3 research outputs found
Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity
Although
peptide-based reporters of protein tyrosine kinase (PTK)
activity have been used to study PTK enzymology <i>in vitro</i>, the application of these reporters to intracellular conditions
is compromised by their dephosphorylation, preventing PTK activity
measurements. Nonproteinogenic amino acids may be utilized to rationally
design selective peptidic ligands by accessing greater chemical and
structural diversity than is available using the native amino acids.
We describe a peptidic reporter that, upon phosphorylation by the
epidermal growth factor receptor (EGFR), is resistant to dephosphorylation
both <i>in vitro</i> and <i>in cellulo</i>. The
reporter contains a conformationally constrained phosphorylatable
moiety (7-(<i>S</i>)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid) in the place of l-tyrosine and is efficiently phosphorylated
in A431 epidermoid carcinoma cells. Dephosphorylation of the reporter
occurs 3 orders of magnitude more slowly compared with that of the
conventional tyrosine-containing reporter
Self-renewing Monolayer of Primary Colonic or Rectal Epithelial CellsSummary
Background & Aims: Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished. Methods: The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified. Results: The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells. Conclusions: This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies. Keywords: Colonic Epithelial Cells, Monolayer, Organoids, Compound Screenin