scholarly journals A Genetic Screen for Hedgehog Targets Involved in the Maintenance of the Drosophila Anteroposterior Compartment Boundary

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1427-1438 ◽  
Author(s):  
Mátyás Végh ◽  
Konrad Basler
Keyword(s):  
Cell Sorting ◽  
Genetic Screen ◽  
Cubitus Interruptus ◽  
Adhesion Properties ◽  
Compartment Boundary ◽  
Dependent Cell ◽  
A Cell ◽  
Hh Signaling ◽  
Cell Segregation ◽  
Multicellular Organisms

Abstract The development of multicellular organisms requires the establishment of cell populations with different adhesion properties. In Drosophila, a cell-segregation mechanism underlies the maintenance of the anterior (A) and posterior (P) compartments of the wing imaginal disc. Although engrailed (en) activity contributes to the specification of the differential cell affinity between A and P cells, recent evidence suggests that cell sorting depends largely on the transduction of the Hh signal in A cells. The activator form of Cubitus interruptus (Ci), a transcription factor mediating Hh signaling, defines anterior specificity, indicating that Hh-dependent cell sorting requires Hh target gene expression. However, the identity of the gene(s) contributing to distinct A and P cell affinities is unknown. Here, we report a genetic screen based on the FRT/FLP system to search for genes involved in the correct establishment of the anteroposterior compartment boundary. By using double FRT chromosomes in combination with a wing-specific FLP source we screened 250,000 mutagenized chromosomes. Several complementation groups affecting wing patterning have been isolated, including new alleles of most known Hh-signaling components. Among these, we identified a class of patched (ptc) alleles exhibiting a novel phenotype. These results demonstrate the value of our setup in the identification of genes involved in distinct wing-patterning processes.

scholarly journals A Numerical Simulation of Cell Separation by Simplified Asymmetric Pinched Flow Fractionation

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Jing-Tao Ma ◽  
Yuan-Qing Xu ◽  
Xiao-Ying Tang
Keyword(s):  
Lattice Boltzmann ◽  
Cell Sorting ◽  
Cell Separation ◽  
Immersed Boundary ◽  
Flow Cells ◽  
Size Dependent ◽  
Sorting Technique ◽  
Dependent Cell ◽  
A Cell ◽  
Boltzmann Method

As a typical microfluidic cell sorting technique, the size-dependent cell sorting has attracted much interest in recent years. In this paper, a size-dependent cell sorting scheme is presented based on a controllable asymmetric pinched flow by employing an immersed boundary-lattice Boltzmann method (IB-LBM). The geometry of channels consists of 2 upstream branches, 1 transitional channel, and 4 downstream branches (D-branches). Simulations are conducted by varying inlet flow ratio, the cell size, and the ratio of flux of outlet 4 to the total flux. It is found that, after being randomly released in one upstream branch, the cells are aligned in a line close to one sidewall of the transitional channel due to the hydrodynamic forces of the asymmetric pinched flow. Cells with different sizes can be fed into different downstream D-branches just by regulating the flux of one D-branch. A principle governing D-branch choice of a cell is obtained, with which a series of numerical cases are performed to sort the cell mixture involving two, three, or four classes of diameters. Results show that, for each case, an adaptive regulating flux can be determined to sort the cell mixture effectively.

Proximal to distal cell communication in the Drosophila leg provides a basis for an intercalary mechanism of limb patterning

Development ◽  
1999 ◽  
Vol 126 (15) ◽  
pp. 3407-3413 ◽  
Author(s):  
S. Goto ◽  
S. Hayashi
Keyword(s):  
Cell Sorting ◽  
Planar Cell Polarity ◽  
Cell Communication ◽  
Limb Patterning ◽  
Circular Pattern ◽  
Compartment Boundary ◽  
A Cell ◽  
Circular Arrangement ◽  
Distal Cell ◽  
Anterior Posterior

Proximodistal patterning in the Drosophila leg is elaborated from the circular arrangement of the proximal domain expressing escargot and homothorax, and the distal domain expressing Distal-less that are allocated during embryogenesis. The distal domain differentiates multiply segmented distal appendages by activating additional genes such as dachshund. Secreted signaling molecules Wingless and Decapentaplegic, expressed along the anterior-posterior compartment boundary, are required for activation of Distal-less and dachshund and repression of homothorax in the distal domain. However, whether Wingless and Decapentaplegic are sufficient for the circular pattern of gene expression is not known. Here we show that a proximal gene escargot and its activator homothorax regulate proximodistal patterning in the distal domain. Clones of cells expressing escargot or homothorax placed in the distal domain induce intercalary expression of dachshund in surrounding cells and reorient planar cell polarity of those cells. Escargot and homothorax-expressing cells also sort out from other cells in the distal domain. We suggest that inductive cell communication between the proximodistal domains, which is maintained in part by a cell-sorting mechanism, is the cellular basis for an intercalary mechanism of the proximodistal axis patterning of the limb.

scholarly journals A cell-sorting-based nano-scale pipeline for cell-type-specific proteomics in plants

2021 ◽  
Author(s):  
Hongliang Wang ◽  
Rongxia Li ◽  
Jingkai Rong ◽  
Tian Sang ◽  
Chanhong Kim ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Guard Cells ◽  
Specific Cell ◽  
Root Tips ◽  
Mesophyll Cells ◽  
Cell Functions ◽  
Nano Scale ◽  
A Cell ◽  
Multiple Cell ◽  
Multicellular Organisms

Multicellular organisms such as plants contain different types of cells with specialized functions. Analyzing the characteristics of each type of cells will reveal specific cell functions and enhance understanding of how an organism organizes and works. Due to the lack of a method to enrich the particular type of cells and the limited sensitivity and capability of mass spectrometry-based technologies, comprehensive analyses of the global proteome in a single plant cell are still impossible. Here we report a high-sensitive and efficient single-cell-proteomic pipeline, combining simplified flow cytometry-based fluorescent cell-sorting for fluorescent protoplasts and an optimized nano-scale proteomics method, which allows us to identify more than 1,000 unique proteins from 500 guard cell protoplasts. This method was applied to profile the proteome of guard cells and mesophyll cells in Arabidopsis leaves, and epidermal cells in root tips of Arabidopsis and rice seedlings. We also performed an in-depth, quantitative comparison between the proteomics of guard cells and mesophyll cells and revealed that the enrichment of signal transduction-related proteins enables guard cells to respond to various environmental stimuli quickly. This method is applicable to other types of cells in plant or non-plant systems to acquire systemic knowledge of how cells work specifically and in highly organized multiple cell organisms.

Freezing

2017 ◽  
Author(s):  
Andrew Clarke
Keyword(s):  
Thermal Hysteresis ◽  
Higher Plants ◽  
Terrestrial Environment ◽  
Cold Temperatures ◽  
Cellular Dehydration ◽  
A Cell ◽  
Unicellular Organisms ◽  
Cell Cytosol ◽  
Freezing Temperatures ◽  
Multicellular Organisms

Freezing is a widespread ecological challenge, affecting organisms in over half the terrestrial environment as well as both polar seas. With very few exceptions, if a cell freezes internally, it dies. Polar teleost fish in shallow waters avoid freezing by synthesising a range of protein or glycoprotein antifreezes. Terrestrial organisms are faced with a far greater thermal challenge, and exhibit a more complex array of responses. Unicellular organisms survive freezing temperatures by preventing ice nucleating within the cytosol, and tolerating the cellular dehydration and membrane disruption that follows from ice forming in the external environment. Multicellular organisms survive freezing temperatures by manipulating the composition of the extracellular body fluids. Terrestrial organisms may freeze at high subzero temperatures, often promoted by ice nucleating proteins, and small molecular mass cryoprotectants (often sugars and polyols) moderate the osmotic stress on cells. A range of chaperone proteins (dehydrins, LEA proteins) help maintain the integrity of membranes and macromolecules. Thermal hysteresis (antifreeze) proteins prevent damaging recrystallisation of ice. In some cases arthropods and higher plants prevent freezing in their extracellular fluids and survive by supercooling. Vitrification of extracellular water, or of the cell cytosol, may be a more widespread response to very cold temperatures than recognised to date.

scholarly journals Differential Sorting of Microparticles Using Spiral Microchannels with Elliptic Configurations

Micromachines ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 412
Author(s):  
Kaan Erdem ◽  
Vahid Ebrahimpour Ahmadi ◽  
Ali Kosar ◽  
Lütfullah Kuddusi
Keyword(s):  
Cell Sorting ◽  
Microfluidic Channel ◽  
Major Axis ◽  
Label Free ◽  
Flow Rates ◽  
Inertial Focusing ◽  
Size Dependent ◽  
Curved Channels ◽  
Aspect Ratios ◽  
Dependent Cell

Label-free, size-dependent cell-sorting applications based on inertial focusing phenomena have attracted much interest during the last decade. The separation capability heavily depends on the precision of microparticle focusing. In this study, five-loop spiral microchannels with a height of 90 µm and a width of 500 µm are introduced. Unlike their original spiral counterparts, these channels have elliptic configurations of varying initial aspect ratios, namely major axis to minor axis ratios of 3:2, 11:9, 9:11, and 2:3. Accordingly, the curvature of these configurations increases in a curvilinear manner through the channel. The effects of the alternating curvature and channel Reynolds number on the focusing of fluorescent microparticles with sizes of 10 and 20 µm in the prepared suspensions were investigated. At volumetric flow rates between 0.5 and 3.5 mL/min (allowing separation), each channel was tested to collect samples at the designated outlets. Then, these samples were analyzed by counting the particles. These curved channels were capable of separating 20 and 10 µm particles with total yields up to approximately 95% and 90%, respectively. The results exhibited that the level of enrichment and the focusing behavior of the proposed configurations are promising compared to the existing microfluidic channel configurations.

Structural features and functional domains of amassin-1, a cell-binding olfactomedin protein

2007 ◽  
Vol 85 (5) ◽  
pp. 552-562 ◽  
Author(s):  
Brian J. Hillier ◽  
Victor D. Vacquier
Keyword(s):  
Disulfide Bonds ◽  
Biological Activities ◽  
Structural Features ◽  
Body Cavity ◽  
Binding Activity ◽  
Coiled Coils ◽  
Cell Binding ◽  
Calcium Dependent ◽  
Dependent Cell ◽  
A Cell

Amassin-1 mediates a rapid cell adhesion that tightly adheres sea urchin coelomocytes (body cavity immunocytes) together. Three major structural regions exist in amassin-1: a short β region, 3 coiled coils, and an olfactomedin domain. Amassin-1 contains 8 disulfide-bonded cysteines that, upon reduction, render it inactive. Truncated forms of recombinant amassin-1 were expressed and purified from Pichia pastoris and their disulfide bonding and biological activities investigated. Expressed alone, the olfactomedin domain contained 2 intramolecular disulfide bonds, existed in a monomeric state, and inhibited amassin-1-mediated clotting of coelomocytes by a calcium-dependent cell-binding activity. The N-terminal β region, containing 3 cysteines, was not required for clotting activity. The coiled coils may dimerize amassin-1 in a parallel orientation through a homodimerizing disulfide bond. Neither amassin-1 fragments that were disulfide-linked as dimers or that were engineered to exist as dimers induced coelomocytes clotting. Clotting required higher multimeric states of amassin-1, possibly tetramers, which occurred through the N-terminal β region and (or) the first segment of coiled coils.

Intracellular acidification induces apoptosis by stimulating ICE-like protease activity

1997 ◽  
Vol 110 (5) ◽  
pp. 653-661 ◽  
Author(s):  
I.J. Furlong ◽  
R. Ascaso ◽  
A. Lopez Rivas ◽  
M.K. Collins
Keyword(s):  
Cell Line ◽  
Dna Fragmentation ◽  
Intracellular Ph ◽  
Protease Activity ◽  
Intracellular Acidification ◽  
Over Expression ◽  
Protease Activation ◽  
Dependent Cell ◽  
A Cell ◽  
Ph Decrease

ICE-like protease activation and DNA fragmentation are preceded by a decrease in intracellular pH (pHi) during apoptosis in the IL-3 dependent cell line BAF3. Acidification occurs after 7 hours in cells deprived of IL-3 and after 4 hours when cells are treated with etoposide, close to the time of detection of ICE-like protease activity. Increasing extracellular pH reduces ICE-like protease activation and DNA fragmentation. Bcl-2 over-expression both delays acidification and inhibits ICE-like protease activation. Generation of a rapid intracellular pH decrease, using the ionophore nigericin, induces ICE-like protease activation and apoptosis. ZVAD, a cell permeable inhibitor of ICE-like proteases, does not affect acidification but inhibits apoptosis induced by IL-3 removal or nigericin treatment. These data suggest that intracellular acidification triggers apoptosis by directly or indirectly activating ICE-like proteases.

scholarly journals Novel compound inhibitors of HIV-1NL4-3 Vpu

2021 ◽  
Author(s):  
Carolyn A Robinson ◽  
Terri D Lyddon ◽  
Hwi Min Gil ◽  
David T. Evans ◽  
Yury V Kuzmichev ◽  
...  
Keyword(s):  
Host Cell ◽  
Particle Production ◽  
Viral Glycoprotein ◽  
New Class ◽  
Host Cell Proteins ◽  
Viral Particles ◽  
Dependent Cell ◽  
A Cell ◽  
Hiv 1

HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we developed a cell-based 'gain of function' assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. It was conceivable that the compounds inhibited the formation of infectious virions by targeting host cell proteins instead of Vpu directly, so we developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu.

Role of knot (kn) in Wing Patterning in Drosophila

Genetics ◽  
1997 ◽  
Vol 147 (3) ◽  
pp. 1203-1212 ◽  
Author(s):  
Katerina Nestoras ◽  
Helena Lee ◽  
Jym Mohler
Keyword(s):  
Hedgehog Signaling ◽  
Imaginal Disc ◽  
Hedgehog Signaling Pathway ◽  
Posterior Compartment ◽  
Drosophila Wing ◽  
Compartment Boundary ◽  
Hh Signaling ◽  
Embryonic Segmentation ◽  
Anterior Posterior

We have undertaken a genetic analysis of new strong alleles of knot (kn). The original kn1 mutation causes an alteration of wing patterning similar to that associated with mutations of fused (fu), an apparent fusion of veins 3 and 4 in the wing. However, unlike fu, strong kn mutations do not affect embryonic segmentation and indicate that kn is not a component of a general Hh (Hedgehog)-signaling pathway. Instead we find that kn has a specific role in those cells of the wing imaginal disc that are subject to ptc-mediated Hh-signaling. Our results suggest a model for patterning the medial portion of the Drosophila wing, whereby the separation of veins 3 and 4 is maintained by kn activation in the intervening region in response to Hh-signaling across the adjacent anterior-posterior compartment boundary.

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