26 research outputs found
Click to enter:activation of oligo-arginine cell-penetrating peptides by bioorthogonal tetrazine ligations
\u3cp\u3eCell-penetrating peptides are able to transport a wide variety of cargo across cell membranes. Although promising, they are not often considered for therapeutic purposes as they lack controllable activity and cell selectivity. We have developed an activation strategy based on a split octa-arginine cell-penetrating peptide (CPP) that can be activated by means of bioorthogonal ligation. To this end we prepared two non-penetrating tetra-arginine halves, functionalized either with a tetrazine or with a complementary bicyclo[6.1.0]nonyne (BCN) group. We demonstrate that an active octa-arginine can be reconstituted in situ upon mixing the complementary split peptides. The resulting activated peptide is taken up as efficiently as the well-established cell-penetrating peptide octa-arginine. The activation of the oligo-arginines can also be achieved using trans-cyclooctene (TCO) as a ligation partner, while norbornene appears too kinetically slow for use in situ. We further show that this strategy can be applied successfully to transport a large protein into living cells. Our results validate a promising first step in achieving control over cell penetration and to use CPPs for therapeutic approaches.\u3c/p\u3
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Using light to shape chemical gradients for parallel and automated analysis of chemotaxis.
Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G-protein Giα subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low- and high-resolution fluorescence microscopy
Vinylboronic Acids as Efficient Bioorthogonal Reactants for Tetrazine Labeling in Living Cells
Using light to shape chemical gradients for parallel and automated analysis of chemotaxis
Numerous molecular components have been identified that regu-late the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemo-attractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis pheno-types for 285 siRNA perturbations. A key finding was that the G-protein Gia subunit selectively controls the direction of migration while the receptor and Gb subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutro
Subcellular Protein Labeling by a Spatially Restricted Arylamine <i>N</i>‑Acetyltransferase
Mapping proteins at a specific subcellular
location is essential to gaining detailed insight on local protein
dynamics. We have developed an enzymatic strategy to label proteins
on a subcellular level using arylamine <i>N</i>-acetyltransferase
(NAT). The NAT enzyme activates an arylhydroxamic acid functionality
into a nitrenium ion that reacts fast, covalently, and under neutral
conditions with nucleophilic residues of neighboring proteins. The
electron density on the aromatic ring proved important for probe activation
as strong labeling was only observed with an arylhydroxamic acid bearing
an electron donating substituent. We further demonstrate that, using
this electron rich arylhydroxamic acid, clear labeling was achieved
on a subcellular level in living cells that were transfected with
a genetically targeted NAT to the nucleus or the cytosol