scholarly journals Resolving Cell Cycle Speed in One Snapshot with a Live-Cell Fluorescent Reporter

Cell Reports ◽  
2020 ◽  
Vol 31 (12) ◽  
pp. 107804 ◽  
Author(s):  
Anna E. Eastman ◽  
Xinyue Chen ◽  
Xiao Hu ◽  
Amaleah A. Hartman ◽  
Aria M. Pearlman Morales ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Live Cell ◽  
Fluorescent Reporter

scholarly journals Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

2018 ◽  
Author(s):  
Anna E. Eastman ◽  
Xinyue Chen ◽  
Xiao Hu ◽  
Amaleah A. Hartman ◽  
Aria M. Pearlman Morales ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Fluorescent Reporter ◽  
Native Cell ◽  
Active Locus ◽  
Solid Tissues ◽  
Cell Cycle Dynamics ◽  
Intracellular Fluorescence

SummaryCell proliferation changes concomitantly with fate transitions during reprogramming, differentiation, regeneration, and oncogenesis. Methods to resolve cell cycle length heterogeneity in real-time are currently lacking. Here, we describe a genetically encoded fluorescent reporter that captures live cell cycle speed using a single measurement. This reporter is based on the color-changing Fluorescent Timer (FT) protein, which emits blue fluorescence when newly synthesized before maturing into a red fluorescent protein. We generated a mouse strain expressing an H2B-FT fusion reporter from a universally active locus, and demonstrate that faster-cycling cells can be distinguished from slower-cycling ones based on the intracellular fluorescence ratio between the FT’s blue and red states. Using this reporter, we reveal the native cell cycle speed distributions of fresh hematopoietic cells, and demonstrate its utility in analyzing cell proliferation in solid tissues. This system is broadly applicable for dissecting functional heterogeneity associated with cell cycle dynamics in complex tissues.

scholarly journals Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations

2016 ◽  
Vol 124 (6) ◽  
pp. 1780-1787 ◽  
Author(s):  
Zhenjun Zhao ◽  
Michael S. Johnson ◽  
Biyi Chen ◽  
Michael Grace ◽  
Jaysree Ukath ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Endothelial Cells ◽  
Ionizing Radiation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Vascular Targeting ◽  
Radiation Doses ◽  
Radiation Induced ◽  
Polarity Sensitive

OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation causes remarkable cellular changes in endothelial cells. Significant PS externalization is induced by radiation at doses of 15 Gy or higher, concomitant with a block in the cell cycle. Radiation-induced markers/targets may have high discriminating power to be harnessed in vascular targeting for AVM treatment.

scholarly journals Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

Methods ◽  
2018 ◽  
Vol 133 ◽  
pp. 81-90 ◽  
Author(s):  
Katja M. Piltti ◽  
Brian J. Cummings ◽  
Krystal Carta ◽  
Ayla Manughian-Peter ◽  
Colleen L. Worne ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Neural Stem Cells ◽  
Single Cell ◽  
Cell Tracking ◽  
Time Lapse ◽  
Live Cell ◽  
Time Lapse Imaging

scholarly journals Fucci2a mouse upgrades live cell cycle imaging

Cell Cycle ◽  
2015 ◽  
Vol 14 (7) ◽  
pp. 948-949 ◽  
Author(s):  
Alessandro Bertero ◽  
Ludovic Vallier
Keyword(s):  
Cell Cycle ◽  
Live Cell

scholarly journals Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
H. M. van der Schaar ◽  
C. E. Melia ◽  
J. A. C. van Bruggen ◽  
J. R. P. M. Strating ◽  
M. E. D. van Geenen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Lipid Composition ◽  
Viral Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Living Cells ◽  
Live Cell ◽  
Genome Replication ◽  
Fluorescent Reporter ◽  
Unique Protein

ABSTRACT Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells. Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.

scholarly journals Live Cell Imaging of Telomerase RNA Dynamics Reveals Cell Cycle-Dependent Clustering of Telomerase at Elongating Telomeres

2011 ◽  
Vol 44 (5) ◽  
pp. 819-827 ◽  
Author(s):  
Franck Gallardo ◽  
Nancy Laterreur ◽  
Emilio Cusanelli ◽  
Faissal Ouenzar ◽  
Emmanuelle Querido ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Telomerase Rna ◽  
Rna Dynamics ◽  
Cycle Dependent

scholarly journals Classic “broken cell” techniques and newer live cell methods for cell cycle assessment

2013 ◽  
Vol 304 (10) ◽  
pp. C927-C938 ◽  
Author(s):  
Lindsay Henderson ◽  
Dante S. Bortone ◽  
Curtis Lim ◽  
Alexander C. Zambon
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Single Cell ◽  
Living Cells ◽  
Cell Division Cycle ◽  
Live Cell ◽  
Nucleotide Analogs ◽  
Protein Ubiquitination ◽  
Cell Cycle Pathway ◽  
Cell Niche

Many common, important diseases are either caused or exacerbated by hyperactivation (e.g., cancer) or inactivation (e.g., heart failure) of the cell division cycle. A better understanding of the cell cycle is critical for interpreting numerous types of physiological changes in cells. Moreover, new insights into how to control it will facilitate new therapeutics for a variety of diseases and new avenues in regenerative medicine. The progression of cells through the four main phases of their division cycle [G0/G1, S (DNA synthesis), G2, and M (mitosis)] is a highly conserved process orchestrated by several pathways (e.g., transcription, phosphorylation, nuclear import/export, and protein ubiquitination) that coordinate a core cell cycle pathway. This core pathway can also receive inputs that are cell type and cell niche dependent. “Broken cell” methods (e.g., use of labeled nucleotide analogs) to assess for cell cycle activity have revealed important insights regarding the cell cycle but lack the ability to assess living cells in real time (longitudinal studies) and with single-cell resolution. Moreover, such methods often require cell synchronization, which can perturb the pathway under study. Live cell cycle sensors can be used at single-cell resolution in living cells, intact tissue, and whole animals. Use of these more recently available sensors has the potential to reveal physiologically relevant insights regarding the normal and perturbed cell division cycle.

scholarly journals Precise detection of S phase onset reveals decoupled G1/S transition events

2018 ◽  
Author(s):  
Gavin D. Grant ◽  
Katarzyna M. Kedziora ◽  
Juanita C. Limas ◽  
Jeremy E. Purvis ◽  
Jeanette Gowen Cook
Keyword(s):  
Cell Cycle ◽  
Genome Stability ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Cycle Phase ◽  
Reporter System ◽  
Phase Boundaries ◽  
Fluorescent Reporter ◽  
Molecular Events ◽  
Cell Cycle Phases

AbstractThe eukaryotic cell division cycle is the process by which cells duplicate their genomes and proliferate. Transitions between sequential cell cycle phases are tightly orchestrated to ensure precise and efficient cell cycle progression. Interrogating molecular events at these transitions is important for understanding normal and pathological cell proliferation and mechanisms that ensure genome stability. A popular fluorescent reporter system known as “FUCCI” has been widely adopted for identifying cell cycle phases. Using time-lapse fluorescence microscopy, we quantitatively analyzed the dynamics of the FUCCI reporters relative to the transitions into and out of S phase. Although the original reporters reflect the E3 ubiquitin ligase activities for which they were designed, SCFSkp2 and APCCdh1, their dynamics are significantly and variably offset from actual S phase boundaries. To precisely mark these transitions, we generated and thoroughly validated a new reporter containing a PCNA-interacting protein degron whose oscillations are directly coupled to the process of DNA replication itself. We combined this reporter with the geminin-based APCCdh1 reporter to create “PIP-FUCCI.” PIP degron reporter dynamics closely correlate with S phase transitions irrespective of reporter expression levels. Using PIP-FUCCI, we made the unexpected observation that the apparent timing of APCCdh1 inactivation frequently varies relative to the onset of S phase. We demonstrate that APCCdh1 inactivation is not a strict pre-requisite for S phase entry, though delayed APCCdh1 inactivation correlates with longer S phase. Our results illustrate the benefits of precise delineation of cell cycle phase boundaries for uncovering the sequences of molecular events at critical cell cycle transitions.

scholarly journals Expression of Separate Foreign Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart Element

2020 ◽  
Author(s):  
Asha A. Philip ◽  
John T. Patton
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Viral Proteins ◽  
Live Cell ◽  
Nuclear Accumulation ◽  
Foreign Protein ◽  
Fluorescent Reporter ◽  
Recombinant Viruses ◽  
Full Complement ◽  
Foreign Proteins

AbstractThe segmented 18.5-kB dsRNA genome of rotavirus expresses 6 structural and 6 nonstructural proteins. We investigated the possibility of using the recently-developed plasmid-based rotavirus reverse genetics (RG) system to generate recombinant viruses that express a separate foreign protein, in addition to the 12 viral proteins. To address this, we replaced the NSP3 open-reading-frame (ORF) of the segment 7 (pT7/NSP3) transcription vector used in the RG system with an ORF encoding NSP3 fused to a fluorescent reporter protein (i.e., UnaG, mRuby, mKate, or TagBFP). Inserted at the fusion junction was a teschovirus 2A-like self-cleaving element designed to direct the separate expression of NSP3 and the fluorescent protein. Recombinant rotaviruses made with the modified pT7/NSP3 vectors were well growing, generally genetically stable, and expressed NSP3 and a separate fluorescent protein detectable by live cell imaging. NSP3 made by the recombinant viruses was functional, inducing nuclear accumulation of cellular poly(A)-binding protein. Further modification of the NSP3 ORF showed that it was possible to generate recombinant viruses encoding 2 foreign proteins (mRuby and UnaG) in addition to NSP3. Our results demonstrate that, through modification of segment 7, the rotavirus genome can be increased in size to at least 19.8 kB and can be used to produce recombinant rotaviruses expressing a full complement of viral proteins and multiple foreign proteins. The generation of recombinant rotaviruses expressing fluorescent proteins will be valuable for the study of rotavirus replication and pathogenesis by live cell imagining and suggest that rotaviruses may prove useful as expression vectors.ImportanceRotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate foreign protein, in addition to the full complement of viral proteins. Through this approach, we have generated wildtype-like rotaviruses that express various fluorescent reporter proteins, including UnaG (green), mRuby (far red), mKate (red), and TagBFP (blue). Such strains will be of value in probing rotavirus biology and pathogenesis by live-cell imagining techniques. Notably, our work indicates that the rotavirus genome is remarkably flexible and able to accommodate significant amounts of foreign RNA sequence, raising the possibility of using the virus as vaccine expression vector.

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