Zakharenko S and Popov S (1998), Dynamics of axonal microtubules regulate the to...

XB-ART-13965

J Cell Biol 1998 Nov 16;1434:1077-86. doi: 10.1083/jcb.143.4.1077.

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Dynamics of axonal microtubules regulate the topology of new membrane insertion into the growing neurites.

Zakharenko S , Popov S .

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Nerve growth depends on the delivery of cell body-synthesized material to the growing neuronal processes. The cellular mechanisms that determine the topology of new membrane addition to the axon are not known. Here we describe a technique to visualize the transport and sites of exocytosis of cell body- derived vesicles in growing axons. We found that in Xenopus embryo neurons in culture, cell body-derived vesicles were rapidly transported all the way down to the growth cone region, where they fused with the plasma membrane. Suppression of microtubule (MT) dynamic instability did not interfere with the delivery of new membrane material to the growth cone region; however, the insertion of vesicles into the plasma membrane was dramatically inhibited. Local disassembly of MTs by focal application of nocodazole to the middle axonal segment resulted in the addition of new membrane at the site of drug application. Our results suggest that the local destabilization of axonal MTs is necessary and sufficient for the delivery of membrane material to specific neuronal sites.

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Species referenced: Xenopus
Genes referenced: dnai1

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	Figure 2. Progressive staining of the distal axon after labeling of cell body–derived vesicles. DIC and fluorescence images of the distal axon at different times (marked in minutes) after local labeling of cell body– derived vesicles with DiIC12 molecules. The length of the axon was ∼1,900 μm. DiIC12-labeled vesicles gradually accumulated at the central domain of the growth cone. The delay between the staining of the cell body and accumulation of the vesicles at the distal axon is likely to reflect the time required for the fast axonal transport of the vesicles. Notice a gradual increase in the diffuse staining of the vesicle-free peripheral growth cone.
	Figure 3. Individual DiIC12-labeled vesicles at the growth cone region. Two fluorescence images (time in seconds) of the growth cone region taken ∼20 min after the labeling of cell body– derived vesicles with DiIC12 molecules. Individual DiIC12-labeled vesicles are detected as bright puncta. Vesicles (arrowheads) are able to move within the growth cone. Although occasionally DiIC12-labeled vesicles could be found in the filopodia (arrows), most of the vesicles were excluded from the filopodia. Note uniform diffuse staining of the vesicle-free filopodia and flat lamellipodium-like protrusions. Since individual vesicles are excluded from these structures, the diffuse staining is likely to reflect the incorporation of DiIC12 molecules into the plasmalemma.
	Figure 4. Preferential insertion of cell body–derived vesicles into the growth cone region. (A and B) DIC (top) and fluorescence (bottom) images of the middle (A) and distal (B) segments of the same axon 30 min after staining of the cell body. The length of the axon was ∼1,600 μm, and the middle segment was ∼900 μm away from the soma. Staining of the plasma membrane with DiIC12 molecules, reflecting the insertion of DiIC12- labeled vesicles into the plasmalemma, could be observed at the growth cone (B). No plasma membrane staining was detected at the middle axon, as well as at sufficiently large distances from the growth cone in the distal segment (A and B). (C–E) Differential interference contrast and fluorescent micrographs of the distal axons 60 min after the staining of the cell body. Before the staining of the soma, neuronal cultures were pretreated for 1 h with brefeldin A (10 μg/ml, C), nocodazole (5 μg/ ml, D), or cytochalasin D (5 μM, E). The drugs were present in the culture medium throughout the experiment. Very few (C) or no (D) fluorescent vesicles and no plasma membrane staining (C and D) could be detected. Cytochalasin treatment (E) had no obvious effect on the delivery of the vesicles to the growth cone and their incorporation into the plasma membrane. Bars, 30 μm.
	Figure 5. Dynamic instability of MTs is required for the insertion of new membrane into the distal axon. (A–D) DIC (top) and fluorescence images of the distal axon at two different times (marked in minutes) after the staining of the soma. 7 nM taxol (A) or 3 nM vinblastine (B and C) was added to the culture medium 30 min before the staining of the soma. Fluorescent vesicles accumulated at the growth cone region. Filopodia staining in A and B was drastically reduced in comparison with control (D) neurons (P < 0.001, t test). In C, 30 min after the staining of the cell body, the concentration of vinblastine was increased to 1 μM. This induced a rapid insertion of the vesicles accumulated at the distal axon into the plasma membrane and staining of the filopodia. (E) Quantitative analysis of the plasma membrane staining. For each neuron the intensity of the plasma membrane staining at the growth cone region was determined as an average for at least 20 filopodia. The data are presented as a mean ± SEM for five to seven different neurons. *P < 0.001, t test. Bar, 30 μm.
	Figure 6. Local disruption of axonal MTs is sufficient for the insertion of cell body–derived vesicles along the axon. (A) Cell body– derived vesicles were stained with DiIC12 molecules as in Fig. 1. Local perfusion of the middle axonal segment with a culture medium containing 5 μg/ml nocodazole was started 30 min before the soma staining. (B) Fluorescent images of the superfused site at different times (marked in minutes) after the staining of the cell body with DiIC12. The staining of filopodia, which reflects the insertion of soma-derived vesicles into the plasmalemma, could be detected as soon as 10 min after the onset of cell body staining. The bright staining of the axon proximal to the perfusion site reflects an accumulation of fluorescent vesicles in this region. (C) Fluorescence intensity profiles of the filopodia staining 10 min (filled triangles) and 50 min (open squares) after the soma staining. The intensity of individual filopodia staining (arbitrary units) is plotted vs. the distance from the center of the profile. The center of the superfused zone was located ∼50 μm distal from the center of the fluorescence profile. The widening of the profiles reflects the lateral diffusion of DiIC12 molecules incorporated into the plasma membrane along the axon. Data from three representative experiments are combined together. (D) Representative immunofluorescent micrograph of the MT array in the axon near the site locally superfused with nocodazole. The cell was fixed and stained for MTs 30 min after the onset of perfusion. (E) Quantitative analysis of immunofluorescence data. For each axon, the intensity of fluorescence along the axon was normalized to that ∼100 μm proximal to the center of the superfused zone. Data are presented as mean ± SEM for 10 different axons. Bars: (B) 50 μm; (D) 30 μm.

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