The Journal of Neuroscience, October 22, 2003, ():
The Role of Microtubule-Associated Protein 2c in the Reorganization of Microtubules and Lamellipodia during Neurite Initiation
J. Neurosci.
Dehmelt et al. 23 (29): 9479.
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Files in this Data Supplement:
- Suppl. Fig S1
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Additional observations on drug-induced neurite like processes.
Cells were stained for a-tubulin (green) and F-actin (red) as described in Methods.
A: Drug-induced formation of neurite-like processes is not limited to neuroblastoma cells. HEK-293 cells were treated with 1mM taxol and/or 40nM jasplakinolide for 4h and imaged using an epifluorescence microscope. Percentages in the panels represent the fraction of cells exhibiting neurite like processes (n>100).
B: Drug-induced initiation of neurite-like processes in Neuro2a cells is not limited to specific conditions of taxol application. A wide range of taxol concentrations (0.1-20mM) effectively induced process formation in combination with 40nM jasplakinolide. Drugs were applied either for a total of 4h, or taxol (20mM) was preincubated overnight (ON), followed by 4h incubation with 20mM taxol + 40nM jasplakinolide, similar to conditions used previously (Edson et al. 1993 Development 117:689). Percentages in the panels represent the fraction of cells exhibiting neurite like processes (n>100)
- Fig1.mov
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Spontaneous neurite formation by primary hippocampal neurons.
Neurons were observed at 1 frame per 9 seconds using DIC optics (elapsed time indicated as hours:min). As shown in Figure 1C, this neuron initially formed a fairly uniform lamellum. At the beginning of this movie (0:00), this lamellum is already segregated into several smaller lamellipodia, of which one further segmented to form two separate lamellipodia (0:40). Subsequently, these lamellipodia slowly separate from the cell soma to become growth cones at the tips of newly formed neurites (1:10 to 6:01; frame rate is 15Hz).
- Fig3.mov
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MAP2c-induced neurite formation.
Single confocal planes of Neuro2a cells expressing GFP-MAP2c were recorded at 1 frame every 20 seconds (elapsed time indicated in hours:min). At the beginning of the series, the cell was characterized by a large, weakly polarized lamellipodium (see Figure 3). Small bundles of MAP2c-decorated microtubules invaded this large lamellipodium during the first minutes of the timelapse without inducing neurites (0:00 to 0:28). The large lamellipodium quickly became segmented into smaller lamellipodia and a stable neurite formed by invasion of multiple microtubules into one condensed lamellipodium (between 0:30 and 0:55; frame rate is 15Hz).
- Fig4.mov
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Alignment of MAP2c-decorated microtubules and actin bundles in lamellipodia.
Single confocal planes of Neuro2a cells expressing GFP-g-actin and dsRED2-MAP2c were recorded at 1 frame every 10 seconds (elapsed time indicated in h:min). MAP2c-decorated microtubules often invade peripheral protrusions (arrow) or lamellipodia (arrowhead). In lamellipodia, these microtubules frequently align with actin bundles (arrowhead). In this example, the association between the networks is additionally emphasized by the simultaneous breakage of a microtubule and an actin bundle (arrowhead). The lower protrusion later goes on to form a neurite, as shown in Figure 4B (frame rate is 15Hz).
- Fig5.mov
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Dynamic properties of MAP2c-induced neurites.
MAP2c-induced neurite formation in Neuro2a cells expressing GFP-MAP2c was recorded using a confocal microscope via simultaneous DIC and single plane fluorescence imaging. Images were collected every 30 seconds (elapsed time indicated as hours:min). Growth cones are rapidly formed at the tips of nascent MAP2c-induced neurites. In this example the newly formed neurite retracted upon contact with a non-permissive substrate (a scratch on the coverslip; frame rate is 5Hz).
- Fig6.mov
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Process formation in Neuro2a cells induced by combined incubation with taxol and jasplakinolide.
Images were collected before and during application of drugs at 1 frame every 5 seconds using DIC optics (elapsed time indicated in hours:min). Before drug application (0:04), cells characteristically exhibited broad lamellipodia undergoing rapid retrograde flow. These lamellipodia frequently collapsed after drug application; however, rapid retrograde flow was still observed in remaining lamellipodia for several minutes (in this example until 0:28). Shortly after retrograde flow became undetectable, short, dynamic processes began to form, which further elongated (0:29 to 1:23) after retrograde flow ceased (See Fig.6C for a kymograph; frame rate is 15Hz).
