Vesicle Transport Assay

Abstract

Eukaryotic cells contain an extensive cytoplasmic network of actin filaments and microtubules that function as tracks for vesicle transport. The ability to visualise vesicle movement along both sets of filaments advanced rapidly when in vitro vesicle transport (motility) assays were developed. These assays utilise super‐resolution, contrast‐enhanced DIC microscopy to detect structures below the resolution of the light microscope including vesicles as small as 100 nm and microtubules 25 nm in diameter. Images are captured in real time and stored digitally for subsequent analysis. The standard motion analysis parameters include vesicle type, velocity, distances and path length. In vitro motility assays enabled the identification of important factors that are essential for the regulation of intracellular transport including motor proteins and their membrane receptors, the formation of hetero‐motor complexes and transported cargoes. Key Concepts The cytoskeleton contains actin filaments and microtubules, both of which serve as tracks for vesicle transport. Vesicle transport is the directed movement of membrane vesicles on filaments of the cytoskeleton. Microtubules function as intracellular tracks for transport of vesicles by two classes of motor proteins, kinesins and dyneins. Actin filaments serve as tracks for vesicle transport by myosin motors. Motor proteins are mechanochemical nanomachines that use the energy of ATP for muscle contraction, cell motility, cell division and transport of different cargoes along the cytoskeleton. The superfamily of myosin motor proteins found in eukaryotic cells is known to contain over 20 different classes and of these, 12 classes are found in vertebrates, including humans. The kinesin superfamily is subdivided into 15 classes and the dynein family is grouped into axonemal and cytoplasmic dyneins. Motor protein defects are associated with diseases including Griscelli syndrome, Usher syndrome, myopathies, deafness, tumour progression and metastasis. Differential interference microscopy, also known as Nomarski microscopy, uses polarised light to enhance gradients in the optical path length and phase shifts in unstained, transparent biological specimens. Cell‐free extracts are important tools for cell biologists and have a variety of applications including cell cycle studies, intracellular transport mechanisms, signal transduction events and maintenance of cell architecture.