Rapid Assembly of Cellular Aggregation Using Micro-Nano Technologies

2014 ◽  
pp. 43-55
Author(s):  
Taisuke Masuda ◽  
Fumihito Arai
Keyword(s):  
Cellular Aggregation

scholarly journals The AGA1 product is involved in cell surface attachment of the Saccharomyces cerevisiae cell adhesion glycoprotein a-agglutinin.

1991 ◽  
Vol 11 (8) ◽  
pp. 4196-4206 ◽  
Author(s):  
A Roy ◽  
C F Lu ◽  
D L Marykwas ◽  
P N Lipke ◽  
J Kurjan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Signal Sequence ◽  
Complementation Group ◽  
Alpha Cells ◽  
Specific Expression ◽  
Surface Attachment ◽  
Cellular Aggregation ◽  
Specific Regulation ◽  
Binding Subunit

Saccharomyces cerevisiae a and alpha cells express the complementary cell surface glycoproteins a-agglutinin and alpha-agglutinin, respectively, which interact with one another to promote cellular aggregation during mating. Treatment of S. cerevisiae a cells with reducing agents releases the binding subunit of a-agglutinin, which has been purified and characterized; little biochemical information on the overall structure of a-agglutinin is available. To characterise a-agglutinin structure and function, we have used a genetic approach to clone an a-agglutinin structural gene (AGAI). Mutants with a-specific agglutination defects were isolated, the majority of which fell into a single complementation group, called aga1. The aga1 mutants showed wild-type pheromone production and response, efficient mating on solid medium, and a mating defect in liquid medium; these phenotypes are characteristic of agglutinin mutants. The AGA1 gene was cloned by complementation; the gene sequence indicated that it could encode a protein of 725 amino acids with high serine and threonine content, a putative N-terminal signal sequence, and a C-terminal hydrophobic sequence similar to signals for the attachment to glycosyl phosphatidylinositol anchors. Active a-agglutinin binding subunit is secreted by aga1 mutants, indicating that AGA1 is involved in cells surface attachment of a-agglutinin. This result suggests that AGA1 encodes a protein with functional similarity to the core subunits of a-agglutinin analogs from other budding yeasts. Unexpectedly, the AGA1 transcript was expressed and induced by pheromone in both a and alpha cells, suggesting that the a-specific expression of active a-agglutinin results only from a-specific regulation of the a-agglutinin binding subunit.

scholarly journals Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Lisa Grossman ◽  
Chris Chang ◽  
Joanne Dai ◽  
Pavel A. Nikitin ◽  
Dereje D. Jima ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Cellular Aggregation ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out. Epstein-Barr virus (EBV), an oncogenic herpesvirus, infects and transforms primary B cells into immortal lymphoblastoid cell lines (LCLs), providing a model for EBV-mediated tumorigenesis. EBV transformation stimulates robust homotypic aggregation, indicating that EBV induces molecules that mediate cell-cell adhesion. We report that EBV potently induced expression of the adhesion molecule CD226, which is not normally expressed on B cells. We found that early after infection of primary B cells, EBV promoted an increase in CD226 mRNA and protein expression. CD226 levels increased further from early proliferating EBV-positive B cells to LCLs. We found that CD226 expression on B cells was independent of B-cell activation as CpG DNA failed to induce CD226 to the extent of EBV infection. CD226 expression was high in EBV-infected B cells expressing the latency III growth program, but low in EBV-negative and EBV latency I-infected B-lymphoma cell lines. We validated this correlation by demonstrating that the latency III characteristic EBV NF-κB activator, latent membrane protein 1 (LMP1), was sufficient for CD226 upregulation and that CD226 was more highly expressed in lymphomas with increased NF-κB activity. Finally, we found that CD226 was not important for LCL steady-state growth, survival in response to apoptotic stress, homotypic aggregation, or adhesion to activated endothelial cells. These findings collectively suggest that EBV induces expression of a cell adhesion molecule on primary B cells that may play a role in the tumor microenvironment of EBV-associated B-cell malignancies or facilitate adhesion in the establishment of latency in vivo. IMPORTANCE Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out.

scholarly journals Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies

2021 ◽  
Author(s):  
David Choy Buentello ◽  
Lina Sophie Koch ◽  
Grissel Trujillo-de Santiago ◽  
Mario Moisés Alvarez ◽  
Kerensa Broersen
Keyword(s):  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Cost Effective ◽  
Embryoid Bodies ◽  
High Yield ◽  
Final Size ◽  
Surface Attachment ◽  
Cellular Aggregation ◽  
Centrifugation Step ◽  
Lower Variability

The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an anti-adherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 ×10 3 cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11×10 3 cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their non-centrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7×10 3 and 11×10 3 and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages

scholarly journals Bistable Expression of CsgD in Biofilm Development of Salmonella enterica Serovar Typhimurium

2009 ◽  
Vol 192 (2) ◽  
pp. 456-466 ◽  
Author(s):  
Nina Grantcharova ◽  
Verena Peters ◽  
Claudia Monteiro ◽  
Katherina Zakikhany ◽  
Ute Römling
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Population Based ◽  
Quantitative Expression ◽  
Serovar Typhimurium ◽  
Cellular Aggregation ◽  
Green Fluorescent ◽  
Biofilm Development ◽  
Biofilm Structure

ABSTRACT Bacterial persistence in the environment and in the infected host is often aided by the formation of exopolymer-enclosed communities known as biofilms. Heterogeneous gene expression takes place in microcompartments formed within the complex biofilm structure. This study describes cell differentiation within an isogenic bacterial cell population based on the example of biofilm formation by Salmonella enterica serovar Typhimurium. We analyzed the expression of the major biofilm regulator CsgD at the single-cell level with a chromosomal CsgD-green fluorescent protein (GFP) translational fusion. In individual cells, CsgD-GFP expression is mostly found in the cytoplasm. Quantitative expression analysis and results from three different models of S. Typhimurium biofilms demonstrated that CsgD is expressed in a bistable manner during biofilm development. CsgD expression is, however, monomodal when CsgD is expressed in larger amounts due to a promoter mutation or elevated levels of the secondary signaling molecule c-di-GMP. High levels of CsgD-GFP are associated with cellular aggregation in all three biofilm models. Furthermore, the subpopulation of cells expressing large amounts of CsgD is engaged in cellulose production during red, dry, and rough (rdar) morphotype development and in microcolony formation under conditions of continuous flow. Consequently, bistability at the level of CsgD expression leads to a corresponding pattern of task distribution in S. Typhimurium biofilms.

scholarly journals Pattern Formation in Keller–Segel Chemotaxis Models with Logistic Growth

2016 ◽  
Vol 26 (02) ◽  
pp. 1650033 ◽  
Author(s):  
Ling Jin ◽  
Qi Wang ◽  
Zengyan Zhang
Keyword(s):  
Steady State ◽  
Pattern Formation ◽  
Neumann Boundary ◽  
Steady States ◽  
Local Theory ◽  
Logistic Growth ◽  
Homogeneous Steady State ◽  
Cellular Aggregation ◽  
Chemotaxis Models ◽  
Steady State Stability

In this paper, we investigate pattern formation in Keller–Segel chemotaxis models over a multidimensional bounded domain subject to homogeneous Neumann boundary conditions. It is shown that the positive homogeneous steady state loses its stability as chemoattraction rate [Formula: see text] increases. Then using Crandall–Rabinowitz local theory with [Formula: see text] being the bifurcation parameter, we obtain the existence of nonhomogeneous steady states of the system which bifurcate from this homogeneous steady state. Stability of the bifurcating solutions is also established through rigorous and detailed calculations. Our results provide a selection mechanism of stable wavemode which states that the only stable bifurcation branch must have a wavemode number that minimizes the bifurcation value. Finally, we perform extensive numerical simulations on the formation of stable steady states with striking structures such as boundary spikes, interior spikes, stripes, etc. These nontrivial patterns can model cellular aggregation that develop through chemotactic movements in biological systems.

Effect of glycosylation on the extracellular domain of the Ag43 bacterial autotransporter: enhanced stability and reduced cellular aggregation

2008 ◽  
Vol 412 (3) ◽  
pp. 563-577 ◽  
Author(s):  
Stine K. Knudsen ◽  
Allan Stensballe ◽  
Magnus Franzmann ◽  
Uffe B. Westergaard ◽  
Daniel E. Otzen
Keyword(s):  
Mammalian Cells ◽  
Bacterial Attachment ◽  
Passenger Domain ◽  
Glycosylation Sites ◽  
Structural And Functional Properties ◽  
Cellular Aggregation ◽  
Membrane Bound ◽  
Extracellular Environment ◽  
Resistant Structure ◽  
Different Levels

Autotransporters constitute the biggest group of secreted proteins in Gram-negative bacteria and contain a membrane-bound β-domain and a passenger domain secreted to the extracellular environment via an unusually long N-terminal sequence. Several passenger domains are known to be glycosylated by cytosolic glycosyl transferases, promoting bacterial attachment to mammalian cells. In the present study we describe the effect of glycosylation on the extracellular passenger domain of the Escherichia coli autotransporter Ag43α, which induces frizzy colony morphology and cell settling. We identify 16 glycosylation sites and suggest two possible glycosylation motifs for serine and threonine residues. Glycosylation stabilizes against thermal and chemical denaturation and increases refolding kinetics. Unexpectedly, glycosylation also reduces the stabilizing effect of Ca2+ ions, removes the ability of Ca2+ to promote cell adhesion, reduces the ability of Ag43α-containing cells to form bacterial amyloid and increases the susceptibility of the resulting amyloid to proteolysis. In addition, our results indicate that Ag43α folds without a stable intermediate, unlike pertactin, indicating that autotransporters may arrive at the native state by a variety of different mechanisms despite a common overall structure. A small but significant fraction of Ag43α can survive intact in the periplasm if expressed without the β-domain, suggesting that it is able to adopt a protease-resistant structure prior to translocation across the membrane. The present study demonstrates that glycosylation may play significant roles in structural and functional properties of bacterial autotransporters at many different levels.

A potential role for the plasmin(ogen) system in the posttranslational cleavage of the neural cell adhesion molecule L1

1999 ◽  
Vol 112 (24) ◽  
pp. 4739-4749 ◽  
Author(s):  
N. Nayeem ◽  
S. Silletti ◽  
X. Yang ◽  
V.P. Lemmon ◽  
R.A. Reisfeld ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Amino Terminal ◽  
Neural Cell Adhesion ◽  
Cellular Aggregation ◽  
Important Regulator ◽  
Cell Adhesion Molecule L1 ◽  
And Function ◽  
Dose Dependent ◽  
Amino Terminal Fragment

L1 is a neural recognition molecule that promotes neural developmental and regenerative processes. Posttranslational cleavage of L1 is believed to be important for regulating its function in vivo, but little is known of the proteolytic systems responsible. In this study we present evidence that plasmin can regulate both L1 expression and function. The addition of plasmin to cell lines results in a dose-dependent loss of surface L1 expression, with the simultaneous appearance of soluble L1 species. The addition of plasminogen to primary neurons and melanoma cells also resulted in the generation of plasmin and the concomitant release of L1. One product of plasmin-mediated cleavage is an amino-terminal fragment of approximately 140 kDa that has been previously described as a natural posttranslational cleavage product in vivo. This fragment was confirmed to result from cleavage at two sites in the middle of the third fibronectin-like domain of L1. Cleavage at a further site, proximal to the transmembrane domain of L1, was also observed at higher plasmin concentrations. Plasmin was further confirmed to abrogate homophilic L1 interactions required for cellular aggregation. Based on these findings we propose that plasmin is likely to be an important regulator of L1-mediated processes including those documented in the nervous system.

Global and exponential attractor of the repulsive Keller–Segel model with logarithmic sensitivity

2020 ◽  
pp. 1-19
Author(s):  
LIN CHEN ◽  
FANZE KONG ◽  
QI WANG
Keyword(s):  
Initial Data ◽  
Steady States ◽  
Sensitivity Function ◽  
Exponential Attractor ◽  
Physical Stimulus ◽  
Chemotaxis Model ◽  
Constant Solution ◽  
Cellular Aggregation ◽  
Logarithmic Sensitivity ◽  
Well Posed

We consider a Keller–Segel model that describes the cellular chemotactic movement away from repulsive chemical subject to logarithmic sensitivity function over a confined region in ${{\mathbb{R}}^n},\,n \le 2$ . This sensitivity function describes the empirically tested Weber–Fecher’s law of living organism’s perception of a physical stimulus. We prove that, regardless of chemotaxis strength and initial data, this repulsive system is globally well-posed and the constant solution is the global and exponential in time attractor. Our results confirm the ‘folklore’ that chemorepulsion inhibits the formation of non-trivial steady states within the logarithmic chemotaxis model, hence preventing cellular aggregation therein.

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