larger synaptic size and increased numbers of synaptic boutons. Here we show that Hiw binds to the Smad protein Medea (Med). Med is part of a presynaptic bone morphogenetic protein (BMP) signaling cascade consisting of three receptor subunits, Wit, Tkv, and Sax, in addition to the Smad transcription factor Mad. When compared to wild-type, mutants of BMP signaling components have smaller NMJ size, reduced neurotransmitter release, and aberrant synaptic ultrastructure. BMP signaling mutants suppress the excessive synaptic growth in hiw mutants. Activation of BMP signaling, which in wild-type does not cause additional growth, in hiw mutants does lead to further synaptic expansion. These results reveal a balance between positive BMP signaling and negative regulation by Highwire, governing the growth of neuromuscular synapses.

Background: Pulmonary papillary adenoma is a benign pulmonary neoplasm. Previously pulmonary papillary adenoma was described in terms of immunohistochemistry and ultrastructure. However, there are no previous reports describing the cytologic characteristics of pulmonary papillary adenoma., Case: A 50-year-old male was admitted for evaluation of a coin lesion in the left upper lung field. Radiologic images showed a solid, round tumor approximately 25 mm in diameter in the left upper lung. Transbronchial needle aspiration biopsy (TBNA) was performed, and small numbers of atypical cells were collected. Adenocarcinoma was suggested clinically, and left upper segmentectomy was performed. The histologic diagnosis was pulmonary papillary adenoma. Imprint cytology of the cut surface of the tumor showed tumor cells arranged in sheets that contained scant or vesicular cytoplasm. The nuclei were oval or round, without obvious anisokaryosis, and their chromatin was fine, without hyperchromasia. Cytologically, the nuclei of the tumor cells in the imprint specimen (38.70 +/- 8.69 microns 2) were uniform in size and similar to the atypical cells in the TBNA specimen (38.29 +/- 11.56 microns 2) but significantly larger than the nuclei of the bronchial cells (23.61 +/- 5.98 microns 2) (P < .0001)., Conclusion: The cytologic appearance of pulmonary papillary adenoma was characterized morphologically and morphometrically. The possibility of cytodiagnosis by TBNA was suggested.

Heat shock protein (hsp) 56 (hsp56) is an immunophilin that acts as a cofactor with hsp90 and exhibits both peptidyl-prolyl isomerase activity and chaperone activity. Previous studies have shown that the hypertrophic effect of cardiotrophin-1 (CT-1) in cardiac cells is dependent on hsp56 induction. CT-1 activates a number of signal-transduction pathways. Therefore, we sought to determine whether these pathways were also important for hsp56-induced hypertrophy using overexpression with transiently transfected plasmid vectors in rat neonatal cardiomyocytes. Here we show that multiple signalling pathways are involved in hsp56-induced hypertrophy, namely the Janus kinase-signal-transducer and activator of transcription, extracellular signal-regulated protein kinase and PI3-kinase/Akt signalling pathways. Dominant-negative mutants and inhibitors of these pathways were able to block the hypertrophy observed as a result of hsp56 overexpression. However, an inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway was not able to block the hypertrophic effect of hsp56 overexpression. Furthermore, we show that domains I, II and IV of the hsp56 protein may be involved in its hypertrophic effect. These studies show for the first time that multiple signalling pathways are involved in the hypertrophic effect of hsp56 and that overexpression of hsp56 itself is able to activate the necessary signalling pathways, which induce hypertrophy.

The molecular mechanisms that control dendritic development are still unclear. Semaphorin 3A, a class III semaphorin, has been shown to regulate the radial orientation of pyramidal neurons in the developing neocortex. Here, we investigate the effects of Sema3A on the development of dendritic topology. Neocortical slices from Sema3A null mutant mice were cultured and neurons were transfected with GFP, reconstructed, and compared with neurons from wild-type and heterozygote littermates. We also added exogenous Sema3A to cultured wild-type neocortical slices to further test its effects on dendritic development. We document reductions in dendritic length and branching in Sema3A null mice and increases in dendritic length and branching after the addition of exogenous Sema3A to wild-type neurons. We conclude that Sema3A is necessary for the elaboration of second and third order dendritic branches in pyramidal neurons., (Copyright 2003 Wiley Periodicals, Inc. J Neurobiol 58: 403-412, 2004)

Mouse embryonic stem (ES) cells are isolated from the inner cell mass (ICM)/epiblast of preimplantation embryos and are widely used in cell differentiation studies. We have previously observed differences in transcript and antigen expression following differentiation of ES cells lines in vitro. We have investigated this further by comparing the differentiation characteristics of five independently derived ES cell lines cultured and differentiated under defined conditions. Undifferentiated ES cell lines exhibited similar morphology and antigen/transcript marker expression. However, upon differentiation in monolayer culture by LIF withdrawal, only two of the lines expressed similar germ layer transcript profiles, and these were significantly altered compared to differentiation in serum-supplemented media. Neurofilament-68k was the only transcript marker common to all cell lines, however, induction of neuroectoderm lineages using 1 microM all-trans retinoic acid (RA) resulted in significant variations in cell number and morphology between the lines. Furthermore, neurons were only formed from clones of the two cell lines that exhibited similar transcript profiles, although the morphology was different between the two. We conclude that the independent ES cell lines in this study differ in their response to alterations in culture conditions in vitro, and the use of an appropriate cell line enables relatively homogeneous neuronal populations to be achieved in monolayer culture under defined conditions.

Morphological features of interneuronal adaptation to an altered, more complex neuronal architecture have been investigated in p21H-Ras(Val12) transgenic mice. This transgenic strain serves as a model for studying the morphogenetic role of the G-protein p21Ras on cortical principal neurons. We have recently demonstrated that postmitotic expression of constitutively active p21H-Ras(Val12) in the neocortical pyramidal cell population results in increased size and dendritic complexity of the affected neurons, leading to an enlarged cortical volume. Interneurons do not express the transgene and are therefore excluded from direct, intrinsic p21H-Ras(Val12) effects. In the present study, immunolabelling of gamma-amino-butyric-acid (GABA), and of the calcium-binding proteins parvalbumin, calbindin and calretinin revealed that in the transgenic mice local circuit neurons are not increased in either somal size or number and their main morphological characteristics are preserved. However, the dendritic arbour of interneurons was found to be extended, at least in the vertical dimension, to follow the cortical expansion. Immunostaining for the vesicular GABA transporter revealed a denser inhibitory innervation of p21H-Ras(Val12)-expressing pyramidal cell perikarya than in those of wild-type animals, while the overall density of inhibitory axon terminals within the cortex was decreased in the transgenic animals as a consequence of cortical expansion. The findings of the present study demonstrate the morphogenetic capacity of interneurons for adapting to morphological alterations of principal neurons in the cerebral cortex.

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.

Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell-cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca(2+)-dependent cell-cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

Oxidative stress is thought to be a major contributor to the progress of the Parkinson's Disease (PD) because of the high vulnerability of dopaminergic cells against oxidative stress. The present work demonstrates that with the expression of the baculovirus p35 gene, PC12 cells could gain a high resistance against oxidative toxicants, hydrogen peroxide (H(2)O(2)) and 6-hydroxydopamine (6-OHDA). The DNA fragmentation analysis showed that PC12 cells underwent apoptosis after exposure to H(2)O(2) or 6-OHDA, while PP35 cells, a p35-expressing PC12 cell line, did not. Flow cytometric analysis showed that treatment with 150 microM H(2)O(2) or 120 microM 6-OHDA for 24 h caused 52.86% or 66.36% apoptotic cell, respectively, in PC 12 cells, but only 4.26% or 5.80% in PP35 cells. The cell viability measured by 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay indicated that H(2)O(2) and 6-OHDA induced a dose-dependent cell death on PC12 cells that were greatly remitted on PP35 cells. The viability of PP35 cells was even stronger than that of PC12 cells protected by glial cell line deprived neurotrophic factor (GDNF). The surviving PP35 cells remained normal cell morphology and showed positive with tyrosine hydroxylase (TH) immunocytochemical staining. These results indicate that baculovirus p35 gene possesses remarkable ability to rescue PC12 cells from death in experimental paradigms associated with oxidative stress.

The small G protein p21Ras is a key signal transducer mediating cellular growth and proliferation responses to extracellular stimuli. We investigated by electron microscopy the effects of augmented p21Ras activity on neuronal processes and microtubule arrangement in vivo. We used transgenic mice with a neuron-specific overexpression of p21H-RasVal12, which starts postnatally around Day 15. Axonal and dendritic diameters and the numerical density of dendritic microtubules were analyzed at postnatal Day 12 before the onset of transgene expression and in adult mice. In adult transgenic mice, calibers of both axons (corpus callosum) and dendrites (layers II/III of somatosensory cortex) were enlarged by about 57% and 79%, respectively. The increase in dendritic calibers was associated with an increment in the amount of microtubules. Even in dendrites of equivalent diameters, the number of microtubules was higher in transgenic mice compared to that in wild-type mice suggesting an elevated microtubule density. Changes in process diameters or microtubule density were not observed at postnatal Day 12 before relevant transcription of transgenic p21H-RasVal12. The present results extend previous findings on neuronal hypertrophy as a consequence of p21H-RasVal12 expression and suggest a profound influence on the dendritic microtubule network., (Copyright 2003 Wiley-Liss, Inc.)

Cells undergoing endoreduplication replicate chromosomal DNA without intervening mitoses. The resulting larger, higher-ploidy nucleus is often associated with an increase in cell size, but the molecular basis for this correlation remains poorly understood. Recent advances in characterising various mutants and transgenic plants are beginning to unravel how this unique type of cell cycling is regulated and how it contributes to cell-size control. Both cell growth (i.e. increase in cytoplasmic macromolecular mass) and cell expansion (i.e. increase in cell volume through vacuolation) contribute independently to increases in cell size in plants. A total organ-size checkpoint may also help to coordinate cell size and cell number within an organ, and can contribute to final cell-size determination in plants.

Accumulation of noncoding DNA and therefore genome size (C-value) may be under strong selection toward increase of body size accompanied by low metabolic costs. C-value directly affects cell size and specific metabolic rate indirectly. Body size can enlarge through increase of cell size and/or cell number, with small cells having higher metabolic rates. We argue that scaling exponents of interspecific allometries of metabolic rates are by-products of evolutionary diversification of C-values within narrow taxonomic groups, which underlines the participation of cell size and cell number in body size optimization. This optimization leads to an inverse relation between slopes of interspecific allometries of metabolic rates and C-value. To test this prediction we extracted literature data on basal metabolic rate (BMR), body mass, and C-value of mammals and birds representing six and eight orders, respectively. Analysis of covariance revealed significant heterogeneity of the allometric slopes of BMR and C-value in both mammals and birds. As we predicted, the correlation between allometric exponents of BMR and C-value was negative and statistically significant among mammalian and avian orders.

DNA repair is essential for the maintenance of genomic integrity and stability. Nucleotide excision repair (NER) is a major pathway responsible for remediation of damage caused by UV light, bulky adducts, and cross-linking agents. We now show that NER capacity is differentially expressed in human tissues. We established primary cultures of peripheral blood lymphocytes (PBLs: N = 33) and foreskin fibroblasts (FF: N = 6), as well as adult breast tissue (N = 22) using a unique culture system, and measured their NER capacity using the unscheduled DNA synthesis (UDS) functional assay. Relative to FF, primary cultures of breast cells exhibited only 24.6 +/- 2.1% of NER capacity and PBLs only 8.9 +/- 1.2%. Cells from the breast therefore have a unique and distinctive DNA repair capacity. The NER capacities of all three cell types had similar coefficients of variation in the range of 10%-15%, which should be taken into account when running controls for this contextual assay. Unlike previous studies and speculation in the field, we found that NER was not affected by cell morphology, donor age, or proliferation as measured by the S phase index. While the NER capacity of the transformed lymphoblastoid cell line TK6 was within the range of our PBL samples, the breast tumor-derived MDA MB-231 cell line was four-fold higher than normal breast tissue. These studies show that analysis of baseline DNA repair in normal human cell types is critical as a basis for evaluation of the effects of "mutator" genes as etiological factors in the development of cancer.

Previously, we identified a novel rice gene, GROWTH-REGULATING FACTOR1 (OsGRF1), which encodes a putative transcription factor that appears to play a regulatory role in stem elongation. We now describe the GRF gene family of Arabidopsis thaliana (AtGRF), which comprises nine members. The deduced AtGRF proteins contain the same characteristic regions--the QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) domains--as do OsGRF1 and related proteins in rice, as well as features indicating a function in transcriptional regulation. Most of the AtGRF genes are strongly expressed in actively growing and developing tissues, such as shoot tips, flower buds, and roots, but weakly in mature stem and leaf tissues. Overexpression of AtGRF1 and AtGRF2 resulted in larger leaves and cotyledons, as well as in delayed bolting of the inflorescence stem when compared to wild-type plants. In contrast, triple insertional null mutants of AtGRF1-AtGRF3 had smaller leaves and cotyledons, whereas single mutants displayed no changes in phenotype and double mutants displayed only minor ones. The alteration of leaf growth in overexpressors and triple mutants was based on an increase or decrease in cell size, respectively. These results indicate that AtGRF proteins play a role in the regulation of cell expansion in leaf and cotyledon tissues.

The polycotyledon mutant of tomato (Lycopersicon esculentum L. cv Ailsa Craig) showed altered development during embryogenesis and during vegetative and reproductive phases. The phenotype was pleiotropic and included the formation of extra cotyledons, changes in leaf shape, increased number of flowers (indeterminacy) with abnormal floral organs, the formation of epiphyllous structures, and altered gravitropism. The earliest defects were observed at the transition from the globular to the heart stage of embryogenesis with the formation of multiple cotyledons. Epidermal cells in the mutant embryo were smaller and less expanded compared with wild type. Examination of polar auxin transport (PAT) showed a striking enhancement in the case of the mutant. Increase in PAT did not appear to be caused by a decrease in flavonoids because the mutant had normal flavonoid levels. Application of 2,3,5-triiodobenzoic acid, an inhibitor of polar transport of auxin, rescued postgermination phenotypes of young seedlings. Our analysis reveals a level of control that negatively regulates PAT in tomato and its contribution to plant development and organogenesis.

We have identified a novel form of cardiac myocyte enlargement in nonmuscle myosin heavy chain II-B (NMHC II-B) ablated mice, based on a partial failure in cytokinesis. In contrast to most cells, cardiac myocytes lack NMHC II-A, and ablation of NMHC II-B results in a heart with 70% fewer myocytes at embryonic day 14.5 (E14.5) than control mice (B+/B- and B+/B+). In addition, B-/B- cardiac myocytes show a marked increase in binucleation at E12.5, reflecting the occurrence of karyokinesis in the absence of cytokinesis. An increase in binucleation and cell size is also found in hypomorphic, homozygous mice harboring a single amino acid mutation (R709C) in the gene encoding NMHC II-B. The nonmyocytes in B-/B- hearts and homozygous mutant hearts, all of which contain NMHC II-A, do not show either of these abnormalities. B-/B- cardiac myocytes at E14.5 show a decreased bromodeoxyuridine (BrdU) labeling index compared with controls, consistent with the decrease in myocyte proliferation. This decreased BrdU labeling is not seen in nonmyocyte cells in the heart. In addition to these changes, both B-/B- mice as well as homozygous mutated mice show an increase in cyclin D2 and D3 reflecting an abnormality in earlier steps in the cell cycle. Whereas cardiac myocytes completely ablated for NMHC II-B show enlargement and binucleation, mice expressing as little as 6% of the normal amount of wild-type NMHC II-B in the heart do not show these abnormalities.

Astrocytes respond to injury of the CNS with a dramatic change in morphology, contributing to the formation of a glial scar. We recently identified a novel actin-associated protein named palladin, which possesses the features of a potent cytoskeletal scaffold. Palladin expression was assayed in two populations of cultured astrocytes, polygonal versus stellate, and was detected at high levels in polygonal astrocytes and low levels in stellate astrocytes. When stellate astrocyte monolayers were wounded, palladin was rapidly upregulated along the edge of the wound, coordinate with an increase in actin assembly. Palladin upregulation occurred along a similar rapid time course following injury to the cerebral cortex of adult rats. To explore palladin function more directly, palladin cDNA was transfected into stellate astrocytes, which acquired a spread morphology and prominent actin bundles. These results suggest that palladin upregulation following injury may be a key step in the acquisition of the reactive astrocyte morphology.

The Src family kinase Lyn has been shown to play both stimulatory and inhibitory roles within several hemopoietic cell types. In this study, we investigated the role played by Lyn in neutrophil integrin signaling. Loss of Lyn resulted in a hyperresponsive phenotype on engagement of surface integrins at low valency. Lyn(-/-) neutrophils displayed enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype when adherent to surfaces coated with either cellular counterreceptors or extracellular matrix proteins. In contrast, Lyn-deficient and wild-type cells expressed similar levels of surface integrins and responded equivalently to activating agents in suspension, indicating that the enhanced responses of lyn(-/-) cells was specific to the integrin signaling pathways. Lyn-deficient macrophages also displayed a hyperadhesive phenotype. Biochemical analysis of macrophages from lyn(-/-) mice revealed that Lyn plays an essential role in the adhesion-dependent phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of the inhibitory receptors SIRP1alpha and PIR-B, which in turn recruit the phosphatase SHP-1. These observations suggest that reduced mobilization of SHP-1 to the membrane in lyn(-/-) neutrophils results in a hyperadhesive and hyperactive phenotype. This hypothesis is further supported by the fact that neutrophils from me(v)/me(v) mice, which have significantly reduced SHP-1 activity, are also hyperresponsive following integrin engagement. This is the first direct evidence using primary leukocytes from lyn(-/-) mice that this kinase functions as a negative regulator in integrin signaling.

During its life cycle, the parasitic protozoon Leishmania mexicana differentiates from a flagellated form, the promastigote, to an amastigote form carrying a rudimentary flagellum. Besides biochemical changes, this process involves a change in overall cell morphology including flagellar shortening. A mitogen-activated protein kinase kinase homologue designated LmxMKK was identified in a homology screening and found to be critically involved in the regulation of flagellar assembly and cell size. LmxMKK is exclusively expressed in the promastigote stage and is likely to be regulated by posttranslational mechanisms such as phosphorylation. A deletion mutant for the single-copy gene revealed motile flagella dramatically reduced in length and lacking the paraflagellar rod, a structure adjacent to the axoneme in kinetoplastid flagella. Moreover, a fraction of the cells showed perturbance of the axonemal structure. Complementation of the deletion mutant with the wild-type gene restored typical promastigote morphology. We propose that LmxMKK influences anterograde intraflagellar transport to maintain flagellar length in Leishmania promastigotes; as such, it is the first protein kinase known to be involved in organellar assembly.