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. 2008 Oct 14:9:58.
doi: 10.1186/1471-2121-9-58.

A role for SNX5 in the regulation of macropinocytosis

Jet Phey Lim  1 , Jack T H Wang, Markus C Kerr, Rohan D Teasdale, Paul A Gleeson
Affiliations

A role for SNX5 in the regulation of macropinocytosis

Jet Phey Lim et al. BMC Cell Biol. .

Abstract

Background: The mechanisms and components that regulate macropinocytosis are poorly understood. Here we have investigated the role of sorting nexin 5 (SNX5) in the regulation of macropinocytic activity.

Results: SNX5 is abundantly expressed in macrophages, cells very active in macropinocytosis, and is recruited onto newly-formed macropinosomes. LPS treatment of bone marrow-derived macrophages resulted in a 2.5 fold decrease in macropinosome formation that correlates with a reduction in the levels of SNX5. To investigate the relationship between SNX5 levels and macropinocytic activity we examined the formation of macropinosomes in HEK-FlpIn cells stably expressing GFP-SNX5. Constitutive macropinocytosis was increased approximately 2 fold in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. Furthermore, EGF stimulation resulted in a significant increase in macropinocytosis and there was also a 2.0 fold increase in the generation of macropinosomes in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. SNX5, which interacts specifically with PtdIns(3)P and PtdIns(3,4)P2 through its PX domain, was recruited to regions on the plasma membrane containing EGF receptor or positive for PtdIns(3,4)P2 as detected with the PH domain of TAPP1. Treatment with AG1478, an EGF receptor specific tyrosine kinase inhibitor, prevented the recruitment of SNX5 to the cytosolic face of the plasma membrane and inhibited the formation of macropinosomes in response to EGF treatment.

Conclusion: Based on these data, we propose that SNX5 requires the generation of phosphoinositides for recruitment to the plasma membrane and, moreover, influences the level of macropinocytic activity.

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Figures

Figure 1
Figure 1
LPS stimulation of primary macrophages results in a decrease in macropinocytic activity and SNX5 levels. (A-D) Bone marrow-derived macrophages were grown for 7 days in M-CSF and then analysed as follows. (A) Cells were cultured in the presence of 100 μg/ml TR-conjugated dextran (10,000 MW) for 3 min. The cells were washed at 4°C and fixed in 4% PFA and stained with affinity-purified SNX5 antibodies followed by Alexa488-conjugated goat anti-rabbit IgG. Bar = 10 μm. (B) Macrophages were seeded at 1 × 107 cells/ml and incubated with 10 ng/ml LPS for up to 24 h, as indicated. Samples (15 μg protein) were separated by SDS-PAGE and probed by immunoblotting using the anti-SNX5 antibody. (C-D) Macrophages were either left untreated or treated with 10 ng/ml LPS for 24 h and then pulsed with 1 mg/ml Alexa 488-conjugated dextran for 3 minutes at 37°C and fixed in 4% PFA. The samples were then stained with Texas-red-X conjugated phalloidin. Images were captured using identical settings for -/+ LPS conditions and counted as described in Methods. At least 180 cells were analysed for each condition.
Figure 2
Figure 2
SNX5 levels influence macropinocytosis activity. (A) HEK-FlpIn parental (white bars) and HEK-GFP-SNX5 (grey bars) cells were serum-starved for 16 h and pulsed with 100 μg/ml dextran (10,000 Da) conjugated to tetramethylrhodamine for 5 min in the presence or absence of 100 ng/ml EGF at 37°C prior to fixation in 4% PFA. Macropinosomes were identified as dextran-positive structures > 0.5 μm in diameter, and counted using an automated image analysis protocol as described in Methods. The mean number of macropinosomes per 100 cells was determined over 500 cell triplicates for each condition (B) HEK-GFP-SNX5 cells were serum-starved overnight and either left untreated or treated with 100 ng/ml EGF in the presence and absence of 100 nM of AG1478, or in the presence of 0.001% DMSO (carrier control), as indicated. Cells were fixed and permeabilised and stained with anti-human EEA1 and Alexa568-conjugated goat anti-mouse IgG. Macropinosomes were identified as large >1 μm EEA1 positive-structures, as described in text. The mean number of macropinosomes from a triplicate of 100 cells was determined without knowledge of the identity of sample. Each experiment was repeated twice. Shown is mean and error bars represent standard deviation. ** p < 0.05, *** p < 0.005.
Figure 3
Figure 3
EGF-R is internalized into macropinosomes in HEK-GFP-SNX5 cells. (A) HEK-GFP-SNX5 cells were transfected with EGF-R and 24 h later incubated with Alexa555-conjugated EGF on ice. After washing, cells were incubated at 37°C for 15 min, then fixed and permeabilised and stained with human anti-EEA1 antibody followed by Alexa647-conjugated anti-human IgG. Insert shows overlay of GFP, SNX5 and EEA1. In inset, bar = 5 μm.
Figure 4
Figure 4
Elevated SNX5 does not impact on macropinosome maturation. HEK-FlpIn parental (white bars) or HEK-GFP-SNX5 (black bars) cell monolayers were cultured in the presence of 100 μg/ml Alexa647-conjugated dextran (10,000 MW) for 4 h at 37°C, washed and incubated overnight to label the late endosomes and lysosomes. The cells were then incubated with 100 μg/ml TR-labeled dextran (10,000 MW) for 5 min at 37°C, washed and then incubated at 37°C in serum free media for up to 20 min, as indicated, to internalize the TR-labeled dextran. Monolayers were fixed in 4% PFA and the samples mounted and images captured using a confocal microscope. The proportion of macropinosomes that had fused and acquired content from the late endosomes/lysosomes was scored.
Figure 5
Figure 5
SNX5 is recruited to regions of the plasma membrane rich in PI(3,4)P2. (A) HEK-GFP-SNX5 cells were transfected with EGF-R and 24 h later serum-starved overnight and either left untreated or treated with 100 ng/ml EGF for 3 min at 37°C, as indicated. Cells were fixed and stained with anti-human EGF-R antibody followed by Alexa568-conjugated goat anti-mouse IgG for detection of surface EGF-R. Optical sections were taken to highlight the plasma membrane. Arrows indicate membrane ruffles showing co-localization of GFP-SNX5 and EGF-R (B) HEK-GFP-SNX5 cells were transfected with mCherry-TAPP1-PH and 24 h later serum-starved overnight then either left untreated or treated with 100 ng/ml EGF for 5 min at 37°C and then fixed. Arrows indicate co-localization of GFP-SNX5 and mCherry-TAPP1-PH (C) HEK-GFP-SNX5 cells were serum-starved overnight and either left untreated or treated with 100 ng/ml EGF for 3 min at 37°C, in the presence or absence of 100 nM AG1478, as indicated, and fixed. Boxed images are shown magnified on upper right hand corner. (D). Cells from (C) were scored for plasma membrane GFP-SNX5 by epifluorescence microscopy. 100 cells in triplicate were analysed. Shown is mean and error bars represent standard deviation. *** p <0.005. Bars = 10 μm.
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