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. 2002 Oct 15;21(20):5331-42.
doi: 10.1093/emboj/cdf550.

Functions of LIM proteins in cell polarity and chemotactic motility

Bharat Khurana  1 , Taruna Khurana, Nandkumar Khaire, Angelika A Noegel
Affiliations

Functions of LIM proteins in cell polarity and chemotactic motility

Bharat Khurana et al. EMBO J. .

Abstract

LimC and LimD are two novel LIM proteins of Dictyostelium, which are comprised of double and single LIM domains, respectively. Green fluorescent protein-fused LimC and LimD proteins preferentially accumulate at areas of the cell cortex where they co-localize with actin and associate transiently with cytoskeleton-dependent dynamic structures like phagosomes, macropinosomes and pseudopods. Furthermore, both LimC and LimD interact directly with F-actin in vitro. Mutant cells that lack either LimC or LimD, or both, exhibit normal growth. They are, however, significantly impaired in growth under stress conditions and are highly sensitive to osmotic shock, suggesting that LimC and LimD contribute towards the maintenance of cortical strength. Moreover, we noted an altered morphology and F-actin distribution in LimD(-) and LimC(-)/D(-) mutants, and changes in chemotactic motility associated with an increased pseudopod formation. Our results reveal both unique and overlapping roles for LimC and LimD, and suggest that both act directly on the actin cytoskeleton and provide rigidity to the cortex.

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Figures

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Fig. 1. (A) Deduced amino acid sequence of LimC and LimD. The conserved cysteine, histidine or aspartate residues of the N-terminal LIM domains of LimC and LimD (bold, circled letters) and the C-terminal LIM domain of LimC (bold, shaded and circled letters) are shown. The intervening sequence between the two LIM domains of LimC exhibiting low compositional complexity is shaded, the proline residues are underlined. The two nearly perfect repeat motifs of 25 amino acid residues (shown in bold, underlined letters) and two shorter versions of this repeat motif (shown in bold letters) in the C-terminal region of LimD are indicated. (B) Alignment of the LimC and LimD amino acid sequences with members of the CRP family, CRP1, CRP2 and CRP3. Residues that are identical between LimC and/or LimD and any of the other members of the CRP family are shadowed in dark gray. Asterisks and dashes below the aligned sequences indicate the N-terminal LIM consensus (in all sequences) and C-terminal LIM consensus (in all sequences except LimD), respectively. (C) Alignment of the LimC and LimD amino acid sequences with Dictyostelium DdLim. Residues that are identical between any two sequences are shadowed in dark gray, and residues that are similar are shadowed in gray. Asterisks below the aligned sequences indicate the N-terminal LIM consensus. Sequences were aligned using the Clustal_W program (Wisconsin package version 9.0). Dashes indicate gaps introduced in the sequence for optimal alignment. DDBJ/EMBL/GenBank accession Nos: LimC, AF348466; LimD, AF348467; DdLim, U97699.1; CRP1, P32965; CRP2, P50460; CRP3, P50463.
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Fig. 2. Presence of limC and limD mRNAs and protein during development. The same amount (30 µg) of total RNA isolated from cells developed on phosphate-buffered agar plates at different hours of development as indicated was loaded in each lane. Hybridization was with 32P-labeled limC or limD cDNA. mRNA sizes are given in kb. For detection of LimD protein, total cellular extracts from AX2 cells (4 × 105 cells per lane) harvested at different time points were separated by SDS–PAGE (15% acrylamide). The resulting western blot was probed with LimD-specific monoclonal antibody K4-353-6.
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Fig. 3. Distribution of GFP–LimC and GFP–LimD fusion proteins. (A) Both the fusion proteins accumulate to high levels at distinct areas of the cell cortex and are distributed throughout the cytoplasm. Occasional localization of both the fusion proteins in the nucleus was also observed (arrowheads). Bars, 10 µm. (B) GFP–LimC (upper panels) and GFP–LimD (lower panels) localization coincides with that of actin in the cell cortex. The cells were fixed with cold methanol and immunolabeled with anti-actin monoclonal antibody followed by Cy3-conjugated anti-mouse secondary antibody. Overlay images show the co-localization of GFP–LimC or GFP–LimD fluorescence with the actin staining in the cell cortex. Bars, 10 µm.
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Fig. 4. GFP–LimC and GFP–LimD redistribution during fluid-phase endocytosis. Cells expressing GFP–LimC (A) or GFP–LimD (B) were allowed to adhere to glass coverslips and the supernatant was replaced by phosphate buffer containing 1 mg/ml TRITC–dextran. Confocal sections were taken at the times indicated. Arrowheads in the 0 s panels indicate the site of formation of pinocytic vesicles and arrows in subsequent images indicate newly formed pinosomes. Bars, 10 µm.
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Fig. 5. LimC and LimD in phagocytosis and exocytosis. (A and B) Dynamics of GFP–LimC and GFP–LimD redistribution during phagocytosis. GFP–LimC (A) or GFP–LimD (B) expressing cells were incubated with TRITC-labeled, heat-killed yeast cells. Confocal sections were taken at the times indicated. Arrowheads in the 0 s panel (A and B) mark the site of formation of the phagosomes. Bars, 10 µm. (C and D) Co-localization of GFP–LimC and GFP–LimD with actin at the phagocytic cups. GFP–LimC (C) or GFP–LimD (D) expressing cells were incubated for 10 min with heat-killed yeast cells and labeled after methanol fixation with anti-actin monoclonal antibody. Fluorescence patterns of the GFP–LimC (C) and the GFP–LimD (D) fusion proteins at the phagocytic cups as well as the cell cortex coincide with actin staining (overlay). The arrowheads in the GFP panels mark the position of the yeast cell being phagocytosed. Bars, 10 µm. (E) Dynamics of GFP–LimC redistribution during exocytosis. GFP–LimC cells were incubated with TRITC-labeled, heat-killed yeast cells for ∼1 h. Confocal sections were obtained after every 6 s. Selected images are shown. Asterisks (in all the frames) mark the yeast cell of interest and the arrowhead (in 0 s panel) marks the yeast that the cell is trying to phagocytose. Note the accumulation of GFP–LimC around the exocytotic vacuole as the cell prepares for exocytosis of the phagocytosed yeast (marked with an asterisk). Arrowheads in 6 min 30 s and 8 min 42 s panels show the simultaneous accumulation of GFP–LimC fusion protein on the phagocytic cup. Bar, 10 µm. Localization of endogenous LimC (F) and LimD (G) during uptake of yeast particles. Fixation and immunofluorescence detection with the respective monoclonal antibodies was carried out as for Figure 3. The corresponding phase-contrast images are shown. Bars, 5 µm.
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Fig. 6. LimC and LimD during cell motility. Cells starved for 6 h aggregate and, in general, acquire an elongated shape with a well-defined front and tail. Fluorescence images of aggregation-competent GFP–LimC (A) and GFP–LimD (C) cells and their respective phase-contrast images are shown. The arrows point to an extending pseudopod. Time series of aggregation-competent GFP–LimC (A′) and GFP–LimD (C′) expressing cells, which are rapidly extending pseudopods. Confocal images were taken at the times indicated. Both GFP–LimC and GFP–LimD accumulate to high levels in the pseudopods, as do endogenous LimC (B) and LimD (D) in aggregating AX2 cells. Detection was as described in Figure 5. Bars, 10 µm.
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Fig. 7. Localization of N- and C-terminal LimC deletion products. (A) Schematic representation of terminally deleted LimC proteins used for localization studies. Domain organization of LimC is shown. NLIM and CLIM represent the N- and C-terminal LIM domain, respectively. ‘P-rich seq.’ represents the intervening region between the two LIM domains that is rich in proline residues. Arrows indicate the position and orientation of oligonucleotide primers used for amplification. (B) Distribution of GFP–NLIM, GFP–NLIM-P, GFP–CLIM and GFP–CLIM-P during phagocytosis. Cells expressing these proteins exhibit a fluorescence pattern that is identical to that of the parent GFP–LimC fusion protein (see Figure 5C). All the truncated GFP fusion proteins accumulate at the phagocytic cup (arrowheads) during uptake of the yeast cell and their fluorescence pattern coincides with actin staining (overlay). Bars, 10 µm.
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Fig. 8. Binding of LimC and LimD to F-actin. Co-sedimentation of (A)  GST–LimC, GST–LimD and (B) GST–LimC deletion constructs GST–CLIM and GST–NLIM-P with F-actin. G-actin (5 µM) from D.discoideum was incubated with the GST fusion proteins in polymerization buffer containing 2 mM MgCl2, 100 mM KCl and 1 mM EGTA. Pellets (P) and supernatants (S) were separated by high-speed centrifugation, and the proteins in these fractions were analyzed by SDS–PAGE (12% acrylamide) and staining with Coomassie Blue. Controls for the fusion proteins alone were performed without the addition of G-actin in the reaction mixture. The presence (+) or absence (–) of G-actin in the reaction mixture is indicated on the top of the lanes. Arrows marked with C and D indicate GST–LimC and GST–LimD, respectively. Varying amounts of polymerized (P) and unpolymerized actin (S) in GST–LimC and GST–LimD panels might be due to different actin preparations.
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Fig. 8. Binding of LimC and LimD to F-actin. Co-sedimentation of (A)  GST–LimC, GST–LimD and (B) GST–LimC deletion constructs GST–CLIM and GST–NLIM-P with F-actin. G-actin (5 µM) from D.discoideum was incubated with the GST fusion proteins in polymerization buffer containing 2 mM MgCl2, 100 mM KCl and 1 mM EGTA. Pellets (P) and supernatants (S) were separated by high-speed centrifugation, and the proteins in these fractions were analyzed by SDS–PAGE (12% acrylamide) and staining with Coomassie Blue. Controls for the fusion proteins alone were performed without the addition of G-actin in the reaction mixture. The presence (+) or absence (–) of G-actin in the reaction mixture is indicated on the top of the lanes. Arrows marked with C and D indicate GST–LimC and GST–LimD, respectively. Varying amounts of polymerized (P) and unpolymerized actin (S) in GST–LimC and GST–LimD panels might be due to different actin preparations.
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Fig. 9. F-actin distribution and cell polarization in mutant cells. (A) LimD– cells have a polarization defect and have F-actin acumulated in patches at the cortex. (B) LimC– cells are polarized and show F-actin accumulation at cell–cell contacts comparable to AX2 (D). (C) LimC–/LimD– cells resemble LimD– single mutants. Aggregation stage cells (t6) were fixed with picric acid/paraformaldehyde and stained with TRITC–phalloidin. Bar, 10 µm.
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Fig. 10. Expression of GFP–LimD rescues the polarity defect in LimD– cells. Phase-contrast images of (A) aggregating (t6) LimD– cells expressing GFP–LimD and (B) aggregating LimD– cells. Bar, 20 µm.
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