Dense Bicoid hubs accentuate binding along the morphogen gradient

Abstract

Morphogen gradients direct the spatial patterning of developing embryos; however, the mechanisms by which these gradients are interpreted remain elusive. Here we used lattice light-sheet microscopy to perform in vivo singlemolecule imaging in early Drosophila melanogaster embryos of the transcription factor Bicoid that forms a gradient and initiates patterning along the anteroposterior axis. In contrast to canonical models, we observed that Bicoid binds to DNA with a rapid off rate throughout the embryo such that its average occupancy at target loci is on-ratedependent. We further observed Bicoid forming transient "hubs" of locally high density that facilitate binding as factor levels drop, including in the posterior, where we observed Bicoid binding despite vanishingly low protein levels. We propose that localized modulation of transcription factor on rates via clustering provides a general mechanism to facilitate binding to low-affinity targets and that this may be a prevalent feature of other developmental transcription factors.

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

“…Clustering of a range of nuclear factors has been observed previously using single-molecule techniques, such as transient RNA Polymerase II cluster dynamics in living cells using time-correlated PALM (tc-PALM) [73,74]. Also functional nuclear protein clusters have been seen [75] and the Bicoid transcription factor in fruit fly embryos has been observed to form clusters mediated in part mediated by intrinsically disordered peptide sequences [76]. …”

Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy

“…For example, 100 ms or longer, where freely diffusing molecules largely “motion blur” into the background, but bound molecules are readily detected, for residence/binding time [2, 5, 9] measurements. For capturing the dynamics of both the diffusing and bound molecules, typically an exposure time of 10 ms or shorter is appropriate ( see Note 23).…”

“…For these reasons primarily, single molecule imaging studies have been restricted to regions of the sample within just a few microns of the coverslip. Lattice light-sheet microscopy (LLSM) [8] addresses this technological limitation and enables single molecule imaging in thicker specimens such as living embryos [9]. …”

“…We have recently demonstrated the first application of LLSM to perform single molecule imaging in embryos by tracking single molecules of the transcription factor Bicoid in early Drosophila melanogaster embryos [9]. The ability to quantify single molecule kinetics during development in terms of off-rates of DNA binding, fraction of molecules in a bound or mobile state, diffusion coefficients, and spatial distributions has a huge discovery potential as highlighted by our findings on Bicoid [9] .…”

“…The ability to quantify single molecule kinetics during development in terms of off-rates of DNA binding, fraction of molecules in a bound or mobile state, diffusion coefficients, and spatial distributions has a huge discovery potential as highlighted by our findings on Bicoid [9] . We expect that extending these live-embryo single molecule imaging approaches to other factors including proteins [5], RNA [10], and DNA [11] will contribute greatly to our understanding of Drosophila development.…”