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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Electronic supplementary material
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 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 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
- Cell Periphery
- Tubule Bundle
- Cytoplasmic Dynein
- Primary Hippocampal Neuron