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. 2010 Oct 29;5(10):e13763.
doi: 10.1371/journal.pone.0013763.

The SNX-PX-BAR family in macropinocytosis: the regulation of macropinosome formation by SNX-PX-BAR proteins

Jack T H Wang  1 , Markus C Kerr, Seetha Karunaratne, Angela Jeanes, Alpha S Yap, Rohan D Teasdale
Affiliations

The SNX-PX-BAR family in macropinocytosis: the regulation of macropinosome formation by SNX-PX-BAR proteins

Jack T H Wang et al. PLoS One. .

Abstract

Background: Macropinocytosis is an actin-driven endocytic process, whereby membrane ruffles fold back onto the plasma membrane to form large (>0.2 µm in diameter) endocytic organelles called macropinosomes. Relative to other endocytic pathways, little is known about the molecular mechanisms involved in macropinocytosis. Recently, members of the Sorting Nexin (SNX) family have been localized to the cell surface and early macropinosomes, and implicated in macropinosome formation. SNX-PX-BAR proteins form a subset of the SNX family and their lipid-binding (PX) and membrane-curvature sensing (BAR) domain architecture further implicates their functional involvement in macropinosome formation.

Methodology/principal findings: We exploited the tractability of macropinosomes through image-based screening and systematic overexpression of SNX-PX-BAR proteins to quantitate their effect on macropinosome formation. SNX1 (40.9+/-3.19 macropinosomes), SNX5 (36.99+/-4.48 macropinosomes), SNX9 (37.55+/-2.4 macropinosomes), SNX18 (88.2+/-8 macropinosomes), SNX33 (65.25+/-6.95 macropinosomes) all exhibited statistically significant (p<0.05) increases in average macropinosome numbers per 100 transfected cells as compared to control cells (24.44+/-1.81 macropinosomes). SNX1, SNX5, SNX9, and SNX18 were also found to associate with early-stage macropinosomes within 5 minutes following organelle formation. The modulation of intracellular PI(3,4,5)P(3) levels through overexpression of PTEN or a lipid phosphatase-deficient mutant PTEN(G129E) was also observed to significantly reduce or elevate macropinosome formation respectively; coexpression of PTEN(G129E) with SNX9 or SNX18 synergistically elevated macropinosome formation to 119.4+/-7.13 and 91.4+/-6.37 macropinosomes respectively (p<0.05).

Conclusions/significance: SNX1, SNX5, SNX9, SNX18, and SNX33 were all found to elevate macropinosome formation and (with the exception of SNX33) associate with early-stage macropinosomes. Moreover the effects of SNX9 and SNX18 overexpression in elevating macropinocytosis is likely to be synergistic with the increase in PI(3,4,5)P(3) levels, which is known to accumulate on the cell surface and early-stage macropinocytic cups. Together these findings represent the first systematic functional study into the impact of the SNX-PX-BAR family on macropinocytosis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Macropinosome formation screening assay validation.
A: 24 hours post transfection, HEK-Flp-In cell monolayers were pulsed for 5 minutes with 100 µg/mL dextran (10,000 MW) conjugated to tetramethylrhodamine (dextran-TR) at 37°C. The samples were then washed in 4°C PBS, fixed in 4% PFA and imaged and processed as described in Materials and Methods. Briefly Z-stack images comprising of 3×5 µm Z slices were merged into a single RGB image of transfected cells (green) stained with dextran-TR (red) (Ai). The red channel from the RGB image was isolated and converted to an 8-bit grayscale image (Aii). Dextran-positive macropinosomes were selected based on size (>0.5 µm in diameter) and fluorescent intensity (>100). Selected macropinosomes are shown in the foreground as black (Aiii). This binary image was then converted to a mask and superimposed onto the Green channel of the original RGB image to measure the green fluorescent intensity of the area occupied by each macropinosome in the image (Aiv). The particles with green fluorescence intensity higher than background signal (>20) were considered to be macropinosomes within a transfected cell, represented in green. Red particles represent discarded macropinosomes determined to be outside of a transfected cell. Scale  = 10 µm. B, C, D: HEK-Flp-In cell monolayers were either serum-starved for 16 hours and treated with dextran-TR in the presence or absence of 100 ng/mL EGF for 5 minutes at 37°C (B), treated with 1 mM amiloride or carrier (0.6% Methanol) for 30 minutes at 37°C before pulsing with dextran-TR (C), or transiently transfected with pEGFP-C1 or pEGFP-SNX5 before pulsing with dextran-TR (D). The samples in each case were assayed for macropinosome formation as described in Materials and Methods, quantitating the mean number of macropinosomes/100 transfected cells over 3 replicates of 500 transfected cells for each condition. * denotes statistical significance (p<0.05) using the Student's T-test. Error bars denote Standard Error of the Mean (S.E.M).
Figure 2
Figure 2. Rab5 and Rabankyrin5 overexpression increases macropinosome formation.
The distribution of Rab5, Rabankyrin-5 (A) or Rab5(Q79L) (B) in green relative to dextran-positive macropinosomes (red) Scale bar  = 10 µm. (C) HEK-Flp-In cells were transiently transfected with pEGFP-C1, pEGFP-Rab5, pEGFP-Rab5-Q79L and pEYFP-Rabankyrin-5. The mean number of macropinosomes/100 transfected cells was quantitated over 3 replicates of 500 transfected cells for each condition. * denotes statistical significance (p<0.05) using the Student's T-test, performing pairwise analyses relative to cells transfected with pEGFP-C1 alone. Error bars denote S.E.M.
Figure 3
Figure 3. The SNX-PX-BAR family is involved in macropinosome formation.
HEK-Flp-In cells transiently overexpressing GFP-tagged members of the SNX-PX-BAR family were assayed for macropinosome formation as described in Materials and Methods. A: Dextran-TR labeling of cells transfected with the specified constructs. Scale bar  = 10 µm. B: The mean number of macropinosomes/100 transfected cells was quantitated over 8 replicates of 500 transfected cells for each condition. * denotes statistical significance (p<0.05) using the Student's T-test, performing pairwise analyses relative to cells transfected with pEGFP-C1 alone. Error bars denote S.E.M.
Figure 4
Figure 4. SNX1, SNX5, SNX9 and SNX18 associate with early macropinosomes.
A: HEK-Flp-In cells were pulsed with 100 µg/mL dextran (10,000 MW) conjugated to Alexa-647 (dextran-647) for 5 minutes before being transferred to 4°C and washed with 0.45 mM CaCl2 1 mM MgCl2 PBS. The cells were then treated with 80 U/mL Streptolysin O for 5 minutes at 4°C to only permeabilize the plasma membrane before washing in 0.45 mM CaCl2 1 mM MgCl2 PBS and incubating with 37°C PBS for 5 minutes. Following fixation with 4% PFA for 30 minutes at 4°C, cell monolayers are incubated with monoclonal and polyclonal antibodies against SNX1 and SNX5 respectively, followed by Alexa-488-conjugated goat-anti-mouse and Cy3-conjugated goat-anti-rabbit IgG secondary antibodies. B, C, and D: 24 hours post transfection, HEK-Flp-In cells transfected with pEGFP-SNX9 (B), pEGFP-SNX18 (C) and pEGFP-SNX33 (D) were pulsed with dextran-TR for 5 minutes at 37°C prior to fixation at 4°C in 4% PFA. Images were collected on an LSM 510 Meta confocal microscope. Scale bar  = 5 µm.
Figure 5
Figure 5. The SH3 domain of SNX18 is required for elevation of macropinosome formation.
HEK-Flp-In cells transiently overexpressing pEGFP, pEGFP-SNX18, or pEGFP-ΔSH3-SNX18 were assayed for macropinosome formation as described in Materials and Methods. The mean number of macropinosomes/100 transfected cells was quantitated over 3 replicates of 500 transfected cells for each condition. Error bars denote S.E.M.
Figure 6
Figure 6. The modulation of PI(3,4,5)P3 levels affects macropinosome formation.
A: HEK-Flp-In cell monolayers were treated with either 65 µM LY294002 or carrier (0.2% Ethanol) for 30 minutes at 37°C prior to pulsing with dextran-TR as described in Materials and Methods in the continued presence of the drug or carrier. The mean number of macropinosomes/100 cells was quantitated as described in Materials and Methods. Error bars represent S.E.M. B: HEK-Flp-In cell monolayers transfected with pEGFP-Grp1-PH for 24 hours were imaged using the 100× oil immersion objective of an Olympus IX-81 OBS Real Time microscope before and after treatment with 65 µM LY294002. Time following LY294002 treatment is indicated in bottom right hand corner of each panel. C: HEK-Flp-In cells transiently transfected with pIRES2-EGFP, pIRES2-EGFP-PTEN, or pIRES-EGFP-PTEN(G129E) were co-transfected with pmCherry, pmCherry-SNX9, or pmCherry-SNX18 and assayed for macropinosome formation as described in Materials and Methods. The mean number of macropinosomes/100 transfected cells was quantitated over 3 replicates of 500 transfected cells for each condition. Error bars denote S.E.M.
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