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Molecular Biology of the Cell

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https://www.readbyqxmd.com/read/28637769/potential-contribution-of-tandem-circadian-enhancers-to-nonlinear-oscillations-in-clock-gene-expression
#1
Isao T Tokuda, Akihiko Okamoto, Ritsuko Matsumura, Toru Takumi, Makoto Akashi
Limit cycle oscillations require the presence of nonlinear processes. Although mathematical studies have long suggested that multiple nonlinear processes are required for autonomous circadian oscillation in clock gene expression, the underlying mechanism remains controversial. Here, we experimentally show that cell-autonomous circadian transcription of a mammalian clock gene requires a functionally interdependent tandem E-box motif; the lack of either of the two E-boxes results in arrhythmic transcription. Although previous studies have indicated the role of the tandem motifs in increasing circadian amplitude, enhancing amplitude does not explain the mechanism for limit cycle oscillations in transcription...
June 21, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28637768/vrk2a-is-an-a-type-lamin-dependent-nuclear-envelope-kinase-that-phosphorylates-baf
#2
K C Birendra, Danielle G May, Benjamin V Benson, Dae In Kim, Winnie G Shivega, Manaal H Ali, Randolph S Faustino, Alexandre R Campos, Kyle J Roux
The nuclear envelope (NE) is critical for numerous fundamental cellular functions and mutations in several NE constituents can lead to a heterogeneous spectrum of diseases. We used proximity biotinylation to uncover new constituents of the inner nuclear membrane (INM) by comparative BioID analysis of lamin-A, Sun2 and a minimal INM-targeting motif. These studies identified vaccinia-related kinase-2 (VRK2) as a candidate constituent of the INM. We determined that the transmembrane VRK2A isoform is retained at the NE by association with A-type lamins...
June 21, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28637767/a-short-region-upstream-of-the-yeast-vacuolar-qa-snare-heptad-repeats-promotes-membrane-fusion-through-enhanced-snare-complex-assembly
#3
Hongki Song, William Wickner
While SNARE heptad-repeats are well studied, SNAREs also have upstream N-domains of indeterminate function. The assembly of yeast vacuolar SNAREs into complexes for fusion can be studied in chemically-defined reactions. Complementary proteoliposomes bearing a Rab:GTP and either the vacuolar R-SNARE or one of the 3 integrally-anchored Q-SNAREs were incubated with the tethering/SM protein complex HOPS and the two other soluble SNAREs (lacking a trans-membrane anchor) or their SNARE heptad-repeat domains. Fusion required a trans-membrane anchored R-SNARE on one membrane and an anchored Q-SNARE on the other...
June 21, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28637766/emery-dreifuss-muscular-dystrophy-linked-genes-and-centronuclear-myopathy-linked-genes-regulate-myonuclear-movement-by-distinct-mechanisms
#4
Mary Ann Collins, Torrey R Mandigo, Jaclyn M Camuglia, Gabriella A Vazquez, Alyssa J Anderson, Christine H Hudson, John L Hanron, Eric S Folker
Muscle cells are a syncytium in which the many nuclei are positioned to maximize the distance between adjacent nuclei. Although mispositioned nuclei are correlated with many muscle disorders, it is not known whether this common phenotype is the result of a common mechanism. To answer this question, the expression of genes linked to Emery-Dreifuss Muscular Dystrophy (EDMD) and Centronuclear myopathy (CNM) was disrupted in Drosophila, and the position of the nuclei was evaluated. We found that the genes linked to EDMD and CNM were each necessary to properly position nuclei...
June 21, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28637765/electrostatic-interaction-between-polyglutamylated-tubulin-and-the-nexin-dynein-regulatory-complex-regulates-flagellar-motility
#5
Tomohiro Kubo, Toshiyuki Oda
Tubulins undergo various posttranslational modifications. Among them, polyglutamylation is involved in the motility of eukaryotic flagella and the stability of the axonemal microtubules. However, it remains unclear where polyglutamylated tubulin localizes precisely within the axoneme, and how tubulin polyglutamylation affects the flagellar motility. In this study, we identified the three-dimensional (3D) localization of the polyglutamylated tubulin in Chlamydomonas flagella using antibody labeling and cryo-electron tomography...
June 21, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28615324/hihimap-single-cell-quantitation-of-histones-and-histone-post-translational-modifications-across-the-cell-cycle-by-high-throughput-imaging
#6
Linda Zane, Fleur Chapus, Gianluca Pegoraro, Tom Misteli
We describe HiHiMap (High-throughput Histone Mapping), a high-throughput imaging method to measure histone and histone post-translational modifications (PTM) in single cells. HiHiMap uses imaging-based quantification of DNA and Cyclin A to stage individual cells in the cell cycle to determine the levels of histone or histone PTMs in each stage of the cell cycle. As proof-of-principle, we apply HiHiMap to measure the level of 21 core histones, histone variants and PTMs in primary, immortalized and transformed cells...
June 14, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28615323/generic-membrane-spanning-features-endow-ire1%C3%AE-with-responsiveness-to-membrane-aberrancy
#7
Nozomu Kono, Niko Amin-Wetzel, David Ron
Altered cellular lipid composition activates the endoplasmic reticulum unfolded protein response (UPR) and UPR signaling effects important changes in lipid metabolism. Secondary effects on protein folding homeostasis likely contribute to UPR activation, but deletion of the unfolded protein stress-sensing luminal domain of the UPR transducers PERK and IRE1α does not abolish their responsiveness to lipid perturbation. This finding suggests that PERK and IRE1α also directly recognize the membrane aberrancy wrought by lipid perturbation...
June 14, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28615322/in-vivo-manipulation-of-the-extracellular-matrix-induces-vascular-regression-in-a-basal-chordate
#8
Delany Rodriguez, Brian P Braden, Scott W Boyer, Daryl A Taketa, Leah Setar, Chris Calhoun, Alessandro Di Maio, Adam Langenbacher, Megan T Valentine, Anthony W De Tomaso
We investigated the physical role of the extracellular matrix in vascular homeostasis in the basal chordate Botryllus schlosseri, which has a large, transparent extracorporeal vascular network encompassing areas >100 cm(2) We found that the collagen crosslinking enzyme lysyl oxidase is expressed in all vascular cells, and that in vivo inhibition using ß-aminopropionitrile (BAPN) caused a rapid, global regression of the entire network, with some vessels regressing >10 mm within 16 hours. BAPN treatment changed the ultrastructure of collagen fibers in the vessel basement membrane, and the kinetics of regression were dose-dependent...
June 14, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28615321/par-proteins-regulate-maintenance-phase-myosin-dynamics-during-c-elegans-zygote-polarization
#9
Lawrence E Small, Adriana T Dawes
Establishment of anterior-posterior polarity in the C. elegans zygote requires two different processes: mechanical activity of the actin-myosin cortex and biochemical activity of partitioning defective (PAR) proteins. Here, we have analyzed how PARs regulate the behavior of the cortical motor protein non-muscle myosin (NMY-2) to complement recent efforts that investigate how PARs regulate the Rho GTPase CDC-42, which in turn regulates the actin-myosin cortex. We found that PAR-3 and PAR-6 concentrate CDC-42-dependent NMY-2 in the anterior cortex, while PAR-2 inhibits CDC-42-dependent NMY-2 in the posterior by inhibiting PAR-3 and PAR-6...
June 14, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28615320/differential-phosphorylation-signals-control-endocytosis-of-gpr15
#10
Yukari Okamoto, Sojin Shikano
GPR15 is an orphan G protein-coupled receptor (GPCR) that serves for an HIV coreceptor and was also recently found as a novel homing receptor for T cells implicated in colitis. We show that GPR15 undergoes a constitutive endocytosis in the absence of ligand. The endocytosis was clathrin-dependent and partially dependent on β-arrestin in HEK293 cells, and nearly half of the internalized GPR15 receptors were recycled to the plasma membrane. Ala mutation of the distal C-terminal Arg(354) or Ser(357), which forms a consensus phosphorylation site for basophilic kinases, markedly reduced the endocytosis while phosphomimetic mutation of Ser(357) to Asp did not...
June 14, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28615319/retinoschisin-is-linked-to-retinal-na-k-atpase-signaling-and-localization
#11
Karolina Plössl, Melanie Royer, Sarah Bernklau, Neslihan N Tavraz, Thomas Friedrich, Jens Wild, Bernhard H F Weber, Ulrike Friedrich
Mutations in the RS1 gene cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy. We have recently shown that retinoschisin, the protein encoded by RS1, regulates ERK signaling and apoptosis in retinal cells. In this study, we explored an influence of retinoschisin on the functionality of the Na/K-ATPase, its interaction partner at retinal plasma membranes. We show that retinoschisin binding requires the β2-subunit of the Na/K-ATPase while the α-subunit is exchangeable. Our investigations revealed no impact of retinoschisin on Na/K-ATPase mediated ATP hydrolysis and ion transport...
June 14, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28615318/miro1-mediated-mitochondrial-positioning-shapes-intracellular-energy-gradients-required-for-cell-migration
#12
Max-Hinderk Schuler, Agnieszka Lewandowska, Giuseppe Di Caprio, Wesley Skillern, Srigokul Upadhyayula, Tom Kirchhausen, Janet M Shaw, Brian Cunniff
It has long been postulated, though never directly demonstrated that mitochondria are strategically positioned in the cytoplasm to meet local requirements for energy production. Here we show that positioning of mitochondria in mouse embryonic fibroblasts (MEFs) determines the shape of intracellular energy gradients in living cells. Specifically, the ratio of ATP to ADP was highest at perinuclear areas of dense mitochondria and gradually declined approaching more peripheral sites. Furthermore, the majority of mitochondria were positioned at the ventral surface of the cell, correlating with high ATP:ADP ratios close to the ventral membrane that rapidly decreased towards the dorsal surface...
June 14, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28615317/orientation-and-repositioning-of-chromosomes-correlate-with-cell-geometry-dependent-gene-expression
#13
Yejun Wang, Mallika Nagarajan, Caroline Uhler, G V Shivashankar
Extracellular matrix signals from the microenvironment regulate gene expression patterns and cell behavior. Using a combination of experiments and geometric models, we demonstrate correlations between cell geometry, 3D organization of chromosome territories and gene expression. Fluorescence in situ hybridization experiments showed that micropatterned fibroblasts cultured on anisotropic (AP) versus isotropic (IP) substrates resulted in repositioning of specific chromosomes, which contained genes that were differentially regulated by cell geometries...
June 14, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28592635/a-cytoskeletal-clutch-mediates-cellular-force-transmission-in-a-soft-3d-extracellular-matrix
#14
Leanna M Owen, Arjun S Adhikari, Mohak Patel, Peter Grimmer, Natascha Leijnse, Min Cheol Kim, Jacob Notbohm, Christian Franck, Alexander R Dunn
The ability of cells to impart forces and deformations on their surroundings underlies cell migration and extracellular matrix (ECM) remodeling, and is thus an essential aspect of complex, metazoan life. Previous work has resulted in a refined understanding, commonly termed the molecular clutch model, of how cells adhering to flat surfaces such as a microscope coverslip transmit cytoskeletally generated forces to their surroundings. Comparatively less is known about how cells adhere to and exert forces in soft, three-dimensional, and structurally heterogeneous ECM environments such as occur in vivo We used timelapse 3D imaging and quantitative image analysis to determine how the actin cytoskeleton was mechanically coupled to the surrounding matrix for primary dermal fibroblasts embedded in a 3D fibrin matrix...
June 7, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28592634/high-quality-frozen-extracts-of-xenopus-laevis-eggs-reveal-size-dependent-control-of-metaphase-spindle-micromechanics
#15
Jun Takagi, Yuta Shimamoto
Cell-free extracts from unfertilized Xenopus laevis eggs offer the opportunity for a variety of biochemical assays for analyzing essential cell cycle events, such as metaphase spindle assembly. However, the extracts often exhibit substantial variation in quality and have low storage stability, which mask important information related to their intrinsic heterogeneity. Here, we report a simple two-step method for preparing frozen egg extracts that retain spindle assembly activity levels that are similar to those of freshly prepared extracts...
June 7, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28592633/the-phospho-caveolin-1-scaffolding-domain-dampens-force-fluctuations-in-focal-adhesions-and-promotes-cancer-cell-migration
#16
Fanrui Meng, Sandeep Saxena, Youtao Liu, Bharat Joshi, Timothy H Wong, Jay Shankar, Leonard J Foster, Pascal Bernatchez, Ivan R Nabi
Caveolin-1 (Cav1), a major Src kinase substrate phosphorylated on tyrosine-14 (Y14), contains the highly-conserved membrane-proximal caveolin scaffolding domain (CSD; amino acids 82-101). Here we show, using CSD mutants (F92A/V94A) and membrane-permeable CSD competing peptides, that Src kinase-dependent pY14Cav1 regulation of focal adhesion protein stabilization, focal adhesion tension and cancer cell migration is CSD-dependent. Quantitative proteomic analysis of Cav1-GST (1-101 amino acids) pulldowns showed 6-fold increased binding of vinculin and, to a lesser extent, α-actinin, talin and filamin, to phosphomimetic Cav1Y14D relative to non-phosphorylatable Cav1Y14F...
June 7, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28592632/rear-polarized-wnt5a-receptor-actin-myosin-polarity-wramp-structures-promote-the-speed-and-persistence-of-directional-cell-migration
#17
Mary Katherine Connacher, Jian Wei Tay, Natalie G Ahn
In contrast to events at the cell leading edge, rear-polarized mechanisms that control directional cell migration remain poorly defined. Previous work described a new intracellular complex, named the "Wnt5a-receptor-actomyosin polarity (WRAMP) structure", which coordinates the polarized localization of MCAM, actin, and myosin IIB, in a Wnt5a-induced manner. However, the polarity and function for the WRAMP structure during cell movement has not been determined. Here, we characterize WRAMP structures during extended cell migration using live cell imaging...
June 7, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28566555/cross-linked-matrix-rigidity-and-soluble-retinoids-synergize-in-nuclear-lamina-regulation-of-stem-cell-differentiation
#18
Irena L Ivanovska, Joe Swift, Kyle Spinler, Dave Dingal, Sangkyun Cho, Dennis E Discher
Synergistic cues from extracellular matrix and soluble factors are often obscure in differentiation. Here, the rigidity of crosslinked collagen synergizes with retinoids in the osteogenesis of human marrow mesenchymal stem cells (MSCs). Collagen nano-films serve as a model matrix that MSCs can easily deform unless the film is enzymatically crosslinked, which promotes the spreading of cells and the stiffening of nuclei as both actomyosin assembly and nucleoskeletal lamin-A increase. Expression of lamin-A is known to be controlled by Retinoic Acid Receptor (RAR) transcription factors, but rigid matrix is needed to induce rapid nuclear accumulation of the RARG isoform and subsequent expression of osteogenic markers...
May 31, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28566554/pi3k-class-ii-%C3%AE-regulates-%C3%AE-opioid-receptor-export-from-the-trans-golgi-network
#19
Daniel J Shiwarski, Marlena Darr, Cheryl A Telmer, Marcel P Bruchez, Manojkumar A Puthenveedu
The interplay between signaling and trafficking by G protein-coupled receptors (GPCRs) has focused mainly on endocytic trafficking. Whether and how surface delivery of newly synthesized GPCRs is regulated by extracellular signals is less understood. Here we define a signaling-regulated checkpoint at the trans-Golgi network (TGN) that controls the surface delivery of the delta opioid receptor (δR). In PC12 cells, inhibition of Phosphoinositide-3 Kinase (PI3K) activity blocked export of newly synthesized δR from the Golgi and delivery to the cell surface, similar to treatment with Nerve Growth Factor (NGF)...
May 31, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28566553/systematic-analysis-of-ca-2-homoeostasis-in-saccharomyces-cerevisiae-based-on-chemical-genetic-interaction-profiles
#20
Farzan Ghanegolmohammadi, Mitsumori Yoshida, Shinsuke Ohnuki, Yuko Sukegawa, Hiroki Okada, Keisuke Obara, Akio Kihara, Kuninori Suzuki, Tetsuya Kojima, Nozomu Yachie, Dai Hirata, Yoshikazu Ohya
We investigated the global landscape of Ca(2+) homeostasis in the budding yeast based on high-dimensional chemical-genetic interaction profiles. The morphological responses of 62 Ca(2+)-sensitive (cls) mutants were quantitatively analyzed with the image processing program CalMorph following exposure to a high concentration of Ca(2+) After a generalized linear model was applied, an analysis of covariance model was used to detect significant Ca(2+)-cls interactions. We found that high-dimensional, morphological Ca(2+)-cls interactions were mixed with positive (86%) and negative (14%) chemical-genetic interactions, while one-dimensional fitness Ca(2+)-cls interactions were all negative in principle...
May 31, 2017: Molecular Biology of the Cell
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