scholarly journals Migration of primordial germ cells during late embryogenesis of pikeperch Sander lucioperca relative to blastomere transplantation

2017 ◽  
Vol 62 (No. 3) ◽  
pp. 121-129 ◽  
Author(s):  
H. Güralp ◽  
K. Pocherniaieva ◽  
M. Blecha ◽  
T. Policar ◽  
M. Pšenička ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Fluorescent Protein ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Somatic Cells ◽  
Time Lapse ◽  
Sander Lucioperca ◽  
Initial Appearance ◽  
Green Fluorescent ◽  
Time Lapse Imaging

Pikeperch Sander lucioperca is a valuable fish in Europe, and basic information about its embryonic development, especially primordial germ cell (PGC) migration, is important for use in biotechnology. We categorized pikeperch embryonic development into six stages as in other fish species: zygote, cleavage, blastula, gastrula, segmentation, and hatching and described PGC migration. PGCs were visualized by injection of synthesized green fluorescent protein (GFP) within the 3’untranslated region (UTR) mRNA of nanos3. GFP-positive PGCs appeared in all embryos at approximately 100% epiboly. Time-lapse imaging revealed the PGC migration pattern from their initial appearance to location at the gonadal ridge. We conducted blastomere transplantation (BT) at the blastula stage. Donor embryos were labelled with GFP-nos3 3’UTR mRNA and tetramethylrhodamine dextran to label PGCs and somatic cells, respectively. Twelve BT chimeras were produced, with eight surviving to hatching. All exhibited donor-derived somatic cells in the developing body. The PGCs from donor embryos were observed to migrate towards the gonad region of the host embryos. Our results indicated that BT can be successfully applied in pikeperch, and these findings may be useful to produce germline chimeras in percids.

Lumenal targeted GFP, used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network

2000 ◽  
Vol 113 (18) ◽  
pp. 3151-3159 ◽  
Author(s):  
R. Blum ◽  
D.J. Stephens ◽  
I. Schulz
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Temperature Sensitive ◽  
Long Distance ◽  
Anterograde Transport ◽  
Network Transport ◽  
Vesicular Stomatitis ◽  
Green Fluorescent ◽  
Time Lapse Imaging ◽  
Tubular Membranes

The mechanism by which soluble proteins without sorting motifs are transported to the cell surface is not clear. Here we show that soluble green fluorescent protein (GFP) targeted to the lumen of the endoplasmic reticulum but lacking any known retrieval, retention or targeting motifs, was accumulated in the lumen of the ERGIC if cells were kept at reduced temperature. Upon activation of anterograde transport by rewarming of cells, lumenal GFP stained a microtubule-dependent, pre-Golgi tubulo-vesicular network that served as transport structure between peripheral ERGIC-elements and the perinuclear Golgi complex. Individual examples of these tubular elements up to 20 microm in length were observed. Time lapse imaging indicated rapid anterograde flow of soluble lumenal GFP through this network. Transport tubules, stained by lumenal GFP, segregated rapidly from COPI-positive membranes after transport activation. A transmembrane cargo marker, the temperature sensitive glycoprotein of the vesicular stomatitis virus, ts-045 G, is also not present in tubules which contained the soluble cargo marker lum-GFP. These results suggest a role for pre-Golgi vesicular tubular membranes in long distance anterograde transport of soluble cargo. http://www.biologists.com/JCS/movies/jcs1334.html

scholarly journals Two-Color Green Fluorescent Protein Time-Lapse Imaging

BioTechniques ◽  
1998 ◽  
Vol 25 (5) ◽  
pp. 838-846 ◽  
Author(s):  
Jan Ellenberg ◽  
Jennifer Lippincott-Schwartz ◽  
John F. Presley
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Green Fluorescent ◽  
Time Lapse Imaging

Regulation of zebrafish primordial germ cell migration by attraction towards an intermediate target

Development ◽  
2002 ◽  
Vol 129 (1) ◽  
pp. 25-36
Author(s):  
Gilbert Weidinger ◽  
Uta Wolke ◽  
Marion Köprunner ◽  
Christine Thisse ◽  
Bernard Thisse ◽  
...  
Keyword(s):  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Somatic Cells ◽  
Time Lapse ◽  
Intermediate Target ◽  
Target Time ◽  
Mesoderm Development ◽  
Somatic Tissues ◽  
Germ Cell Migration ◽  
Anterior Trunk

Migration of primordial germ cells (PGCs) from their site of specification towards the developing gonad is controlled by directional cues from somatic tissues. Although in several animals the PGCs are attracted by signals emanating from their final target, the gonadal mesoderm, little is known about the mechanisms that control earlier steps of migration. We provide evidence that a key step of zebrafish PGC migration, in which the PGCs become organized into bilateral clusters in the anterior trunk, is regulated by attraction of PGCs towards an intermediate target. Time-lapse observations of wild-type and mutant embryos reveal that bilateral clusters are formed at early somitogenesis, owing to migration of PGCs towards the clustering position from medial, posterior and anterior regions. Furthermore, PGCs migrate actively relative to their somatic neighbors and they do so as individual cells. Using mutants that exhibit defects in mesoderm development, we show that the ability to form PGC clusters depends on proper differentiation of the somatic cells present at the clustering position. Based on these findings, we propose that these somatic cells produce signals that attract PGCs. Interestingly, fate-mapping shows that these cells do not give rise to the somatic tissues of the gonad, but rather contribute to the formation of the pronephros. Thus, the putative PGC attraction center serves as an intermediate target for PGCs, which later actively migrate towards a more posterior position. This final step of PGC migration is defective in hands off mutants, where the intermediate mesoderm of the presumptive gonadal region is mispatterned. Our results indicate that zebrafish PGCs are guided by attraction towards two signaling centers, one of which may represent the somatic tissues of the gonad.Movies available on-line

scholarly journals Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles

2000 ◽  
Vol 113 (24) ◽  
pp. 4421-4426 ◽  
Author(s):  
M. Kaksonen ◽  
H.B. Peng ◽  
H. Rauvala
Keyword(s):  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Time Lapse ◽  
Actin Binding ◽  
Common Mechanism ◽  
Membrane Protrusions ◽  
Green Fluorescent ◽  
Time Lapse Imaging ◽  
Endosomal Vesicles

We have used fluorescent protein tagging to study the localization and dynamics of the actin-binding protein cortactin in living NIH 3T3 fibroblast cells. Cortactin was localized to active lamellipodia and to small cytoplasmic spots. Time-lapse imaging revealed that these cortactin labeled structures were very dynamic. In the lamellipodia, cortactin labeled structures formed at the leading edge and then moved toward the cell center. Experiments with green fluorescent protein (GFP)-tagged actin showed that cortactin movement was coincident with the actin retrograde flow in the lamellipodia. Cytoplasmic cortactin spots also contained F-actin and were propelled by actin polymerization. Arp3, a component of the arp2/3 complex which is a key regulator of actin polymerization, co-localized with cortactin. Cytoplasmic cortactin-labeled spots were found to be associated with endosomal vesicles. Association was asymmetric and approximately half of the endosomes were associated with cortactin spots. Time-lapse imaging suggested that these cortactin and F-actin-containing spots propelled endosomes. Actin polymerization based propulsion may be a common mechanism for endomembrane trafficking in the same manner as used in the plasma membrane protrusions. As cortactin is known to interact with membrane-associated signaling proteins it could have a role in linking signaling complexes with dynamic actin on endosomes and in lamellipodia.

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.

scholarly journals A Ras-like GTPase Is Involved in Hyphal Growth Guidance in the Filamentous Fungus Ashbya gossypii

2004 ◽  
Vol 15 (10) ◽  
pp. 4622-4632 ◽  
Author(s):  
Yasmina Bauer ◽  
Philipp Knechtle ◽  
Jürgen Wendland ◽  
Hanspeter Helfer ◽  
Peter Philippsen
Keyword(s):  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Time Lapse ◽  
Growth Direction ◽  
Ashbya Gossypii ◽  
Growth Guidance ◽  
Characteristic Features ◽  
Green Fluorescent ◽  
Hyphal Tip

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Δ deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.

scholarly journals Proteins P24 and P41 Function in the Regulation of Terminal-Organelle Development and Gliding Motility in Mycoplasma pneumoniae

2007 ◽  
Vol 189 (20) ◽  
pp. 7442-7449 ◽  
Author(s):  
Benjamin M. Hasselbring ◽  
Duncan C. Krause
Keyword(s):  
Cell Division ◽  
Mycoplasma Pneumoniae ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Gliding Motility ◽  
Atypical Pneumonia ◽  
Reading Frame ◽  
Spatial Positioning ◽  
Time Lapse Imaging ◽  
Gliding Mechanism

ABSTRACT Mycoplasma pneumoniae is a major cause of bronchitis and atypical pneumonia in humans. This cell wall-less bacterium has a complex terminal organelle that functions in cytadherence and gliding motility. The gliding mechanism is unknown but is coordinated with terminal-organelle development during cell division. Disruption of M. pneumoniae open reading frame MPN311 results in loss of protein P41 and downstream gene product P24. P41 localizes to the base of the terminal organelle and is required to anchor the terminal organelle to the cell body, but during cell division, MPN311 insertion mutants also fail to properly regulate nascent terminal-organelle development spatially or gliding activity temporally. We measured gliding velocity and frequency and used fluorescent protein fusions and time-lapse imaging to assess the roles of P41 and P24 individually in terminal-organelle development and gliding function. P41 was necessary for normal gliding velocity and proper spatial positioning of new terminal organelles, while P24 was required for gliding frequency and new terminal-organelle formation at wild-type rates. However, P41 was essential for P24 function, and in the absence of P41, P24 exhibited a dynamic localization pattern. Finally, protein P28 requires P41 for stability, but analysis of a P28− mutant established that the MPN311 mutant phenotype was not a function of loss of P28.

scholarly journals Differential roles of microtubule assembly and sliding in proplatelet formation by megakaryocytes

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4076-4085 ◽  
Author(s):  
Sunita R. Patel ◽  
Jennifer L. Richardson ◽  
Harald Schulze ◽  
Eden Kahle ◽  
Niels Galjart ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Average Rate ◽  
Blood Platelets ◽  
Time Lapse ◽  
Microtubule Assembly ◽  
Alternative Mechanism ◽  
Differentiated Cells ◽  
Continuous Polymerization ◽  
Regulatory Complex ◽  
Green Fluorescent

Megakaryocytes are terminally differentiated cells that, in their final hours, convert their cytoplasm into long, branched proplatelets, which remodel into blood platelets. Proplatelets elongate at an average rate of 0.85 μm/min in a microtubule-dependent process. Addition of rhodamine-tubulin to permeabilized proplatelets, immunofluorescence microscopy of the microtubule plus-end marker end-binding protein 3 (EB3), and fluorescence time-lapse microscopy of EB3–green fluorescent protein (GFP)–expressing megakaryocytes reveal that microtubules, organized as bipolar arrays, continuously polymerize throughout the proplatelet. In immature megakaryocytes lacking proplatelets, microtubule plus-ends initiate and grow by centrosomal nucleation at rates of 8.9 to 12.3 μm/min. In contrast, plus-end growth rates of microtubules within proplatelets are highly variable (1.5-23.5 μm/min) and are both slower and faster than those seen in immature cells. Despite the continuous assembly of microtubules, proplatelets continue to elongate when net microtubule assembly is arrested. One alternative mechanism for force generation is microtubule sliding. Triton X-100–permeabilized proplatelets containing dynein and its regulatory complex, dynactin, but not kinesin, elongate with the addition of adenosine triphosphate (ATP) at a rate of 0.65 μm/min. Retroviral expression in megakaryocytes of dynamitin (p50), which disrupts dynactindynein function, inhibits proplatelet elongation. We conclude that while continuous polymerization of microtubules is necessary to support the enlarging proplatelet mass, the sliding of overlapping microtubules is a vital component of proplatelet elongation.

scholarly journals The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

2001 ◽  
Vol 152 (2) ◽  
pp. 385-400 ◽  
Author(s):  
Patrick Heun ◽  
Thierry Laroche ◽  
M.K. Raghuraman ◽  
Susan M. Gasser
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Structure ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Yeast Cells ◽  
G1 Phase ◽  
Nuclear Pore ◽  
Origin Firing ◽  
Green Fluorescent

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

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