ENDOPLASMIC-RETICULUM MEMBRANE TUBULES ARE DISTRIBUTED BY MICROTUBULES IN LIVING CELLS USING 3 DISTINCT MECHANISMS

Background: The microtubule-dependent motility of endoplasmic reticulu m (ER) tubules is fundamental to the structure and function of the ER. From in vitro assays, three mechanisms for ER tubule motility have ar isen: the 'membrane sliding mechanism' in which ER tubules slide along microtubules using microtubule motor activity; the 'microtubule movem ent mechanism' in which ER attaches to moving microtubules; and the 't ip attachment complex (TAC) mechanism' in which ER tubules attach to g rowing plus ends of microtubules. Results: We have used multi-waveleng th time-lapse epifluorescence microscopy to image the dynamic interact ions between microtubules (by microinjection of X-rhodamine-labeled tu bulin) and ER (by DiOC(6)(3) staining) in living cells to determine wh ich 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, statical ly attached to microtubules, moved towards the cell center with microt ubules through actomyosin-based retrograde flow. TACs did not change m icrotubule growth and shortening velocities, but reduced transitions b etween these states. Treatment of cells with 100 nM nocodazole to inhi bit plus-end microtubule dynamics demonstrated that TAC motility requi red microtubule assembly dynamics, whereas membrane sliding and retrog rade-flow-driven ER motility did not. Conclusions: Both plus-end-direc ted 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 modulat e plus-end microtubule dynamics.