5 research outputs found
Genetic Targeting of a Small Fluorescent Zinc Indicator to Cell Surface for Monitoring Zinc Secretion
Numerous mammalian cells contain
Zn<sup>2+</sup> in their secretory
granules. During secretion, Zn<sup>2+</sup> is coreleased with granular
cargos into extracellular medium so Zn<sup>2+</sup> serves as a convenient
surrogate marker for tracking the dynamics of secretion. Fluorescent
Zn<sup>2+</sup> sensors that can be selectively targeted to cells
of interest would be invaluable tools for imaging Zn<sup>2+</sup> release
in multicellular systems including tissues and live animals. Exploiting
the HaloTag labeling technology and using an optimized linker, we
have engineered a fluorescent Zn<sup>2+</sup> indicator that displayed
a 15-fold fluorescence enhancement upon Zn<sup>2+</sup> binding while
reacting efficiently with a HaloTag enzyme in a cellular environment.
Two-color imaging of ZIMIR-HaloTag and a red-emitting calcium indicator
in pancreatic islet beta cells demonstrated that photoactivation of
a channelrhodopsin was able to induce exocytosis of Zn<sup>2+</sup>/insulin granules and revealed heterogeneity in secretory activity
along the cell membrane that was uncoupled from cellular Ca<sup>2+</sup> activity. This integrated photonic approach for imaging and controlling
the release of large dense core granules provides exquisite cellular
selectivity and should facilitate future studies of stimulus-secretion
coupling and paracrine signaling in secretory cells
GLP‑1 Receptor Mediated Targeting of a Fluorescent Zn<sup>2+</sup> Sensor to Beta Cell Surface for Imaging Insulin/Zn<sup>2+</sup> Release
The pancreatic islet beta cell plays
an essential role in maintaining
the normal blood glucose level by releasing insulin. Loss of functional
beta cell mass leads to diabetesa disease affecting ∼9%
of the population worldwide. There has been great interest and intense
effort in developing imaging probes for monitoring islet beta cells,
and glucagon-like peptide-1 receptor (GLP-1R) has emerged as a valuable
biomarker for targeting beta cells. However, efforts thus far in GLP-1R
mediated beta cell labeling and imaging has largely, if not exclusively,
focused on developing imaging probes for monitoring beta cell mass,
and few studies have investigated imaging beta cell function (insulin
release) through GLP-1R. We now report the design and synthesis of
a bioconjugate, ZIMIR-Ex4(9–39), that consists of a fluorescent
Zn<sup>2+</sup> sensor and a truncated exendin 4 peptide for imaging
insulin/Zn<sup>2+</sup> release in islet beta cells. In vitro, the
conjugate bound to Zn<sup>2+</sup> with high affinity and displayed
a robust fluorescence enhancement upon Zn<sup>2+</sup> chelation.
When added to beta cells at submicromolar concentration, ZIMIR-Ex4(9–39)
rapidly labeled cell surface in minutes to report the dynamics of
insulin/Zn<sup>2+</sup> release with high spatiotemporal resolution.
Future explorations of this approach may lead to probes for tracking
beta cell function using different imaging modalities
TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells
<p>MicroRNAs are small non-coding RNAs acting as posttranscriptional repressors of gene expression. Identifying mRNA targets of a given miRNA remains an outstanding challenge in the field. We have developed a new experimental approach, TargetLink, that applied locked nucleic acid (LNA) as the affinity probe to enrich target genes of a specific microRNA in intact cells. TargetLink also consists a rigorous and systematic data analysis pipeline to identify target genes by comparing LNA-enriched sequences between experimental and control samples. Using miR-21 as a test microRNA, we identified 12 target genes of miR-21 in a human colorectal cancer cell by this approach. The majority of the identified targets interacted with miR-21 via imperfect seed pairing. Target validation confirmed that miR-21 repressed the expression of the identified targets. The cellular abundance of the identified miR-21 target transcripts varied over a wide range, with some targets expressed at a rather low level, confirming that both abundant and rare transcripts are susceptible to regulation by microRNAs, and that TargetLink is an efficient approach for identifying the target set of a specific microRNA in intact cells. C20orf111, one of the novel targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence.</p
Iron-Catalyzed Dehydrative Alkylation of Propargyl Alcohol with Alkyl Peroxides To Form Substituted 1,3-Enynes
This paper reports
a new method for the generation of substituted
1,3-enynes, whose synthesis by other methods could be a challenge.
The dehydrative decarboxylative cascade coupling reaction of propargyl
alcohol with alkyl peroxides is enabled by an iron catalyst and alkylating
reagents. Primary, secondary, and tertiary alkyl groups can be introduced
into 1,3-enynes, affording various substituted 1,3-enynes in moderate
to good yields. Mechanistic studies suggest the involvement of a radical-polar
crossover pathway
Copper-Catalyzed Ligand-Free Diazidation of Olefins with TMSN<sub>3</sub> in CH<sub>3</sub>CN or in H<sub>2</sub>O
An environmentally
benign, copper-catalyzed diazidation of a broad
range of olefins, including vinylarenes, unactivated alkenes, allene,
and dienes, under mild conditions with TMSN<sub>3</sub> (trimethylazidosilane)
as azido source, has been developed. This reaction can be carried
out in organic solvent or in aqueous solution where water is the sole
solvent. The functional group compatibility of this reaction is good,
which is proved by late-stage functionalizations of complex substrates