Highlights d A PCR-based electroporation screen yielded an improved voltage indicator, ASAP3 d ASAP3 shows larger voltage responses than other fluorescent protein-based sensors d Ultrafast local volume excitation (ULoVE) boosts randomaccess two-photon signals d ASAP3 and ULoVE report subthreshold and spiking potentials in deep brain regions

NKX2.5 is a homeodomain containing transcription factor regulating cardiac formation and function, and its mutations are linked to congenital heart disease. Here we provide the first report of the crystal structure of the NKX2.5 homeodomain in complex with double-stranded DNA of its endogenous target, locating within the proximal promoter –242 site of the atrial natriuretic factor gene. The crystal structure, determined at 1.8 Å resolution, demonstrates that NKX2.5 homeodomains occupy both DNA binding sites separated by five nucleotides without physical interaction between themselves. The two homeodomains show identical conformation despite the differences in the DNA sequences they bind, and no significant bending of the DNA was observed. Tyr54, absolutely conserved in NK2 family proteins, mediates sequence-specific interaction with the TAAG motif. This high resolution crystal structure of NKX2.5 protein provides a detailed picture of protein and DNA interactions, which allows us to predict DNA binding of mutants identified in human patients.

Background Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5, in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Since missense mutations in the NKX2-5 homeodomain (DNA binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knock-in model. Methods and Results We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5+/R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein’s malformation of the tricuspid valve, and perimembranous and/or muscular ventricular septal defects. In addition, P10 Nkx2-5+/R52G mice demonstrated atrial septal anomalies, with significant increase in the size of the inter-atrial communication and fossa ovalis, and decrease in the length of the flap valve compared to control Nkx2-5+/+ or Nkx2-5+/− mice. Conclusion The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain (R52G) are highly penetrant, and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knock-in mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.

Heart development in mammalian systems is controlled by combinatorial interactions of master cardiac transcription factors such as TBX5 and NKX2.5. They bind to promoters/enhancers of downstream targets as homo- or heteromultimeric complexes. They physically interact and synergistically regulate their target genes. To elucidate the molecular basis of the intermolecular interactions, a heterodimer and a homodimer of NKX2.5 and TBX5 were studied using X-ray crystallography. Here we report a crystal structure of human NKX2.5 and TBX5 DNA binding domains in a complex with a 19 bp target DNA and a crystal structure of TBX5 homodimer. The ternary complex structure of NKX2.5 and TBX5 with the target DNA shows physical interactions between the two proteins through Lys158 (NKX2.5), Asp140 (TBX5), and Pro142 (TBX5), residues that are highly conserved in TBX and NKX families across species. Extensive homodimeric interactions were observed between the TBX5 proteins in both crystal structures. In particular, in the crystal structure of TBX5 protein that includes the N-terminal and DNA binding domains, intermolecular interactions were mediated by the N-terminal domain of the protein. The N-terminal domain of TBX5 was predicted to be "intrinsically unstructured", and in one of the two molecules in an asymmetric unit, the N-terminal domain assumes a β-strand conformation bridging two β-sheets from the two molecules. The structures reported here may represent general mechanisms for combinatorial interactions among transcription factors regulating developmental processes.

Imaging of transmembrane voltage deep in brain tissue with cellular resolution has the potential to reveal information processing by neuronal circuits in living animals with minimal perturbation. Multi-photon voltage imaging in vivo, however, is currently limited by speed and sensitivity of both indicators and imaging methods. Here, we report the engineering of an improved genetically encoded voltage indicator, ASAP3, which exhibits up to 51% fluorescence responses in the physiological voltage range, sub-millisecond activation kinetics, and full responsivity under two-photon illumination. We also introduce an ultrafast local volume excitation (ULOVE) two-photon scanning method to sample ASAP3 signals in awake mice at kilohertz rates with increased stability and sensitivity. ASAP3 and ULOVE allowed continuous single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution, and with repeated sampling over multiple days. By imaging voltage in visual cortex neurons, we found evidence for cell type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULOVE enable continuous high-speed highresolution imaging of electrical activity in deeply located genetically defined neurons during awake behavior.shaping 7,8 . However, parallel excitation is problematic for imaging, as scattered fluorescence signals from simultaneously excited cells would become intermixed, hindering detection of small responses. Furthermore, as GEVIs reside in the membrane rather than the cytosol, the number of indicator molecules that can be excited in an optical section through a mammalian cell body is typically smaller for GEVIs than for GECIs 4,6 .Given these limitations, the development of GEVIs with larger two-photon responses to electrical events of interest is highly desirable. Currently, the GEVIs with the largest responses to both subthreshold changes and APs are based on two types of voltage-sensing domains: seven-transmembrane helix opsins and four-transemembrane helix voltage-sensing domains (VSDs). Opsin-based GEVIs have been used in vivo with one-photon excitation to report electrical activity of superficially located neurons 9,10 , but their responsivity is severely attenuated under twophoton excitation 4,11 . In contrast, ASAP-family GEVIs, composed of a circularly permuted green fluorescent protein variant inserted within the VSD of G. gallus voltage-sensing phosphatase (Fig. 1a), are fully responsive under twophoton excitation 11 . In particular, ASAP2s demonstrates the largest response per AP of fluorescent protein-based GEVIs, but its kinetics are actually slower than earlier ASAP variants 11 . If ASAP-family kinetics and/or overall responsivity could be improved, then electrical events could be more easily detected by two-photon imaging.Here we report an improved indicator, ASAP3, resulting from novel methods for generation and screening of GEVI libraries in mammalian cells. ASAP3 features the largest responses of fluorescent GEVIs to either steady-state voltages or ...

The epithelial-mesenchymal transition (EMT) has been suggested as a new target for therapeutic intervention of metastatic cancer. Forkhead box protein C2 (FOXC2) is known to be necessary for initiating and maintaining EMT, and therefore bestows on cancer cells metastatic and cancer stem cell (CSC)-like phenotypes, allowing cells to acquire higher motility, invasiveness, self-renewal, and therapy resistance. Here, we describe the first inhibitor of FOXC2, MC-1-F2. MC-1-F2 was able to induce cadherin switching and reverse EMT through the degradation of FOXC2 and blocking of its nuclear localization. In addition, MC-1-F2 was very effective in inhibiting cancer cell migration and invasion. As the first small-molecule inhibitor of FOXC2 and the first compound targeting EMT-associated transcription factor, MC-1-F2 will pave the way for a new anticancer therapeutic agent targeting metastatic cancer and help to elucidate the network of EMT signaling pathways.

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Growing numbers of protein and nucleic acid complex structures are being determined and deposited in the Protein Data Bank and the Nucleic Acid Database. With the increasing complexity of these structures, it is challenging to analyse and visualize the three-dimensional interactions. The currently available programs for such analysis and visualization are limited in their applications. They can only analyse a subset of protein-nucleic acid complexes and require multiple iterations before obtaining plots that are suitable for presentation. An interactive web-based program, NuProPlot (http://www.nuproplot.com), has been developed which can automatically identify hydrogen, electrostatic and van der Waals interactions between proteins and nucleic acids and generate a plot showing all of the interactions. Protein-DNA and protein-RNA interactions can be visualized in simple two-dimensional schematics. Interactive schematic drawing options allow selection of the plotted area and repositioning of the individual interactions for better legibility. NuProPlot is a fully automated and user-friendly program providing various custom options. NuProPlot represents a greatly improved option for analysis and presentation of protein-nucleic acid interactions.