Endoplasmic Reticulum Motility Research Articles

TAOK2 is an ER kinase that catalyzes the dynamic tethering of ER to microtubules

The endoplasmic reticulum (ER) extensively associates with the microtubule cytoskeleton. Although integral to its many functions, mechanisms through which the ER tethers to microtubules and regulates its coupling dynamics with the cytoskeleton are poorly understood. Herein we report that TAOK2 is an ER-resident multifunctional protein kinase required for tethering of ER membranes to microtubules. Using biochemical and super-resolution imaging techniques, we show that TAOK2 is a multipass membrane-spanning kinase localized in distinct ER domains via four transmembrane and amphipathic helices. Using in vitro and cellular assays, we find that TAOK2 directly binds microtubules with high affinity. We define the minimal TAOK2 determinants that induce ER-microtubule tethering. While TAOK2 knockout cells exhibit decreased ER stability and disrupted movement of ER along growing microtubule plus-ends, motor-mediated ER motility is not affected. ER-microtubule tethering is tightly regulated by the catalytic activity of TAOK2 in interphase and mitotic cells. Perturbation of TAOK2 activity leads to defects in ER morphology and cell division. Our study identifies TAOK2 as an ER-microtubule tether, and reveals a kinase-regulated mechanism for control of ER dynamics critical for cell growth and division.

Endoplasmic reticulum visits highly active spines and prevents runaway potentiation of synapses

In hippocampal pyramidal cells, a small subset of dendritic spines contain endoplasmic reticulum (ER). In large spines, ER frequently forms a spine apparatus, while smaller spines contain just a single tubule of smooth ER. Here we show that the ER visits dendritic spines in a non-random manner, targeting spines during periods of high synaptic activity. When we blocked ER motility using a dominant negative approach against myosin V, spine synapses became stronger compared to controls. We were not able to further potentiate these maxed-out synapses, but long-term depression (LTD) was readily induced by low-frequency stimulation. We conclude that the brief ER visits to active spines have the important function of preventing runaway potentiation of individual spine synapses, keeping most of them at an intermediate strength level from which both long-term potentiation (LTP) and LTD are possible.

Intracellular calcium stores mediate metaplasticity at hippocampal dendritic spines.

Key points Calcium (Ca2+) entry mediated by NMDA receptors is considered central to the induction of activity‐dependent synaptic plasticity in hippocampal area CA1; this description does not, however, take into account the potential contribution of endoplasmic reticulum (ER) Ca2+ stores.The ER has a heterogeneous distribution in CA1 dendritic spines, and may introduce localized functional differences in Ca2+ signalling between synapses, as suggested by experiments on metabotropic receptor‐dependent long‐term depression.A physiologically detailed computational model of Ca2+ dynamics at a CA3–CA1 excitatory synapse characterizes the contribution of spine ER via metabotropic signalling during plasticity induction protocols.ER Ca2+ release via IP3 receptors modulates NMDA receptor‐dependent plasticity in a graded manner, to selectively promote synaptic depression with relatively diminished effect on LTP induction; this may temper further strengthening at the stronger synapses which are preferentially associated with ER‐containing spines.Acquisition of spine ER may thus represent a local, biophysically plausible ‘metaplastic switch’ at potentiated CA1 synapses, contributing to the plasticity–stability balance in neural circuits. Long‐term plasticity mediated by NMDA receptors supports input‐specific, Hebbian forms of learning at excitatory CA3–CA1 connections in the hippocampus. There exists an additional layer of stabilizing mechanisms that act globally as well as locally over multiple time scales to ensure that plasticity occurs in a constrained manner. Here, we investigated the role of calcium (Ca2+) stores associated with the endoplasmic reticulum (ER) in the local regulation of plasticity at individual CA1 synapses. Our study was spurred by (1) the curious observation that ER is sparsely distributed in dendritic spines, but over‐represented in larger spines that are likely to have undergone activity‐dependent strengthening, and (2) evidence suggesting that ER motility at synapses can be rapid, and accompany activity‐regulated spine remodelling. We constructed a physiologically realistic computational model of an ER‐bearing CA1 spine, and examined how IP3‐sensitive Ca2+ stores affect spine Ca2+ dynamics during activity patterns mimicking the induction of long‐term potentiation and long‐term depression (LTD). Our results suggest that the presence of ER modulates NMDA receptor‐dependent plasticity in a graded manner that selectively enhances LTD induction. We propose that ER may locally tune Ca2+‐based plasticity, providing a braking mechanism to mitigate runaway strengthening at potentiated synapses. Our study provides a biophysically accurate description of postsynaptic Ca2+ regulation, and suggests that ER in the spine may promote the re‐use of hippocampal synapses with saturated strengths.

Peroxisomes, lipid droplets, and endoplasmic reticulum "hitchhike" on motile early endosomes.

Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3- and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell.

ER homeostasis and motility of NSCLC cell lines can be therapeutically targeted with combined Hsp90 and HDAC inhibitors

Background and objectiveLung cancer remains the most common cause of cancer-related death in the world for which novel systemic treatments are urgently needed.Protein homeostasis that regulates protein levels and their fold is critical for cancer cell proliferation and survival. A complex network of cellular organelles and signaling cascades is involved in control of protein homeostasis including endoplasmic reticulum (ER). Thus, proteins in control of ER homeostasis are increasingly recognized as potential therapeutic targets.Molecular chaperone heat shock protein 90 (Hsp90) and histone deacetylase (HDAC) play an important role in ER homeostasis. Previous studies demonstrate that Hsp90 and HDAC inhibitors are individually functional against lung cancer. In this work we suggested that combined Hsp90 and HDAC inhibitors may elevate ER stress thereby enhancing the anti non small lung cancer (NSCLC) activity. Methods and resultsUsing an in vitro cell line model we demonstrated that 17-DMAG (HSP90 inhibitor) co-administration with PTACH (HDAC inhibitor) caused elevated ER stress (immunoblotting) (more than 110%↑, p < 0.05) accompanied by apoptotic cell death (Annexin V) (7–21%↑, p < 0.05). Moreover, 17-DMAG/PTACH treated cells lost the ability to migrate (scratch test) (57–85%↓ of scratch closure, p < 0.05). ConclusionsOur findings provide proof-of-concept that targeting ER homeostasis is therapeutically beneficial in lung cancer cell lines. Indeed, the elevated ER stress caused by 17-DMAG/PTACH combined treatment leads to increased cell death of NSCLC cell lines compared to the application of the drugs separately.

Mutations to the Formin Homology 2 Domain of INF2 Protein Have Unexpected Effects on Actin Polymerization and Severing

INF2 (inverted formin 2) is a formin protein with unusual biochemical characteristics. As with other formins, the formin homology 2 (FH2) domain of INF2 accelerates actin filament assembly and remains at the barbed end, modulating elongation. The unique feature of INF2 is its ability to sever filaments and enhance depolymerization, which requires the C-terminal region. Physiologically, INF2 acts in the secretory pathway and is mutated in two human diseases, focal and segmental glomerulosclerosis and Charcot-Marie-Tooth disease. In this study, we investigate the effects of mutating two FH2 residues found to be key in other formins: Ile-643 and Lys-792. Surprisingly, neither mutation abolishes barbed end binding, as judged by pyrene-actin and total internal reflection (TIRF) microscopy elongation assays. The I643A mutation causes tight capping of a subset of filaments, whereas K792A causes slow elongation of all filaments. The I643A mutation has a minor inhibitory effect on polymerization activity but causes almost complete abolition of severing and depolymerization activity. The K792A mutation has relatively small effects on polymerization, severing, and depolymerization. In cells, the K792A mutant causes actin accumulation around the endoplasmic reticulum to a similar extent as wild type, whereas the I643A mutant causes no measurable polymerization. The inability of I643A to induce actin polymerization in cells is explained by its inability to promote robust actin polymerization in the presence of capping protein. These results highlight an important point: it is dangerous to assume that mutation of conserved FH2 residues will have equivalent effects in all formins. The work also suggests that both mutations have effects on the mechanism of processive elongation.

Opaque1 Encodes a Myosin XI Motor Protein That Is Required for Endoplasmic Reticulum Motility and Protein Body Formation in Maize Endosperm

Myosins are encoded by multigene families and are involved in many basic biological processes. However, their functions in plants remain poorly understood. Here, we report the functional characterization of maize (Zea mays) opaque1 (o1), which encodes a myosin XI protein. o1 is a classic maize seed mutant with an opaque endosperm phenotype but a normal zein protein content. Compared with the wild type, o1 endosperm cells display dilated endoplasmic reticulum (ER) structures and an increased number of smaller, misshapen protein bodies. The O1 gene was isolated by map-based cloning and was shown to encode a member of the plant myosin XI family (myosin XI-I). In endosperm cells, the O1 protein is associated with rough ER and protein bodies. Overexpression of the O1 tail domain (the C-terminal 644 amino acids) significantly inhibited ER streaming in tobacco (Nicotiana benthamiana) cells. Yeast two-hybrid analysis suggested an association between O1 and the ER through a heat shock protein 70-interacting protein. In summary, this study indicated that O1 influences protein body biogenesis by affecting ER morphology and motility, ultimately affecting endosperm texture.

Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons

Extension of the endoplasmic reticulum (ER) into dendritic spines of Purkinje neurons (PNs) is required for cerebellar synaptic plasticity and is disrupted in animals with null mutations in Myo5a, the gene encoding myosin-Va1–3. Notably, the mechanism ensuring the ER's localization to spines has not been unraveled. While it has been proposed that animal class V myosins localize organelles by tethering them to the actin cytoskeleton4–7, we demonstrate here that myosin-Va acts as a point-to-point organelle transporter to pull ER as cargo into PN spines. Specifically, the myosin accumulates at the ER tip as the organelle moves into spines, and the myosin's ability to hydrolyze ATP is required for spine ER targeting. Moreover, myosin-Va is responsible for the vast majority of spine ER insertional events. Finally, attenuation of the myosin's ability to move along actin filaments reduces the maximum velocity of ER movement into spines, providing direct evidence that myosin-Va drives ER motility. Thus, we establish that an actin-based motor moves ER within animal cells, and we uncover the mechanism that mediates ER localization to PN spines, a prerequisite for synaptic plasticity.

Role of kinesin-1 and cytoplasmic dynein in endoplasmic reticulum movement in VERO cells

Generating the extended endoplasmic reticulum (ER) network depends on microtubules, which act as tracks for motor-driven ER tubule movement, generate the force to extend ER tubules by means of attachment to growing microtubule plus-ends and provide static attachment points. We have analysed ER dynamics in living VERO cells and find that most ER tubule extension is driven by microtubule motors. Surprisingly, we observe that approximately 50% of rapid ER tubule movements occur in the direction of the centre of the cell, driven by cytoplasmic dynein. Inhibition of this movement leads to an accumulation of lamellar ER in the cell periphery. By expressing dominant-negative kinesin-1 constructs, we show that kinesin-1 drives ER tubule extension towards the cell periphery and that this motility is dependent on the KLC1B kinesin light chain splice form but not on KLC1D. Inhibition of kinesin-1 promotes a shift from tubular to lamellar morphology and slows down the recovery of the ER network after microtubule depolymerisation and regrowth. These observations reconcile previous conflicting studies of kinesin-1 function in ER motility in vivo. Furthermore, our data reveal that cytoplasmic dynein plays a role in ER motility in a mammalian cultured cell, demonstrating that ER motility is more complex than previously thought.

Microtubule‐dependent motility and orientation of the cortical endoplasmic reticulum in elongating characean internodal cells

Motility of the endoplasmic reticulum (ER) is predominantly microtubule- dependent in animal cells but thought to be entirely actomyosin-dependent in plant cells. Using live cell imaging and transmission electron microscopy to examine ER motility and structural organization in giant internodal cells of characean algae, we discovered that at the onset of cell elongation, the cortical ER situated near the plasma membrane formed a tight meshwork of predominantly transverse ER tubules that frequently coaligned with microtubules. Microtubule depolymerization increased mesh size and decreased the dynamics of the cortical ER. In contrast, perturbing the cortical actin array with cytochalasins did not affect the transverse orientation but decreased mesh size and increased ER dynamics. Our data suggest that myosin-dependent ER motility is confined to the ER strands in the streaming endoplasm, while the more sedate cortical ER uses microtubule-based mechanisms for organization and motility during early stages of cell elongation. We show further that the ER has an inherent, NEM-sensitive dynamics which can be altered via interaction with the cytoskeleton and that tubule formation and fusion events are cytoskeleton-independent.

Cargo selection by specific kinesin light chain 1 isoforms.

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures.

Differential distribution of the endoplasmic reticulum network as visualized by the BipA–EGFP fusion protein in hyphal compartments across the septum of the filamentous fungus, Aspergillus oryzae

We visualized the endoplasmic reticulum (ER) network by expression of the BipA–EGFP fusion protein in the filamentous fungus, Aspergillus oryzae, and focused on the spatial difference of the ER distribution throughout hyphae. The ER formed an interconnected network with motility and displayed a gradient distribution from the apical region. The ER was also found as a tubular network along the septum, which was formed soon after the completion of septation. Discontinuity of the ER network distribution was noticed between the adjacent compartments across the septum, suggesting that the cellular activities in these compartments were independently regulated although they are considered to communicate with each other through the septal pore. Moreover, the ER-visualized strain was subjected to a hypotonic shock, leading to hyphal tip bursting where the Woronin body plugs septal pores and prevents excessive loss of the cytoplasm. In the compartment adjacent to the burst apical tip, the ER network structure and motility were still retained. We also observed re-growth of hyphae from the plugged septa forming intrahyphal hyphae in which the ER network distribution, specialized for apical growth, was regenerated.

Agonist-induced regulation of mitochondrial and endoplasmic reticulum motility

Using fluorescently tagged markers for organelles in conjunction with confocal microscopy, we have studied the effects of agonist-induced Ca2+ signals on the motility of mitochondria and the ER (endoplasmic reticulum). We observed that the muscarinic agonist carbachol produced a rapid, simultaneous and reversible cessation of the movements of both organelles, which was dependent on a rise in cytosolic Ca2+. This rise in Ca2+ was shown to cause a fall in cellular ATP levels, and the effect of carbachol on organelle movement could be mimicked by depleting ATP with metabolic inhibitors in the absence of any such rise in Ca2+. However, a Ca2+-sensing process independent of ATP appears also to be involved, because we identified conditions where the ATP depletion was blocked (by inhibitors of the Ca2+ pumps), but the organelle movements still ceased following a rise in cytosolic Ca2+. We conclude that the co-ordinated cessation of mitochondria and ER motility is a process regulated by the cytosolic concentration of both Ca2+ and ATP, and that these two parameters are likely to synergize to regulate the localization of the two organelles, and to facilitate the transfer of Ca2+ between them.

Dynein supports motility of endoplasmic reticulum in the fungus Ustilago maydis.

The endoplasmic reticulum (ER) of most vertebrate cells is spread out by kinesin-dependent transport along microtubules, whereas studies in Saccharomyces cerevisiae indicated that motility of fungal ER is an actin-based process. However, microtubules are of minor importance for organelle transport in yeast, but they are crucial for intracellular transport within numerous other fungi. Herein, we set out to elucidate the role of the tubulin cytoskeleton in ER organization and dynamics in the fungal pathogen Ustilago maydis. An ER-resident green fluorescent protein (GFP)-fusion protein localized to a peripheral network and the nuclear envelope. Tubules and patches within the network exhibited rapid dynein-driven motion along microtubules, whereas conventional kinesin did not participate in ER motility. Cortical ER organization was independent of microtubules or F-actin, but reformation of the network after experimental disruption was mediated by microtubules and dynein. In addition, a polar gradient of motile ER-GFP stained dots was detected that accumulated around the apical Golgi apparatus. Both the gradient and the Golgi apparatus were sensitive to brefeldin A or benomyl treatment, suggesting that the gradient represents microtubule-dependent vesicle trafficking between ER and Golgi. Our results demonstrate a role of cytoplasmic dynein and microtubules in motility, but not peripheral localization of the ER in U. maydis.

Microtubule-based endoplasmic reticulum motility in Xenopus laevis: activation of membrane-associated kinesin during development.

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.

Endoplasmic reticulum membrane tubules are distributed by microtubules in living cells using three distinct mechanisms.

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. 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. 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.

Immunolocalization of a putative unconventional myosin on the surface of motile mitochondria in locust photoreceptors.

Light stimulation of locust (Schistocerca gregaria) photoreceptors results in an actin-dependent translocation of mitochondria towards the photoreceptive microvilli and an antagonistic movement of endoplasmic reticulum towards the cell body. Using immunocytochemical techniques, we have tried to identify myosin-like motors that may drive the light-induced organelle motility. A monoclonal antibody against the motor domain of Acanthamoeba myosin identifies a prominent 110-kDa protein on Western blots of locust retina. Cross-reactivity with two polyclonal anti-myosin antibodies and a monoclonal anti-myosin-I-antibody, together with ATP-dependent binding to actin filaments, provides evidence that the 110-kDa protein is an unconventional myosin. By indirect immunofluorescence, the 110-kDa protein has been localized to both photoreceptors and pigment cells within the retina. In the photoreceptor cells, the 110-kDa protein is bound to the surface of mitochondria. This putative unconventional myosin may thus be a motor protein involved in the light-induced translocation of mitochondria in photoreceptors.

Cytoskeleton and PCH-induced pigment aggregation in Macrobrachium potiuna erythrophores.

Herein we report the effects of microtubule- and actin-like filament disrupting drugs, as well as the microtubule stabilizer taxol, on PCH-induced pigment granule aggregation within erythrophores of the freshwater crustacean Macrobrachium potiuna. Dose-response curves (DRCs) to the pigment-concentrating hormone PCH were determined under control and experimental conditions to evaluate the effects elicited by the cytoskeleton-affecting drugs. Colchicine, at temperatures 22 degrees C and 4 degrees C, and vinblastine significantly inhibited the aggregating response to PCH and affected the dynamics of the process, as shown by the change in the slope of the regression curve calculated from the DRCs. Lumicolchicine, a colchicine analogue with no affinity for tubulin, also inhibited pigment migration, though no change in the slope of the regression curve was observed. The inhibitory effects of lumicolchicine demonstrate that changes in sites other than cytoskeleton, such as membrane permeability, may also cause a decrease in the PCH-induced aggregating responses and that the colchicine effects may result from its action on cellular sites additional to the cytoskeleton. Taxol, a microtubule stabilizer, did not affect the DRC to PCH, and DMSO improved the PCH-evoked responses, pointing out to the maintenance of tubulin in the polymerized state as the appropriate condition for aggregation. Cytochalasin B, an actin-like filament disrupter, diminished the aggregating responses to the hormone, with no change in the slope of the regression curve, indicating that these elements take part in the process and that cytosolic calcium rise, sol/gel transformations and endoplasmic reticulum motility may well play an important role in granule migration. It is suggested that microtubules are steadily polymerized as a requirement for pigment aggregation and that process is biphasic, the initial phase being dependent on the microtubule integrity.

Dynamic behavior of endoplasmic reticulum in living cells

Endoplasmic reticulum (ER) was studied by fluorescence microscopy of living CV-1 cells treated with the fluorescent carbocyanine dye DiOC 6(3). Using video recording and image processing techniques, several distinct forms of highly localized movements of ER were documented, categorized, and analyzed in terms of mechanism and structural implications. These include tubule branching, ring closure, and sliding. These localized movements have been observed to generate the basic elements of ER: linear tubules, polygonal reticulum, and triple junctions. We propose that as such they act as the mechanism for constructing the polygonal lattice of interconnected membrane tubules that constitutes ER. The nature of these movements suggests possible involvement of the cytoskeleton, and, in view of the close correlations in the distributions of ER and microtubules, and the accompanying paper (Dabora and Sheetz), it is possible that microtubules may play a role in generating ER motility and in constructing and maintaining the ER network in living cells.