Ludovic Jullien

How to control proteins with light in living systems

The possibility offered by photocontrolling the activity of biomolecules in vivo while recording physiological parameters is opening up new opportunities for the study of physiological processes at the single-cell level in a living organism. For the last decade, such tools have been mainly used in neuroscience, and their application in freely moving animals has revolutionized this field. New photochemical approaches enable the control of various cellular processes by manipulating a wide range of protein functions in a noninvasive way and with unprecedented spatiotemporal resolution. We are at a pivotal moment where biologists can adapt these cutting-edge technologies to their system of study. This user-oriented review presents the state of the art and highlights technical issues to be resolved in the near future for wide and easy use of these powerful approaches.

Self-Immolative Spacers: Kinetic Aspects, Structure–Property Relationships, and Applications

Self-immolative spacers are covalent assemblies tailored to correlate the cleavage of two chemical bonds after activation of a protective part in a precursor: Upon stimulation, the protective moiety is removed, which generates a cascade of disassembling reactions leading to the temporally sequential release of smaller molecules. Originally introduced to overcome limitations for drug delivery, self-immolative spacers have gained wide interest in medicinal chemistry, analytical chemistry, and material science. For most applications, the kinetics of the disassembly of the activated self-immolative spacer governs functional properties. This Review addresses kinetic aspects of self-immolation. It provides information for selecting a particular self-immolative motif for a specific demand. Moreover, it should help researchers design kinetic experiments and fully exploit the rich perspectives of self-immolative spacers.

A critique of methods for temperature imaging in single cells

We argue that standard thermodynamic considerations and scaling laws show that a single cell cannot substantially raise its temperature by endogenous thermogenesis. This statement seriously questions the interpretations of recent work reporting temperature heterogeneities measured in single living cells.

Coiling of Cylindrical Membrane Stacks with Anchored Polymers

(Received 22 April 1999) We study experimentally a coiling instability of cylindrical multilamellar stacks of phospholipid membranes, induced by polymers with hydrophobic anchors grafted along their hydrophilic backbone. We interpret our experimental results in terms of a model in which local membrane curvature and polymer concentration are coupled. The model predicts the occurrence of maximally tight coils above a threshold polymer concentration. Indeed, only maximally tight coils are observed experimentally. Our system is unique in that coils form in the absence of twist and adhesion. The coil motif is ubiquitous in a wide range of natural contexts. One-dimensional filaments of mutant bacteria [1], supercoiled DNA molecules [2], and tendrils of climbing plants [3] all exhibit a writhing instability as a result of forcing or interaction with an external agent. Such systems are dominated by elastic properties, and the appearance of coils is a result of the relief of twist. In this paper we show that coiling can also be effected in cylindrical multilamellar tubes of phospholipid bilayers, by anchoring hydrophilic polymers with hydrophobic side groups grafted along the backbone. This system is unique in that, in contrast with the above examples, fluid membranes cannot support any twist. Yet coils are

A Blue-Absorbing Photolabile Protecting Group for in Vivo Chromatically Orthogonal Photoactivation

The small and synthetically easily accessible 7-diethylamino-4-thiocoumarinylmethyl photolabile protecting group has been validated for uncaging with blue light. It exhibits a significant action cross-section for uncaging in the 470-500 nm wavelength range and a low light absorption between 350 and 400 nm. These attractive features have been implemented in living zebrafish embryos to perform chromatic orthogonal photoactivation of two biologically active species controlling biological development with UV and blue-cyan light sources, respectively.

Channel-type molecular structures. Part 4. Transmembrane transport of alkali-metal ions by ‘bouquet’ molecules

This report describes transport experiments with ‘bouquet’ molecules designed to act as artificial ion channels. The ‘bouquets’ are based on a central macrocycle which is either an 18-crown-6 (BM) or a cyclodextrin derivative (BCD) to which are attached polyethylene oxide [poly(oxyethylene)] chains (BMo and BCDo) or polyalkyl chains (BMc and BCDc) tipped with carboxylate endgroups. The ‘bouquets’ were studied in liposomes prepared from egg phosphatidylcholine (egg PC), dipalmitoyl phosphatidylcholine (DPPC) and a mixture of egg PC, stearylamine and cholesterol. Opposing gradients in Li+ and Na+ concentration were created and the transport of alkali-metal ions down their concentration gradients was followed directly by 7Li and 23Na NMR spectroscopy. ‘Bouquets’ were found to cause a one-for-one exchange of Na+ for Li+(antiport). In order to estimate transport rates, the extent of Na+ entry into liposomes was followed as a function of time. All ‘bouquets’ transported ions at similar rates in fluid membranes. Comparison of transport rates in fluid-(egg PC) and gel-state membranes (DPPC) was used to distinguish carrier and channel mechanisms. Control experiments demonstrated that a known carrier (monensin A) gave significantly lower transport rates in gel-state membranes. Two ‘bouquets’, BMc and BCDc, were found to transport Na+ at similar rates in fluid- and gel-state membranes; this suggests that ion passage occurs preferentially by the channel mechanism and not by the carrier mechanism. Variation of transport rate with ‘bouquet’ concentration was probed for BMo and BMc and the rates were found to increase with BMc concentration but not with BMo concentration. Since the transport rate is expected to be proportional to transporter concentration in both the carrier and channel mechanisms, this indicates that BMo uses neither a carrier nor a channel mechanism. The mechanism by which ‘bouquet’ molecules operate and the criteria which may be used to decide whether functioning channels have been created are discussed.

Excited-state relaxation dynamics of a PYP chromophore model in solution: influence of the thioester group

Abstract Cis–trans photoisomerization of a photoactive yellow protein chromophore model, the deprotonated trans S -phenyl thio- p -hydroxycinnamate, is studied in aqueous solution by subpicosecond transient absorption and gain spectroscopy. The excited-state deactivation is found to involve the formation, in 1.7 ps, of an intermediate state which decays in 2.8 ps. A persistent bleaching signal is observed at longer times indicating that the excited state not only relaxes to the ground state but also partly forms a stable photoproduct, possibly the cis isomer. This behavior is analogous to that of the native photoactive yellow protein.

Photocontrol of protein activity in cultured cells and zebrafish with one- and two-photon illumination.

We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen‐OH, a photochemically stable inducer of the receptor specific for 4‐hydroxy‐tamoxifen (ERT2). Cyclofen‐OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ERT2 receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen‐OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two‐photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.

Photoactivation of the CreERT2 Recombinase for Conditional Site-Specific Recombination with High Spatiotemporal Resolution

We implemented a noninvasive optical method for the fast control of Cre recombinase in single cells of a live zebrafish embryo. Optical uncaging of the caged precursor of a nonendogeneous steroid by one- or two-photon illumination was used to restore Cre activity of the CreER(T2) fusion protein in specific target cells. This method labels single cells irreversibly by inducing recombination in an appropriate reporter transgenic animal and thereby can achieve high spatiotemporal resolution in the control of gene expression. This technique could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration, or carcinogenesis) with high spatiotemporal resolution (single cell and 10-min scales).

Coumarinylmethyl Caging Groups with Redshifted Absorption

The small and synthetically easily accessible coumarinylmethyl backbone has been modified to generate a family of photolabile protecting groups with redshifted absorption. We relied on introducing electron-donating groups in the 7 position and electron-withdrawing groups in the 2-, and 2- and 3 positions. In particular, we showed that the diethylamino-thiocoumarylmethyl and the diethylamino-coumarylidenemalononitrilemethyl are relevant for uncaging with cyan light. They both exhibit a significant action cross section for uncaging in the 470-500 nm wavelength range and a low light absorption between 350 and 400 nm. These attractive features are favorable to perform chromatic orthogonal photoactivation with UV and blue-cyan light sources, respectively.