Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer ...from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.
The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not ...converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles.
Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. ...Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification.
In migrating cells, the cytoskeleton coordinates signal transduction and redistribution of transmembrane proteins, including integrins and growth factor receptors. Supervillin is an F-actin- and ...myosin II-binding protein that tightly associates with signaling proteins in cholesterol-rich, 'lipid raft' membrane microdomains. We show here that supervillin also can localize with markers for early and sorting endosomes (EE/SE) and with overexpressed components of the Arf6 recycling pathway in the cell periphery. Supervillin tagged with the photoswitchable fluorescent protein, tdEos, moves both into and away from dynamic structures resembling podosomes at the basal cell surface. Rapid integrin recycling from EE/SE is inhibited in supervillin-knockdown cells, but the rates of integrin endocytosis and recycling from the perinuclear recycling center (PNRC) are unchanged. A lack of synergy between supervillin knockdown and the actin filament barbed-end inhibitor, cytochalasin D, suggests that both treatments affect actin-dependent rapid recycling. Supervillin also enhances signaling from the epidermal growth factor receptor (EGFR) to extracellular signal-regulated kinases (ERKs) 1 and 2 and increases the velocity of cell translocation. These results suggest that supervillin, F-actin and associated proteins coordinate a rapid, basolateral membrane recycling pathway that contributes to ERK signaling and actin-based cell motility.
The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in ...replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties.
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•Stable transcriptional reprogramming toward endoderm within one cell cycle•Replication timing can change in the first S phase of a cell fate change•Early discordance between Replication timing and Hi-C compartments•Alignment of replication timing to compartments increases in later cell cycles
The temporal order of DNA replication is regulated during development, and is highly correlated with gene expression, chromatin structure, and 3D-genome architecture. Gilbert and colleagues find that stable transcriptional changes of human embryonic stem cells to endoderm can occur within a single cell cycle, accompanied by discordance in replication timing and chromatin compartment that align in later differentiation stages.
Many community pharmacists are uncomfortable educating patients about naloxone, an opioid reversal agent.
To examine whether training materials prepare pharmacists to counsel patients and caregivers ...about naloxone, online naloxone education materials for pharmacists in the 13 states with standing orders were analyzed.
Two coders reviewed 12 naloxone training programs and extracted data for 15 topics that were clustered in four categories: background/importance, naloxone products, business/operations, and communication. Programs that included communication content were coded for whether they: 1) suggested specific verbiage for naloxone counseling; 2) recommended evidence-based communication practices; and 3) included example naloxone conversations.
Most programs covered the majority of topics, with the exception of extended treatment for individuals who overdose and naloxone storage/expiration information. Eleven programs addressed pharmacist-patient communication, although information on communication was often limited. Only one program included an example pharmacist-patient naloxone conversation, but the conversation was 10 min long and occurred in a private room, limiting its applicability to most community pharmacies.
Online naloxone training materials for pharmacists include limited content on how to communicate with patients and caregivers. Training materials that include more in-depth content on communication may increase pharmacists' confidence to discuss the topics of overdose and naloxone.
Lipid multilayer microarrays are a promising
approach to miniaturize laboratory procedures by taking
advantage of the microscopic compartmentalization
capabilities of lipids. Here, we demonstrate a ...new method
to pattern lipid multilayers on surfaces based on solvent
evaporation along the edge where a stencil contacts a
surface called evaporative edge lithography (EEL). As an
example of an application of this process, we use EEL
to make microarrays suitable for a cell-based migration
assay. Currently existing cell migration assays require a
separate compartment for each drug which is dissolved
at a single concentration in solution. An advantage of
the lipid multilayer microarray assay is that multiple
compounds can be tested on the same surface. We
demonstrate this by testing the effect of two different
lipophilic drugs, Taxol and Brefeldin A, on collective cell
migration into an unpopulated area. This particular assay
should be scalable to test of 2000 different lipophilic
compounds or dosages on a standard microtiter plate
area, or if adapted for individual cell migration, it would
allow for high-throughput screening of more than 50,000
compounds per plate.
The temporal order in which segments of the genome are duplicated is referred to as the replication timing (RT) program. RT is established in each cell cycle coincident with the repositioning and ...anchorage of chromosomes in early G1. In general, segments that replicate in early S are organized into transcriptionally permissive chromatin, and segments that replicate in late S are assembled into repressive chromatin. During human embryonic stem cell (hESC) differentiation, segments of the genome undergo changes in RT, which are accompanied by changes in chromatin compartments, and transcriptional activity. Determining the order these changes occur during hESC differentiation required defining cell cycle parameters for hESCs. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S cell cycle transitions. Instead, we employed a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replicating DNA and track 3D organization. We find that re-localization and anchorage of chromosomes were completed prior to the onset of S phase, even in the context of an abbreviated G1 phase. Furthermore, we find that single hESCs preferentially differentiate from G1. We show changes in RT are remarkably coincident with transcription; although, neither is sufficient for the other to occur. We also show changes in RT accompany cell commitment during the first cell cycle and precede changes in chromatin compartments. Finally, we find that in hESCs, domains that switch from early to late replication interact more frequently with late replicating chromatin, suggesting hESCs may be poised to quickly repress early to late switching domains upon stimulation.
Net resources WILSON, KOREY A
Black enterprise,
November 1999, 19991101, Volume:
30, Issue:
4
Magazine Article
Several reference Web sites that can help you handle your finances better, faster and more efficiently are highlighted, including: 1. www. investorguide.com, 2. www.stocksense.com, 3. ...www.invest-faq.com, 4. www.findafund.com, 5. www.irs.ustreas.gov, 6. www.loanpage.com, 7. www.insweb.com, and 8. www.financenter.com.
Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. ...Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification.
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