Correspondence to W. Mothes: walther.mothes{at}yale.edu
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Abbreviations used: ALV, avian leukosis virus; Env, viral envelope glycoprotein; HIV, human immunodeficiency virus; MLV, murine leukemia virus; SEM, scanning electron microscopy; TEM, transmission electron microscopy; VSV, vesicular stomatitis virus.
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Introduction |
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Results |
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Cell surfing is shared by other viruses
Surfing was not restricted to MLV. Gag (viral capsid polyprotein precursor)YFP-labeled MLV capsids containing the envelope glycoprotein of the avian leukosis virus (ALV) surfed along filopodia of HEK 293 cells when they expressed the ALV receptor Tva fused to CFP (Bates et al., 1993; Fig. 2 I; Video 4 available at http://www.jcb.org/cgi/content/full/jcb.200503059/DC1). ALV surfed at a slower speed than MLV, averaging 1 µm/min, and attaching particles lacked the initial random movement seen for MLV (Fig. 2 J). Human immunodeficiency virus (HIV) similarly surfed when cells were expressing the HIV receptors CD4 and CXCR4 (Fig. 2 K; Video 5 available at http://www.jcb.org/cgi/content/full/jcb.200503059/DC1). Surfing was not restricted to retroviruses. Particles bearing the envelope protein of vesicular stomatitis virus (VSV) also surfed along filopodia of rat fibroblasts (Fig. 2 L; Video 6 available at http://www.jcb.org/cgi/content/full/jcb.200503059/DC1). VSVG-containing viruses were found to move along filopodia and then continued to surf along the plasma membrane until they disappeared from the confocal microscope plane, likely due to endocytosis. Parallel immunofluorescence for actin in these cells showed that the areas that support virus movement are rich in actin filaments (Fig. S2, A and B available at http://www.jcb.org/cgi/content/full/jcb.200503059/DC1). Co-expression of clathrinlight chainYFP confirmed colocalization of viruses with clathrin 15 min after infection (Fig. S2 C). Video microscopy revealed that clathrin-recruitment to surfing viruses was initiated as soon as the virus reached the cell body (Video 7 available at http://www.jcb.org/cgi/content/full/jcb.200503059/DC1). Interestingly, surfing continued during clathrin recruitment (Video 7). These experiments suggest that pH-dependent viruses such as VSV surf along filopodia and actin filaments to reach endocytic hot spots.
Virus cell surfing is actin and myosin dependent
The association of viral particles with actin-rich filaments and the observed highly ordered movement of viral particles implied an actin and motor-driven process. Addition of cytochalasin D, a reagent that blocks the barbed ends of actin filaments, inhibited surfing (Fig. 4, compare B with A; Video 8 available at http://www.jcb.org/cgi/content/full/jcb.200503059/DC1), whereas treatment with nocodazole, which disassembles microtubules, had no effect (Figs. 4 C and 5 C; unpublished data). Addition of the ATPase inhibitor sodium azide immediately blocked particle movement toward the cell body (Fig. 4 D; unpublished data). Quantitative analysis revealed that cytochalasin D or sodium azide treatment led to a diffusive random movement (Fig. 5, compare B and D with A; see Fig. S1 for tracks of individual particles) and a complete loss of directional motility toward the cell body (Fig. 5, H and I). Thus, surfing is an energy-dependent process mediated by the underlying actin cytoskeleton.
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Inhibitors that block virus cell surfing interfere with infection of filopodia and microvilli-rich cells
If virus cell surfing represents a physiologically relevant pathway of viral infection, our observed visual block to surfing by blebbistatin and cytochalasin D should result in reduced infectivity. To address this question, we applied an assay that allowed us to test the role of these reversible inhibitors specifically in the earliest stages of infection (Kizhatil and Albritton, 1997). Viruses were added to cells in the presence or absence of drugs for periods of time as short as 5 min before the drugs were washed out and viral particles that had failed to enter cells were inactivated by either a 1-min acid wash or a 10-min treatment with methyl-ß-cyclodextrin. Infection was continued in the absence of inhibitors. Applying this assay, we observed significant inhibitory effects of blebbistatin on MLV and VSV infection only when cells were cultured at low confluencies, conditions where cells exhibited abundant filopodia (Table I). At higher confluencies, when cells contacted each other, blebbistatin had reduced effects. Moreover, when cells were rounded completely due to trypsinization (receptor mCAT-1 is trypsin resistant), blebbistatin had no effect on MLV entry as compared with the untreated control. These data indicate that blebbistatin can only affect the earliest steps of MLV entry when cells exhibit abundant filopodia, suggesting a contribution of virus cell surfing to infection.
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Because of the relevance of mucosal epithelia to viral infection, we tested if polarized epithelial cells covered with dense microvilli would require surfing for efficient infection. Indeed, infection of polarized MDCK cells by MLV or VSVG-containing viruses was reduced at least fivefold by blebbistatin and cytochalasin D when virus was added to the microvilli-rich apical side but not to the basolateral side (Table I). As was the case for fibroblasts, no inhibitory effects of blebbistatin were observed for trypsinized MDCK cells that exhibited a highly folded surface but lacked microvilli as visualized by scanning electron microscopy (SEM; Table I; Fig. S4 C). In fact, blebbistatin caused a slight enhancement in infection of trypsinized MDCK cells as well as if the virus was applied to the basolateral side of a monolayer of polarized cells. Although these observations suggest an additional role for myosin II in virus entry, inhibitory effects were strictly associated with the microvilli-rich apical side of polarized epithelial cells.
Cytochalasin D behaved identically to blebbistatin in its ability to inhibit the entry of MLV via microvilli-rich surfaces suggesting that the role of actin is limited to the movement along microvilli (Table I). In contrast, infection of MDCK cells by VSV, which uses a pH-dependent endocytic entry route, remained moderately sensitive to cytochalasin D even on trypsinized MDCK cells.
In contrast to the filopodia of fibroblasts, microvilli are too small to be imaged by live microscopy. To test if the observed inhibitory effects of blebbistatin were due to a block in virus cell surfing, we applied SEM to visualize microvilli-rich surfaces and viruses at magnifications of 20,000 and 40,000x. To this end, viruses were bound on ice to polarized MDCK cells. After 30 min, the samples were washed with cold media to remove unbound virus. One sample was immediately prepared for SEM while the other two were incubated for an additional 30 min at 37°C in the absence or presence of blebbistatin. Scanning electron micrographs of all samples revealed efficient binding of viruses to the microvilli of MDCK cells. A clustering and bundling of tips of microvilli and viruses, both of similar size, was observed (Fig. 6 A, upper panel). This effect was not due to aggregation of virions, rather, as revealed by parallel TEM, was caused by the multivalency of virus/microvilli interactions (Fig. 6 B). Incubation at 37°C in the absence of blebbistatin resulted in a complete loss of microvilli-associated viruses (Fig. 6 A, middle panel). Parallel TEM demonstrated that under these conditions viruses had entered cells (Fig. 6 C). In contrast, the presence of blebbistatin completely blocked virus movement along microvilli, arresting the process at the step of virus binding (Fig. 6 A, lower panel). These data demonstrate that viruses efficiently bind to the microvilli of polarized epithelial cells, suggest that they use surfing to enter cells at the cell body and indicate that surfing is controlled by myosin II.
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Discussion |
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With virus cell surfing, our understanding of viral entry is now modified by an early stage involving virus attachment, receptor recruitment, the establishment of a link to the underlying actin cytoskeleton and myosin IImediated transport of the virus to the cell body. This process lies upstream of cell entry regardless of whether the virus enters cells by pH-independent (MLV, HIV) or pH-dependent (ALV, VSV) mechanisms (Stein et al., 1987; Maddon et al., 1988; Mothes et al., 2000; Kolokoltsov and Davey, 2004; Matsuyama et al., 2004). Surfing may have evolved to avoid having to penetrate the dense cortical actin cytoskeleton by moving along the plasma membrane to reach endocytic hot spots, which are areas of active actin remodeling. MLV and HIV, which do not require endocytosis for fusion, also surf to these areas before fusion, thereby avoiding the delivery of capsid into actin filament-dense areas.
Virus cell surfing depends on cognate Envreceptor interactions. Visually, receptor recruitment precedes the onset of surfing. The initial random movements of MLV observed within the first 10 s of attachment to filopodia likely reflects a period of mCAT-1 receptor recruitment as well as the establishment of a link between oligomerized receptor and actin filaments.
Transport along filopodia is mediated by the underlying actin cytoskeleton and is controlled by myosin II. Because myosin II is a plus end motor that apparently mediates minus end motility toward the cell body, it must regulate the movement of entire actin filaments, a process called retrograde F-actin flow (Forscher and Smith, 1988; Mitchison and Kirschner, 1988; Sheetz et al., 1989; Lin and Forscher, 1993). Thus, our data suggest a role for myosin II in retrograde flow. How myosin II controls retrograde flow remains to be determined. Although myosin II clearly localizes to retraction fibers, it is absent from filopodia. In the latter case, myosin II may control actin filament movement at the base of filopodia by regulating actin filament disassembly. Although we would anticipate the involvement of other myosins in retrograde flow, azide does not block virus cell surfing beyond the level observed for blebbistatin, suggesting that myosin II plays a predominant role.
How viruses engage retrograde F-actin flow remains to be determined. Signaling from cytoplasmic tails of oligomerized receptor appears to be a prerequisite for the establishment of a link to the actin cytoskeleton (Felsenfeld et al., 1996; Suter et al., 1998). Tva receptors carrying a GPI anchor instead of a transmembrane domain do not support efficient surfing of ALV (unpublished data), consistent with the observed slow kinetics of ALV infection mediated by these receptors (Narayan et al., 2003). The link to the actin cytoskeleton may only briefly be tight when viruses travel at maximum speed. In general, however, surfing viruses appear to engage a molecular clutch resulting in a fast-and-slow or slippage mode of movement rather than a rigid link to actin filaments. The ability of different viruses and their receptors to engage retrograde flow varies. MLV and ALV move at different average speeds along filopodia of the same cell type. In addition, movement of individual viral particles along the same filopodium occurs independently with no apparent coordination (unpublished data).
Virus cell surfing was observed on a number of different actin-rich protrusions such as filopodia, retraction fibers, and microvilli. In each case, surfing was mediated by the underlying actin cytoskeleton and was dependent on myosin II. Even in the case of short-lived filopodia that were observed to occasionally capture viral particles, particles were brought to the cell body by retrograde flow of actin filaments. These observations suggest that all instances of virus cell surfing are based on a related basic mechanism despite being morphologically different.
It is the nature of hostpathogen interactions that pathogens exploit existing cellular pathways. In this case, viruses likely engage retrograde F-actin flow, a process that was discovered and has been studied extensively using artificial beads (Abercrombie et al., 1970; Harris and Dunn, 1972; Albrecht-Buehler and Lancaster, 1976; Forscher and Smith, 1988; Mitchison and Kirschner, 1988; Sheetz et al., 1989). The ability of beads to establish a link to the actin cytoskeleton had been used to study how the forces of the actin cytoskeleton can translate into cell motility if beads were immobilized. As such, the field has focused on adhesion molecules such as integrins and members of the immunoglobulin family of CAM proteins (Felsenfeld et al., 1996; Suter et al., 1998). Our work with viruses now reemphasizes the role of the actin cytoskeleton in ligand uptake, transport, and endocytosis and offers viruses as physiological "beads" to study this process. In addition to adhesion molecules we extend the list of receptors capable of promoting rearward movement toward the cell body to also include transporters such as mCAT-1 for MLV (Albritton et al., 1989), receptors of the LDL receptor-like family Tva for ALV (Bates et al., 1993) as well as CD4/chemokine receptors in the case of HIV (Littman, 1998). As such, virus cell surfing likely reflects the fundamental ability of receptors to transport ligands along filopodia. Movement of EGF quantum dots along filopodia has recently been reported (Lidke et al., 2004). Ferritin and toxins have also been observed to travel along microvilli in an actin-dependent fashion (Gottlieb et al., 1993; Shurety et al., 1996). Thus, viral cell surfing appears to represent the exploitation of retrograde F-actin flow to efficiently transport signaling molecules from the periphery toward endocytic zones at the cell body. One prediction of this hypothesis is that signaling cascades are spatially separated into peripheral events that induce cell surfing and downstream events in the cell body that trigger signaling toward the nucleus. As such, cell surfing, powered by the underlying F-actin flow, and endocytosis in areas of actin remodeling, likely represent two steps of a coordinated process of ligand signaling and uptake.
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Materials and methods |
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Generation of fluorescently labeled viruses
Most reagents for the generation of fluorescent viruses have previously been described (Sherer et al., 2003). EnvYFP-labeled MLV is a replication competent virus produced in large quantities from infected DFJ8 cells cultured in roller bottles. Viruses obtained from filtered supernatants were spun through a 15% sucrose cushion, resuspended in DME containing 10% FBS, filtered through 0.2 µm filters (Nalgene) and stored at 80°C. GagYFP-labeled MLV cores (Sherer et al., 2003) were used for the generation of viruses bearing fusion-defective MLV Env (delta H8; James Cunningham, Harvard Medical School, Boston, MA), ALV-A Env, and VSVG (Mothes et al., 2000). Fluorescent HIV particles were generated by cotransfection of a 10 cm plate of 293 cells with plasmids encoding HIVBRU3, Env (5 µg; McDonald et al., 2002), HIV-Env (5 µg), and HIV-GagYFP (1 µg; Sherer et al., 2003). Medium was replaced 36 h after transfection. Virus-containing supernatants were collected at 40 and 44 h and directly used for imaging experiments.
Imaging
Transfection and live cell imaging of 293, DFJ8, and XC cells were as previously described (Sherer et al., 2003) using the 100x oil objective (numerical aperture 1.4) of an LSM 510 confocal microscope equipped with a Zeiss axiovert 100 M base (Zeiss MicroImaging, Inc.). For live cell imaging, cells were grown on poly-L-lysinecoated 35-mm glass-bottom plates (MatTek). Before imaging at 37°C, media was replaced with DME/10% FBS supplemented with 10 mM Hepes, pH 7.4. In most imaging experiments, a high viral multiplicity of infection of 1001,000 was used in order to capture a suitable number of particles in an individual confocal plane within a short period of time. In time-lapse videos, CFP and YFP channels were imaged every 10 s and pseudocolored in green and red, respectively. All videos were exported from the LSM 510 as QuickTime videos, edited using Openlab (Improvision) or Photoshop software (Adobe) and compressed using Apple video (Videos 16 and 810) or the Apple animation mode (Video 7). For quantitative analysis, the distance each individual particle traveled within 10 s was determined using LSM 510 software.
In some experiments MatTek glass-bottom plates were pretreated with 1 mg/ml fibronectin (Invitrogen) for 10 min at room temperature and spun at 1,000 rpm to generate an evenly coated surface. Fluorescent viruses were allowed to bind for 30 min at room temperature. After removal of excess virus, HEK 293 cells expressing viral receptors were seeded for 30 min at 37°C before fixation and imaging.
For myosin IIindirect fluorescence, XC cells stably expressing actinYFP were incubated with 0.02% saponinPBS for 30 s before 10 min fixation in 4% paraformaldehyde/0.25% Triton X-100. Cells were washed with PBS, quenched with 50 mM NH4Cl, and washed with 0.05% Triton X-100PBS before incubation for 1 h with rabbit antimyosin II antisera (Biomedical Technologies Inc.). After washing, cells were subsequently incubated for 40 min with Alexa Fluor 568conjugated antirabbit secondary antisera (Invitrogen), washed, and mounted on glass slides with Gelmount (Biomeda). For three-colored imaging of virus, clathrin, and actin, VSV-Gcarrying MLV GagCFP virions were added to XC cells stably transfected with clathrin light chainYFP and fixed at various times after infection using 4% paraformaldehyde. After permeabilization using 20°C methanol immunostaining was as described above using rabbit antiactin antisera (Sigma-Aldrich) and Alexa Fluor 568conjugated antirabbit secondary antisera (Invitrogen). All immunofluorescence was imaged using the 60x oil objective (numerical aperture 1.4) of the Nikon TE2000 microscope and Openlab acquisition software (Improvision).
Infection assays
To gauge the effects of various drugs on viral infection, MLV particles carrying a viral genome for the expression of ß-galactosidase (pMMP-LTR-LacZ; Richard Mulligan, Harvard Medical School, Boston, MA) were generated as previously described (Mothes et al., 2000; Sherer et al., 2003). Viruses were added in the presence of polybrene to cells either untreated or pretreated for 5 min with 550 µM cytochalasin D or 3050 µM blebbistatin. Infection was performed for 5, 30, or 120 min before nonfused virus was inactivated using either a 1-min acid wash (Kizhatil and Albritton, 1997) or a 15-min incubation with 10 mM methyl-ß-cyclodextrin (MCD). MCD-treated MLV was five orders of magnitude less infectious. Cells were cultured for 2 d before infectivity was scored by X-gal staining.
Polarized MDCK cells were prepared for infection experiments by growing a confluent cell layer for at least 12 d on transwell filters (pore size 0.4 µm; Corning) until the transepithelial electrical resistance (TER) plateaued (Fig. S4 A). Addition of inhibitors for up to 2 h did not affect the TER. TEM of fully polarized MDCK cells revealed the apical formation of a typical dense meshwork of microvilli (Fig. S4 B). Infection was performed as described above except that virus was added to either the apical or basolateral side of the transwell. Cells were transferred into six well dishes after MCD treatment and infection was scored as above.
Electron microscopy
Cells were rinsed with serum-free buffer and then quick-fixed with 1% osmium tetroxide for 10 s, immediately followed by aldehyde fixation for 1 h (2.5% glutaraldehyde, 2% paraformaldehyde in 100 mM cacodylate buffer, pH 7.4). Cells were rinsed three times for 5 min with 100 mM cacodylate buffer, postfixed for 1 h in 1% osmium tetroxide, rinsed three times with HPLC water, en bloc uranyl acetate stained, dehydrated through a graded ethanol series, and finally embedded using EMBed 812 (EMS).
293 cells were cut en face and 7090 nm sections were collected within the first 200 nm of the coverglass surface (Fig. 3). Scraped transwell plate samples of MDCK cells were cut perpendicular to the substrate, at the same section thickness (Fig. S4 B). In both cases, sections were counterstained with either 2% (wt/vol) or 4% (wt/vol) uranyl acetate followed by lead citrate. All samples were imaged on an FEI Tecnai 12 (Philips).
For analysis by SEM, cells were fixed for 30 min with 2.5% glutaraldehyde/2% paraformaldehyde in 100 mM cacodylate buffer (pH 7.4), rinsed three times with 100 mM cacodylate buffer, and dehydrated through a graded ethanol series. After washing three times with hexamethyldisilazane (EMS), cells were dried for 5 min at 60°C, coated with platinum, and analyzed on a FEI ESEM scanning electron microscope (Philips).
Online supplemental material
Descriptions of the data presented in supplemental figures and videos are introduced upon citation in the text. Online supplemental materials are available at http://www.jcb.org/cgi/content/full/jcb.200503059/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health grant R01CA098727 and the Searle Scholars Program to W. Mothes, as well as an Anna Fuller Fund Fellowship and a Leopoldina Fellowship BMBF-LPD 9901/8-75 to M. Lehmann.
Submitted: 14 March 2005
Accepted: 8 June 2005
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References |
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Abercrombie, M., J.E. Heaysman, and S.M. Pegrum. 1970. The locomotion of fibroblasts in culture. 3. Movements of particles on the dorsal surface of the leading lamella. Exp. Cell Res. 62:389398.[CrossRef][Medline]
Albrecht-Buehler, G., and R.M. Lancaster. 1976. A quantitative description of the extension and retraction of surface protrusions in spreading 3T3 mouse fibroblasts. J. Cell Biol. 71:370382.[Abstract]
Albritton, L.M., L. Tseng, D. Scadden, and J.M. Cunningham. 1989. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell. 57:659666.[CrossRef][Medline]
Bates, P., J.A. Young, and H.E. Varmus. 1993. A receptor for subgroup A Rous sarcoma virus is related to the low density lipoprotein receptor. Cell. 74:10431051.[CrossRef][Medline]
Bomsel, M., and A. Alfsen. 2003. Entry of viruses through the epithelial barrier: pathogenic trickery. Nat. Rev. Mol. Cell Biol. 4:5768.[CrossRef][Medline]
Bukrinskaya, A., B. Brichacek, A. Mann, and M. Stevenson. 1998. Establishment of a functional human immunodeficiency virus type 1 (HIV-1) reverse transcription complex involves the cytoskeleton. J. Exp. Med. 188:21132125.
Duus, K.M., V. Lentchitsky, T. Wagenaar, C. Grose, and J. Webster-Cyriaque. 2004. Wild-type Kaposi's sarcoma-associated herpesvirus isolated from the oropharynx of immune-competent individuals has tropism for cultured oral epithelial cells. J. Virol. 78:40744084.
Felsenfeld, D.P., D. Choquet, and M.P. Sheetz. 1996. Ligand binding regulates the directed movement of beta1 integrins on fibroblasts. Nature. 383:438440.[CrossRef][Medline]
Forscher, P., and S.J. Smith. 1988. Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J. Cell Biol. 107:15051516.[Abstract]
Gaidarov, I., F. Santini, R.A. Warren, and J.H. Keen. 1999. Spatial control of coated-pit dynamics in living cells. Nat. Cell Biol. 1:17.[CrossRef][Medline]
Gottlieb, T.A., I.E. Ivanov, M. Adesnik, and D.D. Sabatini. 1993. Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells. J. Cell Biol. 120:695710.[Abstract]
Hammond, C., L.K. Denzin, M. Pan, J.M. Griffith, H.J. Geuze, and P. Cresswell. 1998. The tetraspan protein CD82 is a resident of MHC class II compartments where it associates with HLA-DR, -DM, and -DO molecules. J. Immunol. 161:32823291.
Harris, A., and G. Dunn. 1972. Centripetal transport of attached particles on both surfaces of moving fibroblasts. Exp. Cell Res. 73:519523.[CrossRef][Medline]
Helenius, A., J. Kartenbeck, K. Simons, and E. Fries. 1980. On the entry of Semliki forest virus into BHK-21 cells. J. Cell Biol. 84:404420.
Hernandez, L.D., L.R. Hoffman, T.G. Wolfsberg, and J.M. White. 1996. Virus-cell and cell-cell fusion. Annu. Rev. Cell Dev. Biol. 12:627661.[CrossRef][Medline]
Kizhatil, K., and L.M. Albritton. 1997. Requirements for different components of the host cell cytoskeleton distinguish ecotropic murine leukemia virus entry via endocytosis from entry via surface fusion. J. Virol. 71:71457156.[Abstract]
Klement, V., W.P. Rowe, J.W. Hartley, and W.E. Pugh. 1969. Mixed culture cytopathogenicity: a new test for growth of murine leukemia viruses in tissue culture. Proc. Natl. Acad. Sci. USA. 63:753758.
Kolokoltsov, A.A., and R.A. Davey. 2004. Rapid and sensitive detection of retrovirus entry by using a novel luciferase-based content-mixing assay. J. Virol. 78:51245132.
Lidke, D.S., P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H.E. Grecco, E.A. Jares-Erijman, and T.M. Jovin. 2004. Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction. Nat. Biotechnol. 22:198203.[CrossRef][Medline]
Lin, C.H., and P. Forscher. 1993. Cytoskeletal remodeling during growth cone-target interactions. J. Cell Biol. 121:13691383.[Abstract]
Littman, D.R. 1998. Chemokine receptors: keys to AIDS pathogenesis? Cell. 93:677680.[CrossRef][Medline]
Maddon, P.J., J.S. McDougal, P.R. Clapham, A.G. Dalgleish, S. Jamal, R.A. Weiss, and R. Axel. 1988. HIV infection does not require endocytosis of its receptor, CD4. Cell. 54:865874.[CrossRef][Medline]
Masuda, M., N. Kakushima, S.G. Wilt, S.K. Ruscetti, P.M. Hoffman, and A. Iwamoto. 1999. Analysis of receptor usage by ecotropic murine retroviruses, using green fluorescent protein-tagged cationic amino acid transporters. J. Virol. 73:86238629.
Matsuyama, S., S.E. Delos, and J.M. White. 2004. Sequential roles of receptor binding and low pH in forming prehairpin and hairpin conformations of a retroviral envelope glycoprotein. J. Virol. 78:82018209.
McDonald, D., M.A. Vodicka, G. Lucero, T.M. Svitkina, G.G. Borisy, M. Emerman, and T.J. Hope. 2002. Visualization of the intracellular behavior of HIV in living cells. J. Cell Biol. 159:441452.
Miller, M., E. Bower, P. Levitt, D. Li, and P.D. Chantler. 1992. Myosin II distribution in neurons is consistent with a role in growth cone motility but not synaptic vesicle mobilization. Neuron. 8:2544.[CrossRef][Medline]
Mitchison, T., and M. Kirschner. 1988. Cytoskeletal dynamics and nerve growth. Neuron. 1:761772.[CrossRef][Medline]
Mitchison, T.J. 1992. Actin based motility on retraction fibers in mitotic PtK2 cells. Cell Motil. Cytoskeleton. 22:135151.[CrossRef][Medline]
Mothes, W., A.L. Boerger, S. Narayan, J.M. Cunningham, and J.A. Young. 2000. Retroviral entry mediated by receptor priming and low pH triggering of an envelope glycoprotein. Cell. 103:679689.[CrossRef][Medline]
Narayan, S., R.J. Barnard, and J.A. Young. 2003. Two retroviral entry pathways distinguished by lipid raft association of the viral receptor and differences in viral infectivity. J. Virol. 77:19771983.
Ponti, A., M. Machacek, S.L. Gupton, C.M. Waterman-Storer, and G. Danuser. 2004. Two distinct actin networks drive the protrusion of migrating cells. Science. 305:17821786.
Rochlin, M.W., K. Itoh, R.S. Adelstein, and P.C. Bridgman. 1995. Localization of myosin II A and B isoforms in cultured neurons. J. Cell Sci. 108:36613670.
Sheetz, M.P., S. Turney, H. Qian, and E.L. Elson. 1989. Nanometre-level analysis demonstrates that lipid flow does not drive membrane glycoprotein movements. Nature. 340:284288.[CrossRef][Medline]
Sherer, N.M., M.J. Lehmann, L.F. Jimenez-Soto, A. Ingmundson, S.M. Horner, G. Cicchetti, P.G. Allen, M. Pypaert, J.M. Cunningham, and W. Mothes. 2003. Visualization of retroviral replication in living cells reveals budding into multivesicular bodies. Traffic. 4:785801.[CrossRef][Medline]
Shurety, W., N.A. Bright, and J.P. Luzio. 1996. The effects of cytochalasin D and phorbol myristate acetate on the apical endocytosis of ricin in polarised Caco-2 cells. J. Cell Sci. 109:29272935.
Smith, A.E., and A. Helenius. 2004. How viruses enter animal cells. Science. 304:237242.
Stein, B.S., S.D. Gowda, J.D. Lifson, R.C. Penhallow, K.G. Bensch, and E.G. Engleman. 1987. pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane. Cell. 49:659668.[CrossRef][Medline]
Straight, A.F., A. Cheung, J. Limouze, I. Chen, N.J. Westwood, J.R. Sellers, and T.J. Mitchison. 2003. Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor. Science. 299:17431747.
Suter, D.M., L.D. Errante, V. Belotserkovsky, and P. Forscher. 1998. The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling. J. Cell Biol. 141:227240.
Svitkina, T.M., A.B. Verkhovsky, K.M. McQuade, and G.G. Borisy. 1997. Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation. J. Cell Biol. 139:397415.
Verkhovsky, A.B., T.M. Svitkina, and G.G. Borisy. 1995. Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles. J. Cell Biol. 131:9891002.[Abstract]
Young, J.A. 2001. Virus entry and uncoating. Fields Virology. Vol. 1. D.M. Knipe, editor. Lippincott Williams & Wilkins, Philadelphia. 87103.
Zavorotinskaya, T., Z. Qian, J. Franks, and L.M. Albritton. 2004. A point mutation in the binding subunit of a retroviral envelope protein arrests virus entry at hemifusion. J. Virol. 78:473481.
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