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Molecular Biology of the Cell

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https://www.readbyqxmd.com/read/29444959/the-synaptotagmin-c2b-domain-calcium-binding-loops-modulate-the-rate-of-fusion-pore-expansion
#1
Mounir Bendahmane, Kevin P Bohannon, Mazdak M Bradberry, Tejeshwar C Rao, Michael W Schmidtke, Prabhodh S Abbineni, Nara L Chon, Sherleen Tran, Hai Lin, Edwin R Chapman, Jefferson D Knight, Arun Anantharam
In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here, we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca 2+ -binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca 2+ -binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca 2+ -binding loops of Syt-7 (Syt-1:7C2B 123 ) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1...
February 14, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29444958/cdc48-and-ubiquilins-confer-selective-anterograde-protein-sorting-and-entry-into-the-multivesicular-body-in-yeast
#2
Rachel Kama, Galina Gabriely, Vydehi Kanneganti, Jeffrey E Gerst
Cdc48/p97 is known primarily for the retro-translocation of misfolded proteins in ER-associated protein degradation (ERAD). Here, we uncover a novel function for both Cdc48 and the conserved UBA-UBL ubiquitin receptor (ubiquilin) proteins in yeast ( e.g. Ddi1, Dsk2, Rad23), which deliver ubiquitinated proteins to the proteasome for degradation. We show that Cdc48, its core adaptors Npl4 and Ufd1, and the ubiquilins confer the constitutive anterograde delivery of carboxypeptidase S (Cps1), a membranal hydrolase, to the multivesicular body (MVB) and vacuolar lumen...
February 14, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29444957/p38-activation-induces-the-dissociation-of-tristetraprolin-from-argonaute-2-to-increase-are-mrna-stabilization
#3
Mei-Yan Qi, Jing-Wen Song, Zhuo Zhang, Shuang Huang, Qing Jing
Tristetraprolin (TTP) destabilizes AU-rich element (ARE)-containing mRNA by directly binding with their 3'UTR. P38 stimulation substantially increases ARE-mRNA stability, at least through repressing TTP. However, the mechanism by which P38 keeps TTP inactive has not been fully understood. TTP and ARE-mRNA localize to processing bodies (PBs), the mRNA granules associated with mRNA silencing. Here, we detected the influence of P38 upon TTP localization within PBs and found that P38 regulates TTP localization within PBs...
February 14, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29444956/dual-color-metal-induced-and-f%C3%A3-rster-resonance-energy-transfer-for-cell-nanoscopy
#4
Anna M Chizhik, Carina Wollnik, Daja Ruhlandt, Narain Karedla, Alexey I Chizhik, Lara Hauke, Dirk Hähnel, Ingo Gregor, Jörg Enderlein, Florian Rehfeldt
We report a novel method, dual color axial nanometric localization by Metal Induced Energy Transfer (dcMIET), and combine it with Förster Resonant Energy Transfer (FRET) for resolving structural details in cells on the molecular level. We demonstrate the capability of this method on cytoskeletal elements and adhesions in human mesenchymal stem cells (hMSCs). Our approach is based on Fluorescence-Lifetime-Imaging Microscopy (FLIM), and allows for precise determination of the 3D architecture of stress fibers anchoring at focal adhesions, thus yielding crucial information to understand cell-matrix mechanics...
February 14, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29444955/common-and-divergent-features-of-galactose-1-phosphate-and-fructose-1-phosphate-toxicity-in-yeast
#5
Patrick A Gibney, Ariel Schieler, Jonathan C Chen, Jessie M Bacha-Hummel, Maxim Botstein, Matthew Volpe, Sanford J Silverman, Yifan Xu, Bryson D Bennett, Joshua D Rabinowitz, David Botstein
Toxicity resulting from accumulation of sugar-phosphate molecules is an evolutionarily conserved phenomenon, observed in multiple bacterial and eukaryotic systems, including a number of human diseases. However, the molecular mechanisms involved in sugar-phosphate toxicity remain unclear. Using the model eukaryote S. cerevisiae , we developed two systems to accumulate human disease-associated sugar-phosphate species. One system utilizes constitutive expression of galactose permease and galactose kinase to accumulate galactose-1-phosphate, while the other system utilizes constitutive expression of a mammalian ketohexokinase gene to accumulate fructose-1-phosphate...
February 14, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29437907/mitochondrial-versus-nuclear-gene-expression-and-membrane-protein-assembly-the-case-of-subunit-2-of-yeast-cytochrome-c-oxidase
#6
Diana Rubalcava-Gracia, Miriam Vázquez-Acevedo, Soledad Funes, Xochitl Pérez-Martínez, Diego González-Halphen
Deletion of the yeast mitochondrial gene COX2 , encoding subunit 2 (mtCox2) of cytochrome c oxidase (C c O), results in a respiratory-incompetent Δcox2 strain. For a cytosol-synthesized Cox2 to restore respiratory growth, it must carry the W56R mutation (cCox2W56R). Nevertheless, only a fraction of cCox2W56Ris matured in mitochondria, allowing ∼60% steady-state accumulation of C c O. This can be attributed either to the point mutation or to an inefficient biogenesis of cCox2W56RWe generated a strain expressing the mutant protein mtCox2W56Rinside mitochondria which should follow the canonical biogenesis of mitochondria-encoded Cox2...
February 7, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29386297/dynein-pulling-forces-counteract-lamin-mediated-nuclear-stability-during-nuclear-envelope-repair
#7
Lauren Penfield, Brian Wysolmerski, Michael Mauro, Reza Farhadifar, Michael A Martinez, Ronald Biggs, Hai-Yin Wu, Curtis Broberg, Daniel Needleman, Shirin Bahmanyar
Recent work done exclusively in tissue culture cells revealed that the nuclear envelope (NE) ruptures and repairs in interphase. The duration of NE ruptures depends on lamins, however the underlying mechanisms and relevance to in vivo events is not known. Here, we use the C. elegans zygote to analyze lamin's role in NE rupture and repair in vivo Transient NE ruptures and subsequent NE collapse are induced by weaknesses in the nuclear lamina caused by expression of an engineered hypomorphic C. elegans lamin allele...
January 31, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29386296/respiratory-chain-supercomplexes-associate-with-the-cysteine-desulfurase-complex-of-the-iron-sulfur-cluster-assembly-machinery
#8
Lena Böttinger, Christoph U Mårtensson, Jiyao Song, Nicole Zufall, Nils Wiedemann, Thomas Becker
Mitochondria are the powerhouses of eukaryotic cells. The activity of the respiratory chain complexes generates a proton gradient across the inner membrane, which is used by the F1FO-ATP synthase to produce ATP for cellular metabolism. In baker's yeast Saccharomyces cerevisiae the cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) associate in respiratory chain supercomplexes. Iron-sulfur clusters (ISC) form reactive centers of respiratory chain complexes. The assembly of ISC occurs in the mitochondrial matrix and is essential for cell viability...
January 31, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29386295/dynamics-of-human-telomerase-recruitment-depend-on-template-telomere-base-pairing
#9
Jens C Schmidt, Arthur J Zaug, Regina Kufer, Thomas R Cech
The reverse transcriptase telomerase adds telomeric repeats to chromosome ends to counteract telomere shortening and thereby assures genomic stability in dividing human cells. Key parameters in telomere homeostasis are the frequency with which telomerase engages the chromosome end and the number of telomeric repeats it adds during each association event. To study telomere elongation in vivo we have established a live-cell imaging assay to track individual telomerase RNPs in CRISPR-edited HeLa cells. Using this assay and the drug imetelstat, which is a competitive inhibitor of telomeric DNA binding, we demonstrate that stable association of telomerase with the single-stranded overhang of the chromosome end requires telomerase-DNA base pairing...
January 31, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29386294/the-non-motor-adaptor-hmmr-dampens-eg5-mediated-forces-to-preserve-the-kinetics-and-integrity-of-chromosome-segregation
#10
Helen Chen, Marisa Connell, Lin Mei, Gregor S D Reid, Christopher A Maxwell
Mitotic spindle assembly and organization require forces generated by motor proteins. The activity of these motors is regulated by non-motor adaptor proteins. However, there are limited studies reporting the functional importance of adaptors on the balance of motor forces and the promotion of faithful and timely cell division. Here, we show that genomic deletion or siRNA silencing of the non-motor adaptor Hmmr/HMMR disturbs spindle microtubule organization and bipolar chromosome-kinetochore attachments with a consequent elevated occurrence of aneuploidy...
January 31, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29367436/differential-equation-methods-for-simulation-of-gfp-kinetics-in-non-steady-state-experiments
#11
Robert D Phair
Genetically encoded fluorescent proteins, combined with fluorescence microscopy, are widely used in cell biology to collect kinetic data on intracellular trafficking. Methods for extraction of quantitative information from these data are based on the mathematics of diffusion and tracer kinetics. Current methods, though useful and powerful, depend on the assumption that the cellular system being studied is in a steady state, that is, the assumption that all the molecular concentrations and fluxes are constant for the duration of the experiment...
January 24, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29367435/interaction-between-the-c-elegans-centriolar-protein-sas-5-and-microtubules-facilitates-organelle-assembly
#12
Sarah Bianchi, Kacper B Rogala, Nicola J Dynes, Manuel Hilbert, Sebastian A Leidel, Michel O Steinmetz, Pierre Gönczy, Ioannis Vakonakis
Centrioles are microtubule-based organelles that organise the microtubule network and seed the formation of cilia and flagella. New centrioles assemble through a step-wise process dependent notably on the centriolar protein SAS-5 in Caenorhabditis elegans SAS-5 and its functional homologues in other species form oligomers that bind the centriolar proteins SAS-6 and SAS-4, thereby forming an evolutionarily conserved structural core at the onset of organelle assembly. Here, we report a novel interaction of SAS-5 with microtubules...
January 24, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29343552/constitutive-centromere-associated-network-contacts-confer-differential-stability-on-cenp-a-nucleosomes-in-vitro-and-in-the-cell
#13
Shengya Cao, Keda Zhou, Zhening Zhang, Karolin Luger, Aaron F Straight
Eukaryotic centromeres are defined by the presence of nucleosomes containing the histone H3 variant, Centromere Protein A (CENP-A). Once incorporated at centromeres, CENP-A nucleosomes are remarkably stable, exhibiting no detectable loss or exchange over many cell cycles. It is currently unclear whether this stability is an intrinsic property of CENP-A containing chromatin or whether it arises from proteins that specifically associate with CENP-A chromatin. Two proteins, CENP-C and CENP-N, are known to bind CENP-A human nucleosomes directly...
January 17, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29343551/two-subunits-of-the-exocyst-sec3p-and-exo70p-can-function-exclusively-on-the-plasma-membrane
#14
Dongmei Liu, Xia Li, David Shen, Peter Novick
The exocyst is an octameric complex that tethers secretory vesicles to the plasma membrane in preparation for fusion. We anchored each subunit with a transmembrane (TM) domain at its N- or C-terminus. Only N-terminally anchored TM-Sec3p and C-terminally anchored Exo70p-TM proved functional. These findings orient the complex with respect to the membrane and establish that Sec3p and Exo70p can function exclusively on the membrane. The functions of TM-Sec3p and Exo70p-TM were largely unaffected by blocks in endocytic recycling suggesting that they act on the plasma membrane, rather than on secretory vesicles...
January 17, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29343550/cdk1-dependent-phospho-inhibition-of-a-formin-f-bar-interaction-opposes-cytokinetic-contractile-ring-formation
#15
Alaina H Willet, K Adam Bohnert, Kathleen L Gould
In Schizosaccharomyces pombe, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). A single essential formin, Cdc12, localizes to the cell middle upon mitotic onset and nucleates the F-actin of the CR. Cdc12 medial recruitment is mediated in part by its direct binding to the F-BAR scaffold Cdc15. Given that Cdc12 is hyper-phosphorylated in M phase, we explored whether Cdc12 phospho-regulation impacts its association with Cdc15 during mitosis. We found that Cdk1, a major mitotic kinase, phosphorylates Cdc12 on six N-terminal residues near the Cdc15-binding site...
January 17, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29343549/intracellular-vesicle-trafficking-plays-an-essential-role-in-mitochondrial-quality-control
#16
Mike Gerards, Giuseppe Cannino, Jose M González de Cózar, Howard T Jacobs
The Drosophila gene products Bet1, Slh and CG10144, predicted to function in intracellular vesicle trafficking, were previously found to be essential for mitochondrial nucleoid maintenance. Here we show that Slh and Bet1 co-operate to maintain mitochondrial functions. In their absence, mitochondrial content, membrane potential and respiration became abnormal, accompanied by mitochondrial proteotoxic stress, but without direct effects on mtDNA. Immunocytochemistry showed that both Slh and Bet1 are localized at the Golgi, together with a proportion of Rab5-positive vesicles...
January 17, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29321253/role-of-the-hof1-cyk3-interaction-in-cleavage-furrow-ingression-and-primary-septum-formation-during-yeast-cytokinesis
#17
Meng Wang, Ryuichi Nishihama, Masayuki Onishi, John R Pringle
In Saccharomyces cerevisiae, it is well established that Hof1, Cyk3, and Inn1 contribute to septum formation and cytokinesis. Because hof1∆ and cyk3∆ single mutants have relatively mild defects but hof1∆ cyk3∆ double mutants are nearly dead, it has been hypothesized that these proteins contribute to parallel pathways. However, there is also evidence that they interact physically. In this study, we examined this interaction and its functional significance in detail. Our data indicate that the interaction (i) is mediated by a direct binding of the Hof1 SH3 domain to a proline-rich motif in Cyk3; (ii) occurs specifically at the time of cytokinesis but is independent of the (hyper)phosphorylation of both proteins that occurs at about the same time; (iii) is dispensable for the normal localization of both proteins; (iv) is essential for normal primary-septum formation and a normal rate of cleavage-furrow ingression; and (v) becomes critical for growth when either Inn1 or the type II myosin Myo1 (a key component of the contractile actomyosin ring) is absent...
January 10, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29321252/analysis-of-the-thresholds-for-transcriptional-activation-by-the-yeast-map-kinases-fus3-and-kss1
#18
Matthew J Winters, Peter M Pryciak
Signaling in the pheromone response pathway of budding yeast activates two distinct MAP kinases (MAPKs), Fus3 and Kss1. Either MAPK alone can mediate pheromone-induced transcription, but it has been unclear to what degree each one contributes to transcriptional output in wild-type cells. Here, we report that transcription reflects the ratio of active to inactive MAPK, and not simply the level of active MAPK. For Kss1 the majority of MAPK molecules must be converted to the active form, whereas for Fus3 only a small minority must be activated...
January 10, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29321251/control-of-septin-filament-flexibility-and-bundling-by-subunit-composition-and-nucleotide-interactions
#19
Anum Khan, Jay Newby, Amy S Gladfelter
Septins self-assemble into heteromeric rods and filaments to act as scaffolds and modulate membrane properties. How cells tune the biophysical properties of septin filaments to control filament flexibility and length, and in turn the size, shape, and position of higher-order septin structures is not well understood. We examined how rod composition and nucleotide availability influence physical properties of septins such as annealing, fragmentation, bundling and bending. We found that septin complexes have symmetric termini, even when both Shs1 and Cdc11 are coexpressed...
January 10, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29321250/myosin-ii-interacting-guanine-nucleotide-exchange-factor-myogef-promotes-bleb-retraction-via-stimulating-cortex-reassembly-at-the-bleb-membrane
#20
Meng Jiao, Di Wu, Qize Wei
Blebs are involved in various biological processes such as cell migration, cytokinesis, and apoptosis. While the expansion of blebs is largely an intracellular pressure-driven process, the retraction of blebs is believed to be driven by RhoA activation that leads to the reassembly of the actomyosin cortex at the bleb membrane. However, it is still poorly understood how RhoA is activated at the bleb membrane. Here, we provide evidence demonstrating that nonmuscle myosin II-interacting guanine nucleotide exchange factor (MYOGEF) is implicated in bleb retraction via stimulating RhoA activation and the reassembly of an actomyosin network at the bleb membrane during bleb retraction...
January 10, 2018: Molecular Biology of the Cell
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