scholarly journals A yeast-optimized single-cell transcriptomics platform elucidates how mycophenolic acid and guanine alter global mRNA levels

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Guste Urbonaite ◽  
Jimmy Tsz Hang Lee ◽  
Ping Liu ◽  
Guillermo E. Parada ◽  
Martin Hemberg ◽  
...  
Keyword(s):  
Single Cell ◽  
Mycophenolic Acid ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Mrna Levels ◽  
A Cell ◽  
Size And Morphology ◽  
And Function ◽  
Morphology Characteristics

AbstractStochastic gene expression leads to inherent variability in expression outcomes even in isogenic single-celled organisms grown in the same environment. The Drop-Seq technology facilitates transcriptomic studies of individual mammalian cells, and it has had transformative effects on the characterization of cell identity and function based on single-cell transcript counts. However, application of this technology to organisms with different cell size and morphology characteristics has been challenging. Here we present yeastDrop-Seq, a yeast-optimized platform for quantifying the number of distinct mRNA molecules in a cell-specific manner in individual yeast cells. Using yeastDrop-Seq, we measured the transcriptomic impact of the lifespan-extending compound mycophenolic acid and its epistatic agent guanine. Each treatment condition had a distinct transcriptomic footprint on isogenic yeast cells as indicated by distinct clustering with clear separations among the different groups. The yeastDrop-Seq platform facilitates transcriptomic profiling of yeast cells for basic science and biotechnology applications.

scholarly journals Reconstitution of functional mRNA-protein complexes in a rabbit reticulocyte cell-free translation system.

1985 ◽  
Vol 5 (2) ◽  
pp. 342-351 ◽  
Author(s):  
J R Greenberg ◽  
E Carroll
Keyword(s):  
Mammalian Cells ◽  
Uv Light ◽  
Protein Complexes ◽  
Micrococcal Nuclease ◽  
Translation System ◽  
Cytoplasmic Process ◽  
Free Translation ◽  
Associated Proteins ◽  
A Cell ◽  
And Function

A variety of evidence suggests that the cytoplasmic mRNA-associated proteins of eucaryotic cells are derived from the cytoplasm and function there, most likely in protein synthesis or some related process. Furthermore, the evidence suggests that protein-free mRNA added to a cell-free translation system should become associated with a set of proteins similar to those associated with mRNA in native polyribosomes. To test this hypothesis, we added deproteinized rabbit reticulocyte mRNA to a homologous cell-free translation system made dependent on exogenous mRNA by treatment with micrococcal nuclease. The resulting reconstituted complexes were irradiated with UV light to cross-link the proteins to mRNA, and the proteins were analyzed by gel electrophoresis. The proteins associated with polyribosomal mRNA in the reconstituted complexes were indistinguishable from those associated with polyribosomal mRNA in intact reticulocytes. Furthermore, reticulocyte mRNA-associated proteins were very similar to those of cultured mammalian cells. The composition of the complexes varied with the translational state of the mRNA; that is, certain proteins present in polyribosomal mRNA-protein complexes were absent or reduced in amount in 40S to 80S complexes and in complexes formed in the absence of translation. However, other proteins, including a 78-kilodalton protein associated with polyadenylate, were present irrespective of translational state, or else they were preferentially associated with untranslated mRNA. These findings are in agreement with previous data suggesting that proteins associated with cytoplasmic mRNA are derived from the cytoplasm and that they function in translation or some other cytoplasmic process, rather than transcription, RNA processing, or transport from the nucleus to the cytoplasm.

scholarly journals Characterization of constitutive ER-phagy of excess membrane proteins

PLoS Genetics ◽  
2020 ◽  
Vol 16 (12) ◽  
pp. e1009255
Author(s):  
Zhanna Lipatova ◽  
Valeriya Gyurkovska ◽  
Sarah F. Zhao ◽  
Nava Segev
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Human Health ◽  
Mammalian Cells ◽  
Normal Growth ◽  
Integral Membrane Proteins ◽  
Yeast Cells ◽  
Cellular Proteins

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.

scholarly journals Single-cell replication profiling reveals stochastic regulation of the mammalian replication-timing program

2017 ◽  
Author(s):  
Vishnu Dileep ◽  
David M. Gilbert
Keyword(s):  
Single Cell ◽  
Mammalian Cells ◽  
Replication Timing ◽  
Chromatin Interaction ◽  
Similar Degree ◽  
Cell Replication ◽  
Stochastic Variation ◽  
Cell Cycles ◽  
And Function ◽  
Cell Variation

AbstractIn mammalian cells, distinct replication domains (RDs), corresponding to structural units of chromosomes called topologically-associating domains (TADs), replicate at different times during S-phase1–4. Further, early/late replication of RDs corresponds to active/inactive chromatin interaction compartments5,6. Although replication origins are selected stochastically, such that each cell is using a different cohort of origins to replicate their genomes7–12, replication-timing is regulated independently and upstream of origin selection13 and evidence suggests that replication timing is conserved in consecutive cell cycles14. Hence, quantifying the extent of cell-to-cell variation in replication timing is central to studies of chromosome structure and function. Here we devise a strategy to measure variation in single-cell replication timing using DNA copy number. We find that borders between replicated and un-replicated DNA are highly conserved between cells, demarcating active and inactive compartments of the nucleus. Nonetheless, measurable variation was evident. Surprisingly, we detected a similar degree of variation in replication timing from cell-to-cell, between homologues within cells, and between all domains genome-wide regardless of their replication timing. These results demonstrate that stochastic variation in replication timing is independent of elements that dictate timing or extrinsic environmental variation.

scholarly journals A new protocol for single-cell RNA-seq reveals stochastic gene expression during lag phase in budding yeast

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Abbas Jariani ◽  
Lieselotte Vermeersch ◽  
Bram Cerulus ◽  
Gemma Perez-Samper ◽  
Karin Voordeckers ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Lag Phase ◽  
Rna Seq ◽  
Molecular Processes ◽  
Sequencing Technology ◽  
Single Cell Rna Sequencing ◽  
Order Of Magnitude

Current methods for single-cell RNA sequencing (scRNA-seq) of yeast cells do not match the throughput and relative simplicity of the state-of-the-art techniques that are available for mammalian cells. In this study, we report how 10x Genomics’ droplet-based single-cell RNA sequencing technology can be modified to allow analysis of yeast cells. The protocol, which is based on in-droplet spheroplasting of the cells, yields an order-of-magnitude higher throughput in comparison to existing methods. After extensive validation of the method, we demonstrate its use by studying the dynamics of the response of isogenic yeast populations to a shift in carbon source, revealing the heterogeneity and underlying molecular processes during this shift. The method we describe opens new avenues for studies focusing on yeast cells, as well as other cells with a degradable cell wall.

INTRODUCTORY REMARKS

PEDIATRICS ◽  
1960 ◽  
Vol 26 (3) ◽  
pp. 452-453
Author(s):  
Mitchell I. Rubin
Keyword(s):  
Single Cell ◽  
Cell Function ◽  
Clinical Medicine ◽  
Human Infant ◽  
Basic Unit ◽  
Organ Function ◽  
Chemical Factory ◽  
A Cell ◽  
Striking Change ◽  
And Function

It May be appropriate to a pediatric audience in a consideration of cellular structure and function to quote from an old nursery rhyme, familiar to most of us: Little girls are made of Sugar and spice and all that's nice— And little boys are made of Frogs and snails and puppy dog tails. Actually, we know that this is not the case, and that little boys and girls are made of cells—millions and millions of them. The human infant at birth is said to contain 10 trillion cells. It, therefore, seems reasonable that prior to a discussion of the numerous diseases which affect children ("bundles of cells"), their nutritional and other requirements, that we consider a basic unit of life—the cell. To the medical student of a quarter of a century ago, and for many years thereafter, the study of the cell held little fascination. Structurally, it was recognized that there was a cell membrane which enclosed a cytoplasmic mass and nucleus, and there were structural substances such as mitochondria within the cytoplasm. The cell as a "chemical factory" was known to biologists, biochemists and geneticists for many years, but this knowledge had very limited influence on medical practice. Physicians thought more in terms of organ function, rather than cell function. This attitude has undergone striking change with the widespread therapeutic usage of vitamins, hormones (particularly the steroid hormones), antibiotics and antimetabolites in clinical medicine. With the recognition of the unity of nature, the biologic similarity between human cells and single-cell organisms and the biologic information gained in the study of single-cell organisms, there has been kindled a widespread and excited inquiry into the functions of the cells of humans.

scholarly journals Structural biology of laminins

2019 ◽  
Vol 63 (3) ◽  
pp. 285-295 ◽  
Author(s):  
Erhard Hohenester
Keyword(s):  
Structural Biology ◽  
Coiled Coil ◽  
Large Cell ◽  
Basement Membranes ◽  
Cell Adhesive ◽  
A Cell ◽  
The Cross ◽  
And Function ◽  
Polypeptide Chains

Abstract Laminins are large cell-adhesive glycoproteins that are required for the formation and function of basement membranes in all animals. Structural studies by electron microscopy in the early 1980s revealed a cross-shaped molecule, which subsequently was shown to consist of three distinct polypeptide chains. Crystallographic studies since the mid-1990s have added atomic detail to all parts of the laminin heterotrimer. The three short arms of the cross are made up of continuous arrays of disulphide-rich domains. The globular domains at the tips of the short arms mediate laminin polymerization; the surface regions involved in this process have been identified by structure-based mutagenesis. The long arm of the cross is an α-helical coiled coil of all three chains, terminating in a cell-adhesive globular region. The molecular basis of cell adhesion to laminins has been revealed by recent structures of heterotrimeric integrin-binding fragments and of a laminin fragment bound to the carbohydrate modification of dystroglycan. The structural characterization of the laminin molecule is essentially complete, but we still have to find ways of imaging native laminin polymers at molecular resolution.

scholarly journals Cell-Based Assays to Detect Inhibitors of Fungal mRNA Capping Enzymes and Characterization of Sinefungin as a Cap Methyltransferase Inhibitor

2005 ◽  
Vol 10 (4) ◽  
pp. 355-364 ◽  
Author(s):  
Gary L. Chrebet ◽  
Douglas Wisniewski ◽  
Ann L. Perkins ◽  
Qiaolin Deng ◽  
Myra B. Kurtz ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Catalytic Mechanism ◽  
Antiviral Agents ◽  
Yeast Cells ◽  
Transcript Stability ◽  
Mrna Capping ◽  
Genes Encoding ◽  
Rna Triphosphatases ◽  
Eukaryotic Mrnas

The m7GpppN cap at the 5′ end of eukaryotic mRNAs is important for transcript stability and translation. Three enzymatic activities that generate the mRNA cap include an RNA 5′-triphosphatase, an RNA guanylyltransferase, and an RNA (guanine-7-) -methyltransferase. The physical organization of the genes encoding these enzymes differs between mammalian cells and yeast, fungi, or viruses. The catalytic mechanism used by the RNA triphosphatases of mammalian cells also differs from that used by the yeast, fungal, or viral enzymes. These structural and functional differences suggest that inhibitors of mRNA capping might be useful antifungal or antiviral agents. The authors describe several whole-cell yeast-based assays developed to identify and characterize inhibitors of fungal mRNA capping. They also report the identification and characterization of the natural product sinefungin in the assays. Their characterization of this S-adenosylmethionine analog suggests that it inhibits mRNA cap methyltransferases and exhibits approximately 5- to 10-fold specificity for the yeast ABD1 and fungal CCM1 enzymes over the human Hcm1 enzyme expressed in yeast cells.

scholarly journals Molecular characterization of bovine placental and ovarian 20α-hydroxysteroid dehydrogenase

Reproduction ◽  
2011 ◽  
Vol 142 (5) ◽  
pp. 723-731 ◽  
Author(s):  
Purevjargal Naidansuren ◽  
Cha-Won Park ◽  
Sang-Hwan Kim ◽  
Tseeleema Nanjidsuren ◽  
Jong-Ju Park ◽  
...  
Keyword(s):  
Estrous Cycle ◽  
Mammalian Cells ◽  
Critical Role ◽  
Immunohistochemical Analysis ◽  
Hydroxysteroid Dehydrogenase ◽  
Mrna Levels ◽  
Luteal Function ◽  
Inactive Form ◽  
Functional Analyses

The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form, 20α-hydroxyprogesterone. This enzyme plays a critical role in the regulation of luteal function in female mammals. In this study, we conducted the characterization and functional analyses of bovine 20α-HSD from placental and ovarian tissues. The nucleotide sequence of bovine 20α-HSD showed significant homology to that of goats (96%), humans (84%), rabbits (83%), and mice (81%). The mRNA levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. Northern blot analysis revealed a 1.2 kb mRNA in the bovine placental and ovarian tissues. An antibody specific to bovine 20α-HSD was generated in a rabbit immunized with the purified, recombinant protein. Recombinant 20α-HSD protein produced in mammalian cells had a molecular weight of ∼37 kDa. Bacterially expressed bovine 20α-HSD protein showed enzymatic activity. The expression pattern of the 20α-HSD protein in the pre-parturition placenta and the CL1 stage of the estrous cycle was similar to the level of 20α-HSD mRNA expression. Immunohistochemical analysis also revealed that bovine 20α-HSD protein was intensively localized in the large luteal cells during the late estrous cycle.

scholarly journals Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Emeline Bonsergent ◽  
Eleonora Grisard ◽  
Julian Buchrieser ◽  
Olivier Schwartz ◽  
Clotilde Théry ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Extracellular Vesicle ◽  
Content Delivery ◽  
Bafilomycin A1 ◽  
Quantitative Characterization ◽  
Endosomal Acidification ◽  
A Cell ◽  
Dose Responses

AbstractExtracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (~1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (~30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion.

Sign in / Sign up

Export Citation Format

Share Document