Single cell physiology

Optical control of protein expression and activity at the single cell level: applications to morphogenesis in zebrafish

Living organisms are made of cells that are capable of responding to external signals by modifying their internal state (gene expression or protein phosphorylation patterns) and subsequently their external environment by the release of signaling molecules.  In multicellular organisms in particular, cellular differentiation and signaling is essential for the development of the organism. While many of the key actors of these processes are known (morphogens in development, kinases in signal transduction) much less is known of the quantitative rules that govern their interaction with one another and with other cellular players (such as the type of complexes, rate constants, strength of feedback or feedforward loops, etc).

We initially investigated the development of means to optically control the expression and activity of small molecules and proteins at the single cell level in a live organism and to use that method to study morphogenesis in zebrafish. The idea is to photo-activate locally with two-photon excitation small soluble biomolecules. We can thus control the activity of proteins and the local development of the organism

We first tested our idea on retinoic acid (RA) a potent teratogen required in many developmental pathways, among which somitogenesis (the formation of the anterio-posterior axis). RA can be controlled by isomerization of its active trans-form to its biologically inactive cis-form (and vice-versa). We have shown that activation of nM of RA (in embryos whose endogenous RA pathway was impaired) hours before the onset of somitogenesis could rescue this important developmental pathway. This finding suggests that RA can be stored in the embryo for hours before it is required. We have further shown that local isomerization of RA (in the anterior part of the embryo) is enough to rescue its development.

We have developed a novel way to photo-control the activity of many different proteins.  By fusing a protein with the estrogen receptor (ERT), it can be sequestered by cyctoplasmic chaperones. Upon uncaging the receptor ligand (cyclofen), the construct is released from its complex with chaperones, thereby activating the protein. Various proteins have thus been controlled at the single cell level in a live embryo: the Cre-recombinase, a nls-GFP (see movie), the Gal4 transcription factor, a homeogene (Engrailed), etc.   We are using that approach to control morphogen gradients in a live embryo and to turn on the activity of oncogenes in order to respectively study development and cancer at the single cell level in a live organism. The goal is to compare the resulting observations with the predictions of models of development and cancer

Ref.: “How to control proteins with light in living systems”, A.Gautier, C.Gauron, M.Volovitch, D.Bensimon, L.Jullien, S.Vriz, invited review, Nat.Chem.Biol.  10, 533, 2014


Fig.1: Two-photon uncaging of cyclofen in a single cell of a fish retina induces activity of a Cre recombinase that excises a GFP gene and turns on a RFP gene in an appropriate transgenic fish line.

Following is an overnight confocal movie of the development of a zebrafish line which is expressing GFP in rhombomeres 3 and 5 of its hindbrain and a confocal movie of a (nuclear localized) GFP fused to the ERT receptor and activated in a single cell of a zebrafish tail by cyclofen uncaging.

development timelapse of embryo expressing GFP in rhombomeres 3 and 5.wmv2.65 MB
confocal image of GFP labelled single cell in fish tail.mov1.08 MB