Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons - PubMed (original) (raw)

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

Wolfgang Wagner et al. Nat Cell Biol. 2011 Jan.

Abstract

Extension of the endoplasmic reticulum (ER) into dendritic spines of Purkinje neurons is required for cerebellar synaptic plasticity and is disrupted in animals with null mutations in Myo5a, the gene encoding myosin-Va. We show here that myosin-Va acts as a point-to-point organelle transporter to pull ER as cargo into Purkinje neuron spines. Specifically, myosin-Va accumulates at the ER tip as the organelle moves into spines, and hydrolysis of ATP by myosin-Va is required for spine ER targeting. Moreover, myosin-Va is responsible for almost all of the spine ER insertion events. Finally, attenuation of the ability of myosin-Va 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 have established that an actin-based motor moves ER within animal cells, and have uncovered the mechanism for ER localization to Purkinje neuron spines, a prerequisite for synaptic plasticity.

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Figures

Figure 1

Figure 1. Absence of the delayed mGluR1-dependent Ca2+ transient in d l20J/d l20J PN spines and hypothetical models for how myosin-Va might function to localize the ER Ca2+ store to spines

(a) Images of a PN (upper panel; size bar, 30 μm) and a PN dendrite and spine (lower panel; size bar, 1 μm). PNs in cerebellar slices were voltage clamped at −70 mV, loaded with Ca2+ indicator Fluo-4 (150 μM) and red fluorophore Alexa-594 (20 μM) through the recording electrode, and imaged by two-photon laser scanning microscopy. In the lower panel, the white circle indicates the location of two-photon laser uncaging of MNI-glutamate (3.75 mM), and the yellow line indicates the line scan region. (b) Ca2+ transients in d v/d l20J PN spines (red traces) and adjacent dendritic shafts (blue traces) evoked by glutamate uncaging at the spine head at the time indicated (arrowhead), in the presence of DNQX (10 μM; left and middle panels), or in the additional presence of mGluR1 antagonist CPCCOEt (100 μM; right panel). Left panel: traces from a single trial from the spine depicted in a. Middle and right panels: group data for 23 spines (4 PNs) and 20 spines (3 PNs), respectively. The solid traces indicate the mean and the shaded areas indicate SEM. (c) CPCCOEt-sensitive Ca2+ transients are not observed in d l20J/d l20J PN spines following glutamate uncaging in the presence of DNQX (compare with b, middle panel). The mean Ca2+ traces (± SEM) from group data for 45 spines (4 PNs) are shown. (d) Shown are the average Ca2+ transient peak magnitudes (ΔF/F mean ± SEM) in spines (red bars) and dendrites (blue bars) of d v/d l20J and d l20J/d l20J PNs. (e) Mechanism 1 (non-cell autonomous model): Myosin-Va present within another cerebellar cell type such as a granule neuron facilitates the release of a diffusible factor that confers upon the PN the ability to target ER to its spines. Relevant to this model, myosin-Va is a synaptic vesicle-associated protein that plays a role in regulating exocytosis, , , and is widely expressed throughout the brain, including within granule neurons. (f) Mechanism 2 (tethering model): Myosin-Va delivers a tethering factor to the spine tip that links the ER to the spine tip following the myosin-Va-independent transport of ER into the spine. In this case, ER motility might be microtubule-based, as microtubules transiently enter the spines of hippocampal neurons, and microtubule plus end-directed motors are known to transport ER, . Alternatively, a direct interaction between the microtubule plus end tracking proteins EB1/3 and the integral ER membrane protein STIM1 might drive the movement of ER tubules into spines. With regard to myosin-Va-dependent tethering, candidates for tethering proteins that might link the ER to the spine tip, such as Homer, are present in PN spines. Alternatively, the myosin itself could be an integral component of the tethering mechanism–. (g) Mechanism 3 (transport model): Myosin-Va associates with the ER and transports it along actin filaments into spines. In the simplest case, the ER is then maintained at the spine tip via the continued effort of myosin-Va to carry it to the barbed end of actin filaments. We note, however, that the myosin-Va-mediated transport of ER into spines envisioned by Mechanism 3 could be followed by the maintenance of the ER within spines via a secondary tethering mechanism that is by definition myosin-Va-dependent either because it occurs only after the myosin has delivered the ER to the vicinity of the spine tip, or because once there, the myosin is an essential component of the tethering mechanism.

Figure 2

Figure 2. The translocation of ER into spines is disrupted in d l20J/d l20J PNs

Shown are cultured, live PNs from WT mice at 15 DIV (a, b) and at 10 DIV (c, d), and from d l20J/d l20J mice at 15 DIV (e, f) and at 10 DIV (g, h). Cultures were transfected with pL7-mRFP-ER-IRES-EGFP to visualize cell volume (Volume) and ER (ER). Superimpositions are also shown (Overlay). The upper panels in a, c, e, and g are images reconstructed from confocal Z-stacks (size bar, 20 μm), whereas the lower panels show magnified images of a single confocal plane of PN dendrites (size bar, 2 μm). The latter images were taken from a time series (Supplementary Movies 1, 3). b, d, f, and h show montages of 30 consecutive frames of a time series and depict the spines in the white boxes in a, c, e, and g, respectively, at higher magnification (size bar, 2 μm; see also Supplementary Movies 2, 4).

Figure 3

Figure 3. mGFP-myosin-Va expressed in d l20J/d l20J PNs rescues ER targeting and accumulates at the tip of the spine ER

(a–c) Shown are live d l20J/d l20J PNs transfected with pL7-mRFP-ER and pL7-mGFP-myosin-Va to visualize the ER (ER) and the myosin (MVa-WT), respectively. Size bars, 2 μm. a shows a maximum projection of a confocal Z-stack of a section of a PN dendrite at 15 DIV (see Supplementary Movie 6 for the corresponding three-dimensional reconstruction). b shows images corresponding to single confocal planes of PN dendrites at 10 DIV or 15 DIV. Three examples are shown for each time point. The images were taken from time series (Supplementary Movies 7, 8). The white boxes indicate the spines from which the kymograph images shown in c were obtained. The kymographs in c show that the dot of mGFP-myosin-Va fluorescence remains present at the distal tip of the ER tubule as the tubule extends or shortens over time. The _x_-axis corresponds to time, the _y_-axis corresponds to size. (d) Shown are live d l20J/d l20J PNs transfected with pL7-mRFP-ER to visualize the ER (ER) and either pL7-mGFP-myosin-Va (MVa-WT), pL7-mGFP-myosin-Va-R219A (MVa-R219A), pL7-mGFP-myosin-Va-G440A (MVa-G440A) or pL7-mGFP-myosin-Va-E442A (MVa-E442A). Images were reconstructed from confocal Z-stacks and depict cells at 13 DIV. Superimposition of images is also shown (Overlay). Size bar, 20 μm. Each insert in the overlay images shows a single confocal plane image of a dendrite from a PN transfected with the respective plasmids and depicts ER (red) and myosin (green). Images were taken from a time series (Supplementary Movie 9). Size bar, 2 μm.

Figure 4

Figure 4. Myosin-Va is present at the leading tip of the ER tubule as the organelle translocates into a spine

Shown is a live d l20J/d l20J PN transfected with pL7-mCerulean, pL7-mGFP-myosin-Va and pL7-mRFP-ER to visualize cell volume (Volume), myosin-Va (MVa) and ER (ER), respectively. Superimpositions of volume and myosin-Va (Volume MVa) and myosin-Va and ER (MVa ER) are also shown. Images show a single confocal plane and were taken from a time series recorded at a rate of 0.5 frames per second (fps) at DIV 8 (see also Supplementary Movie 12). Size bar, 1 μm.

Figure 5

Figure 5. Decreasing the step size or ATPase activity of myosin-Va reduces the efficiency of ER targeting to PN spines and the maximum velocity of ER movement into spines

WT, d v/d l20J, and d l20J/d l20J PNs expressing mRFP-ER to visualize ER and free GFP to visualize cell volume, as well as d l20J/d l20J PNs expressing GFP-tagged versions of WT myosin-Va (MVa-WT), myosin-Va4IQ (MVa-4IQ), myosin-Va2IQ (MVa-2IQ) or myosin-VaS217A (MVa-S217A) in addition to mRFP-ER and free GFP were observed using confocal live microscopy. (a) The graph shows the fraction of spines (mean ± SEM) that contain ER, as determined from images of PN dendrites recorded at DIV 15. The numbers of analyzed PNs were 15 (WT), 18 (d v/d l20J), 16 (d l20J/d l20J), 21 (MVa-WT), 6 (MVa-4IQ), 22 (MVa-2IQ), and 17 (MVa-S217A). On average, 23.5 spines/PN were analyzed. *p< 0.0001; p-values calculated using the Student t-test. See Supplementary Movie 10 for examples. (b) Instantaneous velocities of ER movements were measured at DIV 10, as described in Methods and Supplementary Information, Fig. S9. The graph shows the maximum velocity calculated by averaging the fastest 10% of instantaneous movements directed towards the spine tip (error bars indicate SEM). The total numbers of instantaneous velocities determined were 91 (WT), 207 (d l20J/d l20J), 264 (MVa-4IQ), 350 (MVa-2IQ), and 133 (MVa-S217A). *p< 0.05; **p<0.0005, p-values calculated using the Student t-test. Given the motility and processivity defects observed for myosin-Va2IQ and myosin-VaS217A _in vitro_–, it is notable how well these mutants function to move ER in PNs. This could be indicative of ER transport being mediated by an ensemble of myosin-Va molecules.

Comment in

References

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