Microtubule-based Endoplasmic Reticulum Motility inXenopus laevis: Activation of Membrane-associated Kinesin during Development

Abstract

The endoplasmic reticulum (ER) in animal cells uses microtubule motor proteins to adopt and maintain its extended, reticular organization. Although the orientation of microtubules in many somatic cell types predicts that the ER should move toward microtubule plus ends, motor-dependent ER motility reconstituted in extracts of Xenopus laevis eggs is exclusively a minus end-directed, cytoplasmic dynein-driven process. We have used Xenopus egg, embryo, and somatic Xenopus tissue culture cell (XTC) extracts to study ER motility during embryonic development in Xenopus by video-enhanced differential interference contrast microscopy. Our results demonstrate that cytoplasmic dynein is the sole motor for microtubule-based ER motility throughout the early stages of development (up to at least the fifth embryonic interphase). When egg-derived ER membranes were incubated in somatic XTC cytosol, however, ER tubules moved in both directions along microtubules. Data from directionality assays suggest that plus end-directed ER tubule extensions contribute approximately 19% of the total microtubule-based ER motility under these conditions. In XTC extracts, the rate of ER tubule extensions toward microtubule plus ends is lower ( approximately 0.4 microm/s) than minus end-directed motility ( approximately 1.3 microm/s), and plus end-directed motility is eliminated by a function-blocking anti-conventional kinesin heavy chain antibody (SUK4). In addition, we provide evidence that the initiation of plus end-directed ER motility in somatic cytosol is likely to occur via activation of membrane-associated kinesin.

Functional coupling of microtubules to membranes – implications for membrane structure and dynamics

“…The physiological relevance of a dynamic ER remains unclear. Possible functions include roles in development (Lane and Allan, 1999), Ca 2+ signalling [for example, in terms of ER-plasma-membrane coupling for capacitative Ca 2+ entry (Grigoriev et al, 2008;Orci et al, 2009)], spatial organization of protein synthesis or trafficking, or perhaps in metabolic sensing [through coupling of the ER to the mitochondrial network (Friedman et al, 2010)]. In addition, the link between ER and mitochondrial dynamics is an area of great interest at present, because the ER defines sites of mitochondrial fission (Friedman et al, 2011) and mitochondrial morphology is directly linked to autophagy (Gomes and Scorrano, 2008;Gomes et al, 2011;Rambold et al, 2011).…”

Dynamics and inheritance of the endoplasmic reticulum

“…Microtubule-dependent ER movement results from the activity of microtubule-associated motors as well as the polymerization of microtubules (Dabora and Sheetz, 1988;Vale and Hotani, 1988;Dailey and Bridgeman, 1989;Waterman-Storer et al, 1995;Waterman-Storer and Salmon, 1998;Lane and Allan, 1999). Motors can either drag membranes along underlying microtubules or drive the sliding of membrane-associated microtubules, whereas microtubule polymerization can promote the movement of an ER tubule bound to the dynamic microtubule tip (Waterman-Storer and Salmon, 1998).…”

“…Cytoplasmic dynein, but not conventional kinesin, is required for cortical ER dynamics and reformation in the fungus U. maydis (WedlichSoldner et al, 2002). However, a combination of both dyneinand kinesin-dependent bidirectional movements of ER tubules along microtubules has been observed when membranes from Xenopus eggs are incubated with Xenopus tissue culture cell (XTC) cytosol extracts (Lane and Allan, 1999). The level of membrane-associated kinesin in egg microsomes is comparable with that in somatic cell microsomes.…”

Bidirectional membrane tube dynamics driven by nonprocessive motors

“…A trademark of these cytoskeletal-membrane interactions is the presence of continuously changing membrane tube networks. For example, in the endoplasmic reticulum in vivo (1,2) and in cell-free extracts (3)(4)(5)(6), new membrane tubes are constantly formed as old ones disappear. Colocalization of these membrane tubes with the underlying cytoskeleton has led to the finding that cytoskeletal motor proteins can extract membrane tubes (6).…”

“…For example, in the endoplasmic reticulum in vivo (1,2) and in cell-free extracts (3)(4)(5)(6), new membrane tubes are constantly formed as old ones disappear. Colocalization of these membrane tubes with the underlying cytoskeleton has led to the finding that cytoskeletal motor proteins can extract membrane tubes (6). Motors must work collectively to extract membrane tubes (7,8), because the force needed to form a tube, F tube (9), is larger than the mechanical stall force of an individual motor (10).…”