Damien Baigl

From Convective Assembly to Landau−Levich Deposition of Multilayered Phospholipid Films of Controlled Thickness

In this letter, we describe a method to control the organization and thickness of multilayered phospholipid films. The meniscus of an organic solution of phospholipid molecules was dragged at a speed v on a solid substrate under controlled temperature and forced convection, leading to the deposition of a dried multilayered phospholipid film with a thickness h in the range of 20-200 nm. We found two distinct regimes dominating the film deposition. At low speeds, phospholipid molecules accumulate near the contact line and form a dry film behind the meniscus (evaporation regime). At high speed, viscous forces become predominant and pull out a liquid film that will dry afterward (Landau-Levich regime). Both regimes show robust scaling h infinity v(alpha) with alpha = -1.1 and 0.76, respectively. Although these regimes have been observed separately in the past, they have not been demonstrated in the same material system. Moreover, we present models whose scalings (alpha = -1 and 2/3) are in close agreement with the observed values. The microscale organization of the resulting film is independent of v for a given regime but differs from one regime to another. In the Landau-Levich regime, h is very homogeneous on the microscale with discrete variations of +/- 5 nm, that is, the thickness of one bilayer.

DNA compaction: fundamentals and applications

Compaction is the process in which a large DNA molecule undergoes a transition between an elongated conformation and a very compact form. In nature, DNA compaction occurs to package genomic material inside tiny spaces such as viral capsids and cell nuclei. In vitro, several strategies exist to compact DNA. In this review, we first provide a physico-chemical description of this phenomenon, focusing on the modes of compaction, the types of compaction agents and the chemical and physical parameters that control compaction and its reverse process, decompaction. We then describe three main kinds of applications. First, we show how regulated compaction/decompaction can be used to control gene activity in vitro, with a particular emphasis on the use of light to reversibly control gene expression. Second, we describe several approaches where compaction is used as a way to reversibly protect DNA against chemical, biochemical, or mechanical stresses. Third, we show that compact DNA can be used as a nanostructure template to generate nanomaterials with a well-defined size and shape. We conclude by proposing some perspectives for future biochemical and biotechnological applications and enumerate some remaining challenges that we think worth being undertaken.

Physical Mechanisms Redirecting Cell Polarity and Cell Shape in Fission Yeast

The cylindrical rod shape of the fission yeast Schizosaccharomyces pombe is organized and maintained by interactions between the microtubule, cell membrane, and actin cytoskeleton [1]. Mutations affecting any components in this pathway lead to bent, branched, or round cells [2]. In this context, the cytoskeleton controls cell polarity and thus dictates cell shape. Here, we use soft-lithography techniques to construct microfluidic channels to control cell shape. We show that when wild-type rod-shaped cells are physically forced to grow in a bent fashion, they will reorganize their cytoskeleton and redirect cell polarity to make new ectopic cell tips. Moreover, when bent or round mutant cells are physically forced to conform to the wild-type rod-shape, they will reverse their mutational phenotypes by reorganizing their cytoskeleton to maintain proper wild-type-like localization of microtubules, cell-membrane proteins, and actin. Our study provides direct evidence that the cytoskeleton controls cell polarity and cell shape and demonstrates that cell shape also controls the organization of the cytoskeleton in a feedback loop. We present a model of the feedback loop to explain how fission yeast maintain a rod shape and how perturbation of specific parameters of the loop can lead to different cell shapes.

Time-Resolved Tracking of a Minimum Gene Expression System Reconstituted in Giant Liposomes

Individual expression: We describe a method that allows the observation of real‐time gene expression in a large number of individual giant liposomes encapsulating identical genetic material. We followed the gene expression profiles from DNA and mRNA templates coding for different proteins. Although the average profiles of individual liposomes were similar to those measured in bulk solution, strong variability between individual liposomes was observed at both transcription and translation.

Pre-dispositions and epigenetic inheritance in the Escherichia coli lactose operon bistable switch.

The lactose operon regulation in Escherichia coli is a primary model of phenotypic switching, reminiscent of cell fate determination in higher organisms. Under conditions of bistability, an isogenic cell population partitions into two subpopulations, with the operons genes turned on or remaining off. It is generally hypothesized that the final state of a cell depends solely on stochastic fluctuations of the networks protein concentrations, particularly on bursts of lactose permease expression. Nevertheless, the mechanisms underlying the cell switching decision are not fully understood. We designed a microfluidic system to follow the formation of a transiently bimodal population within growing microcolonies. The analysis of genealogy and cell history revealed the existence of pre‐disposing factors for switching that are epigenetically inherited. Both the pre‐induction expression stochasticity of the lactose operon repressor LacI and the cellular growth rate are predictive factors of the cells response upon induction, with low LacI concentration and slow growth correlating with higher switching probability. Thus, stochasticity at the local level of the network and global physiology are synergistically involved in cell response determination.

Sequence-independent and reversible photocontrol of transcription/expression systems using a photosensitive nucleic acid binder.

To understand non-trivial biological functions, it is crucial to develop minimal synthetic models that capture their basic features. Here, we demonstrate a sequence-independent, reversible control of transcription and gene expression using a photosensitive nucleic acid binder (pNAB). By introducing a pNAB whose affinity for nucleic acids is tuned by light, in vitro RNA production, EGFP translation, and GFP expression (a set of reactions including both transcription and translation) were successfully inhibited in the dark and recovered after a short illumination at 365 nm. Our results indicate that the accessibility of the protein machinery to one or several nucleic acid binding sites can be efficiently regulated by changing the conformational/condensation state of the nucleic acid (DNA conformation or mRNA aggregation), thus regulating gene activity in an efficient, reversible, and sequence-independent manner. The possibility offered by our approach to use light to trigger various gene expression systems in a system-independent way opens interesting perspectives to study gene expression dynamics as well as to develop photocontrolled biotechnological procedures.

Photosensitive surfactants with various hydrophobic tail lengths for the photocontrol of genomic DNA conformation with improved efficiency.

We report the synthesis and characterisation of photosensitive cationic surfactants with various hydrophobic tail lengths. These molecules, called AzoCx, are used as photosensitive nucleic acid binders (pNABs) and are applied to the photocontrol of DNA conformation. All these molecules induce DNA compaction in a photodependent way, originating in the photodependent polarity of their hydrophobic tails. We show that increasing hydrophobicity strongly enhances the compaction efficiencies of these molecules, but reduces the possibility of reversible photocontrol of a DNA conformation. Optimal performance was achieved with AzoC5, which allowed reversible control of DNA conformation with light at a concentration seven times smaller than previously reported.

UV-Induced Bursting of Cell-Sized Multicomponent Lipid Vesicles in a Photosensitive Surfactant Solution

We study the behavior of multicomponent giant unilamellar vesicles (GUVs) in the presence of AzoTAB, a photosensitive surfactant. GUVs are made of an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) and various amounts of cholesterol (Chol), where the lipid membrane shows a phase separation into a DPPC-rich liquid-ordered (Lo) phase and a DOPC-rich liquid-disordered (Ld) phase. We find that UV illumination at 365 nm for 1 s induces the bursting of a significant fraction of the GUV population. The percentage of UV-induced disrupted vesicles, called bursting rate (Yburst), increases with an increase in [AzoTAB] and depends on [Chol] in a non-monotonous manner. Yburst decreases when [Chol] increases from 0 to 10 mol % and then increases with a further increase in [Chol], which can be correlated with the phase composition of the membrane. We show that Yburst increases with the appearance of solid domains ([Chol] = 0) or with an increase in area fraction of Lo phase (with increasing [Chol] ≥ 10 mol %). Under our conditions (UV illumination at 365 nm for 1 s), maximal bursting efficiency (Yburst = 53%) is obtained for [AzoTAB] = 1 mM and [Chol] = 40 mol %. Finally, by restricting the illumination area, we demonstrate the first selective UV-induced bursting of individual target GUVs. These results show a new method to probe biomembrane mechanical properties using light as well as pave the way for novel strategies of light-induced drug delivery.

Electroformation of Giant Phospholipid Vesicles on a Silicon Substrate: Advantages of Controllable Surface Properties

We introduce the use of silicon (Si) as a substrate for the electroformation of giant phospholipid vesicles. By taking advantage of the tunability of silicon surface properties, we varied the organization of the phospholipid film on the electrode and studied the consequences on vesicle formation. In particular, we investigated the effects of Si surface chemistry and microtopology on the organization of the phospholipid film and the properties of the final vesicles. We established correlations between chemical homogeneity, film defects, and resulting vesicle size distribution. By considering phospholipid films that are artificially fragmented by electrode microstructures, we showed that the characteristic size of vesicles decreases with a decrease in microstructure dimensions. We finally proposed a way to control the vesicle size distribution by using a micropatterned silicon dioxide layer on a Si substrate.

Microfluidic mixing triggered by an external LED illumination.

The mixing of confined liquids is a central yet challenging operation in miniaturized devices. Microfluidic mixing is usually achieved with passive mixers that are robust but poorly flexible, or active mixers that offer dynamic control but mainly rely on electrical or mechanical transducers, which increase the fragility, cost, and complexity of the device. Here, we describe the first remote and reversible control of microfluidic mixing triggered by a light illumination simply provided by an external LED illumination device. The approach is based on the light-induced generation of water microdroplets acting as reversible stirrers of two continuous oil phase flows containing samples to be mixed. We demonstrate many cycles of reversible photoinduced transitions between a nonmixing behavior and full homogenization of the two oil phases. The method is cheap, portable, and adaptable to many device configurations, thus constituting an essential brick for the generation of future all-optofluidic chip.