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Whacked and Rab35 polarize dynein-motor-complex-dependent seamless tube growth

Abstract

Seamless tubes form intracellularly without cell–cell or autocellular junctions. Such tubes have been described across phyla, but remain mysterious despite their simple architecture. In Drosophila, seamless tubes are found within tracheal terminal cells, which have dozens of branched protrusions extending hundreds of micrometres. We find that mutations in multiple components of the dynein motor complex block seamless tube growth, raising the possibility that the lumenal membrane forms through minus-end-directed transport of apical membrane components along microtubules. Growth of seamless tubes is polarized along the proximodistal axis by Rab35 and its apical membrane-localized GAP, Whacked. Strikingly, loss of whacked (or constitutive activation of Rab35) leads to tube overgrowth at terminal cell branch tips, whereas overexpression of Whacked (or dominant-negative Rab35) causes formation of ectopic tubes surrounding the terminal cell nucleus. Thus, vesicle trafficking has key roles in making and shaping seamless tubes.

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Figure 1: The lumenal membrane of tracheal terminal cells has apical identity.
Figure 2: The terminal cell microtubule cytoskeleton is polarized.
Figure 3: Seamless tube growth is blocked or overly exuberant in dynein motor complex and wkd mutants, respectively.
Figure 4: Wkd and Rab35 polarize seamless tube growth.
Figure 5: Subcellular distribution of Wkd and Rab35, and the role of minus-end transport in seamless tube growth.

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Acknowledgements

The authors would like to acknowledge: D. Willis, a former Stanford undergraduate student who helped with the rough mapping of wkd; B. Levi, who helped with the third chromosome screen; and M. Krasnow, in whose laboratory the screen and early phases of these studies were carried out. We also thank J. Zhang and the laboratories of M. Scott and H. Bellen (Baylor College of Medicine, USA) for making RabCA and RabDN stocks available to us before publication, the Engels laboratory for sharing deficiency strains, and M. Metzstein (The University of Utah, USA) for sharing 4x-SRF-GAL4 flies. We thank S. DiNardo, C. Burd, E. Bi and members of the Ghabrial and DiNardo laboratories for fruitful discussions. We thank A. S. Burguete, B. Levi and N. Speck for comments on the manuscript. J.S-R. was supported by NIH training grant 5-T32-HD007516-12 and, subsequently, by an NIH postdoctoral fellowship (NRSA—GM090438-01). A.S.G. gratefully acknowledges support from the University of Pennsylvania and the NIH (1R01GM089782-01A1). This work was supported in part by Basil O’Connor Starter Scholar Research Award Grant No. 5-FY09-43 from the March of Dimes Foundation.

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J.S-R. and A.S.G. conceived of and carried out all experiments described here. A.S.G. wrote the manuscript with input from J.S-R. Figures were assembled by J.S-R. and A.S.G.

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Correspondence to Amin S. Ghabrial.

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Schottenfeld-Roames, J., Ghabrial, A. Whacked and Rab35 polarize dynein-motor-complex-dependent seamless tube growth. Nat Cell Biol 14, 386–393 (2012). https://doi.org/10.1038/ncb2454

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