In studies reported here, we demonstrate that modest overexpression of a Rho family GTPase, RacC, results in changes in cell morphology and actin cytoskeleton organization in the simple eukaryote D. discoideum
and leads to a significant increase in phagocytosis rates. Cells overexpressing HA-tagged RacC at a level three- to fourfold higher than endogenous RacC contained F-actin–rich “membrane blebs” that were organized into dynamic dorsal and lateral surface structures that we term petalopodia, because they resemble the petals of a flower. The petalopodia structures described here are not to be confused with earlier defined structures termed “petaloid coelomocytes,” from the sea urchin Strongylocentrotus droebachiensis
), which spontaneously change into filopodia to induce the clotting process in the celom of this organism. Although the petalopodia appearance is reminiscent of coronin-containing “crown-like” structures described previously by Hacker et al. (1997)
, we believe that they are distinct from one another. First of all, crown-like structures are predominantly located on the dorsal surface of the cell, whereas we observed petalopodia forming on both the lateral edges and the dorsal surfaces of RacC WT(+) cells. In addition, crown-like structures were only infrequently observed in RacC WT(+) cells. Finally, crown-like structures are associated with the engulfment of both fluid-phase and particulate matter and open up as a cup-like structure to bring material into the cell. Using video microscopy to examine GFP-labeled ABP-120 in the RacC WT(+) cells, we observed that the cellular F-actin never formed into open crown-like structures (unpublished results). Because the petalopodia are also morphologically different from other previously identified actin-based structures such as lamellipodia or filopodia, they may represent a new form of actin-based structure.
The biochemical mechanisms regulating petalopodia formation are not known, although data presented here suggest that their formation involves RacC and one or more wortmannin- or LY294002-sensitive PI 3-kinase(s). The fact that PI 3-kinase activity appeared necessary for the formation of petalopodia was not unexpected because previous studies have indicated that these enzymes were also necessary for PDGF-induced membrane ruffling in porcine aortic endothelial cells (Wennstrom et al., 1994
) and the Ras-mediated signal transduction pathway that leads to actin cytoskeleton changes (Rodriguez-Viciana et al., 1997
). Rac can also interact with PI 3-kinases, and growth factors can activate Rac through activation of PI 3-kinases (Hawkins et al., 1995
; Bokoch et al., 1996
); however, our results do not distinguish between PI 3-kinase and RacC operating in the same or parallel pathways to regulate the formation of petalopodia, although we favor the first possibility.
RacC WT(+) cells internalized both latex beads and bacteria at three times the rate of control cells. This same phenotype was observed for cells overexpressing non-epitope–tagged RacC, suggesting that the HA epitope does not influence phagocytosis (unpublished results). Interestingly, treating cells with the PI 3-kinase–specific inhibitors Wortmannin and LY294002, which inhibited the formation of petalopodia, only slightly and insignificantly impaired RacC WT(+) cells in their ability to internalize latex beads and bacteria (Figure B). These drugs also did not inhibit phagocytosis in control cells and, in fact, caused a slight stimulation in bead uptake. These results indicate that, although PI 3-kinases may be necessary for RacC-induced morphological and actin cytoskeletal changes, they do not play a major role in RacC-induced phagocytosis, and that RacC-induced actin cytoskeleton changes (petalopodia formation) may not play an important role in the stimulation of phagocytosis. These results are in contrast to other studies that suggested PI 3-kinases were necessary for phagocytosis in macrophages (Araki et al., 1996
). Although our results were unexpected, they are consistent with previous observations indicating that membrane ruffling (dependent on PI 3-kinase activity) could be functionally uncoupled from a stimulation of pinocytosis in mammalian cells. (Kotani et al., 1994
; Li et al., 1997
Interestingly, RacC WT(+) cells were inhibited in their ability to internalize FITC–dextran markers via macropinocytosis, although they internalized particles at rates higher than control cells. This is consistent with previous experiments that indicated that Rac1 and Rho may negatively regulate receptor-mediated endocytosis (Lamaze et al., 1996
), although Rho proteins have been implicated in positively regulating bulk fluid-phase pinocytosis (Schmalzing et al., 1995
). This is an important observation, because it has been proposed previously that phagocytosis and macropinocytosis are biochemically similar processes in macrophages (Araki et al., 1996
) and D. discoideum
(Hacker et al., 1997
). In contrast, our data suggest that these processes are mechanistically distinct in D. discoideum
. Because the formation of crown-like structures is associated with macropinocytosis (Hacker et al., 1997
), this evidence also supports our hypothesis that the actin-based petalopodia structures are functionally distinct from crown-like structures; however, our results presented here do not distinguish between RacC acting as a negative regulator of macropinocytosis or as an inducer of the formation of F-actin structures that are incompatible with macropinocytosis.
Several lines of evidence indicate that PKC may play a role in the regulation of the actin cytoskeleton and/or phagocytosis in mammalian cells such as macrophages (Allen and Aderem, 1996
). For instance, PKC is activated in response to ligation of the Fc receptor in human monocytes (Zheleznyak and Brown, 1992
), suggesting that it may be involved in signal transduction leading to phagocytosis; however, our studies presented here indicated that PKC per se might not play a major role in regulating phagocytosis in Dictyostelium
, because treatment of cells with the PKC inhibitors bis-indolylmaleimide I and chelerythrine did not reduce phagocytosis. Importantly, it has been shown previously that bis-indolylmaleimide I–sensitive forms of PKC do exist in D. discoideum
(Phillips et al., 1997
); however, bacteria and latex bead uptake were significantly inhibited when cells were treated with the inhibitor calphostin C (a drug that competes with phorbol esters for binding to DAG-binding domains), suggesting that a non-PKC protein containing a DAG-binding motif was mediating phagocytosis in Dictyostelium
. Also, calphostin C treatment effectively reversed the formation of petalopodia in RacC WT(+) cells. Whether this unidentified protein is signaling upstream or downstream of RacC remains to be determined at this time and will require the generation of a Dictyostelium
cell line that overexpresses a constitutively active form of RacC. Interestingly, many Rho family GEFs contain DAG-binding domains (Cerione and Zheng, 1996
), suggesting that the target of calphostin C may be an upstream activator of RacC.
In addition to inhibiting macropinocytosis, RacC overexpression also led to a decrease in the rate of efflux of fluid-phase and lysosomal enzymes from the endolysosomal system of D. discoideum
. This block appeared to be imposed at the level of fluid-phase movement from acidic to nonacidic compartments. Although yet to be demonstrated conclusively, this result suggests that RacC overexpression may reduce the transport of fluid phase from lysosomes to postlysosomes, a process that may depend on proper F-actin regulation and the function of other GTPases and lipid kinases. For instance, in addition to RacC, we (and others) have demonstrated that Rab7, a Rab4-like GTPase (RabD), the PI 3-kinases DdPIK1 and DdPIK2, vacuolin B, and actin regulate this step (Bush et al., 1996
; Temesvari et al., 1996
; Buczynski et al., 1997a
; Hacker et al., 1997
; Jenne et al., 1998
). It will be important to determine whether (and how) these proteins functionally interact and, if so, the order of interaction required for the formation of postlysosomes. Why RacC overexpression mediates disparate effects on phagocytosis and fluid-phase pinocytosis and trafficking is unclear at this time, but it raises the question that we are currently experimentally attempting to answer: are the effects mediated by RacC on the movement of fluid-phase matter throughout the endolysosomal system direct, or are they indirect effects caused by a recruitment of F-actin that leads to a stimulation of phagocytosis?
We and others have demonstrated recently that other GTPases, not belonging to the Rho subfamily, can also regulate the distribution of the cellular actin cytoskeleton and endolysosomal processes. For example, overexpressing wild-type or constitutively activated (GTP-bound) forms of a Rap1-like GTPase induced the formation of lamellipodia (Rebstein et al., 1997) and stimulated phagocytosis in D. discoideum, whereas overexpressing dominant negative (GDP-bound) forms of Rap1 inhibited phagocytosis (unpublished results). Taken together, these experiments suggest a possible model in which small GTPases may interact in the signal transduction pathway regulating phagocytosis in Dictyostelium. Defining the biochemical nature of these interactions is a subject of future investigations.