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Open Access Highly Accessed Research article

Examination of actin and microtubule dependent APC localisations in living mammalian cells

Kelly J Langford*, Jon M Askham, Tracy Lee, Matthew Adams and Ewan E Morrison

Author Affiliations

CRUK Clinical Centre at Leeds, Division of Cancer Medicine Research, St James's University Hospital, Leeds, LS9 7TF, UK

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BMC Cell Biology 2006, 7:3  doi:10.1186/1471-2121-7-3

Published: 19 January 2006

Additional files

Additional File 1:

Microtubule-dependent localisation of full-length GFP-APC expressed in subconfluent COS-7 cells. This movie shows a subconfluent cell with GFP-APC associated with microtubule tips at specific regions near the cell periphery, typically in cell vertices. Microtubule tip labelling was comprised of discrete GFP-APC puncta or clusters in conjunction with a dimmer, less specific labelling of microtubule distal segments. Image capture rate was 1frame/5s using a 63× oil immersion lens. Movie is played back at 20 frames/s.

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Additional File 2:

Peripheral deposition of GFP-APC puncta in COS-7 cells. Microtubule-associated GFP-APC puncta can be deposited at the cell periphery by shrinking microtubules. Two microtubules can be seen to grow towards the cell periphery depositing GFP-APC puncta at the cortex (arrowheads). Once the puncta has contacted the cell periphery the microtubule can be seen to shrink away from the cell edge leaving the original puncta at the periphery. Close observation of this image sequence suggested that GFP-APC puncta might remain associated with shrinking microtubule ends; this is examined in more detail in subsequent movies. Image capture rate was 1 frame/5s using a 63× oil immersion lens and the movie is played back at 20 frames/s.

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Additional File 3:

Retrograde movement of GFP-APC puncta in COS-7 cells. Peripheral GFP-APC puncta underwent linear retrograde movements with an average velocity of 21.6 ± 2.4 μm. An example is indicated in this movie by the arrowhead. These movements predominantly occurred over short distances of less than 10 μm. Image capture rate was 1 frame/3s using a 63× oil immersion lens. The movie is played back at 10 frames/s.

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Additional File 4:

Partial deposition of microtubule-associated GFP-APC puncta in COS-7 cells. The movie shows a GFP-APC puncta (arrowhead) undergoing retrograde movement on a shrinking microtubule end within the cytoplasm. Part of this original puncta is deposited within the cytoplasm (arrowhead 2) while the remainder continues retrograde movement (arrowhead 1). Note that close examination of additional files 2 and 3 reveal similar behaviours. Image capture rate was 1 frame/5s using a 63× oil immersion lens. The movie is played back at 10 frames/s.

Format: MOV Size: 614KB Download file

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Additional File 5:

Microtubules possessing GFP-APC puncta could be seen to undergo shrinkage followed by a period of pause and then growth (arrowhead). GFP-APC puncta remained at the end of the microtubule throughout this cycle of shrinkage and growth. Of interest in this sequence is the observation that separate microtubule-associated GFP-APC puncta coalesce into a single large structure at the microtubule tip during shrinkage, then separate out into a distribution resembling beads on a string during microtubule regrowth, before beginning to re-coalesce on the paused microtubule tip at the cell periphery. Image capture was 1 frame/5s using a 63× oil immersion lens, the movie is played back at 10 frames/s.

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Additional File 6:

Comet-like GFP-APC expression in COS-7 cells. Occasionally cells were found with a motile comet-like distribution within the cell interior. These comets moved with an average velocity of 20.4 ± 6 μm/min, essentially indistinguishable from the distribution and behaviour observed for the microtubule tip-tracking APC ligands EB1 and EB3 when expressed as GFP fusion proteins in COS-7 cells. Image capture rate was 1 frame/3s using a 63× oil immersion lens and movie is played back at 20 frames/s.

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Additional File 7:

Fast moving GFP-APC puncta within the cytoplasm of COS-7 cells. GFP-APC puncta were occasionally observed undergoing rapid transport within the cell interior at velocities in the region of 2 μm/s, speeds consistent with microtubule motor-mediated transport. Microtubule and junction-associated GFP-APC populations are also visible at the cell periphery in this sequence. Image capture rate was 1 frame/3s using a 63× oil immersion lens and movie is played back at 20 frames/s.

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Additional File 8:

Cortically-localised GFP-APC in COS-7 cells. In confluent cultures of transfected COS-7 cells GFP-APC was present as a discontinuous array of punctate structures associated with membranes contacting adjacent cells. These puncta were far less dynamic than microtubule-associated GFP-APC distributions, being essentially immobile relative to the cell cortex. Image capture rate was 1 frame/10s using a 63× oil immersion lens and movie is played back at 20 frames/s.

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Additional File 9:

Regional specificity of different GFP-APC localisations in COS-7 cells. In partially confluent COS-7 cells having both contacted and free edges, microtubule and junction-associated GFP-APC populations exist in the same cell but never overlap. Image capture was rate 1 frame/5s using a 63× oil immersion lens and movie is played back at 20 frames/s.

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Additional File 10:

Live imaging of Nocodazole treated COS-7 cell expressing GFP-APC. This movie shows a cell with junction-associated GFP-APC (arrowheads) and microtubule-associated GFP-APC (arrow). After addition of Nocodazole to a final concentration of 5 μg/ml the microtubule-associated GFP-APC population is rapidly lost whereas the junction-associated GFP-APC is largely unaffected. Image capture rate was 1 frame/10s and movie played back at 20 frames/s.

Format: MOV Size: 5.1MB Download file

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Additional File 11:

Live imaging of Cytochalasin D treated COS-7 cell expressing GFP-actin. This movie shows two cells forming cell-cell contacts. A cortical actin belt can be seen around the circumference of the cells. Upon addition of 1 μg/ml Cytochalasin D the cortical actin belt is weakened and contracts along the cell edge (arrowheads). Image capture rate was 1 frame/min, movie played back at 20 frames/s.

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Additional File 12:

Live imaging of Cytochalasin D treated COS-7 cell expressing GFP-APC. This movie shows two GFP-APC expressing cells forming a cell-cell contact. GFP-APC is present along the cortex at the site of cell-cell contact and on microtubules near a free edge in the upper cell. Upon addition of Cytochalasin D the cell-cell contact between the cells is weakened and undergoes breakage (black arrow). The cortical GFP-APC puncta are then seen to contract towards the cell vertices in the direction of the white arrow. The microtubule-associated GFP-APC appears unaffected by Cytochalasin D treatment, within the constraints imposed by retraction of the cell edge. Image capture rate was 1 frame/min, movie played back at 20 frames/s.

Format: MOV Size: 5.3MB Download file

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Additional File 13:

Expression of the short N-terminal APC construct led to the identification of 3 distinct localisations. The first was to the centrosome (arrow), the second to small, sometimes motile puncta within the cytoplasm, and the final localisation was to sites of cell-cell adhesion (arrowheads). Image capture rate was 1 frame/10s using a 63× oil immersion lens and the movie is played back at 20 frames/s.

Format: MOV Size: 4.4MB Download file

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