2010/2011Under physiological conditions in the brain, molecules are released with high spatial and temporal resolution. A lot of efforts have been done in the last years in order to develop techniques that mimic this situation. Among them, we mention the use of micropipettes for the ejection of fluids, the use of AFM (Atomic Force Microscopy), microfluidic devices and optical manipulation. The latter approach exploits light to manipulate the samples, e.g. to create transient pores in the cell membrane or to move small objects carrying a stimulus.
This Thesis concerns with the development of new techniques for the local delivery of molecules based on optical manipulation technologies, and in particular on optical tweezers. Sub-micrometer particles in a compact trap, such as the single-beam gradient or optical tweezers, can be localized within a small fraction of a wavelength of light or moved over long distances of many centimeters without any mechanical contact. A three-dimensional trap is simply created by focusing a laser beam through a microscope objective with high numerical aperture. We studied three types of vectors for local delivery of molecules, which can be optically manipulated: microbeads, micron-sized liposomes and Quantum dots (Qdots).
Silica microbeads can be covalently functionalized on their surface with the protein of interest and placed in contact with the desired part of a cell. In order to validate the technique, we functionalized beads with a secretory molecule, the neurotrophin Brain-derived neurotrophic factor (BDNF). BDNF is a key regulator of neuronal development and plasticity. We showed that single BDNF-coated microbeads can be extracted with optical tweezers from small reservoirs and positioned with submicrometric precision to specific sites on the dendrites of cultured hippocampal neurons. Localized contact of microbeads functionalized with BDNF induced focal increase of Calcium signaling in the stimulated dendrite, specific activation of the TrkB receptor pathway and influenced the development of growth cones. Remarkably, a single BDNF-coated bead positioned on a dendrite was found to be enough for TrkB phosphorylation, an efficient and long-lasting activation of Calcium signaling in the soma, and c-Fos signaling in the nucleus, comparable to bath stimulation conditions. Moreover, since BDNF is covalently cross-linked to the bead surface we could demonstrate that activation of some of the TrkB receptor pathway does not necessarily require BDNF endocytosis.
In the case of liposomes, the molecules of interest were encapsulated within their lumen. Single liposomes were trapped and transported by means of optical tweezers to the site of stimulation on cultured neurons. Finally, the release of liposome content was induced by application of UV-pulses that broke the liposome membrane. In order to test the effect of the UV-induced release, liposomes with a diameter ranging from 1 to 10 μm (fL to pL volumes), were filled with KCl and tested on neuronal cells. Neuronal cultures, loaded with Ca2+ dye, were monitored by imaging intracellular Ca2+. An efficient release from the liposomes was demonstrated by detectable Calcium signals, indicating induced depolarization of the neuronal cells by KCl. Afterwards, this technique was used to address a biological issue, that is the effect of two proteins (Semaphorin 3A and Netrin-1) on growth cones. The growth cone is an intracellular apparatus located at the tip of the neurite of developing neurons. Its motility governs axonal path-finding and the construction of neuronal networks. Growth cones are highly dynamic structures that respond to external stimuli turning towards or away from the chemical gradient. We were able to demonstrate an attractive effect of Netrin-1 on the growth cones of primary hippocampal neurons. On the contrary, Semaphorin 3A showed a repellant behavior.
To correlate the high resolution of vector manipulation with high resolution of imaging we used STimulated Emission Depletion (STED) to investigate the intimate organization of two main cytoskeleton components: actin and tubulin filaments. STED microscopy allowed imaging of actin bundles in the filopodia and organized network in lamellipodia with un-precedent resolution, beyond the diffraction barrier.
Lastly, we used liposomes to encapsulate Quantum dots. Qdots are bright and photostable nanocrystals. Due to their small size, similar to that of proteins, Qdots may be endocyted along the receptor-mediated endocytosis pathway, when they are functionalized with the appropriate ligand. As case study we considered the BDNF-TrkB endocytotic pathway. We optimized the protocol for the direct binding of BDNF to Qdots and we demonstrated the possibility of encapsulating and releasing them from liposomes.
Concluding, two different approaches for local stimulation of neurons, based on optical manipulation of microvectors, were presented and validated in this thesis. Indirect optical manipulation of nanovectors (Qdots) encapsulated in liposomes has been demonstrated as well. The techniques were then successfully applied to address some biological issues, that in turn required the optimization of other imaging tools (super resolution microscopy and Qdots).XXIV Ciclo198
