Optical flow analysis reveals that Kinesin-mediated advection impacts on the orientation of microtubules in theDrosophilaoocyte

Abstract

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...

Kinesin-1 promotes centrosome clustering and nuclear migration in theDrosophilaoocyte

“…Kinesin-1 has been shown to interfere directly with MT organization (Daire et al, 2009; Drechsler et al, 2020; Nieuwburg et al, 2017), especially through its ability to crosslink MTs (Lu et al, 2016; Metzger et al, 2012). Hence, we wondered if the nuclear migration defects observed in absence of Khc and Klc could be explained by defects in MT organization.…”

“…It is quite striking that the delay for nuclear migration does not gradually appear after stage 7, as we would expect from a simple slowing down of the process, but is sharply occurring at stage 9. We and others have previously shown that the MT network in the oocyte undergoes dramatic rearrangement at stage 9 to organize bundles at the cell cortex (Drechsler et al, 2020; Januschke et al, 2006; Lu et al, 2016; Nashchekin et al, 2016; Parton et al, 2011; Trong et al, 2015). At this stage, the MTs generate a cytoplasmic flow which sustains an advection-based transport corresponding to an active transport induced by fluid flow (Drechsler et al, 2017; Drechsler et al, 2020; Ganguly et al, 2012; Loiseau et al, 2010; Williams et al, 2014).…”

“…We and others have previously shown that the MT network in the oocyte undergoes dramatic rearrangement at stage 9 to organize bundles at the cell cortex (Drechsler et al, 2020; Januschke et al, 2006; Lu et al, 2016; Nashchekin et al, 2016; Parton et al, 2011; Trong et al, 2015). At this stage, the MTs generate a cytoplasmic flow which sustains an advection-based transport corresponding to an active transport induced by fluid flow (Drechsler et al, 2017; Drechsler et al, 2020; Ganguly et al, 2012; Loiseau et al, 2010; Williams et al, 2014). This process requires Khc activity, but not Klc, as in absence of Klc the cytoplasmic streaming is unaffected (Loiseau et al, 2010; Palacios and St Johnston, 2002; Williams et al, 2014).…”

“…Then, at mid-oogenesis, concomitantly with nuclear migration, the MT network is progressively reorganized to result in the formation of a dense network with a weak anterior to posterior bias of MT plus-ends. At stage 10, MTs are further reorganized as bundles that are parallel to the cell cortex and generate a cytoplasmic flow (Drechsler et al, 2020; Januschke et al, 2006; Lu et al, 2016; Nashchekin et al, 2016; Parton et al, 2011; Theurkauf et al, 1993; Trong et al, 2015). Throughout oocyte development, MT polymerization occurs from several sources (Januschke et al, 2006; Lu et al, 2021; Nashchekin et al, 2016; Nashchekin et al, 2021) including the centrosomes that are active from the beginning of oogenesis until stage 11 (Pimenta-Marques et al, 2016).…”

Joint Motion Estimation and Source Identification using Convective Regularisation with an Application to the Analysis of Laser Nanoablations

“…Recently, optical flow methods have been used for the quantitative analysis of biological image sequences on cellular and subcellular level. See, for instance, [2,9,10,21,31,34,39,44,50]. While the concept is in practice well-suited for natural scenes, the use of the less restrictive continuity equation, which arises from mass conservation, can be more favourable in certain scenarios.…”