Superresolution imaging enables visualization of the process of cell wall synthase transport in polar growth of ascomycete fungi.

Here we present mGarnet2, a monomeric, far-red fluorescent marker protein derived from mRuby, with absorption and emission bands peaking at 598 and 671 nm, respectively. The protein shows excellent performance as a live-cell fusion marker for STED nanoscopy with 640 nm excitation and 780 nm depletion wavelengths.

Development and homeostasis of multicellular organisms is largely controlled by complex cell-cell signaling networks that rely on specific binding of secreted ligands to cell surface receptors. The Wnt signaling network, as an example, involves multiple ligands and receptors to elicit specific cellular responses. To understand the mechanisms of such a network, ligand-receptor interactions should be characterized quantitatively, ideally in live cells or tissues. Such measurements are possible using fluorescence microscopy yet challenging due to sample movement, low signal-to-background ratio and photobleaching. Here, we present a robust approach based on fluorescence correlation spectroscopy with ultra-high speed axial line scanning, yielding precise equilibrium dissociation coefficients of interactions in the Wnt signaling pathway. Using CRISPR/Cas9 editing to endogenously tag receptors with fluorescent proteins, we demonstrate that the method delivers precise results even with low, near-native amounts of receptors.

The activation of distinct branches of the Wnt signaling network is essential for regulating early vertebrate development. Activation of the canonical Wnt/β-catenin pathway stimulates expression of β-catenin-Lef/Tcf regulated Wnt target genes and a regulatory network giving rise to the formation of the Spemann organizer. Non-canonical pathways, by contrast, mainly regulate cell polarization and migration, in particular convergent extension movements of the trunk mesoderm during gastrulation. By transcriptome analyses, we found caveolin1, caveolin3 and cavin1 to be regulated by Lef1 in the involuting mesoderm of Xenopus embryos at gastrula stages. We show that caveolins and caveolin dependent endocytosis are necessary for proper gastrulation, most likely by interfering with Wnt5a/Ror2 signaling. Wnt5a regulates the subcellular localization of receptor complexes, including Ror2 homodimers, Ror2/Fzd7 and Ror2/dsh heterodimers in an endocytosis dependent manner. Live-cell imaging revealed endocytosis of Ror2/caveolin1 complexes. In Xenopus explants, in the presence of Wnt5a, these receptor clusters remain stable exclusively at the basolateral side, suggesting that endocytosis of non-canonical Wnt/receptor complexes preferentially takes place at the apical membrane. In support of this blocking endocytosis with inhibitors prevents the effects of Wnt5a. Thus, target genes of Lef1 interfere with Wnt5a/Ror2 signaling to coordinate gastrulation movements.

Wnt signaling plays important roles in embryonic development and during tissue homeostasis, and its deregulation contributes to the onset and progression of tumors. β-catenin is a key component of the 'canonical' Wnt pathway. Upon activation of the pathway, β-catenin is stabilized and enters the nucleus to bind transcription factors and activate Wnt target genes. Mutations that activate β-catenin have been found in many gastrointestinal tumors, including colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of β-catenin are not fully understood. Here, we engineered fluorescently tagged alleles of the endogenous βcatenin by CRISPR/Cas9 in a colorectal cancer cell line. We generated a cell model which carries wild-type and the oncogenic 45 mutant allele tagged with different fluorescent proteins, enabling the functional and biochemical analysis of both variants in the same cell. Endogenously tagged β-catenin remains functional, responds to Wnt pathway activation and does not interfere with the role of β-catenin in cell-cell adhesion. We then analyzed their biochemical and biophysical properties using immunoprecipitation, immunofluorescence and fluorescence correlation spectroscopy approaches. Our results show that wild-type and mutant β-catenin have distinct biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3β inhibitor or truncating APC mutation modulated the wild-type allele to mimic the properties of the mutant β-catenin allele found in colorectal cancer. The one-step tagging strategy also demonstrates how CRISPR/Cas9-mediated genome engineering can be employed for the concurrent functional analysis of wild-type and mutant alleles.

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