|Proximity-dependent initiation of hybridization chain reaction.|
Koos, B; Cane, G; Grannas, K; Löf, L; Arngården, L; Heldin, J; Clausson, CM; Klaesson, A; Hirvonen, MK; de Oliveira, FM; Talibov, VO; Pham, NT; Auer, M; Danielson, UH; Haybaeck, J; Kamali-Moghaddam, M; Söderberg, O
Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.
|Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling.|
Wong, VW; Stange, DE; Page, ME; Buczacki, S; Wabik, A; Itami, S; van de Wetering, M; Poulsom, R; Wright, NA; Trotter, MW; Watt, FM; Winton, DJ; Clevers, H; Jensen, KB
Nature cell biology
Maintenance of adult tissues is carried out by stem cells and is sustained throughout life in a highly ordered manner. Homeostasis within the stem-cell compartment is governed by positive- and negative-feedback regulation of instructive extrinsic and intrinsic signals. ErbB signalling is a prerequisite for maintenance of the intestinal epithelium following injury and tumour formation. As ErbB-family ligands and receptors are highly expressed within the stem-cell niche, we hypothesize that strong endogenous regulators must control the pathway in the stem-cell compartment. Here we show that Lrig1, a negative-feedback regulator of the ErbB receptor family, is highly expressed by intestinal stem cells and controls the size of the intestinal stem-cell niche by regulating the amplitude of growth-factor signalling. Intestinal stem-cell maintenance has so far been attributed to a combination of Wnt and Notch activation and Bmpr inhibition. Our findings reveal ErbB activation as a strong inductive signal for stem-cell proliferation. This has implications for our understanding of ErbB signalling in tissue development and maintenance and the progression of malignant disease.