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. 2011 Oct 9;13(12):1431-6.
doi: 10.1038/ncb2353.

An actin-dependent mechanism for long-range vesicle transport

Melina Schuh  1
Affiliations

An actin-dependent mechanism for long-range vesicle transport

Melina Schuh. Nat Cell Biol. .

Abstract

Intracellular transport is vital for the function, survival and architecture of every eukaryotic cell. Long-range transport in animal cells is thought to depend exclusively on microtubule tracks. This study reveals an unexpected actin-dependent but microtubule-independent mechanism for long-range transport of vesicles. Vesicles organize their own actin tracks by recruiting the actin nucleation factors Spire1, Spire2 and Formin-2, which assemble an extensive actin network from the vesicles' surfaces. The network connects the vesicles with one another and with the plasma membrane. Vesicles move directionally along these connections in a myosin-Vb-dependent manner to converge and to reach the cell surface. The overall outward-directed movement of the vesicle-actin network is driven by recruitment of vesicles to the plasma membrane in the periphery of the oocyte. Being organized in a dynamic vesicle-actin network allows vesicles to move in a local random manner and a global directed manner at the same time: they can reach any position in the cytoplasm, but also move directionally to the cell surface as a collective. Thus, collective movement within a network is a powerful and flexible mode of vesicle transport.

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Figures

Figure 1
Figure 1
Vesicles move over long distances to the cell surface (a) Rab11a-EGFP and Transferrin-Cy5 in live cell. Boxed region is magnified in inset. Scale bar: 10 μm. (b) Live cell expressing Rab11a-EGFP. Boxed region is magnified next to overview. Scale bar in magnification: 5 μm. Time: seconds. (c) 3D data sets of live cells expressing Rab11a-EGFP were acquired and isosurfaces of cell, nucleus and vesicles reconstructed. Vesicles were tracked in 3D. Only tracks that were longer than 45 s were evaluated to reliably determine the overall directionality of vesicle movements. The tracks’ colour reflects the mean vesicle speed. Displacement of vesicles towards surface (magenta) or centre (cyan) is plotted. (d) The distance of representative vesicles from the cell’s centre is plotted over time.
Figure 2
Figure 2
Vesicle movements depend on actin instead of microtubules (a-c) 3D data sets of vesicles labelled with Rab11a-EGFP in live control (a), nocodazole (b) and cytochalasin D treated cells (c) were acquired and processed as described for Fig. 1. Vesicle movements in live cells and a time-coloured projection are shown below, with vesicles being coloured if mobile and white if stationary. Time: seconds; Scale bars: 5 μm. (d) Vesicles were tracked as in (a-c) and their speed in control (599 tracks, 10 oocytes), nocodazole (399 tracks, 8 oocytes) and cytochalasin D treated oocytes (1657 tracks, 8 oocytes) is shown. Box plot displays median (line), mean (small square), 1st, 99th (crosses), 5th, 95th (whiskers) and 25th and 75th percentile (boxes) of vesicle speeds. (e) Vesicle displacement to the centre or to the surface of the oocyte was scored. Mean from 10 control, 8 nocodazole and 8 cytochalasin D treated oocytes are shown, with error bars displaying s.d.. P-values were calculated with Student’s t-test.
Figure 3
Figure 3
Spire1/2 and Fmn2 assemble actin tracks (a) Control, cytochalasin D treated, Fmn2−/− and Spire1+2 co-depleted cells were fixed and stained with Alexa488-phalloidin to label F-actin. (b-c) 3D data sets of vesicles labelled with Rab11a-EGFP in live Spire1 and Spire2 co-depleted oocytes (b) and Fmn2−/− oocytes (c) were acquired and processed as described for Fig. 1 and 2. (d) Vesicles were tracked as in b-c and their speed in control (599 tracks, 10 oocytes), Spire1 and Spire2 co-depleted (936 tracks, 6 oocytes) and Fmn2−/− (1605 tracks, 8 oocytes) oocytes is shown. Box plot as in Fig. 2d. (e) Vesicle displacement to the centre or to the surface of the oocyte was scored. Mean from 10 control, 6 Spire1 and Spire2 co-depleted (p<10−3) and 8 Fmn2−/− (p<10−5) oocytes are shown, with error bars displaying s.d.. P-values were calculated with Student’s t-test. (a-c) Scale bars: 5 μm.
Figure 4
Figure 4
Vesicles organize an actin network (a) Cell expressing Rab11a-mCherry was fixed and stained for F-actin. Scale bar: 5 μm. (b) Live cell expressing Spire1-mCherry and Rab11a-mEGFP. Scale bar: 5 μm. (c) Reassembly of F-actin upon washout of cytochalasin D in live cell expressing EGFP-UtrCH (F-actin) and Spire2-mCherry (Vesicle). Time: min:sec; Scale bar: 5 μm.
Figure 5
Figure 5
Mechanism of directional vesicle transport (a) Live cell expressing EGFP-UtrCH (F-actin) and mCherry-Rab11a (Vesicle). Time: seconds; Scale bar: 2 μm. (b) Live cell expressing Rab11a-mEGFP (Rab11a) and Myosin 5b-mCherry (Myosin 5b). (c) Vesicle speed in Myosin 5b (229 tracks, 6 oocytes), Myosin 5b tail (446 tracks, 5 oocytes), rescued (1286 tracks, 13 oocytes), Myosin 5a tail (872 tracks, 12 oocytes) and Myosin 6 tail (606 tracks, 12 oocytes) expressing oocytes is shown. Box plot as in Fig. 2d. (d) Live cell expressing full length Myosin 5b-mCherry (top row) or Myosin 5b tail-mCherry (bottom row) and time-coloured projection, with vesicles being coloured if mobile and white if stationary. Time: seconds. (e) Vesicle displacement to the centre or to the surface of the oocyte was scored. Mean from 10 control and 5 Myosin 5b tail expressing oocytes are shown, with error bars displaying s.d.. P-values were calculated with Student’s t-test. (f) Live cell expressing EGFP-UtrCH. Time: min:sec; Scale bar: 5 μm. (g) Live cells expressing Fmn2-mEGFP, Spire1-mCherry or Spire2-mCherry. (h-j) Mechanistic model for actin-dependent vesicle movements. The different objects are specified in the legend. For details, see text. (b, d, f, g) Scale bars: 5 μm.
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