Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour

Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms

Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50–60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS

Cholesterol-Dependent Cytolysins and Perforin: Similar Pore-Forming Mechanisms in Pathogenic Attack and Human Immune Defense

MACPF/CDCs proteins are a huge family of pore-forming proteins present from the bacteria to the human genera. Cholesterol-dependent cytolysins (CDCs) are a family of toxins that participate in bacterial infection pathway at the membrane level. Great interest in this family is due to their similarity, in structure and in pore-forming mechanism, with some human immune system proteins (MACPF). We focused our attention particularly on two bacterial CDCs, Perfringolysin O and Listeriolysin O, and on the human protein Perforin, which is involved in the apoptotic pathway facilitating Granzyme release. In the literature, two possible configurations of CDCs and Perforin pores are proposed: ring and arc structures that could have different implications on the biological mechanism of action of these pore-forming proteins. By electrophysiological measurements and atomic force microscopy technique on different artificial membrane, we are able to enrich the ring and the arc fraction and demonstrate that both kinds of pore are active, i.e. conduct ions. Thus, my PhD work underlines two physiological structures which are involved in several ways, more than merely by disrupting membrane integrity, in pathogenic attack (bacterial CDCs proteins) as well as in immune response (human Perforin proteins)

Bid binding to negatively charged phospholipids may not be required for its pro-apoptotic activity in vivo

Bid is a ubiquitous pro-apoptotic member of the Bcl-2 family that has been involved in a variety of pathways of cell death. Unique among pro-apoptotic proteins, Bid is activated after cleavage by the apical caspases of the extrinsic pathway; subsequently it moves to mitochondria, where it promotes the release of apoptogenic proteins in concert with other Bcl-2 family proteins like Bak. Diverse factors appear to modulate the pro-apoptotic action of Bid, from its avid binding to mitochondrial lipids (in particular, cardiolipin) to multiple phosphorylations at sites that can modulate its caspase cleavage. This work addresses the question of how the lipid interactions of Bid that are evident in vitro actually impact on its pro-apoptotic action within cells. Using site-directed mutagenesis, we identified mutations that reduced mouse Bid lipid binding in vitro. Mutation of the conserved residue Lys157 specifically decreased the binding to negatively charged lipids related to cardiolipin and additionally affected the rate of caspase cleavage. However, this lipid-binding mutant had no discernable effect on Bid pro-apoptotic function in vivo. The results are interpreted in relation to an underlying interaction of Bid with lysophosphatidylcholine, which is not disrupted in any mutant retaining pro-apoptotic function both in vitro and in vivo

Neuronal firing modulation by a membrane-targeted photoswitch

Light-sensitive azobenzene compounds can be engineered to stably partition into the plasma membrane, thus causing its thinning in the dark and relaxation upon light stimulation. In neurons, the resulting light-dependent change in membrane capacitance induces a transient hyperpolarization followed by rebound depolarization and action potential firing. Optical technologies allowing modulation of neuronal activity at high spatio-temporal resolution are becoming paramount in neuroscience. In this respect, azobenzene-based photoswitches are promising nanoscale tools for neuronal photostimulation. Here we engineered a light-sensitive azobenzene compound (Ziapin2) that stably partitions into the plasma membrane and causes its thinning through trans-dimerization in the dark, resulting in an increased membrane capacitance at steady state. We demonstrated that in neurons loaded with the compound, millisecond pulses of visible light induce a transient hyperpolarization followed by a delayed depolarization that triggers action potential firing. These effects are persistent and can be evoked in vivo up to 7 days, proving the potential of Ziapin2 for the modulation of membrane capacitance in the millisecond timescale, without directly affecting ion channels or local temperature