Developmental Cell

Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria.

Publication Date: 2010 Feb 16 PMID: 20159602
Authors: Robinson, M. S. - Sahlender, D. A. - Foster, S. D.
Journal: Dev Cell

We have developed a method for rapidly inactivating proteins with rapamycin-induced heterodimerization. Cells were stably transfected with siRNA-resistant, FKBP-tagged subunits of the adaptor protein (AP) complexes of clathrin-coated vesicles (CCVs), together with an FKBP and rapamycin-binding domain-containing construct with a mitochondrial targeting signal. Knocking down the endogenous subunit with siRNA, and then adding rapamycin, caused the APs to be rerouted to mitochondria within seconds. Rerouting AP-2 to mitochondria effectively abolished clathrin-mediated endocytosis of transferrin. In cells with rerouted AP-1, endocytosed cation-independent mannose 6-phosphate receptor (CIMPR) accumulated in a peripheral compartment, and isolated CCVs had reduced levels of CIMPR, but normal levels of the lysosomal hydrolase DNase II. Both observations support a role for AP-1 in retrograde trafficking. This type of approach, which we call a "knocksideways," should be widely applicable as a means of inactivating proteins with a time scale of seconds or minutes rather than days.

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The ordered architecture of murine ear epidermis is maintained by progenitor cells with random fate.

Publication Date: 2010 Feb 16 PMID: 20159601
Authors: Doupe, D. P. - Klein, A. M. - Simons, B. D. - Jones, P. H.
Journal: Dev Cell

Typical murine epidermis has a patterned structure, seen clearly in ear skin, with regular columns of differentiated cells overlying the proliferative basal layer. It has been proposed that each column is a clonal epidermal proliferative unit maintained by a central stem cell and its transit amplifying cell progeny. An alternative hypothesis is that proliferating basal cells have random fate, the probability of generating cycling or differentiated cells being balanced so homeostasis is achieved. The stochastic model seems irreconcilable with an ordered tissue. Here we use lineage tracing to reveal that basal cells generate clones with highly irregular shapes that contribute progeny to multiple columns. Basal cell fate and cell cycle time is random. Cell columns form due to the properties of postmitotic cells. We conclude that the ordered architecture of the epidermis is maintained by a stochastic progenitor cell population, providing a simple and robust mechanism of homeostasis.

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The WW domain protein Kibra acts upstream of hippo in Drosophila.

Publication Date: 2010 Feb 16 PMID: 20159600
Authors: Baumgartner, R. - Poernbacher, I. - Buser, N. - Hafen, E. - Stocker, H.
Journal: Dev Cell

The conserved Hippo kinase pathway plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. Whereas the function of the core kinase cascade, consisting of the serine/threonine kinases Hippo and Warts, in phosphorylating and thereby inactivating the transcriptional coactivator Yorkie is well established, much less is known about the upstream events that regulate Hippo signaling activity. The FERM domain proteins Expanded and Merlin appear to represent two different signaling branches that feed into the Hippo pathway. Signaling by the atypical cadherin Fat may act via Expanded, but how Merlin is regulated has remained elusive. Here, we show that the WW domain protein Kibra is a Hippo signaling component upstream of Hippo and Merlin. Kibra acts synergistically with Expanded, and it physically interacts with Merlin. Thus, Kibra predominantly acts in the Merlin branch upstream of the core kinase cascade to regulate Hippo signaling.

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Kibra is a regulator of the salvador/warts/hippo signaling network.

Publication Date: 2010 Feb 16 PMID: 20159599
Authors: Genevet, A. - Wehr, M. C. - Brain, R. - Thompson, B. J. - Tapon, N.
Journal: Dev Cell

The Salvador (Sav)/Warts (Wts)/Hippo (Hpo) (SWH) network controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core of the pathway consists of a MST and LATS family kinase cascade that ultimately phosphorylates and inactivates the YAP/Yorkie (Yki) transcription coactivator. The FERM domain proteins Merlin (Mer) and Expanded (Ex) represent one mode of upstream regulation controlling pathway activity. Here, we identify Kibra as a member of the SWH network. Kibra, which colocalizes and associates with Mer and Ex, also promotes the Mer/Ex association. Furthermore, the Kibra/Mer association is conserved in human cells. Finally, Kibra complexes with Wts and kibra depletion in tissue culture cells induces a marked reduction in Yki phosphorylation without affecting the Yki/Wts interaction. We suggest that Kibra is part of an apical scaffold that promotes SWH pathway activity.

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Kibra functions as a tumor suppressor protein that regulates hippo signaling in conjunction with Merlin and expanded.

Publication Date: 2010 Feb 16 PMID: 20159598
Authors: Yu, J. - Zheng, Y. - Dong, J. - Klusza, S. - Deng, W. M. - Pan, D.
Journal: Dev Cell

The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. Here we identify Kibra as another upstream component of the Hippo signaling pathway. We show that Kibra functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function and implicate Kibra as a potential tumor suppressor with relevance to neurofibromatosis.

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Characterization and in vivo pharmacological rescue of a Wnt2-Gata6 pathway required for cardiac inflow tract development.

Publication Date: 2010 Feb 16 PMID: 20159597
Authors: Tian, Y. - Yuan, L. - Goss, A. M. - Wang, T. - Yang, J. - Lepore, J. J. - Zhou, D. - Schwartz, R. J. - Patel, V. - Cohen, E. D. - Morrisey, E. E.
Journal: Dev Cell

Little is understood about the molecular mechanisms underlying the morphogenesis of the posterior pole of the heart. Here we show that Wnt2 is expressed specifically in the developing inflow tract mesoderm, which generates portions of the atria and atrio-ventricular canal. Loss of Wnt2 results in defective development of the posterior pole of the heart, resulting in a phenotype resembling the human congenital heart syndrome complete common atrio-ventricular canal. The number and proliferation of posterior second heart field progenitors is reduced in Wnt2(-/-) mutants. Moreover, these defects can be rescued in a temporally restricted manner through pharmacological inhibition of Gsk-3beta. We also show that Wnt2 works in a feedforward transcriptional loop with Gata6 to regulate posterior cardiac development. These data reveal a molecular pathway regulating the posterior cardiac mesoderm and demonstrate that cardiovascular defects caused by loss of Wnt signaling can be rescued pharmacologically in vivo.

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Organism-scale modeling of early Drosophila patterning via bone morphogenetic proteins.

Publication Date: 2010 Feb 16 PMID: 20159596
Authors: Umulis, D. M. - Shimmi, O. - O'Connor, M. B. - Othmer, H. G.
Journal: Dev Cell

Advances in image acquisition and informatics technology have led to organism-scale spatiotemporal atlases of gene expression and protein distributions. To maximize the utility of this information for the study of developmental processes, a new generation of mathematical models is needed for discovery and hypothesis testing. Here, we develop a data-driven, geometrically accurate model of early Drosophila embryonic bone morphogenetic protein (BMP)-mediated patterning. We tested nine different mechanisms for signal transduction with feedback, eight combinations of geometry and gene expression prepatterns, and two scale-invariance mechanisms for their ability to reproduce proper BMP signaling output in wild-type and mutant embryos. We found that a model based on positive feedback of a secreted BMP-binding protein, coupled with the experimentally measured embryo geometry, provides the best agreement with population mean image data. Our results demonstrate that using bioimages to build and optimize a three-dimensional model provides significant insights into mechanisms that guide tissue patterning.

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Kinetochores generate microtubules with distal plus ends: their roles and limited lifetime in mitosis.

Publication Date: 2010 Feb 16 PMID: 20159595
Authors: Kitamura, E. - Tanaka, K. - Komoto, S. - Kitamura, Y. - Antony, C. - Tanaka, T. U.
Journal: Dev Cell

In early mitosis, microtubules can be generated at kinetochores as well as at spindle poles. However, the role and regulation of kinetochore-derived microtubules have been unclear. In general, metaphase spindle microtubules are oriented such that their plus ends bind to kinetochores. However, we now have evidence that, during early mitosis in budding yeast, microtubules are generated at kinetochores with distal plus ends. These kinetochore-derived microtubules interact along their length with microtubules that extend from a spindle pole, facilitating kinetochore loading onto the lateral surface of spindle pole microtubules. Once kinetochores are loaded, microtubules are no longer generated at kinetochores, and those that remain disappear rapidly and do not contribute to the metaphase spindle. Stu2 (the ortholog of vertebrate XMAP215/ch-TOG) localizes to kinetochores and plays a central role in regulating kinetochore-derived microtubules. Our work provides insight into microtubule generation at kinetochores and the mechanisms that facilitate initial kinetochore interaction with spindle pole microtubules.

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Broad-minded links cell cycle-related kinase to cilia assembly and hedgehog signal transduction.

Publication Date: 2010 Feb 16 PMID: 20159594
Authors: Ko, H. W. - Norman, R. X. - Tran, J. - Fuller, K. P. - Fukuda, M. - Eggenschwiler, J. T.
Journal: Dev Cell

Recent findings indicate that mammalian Sonic hedgehog (Shh) signal transduction occurs within primary cilia, although the cell biological mechanisms underlying both Shh signaling and ciliogenesis have not been fully elucidated. We show that an uncharacterized TBC domain-containing protein, Broad-minded (Bromi), is required for high-level Shh responses in the mouse neural tube. We find that Bromi controls ciliary morphology and proper Gli2 localization within the cilium. By use of a zebrafish model, we further show that Bromi is required for proper association between the ciliary membrane and axoneme. Bromi physically interacts with cell cycle-related kinase (CCRK), whose Chlamydomonas homolog regulates flagellar length. Biochemical and genetic interaction data indicate that Bromi promotes CCRK stability and function. We propose that Bromi and CCRK control the structure of the primary cilium by coordinating assembly of the axoneme and ciliary membrane, allowing Gli proteins to be properly activated in response to Shh signaling.

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Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish.

Publication Date: 2010 Feb 16 PMID: 20159593
Authors: Yoo, S. K. - Deng, Q. - Cavnar, P. J. - Wu, Y. I. - Hahn, K. M. - Huttenlocher, A.
Journal: Dev Cell

Cell polarity is crucial for directed migration. Here we show that phosphoinositide 3-kinase (PI(3)K) mediates neutrophil migration in vivo by differentially regulating cell protrusion and polarity. The dynamics of PI(3)K products PI(3,4,5)P(3)-PI(3,4)P(2) during neutrophil migration were visualized in living zebrafish, revealing that PI(3)K activation at the leading edge is critical for neutrophil motility in intact tissues. A genetically encoded photoactivatable Rac was used to demonstrate that localized activation of Rac is sufficient to direct migration with precise temporal and spatial control in vivo. Similar stimulation of PI(3)K-inhibited cells did not direct migration. Localized Rac activation rescued membrane protrusion but not anteroposterior polarization of F-actin dynamics of PI(3)K-inhibited cells. Uncoupling Rac-mediated protrusion and polarization suggests a paradigm of two-tiered PI(3)K-mediated regulation of cell motility. This work provides new insight into how cell signaling at the front and back of the cell is coordinated during polarized cell migration in intact tissues within a multicellular organism.

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Structural basis of selective ubiquitination of TRF1 by SCFFbx4.

Publication Date: 2010 Feb 16 PMID: 20159592
Authors: Zeng, Z. - Wang, W. - Yang, Y. - Chen, Y. - Yang, X. - Diehl, J. A. - Liu, X. - Lei, M.
Journal: Dev Cell

TRF1 is a critical regulator of telomere length. As such, TRF1 levels are regulated by ubiquitin-dependent proteolysis via an SCF E3 ligase where Fbx4 contributes to substrate specification. Here, we report the crystal structure of the Fbx4-TRF1 complex at 2.4 A resolution. Fbx4 contains an unusual substrate-binding domain that adopts a small GTPase fold. Strikingly, this atypical GTPase domain of Fbx4 binds to a globular domain of TRF1 through an intermolecular beta sheet, instead of recognizing short peptides/degrons as often seen in other F-box protein-substrate complexes. Importantly, mutations in this interface abrogate Fbx4-dependent TRF1 binding and ubiquitination. Furthermore, the data demonstrate that recognition of TRF1 by SCF(Fbx4) is regulated by another telomere protein, TIN2. Our results reveal an atypical small GTPase domain within Fbx4 as a substrate-binding motif for SCF(Fbx4) and uncover a mechanism for selective ubiquitination and degradation of TRF1 in telomere homeostasis control.

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ATRX partners with cohesin and MeCP2 and contributes to developmental silencing of imprinted genes in the brain.

Publication Date: 2010 Feb 16 PMID: 20159591
Authors: Kernohan, K. D. - Jiang, Y. - Tremblay, D. C. - Bonvissuto, A. C. - Eubanks, J. H. - Mann, M. R. - Berube, N. G.
Journal: Dev Cell

Human developmental disorders caused by chromatin dysfunction often display overlapping clinical manifestations, such as cognitive deficits, but the underlying molecular links are poorly defined. Here, we show that ATRX, MeCP2, and cohesin, chromatin regulators implicated in ATR-X, RTT, and CdLS syndromes, respectively, interact in the brain and colocalize at the H19 imprinting control region (ICR) with preferential binding on the maternal allele. Importantly, we show that ATRX loss of function alters enrichment of cohesin, CTCF, and histone modifications at the H19 ICR, without affecting DNA methylation on the paternal allele. ATRX also affects cohesin, CTCF, and MeCP2 occupancy within the Gtl2/Dlk1 imprinted domain. Finally, we show that loss of ATRX interferes with the postnatal silencing of the maternal H19 gene along with a larger network of imprinted genes. We propose that ATRX, cohesin, and MeCP2 cooperate to silence a subset of imprinted genes in the postnatal mouse brain.

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Organogenesis and development of the liver.

Publication Date: 2010 Feb 16 PMID: 20159590
Authors: Si-Tayeb, K. - Lemaigre, F. P. - Duncan, S. A.
Journal: Dev Cell

Embryonic development of the liver has been studied intensely, yielding insights that impact diverse areas of developmental and cell biology. Understanding the fundamental mechanisms that control hepatogenesis has also laid the basis for the rational differentiation of stem cells into cells that display many hepatic functions. Here, we review the basic molecular mechanisms that control the formation of the liver as an organ.

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Alternative polyadenylation blooms.

Publication Date: 2010 Feb 16 PMID: 20159589
Authors: Rosonina, E. - Manley, J. L.
Journal: Dev Cell

Alternative polyadenylation generates mRNAs with 3' untranscribed regions of different lengths, often affecting transcript stability. Hornyik et al., in this issue of Developmental Cell, and Liu et al. now demonstrate a role for alternative polyadenylation in gene silencing and the regulation of flowering time in Arabidopsis thaliana.

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A PAK-activated linker for EGFR and FAK.

Publication Date: 2010 Feb 16 PMID: 20159588
Authors: Tomar, A. - Schlaepfer, D. D.
Journal: Dev Cell

Transmembrane growth factor and integrin matrix receptors form multiprotein signaling complexes with FAK, a cytoplasmic cell motility-associated kinase. In a recent issue of Molecular Cell, Long et al. now show that a PAK-phosphorylated alternate-spliced isoform of the steroid receptor coactivator-3 (SRC-3Delta4) bridges EGFR and FAK, enhancing breast carcinoma cell migration and metastasis.

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Chromatin modifiers, cognitive disorders, and imprinted genes.

Publication Date: 2010 Feb 16 PMID: 20159587
Authors: Cunningham, M. D. - Kassis, J. A. - Pfeifer, K.
Journal: Dev Cell

In this issue of Developmental Cell, Kernohan et al. link the chromatin regulatory proteins ATRX, MeCP2, CTCF, and cohesin with silencing of H19 and other imprinted genes during critical stages of postnatal brain development, perhaps suggesting a common etiology for several human diseases that exhibit defects in brain development and function.

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Telomeric TuRF1 wars.

Publication Date: 2010 Feb 16 PMID: 20159586
Authors: Sarraf, S. A. - Harper, J. W.
Journal: Dev Cell

The TRF1 subunit of the shelterin complex controls telomere length by regulating telomerase access to chromosome ends. Work from Zeng et al. (2010) now reveals in atomic detail how a battle between the SCF(FBX4) ubiquitin ligase and the shelterin component TIN2 controls TRF1 abundance and telomere length homeostasis.

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The spen family protein FPA controls alternative cleavage and polyadenylation of RNA.

Publication Date: 2010 Feb 16 PMID: 20079695
Authors: Hornyik, C. - Terzi, L. C. - Simpson, G. G.
Journal: Dev Cell

The spen family protein FPA is required for flowering time control and has been implicated in RNA silencing. The mechanism by which FPA carries out these functions is unknown. We report the identification of an activity for FPA in controlling mRNA 3' end formation. We show that FPA functions redundantly with FCA, another RNA binding protein that controls flowering and RNA silencing, to control the expression of alternatively polyadenylated antisense RNAs at the locus encoding the floral repressor FLC. In addition, we show that defective 3' end formation at an upstream RNA polymerase II-dependent gene explains the apparent derepression of the AtSN1 retroelement in fpa mutants. Transcript readthrough accounts for the absence of changes in DNA methylation and siRNA abundance at AtSN1 in fpa mutants, and this may explain other examples of epigenetic transitions not associated with chromatin modification.

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