Abstract
A key event in neurite initiation is the accumulation of microtubule bundles at the neuron periphery. We hypothesized that such bundled microtubules may generate a force at the plasma membrane that facilitates neurite initiation. To test this idea we observed the behavior of microtubule bundles that were induced by the microtubule-associated protein MAP2c. Endogenous MAP2c contributes to neurite initiation in primary neurons, and exogeneous MAP2c is sufficient to induce neurites in Neuro-2a cells. We performed nocodazol washout experiments in primary neurons, Neuro-2a cells and COS-7 cells to investigate the underlying mechanism. During nocodazol washout, small microtubule bundles formed rapidly in the cytoplasm and immediately began to move toward the cell periphery in a unidirectional manner. In neurons and Neuro-2a cells, neurite-like processes extended within minutes and concurrently accumulated bundles of repolymerized microtubules. Speckle microscopy in COS-7 cells indicated that bundle movement was due to transport, not treadmilling. At the periphery bundles remained under a unidirectional force and induced local cell protrusions that were further enhanced by suppression of Rho kinase activity. Surprisingly, this bundle motility was independent of classical actin- or microtubule-based tracks. It was, however, reversed by function-blocking antibodies against dynein. Suppression of dynein expression in primary neurons by RNA interference severely inhibited the generation of new neurites, but not the elongation of existing neurites formed prior to dynein knockdown. Together, these cell biological data suggest that neuronal microtubule-associated proteins induce microtubule bundles that are pushed outward by dynein and locally override inward contraction to initiate neurite-like cell protrusions. A similar force-generating mechanism might participate in spontaneous initiation of neurites in developing neurons.
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Fig. S1.
Fluorescent speckle microscopy: Treadmilling cannot account for microtubule bundle movements. GFP-MAP2c transfected, nocodazole treated COS-7 cells were injected with low concentrations of rhodamine-X-labeled tubulin before drug washout to allow formation of speckle-like fiduciary marks within microtubule bundles. (A) and (B) After drug washout, imaging revealed slow directional flow of speckles within peripheral microtubule bundles towards the cell center (indicated by yellow arrows. See Movie S6). Kymograph analysis was performed in the area marked with the red bar to analyze speckle movements within a stationary microtubule bundle. (C) Likewise, kymograph analysis was performed to measure translocation of moving bundles within a different cell. (D). Identically scaled kymographs derived from panels B) and C) for measuring speckle and bundle movements; green lines are overlaid on individual events to indicate relative velocity. Speckle movements were approximately 50-fold slower than bundle movements (error bars represent standard error of the mean).
Fig. S2.
Rho kinase-dependent contractility inhibits microtubule bundle induced cell protrusion. Similar to Fig 3A, GFP-MAP2c transfected COS-7 cells were treated with nocodazole and imaged after drug washout. 45min following washout, the ROCK inhibitor Y27632 (60 μM) was added. DIC (A) and epi-fluorescence (B) sequences are shown before (−10:00 to 0:00) and after (0:00 to 20:00) addition of Y27632. In GFP-MAP2c transfected cells, more protrusions were formed after Y27632 application (arrowheads in A 20:00 and arrows in B 10:00 and 20:00) compared to the time period before drug addition. Time points represent minutes and seconds.
Fig. S3.
Anti-dynein antibodies m74-2 and m74-1 inhibit dynein-dependent retrograde transport of transferrin in COS-7 cells. COS-7 cells were injected with control antibodies (mouse IgG; n = 26), anti-dynein antibody m74-2 (n = 11) or m74-1 (n = 9, data not shown) supplemented with Alexa-488-Dextran to identify injected cells. After serum starving for 1 h, Texas Red labeled transferrin was applied for 10min, followed by rinsing in PBS (2min) and fixation. Nuclei were stained using DAPI and cells were scored for retrograde transport of internalized transferrin. Whereas perinuclear accumulation of transferrin was detectable in 81% of control cells injected with isotype control antibody, this dynein-dependent process was completely blocked in cells injected with either antibody m74-1 or m74-2.
Fig. S4.
Specificity of dynein function blocking antibodies. (A) COS-7 or (B) Neuro-2a cell extracts were probed with antibodies m74-2 and m74-1 targeted against the dynein intermediate chain in TBS+500 mM NaCl. Both m74-2 and m74-1 recognize a single predominant band in the tested cell types.
Fig. S5.
Estimation of the number of microtubules in MAP2c induced microtubule bundles. (A) GFP-MAP2c transfected COS7 cells or control cells were treated with nocodazole overnight and injected with comparable levels of rhodamine-labeled tubulin. Shortly after washout, microtubules and microtubule bundles were imaged using time-lapse microscopy (see Movie S5). The first two panels show equally scaled frames optimized to depict the smallest detectable microtubule structures. The third panel is identical to panel 2, except that it is displayed at a wider intensity scale to demonstrate that also the thickest bundles were not overexposed in the original acquisition and thus suited for quantitative analysis. Individual frames of these recordings were analyzed by measuring integrated, background corrected intensity profiles along line segments placed across microtubule structures (see small inset in middle panel). Although we do not take into account potential non-linear effects, such as fluorescence quenching, such an analysis can provide a reasonable estimate of microtubule number within bundles. (B) Frequency histograms were used to visualize the distribution of intensity measurements. Peripheral microtubules from control cells all measured within a very similar range of intensities (∼20 arbitrary units). As judged by their dynamic behavior, the peripheral array of microtubule structures in control cells was mostly composed of individual microtubules (in control cells, we avoided measuring regions in which microtubules overlapped with each other). Therefore, we conclude that this intensity range is typical for single microtubules. In GFP-MAP2c transfected cells, we detected a significant number of single microtubules, although these represent only 2% of the total population, as the vast majority of microtubules are contained in bundles, which range from small-sized bundles composed of ∼2–5 microtubules to thicker bundles of up to ∼50 microtubules.
Fig. S6.
Microtubule bundles accumulate at the cell periphery in the absence of microtubule tracks. After nocodazole washout, GFP-MAP2c transfected cells were stained for total tubulin. Untransfected cells displayed radial microtubule networks, which were similar to those seen in control cells not treated with nocodazole. In contrast, transfected cells (*) concentrated nearly all microtubules in bundles at the cell periphery. In transfected cells, no underlying microtubule network was observed that might have facilitated unidirectional microtubule bundle movements.
Fig. S7.
Microtubule-dependent induction of local cell protrusions. (A) Primary hippocampal neurons were co-transfected with vectors coding for GFP-tubulin and mRFP-actin to visualize microtubule and actin reorganization. Five days after plating, neurons were treated with 10 μM nocodazole overnight and imaged during drug washout. During a timeperiod of 2–10 minutes after washout, bundled microtubules accumulate at the cell periphery and induce a local cell protrusion (cyan arrows). Concurrently, a nascent actin rich growth cone forms at the tip of the newly formed neurite (yellow arrows). (B) Primary hippocampal neurons were transfected with GFP-MAP2c treated with 10μM nocodazole one day after plating. The left panel shows the complete neuron shortly after drug washout. The red box indicates a region, which is shown magnified in the middle panel. During the early phases of drug washout, rapid movements of microtubules were observed (orange arrows, see also Movie S11). After washout, cells were stained for the neuronal marker TUJ1 (right panels). (C) After peripheral accumulation, bundles frequently protruded the cell membrane (green arrows, see also Movie S12), suggesting that MAP2c-induced microtubule bundles produce an anterograde force in primary neurons.
Fig. S8.
Validation of dynein heavy chain depletion. Primary hippocampal neurons were transfected with vectors coding for GFP together with control or dynein heavy chain targeted shRNA and stained for dynein heavy chain 4 or 11 days after plating. (A) Representative examples of cells transfected with control or dynein targeted shRNA (11 DIV). (B) Transfection efficiency was below 30%. Thus we measured dynein depletion on a per cell basis by quantification of maximal perinuclear dynein heavy chain immunofluorescence. Dynein levels were strongly reduced in the time interval between 4-11 days post-plating (n ≥ 20; *** = p < 0.0001; Student's T-test, two-tailed; error bars represent standard error of the mean).
Movie S1: Dynamics of Microtubule Bundle Formation in Neuro-2a cells.
GFP-MAP2c transfected Neuro-2a cells were treated with nocodazole overnight and GFP-MAP2c fluorescence was imaged during drug washout. Shortly after nocodazole washout, microtubules repolymerize and quickly form radially oriented microtubule bundles. Time signature represents hours:minutes.
Movie S2: Microtubule Bundle Translocation in Neuro-2a cells.
GFP-MAP2c transfected Neuro-2a cells were treated and imaged as in Movie S1. As shown in this example, we observed microtubule bundle translocation within the cells cytoplasm in some cases. Time signature represents hours:minutes.
Movie S3 and Movie S4: Microtubule Bundle Translocation in COS-7 cells. GFP-MAP2c transfected COS-7 cells were treated with nocodazole overnight and GFP-MAP2c fluorescence was imaged during drug washout. Two examples are shown to exemplify variability in bundle size. However, bundle translocation behavior was similar across all observed cells. Time signature represents hours:minutes.
Movie S4
Movie S5: Microtubule Bundle Translocation in COS-7 cells.
GFP-MAP2c transfected or control COS-7 cells were treated with nocodazole overnight and injected with labeled tubulin. Microtubules were imaged shortly after drug washout in both control and GFP-MAP2c transfected cells. Both panels were scaled identical for comparison. Single microtubules in control cells displayed typical dynamic behavior; single and bundled microtubules in GFP-MAP2c transfected cells were transported rapidly in the absence of microtubule tracks. Time signature represents minutes:seconds.
Movie S6: Speckle Analysis of Microtubule Treadmilling in Peripheral Microtubule Bundles.
GFP-MAP2c transfected COS-7 cells were treated with nocodazole overnight, injected with low amounts of Rhodamine-X-labeled tubulin. One hour after injection, nocodazole was washed out; microtubules were allowed to re-grow and accumulate at the cell periphery. Fluorescent speckles of Rhodamine-X-tubulin were imaged using confocal microscopy. Speckles revealed slow directional treadmilling towards the cell center. Time signature represents minutes:seconds.
Movie S7 and Movie S8: Simultaneous Imaging of Speckle and Bundle Translocation in Nascent Microtubule Bundles.
GFP-MAP2c transfected COS-7 cells were treated with nocodazole and injected with Rhodamine-X-labeled tubulin. GFP-MAP2c (shown in green) and Rhodamine-X-tubulin (shown in red) were imaged in short succession (with a delay between the individual channels of less than 1 second). Two examples are shown. Movie S8 shows a smaller thicker bundle, whereas Movie S7 shows a longer, single microtubule or thin bundle. Both microtubule bundles and speckles moved simultaneously at similar speeds. Time signature represents minutes:seconds.
Movie S9: Microtubule Bundles Induce Membrane Protrusions upon Cytochalasin D Treatment. GFP-MAP2c transfected COS-7 cells or untransfected control cells were treated with nocodazole overnight. After drug washout, microtubules were allowed to re-grow and accumulate at the cell periphery. 4μM cytochalasin D was added while cells were imaged using Nomarski DIC optics. In GFP-MAP2c transfected cells, the plasma membrane was deflected at cell margins shortly after cytochalasin D addition. Time signature represents hours:minutes.
Movie S10: Microtubule Bundle Translocation in Latrunculin A treated COS-7 cells.
GFP-MAP2c transfected COS-7 cells were treated with nocodazole overnight. GFP-MAP2c fluorescence was imaged while cells were first treated with 10μM latrunculin A for 20min to disrupt actin filaments, followed by nocodazole washout in the continued presence of 10μM latrunculin A. Robust bundle movements were observed under these conditions. Time signature represents hours:minutes.
Movie S11: Microtubule Bundle Translocation in Primary Hippocampal Neurons.
GFP-MAP2c transfected primary hippocampal neurons were treated with nocodazole overnight and GFP-MAP2c fluorescence was imaged during drug washout. Shortly after nocodazole washout, microtubules repolymerize and start to move unidirectionally. Time signature represents minutes:seconds.
Movie S12: Microtubule Bundles Induce Local Neurite-Like Protrusions in Primary Hippocampal Neurons. Primary hippocampal neurons were treated with nocodazole overnight and GFP-MAP2c fluorescence was imaged during drug washout. Microtubule bundles accumulate at the neurons periphery and frequently induce local neurite-like protrusions. Time signature represents minutes:seconds.
Movie S13: Model for Peripheral Microtubule Bundle Accumulation by Unidirectional Transport. Bundles were drawn in Photoshop with random orientation. (+) denotes microtubule plus-ends. Unidirectional transport is sufficient to accumulate microtubule bundles in the cell periphery in a polarized array with microtubule plus-ends facing outward.
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Dehmelt, L., Nalbant, P., Steffen, W. et al. A microtubule-based, dynein-dependent force induces local cell protrusions: Implications for neurite initiation. Brain Cell Bio 35, 39–56 (2006). https://doi.org/10.1007/s11068-006-9001-0
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DOI: https://doi.org/10.1007/s11068-006-9001-0