Fabrication of Artificial Nanobasement Membranes for Cell Compartmentalization in 3D Tissues

ABSTRACT Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named “Poribacteria”, to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with “Poribacteria”-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.

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The cell cycle of the planctomycete Gemmata obscuriglobus with respect to cell compartmentalization

BMC Cell Biology ◽

10.1186/1471-2121-10-4 ◽

2009 ◽

Vol 10 (1) ◽

Cited By ~ 46

Author(s):

Kuo-Chang Lee ◽

Rick I Webb ◽

John A Fuerst

Keyword(s):

Cell Cycle ◽

Gemmata Obscuriglobus ◽

Cell Compartmentalization

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DYNAMICS OF ACRIDINE ORANGE-CELL INTERACTION

The Journal of Cell Biology ◽

10.1083/jcb.18.2.237 ◽

1963 ◽

Vol 18 (2) ◽

pp. 237-250 ◽

Cited By ~ 109

Author(s):

Elliott Robbins ◽

Philip I. Marcus

Keyword(s):

Cell Viability ◽

Acridine Orange ◽

Time Course ◽

Physiological State ◽

Vital Staining ◽

Cell Interaction ◽

Extracellular Milieu ◽

Ultimate Fate ◽

Cell Compartmentalization

The in vitro localization of acridine orange (AO) in living cells was monitored by means of fluorescence microscopy, quantitative cell viability studies, and photofluorimetric measurements following dye-cell interaction. The parameters, pH, time, dye concentration, and the metabolic state of the cell were found to exert a profound influence on the time course and distribution of staining. The parameters studied are mutually interdependent, and intracellular dye localization may be predictably altered by their appropriate manipulation. Conditions are defined whereby two morphologically distinct but physiologically interrelated reactions, namely, acridine orange particle (AOP) formation and cytoplasmic reddening (CR) may be caused, prevented, reversed, or modified. These results are explained in terms of the facilitation or inhibition of an intracytoplasmic dye-segregating mechanism, in turn affected by the rate of dye ingress and the physiological state of the cell. Whereas the accumulation of AO in AOP is compatible with cell viability, the appearance of CR is correlated with cell death. It is pointed out that meaningful interpretation of vital staining requires precise regulation of many parameters in the extracellular milieu. A scheme of cell compartmentalization with respect to AO is proposed to satisfactorily account for the effects of environmental variations on the distribution and ultimate fate of intracellular dye. The AOP are viewed as normally present acid phosphatase-positive multivesicular bodies.

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Migration matters: regulatory T-cell compartmentalization determines suppressive activity in vivo

Blood ◽

10.1182/blood-2005-05-1864 ◽

2005 ◽

Vol 106 (9) ◽

pp. 3097-3104 ◽

Cited By ~ 175

Author(s):

Kerstin Siegmund ◽

Markus Feuerer ◽

Christiane Siewert ◽

Saeed Ghani ◽

Uta Haubold ◽

...

Keyword(s):

T Cells ◽

Immune Responses ◽

Conclusive Evidence ◽

Effector Memory ◽

Induction Phase ◽

Inflammatory Reactions ◽

Selectin Ligands ◽

Treg Subset ◽

Cell Compartmentalization

AbstractRegulatory T cells (Tregs) play a fundamental role in the suppression of different immune responses; however, compartments at which they exert suppressive functions in vivo are unknown. Although many groups have described the presence of Tregs within inflammatory sites, it has not been shown that inflamed tissues are, indeed, the sites of active suppression of ongoing immune reactions. Here, by using αE+ effector/memory-like Tregs from fucosyltransferase VII-deficient animals, which lack E/P-selectin ligands and fail to migrate into inflamed sites, we analyzed the functional importance of appropriate Treg localization for in vivo suppressive capacity in an inflammation model. Lack of suppression by Tregs deficient in E/P-selectin ligands demonstrates that immigration into inflamed sites is a prerequisite for the resolution of inflammatory reactions in vivo because these selectin ligands merely regulate entry into inflamed tissues. In contrast, control of proliferation of naive CD4+ T cells during the induction phase of the immune response is more efficiently exerted by the naive-like αE–CD25+ Treg subset preferentially recirculating through lymph nodes when compared with its inflammation-seeking counterpart. Together, these findings provide the first conclusive evidence that appropriate localization is crucial for in vivo activity of Tregs and might have significant implications for anti-inflammatory therapies targeting recruitment mechanisms.

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Epixenosomes: peculiar epibionts of the ciliate Euplotidium itoi: morphological and functional cell compartmentalization

Tissue and Cell ◽

10.1016/s0040-8166(96)80018-8 ◽

1996 ◽

Vol 28 (3) ◽

pp. 313-320 ◽

Cited By ~ 5

Author(s):

G. Rosati ◽

M.A. Giambelluca ◽

E. Taiti

Keyword(s):

Cell Compartmentalization

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Trends in the Development of Tailored Elastin-Like Recombinamer–Based Porous Biomaterials for Soft and Hard Tissue Applications

Frontiers in Materials ◽

10.3389/fmats.2020.601795 ◽

2021 ◽

Vol 7 ◽

Author(s):

Lubinda Mbundi ◽

Miguel González-Pérez ◽

Fernando González-Pérez ◽

Diana Juanes-Gusano ◽

José Carlos Rodríguez-Cabello

Keyword(s):

Extracellular Matrix ◽

Biomedical Applications ◽

Physicochemical Characteristics ◽

Matrix Composition ◽

Natural Polymers ◽

Hard Tissue ◽

Modular Assembly ◽

Cell Compartmentalization ◽

Porous Biomaterials ◽

Synthetic And Natural Polymers

Porous biomaterials are of significant interest in a variety of biomedical applications as they enable the diffusion of nutrients and gases as well as the removal of metabolic waste from implants. Pores also provide 3D spaces for cell compartmentalization and the development of complex structures such as vasculature and the extracellular matrix. Given the variation in the extracellular matrix composition across and within different tissues, it is necessary to tailor the physicochemical characteristics of biomaterials and or surfaces thereof for optimal bespoke applications. In this regard, different synthetic and natural polymers have seen increased usage in the development of biomaterials and surface coatings; among them, elastin-like polypeptides and their recombinant derivatives have received increased advocacy. The modular assembly of these molecules, which can be controlled at a molecular level, presents a flexible platform for the endowment of bespoke biomaterial properties. In this review, various elastin-like recombinamer–based porous biomaterials for both soft and hard tissue applications are discussed and their current and future applications evaluated.

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Design and Optimizations of a High-throughput Valve-based Microfluidic Device for Single Cell Compartmentalization and Analysis

10.21203/rs.3.rs-119917/v1 ◽

2020 ◽

Author(s):

Jonathan Briones ◽

Wilfred Espulgar ◽

Shohei Koyama ◽

Hyota Takamatsu ◽

Eiichi Tamiya ◽

...

Keyword(s):

Protein Expression ◽

Single Cell ◽

Microfluidic Device ◽

High Throughput ◽

Single Cells ◽

Enzymatic Assay ◽

Fluid Exchange ◽

Genomics And Proteomics ◽

Sealing Pressure ◽

Cell Compartmentalization

Abstract The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. However, this technique is throughput limited by the number of compartments in the array. Thus, one big challenge lies in increasing the number of microvalves to several thousand that can be actuated in the microfluidic device to confine enzymes and substrates in picoliter volumes. This work explores the design and optimizations done on a microfluidic platform to achieve high-throughput single cell compartmentalization as applied to single-cell enzymatic assay for protein expression quantification. Design modeling through COMSOL Multiphysics was utilized to determine the circular microvalve’s optimized parameters, which can close thousands of microchambers in an array at lower sealing pressure. Multiphysical modeling results demonstrated the relationships of geometry, valve dimensions, and sealing pressure, which were applied in the fabrication of a microfluidic device comprising of up to 5000 hydrodynamic traps and corresponding microvalves. Comparing the effects of geometry, actuation media and fabrication technique, a sealing pressure as low as 0.04 MPa was achieved. Applying to single cell enzymatic assay, variations in granzyme B activity in Jurkat and human PBMC cells were observed. Improvement in the microfluidic chip’s throughput is significant in single cell analysis applications, especially in drug discovery and treatment personalization.

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Calmodulin is involved in the dual subcellular location of two chloroplast proteins

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010846 ◽

2019 ◽

Vol 294 (46) ◽

pp. 17543-17554 ◽

Cited By ~ 2

Author(s):

Lucas Moyet ◽

Daniel Salvi ◽

Imen Bouchnak ◽

Stéphane Miras ◽

Laura Perrot ◽

...

Keyword(s):

Binding Site ◽

Reverse Genetics ◽

Subcellular Location ◽

Bimolecular Fluorescence Complementation ◽

Long Chain Fatty Acid ◽

Binding Properties ◽

Calmodulin Binding ◽

Plastid Proteins ◽

Cell Compartmentalization ◽

And Control

Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.

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