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1.  A microtubule-associated zinc finger protein, BuGZ, regulates mitotic chromosome alignment by ensuring Bub3 stability and kinetochore targeting 
Developmental cell  2014;28(3):268-281.
Equal chromosome segregation requires proper assembly of many proteins, including Bub3, onto kinetochores to promote kinetochore-microtubule interactions. By screening for mitotic regulators in the Spindle envelope and matrix (Spemix), we identify a conserved Bub3 interacting and GLEBS containing ZNF207 (BuGZ) that associates with spindle microtubules and regulates chromosome alignment. Using its conserved GLE-2-Binding Sequence (GLEBS), BuGZ directly binds and stabilizes Bub3. BuGZ also uses its microtubule-binding domain to enhance the loading of Bub3 to kinetochores that have assumed initial interactions with microtubules in prometaphase. This enhanced Bub3 loading is required for proper chromosome alignment and metaphase to anaphase progression. Interestingly, we show that microtubules are required for the highest kinetochore loading of Bub3, BubR1, and CENP-E during prometaphase. These findings suggest that BuGZ not only serves as a molecular chaperone for Bub3 but also enhances its loading onto kinetochores during prometaphase in a microtubule-dependent manner to promote chromosome alignment.
PMCID: PMC3927447  PMID: 24462186
Mitosis; microtubules; spindle envelope and matrix (Spemix); kinetochore; Bub3; BuGZ; ZFP207; ZNF207
2.  CAMSAPs add to the growing microtubule minus end story 
Developmental cell  2014;28(3):221-222.
Free microtubule minus ends, found in many differentiated cells, contribute to polarized motility. Work from Jiang et al. (2014) in this issue of Developmental Cell show how mammalian CAMSAP proteins stabilize minus ends, providing a key piece to the puzzle of how these minus ends are formed and stabilized.
PMCID: PMC3966109  PMID: 24525183
3.  Lipid Modulation of Calcium Flux through CaV2.3 Regulates Acrosome Exocytosis and Fertilization 
Developmental cell  2014;28(3):310-321.
Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca2+ influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3’s pore-forming α1E subunit showed altered Ca2+ responses, reduced AE, and a strong sub-fertility phenotype. Surprisingly, AE depended on spatio-temporal information encoded by flux through CaV2.3—not merely the presence/ amplitude of Ca2+ waves. Using both studies in sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1’s lipid and sugar components and CaV2.3’s α1E and α2δ subunits. Our results provide mechanistic understanding of membrane lipid regulation of Ca2+ flux and therefore Ca2+-dependent cellular and developmental processes such as exocytosis and fertilization.
PMCID: PMC3947087  PMID: 24525187
4.  BuGZ is required for Bub3 stability, Bub1 kinetochore function, and chromosome alignment 
Developmental cell  2014;28(3):282-294.
During mitosis, the spindle assembly checkpoint (SAC) monitors the attachment of kinetochores (KTs) to the plus ends of spindle microtubules (MTs) and prevents anaphase onset until chromosomes are aligned and KTs are under proper tension. Here, we identify a SAC component, BuGZ/ZNF207, from an RNAi viability screen in human Glioblastoma multiforme (GBM) brain tumor stem cells. BuGZ binds to and stabilizes Bub3 during interphase and mitosis through a highly conserved GLE2p-binding sequence (GLEBS) domain. Inhibition of BuGZ results in loss of both Bub3 and its binding partner Bub1 from KTs, reduction of Bub1-dependent phosphorylation of centromeric histone H2A, attenuation of KT-based Aurora kinase B activity, and lethal chromosome congression defects in cancer cells. Phylogenetic analysis indicates that BuGZ orthologs are highly conserved among eukaryotes, but are conspicuously absent from budding and fission yeasts. These findings suggest BuGZ has evolved to facilitate Bub3 activity and chromosome congression in higher eukaryotes.
PMCID: PMC3995079  PMID: 24462187
BuGZ; ZNF207; Bub3; spindle assembly checkpoint; kinetochore; cancer stem cells; Glioblastoma multiforme
5.  An Expanding World of Small RNAs 
Developmental cell  2014;28(2):111-112.
In ciliate protozans, small RNAs (sRNAs) are integral to guiding large-scale genomic rearrangements after mating. Sandoval et al. (2014) report in this issue of Developmental Cell the discovery of a class of Parameciums RNAs, produced by a unqiue Dicer-like enzyme, that likely provides late stage quality control in this process.
PMCID: PMC3949428  PMID: 24480639
6.  Membrane shape modulates transmembrane protein distribution 
Developmental cell  2014;28(2):212-218.
Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown due to the numerous sorting mechanisms taking place concurrently in cells. To isolate the effect of membrane shape, cellsized Giant Unilamellar Vesicles (GUVs) containing either the potassium channel, KvAP, or water channel, AQP0, were used to form membrane nanotubes with controlled radii. While the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. FRAP measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins, and provides a method for determining the effective shape and flexibility of membrane proteins.
PMCID: PMC3954780  PMID: 24480645
7.  Ankyrin-G Directly Binds to Kinesin-1 to Transport Voltage-Gated Na+ Channels into Axons 
Developmental cell  2014;28(2):117-131.
Action potentials propagating along axons require the activation of voltage-gated Na+ (Nav) channels. How Nav channels are transported into axons is unknown. Here we show KIF5/kinesin-1 directly binds to ankyrin-G (AnkG) to transport Nav channels into axons. KIF5 and Nav1.2 channels bind to multiple sites in the AnkG N-terminal domain that contains 24 ankyrin repeats. Disrupting AnkG-KIF5 binding with siRNA or dominant-negative constructs markedly reduced Nav channel levels at the axon initial segment (AIS) and along entire axons, thereby decreasing action potential firing. Live-cell imaging showed that fluorescently-tagged AnkG or Nav1.2 co-transported with KIF5 along axons. Deleting AnkG in vivo or virus-mediated expression of a dominant-negative KIF5 construct specifically decreased the axonal level of Nav but not Kv1.2 channels in the mouse cerebellum. These results indicate AnkG functions as an adaptor to link Nav channels to KIF5 during axonal transport, before anchoring them to the AIS and nodes of Ranvier.
PMCID: PMC3970081  PMID: 24412576
Developmental cell  2014;28(2):203-211.
Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA Polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation, but that Hox proteins are required to trigger efficient transcription.
PMCID: PMC3930922  PMID: 24480644
9.  Basal cell signaling by p63 controls luminal progenitor function and lactation via NRG1 
Developmental cell  2014;28(2):147-160.
The mammary epithelium is organized as a bi-layer of luminal and basal/myoepithelial cells. During pregnancy the luminal compartment expands for milk production, while basal cells are thought to provide structural and contractile support. Here we reveal an unanticipated, pregnancy-specific role of basal epithelia as a central coordinator of lactogenesis. We demonstrate that genetic deletion of the transcription factor p63 (Trp63) gene exclusively within basal cells of the adult gland during pregnancy leads to dramatic defects in luminal cell proliferation and differentiation, resulting in lactation failure. This phenotype is explained by direct transcriptional activation of the EGF-family ligand gene Nrg1 by p63 selectively in basal cells, which is required for luminal ERBB4/STAT5A activation and consequent luminal progenitor cell maturation. Thus, paracrine basal-to-luminal cell signaling, controlled by p63 via NRG1, orchestrates the entire lactation program. Collectively, these findings redefine the paradigm for cellular interactions specifying the functional maturation of the mammary gland.
PMCID: PMC3951056  PMID: 24412575
p63; basal cell; paracrine signaling; lactation; luminal progenitor cells
10.  Stress-Induced Nuclear to Cytoplasmic Translocation of Cyclin C Promotes Mitochondrial Fission in Yeast 
Developmental cell  2014;28(2):161-173.
Mitochondrial morphology is maintained by the opposing activities of dynamin-based fission and fusion machines. In response to stress, this balance is dramatically shifted toward fission. This study reveals that the yeast transcriptional repressor cyclin C is both necessary and sufficient for stress-induced hyper-fission. In response to oxidative stress, cyclin C translocates from the nucleus to the cytoplasm where it is destroyed. Prior to its destruction, cyclin C both genetically and physically interacts with Mdv1p, an adaptor that links the GTPase Dnm1p to the mitochondrial receptor Fis1p. Cyclin C is required for stress-induced Mdv1p mitochondrial recruitment and the efficient formation of functional Dnm1p filaments. Finally, co-immunoprecipitation studies and fluorescence microscopy revealed an elevated association between Mdv1p and Dnm1p in stressed cells that is dependent on cyclin C. This study provides a mechanism by which stress-induced gene induction and mitochondrial fission are coordinated through translocation of cyclin C.
PMCID: PMC3963397  PMID: 24439911
Cdk8; oxidative stress; programmed cell death
11.  Cyclin C: An Inducer of Mitochondrial Division Hidden in the Nucleus 
Developmental cell  2014;28(2):112-114.
In response to cellular stress, mitochondria remodel their structure by organelle division and fusion. In this issue of Developmental Cell, Cooper et al. (2014) report that a nuclear protein, cyclin C, is recruited from nuclei to mitochondria upon oxidative stress and promotes mitochondrial division and apoptosis of the cell.
PMCID: PMC3971902  PMID: 24480640
12.  Fully Interlocking: A Story of Teamwork amongst Breast Epithelial Cells 
Developmental cell  2014;28(2):114-115.
Reporting in Developmental Cell, Forster et al (2014) show that the basal myoepithelial cell layer (via p63) directs the final maturation of the adjacent luminal cell sheet during pregnancy (Forster et al., 2014). Do all mammary epithelial cells both give and take instructions from others to create the milk machine?
PMCID: PMC3990361  PMID: 24480641
13.  [No title available] 
PMCID: PMC4292982  PMID: 25584796
14.  The plasma membrane flattens out to fuel cell surface growth during Drosophila cellularization 
Developmental cell  2013;27(6):648-655.
Cell shape change demands cell surface growth, but how growth is fueled and choreographed is still debated. Here, we use cellularization, the first complete cytokinetic event in Drosophila embryos, to show that cleavage furrow ingression is kinetically coupled to the loss of surface microvilli. We modulate furrow kinetics with RNAi against the Rho1-GTPase regulator slam, and show that furrow ingression controls the rate of microvillar depletion. Finally, we directly track microvillar membrane and see it move along the cell surface and into ingressing furrows, independent of endocytosis. Together, our results demonstrate that the kinetics of the ingressing furrow regulate the utilization of a microvillar membrane reservoir. Since the membrane of the furrow and microvilli are contiguous, we suggest that ingression drives unfolding of the microvilli and incorporation of microvillar membrane into the furrow. We conclude that plasma membrane folding/unfolding can contribute to the cell shape changes that promote embryonic morphogenesis.
PMCID: PMC3888437  PMID: 24316147
15.  Calpain 2 Activation of P-TEFb Drives Megakaryocyte Morphogenesis and Is Disrupted by Leukemogenic GATA1 Mutation 
Developmental cell  2013;27(6):607-620.
Megakaryocyte morphogenesis employs a “hypertrophy-like” developmental program, dependent on P-TEFb kinase activation and cytoskeletal remodeling. P-TEFb activation classically occurs by a feedback regulated process of signal-induced, reversible release of active Cdk9-cyclin T modules from large inactive 7SK snRNP complexes. Here we have identified an alternative pathway of irreversible P-TEFb activation in megakaryopoiesis, mediated by dissolution of the 7SK snRNP complex. In this pathway calpain 2 cleavage of the core 7SK snRNP component MePCE promoted P-TEFb release and consequent upregulation of a cohort of cytoskeleton remodeling factors, including α-actinin-1. In a subset of human megakaryocytic leukemias, the transcription factor GATA1 undergoes truncating mutation (GATA1s). Here we linked the GATA1s mutation to defects in megakaryocytic upregulation of calpain 2 and of P-TEFb-dependent cytoskeletal remodeling factors. Restoring calpain 2 expression in GATA1s-mutant megakaryocytes rescued normal development, implicating this morphogenetic pathway as a target in human leukemogenesis.
PMCID: PMC3892434  PMID: 24369834
megakaryopoiesis; P-TEFb; 7SK snRNP; calpain 2; GATA1s mutant
16.  Long-Chain Acyl CoA Synthetase 4A regulates Smad activity and dorsoventral patterning in the zebrafish embryo 
Developmental cell  2013;27(6):635-647.
Long-chain polyunsaturated fatty acids (LC-PUFA) and their metabolites are critical players in cell biology and embryonic development. Here we show that long-chain acyl CoA synthetase 4a (Acsl4a), an LC-PUFA activating enzyme, is essential for proper patterning of the zebrafish dorsoventral axis. Loss of Acsl4a results in dorsalized embryos due to attenuated Bmp signaling. We demonstrate that Acsl4a modulates the activity of Smad transcription factors, the downstream mediators of Bmp signaling. Acsl4a promotes the inhibition of p38 MAPK and the Akt-mediated inhibition of glycogen synthase kinase 3 (GSK3), critical inhibitors of Smad activity. Consequently, introduction of a constitutively active Akt can rescue the dorsalized phenotype of Acsl4a deficient embryos. Our results reveal a critical role for Acsl4a in modulating Bmp-Smad activity and provide a potential avenue for LC-PUFAs to influence a variety of developmental processes.
PMCID: PMC3895552  PMID: 24332754
17.  FHOD1 is needed for directed Forces and Adhesion Maturation during Cell Spreading and Migration 
Developmental cell  2013;27(5):545-559.
Matrix adhesions provide critical signals for cell growth or differentiation. They form through a number of distinct steps that follow integrin binding to matrix ligands. In an early step, integrins form clusters that support actin polymerization by an unknown mechanism. This raises the question of how actin polymerization occurs at the integrin clusters. We report here that a major formin in mouse fibroblasts, FHOD1 is recruited to integrin clusters, resulting in actin assembly. Using cell-spreading assays on lipid bilayers, solid substrates and high-resolution force sensing pillar arrays, we find that knockdown of FHOD1 impairs spreading, coordinated application of adhesive force and adhesion maturation. Finally we show that targeting of FHOD1 to the integrin sites depends on the direct interaction with Src family kinases, and is upstream of the activation by Rho Kinase. Thus our findings provide insights into the mechanisms of cell migration with implications for development and disease.
PMCID: PMC3890431  PMID: 24331927
18.  Exo70 Isoform Switching upon Epithelial-Mesenchymal Transition Mediates Cancer Cell Invasion 
Developmental cell  2013;27(5):560-573.
Epithelial-mesenchymal transition (EMT) is an important developmental process hijacked by cancer cells for their dissemination. Here we show that Exo70, a component of the exocyst complex, undergoes isoform switching mediated by ESRP1, a pre-mRNA splicing factor that regulates EMT. Expression of the epithelial isoform of Exo70 affects the levels of key EMT transcriptional regulators such as Snail and ZEB2, and is sufficient to drive the transition to epithelial phenotypes. Differential Exo70 isoforms expression in human tumors correlates with cancer progression, and increased expression of the epithelial isoform of Exo70 inhibits tumor metastasis in mice. At the molecular level, the mesenchymal but not the epithelial isoform of Exo70 interacts with the Arp2/3 complex and stimulates actin polymerization for tumor invasion. Our findings provide a mechanism by which the exocyst function and actin dynamics are modulated for EMT and tumor invasion.
PMCID: PMC3908839  PMID: 24331928
EMT; exocyst; Exo70; the Arp2/3 complex; ESRP; alternative splicing; tumor invasion
19.  Wnt and Bmp fit germ cells to a T 
Developmental cell  2013;27(5):485-487.
Reporting in Developmental Cell, Aramaki et al. (2013) identify T as a key mediator of primordial germ cell (PGC) specification in the embryo. Deconstruction of how Bmp and Wnt signals regulate T expression and targeting to regulatory elements of either mesodermal or PGC genes has implications for differentiation in vitro.
PMCID: PMC3929049  PMID: 24331924
20.  Retinoic Acid Regulation of Hemogenic Endothelial Cell Specification Requires c-Kit, Notch Signaling and p27-mediated Cell Cycle Control 
Developmental cell  2013;27(5):504-515.
Delineating the mechanism(s) that regulate the specification of hemogenic endothelial cells from primordial endothelium is critical for optimizing their derivation from human stem cells for clinical therapies. We previously determined that retinoic acid (RA) is required for hemogenic specification, as well as cell cycle control, of endothelium during embryogenesis. Herein, we define the molecular signals downstream of RA that regulate hemogenic endothelial cell development, and demonstrate that cell cycle control is required for this process. We found that re-expression of c-Kit in RA-deficient (Raldh2−/−) primordial endothelium induced Notch signaling and p27 expression, which restored cell cycle control and rescued hemogenic endothelial cell specification and function. Re-expression of p27 in RA-deficient and Notch-inactivated primordial endothelial cells was sufficient to correct their defects in cell cycle regulation and hemogenic endothelial cell development. Thus, RA regulation of hemogenic endothelial cell specification requires c-Kit, notch signaling and p27-mediated cell cycle control.
PMCID: PMC3994666  PMID: 24331925
21.  aPKC Phosphorylates p27Xic1, Providing a Mechanistic Link between Apicobasal Polarity and Cell-Cycle Control 
Developmental Cell  2014;31(5):559-571.
During the development of the nervous system, apicobasally polarized stem cells are characterized by a shorter cell cycle than nonpolar progenitors, leading to a lower differentiation potential of these cells. However, how polarization might be directly linked to the kinetics of the cell cycle is not understood. Here, we report that apicobasally polarized neuroepithelial cells in Xenopus laevis have a shorter cell cycle than nonpolar progenitors, consistent with mammalian systems. We show that the apically localized serine/threonine kinase aPKC directly phosphorylates an N-terminal site of the cell-cycle inhibitor p27Xic1 and reduces its ability to inhibit the cyclin-dependent kinase 2 (Cdk2), leading to shortening of G1 and S phases. Overexpression of activated aPKC blocks the neuronal differentiation-promoting activity of p27Xic1. These findings provide a direct mechanistic link between apicobasal polarity and the cell cycle, which may explain how proliferation is favored over differentiation in polarized neural stem cells.
Graphical Abstract
•aPKC shortens G1 and S phases of cell cycle by phosphorylating p27Xic1•Phosphorylated p27Xic1 exhibits weaker binding to and inhibition of Cdk2•p27Xic1 promotes neuronal differentiation and elongates cell cycle via G1 phase•Effects of p27Xic1 on neuronal differentiation are rescued by activated aPKC
During embryonic development, apicobasally polarized neuroepithelial cells have a lower propensity to differentiate than nonpolar progenitors. Sabherwal et al. show that the key polarity kinase aPKC directly phosphorylates and inhibits the activity of the cell-cycle inhibitor p27Xic1. This results in shortening of the cell cycle and favors proliferation over differentiation.
PMCID: PMC4262734  PMID: 25490266
22.  Rbx2 regulates neuronal migration through different Cullin5-RING ligase adaptors 
Developmental cell  2013;27(4):10.1016/j.devcel.2013.09.022.
Morphogenesis requires the proper migration and positioning of different cell types in the embryo. Much more is known about how cells start and guide their migrations than how they stop when they reach their destinations. Here we provide evidence that Rbx2, a subunit of the Cullin5-RING E3 ubiquitin ligase (CRL5) complex, stops neocortical projection neurons at their target layers. Rbx2 mutation causes neocortical and cerebellar ectopias dependent on Dab1, a key signaling protein in the Reelin pathway. SOCS7, a CRL5 substrate adaptor protein, is also required for neocortical layering. SOCS7-CRL5 complexes stimulate the ubiquitylation and turnover of Dab1. SOCS7 is up-regulated during projection neuron migration and unscheduled SOCS7 expression stops migration prematurely. Cerebellar development requires Rbx2 but not SOCS7, pointing to the importance of other CRL5 adaptors. Our results suggest that CRL5 adaptor expression is spatio-temporally regulated to modulate Reelin signaling and ensure normal neuron positioning in the developing brain.
PMCID: PMC3851519  PMID: 24210661
23.  miR-219 Regulates Neural Precursor Differentiation by Direct Inhibition of Apical Par Polarity Proteins 
Developmental cell  2013;27(4):10.1016/j.devcel.2013.10.015.
Asymmetric self-renewing division of neural precursors is essential for brain development. Partition defective (Par) proteins promote self-renewal, and their asymmetric distribution provides a mechanism for asymmetric division. Near the end of neural development, most asymmetric division ends and precursors differentiate. This correlates with Par protein disappearance, but mechanisms that cause downregulation are unknown. MicroRNAs can promote precursor differentiation, but have not been linked to Par protein regulation. We tested a hypothesis that microRNA miR-219 promotes precursor differentiation by inhibiting Par proteins. Neural precursors in zebrafish larvae lacking miR-219 function retained apical proteins, remained in the cell cycle and failed to differentiate. miR-219 inhibited expression via target sites within the 3’ untranslated sequence of pard3 and prkci mRNAs, which encode Par proteins, and blocking miR-219 access to these sites phenocopied loss of miR-219 function. We propose that negative regulation of Par protein expression by miR-219 promotes cell cycle exit and differentiation.
PMCID: PMC3862977  PMID: 24239515
24.  Loss of dE2F compromises mitochondrial function 
Developmental cell  2013;27(4):10.1016/j.devcel.2013.10.002.
E2F/DP transcription factors regulate cell proliferation and apoptosis. Here, we investigated the mechanism of the resistance of Drosophilad DP mutants to irradiation-induced apoptosis. Contrary to the prevailing view, this is not due to an inability to induce the apoptotic transcriptional program, since we show that this program is induced, but rather due to a mitochondrial dysfunction of dDP mutants. We attribute this defect to E2F/DP-dependent control of expression of mitochondria associated genes. Genetic attenuation of several of these E2F/DP targets mimics the dDP mutant mitochondrial phenotype and protects from irradiation-induced apoptosis. Significantly, the role of E2F/DP in the regulation of mitochondrial function is conserved between flies and humans. Thus, our results uncovered a role of E2F/DP in the regulation of mitochondrial function and demonstrate that this aspect of E2F regulation is critical for the normal induction of apoptosis in response to irradiation.
PMCID: PMC3866969  PMID: 24286825
dE2f1 transcription factor; Drosophila; apoptosis
25.  The Cytokinin Side Chain Commands Shooting 
Developmental cell  2013;27(4):10.1016/j.devcel.2013.11.008.
Functional distinction between structurally diverse cytokinins as essential plant hormones has remained enigmatic for decades. In this issue of Developmental Cell, Kiba et al. (2013) provide compelling evidence for the central role of CYP735A1/2 in synthesizing trans-hydroxylated cytokinins, which specify shoot growth vital for energy and biomass production.
PMCID: PMC3887519  PMID: 24286823

Results 1-25 (808)