GENERATION OF NEURITE-LIKE PROTRUSIONS FROM NEURONAL SURFACES USING OPTICAL TWEEZERS

The nervous system is highly complex, with neurons playing crucial roles in the formation of neural networks. Neurons have a unique morphology, consisting of a cell body and neurites, which, after differentiation, become axons and dendrites, with the actin cytoskeleton actively participating in this process. Malformations or issues in the shape or structure of neurites can result in diseases. Our group has been able to mechanically generate cylindrical structures from the cell surface using optical  tweezers. In this work, we explore the possibility of generating neurites directly from the surface of neurons. We used primary cultures from 14-day-old embryonic mice, whose cerebral hemispheres were isolated, and neural precursor cells were cultured to form neurospheres. After five days, the neurospheres were dissociated, and the cells were plated for neuronal differentiation. Next, we used an instrument capable of applying point forces (on the order of picoNewtons) to microscopic objects: We placed the cell culture under the microscope and pullet, with ópticas tweezers, a cylindrical nanotube from the cell surface. This entire procedure was monitored by video microscopy and later fluorescence microscopy for the labeling of specific proteins. Our results demonstrate the ability to induce the formation of cylindrical protrusions from the surface of neurons, as well as the generation of protrusions connecting two neuronal cells. These protrusions persist for long periods and exhibit movement of internal structures. Their morphological characterization showed the presence of the same cytoskeletal components found in naturally grown neurites, including β-Tubulin III and F-actin. We conclude that we are capable of stably generating cylindrical protrusions similar to neurites from the surface of neurons. Although more detailed morphofunctional characterizations of the generated protrusions are still necessary, our findings open new perspectives for exploring neurite formation under disease or malformation conditions, thus contributing to the understanding of neurobiological processes from a mechanobiological perspective. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq).