Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Jul 6;24(13):2254-64.
doi: 10.1038/sj.emboj.7600716. Epub 2005 Jun 16.

A Rab21/LIM-only/CH-LIM complex regulates phagocytosis via both activating and inhibitory mechanisms

Taruna Khurana  1 , Joseph A Brzostowski, Alan R Kimmel
Affiliations

A Rab21/LIM-only/CH-LIM complex regulates phagocytosis via both activating and inhibitory mechanisms

Taruna Khurana et al. EMBO J. .

Abstract

We have identified two LIM domain proteins, LimF and ChLim, from Dictyostelium that interact with each other and with the small, Rab5-related, Rab21 GTPase to collectively regulate phagocytosis. To investigate in vivo functions, we generated cell lines that lack or overexpress LimF and ChLim and strains that express activating or inhibiting variants of Rab21. Overexpression of LimF, loss of ChLim, or expression of constitutively active Rab21 increases the rate of phagocytosis above that of wild type. Conversely, loss of LimF, overexpression of ChLim, or expression of a dominant-negative Rab21 inhibits phagocytosis. Our studies using cells carrying multiple mutations in these genes further indicate that ChLim antagonizes the activating function of Rab21-GTP during phagocytosis; in turn, LimF is required for Rab21-GTP function. Finally, we demonstrate that ChLim and LimF localize to the phagocytic cup and phago-lysosomal vesicles. We suggest that LimF, ChLim, and activated Rab21-GTP participate as a novel signaling complex that regulates phagocytic activity.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The interacting partners LimF, ChLim, and Rab21. (A) LimF has three LIM domains. ChLim has an N-terminal CH domain, three LIM domains, and four LRDs. Rab21 is a small GTPase; the dominant-negative (T21N) and constitutively activated (Q66L) mutations are indicated. (B) Interactions of LimF with ChLim and Rab21 were studied in a yeast two-hybrid system. All cells were able to grow in the absence of histidine (H), uracil (U), and tryptophan (W). Positive interaction (+) is indicated by growth in the absence of leucine (L) and by enhanced β-galactosidase activity using X-gal as substrate. ChLim is full-length ChLim, CHChLim is the CH domain of ChLim, and LIMChLim is the Lim domain of ChLim. Images of yeast are representative data for full-length ChLim.
Figure 2
Figure 2
Interactions among LimF, ChLim, and Rab21. (A) Lysates of cells expressing GFP-ChLim were mixed with immobilized GST-LimF and GST control beads, and bound proteins analyzed by immunoblotting (IB) with α-GFP and α-Rab21. (B) Lysates of cells expressing HA-Rab21 were mixed with immobilized GST-ChLim and GST control beads, and bound proteins analyzed by immunoblotting (IB) with α-HA. (C) Total RNA from growing wild-type (WT), limF-null, and chlim-null cells was fractionated on formaldehyde–agarose gels and probed with full-length LimF or ChLim cDNA as indicated. (D) Lysates of limF-null and chlim-null cells expressing HA-Rab21Q66L (CA; constitutively active) or HA-Rab21T21N (DN; dominant-negative) were mixed with immobilized GST-LimF or GST-ChLim, and bound proteins analyzed by immunoblotting (IB) with α-HA. The left panel shows the relative input of equivalent lysate proportions for the indicated cell lines. Constitutively active, GTP-bound Rab21Q66L migrates with a slightly faster mobility than does the dominant-negative Rab21T21N form.
Figure 3
Figure 3
Actin organization in LimF, ChLim, and Rab21 mutant cells. Various strains of Dictyostelium were grown in shaking suspension, fixed with formaldehyde, stained with rhodamine-labeled phalloidin, and analyzed by confocal microscopy. (A) limF- and chlim-null cells display a cortical ring of F-actin similar to wild type (WT). (B) LimFOE and ChLimOE cells show a modest increase in F-actin-rich filopodia. (C) Cells expressing the constitutively active Rab21Q66L have actin-rich, ruffles over their entire surfaces. Cells expressing the dominant-negative Rab21T21N show enhanced actin microspikes; the bar inset represents 10 μm and is comparable for all panels.
Figure 4
Figure 4
Pinocytosis in LimF, ChLim, and Rab21 mutant cells. Log-phase cells were incubated with TRITC-dextran at room temperature at 160 r.p.m. At the indicated times, samples were withdrawn, quenched with Trypan blue, and intracellular rhodamine fluorescence was measured for equivalent cell numbers. Arbitrary fluorescence units were normalized to the maximal value obtained for wild-type (WT) cells within the same experiment. All the experiments were performed 3–4 times.
Figure 5
Figure 5
LimF, ChLim, and Rab21 regulation of phagocytosis. (A) Various strains of Dictyostelium were incubated with TRITC-labeled, heat-killed yeast particles at room temperature under shaking conditions. At the indicated time points, samples were collected and processed for fluorimetric analyses. Arbitrary fluorescence units were normalized to the maximal value obtained for wild-type (WT) cells within the same experiment. All the experiments were performed 3–4 times (see Table II). (B) Comparative analyses of Rab21T21N function for quantitative phagocytosis. (C) Comparative analyses of Rab21Q66L function for quantitative phagocytosis. (D) Comparative analyses for quantitative phagocytosis in limF−/chlim− cells.
Figure 6
Figure 6
Cellular localizations of ChLim, LimF, and Rab21. (A) Live, wild-type cells expressing GFP-ChLim, GFP-CHChLim, GFP-LIMChLim, YFP-LimF, and YFP-Rab21 were resuspended in phosphate buffer, plated in chambered coverglasses, and analyzed by confocal microscopy. (B) Cells expressing GFP-CHChLim were fixed with formaldehyde, stained with rhodamine-phalloidin, and analyzed by confocal microscopy. (C) Cells expressing YFP-LimF were fed TRITC-labeled, heat-killed yeast particles. Individual cells were imaged simultaneously for YFP and rhodamine fluorescence by confocal microscopy.
Figure 7
Figure 7
Dynamic distribution of ChLim during phagocytosis. Live wild-type cells expressing (A) GFP-ChLim, (B) GFP-CHChLim, (C) GFP-LIMChLim, and (D) GFP-ABD were fed TRITC-labeled, heat-killed yeast particles. Individual cells were imaged simultaneously for GFP and rhodamine fluorescence during a time course by confocal microscopy. See Supplementary Figure 5.
Figure 8
Figure 8
The antagonistic action of LimF, ChLim, and Rab21 during phagocytosis. We propose that phagocytosis in Dictyostelium is regulated by activating and inhibitory interactions of LimF, ChLim, and Rab21-GTP and suggest that similar regulatory pathways involving Rab21 are required in other professional phagocytes.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bush J, Franek K, Daniel J, Spiegelman GB, Weeks G, Cardelli J (1993) Cloning and characterization of five novel Dictyostelium discoideum rab-related genes. Gene 136: 55–60 - PubMed
    1. Cardelli J (2001) Phagocytosis and macropinocytosis in Dictyostelium: phosphoinositide-based processes, biochemically distinct. Traffic 2: 311–320 - PubMed
    1. Chien S, Chung CY, Sukumaran S, Osborne N, Lee S, Ellsworth C, McNally JG, Firtel RA (2000) The Dictyostelium LIM domain-containing protein LIM2 is essential for proper chemotaxis and morphogenesis. Mol Biol Cell 11: 1275–1291 - PMC - PubMed
    1. Clemens DL, Lee BY, Horwitz MA (2000) Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate. Infect Immun 68: 2671–2684 - PMC - PubMed
    1. Colicelli J (2004) Human RAS superfamily proteins and related GTPases. Sci STKE 2004: RE13. - PMC - PubMed

LinkOut - more resources