Localised Dynactin protects growing microtubules to deliver<i>oskar</i>mRNA to the posterior cortex of the<i>Drosophila</i>oocyte

Nieuwburg, Ross; Nashchekin, Dmitry; Jakobs, Maximilian Ah; Carter, Andrew; Trong, Philipp Khuc; Goldstein, Raymond E.; Johnston, Daniel St

doi:10.1101/121590

Cited by 6 publications

(15 citation statements)

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“…Secondly, in the presence of Kin-mediated transport, but in the absence of cytoplasmic flows ( khc slow condition), there are more plus ends growing towards the posterior, suggesting that cytoplasmic streaming is necessary for MTs to polarize in the precise pattern observed in control cells. And thirdly, together with cytoplasmic flows, other Kin-mediated processes, such as cargo transport or nucleus anchoring affect the organization of the MT network, supporting previously published findings (Nieuwburg et al, 2017; Zimyanin et al, 2008).…”

Section: Resultssupporting

confidence: 86%

“…To study the spatial orientation of MT filaments in vivo , we used oocytes expressing EB1 (Figures 1, 3, 4 and 5). EB1 constitutes a marker for growing MT plus-tips and has been used in fly oocytes before (Parton et al ., 2011; Nieuwburg et al ., 2017). In these previous studies, wide-field deconvolution microscopy was used to image EB1 dynamics in various areas of st9 oocytes.…”

Section: Resultsmentioning

confidence: 99%

“…To address this question, we used oocytes expressing EB1 (Figure 2a,d and Supplementary Methods). EB1 constitutes a versatile marker for growing plus-tips and has been used in fly oocytes before 3,9 . In these previous studies, widefield deconvolution microscopy was used to image EB1 dynamics in specific areas of the oocyte.…”

Section: Resultsmentioning

confidence: 99%

“…Various MT-associated proteins, such as motors and MT plus end-tracking proteins (+TIPs), are known to regulate dynamic instability (Akhmanova and Steinmetz, 2015). MT plus ends can also be captured at the cell cortex, a process also involving +TIPs and motors, such as the Dynein/Dynactin complex (Nieuwburg et al ., 2017) (and reviewed in (Akhmanova and Steinmetz, 2015)). However, very little is known about how the direction of growth of plus ends, and therefore the orientation of MTs, is controlled in cells.…”

Section: Introductionmentioning

confidence: 99%

“…In order to assess the global MT orientation in st9 oocytes, and to investigate the growth direction of MT plus ends in vivo , we used EB1::GFP (EB1 from here on). EB1 exclusively decorates the growing plus end of MTs, resulting in dynamic ‘comets’ moving through the cytoplasm at a speed of ~100-600 nm/sec across various cell types, including the oocyte (at an average and maximum speeds of 230 nm/sec and 600 nm/s, respectively (Parton et al ., 2011; Nieuwburg et al ., 2017). Analyzing the orientation of MTs in complex networks has proven technically challenging, and requires suitable imaging and image analysis tools.…”

Section: Introductionmentioning

confidence: 99%

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

Drechsler

Lang

al-Khatib

et al. 2019

Preprint

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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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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Drechsler

Lang

al-Khatib

et al. 2019

Preprint

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Symmetry breaking in the female germline cyst

Nashchekin

Busby

Jacobs

et al. 2021

Preprint

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In mammals and flies, only a limited number of cells in a multicellular female germline cyst become oocytes, but how the oocyte is selected is unknown. Here we show that the microtubule minus end-stabilizing protein, Patronin/CAMSAP marks the future Drosophila oocyte and is required for oocyte specification. The spectraplakin, Shot, recruits Patronin to the fusome, a branched structure extending into all cyst cells. Patronin stabilizes more microtubules in the cell with most fusome and this weak asymmetry is amplified by Dynein-dependent transport of Patronin-stabilized microtubules. This forms a polarized microtubule network, along which Dynein transports oocyte determinants into the presumptive oocyte. Thus, Patronin amplifies a weak fusome anisotropy to break cyst symmetry. These findings reveal a molecular mechanism of oocyte selection in the germline cyst.

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Unrestrained growth of correctly oriented microtubules instructs axonal microtubule orientation

Jakobs

Zemel

Franze

2021

Preprint

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In many eukaryotic cells, directed molecular transport occurs along microtubules. Within neuronal axons, transport over vast distances particularly relies on uniformly oriented microtubules, whose plus-ends point towards the distal axon tip (+end out). However, axonal microtubules initially have mixed orientations, and what breaks this orientation symmetry is poorly understood. Using live imaging, physical modeling and simulations, we found that +end out microtubules surpass a growth transition and undergo persistent long-term growth. In contrast, oppositely oriented microtubules remain short. We confirmed experimentally and in silico that the enhanced growth of +end out microtubules is critical for achieving uniform microtubule orientation. Our data suggest that the accelerated growth kinetics of correctly oriented microtubules instructs overall axonal microtubule orientation, thus enabling efficient long-range transport essential for neuronal functioning.

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Localised Dynactin protects growing microtubules to deliveroskarmRNA to the posterior cortex of theDrosophilaoocyte

Cited by 6 publications

References 91 publications

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

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

Symmetry breaking in the female germline cyst

Unrestrained growth of correctly oriented microtubules instructs axonal microtubule orientation

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