Cell-sorting in aggregates of Dictyostelium discoideum

1999 ◽  
Vol 112 (22) ◽  
pp. 3923-3929 ◽  
Author(s):  
A. Nicol ◽  
W. Rappel ◽  
H. Levine ◽  
W.F. Loomis
Keyword(s):  
Cell Sorting ◽  
Fluorescent Protein ◽  
Cell Mass ◽  
Three Dimensional ◽  
Cell Movement ◽  
Time Lapse ◽  
Cell Types ◽  
Cell Type ◽  
Prespore Cell ◽  
Green Fluorescent

When Dictyostelium cells are induced to develop between a coverslip and a layer of agarose, they aggregate normally into groups containing up to a thousand cells but are then constrained to form disks only a few cells thick that appear to be equivalent to the three-dimensional mounds formed on top of agarose. Such vertically restricted aggregates frequently develop into elongated motile structures, the flattened equivalent of three-dimensional slugs. The advantage of using this system is that the restricted z-dimension enables direct microscopic visualization of most of the cells in the developing structure. We have used time lapse digital fluorescence microscopy of Dictyostelium strains expressing green fluorescent protein (GFP) under the control of either prestalk or prespore specific promoters to follow cell sorting in these flattened mounds. We find that prestalk and prespore cells expressing GFP arise randomly in early aggregates and then rotate rapidly around the disk mixed with the other cell type. After a few hours, the cell types sort out by a process which involves striking changes in relative cell movement. Once sorted, the cell types move independently of each other showing very little heterotypic adhesion. When a group of prestalk cells reaches the edge of the disk, it moves out and is followed by the prespore cell mass. We suggest that sorting may result from cell type specific changes in adhesion and the consequent disruption of movement in the files of cells that are held together by end-to-end adhesion.

scholarly journals Nucleocytoplasmic transport in human astrocytes: decreased nuclear uptake of the HIV Rev shuttle protein

2001 ◽  
Vol 114 (9) ◽  
pp. 1717-1729
Author(s):  
M. Neumann ◽  
E. Afonina ◽  
F. Ceccherini-Silberstein ◽  
S. Schlicht ◽  
V. Erfle ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Virus Production ◽  
Time Lapse ◽  
Cell Types ◽  
Nucleocytoplasmic Transport ◽  
Nucleocytoplasmic Trafficking ◽  
Nuclear Uptake ◽  
Strong Shift ◽  
Human Astrocytes ◽  
Green Fluorescent

Astrocytes are cellular targets for the human immunodeficiency virus (HIV) that limit virus production, owing, at least in part, to the diminished functionality of the viral post-transcriptional stimulatory factor Rev. To understand the trafficking process in astrocytes, we compared nucleocytoplasmic transport of Rev and various proteins with well-characterized nucleocytoplasmic transport features in human astrocytes and control cells (HeLa). Localization and trafficking characteristics of several cellular and viral proteins, as well as nuclear trafficking of classical peptide signals upon microinjection were similar in both cell types, indicating maintenance of general features of nucleocytoplasmic transport in astrocytes. Quantification of fluorescence in living cells expressing Rev fused to green fluorescent protein (GFP) indicated a strong shift in intracellular distribution of Rev in astrocytes, with 50–70% of Rev in the cytoplasm, whereas the cytoplasmic proportion of Rev in HeLa cells is around 10%. The dynamics of nucleocytoplasmic trafficking of Rev were compared in astrocytes and Rev-permissive cells by monitoring migration of Rev-GFP in cell fusions using highly sensitive time-lapse imaging. Nuclear uptake of Rev was dramatically retarded in homo-polykaryons of astrocytes compared with control cells. Diminished nuclear uptake of Rev was also observed in hetero-polykaryons of Rev-permissive cells and astrocytes. These results indicate that astrocytes contain a cytoplasmic activity that interferes with nuclear uptake of Rev. Our studies suggest a model in which Rev is prevented from functioning efficiently in astrocytes by specific alterations of its nucleocytoplasmic trafficking properties. http://www.biologists.com/JCS/movies/jcs1709.html

Three Dimensional Immuno-Localization of Green Fluorescent Protein Chimeras Using Rapid Freezing and Freeze-Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 298-299
Author(s):  
Mary Morphew ◽  
David Mastronarde ◽  
Eileen O'Toole ◽  
Mark Ladinsky ◽  
Brad Marsh ◽  
...  
Keyword(s):  
Low Temperature ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Cell Types ◽  
Uranyl Acetate ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Green Fluorescent ◽  
Structural Preservation

All microscopy is limited by the quality of the specimen under study. Three-dimensional (3-D) visualization of antigen localization using the electron microscope (EM) is particularly challenging due to the need to maintain the activity of some epitopes while preserving cellular ultrastructure. We have used rapid freezing to immobilize all cellular constituents almost instantaneously. Freeze-substitution of the frozen samples was used to stabilize the specimen and to accomplish low-temperature dehydration, minimizing perturbation of cellular structure. We have found that high pressure freezing, double jet freezing and plunge freezing are all useful for achieving high quality structural preservation for some cell types or for particular applications. For immunolocalization, we have had most success freeze-substituting into acetone containing 0.2% glutaraldehyde and 0.1 % uranyl acetate. We have utilized low-temperature acrylic embedding resins, Lowicryl HM20 and LRGold, to further maintain structure and decrease protein insolubility. Both of these resins have proven suitable for cutting serial thin sections.

scholarly journals Fluorescence-Activated Cell Sorting Using the D-Root Device and Optimization for Scarce and/or Non-Accessible Root Cell Populations

Plants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 499 ◽  
Author(s):  
Mary-Paz González-García ◽  
Estéfano Bustillo-Avendaño ◽  
Alvaro Sanchez-Corrionero ◽  
Juan C. del Pozo ◽  
Miguel A. Moreno-Risueno
Keyword(s):  
Cell Sorting ◽  
Fluorescent Protein ◽  
Rna Extraction ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Fluorescence Activated Cell Sorting ◽  
Stressful Environment ◽  
Minimum Number ◽  
Green Fluorescent

Fluorescence-activated cell sorting (FACS) is a technique used to isolate specific cell populations based on characteristics detected by flow cytometry. FACS has been broadly used in transcriptomic analyses of individual cell types during development or under different environmental conditions. Different protoplast extraction protocols are available for plant roots; however, they were designed for accessible cell populations, which normally were grown in the presence of light, a non-natural and stressful environment for roots. Here, we report a protocol using FACS to isolate root protoplasts from Arabidopsis green fluorescent protein (GFP)-marked lines using the minimum number of enzymes necessary for an optimal yield, and with the root system grown in darkness in the D-Root device. This device mimics natural conditions as the shoot grows in the presence of light while the roots grow in darkness. In addition, we optimized this protocol for specific patterns of scarce cell types inside more differentiated tissues using the mCherry fluorescent protein. We provide detailed experimental protocols for effective protoplasting, subsequent purification through FACS, and RNA extraction. Using this RNA, we generated cDNA and sequencing libraries, proving that our methods can be used for genome-wide transcriptomic analyses of any cell-type from roots grown in darkness.

scholarly journals Proteomic analysis of V-ATPase-rich cells harvested from the kidney and epididymis by fluorescence-activated cell sorting

2010 ◽  
Vol 298 (6) ◽  
pp. C1326-C1342 ◽  
Author(s):  
Nicolas Da Silva ◽  
Trairak Pisitkun ◽  
Clémence Belleannée ◽  
Lance R. Miller ◽  
Raoul Nelson ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Enzymatic Digestion ◽  
Potential Candidate ◽  
Fluorescence Activated Cell Sorting ◽  
Cytoskeletal Dynamics ◽  
Liquid Chromatography Tandem Mass ◽  
Green Fluorescent ◽  
Extracellular Environment

Proton-transporting cells are located in several tissues where they acidify the extracellular environment. These cells express the vacuolar H+-ATPase (V-ATPase) B1 subunit (ATP6V1B1) in their plasma membrane. We provide here a comprehensive catalog of the proteins that are expressed in these cells, after their isolation by enzymatic digestion and fluorescence-activated cell sorting (FACS) from transgenic B1-enhanced green fluorescent protein (EGFP) mice. In these mice, type A and B intercalated cells and connecting segment cells of the kidney, and narrow and clear cells of the epididymis, which all express ATP6V1B1, also express EGFP, while all other cell types are negative. The proteome of renal and epididymal EGFP-positive (EGFP+) cells was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and compared with their respective EGFP-negative (EGFP−) cell populations. A total of 2,297 and 1,564 proteins were detected in EGFP+ cells from the kidney and epididymis, respectively. Out of these proteins, 202 and 178 were enriched by a factor greater than 1.5 in EGFP+ cells compared with EGFP− cells, in the kidney and epididymis respectively, and included subunits of the V-ATPase (B1, a4, and A). In addition, several proteins involved in intracellular trafficking, signaling, and cytoskeletal dynamics were identified. A novel common protein that was enriched in renal and epididymal EGFP+ cells is the progesterone receptor, which might be a potential candidate for the regulation of V-ATPase-dependent proton transport. These proteomic databases provide a framework for comprehensive future analysis of the common and distinct functions of V-ATPase-B1-expressing cells in the kidney and epididymis.

scholarly journals In Vivo Observations of Myosin II Dynamics Support a Role in Rear Retraction

1999 ◽  
Vol 10 (5) ◽  
pp. 1309-1323 ◽  
Author(s):  
Patricia A. Clow ◽  
James G. McNally
Keyword(s):  
Heavy Chain ◽  
Fluorescent Protein ◽  
Rotational Velocity ◽  
Myosin Ii ◽  
Cell Mass ◽  
Cell Movement ◽  
Posterior Cortex ◽  
Guidance Cues ◽  
Green Fluorescent

To investigate myosin II function in cell movement within a cell mass, we imaged green fluorescent protein-myosin heavy chain (GFP-MHC) cells moving within the tight mound of Dictyostelium discoideum. In the posterior cortex of cells undergoing rotational motion around the center of the mound, GFP-MHC cyclically formed a “C,” which converted to a spot as the cell retracted its rear. Consistent with an important role for myosin in rotation, cells failed to rotate when they lacked the myosin II heavy chain (MHC−) or when they contained predominantly monomeric myosin II (3xAsp). In cells lacking the myosin II regulatory light chain (RLC−), rotation was impaired and eventually ceased. These rotational defects reflect a mechanical problem in the 3xAsp and RLC− cells, because these mutants exhibited proper rotational guidance cues. MHC− cells exhibited disorganized and erratic rotational guidance cues, suggesting a requirement for the MHC in organizing these signals. However, the MHC− cells also exhibited mechanical defects in rotation, because they still moved aberrantly when seeded into wild-type mounds with proper rotational guidance cues. The mechanical defects in rotation may be mediated by the C-to-spot, because RLC− cells exhibited a defective C-to-spot, including a slower C-to-spot transition, consistent with this mutant’s slower rotational velocity.

scholarly journals Influences on neural lineage and mode of division in the zebrafish retina in vivo

2005 ◽  
Vol 171 (6) ◽  
pp. 991-999 ◽  
Author(s):  
Lucia Poggi ◽  
Marta Vitorino ◽  
Ichiro Masai ◽  
William A. Harris
Keyword(s):  
Cell Fate ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Time Lapse ◽  
Retinal Ganglion ◽  
Fixed Tissue ◽  
Environmental Signals ◽  
Neural Lineage ◽  
Green Fluorescent

Cell determination in the retina has been under intense investigation since the discovery that retinal progenitors generate clones of apparently random composition (Price, J., D. Turner, and C. Cepko. 1987. Proc. Natl. Acad. Sci. USA. 84:156–160; Holt, C.E., T.W. Bertsch, H.M. Ellis, and W.A. Harris. 1988. Neuron. 1:15–26; Wetts, R., and S.E. Fraser. 1988. Science. 239:1142–1145). Examination of fixed tissue, however, sheds little light on lineage patterns or on the relationship between the orientation of division and cell fate. In this study, three-dimensional time-lapse analyses were used to trace lineages of retinal progenitors expressing green fluorescent protein under the control of the ath5 promoter. Surprisingly, these cells divide just once along the circumferential axis to produce two postmitotic daughters, one of which becomes a retinal ganglion cell (RGC). Interestingly, when these same progenitors are transplanted into a mutant environment lacking RGCs, they often divide along the central-peripheral axis and produce two RGCs. This study provides the first insight into reproducible lineage patterns of retinal progenitors in vivo and the first evidence that environmental signals influence the orientation of cell division and the lineage of neural progenitors.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

scholarly journals Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

2011 ◽  
Vol 77 (23) ◽  
pp. 8310-8317 ◽  
Author(s):  
Joshua D. Morris ◽  
Jessica L. Hewitt ◽  
Lawrence G. Wolfe ◽  
Nachiket G. Kamatkar ◽  
Sarah M. Chapman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Nile Red ◽  
Temporal Distribution ◽  
Time Lapse ◽  
Content Type ◽  
Rhamnolipid Production ◽  
Serovar Typhimurium ◽  
Surface Motility ◽  
Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

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