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.
According to the textbook view of long-range vesicle transport in animal cells, vesicles move over long distances along polarized microtubules that originate from the centrosome [1] [2] [3] [4] . In a typical animal cell, microtubule plus ends face the cell surface, and minus ends converge at the centre where the centrosome is located. Accordingly, the minus-end-directed motor protein dynein mediates transport to the cell centre, and plus-end-directed kinesins mediate transport to the periphery 5, 6 . However, there is evidence for alternative mechanisms of long-range transport. Indicative of such mechanisms, secretion in various cell types is hardly affected if microtubules are depolymerized 7, 8 . Moreover, some viruses such as HIV are still able to move through the cell if microtubules are absent 9 . The only known microtubuleindependent motor proteins that could alternatively drive long-range transport are myosins, which move along actin filaments. However, the actin cytoskeleton does not seem to provide suitable tracks for long-range transport: actin filaments are randomly oriented in the

cytoplasm of typical animal cells 10 . Only specialized cellular regions such as the actin cortex, microvilli, filopodia or dendritic spines contain polarized actin structures that could potentially be used for myosin-dependent directional transport 10 . Accordingly, actin filaments have been implicated only in local vesicle transport, for instance in the actin cortex and in dendritic spines 1, [11] [12] [13] . This study reveals a mechanism for long-range vesicle transport that is entirely actin dependent. It shows that vesicles organize an extensive actin network that connects them with one another and with the cell surface. Vesicles move along the network in a myosin-Vb-dependent manner that enables them to converge and to collectively move towards the cell surface.
The identity and transport routes of vesicles are defined by members of the large family of Rab GTPases. Different Rab GTPases recruit different effector proteins such as motor proteins, sorting adaptors and tethering factors that guide vesicles to their correct destination 14 . One of the main pathways that mediates transport to the plasma membrane is defined by the small GTPase Rab11a. Rab11a-positive vesicles are, for instance, involved in transporting the iron-carrying protein transferrin, which is one of the best-studied transport cargoes 15 . Whether vesicles use actin filaments for long-range transport is hard to investigate in tissue culture cells because the cells are very flat so that prominent cortical actin structures cover intracellular actin assemblies. Thus, mouse oocytes were used to study the transport of Rab11a-positive vesicles. Cortical and intracellular actin structures are well separated in this cell type 16 , and the large cell volume is ideally suited to analyse quantitatively how vesicles move in three dimensions.
First, it was analysed whether Rab11a-positive vesicles mediate transport to the plasma membrane in this cell type. Indeed, Rab11a co-localized with transferrin (Fig. 1a) , and a dominant-negative variant of Rab11a blocked the transport of transferrin in 22/22 cells (Supplementary Movie S1). Moreover, live-cell imaging of fluorescently labelled Rab11a revealed that vesicles moved over distances of more than 30 µm from the centre towards the surface of the cell, where they merged with the plasma membrane (Fig. 1b) .
To analyse quantitatively how vesicles move, high-resolution threedimensional data sets of vesicles in live cells were recorded, the cell and vesicle surfaces were reconstructed and vesicles were automatically tracked in three dimensions (Fig. 1c,d and Supplementary Movie S2). Vesicles were very mobile, moving in a zigzag pattern with a speed of 12.8±3.4 µm min −1 (Fig. 2a,d ). In contrast, lysosomes and fluorescent beads of similar size moved significantly slower ( Supplementary Fig.  S1a -c and Movie S3), demonstrating that neither cytoplasmic flows nor Brownian motion can account for the observed movements. Vesicle movements were also highly directional, with 76.8 ± 8.8% of vesicles moving towards the cell surface over time (Fig. 2a,e) . The small fraction of vesicles that did not move towards the cell surface was more stationary than other vesicles and mostly located in proximity of the nucleus of the oocyte (Fig. 1c,d ). In contrast to Rab11a-positive vesicles, lysosomes and fluorescent beads moved non-directionally, with equal proportions moving towards the centre and the surface of the cell ( Supplementary Fig. S1a,b,d ). These data show that Rab11a-positive vesicles move directionally over long distances to the plasma membrane. Next, the mechanism by which vesicles were moving was investigated. Long-range vesicle transport is generally thought to be microtubule dependent [1] [2] [3] [4] 10, 17 . To determine whether vesicles were transported along microtubules, cells were treated with 1 µM nocodazole, which depolymerized all microtubules 18 ( Supplementary Fig. S2) . Surprisingly, microtubules were dispensable for vesicle movements (Fig. 2b) : neither the speed (12.8 ± 3.7µm min −1 ; P = 0.92) nor the directionality of vesicle movements (72.5 ± 7.8% towards cell surface; P = 0.39) was significantly different from that in control oocytes (Fig. 2d,e and Supplementary Movie S4). To determine whether vesicles moved along actin filaments instead, cells were treated with cytochalasin D, which depolymerizes F-actin (Fig. 2c) . Without actin filaments, vesicles did not move over long distances anymore. Instead, they moved only locally and their speed was markedly decreased to just 2.7 ± 1.5 µm min −1 (Fig. 2c,d and Supplementary Movie S4).
The directionality of vesicle movements was also lost. Instead, equal proportions of vesicles moved to the surface and to the centre of the cell (Fig. 2e ). These data demonstrate that vesicles move over long distances in an actin-dependent but microtubule-independent manner. That vesicles moved directionally, even if microtubules were absent, indicated that actin filaments might provide the tracks for vesicle movements. Mouse oocytes are filled with a cytoplasmic actin network with an average filament length of 5.7 ± 1.9 µm between branch points, which is required for asymmetric spindle positioning 16 (Fig. 3a) . The actin network was not affected by depolymerizing microtubules (Supplementary Fig. S2 ). Thus, it seemed possible that filaments of the actin network mediate vesicle movements. To test this hypothesis, vesicle movements were analysed in oocytes that lack the actin nucleation factors Spire1 and Spire2 (ref. 19 ; Supplementary Fig. S3a ) or Formin-2 (Fmn2; ref. 20) , which cooperate with one another to assemble the network 16, 19 (Fig. 3a) . When either Fmn2 or Spire1 and Spire2 were absent, the vesicle speed was greatly decreased, to similar levels as in cytochalasin-D-treated cells ( Fig. 3b-d ; RNA interference rescue in Supplementary Fig. S3b,c) . The directionality of vesicle movements was also lost (Fig. 3e) . In contrast, the actin network was still present and vesicle movements were only slightly affected when oocytes were treated with inhibitors of the Arp2/3 complex 21 ( Supplementary Fig. S3d-f) . Together, these data show that vesicle movements require cytoplasmic actin filaments that are nucleated by Spire1, Spire2 and Fmn2.
To investigate how vesicles move within the actin network, the behaviour of vesicles and actin filaments was analysed by highresolution microscopy of live and fixed oocytes. Surprisingly, vesicles were located at nodal points in the network, seemingly serving as organizing centres of the network ( then Spire1, Spire2 and Fmn2 should co-localize with Rab11a-positive vesicles. Indeed, all actin nucleation factors co-localized on the vesicle surface ( Fig. 4b and Supplementary Fig. S4a ). To determine directly whether the actin network was assembled from the vesicle surface, an F-actin regrowth assay was established. Cells were first treated with cytochalasin D to depolymerize F-actin. The drug was then washed out and actin repolymerization was monitored in live cells to determine the sites from where the network was nucleated. This assay confirmed that vesicles served as prominent organizing centres of the network ( Fig. 4c and Supplementary Movie S6). In further support of actin nucleation from the vesicle surface, some vesicles were even pushed through the cell following actin nucleation events ( Supplementary  Fig. S4b) , showing that these nucleation events contribute to local random vesicle movements. The movements of vesicles and associated filaments were also closely correlated (Supplementary Movie S7), with actin filaments moving with a velocity of 10.6 ± 6.8 µm min −1 (49 filaments in 5 oocytes).
Together, these results show that Rab11a-positive vesicles recruit the actin nucleation factors Spire1, Spire2 and Fmn2 to assemble an extensive actin network that fills the cell's cytoplasm and connects vesicles with one another and with the cell surface.
Through an investigation of how vesicles use the actin network to move directionally, two types of directional movement were observed: vesicle convergence (Fig. 5a,d ) and movement of vesicles towards the plasma membrane (Fig. 1) . First, the mechanism of directional movement for vesicle convergence was investigated. High-resolution live-cell imaging revealed that vesicles moved to neighbouring vesicles along actin filaments connecting them (Fig. 5a and Supplementary Movie S8). When the actin network was absent, vesicles could not converge anymore and the average vesicle size was decreased ( Supplementary Fig. S5 ). The lack of convergence and missing transport to the cell surface also significantly increased the number of vesicles per cell (Supplementary Fig. S5 ). Movements along actin filaments are mediated by motor proteins of the myosin family. Most myosins are plus-end-directed. Spire proteins 22, 23 and formins 24, 25 associate with the plus ends of actin filaments. This implies that the plus ends of filaments in the network converge on the vesicle surface, where the nucleators were located (Fig. 5h) . Thus, a myosin-carrying vesicle could attach to an actin filament that is nucleated from a neighbouring vesicle and move towards the neighbouring vesicle along this filament (Fig. 5h) . A good candidate for such a myosin was myosin Vb because it was shown to associate with Rab11a-positive vesicles in other cell types 11, 26 . Indeed, myosin Vb co-localized with Rab11a-positive vesicles in mouse oocytes (Fig. 5b) . To determine whether myosin Vb was required for vesicle convergence, its activity was inhibited by expressing the dominant-negative tail domain of myosin Vb. This blocked vesicle convergence ( Fig. 5d ) and decreased the vesicle speed (Fig. 5c ). Vesicle movements specifically required myosin Vb, because first, vesicle movements could be rescued by overexpressing full-length myosin Vb (Fig. 5c) ; and because second, vesicle movements were not affected by dominant-negative variants of other myosins, such as myosin Va or myosin VI (Fig. 5c) . Moreover, human myosin Vb was reported to have a velocity of 13.2 ± 1.8 µm min −1 in actin gliding assays 27 , which is remarkably similar to the velocity of Rab11a-positive vesicles of 12.8±3.4 µm min −1 that is reported in this study (Fig. 2d) . In summary, these data indicate that vesicles move towards one another along actin filaments that are nucleated from a neighbouring vesicle in a myosin-Vb-dependent manner (Fig. 5h) .
The same transport principle also explains why vesicles move towards the cell surface over time. The plus ends of actin filaments are not enriched only on vesicles, but also at the cell surface. First, Fmn2, Spire1 and Spire2 all localized to the cell surface (Fig. 5g) , from where actin filaments were nucleated (Fig. 5f ) and numerously reached into the cytoplasm (Supplementary Movie S9). Second, filaments in the actin cortex are also polarized so that plus ends face the plasma membrane 28 ( Fig. 5i) . Myosin Vb can bind to these polarized filaments and transport vesicles towards the plasma membrane (Fig. 5i) . Indeed, vesicles attached to these actin filaments and moved along them towards the cell surface ( Supplementary Fig. S6a and Movie S10 and Fig. 5i) . In further support of this model, inhibition of myosin Vb blocked the outward movement of vesicles (Fig. 5e) , and myosin-Vb-tail-carrying vesicles were frequently located below the cell surface ( Supplementary Fig. S6b ). As all vesicles form an interconnected network, short-range directional movements in the periphery of the oocyte are sufficient to drag the entire vesicle-actin network outward (Fig. 5i,j) . The most peripheral vesicles, which bind to polarized actin filaments beneath the plasma membrane, will carry neighbouring vesicles with them, and these will carry their neighbours and so on (Fig. 5i) . In this way, the entire vesicleactin network moves outward over time (Fig. 5j) . Thus, the collective movement of vesicles in a network makes long polarized actin filaments or microtubules dispensable for long-range transport (Fig. 5i,j) . This study reveals an actin-dependent mechanism for long-range vesicle transport. It shows that vesicles recruit the actin nucleation factors Spire1, Spire2 and Fmn2 to organize a cytoplasmic actin network that connects the vesicles with one another and with the cell surface. The filament connections between vesicles are used for myosin-Vb-dependent directional transport that allows vesicles to move towards one another and to reach the cell surface. Directional movements in the cell periphery will automatically drag the network to the surface and thereby determine that vesicles move outward over time. These data uncover a much more flexible transport system than constricted movement along microtubule tracks. 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. This new principle of vesicles as organizers of an extensive actin network is surprising, because vesicle-associated actin nucleators have so far mostly been implicated in membrane remodelling [29] [30] [31] [32] . Interestingly, morphologically similar actin networks that converge at bright actin nodes, which might correspond to vesicles, have been reported in a range of cell types, including Xenopus epithelial cells 33 , Drosophila embryos 34 and oocytes 35 and Caenorhabditis elegans embryos 36 , and Spire proteins co-localize with vesicles in fibroblasts 29 , indicating that network-mediated long-range transport of vesicles might be conserved. Intracellular transport is essential for the existence and function of all eukaryotic cells. This study reveals two new fundamental concepts of how animal cells can achieve this vital task: an unexpected actin-dependent mechanism for long-range transport, and a new principle of intracellular transport, namely the collective movement of vesicles as integral building blocks of a dynamic network.
METHODS
Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturecellbiology 
Preparation and culture of oocytes. All mice were maintained in a specific pathogen-free environment according to UK Home Office regulations. Oocytes were isolated from ovaries of 8-week-old FVB or Fmn2 −/− mice 20 , cultured and microinjected as described in detail previously 18 . Short interfering RNAs (siRNAs) were purchased from Qiagen (Mm_Spire1_1 SI01431591 5 -AAGGTAGAAAGTATAGAAATA-3 , Mm_Spire1_3 SI01421605 5 -CACCATCATTAAGATACAGTA-3 , Mm_Spire2_1 SI01431619 5 -CAGGAACTA-TAAGCTGCGCAA-3 , Mm_Spire2_4 SI01431640 5 -TTCCTGTAGCGTAAAGAT-GAA-3 ). siRNAs were injected to a final concentration of 5.5 nM into follicleenclosed oocytes from 10-12-day-old (C57BL × CBA) F1 females and then cultured for 10 days by methods described in detail previously 19, 37, 38 . For quantitative real-time PCR, messenger RNA was extracted using an RNeasy Mini Kit (Qiagen) and complementary DNA was generated using the High Capacity RNA-to-cDNA Kit from (Applied Biosystems). Real-time PCR was carried out with the 7900 HT Real-Time Fast PCR System (Applied Biosystems) using SYBR Green. GAPDH mRNA was used for normalization. The following primer sets were used: Spire1, forward, 5 -GACAGCCTCTGACTCTGAGGAGG-3 ; Spire1, reverse, 5 -GGGCAAGAATTTTGGAGGCTTCTTC-3 ; Spire2, forward, 5 -TACCACCAGCCAGCTCAGAAG-3 , Spire2, reverse, 5 -TCGATGAGCCTTTCCA-GCTGC-3 ; GAPDH, forward, 5 -AGAGCTGAACGGGAAGCTCACT-3 ; GAPDH, reverse, 5 -TGCCTGCTTCACCACCTTCTTGAT-3 .
In some experiments, oocytes were microinjected with fluorescent beads (Bangs Laboratories; FC03F), treated with Lysotracker Red DND-99 (Molecular Probes), Cy5-labelled transferrin (gift from B. Nichols, MRC LMB, Cambridge, UK) or treated with 1 µM nocodazole or 5 µg ml −1 cytochalasin D (Calbiochem).
Confocal microscopy. Images were acquired with a Zeiss LSM710 confocal microscope equipped with a Zeiss environmental incubator box or a Zeiss LSM780 confocal microscope equipped with a Tokai Hit Stage Top Incubator, with a ×40 C-Apochromat 1.2 NA water-immersion objective lens for live oocytes, and a ×63 C-Apochromat 1.2 NA water-immersion objective for fixed oocytes as previously described 18 . In some images, shot noise was decreased with a Gaussian filter.
Immunofluorescence microscopy. Oocytes were fixed for 30-60 min at 37 • C in 100 mM HEPES (pH 7; titrated with KOH), 50 mM EGTA (pH 7; titrated with KOH), 10 mM MgSO 4 , 2% formaldehyde (methanol free) and 0.2% Triton X-100, based on previously published methods 39 . Oocytes were left in PBS with 0.1% Triton X-100 overnight at 4 • C. Antibody and F-actin staining were carried out in PBS with 0.1% Triton X-100 and 3% BSA. As primary antibody, rat anti-α-tubulin (MCA78S, Serotec; 1:500), and as secondary antibody, Alexa-Fluor-680-labelled anti-rat (Molecular Probes 1:200) were used. F-actin was stained with Alexa-Fluor-488-phalloidin (Molecular Probes; 0.33 µM/10 units ml −1 ). DNA was stained with 5 mg ml −1 Hoechst 33342 (Molecular Probes).
Automated three-dimensional tracking of vesicles, lysosomes and fluorescent beads. To label Rab11a-positive vesicles, oocytes were microinjected with mRNA encoding fluorescently labelled Rab11a. To label lysosomes, oocytes were treated with Lysotracker Red DND-99 (Molecular Probes). To track beads, oocytes were microinjected with fluorescent beads (Bangs Laboratories; FC03F). To track vesicles labelled with myosin Vb or myosin Vb tail, oocytes were microinjected with mRNA encoding fluorescently labelled full-length myosin Vb or myosin Vb tail and arrested in dibutyryl-cAMP-and cytochalasin-D-containing medium until both proteins were expressed. Oocytes were then released into dibutyryl-cAMP-and cytochalasin-D-free medium.
Three-dimensional data sets of vesicles, beads and lysosomes were acquired using Zeiss's MultiTime Series macro (13 sections; spacing: 1.5 µm; time interval: 6.3 s; total duration: ∼5 min; parameters vary slightly in some experiments). Drifts during image acquisition were corrected with the 'correct drift' function of Imaris (Bitplane) after segmenting the cell volume by thresholding on the soluble pool of the fluorescent reporters. Afterwards, vesicles, beads or lysosomes were detected with the spot detection function of Imaris and spots were tracked in three dimensions over time. Vesicles, beads and lysosomes that could be tracked for more than 45 s were evaluated. This was important to ensure that particles could be tracked sufficiently long to determine the overall directionality of their movement. Inclusion of shorter tracks does not alter the vesicle speed however (data not shown). Mean vesicle speeds and vesicle coordinates were exported into Excel. To automatically determine whether vesicles move to the surface or centre of the cell, the centre of mass of the cell volume was determined in Imaris, and the direction of the vector describing the displacement of the vesicle relative to the cell centre was determined. Evaluation of example data sets with higher z-and t -resolution confirmed the obtained results (data not shown).
Statistics. Average (mean), s.d. and statistical significance based on Student's t -test (always two-tailed) were calculated in Excel. All box plots show median (line), mean (small square), 1st, 99th (crosses), 5th, 95th (whiskers) and 25th and 75th percentile (boxes) of vesicle, bead or lysosome speeds.
Measurement of the cytoplasmic network density. To quantify the density of the cytoplasmic actin network in oocytes treated with Arp2/3 inhibitors in Supplementary Fig. S3e , the mean intensity of Alexa-Fluor-488-phalloidin staining was measured in the cytoplasm and in a region outside the oocyte for background subtraction. Images in control and perturbed situations were acquired with identical imaging conditions and care was taken that images were not saturated during acquisition. Average, s.d. and statistical significance based on Student's t -test were calculated in Excel.
Expression constructs and mRNA synthesis. To generate the constructs for in vitro mRNA synthesis, the previously published protein coding sequences were fused with EGFP (Clontech) or mCherry 40 to obtain mEGFP-Rab11a, mCherry-Rab11a, mEGFP-Rab11a S25N (ref. 41) , full-length mCherry-myosin-Vb and mCherry-myosin-Vb-tail 11 , mCherry-myosin-VI-tail 42 and mCherry-myosinVa-tail 43 and inserted into pGEMHE (ref. 44) for in vitro transcription. These constructs as well as pGEMHE-H2B-mRFP1 (ref. 18 ), pGEMHE-Spire1-mCherry and pGEMHE-Spire2-mCherry (ref. 19) were linearized with AscI. Capped mRNA was synthesized using T7 polymerase (mMessage mMachine kit, following the manufacturer's instructions, Ambion), and dissolved in 11 µl water. pCS2-Fmn2-EGFP (ref. 20 ) and pCS2-EGFP-UtrCH (ref. 45 ) were linearized with KpnI or NsiI respectively. Capped mRNAs were synthesized using SP6 polymerase (mMessage mMachine kit, Ambion), polyadenylated (Poly(A) Tailing Kit, Ambion) and dissolved in 6 µl water. mRNA concentrations were determined on ethidium bromide agarose gels by comparison with an RNA standard (Ambion). Supplementary Fig. 6 . Mechanism of vesicle movement to cell surface.
(a) Vesicle moving along actin filament to cell surface. Live cell expressing EGFP-UtrCH (Factin; magenta) and mCherry-Rab11a (Vesicle; cyan). Note that the actin cortex looks much thicker than it actually is because it had to be saturated to visualize intracellular actin filaments. Cross-talk of the actin cortex in the channel for detection of mCherry-Rab11a gives a more precise impression of the position of the surface. The high background in the actin channel in close proximity to the cell surface is due to out of focus light from the bright actin cortex. Time: seconds; Scale bar: 2 µm. 
M E T H O D S


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