Migration through a small pore disrupts inactive chromatin organization in neutrophil-like cells

AbstractBackgroundMammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. Their nucleus must be stiff enough to withstand cytoskeletal forces, but flexible enough to remodel as the cell changes shape. This is particularly important for cells migrating through constricted space, where the nuclear shape must change in order to fit through the constriction. This occurs many times in the life cycle of a neutrophil, which must protect its chromatin from damage and disruption associated with migration.ResultsTotal RNA-sequencing identified that neutrophil migration through 5 or 14μm pores was associated with changes in the transcript levels of inflammation and chemotaxis-related genes, when compared to unmigrated cells. Differentially expressed transcripts specific to migration with constriction were enriched for groups of genes associated with cytoskeletal remodeling.Hi-C was used to capture the genome organization in control and migrated cells. Minimal switching was observed between the active (A) and inactive (B) compartments after migration. However, global depletion of short range contacts was observed following migration with constriction compared to migration without constriction. Regions with disrupted contacts, TADs, and compartments were enriched for inactive chromatin.ConclusionShort range genome organization is preferentially altered in inactive chromatin, possibly protecting transcriptionally active contacts from the disruptive effects of migration with constriction. This is consistent with current hypotheses implicating heterochromatin as the mechanoresponsive form of chromatin. Further investigation concerning the contribution of heterochromatin to stiffness, flexibility, and protection of nuclear function will be important for understanding cell migration in human health and disease.

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In Vitro Immunodetection of Prothymosin Alpha in Normal and Pathological Conditions

Current Medicinal Chemistry ◽

10.2174/0929867326666190807145212 ◽

2020 ◽

Vol 27 (29) ◽

pp. 4840-4854 ◽

Cited By ~ 2

Author(s):

Chrysoula-Evangelia Karachaliou ◽

Hubert Kalbacher ◽

Wolfgang Voelter ◽

Ourania E. Tsitsilonis ◽

Evangelia Livaniou

Keyword(s):

Mammalian Cells ◽

Therapeutic Potential ◽

Prothymosin Alpha ◽

Disease States ◽

Leading Role ◽

Tissue Extracts ◽

Biologic Response ◽

Health And Disease ◽

Almost All

Prothymosin alpha (ProTα) is a highly acidic polypeptide, ubiquitously expressed in almost all mammalian cells and tissues and consisting of 109 amino acids in humans. ProTα is known to act both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similar to molecules termed as “alarmins”. Antibodies and immunochemical techniques for ProTα have played a leading role in the investigation of the biological role of ProTα, several aspects of which still remain unknown and contributed to unraveling the diagnostic and therapeutic potential of the polypeptide. This review deals with the so far reported antibodies along with the related immunodetection methodology for ProTα (immunoassays as well as immunohistochemical, immunocytological, immunoblotting, and immunoprecipitation techniques) and its application to biological samples of interest (tissue extracts and sections, cells, cell lysates and cell culture supernatants, body fluids), in health and disease states. In this context, literature information is critically discussed, and some concluding remarks are presented.

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Tetherin is an exosomal tether

eLife ◽

10.7554/elife.17180 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 64

Author(s):

James R Edgar ◽

Paul T Manna ◽

Shinichi Nishimura ◽

George Banting ◽

Margaret S Robinson

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Short Range ◽

Wild Type ◽

Concomitant Increase ◽

Gpi Anchor ◽

Fold Reduction ◽

Cell Metastasis ◽

Health And Disease ◽

Prion Transmission

Exosomes are extracellular vesicles that are released when endosomes fuse with the plasma membrane. They have been implicated in various functions in both health and disease, including intercellular communication, antigen presentation, prion transmission, and tumour cell metastasis. Here we show that inactivating the vacuolar ATPase in HeLa cells causes a dramatic increase in the production of exosomes, which display endocytosed tracers, cholesterol, and CD63. The exosomes remain clustered on the cell surface, similar to retroviruses, which are attached to the plasma membrane by tetherin. To determine whether tetherin also attaches exosomes, we knocked it out and found a 4-fold reduction in plasma membrane-associated exosomes, with a concomitant increase in exosomes discharged into the medium. This phenotype could be rescued by wild-type tetherin but not tetherin lacking its GPI anchor. We propose that tetherin may play a key role in exosome fate, determining whether they participate in long-range or short-range interactions.

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Mitochondrial Iron in Human Health and Disease

Annual Review of Physiology ◽

10.1146/annurev-physiol-020518-114742 ◽

2019 ◽

Vol 81 (1) ◽

pp. 453-482 ◽

Cited By ~ 14

Author(s):

Diane M. Ward ◽

Suzanne M. Cloonan

Keyword(s):

Human Health ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Innate Immune ◽

Biological Functions ◽

Mitochondrial Homeostasis ◽

Innate Immune Signaling ◽

Mitochondrial Iron ◽

Health And Disease

Mitochondria are an iconic distinguishing feature of eukaryotic cells. Mitochondria encompass an active organellar network that fuses, divides, and directs a myriad of vital biological functions, including energy metabolism, cell death regulation, and innate immune signaling in different tissues. Another crucial and often underappreciated function of these dynamic organelles is their central role in the metabolism of the most abundant and biologically versatile transition metals in mammalian cells, iron. In recent years, cellular and animal models of mitochondrial iron dysfunction have provided vital information in identifying new proteins that have elucidated the pathways involved in mitochondrial homeostasis and iron metabolism. Specific signatures of mitochondrial iron dysregulation that are associated with disease pathogenesis and/or progression are becoming increasingly important. Understanding the molecular mechanisms regulating mitochondrial iron pathways will help better define the role of this important metal in mitochondrial function and in human health and disease.

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TLR4 signalling in the intestine in health and disease

Biochemical Society Transactions ◽

10.1042/bst0351473 ◽

2007 ◽

Vol 35 (6) ◽

pp. 1473-1478 ◽

Cited By ~ 94

Author(s):

M. Fukata ◽

M.T. Abreu

Keyword(s):

Mammalian Cells ◽

Metabolic Energy ◽

Short Term ◽

Receptor Signalling ◽

Health And Disease ◽

Inflammatory Bowel ◽

Term Benefit ◽

Tlr Signalling

The colonic epithelium is lined along its apical membrane with ∼1014 bacteria/g of tissue. Commensal bacteria outnumber mammalian cells in the gut severalfold. The reason for this degree of commensalism probably resides in the recent recognition of the microbiome as an important source of metabolic energy in the setting of poorly digestible nutrients. As in many themes in biology, the host may have sacrificed short-term benefit, i.e. nutritional advantages, for long-term consequences, such as chronic inflammation or colon cancer. In the present review, we examine the role of TLR (Toll-like receptor) signalling in the healthy host and the diseased host. We pay particular attention to the role of TLR signalling in idiopathic IBD (inflammatory bowel disease) and colitis-associated carcinogenesis. In general, TLR signalling in health contributes to homoeostatic functions. These include induction of antimicrobial peptides, proliferation and wound healing in the intestine. The pathogenesis of IBD, ulcerative colitis and Crohn's disease may be due to increased TLR or decreased TLR signalling respectively. Finally, we discuss the possible role of TLR signalling in colitis-associated neoplasia.

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Structural analysis of human neutrophil migration: Centriole, microtubule, and microfilament orientation and function during chemotaxis

The Journal of Cell Biology ◽

10.1083/jcb.75.3.666 ◽

1977 ◽

Vol 75 (3) ◽

pp. 666-693 ◽

Cited By ~ 153

Author(s):

HL Malech ◽

RK Root ◽

JI Gallin

Keyword(s):

Cytochalasin B ◽

Neutrophil Migration ◽

Solid Substrate ◽

Human Neutrophils ◽

Random Migration ◽

Filter Surface ◽

Functional Studies ◽

Small Pore ◽

And Migration ◽

Linear Alignment

Orientation of nucleus, centriole, microtubules, and microfilaments within human neutrophils in a gradient of chemoattractant (5 percent Escherichia coli endotoxin-activated serum) was evaluated by electron microscopy. Purified neutropils (hypaque-Ficoll) were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 μm) micropore filters permitted pseudopod penetration, but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus (activated serum above cells) resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Coordinated orientation of the entire neutrophil population did not occur in buffer (random migration) or in a uniform concentration of activated serum (activated random migration). Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e. cellular asymmetry with linear alignment of nucleus, centriole, microtubule array, and pseudopods. Thus, activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil populayion. In related studies, cytochalasin B and colchicines were used to explore the role of microfilaments and microtubules in the neutrophil orientation and migration response to activated serum. Cytochalasin B (3.0 μg/ml) prevented migration and decreased the microfilaments seen, but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicines, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10(-8) to 10(-7) M. These colchicines effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, a formation of narrow pseudopods within a substrate, and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicines, but not lumicolchicine, minimally decreased activated random migration and markedly inhibited directed migration, but had not effect on random migration. These studies show that, although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are essential for normal pseudopod formation and orientation, and maximal unidirectional migration during chemotaxis.

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Role of autophagy in cancer prevention, development and therapy

Essays in Biochemistry ◽

10.1042/bse0550133 ◽

2013 ◽

Vol 55 ◽

pp. 133-151 ◽

Cited By ~ 23

Author(s):

G. Vignir Helgason ◽

Tessa L. Holyoake ◽

Kevin M. Ryan

Keyword(s):

Quality Control ◽

Clinical Trials ◽

Cancer Prevention ◽

Mammalian Cells ◽

Physiological Role ◽

Anticancer Therapy ◽

Substantial Progress ◽

Health And Disease ◽

Made In

Autophagy is a process that takes place in all mammalian cells and ensures homoeostasis and quality control. The term autophagy [self (auto)-eating (phagy)] was first introduced in 1963 by Christian de Duve, who discovered the involvement of lysosomes in the autophagy process. Since then, substantial progress has been made in understanding the molecular mechanism and signalling regulation of autophagy and several reviews have been published that comprehensively summarize these findings. The role of autophagy in cancer has received a lot of attention in the last few years and autophagy modulators are now being tested in several clinical trials. In the present chapter we aim to give a brief overview of recent findings regarding the mechanism and key regulators of autophagy and discuss the important physiological role of mammalian autophagy in health and disease. Particular focus is given to the role of autophagy in cancer prevention, development and in response to anticancer therapy. In this regard, we also give an updated list and discuss current clinical trials that aim to modulate autophagy, alone or in combination with radio-, chemo- or targeted therapy, for enhanced anticancer intervention.

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Functional correlation of H3K9me2 and nuclear compartment formation

10.1101/2020.08.28.271221 ◽

2020 ◽

Author(s):

Kei Fukuda ◽

Chikako Shimura ◽

Hisashi Miura ◽

Akie Tanigawa ◽

Takehiro Suzuki ◽

...

Keyword(s):

Transcriptional Activation ◽

Genome Organization ◽

Mammalian Cells ◽

Embryonic Stem ◽

Epigenetic Mark ◽

3 Dimensional ◽

3D Genome ◽

Functional Correlation ◽

Genome Wide

AbstractBackgroundHistone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and correlates well with lamina-associated domains and the B compartment. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear.ResultsWe investigated the genome-wide H3K9me2 distribution, the transcriptome and 3D genome organization in mouse embryonic stem cells (mESCs) upon the inhibition or depletion of H3K9 methyltransferases (MTases) G9a/GLP, SETDB1, and SUV39H1/2. We found that H3K9me2 is regulated by these five MTases; however, H3K9me2 and transcription in the A and B compartments were largely regulated by different sets of the MTases: H3K9me2 in the A compartments were mainly regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments were regulated by all five H3K9 MTases. Furthermore, decreased H3K9me2 correlated with the changes to the more active compartmental state that accompanied transcriptional activation.ConclusionOur data showed that H3K9me2 domain formation is functionally linked to 3D genome organization.

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Cohesin-Mediated Genome Architecture Does Not Define DNA Replication Timing Domains

Genes ◽

10.3390/genes10030196 ◽

2019 ◽

Vol 10 (3) ◽

pp. 196 ◽

Cited By ~ 6

Author(s):

Phoebe Oldach ◽

Conrad A. Nieduszynski

Keyword(s):

Dna Replication ◽

Genome Organization ◽

Mammalian Cells ◽

Replication Timing ◽

S Phase ◽

Genome Architecture ◽

3D Genome ◽

Synchronized Cells ◽

Dna Replication Timing ◽

Phase Entry

3D genome organization is strongly predictive of DNA replication timing in mammalian cells. This work tested the extent to which loop-based genome architecture acts as a regulatory unit of replication timing by using an auxin-inducible system for acute cohesin ablation. Cohesin ablation in a population of cells in asynchronous culture was shown not to disrupt patterns of replication timing as assayed by replication sequencing (RepliSeq) or BrdU-focus microscopy. Furthermore, cohesin ablation prior to S phase entry in synchronized cells was similarly shown to not impact replication timing patterns. These results suggest that cohesin-mediated genome architecture is not required for the execution of replication timing patterns in S phase, nor for the establishment of replication timing domains in G1.

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Inhibitors of Mammalian Aquaporin Water Channels

International Journal of Molecular Sciences ◽

10.3390/ijms20071589 ◽

2019 ◽

Vol 20 (7) ◽

pp. 1589 ◽

Cited By ~ 15

Author(s):

Mohammed Abir-Awan ◽

Philip Kitchen ◽

Mootaz Salman ◽

Matthew Conner ◽

Alex Conner ◽

...

Keyword(s):

Structural Biology ◽

Mammalian Cells ◽

Tumor Metastasis ◽

Traumatic Injury ◽

Water Channel ◽

Experimental System ◽

Expression Systems ◽

Novel Treatments ◽

Health And Disease ◽

Heterologous Expression Systems

Aquaporins (AQPs) are water channel proteins that are essential to life, being expressed in all kingdoms. In humans, there are 13 AQPs, at least one of which is found in every organ system. The structural biology of the AQP family is well-established and many functions for AQPs have been reported in health and disease. AQP expression is linked to numerous pathologies including tumor metastasis, fluid dysregulation, and traumatic injury. The targeted modulation of AQPs therefore presents an opportunity to develop novel treatments for diverse conditions. Various techniques such as video microscopy, light scattering and fluorescence quenching have been used to test putative AQP inhibitors in both AQP-expressing mammalian cells and heterologous expression systems. The inherent variability within these methods has caused discrepancy and many molecules that are inhibitory in one experimental system (such as tetraethylammonium, acetazolamide, and anti-epileptic drugs) have no activity in others. Some heavy metal ions (that would not be suitable for therapeutic use) and the compound, TGN-020, have been shown to inhibit some AQPs. Clinical trials for neuromyelitis optica treatments using anti-AQP4 IgG are in progress. However, these antibodies have no effect on water transport. More research to standardize high-throughput assays is required to identify AQP modulators for which there is an urgent and unmet clinical need.

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Organization and Regulation of the Human Genome

Blood ◽

10.1182/blood.v128.22.sci-16.sci-16 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-16-SCI-16

Author(s):

Bing Ren

Keyword(s):

Gene Regulation ◽

Long Range ◽

Genome Organization ◽

Mammalian Cells ◽

Target Genes ◽

Critical Role ◽

Cell Types ◽

Regulatory Elements ◽

Chromatin Organization ◽

Specific Gene

Abstract The 3-dimensional (3D) chromatin organization plays a critical role in gene regulation. Great strides have been made recently to characterize and identify cis regulatory elements from epigenome profiles in different cell types and tissues, but efforts have just begun to functionally characterize these long-range control elements. Mapping interactions between enhancers and promoters, and understanding how the 3D landscape of the genome constrains such interactions is fundamental to our understanding of genome function. I will present recent findings related to 3D genome organization in mammalian cells, with a particular focus on how chromatin organization contributes to transcriptional regulation. I will describe higher-order organizational features that are observed at the level of both the whole chromosome and individual loci. I will highlight changes in genome organization that occur during the course of differentiation, and discuss the functional relationship between chromatin architecture and gene regulation. Taken together, mounting evidence now shows that the genome organization plays an essential role in orchestrating the lineage-specific gene expression programs through modulating long- range interactions between enhancers and target genes. Disclosures Ren: Arima Genomics, Inc.: Equity Ownership, Patents & Royalties; Eli Lilly: Employment.

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