scholarly journals Actin remodeling by Nck regulates endothelial lumen formation

2015 ◽  
Vol 26 (17) ◽  
pp. 3047-3060 ◽  
Author(s):  
Sankar P. Chaki ◽  
Rola Barhoumi ◽  
Gonzalo M. Rivera
Keyword(s):  
Antiangiogenic Therapy ◽  
Three Dimensional ◽  
Resonance Energy Transfer ◽  
Clinical Importance ◽  
Resonance Energy ◽  
Intercellular Junctions ◽  
Time Lapse ◽  
Actin Dynamics ◽  
Actin Remodeling ◽  
Lumen Formation

Multiple angiogenic cues modulate phosphotyrosine signaling to promote vasculogenesis and angiogenesis. Despite its functional and clinical importance, how vascular cells integrate phosphotyrosine-dependent signaling to elicit cytoskeletal changes required for endothelial morphogenesis remains poorly understood. The family of Nck adaptors couples phosphotyrosine signals with actin dynamics and therefore is well positioned to orchestrate cellular processes required in vascular formation and remodeling. Culture of endothelial cells in three-dimensional collagen matrices in the presence of VEGF stimulation was combined with molecular genetics, optical imaging, and biochemistry to show that Nck-dependent actin remodeling promotes endothelial cell elongation and proper organization of VE-cadherin intercellular junctions. Major morphogenetic defects caused by abrogation of Nck signaling included loss of endothelial apical-basal polarity and impaired lumenization. Time-lapse imaging using a Förster resonance energy transfer biosensor, immunostaining with phospho-specific antibodies, and GST pull-down assays showed that Nck determines spatiotemporal patterns of Cdc42/aPKC activation during endothelial morphogenesis. Our results demonstrate that Nck acts as an important hub integrating angiogenic cues with cytoskeletal changes that enable endothelial apical-basal polarization and lumen formation. These findings point to Nck as an emergent target for effective antiangiogenic therapy.

Dye selection for live cell imaging of intact siRNA

2012 ◽  
Vol 393 (1-2) ◽  
pp. 23-35 ◽  
Author(s):  
Markus Hirsch ◽  
Dennis Strand ◽  
Mark Helm
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Live Cell ◽  
High Yield ◽  
Ph Variation ◽  
Rnai Efficiency

Abstract Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.

scholarly journals Recruitment of Eph receptors into signaling clusters does not require ephrin contact

2004 ◽  
Vol 164 (5) ◽  
pp. 661-666 ◽  
Author(s):  
Sabine H. Wimmer-Kleikamp ◽  
Peter W. Janes ◽  
Anthony Squire ◽  
Philippe I.H. Bastiaens ◽  
Martin Lackmann
Keyword(s):  
Binding Capacity ◽  
Cluster Formation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Eph Receptors ◽  
Biological Responses ◽  
Ephrin Ligands ◽  
Membrane Bound ◽  
Large Clusters

Eph receptors and their cell membrane–bound ephrin ligands regulate cell positioning and thereby establish or stabilize patterns of cellular organization. Although it is recognized that ephrin clustering is essential for Eph function, mechanisms that relay information of ephrin density into cell biological responses are poorly understood. We demonstrate by confocal time-lapse and fluorescence resonance energy transfer microscopy that within minutes of binding ephrin-A5–coated beads, EphA3 receptors assemble into large clusters. While remaining positioned around the site of ephrin contact, Eph clusters exceed the size of the interacting ephrin surface severalfold. EphA3 mutants with compromised ephrin-binding capacity, which alone are incapable of cluster formation or phosphorylation, are recruited effectively and become phosphorylated when coexpressed with a functional receptor. Our findings reveal consecutive initiation of ephrin-facilitated Eph clustering and cluster propagation, the latter of which is independent of ephrin contacts and cytosolic Eph signaling functions but involves direct Eph–Eph interactions.

scholarly journals Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure

2011 ◽  
Vol 195 (6) ◽  
pp. 1047-1060 ◽  
Author(s):  
Yoshifumi Yamaguchi ◽  
Naomi Shinotsuka ◽  
Keiko Nonomura ◽  
Kiwamu Takemoto ◽  
Keisuke Kuida ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Neural Tube ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Neural Tube Closure ◽  
Caspase Activation ◽  
Fluorescent Reporter ◽  
Time Lapse Imaging ◽  
Tube Closure

Many cells die during development, tissue homeostasis, and disease. Dysregulation of apoptosis leads to cranial neural tube closure (NTC) defects like exencephaly, although the mechanism is unclear. Observing cells undergoing apoptosis in a living context could help elucidate their origin, behavior, and influence on surrounding tissues, but few tools are available for this purpose, especially in mammals. In this paper, we used insulator sequences to generate a transgenic mouse that stably expressed a genetically encoded fluorescence resonance energy transfer (FRET)–based fluorescent reporter for caspase activation and performed simultaneous time-lapse imaging of apoptosis and morphogenesis in living embryos. Live FRET imaging with a fast-scanning confocal microscope revealed that cells containing activated caspases showed typical and nontypical apoptotic behavior in a region-specific manner during NTC. Inhibiting caspase activation perturbed and delayed the smooth progression of cranial NTC, which might increase the risk of exencephaly. Our results suggest that caspase-mediated cell removal facilitates NTC completion within a limited developmental window.

scholarly journals Grainyhead-like 2 regulates epithelial morphogenesis by establishing functional tight junctions through the organization of a molecular network among claudin3, claudin4, and Rab25

2012 ◽  
Vol 23 (15) ◽  
pp. 2845-2855 ◽  
Author(s):  
Kazunori Senga ◽  
Keith E. Mostov ◽  
Toshihiro Mitaka ◽  
Atsushi Miyajima ◽  
Naoki Tanimizu
Keyword(s):  
Transcription Factor ◽  
Tight Junctions ◽  
Progenitor Cell ◽  
Three Dimensional ◽  
Intercellular Junctions ◽  
Molecular Network ◽  
Epithelial Morphogenesis ◽  
Lumen Formation ◽  
Liver Progenitor Cell ◽  
Progenitor Cell Line

During development, epithelial progenitors establish intercellular junctions, including tight junctions (TJs), and form three-dimensional (3D) tissue structures, which are often associated with luminal structures. Here we identify grainyhead-like 2 (Grhl2) as a transcription factor that regulates the size of luminal space surrounded by polarized epithelial cells. We show that HPPL, a liver progenitor cell line, transfected with Grhl2 cDNA forms remarkably larger cysts than the control cells in 3D cultures. We find that Grhl2 up-regulates claudin (Cldn) 3 and Cldn4, and their functions are necessary for the formation of large cysts. Overexpression of Cldn3 alone induces the cyst expansion. In contrast, expression of Cldn4 alone does not induce expansion, as it is not localized at TJs. Of interest, Rab25, another Grhl2 target, not only increases the Cldn4 protein, but also enhances its localization at TJs. Taken together, the results indicate that Grhl2 regulates epithelial morphogenesis through transcriptional up-regulation of Cldn3 and Cldn4, as well as of Rab25, which increases the Cldn4 protein and its localization at TJs. The results reveal a molecular network regulating epithelial lumen formation organized by Grhl2.

scholarly journals Cohesin-dockerin code in cellulosomal dual binding modes and its allosteric regulation by proline isomerization

2019 ◽  
Author(s):  
Andrés Manuel Vera ◽  
Albert Galera-Prat ◽  
Michał Wojciechowski ◽  
Bartosz Różycki ◽  
Douglas Vinson Laurents ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Modes ◽  
Prolyl Isomerase ◽  
Proline Isomerization ◽  
Allosteric Control ◽  
Dynamics Simulations ◽  
Enzyme Complexes

AbstractCellulose is the most abundant organic molecule on Earth and represents a renewable and practically everlasting feedstock for the production of biofuels and chemicals. Self-assembled owing to the high-affinity cohesin-dockerin interaction, cellulosomes are huge multi-enzyme complexes with unmatched efficiency in the degradation of recalcitrant lignocellulosic substrates. The recruitment of diverse dockerin-borne enzymes into a multicohesin protein scaffold dictates the three-dimensional layout of the complex, and interestingly two alternative binding modes have been proposed. Using single-molecule Fluorescence Resonance Energy Transfer, molecular dynamics simulations and NMR measurements on a range of cohesin-dockerin pairs, we directly detect varying distributions between these binding modes that follow a built-in cohesin-dockerin code. Surprisingly, we uncover a prolyl isomerase-modulated allosteric control mechanism, mediated by the isomerization state of a single proline residue, which regulates the distribution and kinetics of binding modes. Overall, our data provide a novel mechanistic understanding of the structural plasticity and dynamics of cellulosomes.

scholarly journals Pericentrin and γ-Tubulin Form a Protein Complex and Are Organized into a Novel Lattice at the Centrosome

1998 ◽  
Vol 141 (1) ◽  
pp. 163-174 ◽  
Author(s):  
Jason B. Dictenberg ◽  
Wendy Zimmerman ◽  
Cynthia A. Sparks ◽  
Aaron Young ◽  
Charles Vidair ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Sucrose Gradient ◽  
Gel Filtration ◽  
Three Dimensional ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Immunofluorescence Method ◽  
Close Proximity ◽  
Ring Complex ◽  
The Relationship

Pericentrin and γ-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin–γ-tubulin complex was distinct from the previously described γ-tubulin ring complex (γ-TuRC) as purified γ-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and γ-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.

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