Dynamical spatial organization in living cells provides the framework underlying the fundamental genetic and biochemical processes essential to biological life. Importantly, dysfunctions in this process are linked to diseases including neurodegeneration. The dynamic spatial organization cannot be accomplished by passive diffusion alone. Instead, Nature employs molecular motors that are protein machines to actively shuttle materials along biopolymer-based molecular "roads" in cells. Significant advances in single- molecule biophysics have revealed a great deal about how motors function individually in minimal, cell-free environments. Building on these important previous advances, my thesis study aimed at dissecting the physical principles of transport under complex conditions such as those that occur in living cells. With this goal in mind, I tackled three distinct aspects of intracellular transport that are not being considered in single-motor investigations: the number of motors involved (Chapter 2), the condition of the molecular highways that the motors step along (Chapters 3-4), and the physical properties of the lipid membrane that couple the motors to their cargo (Chapters 5-6). My studies to-date have revealed exciting new biophysical mechanisms for potentially correcting the aberrant motor transport that underlies several human pathologies, and established a new, biologically-relevant framework to drive future developments in molecular-motor theory and experiments in cell-free studies.