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Leucine Rich Repeat Transmembrane proteins (LRRTMs) are neuronal cell adhesion molecules involved in synapse development and plasticity. LRRTM2 is the most synaptogenic isoform of the family, and its expression is strongly restricted to excitatory synapses in mature neurons. However, the mechanisms by which LRRTM2 is trafficked and stabilized at synapses remain unknown. Here, we examine the role of LRRTM2 intracellular domain on its membrane expression and stabilization at excitatory synapses, using a knock-down strategy combined to single molecule tracking and super-resolution dSTORM microscopy. We show that LRRTM2 operates an important shift in mobility after synaptogenesis in hippocampal neurons. Knock-down of LRRTM2 during synapse formation reduced excitatory synapse density in mature neurons. Deletion of LRRTM2 C-terminal domain abolished the compartmentalization of LRRTM2 in dendrites and disrupted its synaptic enrichment. Furtheremore, we show that LRRTM2 diffusion is increased in the absence of its intracellular domain, and that the protein is more dispersed at synapses. Surprisingly, LRRTM2 confinement at synapses was strongly dependent on a YxxC motif in the C-terminal domain, but was independent of the PDZ-like binding motif ECEV. Finally, the nanoscale organization of LRRTM2 at excitatory synapses depended on its C-terminal domain, with involvement of both the PDZ-binding and YxxC motifs. Altogether, these results demonstrate that LRRTM2 trafficking and enrichment at excitatory synapses are dependent on its intracellular domain. ispartof: BIOLOGY OF THE CELL vol:113 issue:12 pages:492-506 ispartof: location:England status: published

The precise wiring of the nervous system relies on processes by which axons navigate in a complex environment and are guided by a concerted action of attractive and repulsive factors to reach their target. Investigating these guidance processes depends critically on our ability to control in space and time the microenvironment of neurons. The implementation of microfabrication techniques in cell biology now enables a precise control of the extracellular physical and chemical environment of cultured cells. However, microtechnology is only beginning to be applied in the field of axon guidance due to specific requirements of neuronal cultures. Here we review microdevices specifically designed to study axonal guidance and compare them with the conventional assays used to probe gradient sensing in cell biology. We also discuss how innovative microdevice-based approaches will enable the investigation of important systems-level questions on the gradient sensing properties of nerve cells, such as the sensitivity and robustness in the detection of directional signals or the combinatorial response to multiple cues.

Induced-charge electro-osmosis (ICEO) is demonstrated around an isolated platinum wire in a polymer microchannel filled with lowconcentration KCl, subject to a weak alternating electric field. In contrast to ac electro-osmosis at electrode arrays, which shares the same slip mechanism, ICEO has a more general frequency dependence, including steady flow in the dc limit (since the wire is not an electrode driving the field). The flow profile inside the device, measured by particle image velocimetry, confirms the predicted scaling with the square of the applied voltage, as well as the characteristic cut-off frequency. A quantitative comparison with numerical solutions of the models equations is reported for various equivalent circuit models from the literature. The standard model of linear capacitors captures the basic trends, but systematically over-predicts the velocity. A better fit to experiment can be obtained with a complex impedance, taking into account the frequency dispersion of capacitance, although the microscopic justification of this model is unclear especially in the presence of electro-osmotic flow and applied voltages well into the non-linear regime. © 2005 Elsevier B.V. All rights reserved.

The purification of nucleic acids from microbial and mammalian cells is a crucial step in many biological and medical applications1. We have developed microfluidic chips for automated nucleic acid purification from small numbers of bacterial or mammalian cells. All processes, such as cell isolation, cell lysis, DNA or mRNA purification, and recovery, were carried out on a single microfluidic chip in nanoliter volumes without any pre- or postsample treatment. Measurable amounts of mRNA were extracted in an automated fashion from as little as a single mammalian cell and recovered from the chip. These microfluidic chips are capable of processing different samples in parallel, thereby illustrating how highly parallel microfluidic architectures can be constructed to perform integrated batch-processing functionalities for biological and medical applications.

High-density microfluidic chips contain plumbing networks with thousands of micromechanical valves and hundreds of individually addressable chambers. These fluidic devices are analogous to electronic integrated circuits fabricated using large scale integration (LSI). A component of these networks is the fluidic multiplexor, which is a combinatorial array of binary valve patterns that exponentially increases the processing power of a network by allowing complex fluid manipulations with a minimal number of inputs. These integrated microfluidic networks can be used to construct a variety of highly complex microfluidic devices, for example the microfluidic analog of a comparator array, and a microfluidic memory storage device resembling electronic random access memories.

We present how to make and assemble micro-patterned stickers (microPS) to construct high performance plastic microfluidic devices in a few minutes. We take advantage of soft UV imprint techniques to tailor the geometry, the mechanical properties, and the surface chemistry of 2D and 3D microfluidic circuits. The resulting microfluidic stickers substantially overcome the actual performance of the very popular PDMS devices for a wide range of applications, while sharing their celebrated fast and easy processing. To highlight the intrinsic advantages of this method, three important applications are detailed: (i) we show that both aqueous and organic droplets can be produced and stored in stickers without any specific surface coating. (ii) We report on the outstanding pressure resistance of the microPS, which open the way to the transport of viscous complex fluids. (iii) Finally, a simple design strategy is proposed to generate complex flow patterns in interconnected stacks of microPS.

In the present work, we have measured the messenger RNA expression of specific genes both from total RNA and cells encapsulated in droplets. The microfluidic chip introduced includes the following functionalities: RNA∕cell encapsulation, lysis, reverse transcription and real-time polymerase chain reaction. We have shown that simplex and duplex gene expression measurements can be carried out over a population of 100 purified RNA samples encapsulated simultaneously in 2 nl droplets in less than 2 h. An analysis of 100 samples containing one to three cells has shown excellent consistency with standard techniques regarding average values. The cell-to-cell distributions of the E-cadherin expression suggest fluctuations on the order of 80% in the number of transcripts, which is highly consistent with the general findings from the literature. A mathematical model has also been introduced to strengthen the interpretation of our results. The present work paves the way for the systematic acquisition of such information in biological and biomedical studies.