The work presented here is a comprehensive study for myosin motility on assembling actin filaments. I propose a novel method of reconstructing actin tracks to recapture cytoskeletal complexity in vitro to higher extend than traditional myosin motility assays. By use of polymerizing actin filament and controlling the phosphate state of F-actin I am able to ask questions about intrinsic properties of unconventional myosin motor activity that govern its behavior in vivo. The reconstitution method is building the bridge between the in vitro tests and the experiments done in the cell’s complexity background. By reconstituting cytoskeletal structures to resemble the environments of the cell more faithfully I intended to reveal the behaviors of myosin and actin that are often too subtle, therefore extremely challenging to detect, when investigating cellular interior with its immense amounts of interactions and factors influencing each other. These characteristics of myosin and actin are also not possible to observe in classical in vitro tests, because the experimental set ups omit vast numbers of native cytoskeletal aspects to achieve the desired simplicity of the assay. In here I added back layers of cellular complexity and asked questions about specific effects these extra interactions have on the system. The first question addressed in this dissertation is whether myosin motors and native-like polymerizing actin influence each other in the reconstituted environment that mimics cell interior more faithfully than other assays used currently in the field. The approach taken to answer this question reveals if myosin-5 and myosin-6 properties, such as velocities, run lengths and processive run initiation rates sense the distinct properties of polymerizing actin filament. I also can observe any impact that myosin motors have on actin polymerization dynamics, in terms of increasing up or slowing down polymerization rates of the observed filaments. In this approach I have an opportunity to evaluate the level to which phalloidin could be affecting myosin's activity. The second point addressed in this work deals with elevating the complexity of reconstituted in vitro environment. I introduced, apart from utilizing polymerizing actin filaments and observing motility of one unconventional myosin, additional actin binding protein. A representative of second myosin motor class, either myosin-5 or myosin-6 added to the experimental environment with the other myosin class already present tests the interaction between the two ABPs — myosin-5 and myosin-6. Both of the motors are arguably the best studied unconventional myosins and belong to the classes thought to utilize the same cellular actin filaments for their in vivo activity. As they walk and carry their cargos in opposite directions along the filament, it is not known whether they can share the same exact filament at the same time. If they can, the nuances of how they encounter each other on that shared track are not known. By reconstituting the simultaneous motility of myosin-5 and myosin-6 on the same filament I was able to perform an exhaustive analysis of myosin-5 and myosin-6 crossing mechanisms. This information delineates how the bidirectional transport is achieved in the multi myosin cell interior. I learnt how myosins utilize the same actin track while moving along it in opposite directions. The microscopy observation as well as stochastic simulation model of the bidirectional motility show which of the myosins is dominating in case of an encounter when both myosins trajectories come to spatial and temporal overlap. By dissecting the differences between the two myosin classes I am also able to hypothesize on what are the advantages that allow the more successful myosin pass and what are the implication for the cellular transport effectiveness. Four videos are available as supplementary files.