LETTERS Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming Kenji Kimura 1,2 , Alexandre Mamane 3 , Tohru Sasaki 4 , Kohta Sato 4 , Jun Takagi 5 , Ritsuya Niwayama 1,2,7 , Lars Hufnagel 6 , Yuta Shimamoto 2,5 , Jean-François Joanny 3 , Seiichi Uchida 4 and Akatsuki Kimura 1,2,8 Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types 1–7 . 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 reverses 6 . 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. The direction of cytoplasmic streaming is, in some cases, predefined by the existing cellular polarity 2,4,7–9 . In other cases, the direction of the streaming is not predefined. Examples include MeiCS in C. elegans zygotes 6,10,11 and in stage 6–7 Drosophila oocytes 1 . In these cases, streaming occurs before the polarity of the embryo is established and, interestingly, the direction of the flow occasionally reverses 1,6 . The mechanisms underlying the emergence of collective flow despite the absence of predefined polarity and flow reversal have been elusive. In this study, we quantitatively characterized the dynamics of the ER and microtubules during MeiCS in C. elegans and successfully constructed a theoretical model to explain and predict the streaming behaviour. Previously, it was revealed that MeiCS is driven by the microtubules and a motor protein, kinesin-1 12 . The emergence of MeiCS is explained by the alignment of the microtubules within the cell in the absence of predefined polarity through an unknown mechanism. Microtubules may align through self-organization mechanisms, similar to the alignment of actin filaments in plant cytoplasmic streaming 13 . In plant cells, the motor protein myosin XI-K, which drives cytoplasmic streaming in Arabidopsis thaliana, associates with the ER, and the knockdown of this motor disrupts the alignment of actin filaments 14 . Because the ER forms a network-like structure in the cytoplasm, it may efficiently transmit the forces produced by myosin to the cytoplasm to generate a cell-wide flow, which aligns actin filaments in plant cells. Similarly, in C. elegans, the ER might contribute to the alignment of microtubules and generation of MeiCS. We imaged the ER and cortical granules, in addition to yolk granules, during MeiCS (Fig. 1a and Supplementary Videos 1–3). The direction and speed of flow of these organelles are highly correlated and biased orthogonal to the long axis of the zygote (Fig. 1b,c and Supplementary Fig. 1a), indicating that these organelles move under MeiCS. Our finding that the ER also moves during MeiCS led us to propose an ER-mediated, positive-feedback mechanism (Fig. 1d), similar to that proposed in plant cells 14 . In this model, the ER is associated with cortical microtubules whose minus ends are anchored at the cortex. Kinesin-1 transports the ER towards the plus ends of the microtubules and generates a local flow. The flow of the ER, in turn, aligns microtubules toward the direction of the flow. The network-like structure of the ER is key to this ER-mediated positive-feedback model as its coupling strength to the cytoplasm is greater than that of other objects dispersed in the cytoplasm (for example, yolk granules). If the network structure of the ER is critical to MeiCS, then fragmentation of the ER network should inhibit the streaming. This 1 Cell Architecture Laboratory, Structural Biology Center, National Institute of Genetics, Mishima 411-8540, Japan. 2 Department of Genetics, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Mishima 411-8540, Japan. 3 Physicochimie Curie (Centre National de la Recherche Scientifique-UMR168, UPMC), Institut Curie, PSL Research University, Section de Recherche, Paris 75248, France. 4 Human Interface Laboratory, Department of Advanced Information Technology, Kyushu University, Fukuoka 819-0395, Japan. 5 Quantitative Mechanobiology Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima 411-8540, Japan. 6 Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany. 7 Present address: Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany. 8 Correspondence should be addressed to A.K. (e-mail: akkimura@nig.ac.jp) Received 14 October 2016; accepted 9 February 2017; published online 13 March 2017; DOI: 10.1038/ncb3490 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.