scholarly journals Mammalian cell proliferation requires noncatalytic functions of O-GlcNAc transferase

2021 ◽  
Vol 118 (4) ◽  
pp. e2016778118
Author(s):  
Zebulon G. Levine ◽  
Sarah C. Potter ◽  
Cassandra M. Joiner ◽  
George Q. Fei ◽  
Behnam Nabet ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Mammalian Cell ◽  
Control Cell ◽  
Cell Types ◽  
Cell Physiology ◽  
Enzymatic Reactions ◽  
A Cell ◽  
Glcnac Transferase ◽  
Catalytic Functions

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGT’s myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGT’s enzymatic activities for cell viability. Using genetic complementation, we found that OGT’s glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGT’s catalytic and noncatalytic activities affect protein abundance, we found that OGT’s noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology.

scholarly journals Mammalian Cell Proliferation Requires Noncatalytic Functions of O-GlcNAc Transferase

2020 ◽  
Author(s):  
Zebulon G. Levine ◽  
Sarah C. Potter ◽  
Cassandra M. Joiner ◽  
George Q. Fei ◽  
Behnam Nabet ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Cell Viability ◽  
Mammalian Cell ◽  
Control Cell ◽  
Enzymatic Activities ◽  
Cell Physiology ◽  
Enzymatic Reactions ◽  
Binding Partners ◽  
Glcnac Transferase

AbstractO-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGT’s myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGT’s enzymatic activities for cell viability. Using genetic complementation, we found that OGT’s glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically-inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGT’s catalytic and noncatalytic activities affect protein abundance, we found that OGT’s noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology.SignificanceMammalian cells contain only one glycosyltransferase, OGT, that operates in the nucleus and cytoplasm rather than the secretory pathway. OGT is required for cell proliferation, but a basic unanswered question is what OGT functions are essential. This question is challenging to address because OGT has thousands of glycosylation substrates, two different enzymatic activities, and a large number of binding partners. Here, by establishing genetic tools to replace endogenous OGT with variants that preserve only a subset of its activities, we show that only a low level of glycosylation activity is required to maintain cell viability; however, cell proliferation requires noncatalytic OGT function(s). The ability to replace OGT with variants provides a path to identifying its essential substrates and binding partners.

scholarly journals Long-range memory of growth and cycle progression correlates cell cycles in lineage trees

2018 ◽  
Author(s):  
Erika E Kuchen ◽  
Nils Becker ◽  
Nina Claudino ◽  
Thomas Höfer
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Mammalian Cell ◽  
Latent Variable ◽  
Cycle Length ◽  
Growth Control ◽  
Minimum Size ◽  
Cell Cycles ◽  
Lineage Trees

Mammalian cell proliferation is controlled by mitogens. However, how proliferation is coordinated with cell growth is poorly understood. Here we show that statistical properties of cell lineage trees – the cell-cycle length correlations within and across generations – reveal how cell growth controls proliferation. Analyzing extended lineage trees with latent-variable models, we find that two antagonistic heritable variables account for the observed cycle-length correlations. Using molecular perturbations of mTOR and MYC we identify these variables as cell size and regulatory license to divide, which are coupled through a minimum-size checkpoint. The checkpoint is relevant only for fast cell cycles, explaining why growth control of mammalian cell proliferation has remained elusive. Thus, correlated fluctuations of the cell cycle encode its regulation.

scholarly journals Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death.

1996 ◽  
Vol 133 (5) ◽  
pp. 1041-1051 ◽  
Author(s):  
M D Jacobsen ◽  
M Weil ◽  
M C Raff
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Apoptotic Cell ◽  
Interleukin 1 ◽  
Cell Types ◽  
Cysteine Proteases ◽  
A Cell ◽  
Bona Fide

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS-induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.

scholarly journals miRNA-independent function of long noncoding pri-miRNA loci

2021 ◽  
Vol 118 (13) ◽  
pp. e2017562118
Author(s):  
Daniel He ◽  
David Wu ◽  
Soren Muller ◽  
Lin Wang ◽  
Parna Saha ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Cell Biology ◽  
Cell Types ◽  
Dna Looping ◽  
Gene Promoters ◽  
Functional Studies ◽  
A Genome ◽  
Genome Wide Data ◽  
Cell Propagation

Among the large, diverse set of mammalian long noncoding RNAs (lncRNAs), long noncoding primary microRNAs (lnc-pri-miRNAs) are those that host miRNAs. Whether lnc-pri-miRNA loci have important biological function independent of their cognate miRNAs is poorly understood. From a genome-scale lncRNA screen, lnc-pri-miRNA loci were enriched for function in cell proliferation, and in glioblastoma (i.e., GBM) cells with DGCR8 or DROSHA knockdown, lnc-pri-miRNA screen hits still regulated cell growth. To molecularly dissect the function of a lnc-pri-miRNA locus, we studied LOC646329 (also known as MIR29HG), which hosts the miR-29a/b1 cluster. In GBM cells, LOC646329 knockdown reduced miR-29a/b1 levels, and these cells exhibited decreased growth. However, genetic deletion of the miR-29a/b1 cluster (LOC646329-miR29Δ) did not decrease cell growth, while knockdown of LOC646329-miR29Δ transcripts reduced cell proliferation. The miR-29a/b1–independent activity of LOC646329 corresponded to enhancer-like activation of a neighboring oncogene (MKLN1), regulating cell propagation. The LOC646329 locus interacts with the MKLN1 promoter, and antisense oligonucleotide knockdown of the lncRNA disrupts these interactions and reduces the enhancer-like activity. More broadly, analysis of genome-wide data from multiple human cell types showed that lnc-pri-miRNA loci are significantly enriched for DNA looping interactions with gene promoters as well as genomic and epigenetic characteristics of transcriptional enhancers. Functional studies of additional lnc-pri-miRNA loci demonstrated cognate miRNA-independent enhancer-like activity. Together, these data demonstrate that lnc-pri-miRNA loci can regulate cell biology via both miRNA-dependent and miRNA-independent mechanisms.

FRACTAL FRAGMENTATION IN REPLICATIVE SYSTEMS

Fractals ◽  
1993 ◽  
Vol 01 (02) ◽  
pp. 239-246 ◽  
Author(s):  
GABRIEL LANDINI ◽  
JOHN W. RIPPIN
Keyword(s):  
Cell Growth ◽  
Pattern Formation ◽  
Cell Types ◽  
Active Movement ◽  
Small Perturbations ◽  
Highly Sensitive ◽  
A Cell ◽  
Natural Outcome ◽  
Fractal Fragmentation ◽  
Cell Growth Model

This paper describes a cell growth model formed by two cell types in which the cells are capable of displacing adjacent populations. Evolution of the model gives rise to patches that are fractally distributed (fractal fragmentation). The fragmentation of the system is not highly sensitive to the relative proportions of the two cell types, and it reveals new insights into fractal pattern formation. It is suggested that the fractal fragmentation is the natural outcome of multiple small perturbations in spatial rearrangement of the cells during multiplication. In addition, the model could prove useful in explaining both the development and spread of clones in a population of cells, and pattern formation in mosaic animal organs, in neither of which active movement of cells is implicit.

Thermal ‘Fingerprinting’ of Cells Using FTIR

2007 ◽  
Author(s):  
Saravana Kumar Balasubramanian ◽  
Williem F. Wolkers ◽  
John C. Bischof
Keyword(s):  
Protein Denaturation ◽  
Control Cell ◽  
Cell Types ◽  
Relevant Information ◽  
Dermal Fibroblasts ◽  
Chemical Fingerprint ◽  
Multiple Protein ◽  
Chemical Fingerprinting ◽  
A Cell ◽  
Mechanistic Basis

Every cell is composed of a combination of proteins and lipids that define the cell. The biomolecular composition of a cell can yield a unique IR spectral profile, termed chemical “fingerprint”, in the FTIR. Even though it provides overall information on the state and composition, the challenge is in obtaining a more sensitive and mechanistically relevant “fingerprint” of a cell particularly in the protein regime where numerous detection assays are now based [1]. Heat induced denaturation of proteins in a cell, which can be termed as thermal “fingerprint”, offers detailed and mechanistically relevant information for identification and characterization of different cells. For this study, the chemical and the thermal “fingerprint” of four different cell types are characterized and compared — human dermal fibroblasts (HDFs), LNCaP prostate tumor cells, smooth muscle cells (SMCs) and microvascular endothelial cells (MVECs). Multiple protein denaturation peaks were observed during heating from room temperature to 90°C at 1°C/min that are cell specific. A correlation coefficient (r) was used to compare responses between the cell types (higher ‘r’ implies closer resemblance). For the chemical “fingerprint”, r values of cells compared to HDFs are 0.97 for MVECs, 0.91 for LNCaPs and 0.70 for SMCs. Similarly, for the thermal “fingerprint”, r values of cells compared to HDFs are 0.86 for MVECs, 0.73 for LNCaPs and 0.54 for SMCs. The deviation of ‘r’ values from 1.00 is wider in the case of the thermal “fingerprint” than the chemical “fingerprint”. This result demonstrates that thermal “fingerprinting”, based on protein denaturation, offers a mechanistic basis for enhanced differentiation between cells as compared to chemical “fingerprinting”. Extensive applications ranging from rapid disease diagnostics, forensics, cell culture quality control, cell sorting techniques etc. may benefit from this approach.

scholarly journals Taraxasterol acetate targets RNF31 to inhibit RNF31/p53 axis-driven cell proliferation in colorectal cancer

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Chao-Tao Tang ◽  
Jing Yang ◽  
Zi-De Liu ◽  
Youxiang Chen ◽  
Chunyan Zeng
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Cell Model ◽  
Tissue Samples ◽  
The Third ◽  
Common Cancer ◽  
A Cell ◽  
Growth Of Tumor

AbstractColorectal cancer (CRC) is the third most common cancer worldwide. Several studies have suggested that taraxasterol acetate (TA) can inhibit the growth of tumor cells. However, to date, it remains unclear how TA inhibits cell growth and how RNF31 functions as an oncogene. We examined the expression of RNF31 in CRC tissue samples via immunohistochemistry and elucidated the function of RNF31 in CRC cells by constructing a cell model with RNF31 depletion. A cycloheximide (CHX)-chase analysis and immunofluorescence assays were conducted to demonstrate that TA can promote RNF31 degradation by activating autophagy. We used the PharmMapper website to predict targets of TA and identified RNF31. CHX-chase experiments showed that TA could facilitate RNF31 degradation, which was inhibited by the administration of chloroquine. Immunofluorescence assays showed that RNF31 protein was colocalized with LC3I/II and p62, suggesting that TA promoted RNF31 degradation by activating autophagy. We also found that CRC patients with RNF31 overexpression had poorer survival than those with low RNF31 expression. The results of the CHX-chase experiment showed that depletion of RNF31 alleviated p53 degradation, which was inhibited by MG132. A series of co-immunoprecipitation (Co-IP) assays revealed that RNF31 interacts with p53 and promotes p53 ubiquitination and degradation. A Co-IP assay performed with a truncated RNF31 plasmid showed that the PUB domain interacts with p53. Moreover, the PUB domain is the key structure in the induction of p53 ubiquitination. Our findings reveal a key role of RNF31 in CRC cell growth and indicate a mechanism through which TA inhibits cell growth.

Controlling Cell Growth by Nanoparticles

2006 ◽  
Vol 950 ◽  
Author(s):  
Sergiy Zankovych ◽  
Joerg Bossert ◽  
Ines Thiele ◽  
Klaus D. Jandt ◽  
Liga Berzina-Cimdina
Keyword(s):  
Surface Roughness ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Titanium Surface ◽  
Cell Attachment ◽  
Microcontact Printing ◽  
Control Cell ◽  
Superficial Layer ◽  
Titanium Surfaces ◽  
Titanium Substrates

ABSTRACTWe report preliminary results of using nanoparticles to control cell attachment and growth. We present the way to create titanium surfaces with different roughness in a rage between 2 nm and 117 nm by using nanoparticles as a superficial layer and varying the evaporation parameters. We examined cell proliferation on titanium substrates with increased surface roughness compared to smooth titanium surface. We used nanoparticles to create a micrometer-sized lateral layout onto substrates preliminary structured by microcontact printing. We demonstrate controlled cell growth on substrates laterally structured with nanoparticles.

scholarly journals Monocarboxylate Transporter 8 in Neuronal Cell Growth

Endocrinology ◽  
2008 ◽  
Vol 150 (4) ◽  
pp. 1961-1969 ◽  
Author(s):  
S. R. James ◽  
J. A. Franklyn ◽  
B. J. Reaves ◽  
V. E. Smith ◽  
S. Y. Chan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Thyroid Hormone ◽  
Cell Growth ◽  
Neural Development ◽  
Neuronal Cell ◽  
Normal Growth ◽  
Cell Types ◽  
Monocarboxylate Transporter ◽  
Dependent Manner ◽  
Nt2 Cells

Thyroid hormones are essential for the normal growth and development of the fetus, and even small alterations in maternal thyroid hormone status during early pregnancy may be associated with neurodevelopmental abnormalities in childhood. Mutations in the novel and specific thyroid hormone transporter monocarboxylate transporter 8 (MCT8) have been associated with severe neurodevelopmental impairment. However, the mechanism by which MCT8 influences neural development remains poorly defined. We have therefore investigated the effect of wild-type (WT) MCT8, and the previously reported L471P mutant, on the growth and function of human neuronal precursor NT2 cells as well as MCT8-null JEG-3 cells. HA-tagged WT MCT8 correctly localized to the plasma membrane in NT2 cells and increased T3 uptake in both cell types. In contrast, L471P MCT8 was largely retained in the endoplasmic reticulum and displayed no T3 transport activity. Transient overexpression of WT and mutant MCT8 proteins failed to induce endoplasmic reticular stress or apoptosis. However, MCT8 overexpression significantly repressed cell proliferation in each cell type in both the presence and absence of the active thyroid hormone T3 and in a dose-dependent manner. In contrast, L471P MCT8 showed no such influence. Finally, small interfering RNA depletion of endogenous MCT8 resulted in increased cell survival and decreased T3 uptake. Given that T3 stimulated proliferation in embryonic neuronal NT2 cells, whereas MCT8 repressed cell growth, these data suggest an entirely novel role for MCT8 in addition to T3 transport, mediated through the modulation of cell proliferation in the developing brain.

scholarly journals Bacterial cell proliferation: from molecules to cells

2020 ◽  
Author(s):  
Alix Meunier ◽  
François Cornet ◽  
Manuel Campos
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Growth ◽  
Bacterial Cell ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Cell Physiology ◽  
Precise Knowledge

ABSTRACT Bacterial cell proliferation is highly efficient, both because bacteria grow fast and multiply with a low failure rate. This efficiency is underpinned by the robustness of the cell cycle and its synchronization with cell growth and cytokinesis. Recent advances in bacterial cell biology brought about by single-cell physiology in microfluidic chambers suggest a series of simple phenomenological models at the cellular scale, coupling cell size and growth with the cell cycle. We contrast the apparent simplicity of these mechanisms based on the addition of a constant size between cell cycle events (e.g. two consecutive initiation of DNA replication or cell division) with the complexity of the underlying regulatory networks. Beyond the paradigm of cell cycle checkpoints, the coordination between the DNA and division cycles and cell growth is largely mediated by a wealth of other mechanisms. We propose our perspective on these mechanisms, through the prism of the known crosstalk between DNA replication and segregation, cell division and cell growth or size. We argue that the precise knowledge of these molecular mechanisms is critical to integrate the diverse layers of controls at different time and space scales into synthetic and verifiable models.

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