Materials Designed to Control and Examine The Function of Single Cells

1998 ◽  
Vol 530 ◽  
Author(s):  
C.H. Thomas ◽  
J.B. Lhoest ◽  
D.G. Castner ◽  
C.D. Mcfarland ◽  
K.E. Healy
Keyword(s):  
Mammalian Cells ◽  
Cell Function ◽  
Single Cells ◽  
Polymer Network ◽  
Interpenetrating Polymer Network ◽  
Cytoskeletal Organization ◽  
Spatially Resolved ◽  
Cell Organization ◽  
A Cell ◽  
And Function

AbstractStress levels imparted on a cell have been shown to alter cell organization and function, presumably as a result of morphological cues affecting cytoskeletal organization. Materials with spatially resolved surface chemistry were designed to isolate individual mammalian cells to determine the influence of projected area on cell proliferation and cytoskeletal organization. Surfaces were fabricated using a photolithographic process resulting in islands of cell binding N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) separated by a non-adhesive interpenetrating polymer network [poly acrylamide-co-ethylene glycol; P(AAm-co-EG)]. The surfaces contained over 3800 adhesive islands/cm2, allowing for isolation of single cells with projected areas ranging from 100µm2to 10,000µm2. These surfaces provide a useful tool for researching how cell morphology and mechanical forces affect cell function.

Surfaces Designed to Control the Projected Area and Shape of Individual Cells

1999 ◽  
Vol 121 (1) ◽  
pp. 40-48 ◽  
Author(s):  
C. H. Thomas ◽  
J.-B. Lhoest ◽  
D. G. Castner ◽  
C. D. McFarland ◽  
K. E. Healy
Keyword(s):  
Mammalian Cells ◽  
Cell Function ◽  
Single Cells ◽  
Polymer Network ◽  
Interpenetrating Polymer Network ◽  
Specific Cell ◽  
Cell Binding ◽  
Projected Area ◽  
Cell Functions ◽  
Spatially Resolved

Materials with spatially resolved surface chemistry were designed to isolate individual mammalian cells to determine the influence of projected area on specific cell functions (e.g., proliferation, cytoskeletal organization). Surfaces were fabricated using a photolithographic process resulting in islands of cell binding N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) separated by a nonadhesive interpenetrating polymer network [poly (acrylamide-co-ethylene glycol); P(AAm-co-EG)]. The surfaces contained over 3800 adhesive islands/cm2, allowing for isolation of single cells with projected areas ranging from 100 μm2 to 10,000 μm2. These surfaces provide a useful tool for researching how cell morphology and mechanical forces affect cell function.

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.

Structure and Function of the Urokinase Receptor

1999 ◽  
Vol 82 (S 01) ◽  
pp. 19-22 ◽  
Author(s):  
Massimo Resnati ◽  
Anna Mondino ◽  
Francesco Blasi
Keyword(s):  
Conformational Change ◽  
Tyrosine Kinases ◽  
High Efficiency ◽  
Cytoskeletal Organization ◽  
Surface Molecules ◽  
Experimental Systems ◽  
Recombinant Receptor ◽  
A Cell ◽  
And Function

SummaryThe binding of the urokinase plasminogen activator (uPA) to its receptor (uPAR) regulates cell adhesion, surface proteolysis, chemotaxis and cell extravasation in a number of experimental systems. Recent evidences have suggested that uPAR can by itself mediate chemotaxis of human monocytes and cause profound changes in cytoskeletal organization indicating that this receptor has the properties of a cell-surface regulated chemokine. Indeed, it is likely that upon binding to uPA, uPAR undergoes a conformational change that uncovers a new epitope located in the linker region between domain 1 and 2 of the receptor and is endowed with a potent chemotactic activity. This conformational change can be mimicked in vitro by enzymatic processing of a recombinant receptor. We have shown that chymotrypsin cleaves uPAR between domain 1 and 2 in an area that can be also cleaved by uPA at high efficiency and generate a receptor that can mediate monocytes migration independently of uPA binding. This mechanism is pertussis-toxin sensitive and involves activation of tyrosine kinases and cytoskeletal reorganization events in vitro. These studies indicate that in addition to its receptor function, upon binding to uPA, uPAR becomes a pleiotropic ligand for other still to be identified surface molecules.

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.

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 Bioartificial Therapy of Sepsis: Changes of Norepinephrine-Dosage in Patients and Influence on Dynamic and Cell Based Liver Tests during Extracorporeal Treatments

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Martin Sauer ◽  
Jens Altrichter ◽  
Cristof Haubner ◽  
Annette Pertschy ◽  
Thomas Wild ◽  
...  
Keyword(s):  
Cell Function ◽  
Immune Cell ◽  
Prospective Clinical Study ◽  
Extracorporeal Treatment ◽  
Healthy Donors ◽  
Before And After ◽  
Physiologic Parameters ◽  
A Cell ◽  
And Function ◽  
Liver Tests

Purpose.Granulocyte transfusions have been used to treat immune cell dysfunction in sepsis. A granulocyte bioreactor for the extracorporeal treatment of sepsis was tested in a prospective clinical study focusing on the dosage of norepinephrine in patients and influence on dynamic and cell based liver tests during extracorporeal therapies.Methods and Patients.Ten patients with severe sepsis were treated twice within 72 h with the system containing granulocytes from healthy donors. Survival, physiologic parameters, extended hemodynamic measurement, and the indocyanine green plasma disappearance rate (PDR) were monitored. Plasma of patients before and after extracorporeal treatments were tested with a cell based biosensor for analysis of hepatotoxicity.Results.The observed mortality rate was 50% during stay in hospital. During the treatments, the norepinephrine-dosage could be significantly reduced while mean arterial pressure was stable. In the cell based analysis of hepatotoxicity, the viability and function of sensor-cells increased significantly during extracorporeal treatment in all patients and the PDR-values increased significantly between day 1 and day 7 only in survivors.Conclusion.The extracorporeal treatment with donor granulocytes showed promising effects on dosage of norepinephrine in patients, liver cell function, and viability in a cell based biosensor. Further studies with this approach are encouraged.

scholarly journals ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium assembly and Hedgehog signaling

2021 ◽  
Author(s):  
Fatmanur Tiryaki ◽  
Jovana Deretic ◽  
Elif Nur Firat-Karalar
Keyword(s):  
Primary Cilium ◽  
Mammalian Cells ◽  
Hedgehog Signaling ◽  
Developmental Disorders ◽  
Cell Function ◽  
Microtubule Organization ◽  
Regulatory Relationship ◽  
Cellular Assays ◽  
And Function

Centrioles and cilia are conserved, microtubule-based structures critical for cell function and development. Their structural and functional defects cause cancer and developmental disorders. How microtubules are organized into ordered structures by microtubule-associated proteins (MAPs) and tubulin modifications is best understood during mitosis but is largely unexplored for the centrioles and the ciliary axoneme, which are composed of remarkably stable microtubules that maintain their length at steady state. In particular, we know little about the identity of the centriolar and ciliary MAPs and how they work together during the assembly and maintenance of the cilium and centriole. Here, we identified Enkurin domain containing 1 (ENKD1) as a component of the centriole wall and the axoneme in mammalian cells, and showed that it has extensive proximity interactions with these compartments and MAPs. Using in vitro and cellular assays, we found that ENKD1 is a new MAP that promotes microtubule polymerization and regulates microtubule organization and stability. Consistently, overexpression of ENKD1 increased tubulin polymerization and acetylation and disrupted microtubule organization. Cells depleted for ENKD1 were defective in ciliary length and content regulation and failed to respond to Hedgehog pathway activation. Together, our results establish ENKD1 as a new centriolar and ciliary MAP that regulate primary cilium structure and function, and advances our understanding of the functional and regulatory relationship between MAPs and the primary cilium.

scholarly journals Identification of two pathways mediating protein targeting from ER to lipid droplets

2021 ◽  
Author(s):  
Jiunn Song ◽  
Arda Mizrak ◽  
Chia-Wei Lee ◽  
Marcelo Cicconet ◽  
Zon Weng Lai ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Protein Targeting ◽  
Eukaryotic Cell ◽  
Snare Proteins ◽  
Er Exit Sites ◽  
Cell Organization ◽  
A Cell ◽  
Sites Of Action ◽  
And Function ◽  
Specific Membrane

Pathways localizing proteins to their sites of action within a cell are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, little is known about how proteins, such as key metabolic enzymes, target from the ER to cellular lipid droplets (LDs). Here, we identify two distinct pathways for ER-to-LD (ERTOLD) protein targeting: early ERTOLD, occurring during LD formation, and late ERTOLD, targeting mature LDs after their formation. By using systematic, unbiased approaches, we identified specific membrane-fusion machinery, including regulators, a tether, and SNARE proteins, that are required for late ERTOLD targeting. Components of this fusion machinery localize to LD-ER interfaces and appear to be organized at ER exit sites (ERES) to generate ER-LD membrane bridges. We also identified multiple cargoes for early and late ERTOLD. Collectively, our data provide a new model for how proteins target LDs from the ER.

scholarly journals Click-encoded rolling FISH for visualizing single-cell RNA polyadenylation and structures

2019 ◽  
Vol 47 (22) ◽  
pp. e145-e145 ◽  
Author(s):  
Feng Chen ◽  
Min Bai ◽  
Xiaowen Cao ◽  
Yue Zhao ◽  
Jing Xue ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Molecule ◽  
Rna Processing ◽  
Single Cells ◽  
Quantitative Information ◽  
Spatially Resolved ◽  
A Cell ◽  
Cell Type Specific ◽  
Order Structures ◽  
Rna Polyadenylation

Abstract Spatially resolved visualization of RNA processing and structures is important for better studying single-cell RNA function and landscape. However, currently available RNA imaging methods are limited to sequence analysis, and not capable of identifying RNA processing events and structures. Here, we developed click-encoded rolling FISH (ClickerFISH) for visualizing RNA polyadenylation and structures in single cells. In ClickerFISH, RNA 3′ polyadenylation tails, single-stranded and duplex regions are chemically labeled with different clickable DNA barcodes. These barcodes then initiate DNA rolling amplification, generating repetitive templates for FISH to image their subcellular distributions. Combined with single-molecule FISH, the proposed strategy can also obtain quantitative information of RNA of interest. Finally, we found that RNA poly(A) tailing and higher-order structures are spatially organized in a cell type-specific style with cell-to-cell heterogeneity. We also explored their spatiotemporal patterns during cell cycle stages, and revealed the highly dynamic organization especially in S phase. This method will help clarify the spatiotemporal architecture of RNA polyadenylation and structures.

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