Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Therefore, we aimed to develop a tool to manipulate organelle positioning with spatiotemporal precision. In order to do so, a good understanding of how organelles are being transported and positioned is essential. Organelles are being transported and positioned by molecular motors that walk along a network of motor tracks, called the cytoskeleton. Kinesin motors accomplish fast and long-range transport by walking along microtubules, whereas slow and short-range transport is mediated by myosin motors that walk along the actin cytoskeleton. In fact, a single cargo often travels along both microtubule and actin filaments, but how microtubule and actin-based motors contribute to the final motility and distribution of these cargoes is largely unknown. This thesis aims to illuminate the interplay between kinesin and myosin motors on cargoes that contain both motor types. We next aimed to control organelle positioning with spatiotemporal precision using optogenetics. Molecular motors with known speed and direction were manipulated to bind an organelle of interest, only in the presence of blue light. In other words, targeted blue-light illumination could now be used to trigger selective organelle translocations. Using this tool we showed that the addition of recycling endosomes into the axonal growth cone stimulated axon outgrowth in cultured hippocampal neurons. This data shows that basal cellular processes can be manipulated by changing organelle distributions. With the organelle-positioning tool available, the exact relations between organelle position and function can now be investigated, both in culture and in different animal models.