Here we introduce an optical flow motion estimation approach to study microtubule (MT) orientation in the Drosophila oocyte, a cell displaying substantial cytoplasmic streaming. We show that MT polarity is affected by the regime of these flows and, furthermore, that the presence of flows is necessary for MTs to adopt their proper polarity.
21The polar orientation of microtubule networks is exploited by molecular motors, such as kinesins, to 22 deliver cargoes to specific intracellular destinations, and is thus essential for cell polarity and cell 23 function. Reconstituted in vitro systems have largely contributed to the current understanding of the 24 molecular framework, regulating the behaviour of single microtubule filaments. In cells however, 25 microtubules are subjected to a variety of different biomechanical forces that might impact on their 26 orientation and thus on the organisation of the entire network. Here we implement variational optical 27 flow analysis as a new approach to analyse the polarity of microtubule networks in vivo, and find that 28 cytoplasmic flows impact on the growth direction of microtubule plus ends in the Drosophila oocyte. 29We provide a thorough characterisation of microtubule behaviour and orientation under different 30 kinesin-dependent cytoplasmic flow conditions, and establish that flows are sufficient and necessary 31 to support the overall organisation of the microtubule cytoskeleton. Introduction 33Eukaryotic life depends on many dynamic processes, including for example cell division, cell 34 migration, and cell polarisation. These processes in turn strongly rely on highly organised 35 microtubule (MT) arrays. All MT networks are polarised, with the minus end of each filament linked 36 to a nucleating centre (MT organising centre or MTOC), and the plus end growing away from these 37 centres. This intrinsic polarity is utilised by specific motor proteins to transport cargo along MTs in a 38 defined direction, and is essential for the function of MT networks, and consequently for the function 39 and polarity of cells. 40A number of biophysical studies in reconstituted in vitro systems have helped to understand the 41 mechanical properties of MTs, setting the stage to investigate the behaviour of MTs in vivo. However, 42 much needs to be learnt about the properties of MTs in their natural intracellular environment. For 43 example, a rather new concept emanating from in vivo experiments is that controlling nucleation and 44 the position of minus ends alone is not always sufficient to establish the proper polarity of the 45 network. Thus MT plus ends must be controlled as well in order to allow motor proteins to deliver 46 their cargoes to the correct destination. The plus ends can be regulated at various levels, including 47 dynamic instability, capturing, and direction of growth. Dynamic instability describes a process, in 48 which MT polymerisation is interrupted by a rapid depolymerisation phase, followed by a 'rescue' 49 process 1 . Various MT-associated proteins, such as molecular motors and MT plus end-tracking 50 proteins (+TIPs), are known to regulate dynamic instability 2 . Furthermore, MT plus ends can also be 51 stabilised by cortical capture, also involving +TIPs and other molecules such as the Dynein/Dynactin 52 complex 3 (and as reviewed in 2 ). However, very little is known about how the directi...
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