![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 17, Issue 11, 4866-4875, November 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
*Program in Molecular, Cell, and Developmental Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73121; Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany; Max-Planck-Institut of Molecular Cell Biology and Genetics, 01037 Dresden, Germany; and ||University of Heidelberg, Nikon Imaging Center, 69120 Heidelberg, Germany
Submitted April 28, 2006;
Revised August 15, 2006;
Accepted September 1, 2006
Monitoring Editor: David Drubin
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The Arp2/3 complex, responsible for nucleation and the dendritic assembly of actin filaments (Pollard and Borisy, 2003), is transiently enriched about the nascent phagosome (Insall et al., 2001). Other actin-interacting proteins including coronin (Maniak et al., 1995) and DAip1 (Konzok et al., 1999) are similarly enriched and have been shown by mutational analysis to be involved in phagocytosis. Proper assembly of actin filaments during phagosome formation is likely a G protein–mediated process; the G protein 
-subunit transiently labels nascent phagosomes, and its lack severely impairs phagocytosis (Peracino et al., 1998). The motor protein myosin-IB, also called MyoB, is one of several unconventional myosins that have been shown to be involved in phagocytosis. MyoB binds to actin, to the plasma membrane (Senda et al., 2001) and, through the linker protein CARMIL, to the Arp2/3 complex (Jung et al., 2001). MyoB is concentrated in actin-rich cortical domains (Morita et al., 1996), and its lack impairs lamellipod extension, membrane trafficking, and endocytosis of both fluid and particles (Titus, 2000). Once a phagosome has moved away from the cortex and become acidified, it is devoid of actin and associated proteins, although actin, coronin, and Arp2/3 are recruited once again at a late stage of endocytic transit, after the phagosome has become neutralized (Rauchenberger et al., 1997; Insall et al., 2001).
Here we report that local pressure applied to phagosomes that have already lost their actin coat can trigger actin accumulation in the cell cortex and that asymmetric deposition of the actin is followed by rocketing of the phagosomes. This discovery was made in the course of studies of Dictyostelium cells that had taken up yeast particles and were subsequently overlaid with a thin layer of agar to flatten the cells slightly, a procedure that brings more of the cell into a single focal plane for microscopy (Yumura et al., 1984). To identify proteins involved in phagosome rocketing and to determine the spatiotemporal orders of their recruitment before and during rocketing, we have labeled cells with GFP- and mRFP-tagged proteins including MyoB, members of the Arp2/3 complex, and coronin. As a marker of filamentous actin structures we used a truncated version of the LimE protein of Dictyostelium (LimE
coil), previously demonstrated to provide a brilliant label with low cytoplasmic background (Bretschneider et al., 2004; Diez et al., 2005).
Actin-mediated movement of pathogens has been shown to be powered by the assembly of actin filaments at the phagosome membrane (for review, see Stevens et al., 2006). Our data suggest that in pressure-induced phagosome rocketing the propulsive force is provided by actin assembly at the plasma membrane/phagosome interface. We hypothesize that this local, mechanically induced force production enables cells that are filled with rigid particles to escape from narrow spaces when they are moving in a heterogeneous environment.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Cells of the wild-type strain AX2–214 were transformed by electroporation with vectors expressing one or more of the following fusion proteins: GFP-Arp3 (Insall et al., 2001), mRFPmars-p41-Arc (below), mRFPmars-LimEcoil (Fischer et al., 2004; shortened to "mRFP-LimE" here), LimE-GFP (Bretschneider et al., 2004), VatM-GFP (Clarke et al., 2002a), coronin-GFP (Maniak et al., 1995), GFP-
-tubulin (Neujahr et al., 1998), and GFP-MyoB (a gift from Margaret Titus). The vector encoding mRFPmars-p41-Arc was constructed by cloning the sequence encoding amino acid residues 2–369 of p41-Arc (ArpC1/ArpD; DDB0214932), which was amplified by PCR using cDNA as a template, into the EcoRI-site of a pBsrH-based expression vector encoding mRFPmars (Fischer et al., 2004). A linker encoding GGSGGS was introduced between the mRFPmars and the p41-Arc sequence. Cells of the mutant 2A1 were transformed with the plasmid directing expression of GFP-Arp3.
D. discoideum strains were cultivated at 22°C in nutrient medium containing appropriate selective agents (G418 and/or blasticidin) for maintenance of the plasmids. For the rocketing experiments either living or heat-killed Saccharomyces cerevisiae cells were used. Living yeast cells were S. cerevisiae TH2–1B (Clarke et al., 2002a) and 5288C (whi5::kanR; Jorgensen et al., 2002). The yeast cells were washed in 17 mM K/Na-PO4 buffer, pH 6.0 (PB), before addition to the D. discoideum cells. Heat-killed yeast were prepared from a stock purchased from Sigma (St. Louis, MO; YSC-2) by boiling for 30 min and then labeled with tetramethyl rhodamine isothiocyanate (TRITC) according to Maniak et al. (1995) and stored frozen.
Confocal and Total Internal Reflection Fluorescence Microscopy.
For microscopic observation, D. discoideum cells in the exponential phase of growth were transferred to a chamber consisting of a plastic ring of 19-mm inner diameter and 4-mm height that had been attached to a coverglass with paraffin. Once the cells had settled, the nutrient medium was replaced with PB. After 30–60 min, yeast cells were added. After a period of 30 min to several hours, excess yeast were removed, and the cells were overlaid with a thin layer of agarose (Yumura et al., 1984). The chamber was covered with a second coverglass held in place by silicone grease. Confocal images were collected at 2–4-s intervals using a Zeiss LSM510 laser scanning confocal microscope equipped with a Plan-Apochromat 63x, 1.4 NA DIC objective (Thornwood, NY). S65T-GFP was excited with the 488-nm laser line of an argon laser with a 505–530-nm filter for emission, and mRFP was excited with the 543-nm line of a HeNe laser, with a 560-nm long pass filter for emission. An HFT UV/488/543/633 beam splitter was used.
Three-dimensional confocal time lapse sequences were taken using an Ultra View ERS-FRET system (Perkin Elmer-Cetus LAS; Norwalk, CT) on a TE-2000 microscope equipped with a Plan-Apochromat VC 100x, 1.4 NA objective (Nikon Instruments, Melville, NY). Stacks with 0.3-µm Z-spacing were continuously acquired with the fast sequential mode in which GFP and mRFP were excited sequentially with the 488- and 568-nm lines, respectively. The emission was detected through a triple dichroic and a double bandpass emission filter onto an EM-CCD camera.
Dual-emission total internal reflection fluorescence (TIRF) microscopy was performed essentially as previously described (Gerisch et al., 2004) using an Olympus IX-70 inverted microscope and 100x, 1.45 NA objective (Central Valley, PA). The TIRF condenser ("VisiTirf") was constructed in collaboration with Visitron GmbH. GFP and mRFP were excited simultaneously using for both fluorophores the 488-nm line from a Coherent Innova 70c laser (Santa Clara, CA), which was fiber optically coupled into the microscope after passing through an Opto-Electronique AOTF for intensity modulation. The fluorescence filter cube in the microscope body contained a 488/10 laser cleanup filter, z488 RDC dichroic, and LP 500 emission filter (AHF Analysen Technik, Tübingen, Germany). The green and red emission signals were separated using an Optical Insights (Tucson, AZ) DualView emission splitter, which contained a 595-nm dichroic followed by Chroma HQ 525/50 and HQ 630/60 emission filters (Rockingham, VT). The camera was a Roper Scientific MicroMax 512 BFT (Tucson, AZ) and the entire system was controlled using Metamorph software (Molecular Devices Corporation, Downington, PA).
Measurement of Pressure Required to Induce Actin Accumulation.
The pressure required to induce actin assembly at yeast-containing phagosomes was estimated using cells expressing LimE-GFP. Agar pieces 1 cm2 in area and 0.2 mm thick were covered by an O2 permeable membrane (ibidi, Munich, Germany) of the same size in area. On top of the membrane a PE 390 HD gauze layer (Schweizerische Seidengazefabrik, CH-9425 Thal SG, Switzerland) was placed to allow oxygen to diffuse in, followed by 1-cm2 stainless steel weights. Additional weights were sequentially added until the phagosome-associated accumulation of actin was observed.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
100 nm by an evanescent field generated at the substrate surface (Axelrod, 2001; Weisswange et al., 2005).
In Figure 1A, TIRF images are shown of a cell expressing GFP-tagged LimE, a fluorescent fusion protein that selectively labels filamentous actin structures (Bretschneider et al., 2004; Diez et al., 2005). This cell was fed heat-killed S. cerevisiae labeled with TRITC, and 4 h later, it was covered with a thin layer of agar that was allowed to press against the cells by slight evaporation. In this cell two phagosomes began to move in repeatedly changing directions in accord with the site of strongest actin deposition (Movie 1A). One phagosome moved in a series of runs and pauses. During each pause, an annulus of actin filaments visualized by GFP-LimE accumulated about the region of contact between the phagosome and the plasma membrane. The annulus of actin filaments was initially symmetrical about the stationary phagosome, but this symmetry was broken as the phagosome began to move. Thus, during runs, the moving phagosome left a trail of labeled actin filaments. The comet-like appearance of this trail led us to classify the pressure-induced phagosome movement as "rocketing." Although phagosome movement was commonly preceded by an annular accumulation of actin, sometimes a single focus of actin was observed (Movie 1B).
|
and GFP-MyoB, a class I myosin, dual emission TIRF microscopy allowed us to monitor the spatial and temporal relationship of actin assembly to recruitment of this motor protein close to the plasma membrane. Figure 1B shows images from Movie 2 of a phagosome that experienced multiple cycles of rocketing separated by pauses of a few seconds. When the phagosome paused, a build-up of structures containing GFP-MyoB occurred about the site where the phagosome pressed against the plasma membrane. When the phagosome resumed movement, the green signal of GFP-MyoB dominated at the rear of the phagosome. With increasing distance from the phagosome, the fluorescence became yellow, indicating merged signals, whereas the distal portion of the phagosome trail was labeled red by mRFP-LimE. These data show that MyoB is enriched at the plasma membrane close to the phagosome and that this enrichment precedes the onset of rocketing. The three-dimensional distribution of MyoB is clearly revealed in Z-scans obtained with a spinning disk microscope. The enrichment of MyoB at sites of plasma membrane/phagosome contact is observed not only for rocketing phagosomes, but also for phagosomes prevented from rocketing by strong pressure. Figure 2 shows such a phagosome on which actin extends up to the midregion, whereas MyoB is concentrated at regions of the plasma membrane attached to the glass surface at the bottom and to the agar layer at the top.
|
Dynamics of the Arp2/3 Complex Related to Phagosome Rocketing
Distribution of the Arp2/3 complex in relation to MyoB and actin at rocketing phagosomes was examined using confocal microscopy. A bright ring of GFP-MyoB appeared wherever a yeast-containing phagosome pressed against the plasma membrane; this ring was interior to that of both mRFP-p41-Arc (a subunit of the Arp2/3 complex) and mRFP-LimE (Figure 3, A and B). Several pairs of fluorescent proteins were examined: GFP-MyoB with mRFP-LimE or mRFP-p41-Arc, and GFP-Arp3 with mRFP-LimE. For all images, the focal plane was close to the plasma membrane attached to the substratum unless otherwise specified. For moving phagosomes, GFP-Arp3 and mRFP-LimE exhibited almost complete overlap, although the mRFP-LimE signal was slightly enriched close to the phagosome when a deeper focal plane was examined (Figure 3, C and D; Movie 3).
|
). These cells are defective in cytokinesis because they lack the clathrin light chain, and their large size allows them to phagocytose many yeast cells. When we fed TRITC-labeled yeast to 2A1 cells expressing GFP-Arp3 and induced rocketing, the yeast-containing phagosomes began to move, as shown in Figure 4. A focal plane close to the glass substratum revealed bright rings of GFP-Arp3 that circumscribed areas of phagosome contact with the plasma membrane. A new ring of GFP-Arp3 was formed each time a phagosome paused. When the phagosome moved away, the earlier ring was left behind and finally disappeared (Movie 4; Figure 4A). Often, phagosome movement was back-and-forth, as seen for the cell in Movie 4 and also for another cell in Movie 5 and Figure 4B. In the latter cell, the phagosomes soon converted to a pinwheel motion, and the GFP-Arp3 formed tracks extending between the moving phagosomes (Figure 4B, second panel). Through-focus confocal microscopy revealed enrichment of GFP-Arp3 at both the upper and lower plasma membrane, but not in midcell focal planes displaying equatorial sections of the phagosome (Figure 4B, third panel, and Movie 5B). Thus, GFP-Arp3 was more highly enriched at the plasma membrane than at the membrane of the phagosome.
|
and coronin-GFP. Coronin has been shown in various cell types to bind to and inactivate the Arp2/3 complex (Humphries et al., 2002; Rodal et al., 2005; Cai et al., 2005). Therefore, coronin was expected to localize to sites where net actin polymerization has turned into depolymerization. In the time series shown in Figure 5, images were acquired every 4 s. When the phagosome containing an unlabeled yeast particle was moving in a confined area with frequent pauses and changes of direction, a red patch of mRFP-LimE invariably labeled the rear of the moving phagosome. This red cluster changed to yellow-green (coronin-GFP merged with mRFP-LimE) by the next frame, to pure green (coronin-GFP mostly alone) by the third frame, and disappeared by the fourth frame. In the meantime, the phagosome had developed a new tail labeled with mRFP-LimE, which changed to the green coronin label while the phagosome moved off in a different direction. The conversion of red patches into green occurred repeatedly during this time series (Figure 5, panels 0, 8, and 16, and Movie 6).
|
; Maniak, 2001). The enzyme responsible for acidifying endosomes is the vacuolar H+-ATPase (V-ATPase), which is delivered to new phagosomes within a few minutes after uptake and is retrieved from phagosome membranes before exocytosis (Clarke et al., 2002a). Using cells expressing mRFP-LimE together with GFP-tagged VatM, a subunit of the V-ATPase (Clarke et al., 2002a), we established that the membrane of rocketing phagosomes did contain VatM-GFP, indicating that the phagosomes were in the acidic phase of endocytic transit (Figure 6A; Movie 7). Thus, rocketing is not limited to phagosomes in the very early or late stages of the endocytic cycle.
|
; Clarke and Maddera, 2006). To distinguish pressure-induced rocketing from microtubule-dependent phagosome movement, cells expressing GFP--tubulin and mRFP-LimE were allowed to take up yeast and were thereafter treated with 20 µM nocodazole. After 30 min, the cytoplasmic microtubules had depolymerized, leaving only short stubs attached to the centrosome. When these cells were overlaid with agar, rocketing did occur, as seen in Figure 6B, showing a rocketing phagosome well separated from the microtubule stubs.
Rocketing Phagosomes Generate Forces that Displace the Microtubule System and the Nucleus
Although phagosome rocketing was usually intermittent or back-and-forth, some rocketing phagosomes exhibited long runs about the cell, and these runs often involved collisions with the cell nucleus. Figure 7A shows a cell expressing mRFP-LimE and GFP-MyoB, which contained a phagosome that was moving with a circular trajectory. This rocketing phagosome made three circuits through the cytoplasm during the 232-s time series, traveling at an average velocity of 23 µm/min (Movie 8). In each circuit the phagosome collided with the nucleus, distorting and displacing it. In Figure 7A, frames from one circuit are shown in a focal plane slightly above the plasma membrane. A comet-like flare of actin was evident behind the moving phagosome, whereas GFP-MyoB only rarely entered the focal plane.
|
-tubulin and mRFP-LimE in which a rocketing phagosome was moving about in a rather small area, a frequently observed behavior, suggesting that microtubules were hindering or guiding phagosome movement. Each time the phagosome collided with the nucleus, the force of rocketing was sufficient to displace the nucleus and stretch out the microtubules (Figure 7B, panel 0). As soon as the rocketing phagosome turned away from the nucleus, the microtubules resumed their normal undulating appearance (panel 8). When moving orthogonally to the microtubules, the rocketing phagosome shoved against them, bending the microtubules laterally (panel 20). We infer that rocketing phagosomes are channeled toward the centrosome by microtubules and that rocketing phagosomes strike the centrosome-attached nucleus with considerable force. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). It has been proposed that these pathogens have harnessed a pathway of localized actin polymerization that is normally used for cell motility (see review by Rafelski and Theriot, 2004) and intracellular vesicle movement (Merrifield et al., 1999, Taunton et al., 2000; Kim et al., 2006). Rocketing endosomes have been observed in lanthanum/zinc-treated macrophages (Southwick et al., 2003), in rat basophilic leukemia cells treated with phorbol ester or subjected to hypoosmotic shock (Hayes et al., 2004), and in cells caused to overexpress type I phosphatidylinositol phosphate 5-kinase (Rozelle et al., 2000). In cell-free systems, the mode of actin-driven movement depended on physical parameters such as bead size; beads of <3 µm diameter moved steadily, whereas larger beads displayed a hopping motion (Bernheim-Groswasser et al., 2002).
In this article we have investigated a conditional type of phagosome rocketing, one that is induced in intact cells by mechanical pressure under physiological conditions in the absence of chemical or biological elicitors. In cells that had phagocytosed yeast particles ranging from 3 to 5 µm in diameter, we observed various types of rocketing: phases of straight propagation interrupted by periods of immobility, back-and-forth movements, and persistent travel in circular tracks. Intermittent or back-and-forth rocketing occurred commonly. Long circular runs, while rare, allowed us to determine that the rate of phagosome movement corresponded to that previously reported for rocketing pathogens, which are also propelled by actin polymerization (Theriot et al., 1992).
The large size of yeast-filled phagosomes enabled us to localize the proteins associated with the tail of rocketing phagosomes, including actin itself, predominantly to the plasma membrane-anchored cell cortex rather than to the membrane of the phagosome. In this respect phagosome rocketing in Dictyostelium is distinguished from the previously reported rocketing of pathogens or vesicles, which has always been attributed to the formation of an actin tail solely on the surface of the rocketing particle. The same is true for the cycles of actin assembly called "flashing," which occur on the surface of phagosomes containing Listeria or other particulate matter (Yam and Theriot, 2004).
In cells that had eaten yeast and been flattened by an agar overlay, rings or patches of GFP-MyoB enrichment formed at both the upper and lower plasma membrane where the phagosome pressed against it. MyoB is a class I myosin with a lipid-binding site and multiple protein-interaction domains; it binds to actin, to the plasma membrane (Senda et al., 2001) and, through the linker protein CARMIL, to the Arp2/3 complex (Jung et al., 2001). MyoB accumulated at the phagosome–plasma membrane interface before the onset of rocketing. For phagosomes that did begin to move, TIRF microscopy showed GFP-MyoB on the plasma membrane at the rear of the moving phagosome, with filamentous actin extending behind (Figure 1B). For some large yeast-containing phagosomes, the phagosome was pressed too strongly to allow movement, leading to the accumulation of very high levels of the fluorescent markers (Figure 3A; central phagosome in Movie 8). Rocketing phagosomes typically paused periodically, in which case a new buildup of MyoB occurred at the plasma membrane before another spurt of rocketing (Figure 1B). The correlation between these events suggests that MyoB and other class I myosins may act in the initiation of mechanically induced rocketing. Because there are seven class I myosins in Dictyostelium with considerable overlap in function (reviewed by Uyeda and Titus, 1997; de la Roche and Côté, 2001), single and double gene disruptions tend to cause reduction rather than loss of function (Novak et al., 1995; Jung et al., 1996; Falk et al., 2003). Our preliminary observations of mutant strains conform to this pattern. Although actin-powered phagosome movement did occur in mutant cells lacking MyoB or both MyoA and MyoB, sustained movement appeared to be rare, and little force seemed to be exerted by a moving phagosome (Supplementary Movie 1). In contrast, pressure-induced rocketing in strain JH10, the parent of the myoB– mutant, was quite robust (unpublished data). Additional work is needed to verify and extend these results, in particular to apply defined pressure, as described in this report, and to develop a means of measuring the force generated by a moving phagosome, for instance by the use of magnetic beads (Uhde et al., 2005).
Confocal microscopy of phagosome rocketing in double-labeled strains showed that the Arp2/3 complex extended beyond the outer perimeter of the MyoB (Figure 3B). The greatest enrichment of the Arp2/3 complex was at the plasma membrane, such that bright Arp2/3 complex "prints" of the phagosome were left behind when the phagosome moved away (Figure 4). The third protein studied, coronin, is like Arp2/3 a tail constituent common to rocketing phagosomes and intracellularly moving Listeria (David et al., 1998), where coronin appears to have a regulatory function, one that is not essential for bead propulsion in vitro (Carlier et al., 2003). Similarly, our analysis of coronin-null cells indicated that coronin is not essential for mechanically induced phagosome rocketing (unpublished data). The localization of coronin to the end of actin tails away from the rocketing phagosomes themselves in Dictyostelium suggests a role in actin disassembly, in line with the negative control of Arp2/3 complex activity reported for coronin in yeast and mammalian cells (Humphries et al., 2002; Rodal et al., 2005; Cai et al., 2005).
If one considers the narrow space between phagosome and plasma membrane as an equivalent of the membrane fold at the leading edge of a lamellipod, the similarity of phagosome rocketing and global cell motility becomes obvious. All three components shown here to be associated with rocketing phagosomes, MyoB, the Arp2/3 complex, and coronin, are also enriched at the front of Dictyostelium cells (de Hostos et al., 1991; Fukui et al., 1999; Insall et al., 2001). At both locations it remains open whether MyoB contributes directly through its motor activity to motility, or is only instrumental in the Arp2/3-dependent assembly of a dense network of actin, whose polymerization would then provide the force for phagosome movement. The Arp2/3 complex is an intrinsic component of the dense actin assemblies at the leading edge of chemotaxing Dictyostelium cells (Diez et al., 2005), as it is in other cells (Pollard and Borisy, 2003). Coronin accumulates in a zone separated from the very edge of a migrating cell (K. Anderson, T. Bretschneider, and G. Gerisch, unpublished data), comparable to its lateral separation from the phagosome membrane.
A sequence of events similar to that observed in phagosome rocketing has recently been proposed for the internalization of clathrin-coated vesicles in yeast. Kaksonen et al. (2005) found that during clathrin-mediated endocytosis in S. cerevisiae, a WASP/Myo module at the plasma membrane regulates actin filament nucleation via the Arp2/3 complex and that addition of actin monomers at the plasma membrane drives the clathrin-coated vesicle inward. The behavior of MyoB and Arp2/3 in Dictyostelium during pressure-induced rocketing closely resembles the behavior of their yeast counterparts during clathrin-mediated endocytosis, suggestive of a similar mechanism for site-specific actin recruitment. An open question is how pressure triggers the recruitment of MyoB (or its possible upstream partners) in the signal transduction pathway mediating phagosome rocketing. One possibility is that distortion of the cortical actin network may stimulate MyoB recruitment, just as the stretching of cytoskeletons from detergent-treated cells activates a signaling cascade that results in the binding of specific proteins from the cytoplasm (Tamada et al., 2004). Alternatively, simple contact may suffice. In mammalian cells, the contact of an extracellular vaccinia virus (Frischknecht and Way, 2001; Newsome et al., 2006) or enteropathic Escherichia coli (Swimm et al., 2004) with the cell surface triggers a signaling cascade that involves multiple tyrosine kinases and leads to the recruitment of N-WASP and Arp2/3 at the plasma membrane beneath the pathogen, stimulating actin polymerization there.
What are the functional implications of actin assembly that is stimulated at sites of contact between a phagosome and the plasma membrane? We envisage two functions of mechanically induced rocketing. One is to keep the cell cortex free of trafficking endosomes until exocytosis commences. Indeed, endosomes are concentrated in the inner region of Dictyostelium cells rather than at their periphery. In compressed cells, large phagosomes cannot move away from the plasma membrane, so actin assembly is continuously reiterated at new sites of the plasma membrane. Second, under these conditions of compression, actin-dependent force generation provides a mechanism of propelling large phagosomes filled with rigid particles out of a narrow space, where the particles would hinder the cell's movement. This escape mechanism may be important not only for Dictyostelium cells, which in their natural habitat migrate between soil particles, but also for those cells of higher organisms that penetrate into embryonic or adult tissues.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-04-0365) on September 13, 2006.
Present address: Institute for Cell Biology (ABI), Ludwig Maximilians University Munich, 80336 München, Germany.
Address correspondence to: Margaret Clarke (clarkem{at}omrf.ouhsc.edu)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Axelrod, D. (2001). Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774.[CrossRef][Medline]
Bernheim-Groswasser, A., Wiesner, S., Golsteyn, R. M., Carlier, M. F., Sykes, C. (2002). The dynamics of actin-based motility depend on surface parameters. Nature 417, 308–311.[CrossRef][Medline]
Bretschneider, T., Diez, S., Anderson, K., Heuser, J., Clarke, M., Müller-Taubenberger, A., Köhler, J., Gerisch, G. (2004). Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells. Curr. Biol 14, 1–10.[CrossRef][Medline]
Cai, L., Holoweckyj, N., Schaller, M. D., Bear, J. E. (2005). Phosphorylation of coronin 1B by protein kinase C regulates interaction with Arp2/3 and cell motility. J. Biol. Chem 280, 31913–31923.
Carlier, M.-F., Le Clainche, C., Wiesner, S., Pantaloni, D. (2003). Actin-based motility: from molecules to movement. Bioessays 25, 336–345.[CrossRef][Medline]
Clarke, M. and Maddera, L. (2006). Phagocyte meets prey: uptake, internalization, and killing of bacteria by Dictyostelium amoebae. Eur. J. Cell Biol 85, 1001–1010.[CrossRef][Medline]
Clarke, M., Köhler, J., Arana, Q., Liu, T., Heuser, J., Gerisch, G. (2002a). Dynamics of the vacuolar H(+)-ATPase in the contractile vacuole complex and the endosomal pathway of Dictyostelium cells. J. Cell Sci 115, 2893–2905.
Clarke, M., Köhler, J., Heuser, J., Gerisch, G. (2002b). Endosome fusion and microtubule-based dynamics in the early endocytic pathway of Dictyostelium. Traffic 3, 791–800.[CrossRef][Medline]
David, V., Gouin, E., Van Troys, M., Grogan, A., Segal, A. W., Ampe, C., Cossart, P. (1998). Identification of cofilin, coronin, Rac and capZ in actin tails using a Listeria affinity approach. J. Cell Sci 111, 2877–2884.[Abstract]
de Hostos, E. L., Bradtke, B., Lottspeich, F., Guggenheim, R., Gerisch, G. (1991). Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits. EMBO J 10, 4097–4104.[Medline]
de la Roche, M. A. and Côté, G. P. (2001). Regulation of Dictyostelium myosin I and II. Biochim. Biophys. Acta 1525, 245–261.[Medline]
Diez, S., Gerisch, G., Anderson, K., Müller-Taubenberger, A., Bretschneider, T. (2005). Subsecond reorganization of the actin network in cell motility and chemotaxis. Proc. Natl. Acad. Sci. USA 102, 7601–7606.
Falk, D. L., Wessels, D., Jenkins, L., Pham, T., Kuhl, S., Titus, M. A., Soll, D. R. (2003). Shared, unique, and redundant functions of three members of the class I myosins (MyoA, MyoB, and MyoF) in motility and chemotaxis in Dictyostelium. J. Cell Sci 116, 3985–3999.
Fischer, M., Haase, I., Simmeth, E., Gerisch, G., Müller-Taubenberger, A. (2004). A brilliant monomeric red fluorescent protein to visualize cytoskeleton dynamics in Dictyostelium. FEBS Lett 577, 227–232.[CrossRef][Medline]
Frischknecht, F. and Way, M. (2001). Surfing pathogens and the lessons learned for actin polymerization. Trends Cell Biol 11, 30–38.[CrossRef][Medline]
Fukui, Y., de Hostos, E., Yumura, S., Kitanishi-Yumura, T., Inoué, S. (1999). Architectural dynamics of F-actin in eupodia suggests their role in invasive locomotion in Dictyostelium. Exp. Cell Res 249, 33–45.[CrossRef][Medline]
Gerisch, G., Bretschneider, T., Müller-Taubenberger, A., Simmeth, E., Ecke, M., Diez, S., Anderson, K. (2004). Mobile actin clusters and traveling waves in cells recovering from actin depolymerization. Biophys. J 87, 3493–3503.[Medline]
Hayes, M. J., Merrifield, C. J., Shao, D., Ayala-Sanmartin, J., Schorey, C. D., Levine, T. P., Proust, J., Curran, J., Bailly, M., Moss, S. E. (2004). Annexin 2 binding to phosphatidylinositol 4,5-bisphosphate on endocytic vesicles is regulated by the stress response pathway. J. Biol. Chem 279, 14157–14164.
Humphries, C. L., Balcer, H. I., D'Agostino, J. L., Winsor, B., Drubin, D. G., Barnes, G., Andrews, B. J., Goode, B. L. (2002). Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin. J. Cell Biol 159, 993–1004.
Insall, R., Müller-Taubenberger, A., Machesky, L., Köhler, J., Simmeth, E., Atkinson, S. J., Weber, I., Gerisch, G. (2001). Dynamics of the Dictyostelium Arp2/3 complex in endocytosis, cytokinesis, and chemotaxis. Cell Motil. Cytoskelet 50, 115–128.[Medline]
Jorgensen, P., Nishikawa, J. L., Breitkreutz, B. J., Tyers, M. (2002). Systematic identification of pathways that couple cell growth and division in yeast. Science 297, 395–400.
Jung, G., Remmert, K., Wu, X., Volosky, J. M., Hammer, J. A. III. (2001). The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains. J. Cell Biol 153, 1479–1497.
Jung, G., Wu, X., Hammer, J. A. III. (1996). Dictyostelium mutants lacking classic myosin I isoforms reveal combinations of shared and distinct functions. J. Cell Biol 133, 305–323.
Kaksonen, M., Toret, C. P., Drubin, D. G. (2005). A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell 123, 305–320.[CrossRef][Medline]
Kim, K., Galletta, B. J., Schmidt, K. O., Chang, F. S., Blumer, K. J., Cooper, J. A. (2006). Actin-based motility during endocytosis in budding yeast. Mol. Biol. Cell 17, 1354–1363.
Konzok, A., Weber, I., Simmeth, E., Hacker, U., Maniak, M., Müller-Taubenberger, A. (1999). DAip1, a Dictyostelium homologue of the yeast actin–interacting protein 1, is involved in endocytosis, cytokinesis, and motility. J. Cell Biol 146, 453–464.
Maniak, M. (2001). Fluid-phase uptake and transit in axenic Dictyostelium cells. Biochim. Biophys. Acta 1525, 197–204.[Medline]
Maniak, M., Rauchenberger, R., Albrecht, R., Murphy, J., Gerisch, G. (1995). Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein tag. Cell 83, 915–924.[CrossRef][Medline]
Merrifield, C. J., Moss, S. E., Ballestrem, C., Imhof, B. A., Wunderlich, I., Almers, W. (1999). Endocytic vesicles move at the tips of actin tails in cultured mast cells. Nat. Cell Biol 1, 72–74.[CrossRef][Medline]
Morita, Y. S., Jung, G., Hammer, J. A. III, Fukui, Y. (1996). Localization of Dictyostelium myoB and myoD to filopodia and cell-cell contact sites using isoform-specific antibodies. Eur. J. Cell Biol 71, 371–379.[Medline]
Neujahr, R., Albrecht, R., Köhler, J., Matzner, M., Schwartz, J. M., Westphal, M., Gerisch, G. (1998). Microtubule-mediated centrosome motility and the positioning of cleavage furrows in multinucleate myosin II-null cells. J. Cell Sci 111, 1227–1240.[Abstract]
Newsome, T. P., Weisswange, I., Frischknecht, F., Way, M. (2006). Abl collaborates with Src family kinases to stimulate actin-based motility of vaccinia virus. Cell Microbiol 8, 233–241.[CrossRef][Medline]
Novak, K. D., Peterson, M. D., Reedy, M. C., Titus, M. A. (1995). Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis. J. Cell Biol 131, 1205–1221.
Peracino, B., Borleis, J., Jin, T., Westphal, M., Schwartz, J. M., Wu, L., Bracco, E., Gerisch, G., Devreotes, P., Bozzaro, S. (1998). G protein beta subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton. J. Cell Biol 141, 1529–1537.
Pollard, T. D. and Borisy, G. G. (2003). Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465.[CrossRef][Medline]
Rafelski, S. M. and Theriot, J. A. (2004). Crawling toward a unified model of cell mobility: spatial and temporal regulation of actin dynamics. Annu. Rev. Biochem 73, 209–239.[CrossRef][Medline]
Rauchenberger, R., Hacker, U., Murphy, J., Niewöhner, J., Maniak, M. (1997). Coronin and vacuolin identify consecutive stages of a late, actin-coated endocytic compartment in Dictyostelium. Curr. Biol 7, 215–218.[CrossRef][Medline]
Rodal, A. A., Sokolova, O., Robins, D. B., Daugherty, K. M., Hippenmeyer, S., Riezman, H., Grigorieff, N., Goode, B. L. (2005). Conformational changes in the Arp2/3 complex leading to actin nucleation. Nature Struct. Mol. Biol 12, 26–31.
Rozelle, A. L., Machesky, L. M., Yamamoto, M., Driessens, M. H., Insall, R. H., Roth, M. G., Luby-Phelps, K., Marriott, G., Hall, A., Yin, H. L. (2000). Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3. Curr. Biol 10, 311–320.[CrossRef][Medline]
Senda, S., Lee, S. F., Cote, G. P., Titus, M.A. (2001). Recruitment of a specific amoeboid myosin I isoform to the plasma membrane in chemotactic Dictyostelium cells. J. Biol. Chem 276, 2898–2904.
Southwick, F. S., Li, W., Zhang, F., Zeile, W. L., Purich, D. L. (2003). Actin-based endosome and phagosome rocketing in macrophages: activation by the secretagogue antagonists lanthanum and zinc. Cell Motil. Cytoskelet 54, 41–55.[CrossRef][Medline]
Stevens, J. M., Galyov, E. E., Stevens, M. P. (2006). Actin-dependent movement of bacterial pathogens. Nat. Rev. Microbiol 4, 91–101.[CrossRef][Medline]
Swimm, A., Bommarius, B., Li, Y., Cheng, D., Reeves, P., Sherman, M., Veach, D., Bornmann, W., Kalman, D. (2004). Enteropathogenic Escherichia coli use redundant tyrosine kinases to form actin pedestals. Mol. Biol. Cell 15, 3520–3529.
Tamada, M., Sheetz, M. P., Sawada, Y. (2004). Activation of a signaling cascade by cytoskeleton stretch. Dev. Cell 7, 709–718.[CrossRef][Medline]
Taunton, J., Rowning, B. A., Coughlin, M. L., Wu, M., Moon, R. T., Mitchison, T. J., Larabell, C. A. (2000). Actin-dependent propulsion of endosomes and lysosomes by recruitment of N–WASP. J. Cell Biol 148, 519–530.
Theriot, J. A., Mitchison, T. J., Tilney, L. G., Portnoy, D. A. (1992). The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature 357, 257–260.[CrossRef][Medline]
Titus, M. A. (2000). The role of unconventional myosins in Dictyostelium endocytosis. J. Eukaryot. Microbiol 47, 191–196.[CrossRef][Medline]
Wang, J., Virta, V. C., Riddelle-Spencer, K., O'Halloran, T. J. (2003). Compromise of clathrin function and membrane association by clathrin light chain deletion. Traffic 4, 891–901.[CrossRef][Medline]
Uhde, J., Tr-Oganessian, N., Pink, D. A., Sackmann, E., Boulbitch, A. (2005). Viscoelasticity of entangled actin networks studied by long-pulse magnetic bead microrheometry. Phys. Rev. E 72, 61916–61926.[CrossRef]
Uyeda, T.Q.P. and Titus, M. A. (1997). The myosins of Dictyostelium. In: Dictyostelium: A Model System for Cell and Developmental Biology, ed. Y. Maeda, K. Inouye, I. Takeuchi. Tokyo, Japan: University Academic Press, 43–64.
Weisswange, I., Bretschneider, T., Anderson, K. I. (2005). The leading edge is a lipid diffusion barrier. J. Cell Sci 118, 4375–4380.
Yam, P. T. and Theriot, J. A. (2004). Repeated cycles of rapid actin assembly and disassembly on epithelial cell phagosomes. Mol. Biol. Cell 15, 5647–5658.
Yumura, S., Mori, H., Fukui, Y. (1984). Localization of actin and myosin for the study of amoeboid movement in Dictyostelium using improved immunofluorescence. J. Cell Biol 99, 894–899.
This article has been cited by other articles:
|
|
 |
|
  D. T. Brandt, S. Marion, G. Griffiths, T. Watanabe, K. Kaibuchi, and R. Grosse Dia1 and IQGAP1 interact in cell migration and phagocytic cup formation J. Cell Biol., July 10, 2007; 178(2): 193 - 200. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
