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First published online 14 April 2009
doi: 10.1242/jcs.040691

Journal of Cell Science 122, 1430-1440 (2009)
Published by The Company of Biologists 2009
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INF2 is an endoplasmic reticulum-associated formin protein

Ekta Seth Chhabra1,*, Vinay Ramabhadran1, Scott A. Gerber2 and Henry N. Higgs1,{ddagger}

1 Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA
2 Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA


Figure 1
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  		Fig. 1. Constructs and western blot of INF2. (A) Constructs used in this study: INF2 (full-length protein, 1273 amino acids), INF2-W (full-length protein, triple L->A mutation in DAD/WH2 domain, red star), INF2-C1270S (full-length protein, C1270S mutation, position indicated by blue star), INF2-(FH1-FH2-C) (residues 536-1273), INF2-(DID) (residues 1-424), INF2-(C) (residues 993-1273) and INF2-(C)-W (amino acids 993-1273 with triple L->A mutation in DAD/WH2 domain, red star). (B) Western blot of Swiss 3T3 extract was performed using affinity-purified INF2 antibody [raised against INF2-(C) construct]. (C) INF2 western blots on 10 µg of extract. From left to right: brain (B), heart (H), 3T3 cells (3T3), liver (L) and spleen (S). Upper image shows processing such that none of the INF2 bands are saturated. Lower image shows processing such that brain and 3T3 samples are strongly overprocessed, which reveals lighter bands in heart, liver, and spleen. (D) Cellular fractionation of endogenous INF2. The homogenate (H) was centrifuged at 2000 x g for 10 minutes to create the low-speed pellet (LSP). The supernatant from this centrifugation was centrifuged at 436,000 x g for 20 minutes to create the high-speed pellet (HSP) and high-speed supernatant (HSS). Fractions were separated by SDS-PAGE then probed for INF2 in western blots. (E) Extraction of INF2. The LSS fraction from 3T3 extract was treated with high ionic strength (1 M NaCl), high pH (100 mM CAPS pH 11), or non-ionic detergent (2% thesit), then centrifuged at 436,000 x g for 20 minutes to create a pellet (P) and a supernatant (S) fraction. Fractions were separated by SDS-PAGE then probed for INF2, tubulin, and syntaxin 6 by western blot.

 

Figure 2
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  		Fig. 2. INF2 localizes to ER in Swiss 3T3 cells. Fluorescence microscopy of GFP-Sec61-expressing Swiss 3T3 cell, immunostained for endogenous INF2. (A,B) Images of the same cell were processed in two ways. (A) Basal region in focus, and overprocessed to reveal peripheral ER. Enlarged images on the right represent the boxed region. (B) Focus on region of highest INF2 staining (middle of cell) and processed such that no pixels are saturated for fluorescence intensity, showing that most INF2 staining is perinuclear. Cells were electroporated with GFP-Sec61, plated on glass coverslips and fixed with formaldehyde 16 hours after transfection, followed by staining with Cy3-labeled affinity purified rabbit anti-INF2. (C,D) Microtubules and INF2. (C) Swiss 3T3 cells were fixed and costained for tubulin (monoclonal DM1-{alpha} anti-tubulin) and INF2 (anti-INF2). Close-ups of the boxed region are shown below. (D) Nocodazole treatment results in collapse of ER and INF2. Treatment of 3T3 cells with 33 µM nocodazole-containing medium for 30 minutes at 37°C. Cells were then fixed and stained for tubulin and INF2. Images in C and D were taken with the same exposure times and processed identically. Even when overprocessed, no INF2-stained ER is observed in peripheral regions (arrow).

 

Figure 3
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  		Fig. 3. INF2 is present on the cytoplasmic leaflet of the ER. Swiss 3T3 cells were fixed with formaldehyde, permeabilized with either digitonin or Triton X-100 (TX-100), then stained with anti-calreticulin or anti-INF2. The ER lumen marker calreticulin stains only cells treated with Triton X-100, whereas INF2 staining is present in both types of permeabilized cells. All images for a given staining were taken for the same exposure time and processed identically.

 

Figure 4
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  		Fig. 4. Prenylation of INF2 is required for ER localization. Swiss 3T3 cells were transfected with the GFP-INF2-C1270S mutant along with mCherry-Sec61. Enlarged image at the bottom represent the boxed region. Cells were fixed in formaldehyde 24 hours after transfection. Expression of the full-length GFP protein was confirmed by western blot of cell extracts (not shown).

 

Figure 5
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  		Fig. 5. The C-terminal region of INF2 is sufficient for ER localization. Swiss 3T3 cells were cotransfected with mCherry-Sec61 and the GFP-INF2-(C) construct. INF2-(C) colocalizes with mCherry-Sec61 in a reticular pattern at the cell periphery (arrow). Enlarged images at bottom rows represent the boxed region. Cells were cotransfected with GFP-INF2-(C) and mCherrry-Sec61, plated on glass coverslips and fixed 24 hours after transfection.

 

Figure 6
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  		Fig. 6. Digitonin extraction of GFP-INF2 constructs. mCherry-Sec61 was cotransfected with GFP alone (top row), GFP-INF2 full length (second row), or GFP-INF2-(C) (bottom row) into 3T3 cells. Cells were either fixed in formaldehyde (left images) or permeabilized with digitonin, then fixed (right images). Permeabilization was for 10 minutes [GFP and GFP-INF2-(C)] or 40 minutes (GFP-INF2 full length). All images for mCherry or GFP were taken for the same exposure time and processed identically.

 

Figure 7
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  		Fig. 7. Fractionation of GFP-INF2 constructs. Swiss 3T3 cells, transfected with the constructs indicated on the right, were scraped from plate, then homogenized by Type B dounce in extraction buffer containing 50 mM KCl. The homogenate (Hom) was then centrifuged at 2000 x g for 10 minutes to create the low-speed pellet (LSP). The supernatant from this centrifugation was centrifuged at 436,000 x g for 20 minutes to create the high-speed pellet (HSP) and high-speed supernatant (HSS). Fractions were separated by SDS-PAGE then western blotted for either INF2 [no transfection, GFP-INF2, and GFP-INF2-(C)] or for GFP (GFP alone). Molecular size markers (in kDa) are indicted on the left.

 

Figure 8
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  		Fig. 8. Localization of INF2 with respect to actin in Swiss 3T3 cells. (A) Swiss 3T3 cell co-stained with TRITC-phalloidin and anti-INF2. Cells were fixed with formaldehyde 16 hours after plating on glass coverslips, followed by staining for polymerized actin (TRITC-phalloidin, red) and INF2 (rabbit anti-INF2, green). Enlarged image at right represents the boxed region. (B) Latrunculin A treatment does not disrupt the INF2 reticular staining pattern. Swiss 3T3 cells were treated with 1 µM latrunculin A for 30 minutes, then fixed and stained for INF2 and polymerized actin. Actin image was highly overprocessed to show loss of stress fibers. Enlarged image at right represents boxed region.

 

Figure 9
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  		Fig. 9. The DAD/WH2 of INF2 binds the DID region. (A) Fluorescence anisotropy measurements using fluorescein-labeled INF2-(C) at 20 nM and increasing concentrations of INF2-(DID) reveals an apparent dissociation constant (Kdapp) of 1.1 µM (circles). The fluorescently labeled INF2-(C)-W mutant displays no measurable affinity for DID (squares). Similar results obtained in two independent experiments. (B) INF2-(DID) inhibits INF2-(FH1-FH2-C)-mediated depolymerization, but not polymerization. Pyrene-actin polymerization assays with 1 µM rabbit muscle actin monomers (5% pyrene labeled) and 300 nM of INF2-(FH1-FH2-C) in the absence or presence of 100 µM DID. In independent experiments, this concentration of INF2-(DID) has no effect on actin dynamics. (C) Inhibition of INF2-(FH1-FH2-C)-mediated depolymerization by DID. Actin monomers (1.05 µM, 5% pyrene labeled) were polymerized for 16 hours at 23°C in polymerization buffer, then diluted to 1 µM in the same buffer containing 250 nM INF2-(FH1-FH2-C) and varying concentrations of INF2-(DID). Filament depolymerization was measured by the decrease in pyrene fluorescence intensity with time. F1F2C, INF2-(FH1-FH2-C); D, INF2-(DID).

 

Figure 10
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  		Fig. 10. Expression of GFP-INF2-DAD/WH2 mutant causes collapse of ER around nucleus. Swiss 3T3 cells were transfected with either GFP-INF2-W and mCherry-Sec61 (A), GFP-INF2-W alone (B,C) or GFP-INF2 wild-type (D). Cells were fixed in formaldehyde 24 hours after transfection, stained with DAPI (A) or DAPI with TRITC-phalloidin (B-D). (A) GFP-INF2-W and mCherry-Sec61 co-accumulation in tubular structures around the nucleus (see also supplementary material Fig. S6B). Arrow shows rare region of mCherry-Sec61 staining in the absence of GFP-INF2-W. No reticular staining of either protein is detectable in the periphery. Many cells overexpressing GFP-INF2-W accumulate tubular GFP-containing structures around the nucleus (55%, n=200, from three separate experiments). B and C are panels from a Z-series of the same cell transfected with GFP-INF2-W and stained with TRITC-phalloidin. In B, taken at the base of the cell, GFP-INF2-W and actin filaments colocalize to perinuclear tubular structures. Neither stain is detectable in the periphery (see supplementary material Fig. S6C for overprocessed image). In C, taken 2 µm above the base of the cell, actin filaments and GFP-INF2-W co-accumulate in whorls above the nucleus. 100% of cells with collapsed ER tubules also had actin accumulation (n=200 cells from three experiments). (D) GFP-INF2 wild-type displays reticular staining, and actin filaments display a normal staining pattern in these cells.

 

Figure 11
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  		Fig. 11. INF2 knockdown does not affect ER morphology. Swiss 3T3 cells were transfected with a shRNA vector targeted to INF2, then transfected with GFP-Sec61 56 hours later. At 80 hours after shRNA transfection, cells were fixed in formaldehyde and stained with rabbit anti-INF2. The middle cell is strongly suppressed in INF2 expression, as shown by undetectable INF2 staining. GFP-Sec61 displays a reticular pattern in this cell. Enlarged image at the bottom represents the boxed region in first panel.

 

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© The Company of Biologists Ltd 2009