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Open Access Research article

Single vesicle imaging indicates distinct modes of rapid membrane retrieval during nerve growth

Jacob H Hines12, Steven J Henle1, Lucas P Carlstrom1, Mohammad Abu-Rub13 and John R Henley14*

Author Affiliations

1 Department of Neurologic Surgery, Mayo Clinic, Rochester, MN, USA

2 Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

3 Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, IRL

4 Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA

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BMC Biology 2012, 10:4  doi:10.1186/1741-7007-10-4

Published: 30 January 2012

Additional files

Additional file 1:

Movie 1 - Small endocytic vesicle hot-spots at dorsal ridges of the growth cone. These representative time-lapse movies show examples of rapid membrane retrieval triggered at dorsal ridges. A focal pulse of FM 5-95 transiently labeled the plasma membrane (evident at 00:00). The brief dye pulse was delivered from a micropipette positioned 100 μm in front of the leading edge of the growth cone, which is indicated by the top arrow in frame 1. Confocal images were collected (1 Hz) during the endocytic assay and the time frames (minutes:seconds) are indicated at the top left. Movie 1 corresponds to Figure 2A, B. The scale bar (5 μm) applies to each example. Format: MOV (MPEG4 compression).

Format: MOV Size: 4.5MB Download file

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Additional file 2:

Movie 2 - Small endocytic vesicle hot-spots at the lateral margins of lamellipodia. This representative time-lapse movie demonstrates membrane retrieval triggered at the lateral margins of a lamellipodium. A focal FM 5-95 pulse transiently labelled the growth cone surface membrane and confocal images were acquired as described in Movie 1. The yellow static arrow (00:16) indicates a region undergoing active membrane remodelling and retrieval at the lateral-most lamellipodial margin. Movie 2 corresponds to Figure 3A. Scale bar, 5 μm. Format: MOV (MPEG4 compression).

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Additional file 3:

Movie 3 - Small endocytic vesicle hot-spots at the lateral margins of lamellipodia. This representative time-lapse movie shows membrane remodelling at the lateral margins of the growth cone lamellipodium (black static arrow). The DIC images were acquired at 0.5 Hz and the time (minutes:seconds) is indicated at the top left. Movie 3 corresponds to Figure 3B. Scale bar, 5 μm. Format: MOV (MPEG4 compression).

Format: MOV Size: 6.4MB Download file

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Additional file 4:

Movie 4 - Small endocytic vesicle hot-spots triggered by filopodial - lamellipodial contacts. This representative time-lapse movie demonstrates membrane retrieval triggered by contact between a filopodium and lamellipodium. A focal pulse of FM 5-95 transiently labeled the growth cone surface membrane and confocal images were acquired as described in Movie 1. The yellow arrow (00:09) indicates filopodial detachment, subsequent contact with the growth cone dorsal surface, and the formation of numerous small endocytic vesicles (00:21 to 00:27). Movie 4 corresponds to Figure 4A. Scale bar, 5 μm. Format: MOV (MPEG4 compression).

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Additional file 5:

Movie 5 - Small endocytic vesicle hot-spots triggered by filopodial - lamellipodial contacts. This time-lapse movie shows evidence for the disappearance of growth cone filopodia following contact with an adjacent lamellipodium (black arrows, 00:00 to 00:26, 00:40 to 01:08). The DIC images were acquired at 0.5 Hz and the time (minutes:seconds) is indicated at the top left. Movie 5 corresponds to Figure 4B. Scale bar, 5 μm. Format: MOV (MPEG4 compression).

Format: MOV Size: 9.3MB Download file

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Additional file 6:

Movie 6 - Small endocytic vesicle hot-spots triggered by filopodial contact. This representative time-lapse movie shows membrane retrieval triggered by contact between neighboring filopodia (yellow arrowheads). A focal pulse of FM 5-95 transiently labeled the growth cone surface membrane and confocal images were acquired as described in Movie 1. The blue arrow points toward nascent endocytic vesicles formed at the base of filopodia. Movie 6 corresponds to Figure 5A. Scale bar, 5 μm. Format: MOV (MPEG4 compression).

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Additional file 7:

Movie 7 - Small endocytic vesicle hot-spots triggered by filopodial contact. This time-lapse movie demonstrates contact between adjacent filopodia (black static arrow) similar to that shown by fluorescence images in 6. The DIC images were acquired at 0.5 Hz and the time (minutes:seconds) is indicated at the top left. Movie 7 corresponds to Figure 5B. Scale bar, 5 μm. Format: MOV (MPEG4 compression).

Format: MOV Size: 2.8MB Download file

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Additional file 8:

Movie 8 - Membrane retrieval via lamellipodial contact and peripheral tubules. This representative time-lapse movie demonstrates membrane retrieval triggered upon contact between adjacent regions of lamelipodium (Figure 6A), indicated by the yellow static arrow (00:00 to 00:12). In later frames, an elongated tubule (Figure 9A) is retrieved from a filopodium (blue static arrowhead, 00:07 to- 00:27). Surface labeling and image acquisition was performed as described in Movie 1. Scale bar, 5 μm. Movie 8 corresponds to Figure 6A and 9A. Scale bar, 5 μm. Format: MOV (MPEG4 compression).

Format: MOV Size: 1.6MB Download file

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Additional file 9:

Movie 9 - Examples of membrane remodeling at sites of lamellipodial - lamellipodial contact. This time-lapse DIC movie shows examples of membrane rearrangements at lamellipodial contacts, similar in nature to those shown in Movie 8. Movie 9 corresponds to Figure 6C and 6 the time (minutes:seconds) is indicated at the top left. The time 00:00 corresponds to 0:00 in Figure 6C. The time 03:34 corresponds to 0:00 in Figure 6B. During each respective time period, the black arrow points toward the region of interest highlighted in Figure 6B, whereas the white arrowheads point toward reverse shadowcast structures (00:17 to 00:25) identified in Figure 6C. Scale bar, 5 μm. Format: MOV (MPEG4 compression).

Format: MOV Size: 8MB Download file

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Additional file 10:

Movie 10 - Endocytic vesicle formation at self-membrane contacts requires F-actin. This time-lapse movie shows a representative example of the focal membrane labeling assay performed on a growth cone pre-treated with cytochalasin D (30 nM). Movie 10 corresponds to Figure 7C and 7 the time (minutes:seconds) is indicated at the top left. A filopodium contacts a nearby region of the growth cone periphery (yellow arrow, 00:21 to 00:26). This self-contact fails to elicit vesicle formation at this location. Surface labeling and image acquisition was performed as described in Movie 1. Scale bar, 5 μm. Format: MOV (MPEG4 compression).

Format: MOV Size: 3.6MB Download file

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Additional file 11:

Movie 11 - Sequential dye labeling demonstrates temporally distinct endocytic zones. This time-lapse movie shows that endocytic hot-spots at two separate time-points occur at spatially distinct areas of the same growth cone. The dual FM dye labelling assay was performed as described in Figure 8A. Briefly, a focal pulse of FM 2-10 (green) was applied at 00:00. A second micropipette then applied a subsequent focal pulse of FM 5-95 at 00:38. Movie 11 corresponds to Figure 8B-B''. The time (minutes:seconds) is indicated at the top left. Scale bar, 5 μm. Format: MOV (MPEG4 compression).

Format: MOV Size: 9.6MB Download file

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Additional file 12:

Movie 12 - Membrane retrieval by elongated tubules in the growth cone periphery. This time-lapse movie demonstrates membrane retrieval by elongated tubules in the growth cone periphery. Alexa-488 dextran was focally applied from a micropipette positioned 80 μm in front of the leading edge of the growth cone (top arrow in frame 1). Uninternalized dextran was then rapidly washed away during image acquisition using a second micropipette containing buffered saline (left arrow in frame 1). The time (minutes:seconds) is indicated at the top left. Movie 12 corresponds to Figure 9B. Scale bar, 5 μm. Format: MOV (MPEG4 compression).

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Additional file 13:

Movie 13 - Endocytic tubules and vacuoles in the growth cone central domain. This time-lapse movie demonstrates rapid labeling of elongated tubules and vacuoles within the growth cone central domain. A focal pulse of FM 5-95 transiently labeled the growth cone surface membrane and confocal images were acquired as described in Movie 1. The yellow static arrowhead indicates a central vacuole and the green arrows point toward central tubules. Scale bar, 5 μm. Movie 13 corresponds to Figure 10A. Format: MOV (MPEG4 compression).

Format: MOV Size: 1.3MB Download file

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Additional file 14:

Movie 14 - Vacuole and tubule unloading or recycling back to the plasma membrane. These representative time-lapse movies correspond to Figure 11A-C. The boxed regions at left are shown at higher magnification on the right. The upper time-lapse series was performed using the focal FM 5-95 labeling assay, whereas fluorescent dextran was focally applied to growth cones in the center and lower series (indicated in frame 1). Scale bars, 5 μm (left), 1 μm (right). Format: MOV (MPEG4 compression).

Format: MOV Size: 8.8MB Download file

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