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Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming

Abstract

Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types1,2,3,4,5,6,7. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses6. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

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Figure 1: MeiCS comprises hydrodynamic flow of various organelles and requires an interconnected ER structure.
Figure 2: ER network increases hydrodynamic coupling in the cytoplasm.
Figure 3: ER flow is required for microtubule alignment.
Figure 4: Theoretical framework of MeiCS.
Figure 5: MeiCS contributes to cortical positioning of cortical granules.

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Acknowledgements

C. elegans strain WH327 was kindly provided by A. Spang (University of Basel, Switzerland). The other strains were from the Caenorhabditis Genetics Center. We thank T. Hiiragi (European Molecular Biology Laboratory), K. Nagai (Japan Advanced Institute of Science and Technology), Y. Sumino (Tokyo University of Science), Y. Okada (The University of Tokyo), and the members of the Cell Architecture Laboratory and the Office for Research Development (National Institute of Genetics) for their valuable comments and support. This project was supported by a SOKENDAI Young Faculty Overseas programme grant to A.K., a Japanese Society for the Promotion of Science (JSPS) summer research programme grant to A.M., JSPS Postdoctoral Fellowship to J.T., AMED-PRIME, AMED (JP15665739) to Y.S., and JSPS KAKENHI grant numbers JP26840072 (to K.K.), JP15610795, JP16748896 (to Y.S.), JP15KT0083, JP16H00816, JP16H05119 and JP16H06280 (to A.K.).

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Authors and Affiliations

Authors

Contributions

K.K., A.M. and A.K. conceived the study and wrote the manuscript with support from all authors. K.K. performed all the experiments and data analysis except selective plane illumination microscopy, which was performed by R.N. and L.H., and micromanipulation of reconstituted ER, which was performed by J.T. and Y.S. A.M. and J.-F.J. constructed the theoretical model and performed the calculations. T.S., K.S. and S.U. constructed the image-processing algorithms and processed the data.

Corresponding author

Correspondence to Akatsuki Kimura.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Visualisation and Quantification of MeiCS.

(a) Scatter plots and histograms of the angle and velocity of the ER and cortical granules (CGs) during MeiCS as in Figs 1b, c. The average velocity of the CGs (0.24 ± 0.05 μm s−1, mean ± standard deviation [s.d.], n = 240 frames from 4 zygotes) was larger than that of the ER (0.22 ± 0.05 μm s−1, n = 240 frames from 4 zygotes). This is due to the occasional faster motion of CGs possibly through directed transport along the microtubule independent of the overall streaming. (b) Packing at the internal region of yolk granules (YGs). Left panels show confocal images of untreated and the indicated (RNAi)-treated zygotes expressing GFP::VIT-2 during MeiCS. White dotted lines show embryo outlines. Middle panels show projections of six sequential images (time interval: 2 s). Fluorescence signals at each time point are given in different colours. The boxed region is magnified on the right. White arrows indicate the flow direction. Representative images of at least 6 zygotes are shown. (c) Box plots of velocity of YG flow (n = 720 (untreated), 720 (unc-116 (RNAi)), 720 (yop-1; ret-1 (RNAi)), 1080 (tcc-1 (RNAi)), and 480 (yop-1; ret-1; unc-116 (RNAi)) frames from 6, 6, 6, 9, 4 zygotes, respectively). The boxes display the 25th to 75th percentile range. The lines inside the boxes represent the median. Whiskers extend to the most extreme data point within 1.5 interquartile ranges from the box. The dots are outliers. Flow fields of YG were obtained by optical flow analysis of time-lapse imaging (time interval = 2 s) of zygotes expressing GFP::VIT-2. The length of the resultant vector at each time point is plotted as a velocity. The statistical differences between untreated and RNAi-treated zygotes were determined by Mann–Whitney U test (P < 0.001). (d) The progression of the cell cycle was not delayed in yop-1; ret-1 (RNAi) and unc-116 (RNAi) zygotes. The average time intervals ± s.d. from the exit of the oocyte from the spermatheca to meiotic anaphase I, and from meiotic anaphase I to meiotic anaphase II were determined by the appearance of female chromosomes [untreated (n = 16), unc-116 (RNAi) (n = 12), yop-1; ret-1 (RNAi) (n = 8)]. No statistical difference was found between untreated and RNAi-treated zygotes by Mann–Whitney U test.

Supplementary Figure 2 Orientation of Microtubules during MeiCS.

(ab) Confocal fluorescence images of a zygote expressing both mKate2::α-tubulin and GFP::SP12 during MeiCS. Sequential images of the boxed regions are magnified on the bottom. Yellow arrows indicate vectors of ER-flow (>0.2 μm s−1). Representative images of 10 zygotes are shown. Yellow arrowheads in (b) indicate a microtubule that changes its orientation toward the ER-flow. Scale bars, 5 μm. (c) A majority of the microtubule plus-ends are aligned in a similar direction during MeiCS. Time-lapse confocal images of zygotes expressing GFP::EBP-1, the microtubule plus-end marker, were analysed for the growing direction of microtubules during MeiCS (n = 5 zygotes). The angle differences between the two moving signals at the same time point are grouped every 30 degrees and the ratio of the distribution is plotted. Scale bar, 5 μm.

Supplementary Figure 3 The theoretical model.

(a) Schematic diagrams of the effects of the parameters T and V on MeiCS. T is the squared ratio between the length of the microtubules, and the permeation length, i.e. the thickness necessary to saturate the velocity of the ER. When T is larger, the flow becomes faster and more stable. V is the polarisability ratio that quantifies the sensitivity of the microtubules to the flow. It is the ratio of the depolarisation rate ωcata divided by the polarisation rate um/l. A large V means the polarisation of microtubules is difficult to achieve, because they grow too fast, which makes them too long, or because the kinesin velocity is too small, and thus no collective flow can emerge. (b) Geometry of the model. The cell cortex lies in the x-y plane. The ER and cytoplasm extend along the z axis. r is the vector position in the fluid, x the projection of r on the cell cortex. u is the flow velocity vector at position r. A microtubule is sketched in green, with its unit orientation vector p. (cd) Bifurcation diagrams. Evolution of the flow properties: stationary velocity Ust (blue),Pst (red), and mean reversal time (black) under different conditions. In both situations, we set um = 0.7 μm s−1, total number of microtubules N = 200 and l0 = 5 μm. The two diagrams cut across the coordinates (T, V) = (15,1.43) for l = l0. (c) We varied vg while keeping ωcata and all other parameters constant. (d) We varied ωcata while keeping vg constant. Because microtubule length in the model is defined by l = vg/ωcata, these diagrams were drawn with l on the horizontal axis.

Supplementary Figure 4 Measurement of parameter values.

(a) The length distribution of microtubules in untreated (n = 360 frames from 6 zygotes) and mei-1 (RNAi) zygotes (n = 240 frames from 4 zygotes). The data are identical to those shown in Fig. 4g except for the length unit (1 μm = 7.4 pixels). Fitting lines and equations for both plots are also shown. ‘MT’ refers to ‘microtubule’. (b) Confocal fluorescence image of the cortical ER-network (GFP::SP12). Scale bar, 5 μm. A representative image of at least 10 zygotes is shown. (c) The fluorescence profile of the yellow line (15 μm) in (a) is shown as a typical result of a measurement of ER mesh size. On average, 13.9 ± 1.5 peaks were detected per 15-μm line (n = 60). The mesh size was estimated as 1.1.

Supplementary information

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Yolk granules during MeiCS in an untreated C. elegans zygote.

Time-lapse confocal fluorescence image of a zygote expressing GFP::VIT-2, taken every 2 s. (Latter half) A 3D reconstruction of the flow visualised by selective plane illumination microscopy. (AVI 5241 kb)

Cortical Granules during MeiCS in an Untreated C. elegans Zygote.

Time-lapse confocal fluorescence image of a zygote expressing CAV-1::GFP, taken every 2 s. (AVI 3065 kb)

ER during MeiCS in an untreated C. elegans zygote.

Time-lapse confocal fluorescence image of a zygote expressing GFP::SP12, taken every 2 s. (AVI 2579 kb)

ER during MeiCS and post-meiotic cytoplasmic streaming in a yop-1; ret-1 (RNAi) zygote.

Time-lapse confocal fluorescence image of a zygote expressing GFP::SP12, taken every 2 s. (Former half) MeiCS, taken every 2 s. (Latter half) post-meiotic cytoplasmic streaming, taken every 3 s. The disruption of the network structure by yop-1; ret-1 (RNAi) did not inhibit the post-meiotic cytoplasmic streaming, which demonstrates that the network structure of the ER is not essential for all cytoplasmic streaming. In such ER-independent cytoplasmic streaming, the viscosity of the cytoplasm should be sufficient to generate the cell-wide flow as theoretically demonstrated previously11,12. (AVI 6280 kb)

Yolk granules during MeiCS in a yop-1; ret-1 (RNAi) zygote.

Time-lapse confocal fluorescence image of a zygote expressing GFP::VIT-2, taken every 2 s. (AVI 3022 kb)

Microtubules and ER during MeiCS in an untreated C. elegans zygote.

Time-lapse confocal fluorescence image of a zygote expressing mKate2::α-tubulin (red) and GFP::SP12 (green), taken every 2 s. (AVI 2220 kb)

Microtubules during MeiCS in an untreated C. elegans zygote.

Time-lapse confocal fluorescence image of a zygote expressing GFP::β-tubulin, taken every 2 s. (AVI 2544 kb)

Yolk granules during MeiCS in a mei-1 (RNAi) zygote.

Time-lapse confocal fluorescence image of a zygote expressing GFP::VIT-2, taken every 2 s. (AVI 3053 kb)

ER during MeiCS in a tcc-1 (RNAi) zygote.

Time-lapse confocal fluorescence image of a zygote expressing GFP::SP12, taken every 2 s. (AVI 2646 kb)

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Kimura, K., Mamane, A., Sasaki, T. et al. Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming. Nat Cell Biol 19, 399–406 (2017). https://doi.org/10.1038/ncb3490

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