Wnt signaling biosensors
Final Report Abstract
As a DFG fellow, I worked in the lab of Prof. Fraser at the Biological Imaging Center at the California Institute of Technology in order to develop a diverse imaging toolbox set required for the mechanistic dissection of stem cell and developmental biology problems. My work focused on improving and complementing current imaging approaches in biology. One important consideration was ensuring that these tools would be applicable to extract protein dynamics in four dimensions (x, y, z, t) in vivo. In one project, my work introduces a new type of imaging reagent, SHG nanoprobes, whose photophysical properties are fundamentally different to the fluorescent agents currently used in biomedical research. SHG nanoprobes neither bleach nor blink, and the signal does not saturate with increasing illumination intensity. Furthermore, they are not toxic, and they provide superb signal-to-noise ratio in tissue, readily detectable even with increased imaging depth and in optically challenging, highly scattering environments. The absence of an excited state lifetime and the ability to use brighter illumination to generate more signal permits SHG nanoprobes to offer a window into time regimes of molecular targets to which other techniques are blind. Thus, SHG nanoprobes offer a window into the spatial and temporal dynamics of various biological targets at the molecular level with unmatched sensitivity and temporal resolution for both molecular imaging and molecular diagnostics. In another project, I established a fluorescence decay after photoactivation (FDAP) assay that quantitatively uncovers the kinetics of Oct4, a key transcription factor (TF) controlling pre-implantation development in mammals, fused to photoactivatable GFP (paGFP). Using FDAP, the rates of Oct4-paGFP nuclear export, import, degradation and the immobile fraction were measured within nuclei of single cells of developing mouse embryos. Independently of protein expression levels, two cell populations with distinct Oct4-paGFP kinetics were identified before the first signs of cell lineage patterning. Tracing the lineage of these populations after photoactivation of a membrane protein revealed that single cells maintain their Oct4-paGFP kinetic states after the embryo undergoes compaction, a process where the pluripotent and extraembryonic cell lineages are established. Thus, these findings identify TF kinetics as a measure of developmental heterogeneities that predict cell lineage patterning in the early mouse embryo.
Publications
- (2009) “Second Harmonic Generating (SHG) Nanoprobes: a New Tool for Biomedical Imaging”. Proc. SPIE, 7183(1): 71831-5
Pantazis, P., Pu, Y., Psaltis, D., and Fraser, S.E.
- (2010) “Paramagnetic, silicon quantum dots for magnetic resonance and two photon imaging of macrophages.” J Am Chem Soc. 132(6):2016-23
Tu, C., Ma, X., Pantazis, P., Kauzlarich, S., and Louie, A.
- (2010) “Second Harmonic Generating (SHG) nanoprobes for in vivo imaging.” Proc Natl Acad Sci U S A 107(33):14535-40
Pantazis, P., Maloney, J., Wu, D., and Fraser, S.E.
- (2011) “Intercellular Bridges in Vertebrate Gastrulation.” PLoS ONE 6(5): e20230
Caneparo, L., Pantazis, P., Dempsey, W.P., and Fraser, S.E.
- (2011) “Oct4 kinetics predict cell lineage patterning in the early mammalian embryo.” Nature Cell Biology 13(2):117-23
Plachta, N., Bollenbach, T., Pease, S., Fraser, S.E., and Pantazis, P.
