In vitro reconstitution of Central Spindle Motility

Abstract

Precise localisation of intracellular organelles impacts a myriad of cellular processes including cell motility, polarisation and mitosis. Positioning is actively controlled by organelle specific attachment, often involving dedicated motor proteins, to cytoskeletal network. While much is known about the biophysics of motors on isolated tracks, how the complex cytoskeleton topologies found in cells, such as antiparallel microtubule (MT) overlaps, can influence the steady state distribution of cargoes is not understood molecularly. For example, in dividing sensory organ precursor cells, the antiparallel MT overlap of the central spindle controls the asymmetric segregation of signalling endosomes containing fate determinants in only one daughter cell. A modest asymmetry in the density of microtubules within the antiparallel MT overlap of central spindle, with more MTs on the anterior side of the spindle, generates one order of magnitude higher (than the microtubule asymmetry) bias distribution of endosomes. How such moderate asymmetry in the cytoskeleton is translated by motor proteins to produce strong non-linear effects and how different endosomes respond to this asymmetry is unknown. In this work, I have aimed to address both these questions by using a combination of in vivo imaging and in vitro reconstitution using purified proteins and micropatterning. To address how different endosomes segregate on an asymmetric microtubule track I utilised knock-in Rab-GFP lines and internalised labelled anti-delta antibodies. Remarkably, a class of late endosomes containing anti-delta antibodies indeed partitioned symmetrically during mitosis even on an asymmetric central spindle. This suggested that endosome partitioning is dictated by both the microtubule track and the endosome specific motor proteins in vivo. To gain insight into how the motor content affects endosome behaviour, particularly on defined microtubule network, I attempted to reconstitute the process in vitro. The central spindle, an interdigitated antiparallel microtubule structure, is formed by balanced activity between both motor and non-motor microtubule associating proteins (MAPS). To control the geometry of such antiparallel microtubule tracks I utilised an improved micropatterning technology and purified central spindle proteins. This combined approach successfully led to formation of asymmetric antiparallel overlaps akin to the central spindle for the first time.