Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants. We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely...
The collecting system of the kidney, derived from the ureteric bud (UB), undergoes repetitive bifid branching events during early development followed by a phase of tubular growth and elongation. Although members of the Ras GTPase family control cell growth, differentiation, proliferation, and migration, their role in development of the collecting system of the kidney is unexplored. In this study, we demonstrate that members of the R-Ras family of proteins, R-Ras and TC21, are expressed in the murine collecting system at E13.5, whereas H-Ras is only detected at day E17.5. Using murine UB cells expressing activated H-Ras, R-Ras, and TC21, we demonstrate that R-Ras–expressing cells show increased branching morphogenesis and cell growth, TC21-expressing cells branch excessively but lose their ability to migrate, whereas H-Ras–expressing cells migrated the most and formed long unbranched tubules. These differences in branching morphogenesis are mediated by differential regulation/activation of the Rho family of GTPases and mitogen-activated protein kinases. Because most branching of the UB occurs early in development, it is conceivable that R-Ras and TC-21 play a role in facilitating branching and growth in early UB development, whereas H-Ras might favor cell migration and elongation of tubules...
Septins are filamentous GTPases that associate with cell membranes and the
cytoskeleton and play essential roles in cell division and cellular
morphogenesis. Septins are implicated in many human diseases including cancer
and neuropathies. Small molecules that reversibly perturb septin organization
and function would be valuable tools for dissecting septin functions and could
be used for therapeutic treatment of septin-related diseases. Forchlorfenuron
(FCF) is a plant cytokinin previously shown to disrupt septin localization in
budding yeast. However, it is unknown whether FCF directly targets septins and
whether it affects septin organization and functions in mammalian cells. Here,
we show that FCF alters septin assembly in vitro without affecting
either actin or tubulin polymerization. In live mammalian cells, FCF dampens
septin dynamics and induces the assembly of abnormally large septin
structures. FCF has a low level of cytotoxicity, and these effects are
reversed upon FCF washout. Significantly, FCF treatment induces mitotic and
cell migration defects that phenocopy the effects of septin depletion by small
interfering RNA. We conclude that FCF is a promising tool to study mammalian
septin organization and functions.
In vertebrates, Sonic hedgehog (Shh) and transforming growth factor-β
(TGF-β) signaling pathways occur in an overlapping manner in many
morphogenetic processes. In vitro data indicate that the two pathways
may interact. Whether such interactions occur during embryonic development
remains unknown. Using embryonic lung morphogenesis as a model, we generated
transgenic mice in which exon 2 of the TβRII gene,
which encodes the type II TGF-β receptor, was deleted via a
mesodermal-specific Cre. Mesodermal-specific deletion of
(TβRIIΔ/Δ) resulted in embryonic
lethality. The lungs showed abnormalities in both number and shape of
cartilage in trachea and bronchi. In the lung parenchyma, where
epithelial-mesenchymal interactions are critical for normal development,
deletion of mesenchymal TβRII caused abnormalities in
epithelial morphogenesis. Failure in normal epithelial branching morphogenesis
in the TβRIIΔ/Δ lungs caused
cystic airway malformations. Interruption of the TβRII
locus in the lung mesenchyme increased mRNA for Patched and
Gli-1, two downstream targets of Shh signaling, without alterations
in Shh ligand levels produced in the epithelium. Therefore, we conclude that
TβRII-mediated signaling in the lung mesenchyme modulates transduction of
Shh signaling that originates from the epithelium. To our knowledge...
We have shown that branching morphogenesis of mammary ductal structures
requires the action of the morphogen epimorphin/syntaxin-2. Epimorphin,
originally identified as an extracellular molecule, is identical to
syntaxin-2, an intracellular molecule that is a member of the extensively
investigated syntaxin family of proteins that mediate vesicle trafficking. We
show here that, although epimorphin/syntaxin-2 is highly homologous to
syntaxin-1a, only epimorphin/syntaxin-2 can stimulate mammary branching
morphogenesis. We construct a homology model of epimorphin/syntaxin-2 based on
the published structure of syntaxin-1a, and we use this model to identify the
structural motif responsible for the morphogenic activity. We identify four
residues located within the cleft between helices B and C that differ between
syntaxin-1a and epimorphin/syntaxin-2; through site-directed mutagenesis of
these four amino acids, we confer the properties of epimorphin for cell
adhesion, gene activation, and branching morphogenesis onto the inactive
syntaxin-1a template. These results provide a dramatic demonstration of the
use of structural information about one molecule to define a functional motif
of a second molecule that is related at the sequence level but highly
In order for neurons to perform their function, they must establish a highly polarized morphology characterized, in most of the cases, by a single axon and multiple dendrites. Herein we find that the evolutionarily conserved protein Kidins220 (kinase D-interacting substrate of 220-kDa), also known as ARMS (ankyrin repeat-rich membrane spanning), a downstream effector of protein kinase D and neurotrophin and ephrin receptors, regulates the establishment of neuronal polarity and development of dendrites. Kidins220/ARMS gain and loss of function experiments render severe phenotypic changes in the processes extended by hippocampal neurons in culture. Although Kidins220/ARMS early overexpression hinders neuronal development, its down-regulation by RNA interference results in the appearance of multiple longer axon-like extensions as well as aberrant dendritic arbors. We also find that Kidins220/ARMS interacts with tubulin and microtubule-regulating molecules whose role in neuronal morphogenesis is well established (microtubule-associated proteins 1b, 1a, and 2 and two members of the stathmin family). Importantly, neurons where Kidins220/ARMS has been knocked down register changes in the phosphorylation activity of MAP1b and stathmins. Altogether...
Runx2 is a key transcription factor regulating osteoblast differentiation and skeletal morphogenesis, and FGF2 is one of the most important regulators of skeletal development. The importance of the ERK mitogen-activated protein (MAP) kinase pathway in cranial suture development was demonstrated by the findings that the inhibition of FGF/FGF receptor (FGFR) signaling by a MEK blocker prevents the premature suture closure caused by an Fgfr2 mutation in mice. We previously demonstrated that ERK activation does not affect Runx2 gene expression but that it stimulates Runx2 transcriptional activity. However, the molecular mechanism underlying Runx2 activation by FGF/FGFR or ERK was still unclear. In this study, we found that FGF2 treatment increased the protein level of exogenously overexpressed Runx2 and that this increase is reversed by ERK inhibitors. In contrast, overexpression of constitutively active MEK strongly increased the Runx2 protein level, which paralleled an increase in Runx2 acetylation. As Runx2 protein phosphorylation mediated by ERK directly correlates with Runx2 protein stabilization, acetylation, and ubiquitination, we undertook to identify the ERK-dependent phosphorylation sites in Runx2. Analysis of two C-terminal Runx2 deletion constructs showed that the middle third of the protein is responsible for ERK-induced stabilization and activation. An in silico analysis of highly conserved ERK targets indicated that there are three relevant serine residues in this domain. Site-directed mutagenesis implicated Ser-301 in for ERK-mediated Runx2 stabilization and acetylation. In conclusion...
Focal adhesion kinase (FAK) associates with both integrins and growth factor receptors in the control of cell motility and survival. Loss of FAK during mouse development results in lethality at embryonic day 8.5 (E8.5) and a block in cell proliferation. Because FAK serves as both a scaffold and signaling protein, gene knock-outs do not provide mechanistic insights in distinguishing between these modes of FAK function. To determine the role of FAK activity during development, a knock-in point mutation (lysine 454 to arginine (R454)) within the catalytic domain was introduced by homologous recombination. Homozygous FAKR454/R454 mutation was lethal at E9.5 with defects in blood vessel formation as determined by lack of yolk sac primary capillary plexus formation and disorganized endothelial cell patterning in FAKR454/R454 embryos. In contrast to the inability of embryonic FAK−/− cells to proliferate ex vivo, primary FAKR454/R454 mouse embryo fibroblasts (MEFs) were established from E8.5 embryos. R454 MEFs exhibited no difference in cell growth compared with normal MEFs, and R454 FAK localized to focal adhesions but was not phosphorylated at Tyr-397. In E8.5 embryos and primary MEFs, FAK R454 mutation resulted in decreased c-Src Tyr-416 phosphorylation. R454 MEFs exhibited enhanced focal adhesion formation...
Hypospadias is a common birth defect in humans, yet its etiology and pattern of onset are largely unknown. Recent studies have shown that male mice with targeted ablation of FK506-binding protein-52 (Fkbp52) develop hypospadias, most likely due to actions of Fkbp52 as a molecular co-chaperone of the androgen receptor (AR). Here, we further dissect the developmental and molecular mechanisms that underlie hypospadias in Fkbp52-deficient mice. Scanning electron microscopy revealed a defect in the elevation of prepucial swelling that led to the onset of the ventral penile cleft. Interestingly, expression of Fkbp52 was highest in the ventral aspect of the developing penis that undergoes fusion of the urethral epithelium. Although in situ hybridization and immunohistochemical analyses suggested that Fkbp52 mutants had a normal urethral epithelium signaling center and epithelial differentiation, a reduced apoptotic cell index at ventral epithelial cells at the site of fusion and a defect of genital mesenchymal cell migration were observed. Supplementation of gestating females with excess testosterone partially rescued the hypospadic phenotype in Fkbp52 mutant males, showing that loss of Fkbp52 desensitizes AR to hormonal activation. Direct measurement of AR activity was performed in mouse embryonic fibroblast cells treated with dihydrotestosterone or synthetic agonist R1881. Reduced AR activity at genes controlling sexual dimorphism and cell growth was found in Fkbp52-deficient mouse embryonic fibroblast cells. However...
How cell morphology and the cell cycle are coordinately regulated is a fundamental subject in cell biology. In fission yeast, 2 germinal center kinases (GCKs), Sid1 and Nak1, play an essential role in septation/cytokinesis and cell separation/cell polarity control, respectively, as components of the septation initiation network (SIN) and the morphogenesis Orb6 network (MOR). Here we show that a third GCK, Ppk11, is also required for efficient cell separation particularly, at a high temperature. Although Ppk11 is not essential for cell division, this kinase plays an auxiliary role in concert with MOR in cell morphogenesis. Ppk11 physically interacts with the MOR component Pmo25 and is localized to the septum, by which Ppk11 is crucial for Pmo25 targeting/accumulation to the septum. The conserved C-terminal WDF motif of Ppk11 is essential for both septum accumulation of Pmo25 and efficient cell separation. In contrast its kinase activity is required only for cell separation. Thus, both interaction of Ppk11 with Pmo25 and Ppk11 kinase activity are critical for efficient cell separation.
Sprouting angiogenesis is a multistep process that involves endothelial cell activation, basement membrane degradation, proliferation, lumen formation, and stabilization. In this study, we identified annexin 2 as a regulator of endothelial morphogenesis using a three-dimensional in vitro model where sprouting angiogenesis was driven by sphingosine 1-phosphate and angiogenic growth factors. We observed that sphingosine 1-phosphate triggered annexin 2 translocation from the cytosol to the plasma membrane and its association with vascular endothelial (VE)-cadherin. In addition, annexin 2 depletion attenuated Akt activation, which was associated with increased phosphorylation of VE-cadherin and endothelial barrier leakage. Disrupting homotypic VE-cadherin interactions with EGTA, antibodies to the extracellular domain of VE-cadherin, or gene silencing all resulted in decreased Akt (but not Erk1/2) activation. Furthermore, expression of constitutively active Akt restored reduced endothelial sprouting responses observed with annexin 2 and VE-cadherin knockdown. Collectively, we report that annexin 2 regulates endothelial morphogenesis through an adherens junction-mediated pathway upstream of Akt.
Cdc42 plays an evolutionarily conserved role in promoting cell polarity and is indispensable during epithelial morphogenesis. To further investigate the role of Cdc42, we have used a three-dimensional matrigel model, in which single Caco-2 cells develop to form polarized cysts. Using this system, we previously reported that Cdc42 controls mitotic spindle orientation during cell division to correctly position the apical surface in a growing epithelial structure. In the present study, we have investigated the specific downstream effectors through which Cdc42 controls this process. Here, we report that Par6B and its binding partner, atypical protein kinase C (aPKC), are required to regulate Caco-2 morphogenesis. Depletion or inhibition of Par6B or aPKC phenocopies the loss of Cdc42, inducing misorientation of the mitotic spindle, mispositioning of the nascent apical surface, and ultimately, the formation of aberrant cysts with multiple lumens. Mechanistically, Par6B and aPKC function interdependently in this context. Par6B localizes to the apical surface of Caco-2 cysts and is required to recruit aPKC to this compartment. Conversely, aPKC protects Par6B from proteasomal degradation, in a kinase-independent manner. In addition, we report that depletion or inhibition of aPKC induces robust apoptotic cell death in Caco-2 cells...
The control of cellular growth is central to multicellular patterning. In plants, the encapsulating cell wall literally binds neighbouring cells to each other and limits cellular sliding/migration. In contrast to its developmental importance, growth regulation is poorly understood in plants. Here, we reveal that the phytohormone auxin impacts on the shape of the biggest plant organelle, the vacuole. TIR1/AFBs-dependent auxin signalling posttranslationally controls the protein abundance of vacuolar SNARE components. Genetic and pharmacological interference with the auxin effect on vacuolar SNAREs interrelates with auxin-resistant vacuolar morphogenesis and cell size regulation. Vacuolar SNARE VTI11 is strictly required for auxin-reliant vacuolar morphogenesis and loss of function renders cells largely insensitive to auxin-dependent growth inhibition. Our data suggests that the adaptation of SNARE-dependent vacuolar morphogenesis allows auxin to limit cellular expansion, contributing to root organ growth rates.
Background: The Lrig genes encode a family of transmembrane proteins that have been implicated in tumorigenesis, psoriasis, neural crest development, and complex tissue morphogenesis. Whether these diverse phenotypes reflect a single underlying cellular mechanism is not known. However, Lrig proteins contain evolutionarily conserved ectodomains harboring both leucine-rich repeats and immunoglobulin domains, suggesting an ability to bind to common partners. Previous studies revealed that Lrig1 binds to and inhibits members of the ErbB family of receptor tyrosine kinases by inducing receptor internalization and degradation. In addition, other receptor tyrosine kinase binding partners have been identified for both Lrig1 and Lrig3, leaving open the question of whether defective ErbB signaling is responsible for the observed mouse phenotypes. Methodology/Principal Findings: Here, we report that Lrig3, like Lrig1, is able to interact with ErbB receptors in vitro. We examined the in vivo significance of these interactions in the inner ear, where Lrig3 controls semicircular canal formation by determining the timing and extent of Netrin1 expression in the otic vesicle epithelium. We find that ErbB2 and ErbB3 are present in the early otic epithelium...
Background: Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood. Methodology/Principal Findings: In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an in vivo angiogenesis model of chicken chorioallantoic membrane (CAM). Conclusions/Significance: Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2; it phosphorylates VEGFR-2 at Y1173...
Cells in the wing blade of Drosophila melanogaster exhibit an in-plane polarization causing distal orientation of hairs. Establishment of the Planar Cell Polarity (PCP) involves intercellular interactions as well as a global orienting signal. Many of the genetic and molecular components underlying this process have been experimentally identified and a recently advanced system-level model has suggested that the observed mutant phenotypes can be understood in terms of intercellular interactions involving asymmetric localization of membrane bound proteins. Among key open questions in understanding the emergence of ordered polarization is the effect of stochasticity and the role of the global orienting signal. These issues relate closely to our understanding of ferromagnetism in physical systems. Here we pursue this analogy to understand the emergence of PCP order. To this end we develop a semi-phenomenological representation of the underlying molecular processes and define a “phase diagram” of the model which provides a global view of the dependence of the phenotype on parameters. We show that the dynamics of PCP has two regimes: rapid growth in the amplitude of local polarization followed by a slower process of alignment which progresses from small to large scales. We discuss the response of the tissue to various types of orienting signals and show that global PCP order can be achieved with a weak orienting signal provided that it acts during the early phase of the process. Finally we define and discuss some of the experimental predictions of the model.; Other Research Unit
Patterning, morphogenesis, and cell divisions are distinct processes during development yet are concurrent and likely highly integrated. However, it has been challenging to investigate them as a whole. Recent advances in imaging and labeling tools make it possible to observe live tissues with high coverage and resolution. In this dissertation work, we developed a novel imaging platform that allowed us to fully capture the early neural tube formation process in live zebrafish embryos at cellular resolution. Importantly, these datasets allow us to reliably track single neural progenitors. These tracks carry information on the history of cell movement, shape change, division, and gene expression all together. By comparing tracks of different progenitor fates, we found they show a spatially noisy response to Sonic hedgehog (Shh) and become specified in a positionally mixed manner, in surprising contrast to the "French Flag" morphogen patterning model. Both cell movement and division contribute to cell mixing. In addition, we decoupled the temporal and genetic regulatory network (GRN) noises in Shh interpretation using tracks that carry both Shh signaling and cell fate reporters. Our tracks suggest that, after specification, progenitors undergo sorting to self-assemble a sharp pattern. Consistent with this hypothesis...
Morphogenesis is critical to the development of multicellular organisms. Morphogenesis is also a complex process that requires the coordination of many events, including cell division, cytoskeletal reorganization, cell-cell and cell-matrix interactions. To investigate a driving mechanism of morphogenesis in organ formation, we characterized a group of C. elegans mutants defective in vulval morphogenesis. We established that the morphogenetic defect is caused by disruption in biosynthesis of chondroitin glycosaminoglycans by molecular identification and characterization of five sqv (uashed vulva) genes. Glycosaminoglycans are known to function in development by binding a large repertoire of ligands involved in morphogenesis and wound healing, in some cases by modulating certain conserved signaling pathways, and in providing structural support in the extracellular matrix. Defects in biosynthesis of glycosaminoglycans can cause connective tissue diseases. We found that regulation of sqv-4 likely controls glycosaminoglycan biosynthesis and the aspect of vulval morphogenesis driven by chondroitin. We also characterized specific defects in separation of the plasma membrane from the eggshell and failure to initiate cytokinesis caused by mutations in sqv genes in the one-cell embryo.; by Ho-Yon Hwang.; Thesis (Ph. D.)--Massachusetts Institute of Technology...
Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements...
Despite the importance of airspace integrity in vertebrate gas exchange,
the molecular pathways that instruct distal lung formation are poorly
understood. Recently, we found that fibrillin-1 deficiency in mice impairs
alveolar formation and recapitulates the pulmonary features of human Marfan
syndrome. To further elucidate effectors involved in distal lung formation, we
performed expression profiling analysis comparing the fibrillin-1-deficient
and wild-type developing lung. NeuroD, a basic helix-loop-helix transcription
factor, fulfilled the expression criteria for a candidate mediator of distal
lung development. We investigated its role in murine lung development using
genetically targeted NeuroD-deficient mice. We found that NeuroD deficiency
results in both impaired alveolar septation and altered morphology of the
pulmonary neuroendocrine cells. NeuroD-deficient mice had enlarged alveoli
associated with reduced epithelial proliferation in the airway and airspace
compartments during development. Additionally, the neuroendocrine compartment
in these mice manifested an increased number of neuroepithelial bodies but a
reduced number of solitary pulmonary neuroendocrine cells in the neonatal
lung. Overexpression of NeuroD in a murine lung epithelial cell line conferred
a neuroendocrine phenotype characterized by the induction of neuroendocrine
markers as well as increased proliferation. These results support an
unanticipated role for NeuroD in the regulation of pulmonary neuroendocrine
and alveolar morphogenesis and suggest an intimate connection between the
neuroendocrine compartment and distal lung development.