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Open Access Highly Accessed Research article

Arp2/3 complex activity in filopodia of spreading cells

Simon A Johnston1, Jonathan P Bramble4, Chun L Yeung2, Paula M Mendes2 and Laura M Machesky13*

Author Affiliations

1 University of Birmingham School of Biosciences, Edgbaston, Birmingham, B15 2TT, UK

2 Department of Chemical Engineering, Edgbaston, Birmingham, B15 2TT, UK

3 CRUK Beatson Institute for Cancer Research, Garscube Estate, Switchback Rd, Bearsden, Glasgow, G63 9AE, UK

4 Molecular and Nanoscale Physics Group, School of Physics and Astronomy, University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, UK

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BMC Cell Biology 2008, 9:65  doi:10.1186/1471-2121-9-65

Published: 9 December 2008

Additional files

Additional file 1:

Movie of morphology of mouse embryonic fibroblast spreading on fibronectin. MEFs spread through cycles of filopodia and lamellipodia production making them excellent for the study of both these structures. MEFs in suspension were plated onto fibronectin coated coverslips and a field of view was selected at random. Lines that can be seen on the coverslip are numbered areas for locating cells for correlative fluorescence microscopy. Cells spread identically on marked coverslips compared to unmarked coverslips in all experiments. Frames were captured every 10 seconds. This cell is representative of 30 cells from 4 independent experiments. Movie is displayed at 5 frames per second.

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Additional file 2:

Morphology of mouse embryonic fibroblast spreading on fibronectin. A. Mouse embryonic fibroblasts spread with cycles of filopodia and then lamellipodia. MEFs were plated on fibronectin and a timelapse sequence of DIC images taken up to 60 minutes after plating. Images shown here are frames taken at times indicated after plating. Scale bar 10 μm. B. Total spread area was measured from cells spread for times indicated and then fixed. Data are from three experiments, 15' n = 73, 30' n = 66 and 60' n = 66.

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Additional file 3:

Double antibody labelling Arp2/3 complex in filopodia. Antibodies to the Arp2/3 complex members p34-arc and arp2 both localise Arp2/3 complex to filopodia. Cells were spread on fibronectin for 60 minutes and co-labelled with a rabbit polyclonal antibody to p34-arc and a mouse monoclonal antibody to Arp2. B and D (scale bar 2 μm) are enlargements of boxed area in A and C (scale bar 10 μm) respectively.

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Additional file 4:

Example of protruding filopodia containing Arp2/3 complex. This is an example of a protruding filopodium that contains Arp2/3 complex (still pictures are shown in Figure 2). Movie 3 is a cropped region of interest of a MEF that was spread for 60 minutes on a fibronectin coated coverslip. The tip of the filopodium extends towards the bottom left hand corner up until fixation for correlative fluorescent microscopy. Frames were captured every 10 seconds for 60 minutes. Movie contains the last 3 minutes of this timelapse. Still pictures are shown in Figure 2. Movie is displayed at 5 frames per second.

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Additional file 5:

Movie of localisation of Arp2/3 complex to filopodia. Arp2/3 complex localises to filopodia of live cells at multiple sites. A GFP tagged p21-arc subunit of Arp2/3 complex and mRFP tagged actin were transiently transfected into MEFs. After spreading on fibronectin for 60 minutes low expressing cells were identified and timelapse sequences taken. Movie 3 is representative of 11 cells in 6 independent experiments. Frames were captured every 10 seconds and movie is displayed at 5 frames per second.

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Additional file 6:

Dynamics of Arp2/3 complex in filopodia. Arp2/3 complex patches in filopodia are highly dynamic. Movie 4 is a cropped region of interest from Movie 3. Arrows were added in Openlab 4 (Improvision, UK). Movie is displayed at 5 frames per second.

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Additional file 7:

Arp2/3 localises to dynamic and static filopodia. A) Arp2/3 complex containing filopodium from a cell that is actively protruding. A patch of Arp2/3 complex appears in a filopodium before being overtaken by the extending lamellipodium. Images are taken from timelapse movie frames captured every 10 seconds. Scale bar 5 μm. B) Arp2/3 complex containing filopodium from a cell that is not actively protruding. The Arp2/3 complex in this situation is much less dynamic than that seen in additional file 7. This suggests a correlation between the protrusive activity at the site of an Arp2/3 complex containing filopodium and the dynamics of Arp2/3 complex in that filopodium. Images are taken from timelapse movie frames captured every 10 seconds. Scale bar 5 μm.

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Additional file 8:

Blocking new adhesion formation of spreading mouse embryonic fibroblasts. Lamellipodia but not filopodia are dependent on formation of new adhesions. After 5 minutes of attachment to a glass cover slip, the surface was blocked with denatured BSA and cells were allowed to spread for a further 55 minutes before fixation. Actin was labelled with rhodamine phalloidin and vinculin and Arp2/3 complex with antibodies. Scale bar 10 μm.

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Additional file 9:

Formation of lamellipodia and filopodia on micro-patterned surfaces. Lamellipodia but not filopodia are dependent on an adhesive surface. A. Cells were spread on 5 μm fibronectin and BSA stripes for 60 minutes. Scale bar 10 μm B. Cells were spread on 10 μm fibronectin and BSA stripes for 60 minutes. Scale bar 10 μm. Hollow arrows indicate filopodia. Solid arrows indicate lamellipodia/ruffles. Actin was labelled with Alexa 350 phalloidin. Scale bar 10 μm.

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