Endoplasmic reticulum membrane tubules are distributed by microtubules in living cells using three distinct mechanisms.
BACKGROUND
The microtubule-dependent motility of endoplasmic reticulum (ER) tubules is fundamental to the structure and function of the ER. From in vitro assays, three mechanisms for ER tubule motility have arisen: the 'membrane sliding mechanism' in which ER tubules slide along microtubules using microtubule motor activity; the 'microtubule movement mechanism' in which ER attaches to moving microtubules; and the 'tip attachment complex (TAC) mechanism' in which ER tubules attach to growing plus ends of microtubules.


RESULTS
We have used multi-wavelength time-lapse epifluorescence microscopy to image the dynamic interactions between microtubules (by microinjection of X-rhodamine-labeled tubulin) and ER (by DiOC6(3) staining) in living cells to determine which mechanism contributes to the formation and motility of ER tubules in migrating cells in vivo. Newly forming ER tubules extended only in a microtubule plus-end direction towards the cell periphery: 31.4% by TACs and 68.6% by the membrane sliding mechanism. ER tubules, statically attached to microtubules, moved towards the cell center with microtubules through actomyosin-based retrograde flow. TACs did not change microtubule growth and shortening velocities, but reduced transitions between these states. Treatment of cells with 100 nM nocodazole to inhibit plus-end microtubule dynamics demonstrated that TAC motility required microtubule assembly dynamics, whereas membrane sliding and retrograde-flow-driven ER motility did not.


CONCLUSIONS
Both plus-end-directed membrane sliding and TAC mechanisms make significant contributions to the motility of ER towards the periphery of living cells, whereas ER removal from the lamella is powered by actomyosin-based retrograde flow of microtubules with ER attached as cargo. TACs in the ER modulate plus-end microtubule dynamics.
Waterman-Storer
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CM
Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA.
Salmon
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ED
D000818:Animals; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000502:physiology* / Q000648:ultrastructure; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D007425:Intracellular Membranes / Q000502:physiology* / Q000648:ultrastructure; D008168:Lung / Q000166:cytology / Q000502:physiology*; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D012455:Salamandridae; D013552:Swine; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism
D014404:Tubulin