Dynein-dependent collection of membranes defines the architecture and position of microtubule asters in isolated, geometrically confined volumes of cell-free extracts

It is well established that changes in the underlying architecture of the cell’s microtubule (MT) network can affect organelle organization within the cytoplasm, but it remains unclear whether the spatial arrangement of organelles reciprocally influences the MT network. Here we use a combination of cell-free extracts and hydrogel microenclosures to characterize the relationship between membranes and MTs during MT aster centration. We found that initially disperse ER membranes are collected by the aster and compacted near its nucleating center, all while the whole ensemble moves toward the geometric center of its confining enclosure. Once there, aster MTs adopt a bull’s-eye pattern with a high-density annular ring of MTs surrounding the compacted membrane core of lower MT density. Formation of this pattern was inhibited when dynein-dependent transport was perturbed or when membranes were depleted from the extracts. Asters in membrane-depleted extracts were able to move away from the most proximal wall but failed to center in cylindrical enclosures with diameters greater than or equal to 150 µm. Taken as whole, our data suggest that the dynein-dependent transport of membranes buttresses MTs near the aster center and that this plays an important role in modulating aster architecture and position.