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1.
Dirk Jochmans Maria Anders Inge Keuleers Liesbeth Smeulders Hans-Georg Kräusslich Günter Kraus Barbara Müller 《Retrovirology》2010,7(1):1-14
Background
The gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system.Results
In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability.Conclusions
It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD. 相似文献2.
Mary LaLonde Hilde Janssens Suyong Yun Juan Crosby Olga Redina Virginie Olive Yelena M Altshuller Seok-Yong Choi Guangwei Du Peter J Gergen Michael A Frohman 《BMC developmental biology》2006,6(1):1-13
Background
Cellularization of the Drosophila embryo is an unusually synchronous form of cytokinesis in which polarized membrane extension proceeds in part through incorporation of new membrane via fusion of apically-translocated Golgi-derived vesicles.Results
We describe here involvement of the signaling enzyme Phospholipase D (Pld) in regulation of this developmental step. Functional analysis using gene targeting revealed that cellularization is hindered by the loss of Pld, resulting frequently in early embryonic developmental arrest. Mechanistically, chronic Pld deficiency causes abnormal Golgi structure and secretory vesicle trafficking.Conclusion
Our results suggest that Pld functions to promote trafficking of Golgi-derived fusion-competent vesicles during cellularization. 相似文献3.
Key message
We found that protein trafficking between apical and basal cell can be unidirectional, which reveals the different roles of the two cells in the cell-to-cell communication between them during early embryogenesis.Abstract
In most angiosperm species, asymmetric zygote division results in an apical cell and a basal cell that have distinct cell fates. Much has been speculated about possible communication between these cell types in relation to their cell fate determination. Here, we report on the use of photoactivatable green fluorescent protein (PA-GFP) in tobacco to trace intercellular communication between apical and basal cells during early embryogenesis. We found that PA-GFP was transported between apical and basal cells of a two-celled proembryo, and that protein trafficking was unidirectional toward the apical cell, highlighting different cell communication roles. Further ultrastructural analysis showed numerous plasmodesmata in the walls connecting the apical and basal cells, which may provide channels for protein trafficking. Our data show a possible unique method of cell-to-cell communication between apical and basal cells during early embryogenesis. 相似文献4.
Key message
Wheat low-molecular-weight-glutenin and α-gliadin were accumulated in the endoplasmic reticulum and formed protein body-like structures in tobacco cells, with the participation of BiP chaperone. Possible interactions between these prolamins were investigated.Abstract
Wheat prolamins are the major proteins that accumulate in endosperm cells and are largely responsible for the unique biochemical properties of wheat products. They are accumulated in the endoplasmic reticulum (ER) where they form protein bodies (PBs) and are then transported to the storage vacuole where they form a protein matrix in the ripe seeds. Whereas previous studies have been carried out to determine the atypical trafficking pathway of prolamins, the mechanisms leading to ER retention and PB formation are still not clear. In this study, we examined the trafficking of a low-molecular-weight glutenin subunit (LMW-glutenin) and α-gliadin fused to fluorescent proteins expressed in tobacco cells. Through transient transformation in epidermal tobacco leaves, we demonstrated that both LMW-glutenin and α-gliadin were retained in the ER and formed mobile protein body-like structures (PBLS) that generally do not co-localise with Golgi bodies. An increased expression level of BiP in tobacco cells transformed with α-gliadin or LMW-glutenin was observed, suggesting the participation of this chaperone protein in the accumulation of wheat prolamins in tobacco cells. When stably expressed in BY-2 cells, LMW-glutenin fusion was retained longer in the ER before being exported to and degraded in the vacuole, compared with α-gliadin fusion, suggesting the involvement of intermolecular disulphide bonds in ER retention, but not in PBLS formation. Co-localisation experiments showed that gliadins and LMW-glutenin were found in the same PBLS with no particular distribution, which could be due to their ability to interact with each other as indicated by yeast two-hybrid assays. 相似文献5.
Background
Polyethylenimine (PEI) is one of the most efficient and versatile non-viral vectors available for gene delivery. Despite many advantages over viral vectors, PEI is still limited by lower transfection efficiency compared to its viral counterparts. Considerable investigation is devoted to the modification of PEI to incorporate virus-like properties to improve its efficacy, including the incorporation of the protein transduction domain (PTD) polyarginine (Arg); itself demonstrated to facilitate membrane translocation of molecular cargo. There is, however, limited understanding of the underlying mechanisms of gene delivery facilitated by both PEI and PEI-bioconjugates such as PEI-polyarginine (PEI-Arg) within live cells, which once elucidated will provide valuable insights into the development of more efficient non-viral gene delivery vectors.Methods
PEI and PEI-Arg were investigated for their ability to facilitate DNA internalization and gene expression within live COS-7 cells, in terms of the percentage of cells transfected and the relative amount of gene expression per cell. Intracellular trafficking of vectors was investigated using fluorescent microscopy during the first 5 h post transfection. Finally, nocodazole and aphidicolin were used to investigate the role of microtubules and mitosis, respectively, and their impact on PEI and PEI-Arg mediated gene delivery and expression.Results
PEI-Arg maintained a high cellular DNA uptake efficiency, and facilitated as much as 2-fold more DNA internalization compared to PEI alone. PEI, but not PEI-Arg, displayed microtubule-facilitated trafficking, and was found to accumulate within close proximity to the nucleus. Only PEI facilitated significant gene expression, whereas PEI-Arg conferred negligible expression. Finally, while not exclusively dependant, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression.Conclusion
PEI polyplexes are trafficked by an indirect association with microtubules, following endosomal entrapment. PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes. PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking. This study emphasizes the importance of a holistic approach to investigate the mechanisms of novel gene delivery vectors. 相似文献6.
Background
Protein translocation across the membrane of the Endoplasmic Reticulum (ER) is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized.Results
In the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF) receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect.Conclusion
Based on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER. 相似文献7.
Naoshi Ohta Masato Sato Kiminori Ushida Mami Kokubo Takayuki Baba Kayoko Taniguchi Makoto Urai Koji Kihira Joji Mochida 《BMC biotechnology》2009,9(1):1-11
Background
Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles.Results
Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies.Conclusion
Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells. 相似文献8.
Background
Structural and dynamic studies of chromosomes tagged with green fluorescent protein (GFP) in yeast and cultured animal cells have revealed some surprises. Although this technology can be very powerful, only a few studies using this approach with developed multicellular systems have been reported for the study of chromatin behavior in situ.Results
We established vectors and conditions to visualize tagged loci stably inserted in the Arabidopsis genome via GFP fused to a bacterial DNA-binding protein. Using this system, three-dimensional coordinates for tagged loci within nuclei from cells of a live plant can be directly determined with concomitant visualization of the position of the nucleolus. Chromosome polyploidization in epidermal cells at the elongation zone of the root in transgenic plants can be visualized in situ using this technique.Conclusion
We have established that GFP fusion with DNA-binding proteins can be used in conjunction with concatameric binding-site arrays to track genomic loci in living Arabidopsis plants. It should now be feasible to study the mechanisms of organization and dynamics of chromatin in specific cell types during various times of plant development, taking advantage of the well developed genetic systems and resources available for Arabidopsis. 相似文献9.
Andrew P Jackson 《BMC evolutionary biology》2007,7(1):1-17
Background
In membrane trafficking, the mechanisms ensuring vesicle fusion specificity remain to be fully elucidated. Early models proposed that specificity was encoded entirely by SNARE proteins; more recent models include contributions from Rab proteins, Syntaxin-binding (SM) proteins and tethering factors. Most information on membrane trafficking derives from an evolutionarily narrow sampling of model organisms. However, considering factors from a wider diversity of eukaryotes can provide both functional information on core systems and insight into the evolutionary history of the trafficking machinery. For example, the major Qa/syntaxin SNARE families are present in most eukaryotic genomes and likely each evolved via gene duplication from a single ancestral syntaxin before the existing eukaryotic groups diversified. This pattern is also likely for Rabs and various other components of the membrane trafficking machinery.Results
We performed comparative genomic and phylogenetic analyses, when relevant, on the SM proteins and components of the tethering complexes, both thought to contribute to vesicle fusion specificity. Despite evidence suggestive of secondary losses amongst many lineages, the tethering complexes are well represented across the eukaryotes, suggesting an origin predating the radiation of eukaryotic lineages. Further, whilst we detect distant sequence relations between GARP, COG, exocyst and DSL1 components, these similarities most likely reflect convergent evolution of similar secondary structural elements. No similarity is found between the TRAPP and HOPS complexes and the other tethering factors. Overall, our data favour independent origins for the various tethering complexes. The taxa examined possess at least one homologue of each of the four SM protein families; since the four monophyletic families each encompass a wide diversity of eukaryotes, the SM protein families very likely evolved before the last common eukaryotic ancestor (LCEA).Conclusion
These data further support a highly complex LCEA and indicate that the basic architecture of the trafficking system is remarkably conserved and ancient, with the SM proteins and tethering factors having originated very early in eukaryotic evolution. However, the independent origin of the tethering complexes suggests a novel pattern for increasing complexity in the membrane trafficking system, in addition to the pattern of paralogous machinery elaboration seen thus far. 相似文献10.
Background
Intracellular vesicle fusion is mediated by the interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs). The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments. To fully understand the specific role of each VAMP in vesicle trafficking, it is important to determine if VAMPs have differential membrane fusion activities.Methodology/Principal Findings
In this study, we developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase, and examined fusogenic pairings between the seven VAMPs, i.e., VAMPs 1, 2, 3, 4, 5, 7 and 8, and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 and syntaxin4/SNAP-25. VAMPs 1, 2, 3, 4, 7 and 8 drove fusion efficiently, whereas VAMP5 was unable to mediate fusion with the t-SNAREs. By expressing VAMPs 1, 3, 4, 7 and 8 at the same level, we further compared their membrane fusion activities. VAMPs 1 and 3 had comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 exhibited 30–50% lower fusion activities. Moreover, we determined the dependence of cell fusion activity on VAMP1 expression level. Analysis of the dependence data suggested that there was no cooperativity of VAMP proteins in the cell fusion reaction.Conclusions/Significance
These data indicate that VAMPs have differential membrane fusion capacities, and imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating fusion with plasma membrane t-SNAREs. 相似文献11.
Stephen A Gallo Jacqueline D Reeves Himanshu Garg Brian Foley Robert W Doms Robert Blumenthal 《Retrovirology》2006,3(1):1-8
Background
Human immunodeficiency virus (HIV) enters target cells by a membrane fusion process that involves a series of sequential interactions between its envelope glycoproteins, the CD4 receptor and CXCR4/CCR5 coreceptors. CD4 molecules are expressed at the cell surface of lymphocytes and monocytes mainly as monomers, but basal levels of CD4 dimers are also present at the cell surface of these cells. Previous evidence indicates that the membrane distal and proximal extracellular domains of CD4, respectively D1 and D4, are involved in receptor dimerization.Results
Here, we have used A201 cell lines expressing two CD4 mutants, CD4-E91K, E92K (D1 mutant) and CD4-Q344E (D4 mutant), harboring dimerization defects to analyze the role of CD4 dimerization in HIV-1 entry. Using entry assays based on β-lactamase-Vpr or luciferase reporter activities, as well as virus encoding envelope glycoproteins derived from primary or laboratory-adapted strains, we obtained evidence suggesting an association between disruption of CD4 dimerization and increased viral entry efficiency.Conclusion
Taken together, our results suggest that monomeric forms of CD4 are preferentially used by HIV-1 to gain entry into target cells, thus implying that the dimer/monomer ratio at the cell surface of HIV-1 target cells may modulate the efficiency of HIV-1 entry. 相似文献12.
Corinne Lorenzo Céline Frongia Raphaël Jorand Jérôme Fehrenbach Pierre Weiss Amina Maandhui Guillaume Gay Bernard Ducommun Valérie Lobjois 《Cell division》2011,6(1):1-8
Background
Multicellular tumor spheroids are models of increasing interest for cancer and cell biology studies. They allow considering cellular interactions in exploring cell cycle and cell division mechanisms. However, 3D imaging of cell division in living spheroids is technically challenging and has never been reported.Results
Here, we report a major breakthrough based on the engineering of multicellular tumor spheroids expressing an histone H2B fluorescent nuclear reporter protein, and specifically designed sample holders to monitor live cell division dynamics in 3D large spheroids using an home-made selective-plane illumination microscope.Conclusions
As illustrated using the antimitotic drug, paclitaxel, this technological advance paves the way for studies of the dynamics of cell divion processes in 3D and more generally for the investigation of tumor cell population biology in integrated system as the spheroid model. 相似文献13.
Vera Teixeira Natacha Arede Rui Gardner Joaquín Rodríguez-León Ana T Tavares 《BMC developmental biology》2011,11(1):1-7
Background
Hemangioblasts are known as the common precursors for primitive hematopoietic and endothelial lineages. Their existence has been supported mainly by the observation that both cell types develop in close proximity and by in vitro differentiation and genetic studies. However, more compelling evidence will arise from tracking their cell fates using a lineage-specific marker.Results
We report the identification of a hemangioblast-specific enhancer (Hb) located in the cis-regulatory region of chick Cerberus gene (cCer) that is able to direct the expression of enhanced green fluorescent protein (eGFP) to the precursors of yolk sac blood and endothelial cells in electroporated chick embryos. Moreover, we present the Hb-eGFP reporter as a powerful live imaging tool for visualizing hemangioblast cell fate and blood island morphogenesis.Conclusions
We hereby introduce the Hb enhancer as a valuable resource for genetically targeting the hemangioblast population as well as for studying the dynamics of vascular and blood cell development. 相似文献14.
Man Lung Yeung Yamina Bennasser Timothy G Myers Guojian Jiang Monsef Benkirane Kuan-Teh Jeang 《Retrovirology》2005,2(1):1-8
Background
The HIV-1 Rev regulatory protein binds as an oligomeric complex to viral RNA mediating nuclear export of incompletely spliced and non-spliced viral mRNAs encoding the viral structural proteins. However, the biological significance of the obligatory complex formation of Rev upon the viral RNA is unclear.Results
The activity of various fusion proteins based on the negative oligomerization-defect Rev mutant M4 was tested using Rev dependent reporter constructs. An artificial M4 mutant dimer and an M4 mutant containing an extra basic domain from the HTLV-I Rex protein exhibited nearly full activity when compared to wild type Rev.Conclusion
Rev dimerization appears to be required to expose free basic domains whilst the Rev oligomeric complex remains bound to viral RNA via other basic domains. 相似文献15.
Mingli Yang Guohua Jiang Wenjing Li Kai Qiu Min Zhang Christopher M Carter Samer Z Al-Quran Ying Li 《Journal of hematology & oncology》2014,7(1):1-14
Background
The majority of patients with acute myelogenous leukemia (AML) still die of their disease. In order to improve survival rates in AML patients, new strategies are necessary to discover biomarkers for the detection and targeted therapy of AML. One of the advantages of the aptamer-based technology is the unique cell-based selection process, which allows us to efficiently select for cell-specific aptamers without knowing which target molecules are present on the cell surface.Methods
The NB4 AML cell line was used as the target cell population for selecting single stranded DNA aptamers. After determining the affinity of selected aptamers to leukocytes, the aptamers were used to phenotype human bone marrow leukocytes and AML cells in clinical specimens. Then a biotin-labelled aptamer was used to enrich and identify its target surface protein.Results
Three new aptamers were characterized from the selected aptamer pools (JH6, JH19, and K19). All of them can selectively recognize myeloid cells with Kd in the low nanomole range (2.77 to 12.37 nM). The target of the biotin-labelled K19 aptamer probe was identified as Siglec-5, a surface membrane protein in low abundance whose expression can serve as a biomarker of granulocytic maturation and be used to phenotype AML. More importantly, Siglec-5 expression can be used to detect low concentrations of AML cells in human bone marrow specimens, and functions as a potential target for leukemic therapy.Conclusions
We have demonstrated a pipeline approach for developing single stranded DNA aptamer probes, phenotyping AML cells in clinical specimens, and then identifying the aptamer-recognized target protein. The developed aptamer probes and identified Siglec-5 protein may potentially be used for leukemic cell detection and therapy in our future clinical practice. 相似文献16.
Saiying Hou Jiao Shi Lihong Hao Zhijuan Wang Yalan Liao Hongya Gu Juan Dong Thomas Dresselhaus Sheng Zhong Li-Jia Qu 《The Plant cell》2021,33(9):3042
In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.Arabidopsis VPS18 plays an important role in regulating pollen tube growth through mediating the late endocytic trafficking and secretion of pectin and associated enzymes to the cell wall. 相似文献
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Background
Guanonine-protein (G-protein) is known as molecular switches inside cells, and is very important in signals transmission from outside to inside cell. Especially in transport protein, most of G-proteins play an important role in membrane trafficking; necessary for transferring proteins and other molecules to a variety of destinations outside and inside of the cell. The function of membrane trafficking is controlled by G-proteins via Guanosine triphosphate (GTP) binding sites. The GTP binding sites active G-proteins initiated to membrane vesicles by interacting with specific effector proteins. Without the interaction from GTP binding sites, G-proteins could not be active in membrane trafficking and consequently cause many diseases, i.e., cancer, Parkinson… Thus it is very important to identify GTP binding sites in membrane trafficking, in particular, and in transport protein, in general.Results
We developed the proposed model with a cross-validation and examined with an independent dataset. We achieved an accuracy of 95.6% for evaluating with cross-validation and 98.7% for examining the performance with the independent data set. For newly discovered transport protein sequences, our approach performed remarkably better than similar methods such as GTPBinder, NsitePred and TargetSOS. Moreover, a friendly web server was developed for identifying GTP binding sites in transport proteins available for all users.Conclusions
We approached a computational technique using PSSM profiles and SAAPs for identifying GTP binding residues in transport proteins. When we included SAAPs into PSSM profiles, the predictive performance achieved a significant improvement in all measurement metrics. Furthermore, the proposed method could be a power tool for determining new proteins that belongs into GTP binding sites in transport proteins and can provide useful information for biologists.19.
Background
Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts.Results
In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets.Conclusions
Our software solution, Circadian Gene Express (CGE), is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and efficient recording of large number of cell parameters, including level of reporter protein expression, velocity, direction of movement, and others. CGE proves to be useful for the analysis of widefield fluorescent microscopy datasets, as well as for bioluminescence imaging. Moreover, it might be easily adaptable for confocal image analysis by manually choosing one of the focal planes of each z-stack of the various time points of a time series.Availability
CGE is a Java plugin for ImageJ; it is freely available at: http://bigwww.epfl.ch/sage/soft/circadian/. 相似文献20.