Across the tree of life, cells display remarkable diversity in traits including cellular organization and morphology. These adaptations are critical to the survival of a cell or organism within its unique environment. To perform fundamental cellular processes, each cell must be able to transport cargos such as organelles, vesicles, mRNAs, and protein complexes with spatial and temporal precision. Despite understanding many of the players involved in intracellular transport, little is known about why some organisms use one mode of transport over another. For example, human cells and many filamentous fungi use the microtubule-based motors dynein and kinesin for long-distance transport, while yeast and plants primarily use actin-based myosin motors. Our lab takes a comparative biology approach to uncover the fundamental principles behind cargo transport, how these principles are adapted across species, and when organisms use non-traditional forms of intracellular transport. Together, we use comparative genomics, comparative cell biology, cellular proteomics, and biochemical reconstitution/single molecule TIRF microscopy to understand transport regulation on the evolutionary, cellular, and molecular scales.