Splice variant-specific cellular function of the formin INF2 in maintenance of Golgi architecture

Abstract

INF2 is a unique formin that can both polymerize and depolymerize actin filaments. Mutations in INF2 cause the kidney disease focal and segmental glomerulosclerosis. INF2 can be expressed as two C-terminal splice variants: CAAX and non-CAAX. The CAAX isoform contains a C-terminal prenyl group and is tightly bound to endoplasmic reticulum (ER). The localization pattern and cellular function of the non-CAAX isoform have not been studied. Here we find that the two isoforms are expressed in a cell type-dependent manner, with CAAX predominant in 3T3 fibroblasts and non-CAAX predominant in U2OS, HeLa, and Jurkat cells. Although INF2-CAAX is ER localized in an actin-independent manner, INF2-non-CAAX localizes in an actin-dependent meshwork pattern distinct from ER. INF2-non-CAAX is loosely attached to this meshwork, being extracted by brief digitonin treatment. Suppression of INF2-non-CAAX causes fragmentation of the Golgi apparatus. This effect is counteracted by treatment with the actin monomer-sequestering drug latrunculin B. We also find discrete patches of actin filaments in the peri-Golgi region, and these patches are reduced upon INF2 suppression. Our results suggest that the non-CAAX isoform of INF2 serves a distinct cellular function from that of the CAAX isoform.

FIGURE 1:

INF2 splice variants. (A) Domain architecture of INF2. C-Terminal sequences starting at K1224 of human INF2. The CAAX and non-CAAX splice variants are shown in purple and green amino acids, respectively. (B) Schematic of posttranslational modifications associated with prenylation.

FIGURE 2:

INF2 does not localize to ER in U2OS cells. (A) U2OS cells transfected with mCherry-Sec61β (an ER membrane protein; red) and stained with anti-INF2 antibody (green) and DAPI (blue). (B) NIH 3T3 cells stained with anti-INF2 (green), anti-GRP94 (an ER luminal protein; red) and DAPI (blue). See Supplemental Figure S1B for analogous INF2/ER localization using the ER marker. Scale bar, 20 μm.

FIGURE 3:

Actin depolymerization causes INF2 aggregation in U2OS cells. (A) U2OS cells were treated with 1 μM LatB for 0 and 30 min prior to fixation and staining with anti-INF2 (green), TRITC-phalloidin (red), and DAPI (blue). Arrow indicates one such actin aggregate where INF2 colocalizes. Scale bar, 20 μm. (B) Single confocal slice of U2OS cells stained with anti-INF2 (green) and rhodamine-phalloidin (red). Arrows show examples of regions where INF2 and actin filaments colocalize. Scale bars: 5 μm (leftmost three images) and 20 μm (rightmost image).

FIGURE 4:

3T3 cells express INF2-CAAX, whereas U2OS, Jurkat, and HeLa cells express INF2–non-CAAX. (A) Western blots of whole-cell homogenates from NIH 3T3 cells. CAAX and non-CAAX controls are from U2OS cells transiently transfected with GFP-tagged mouse INF2 isoforms. Western blots were probed with antibodies specific for total mouse INF2, INF2-CAAX, or INF2–non-CAAX. (B) Similar analysis as in A, using homogenates from Jurkat and HeLa cells. CAAX and non-CAAX controls are from U2OS cells transiently transfected with GFP-tagged human INF2 isoforms. (C) Western blots of immunoprecipitations of denatured lysates from U2OS cells either untransfected or stably expressing GFP-INF2-CAAX or GFP-INF2–non-CAAX. Immunoprecipitations were conducted using the total INF2 antibody. Western blots were probed with total INF2, INF2-CAAX, or INF2–non-CAAX antibodies. E, endogenous INF2; G, GFP INF2.

FIGURE 5:

INF2 is rapidly extracted from U2OS cells. U2OS cells were permeabilized with digitonin on ice for 0, 5, and 30 min before fixation. The cells were then stained with anti-INF2 antibody (green) to detect endogenous INF2 and DAPI (blue). mCherry-Sec61β (red) was used as an ER membrane marker. Scale bar, 20 μm.

FIGURE 6:

Stably transfected U2OS cells lines expressing GFP-INF2-CAAX and GFP-INF2–non-CAAX recapitulate endogenous proteins. (A) Localization of GFP-INF2-CAAX and GFP-INF2–non-CAAX in U2OS cells. mCherry-Sec61β (red) was used as an ER marker. Scale bar, 10 μm. mCherry-Sec61β was overexposed to visualize the peripheral ER. (B) Digitonin permeabilization of U2OS INF2-CAAX and non-CAAX stable cell lines. The cells were permeabilized for 0, 5, or 30 min on ice before fixation. Scale bar, 20 μm. (C) Effect of LatB on GFP-INF2-CAAX (C1246S) mutant and GFP-INF2–non-CAAX. U2OS cells were transfected with mouse GFP-INF2-CAAX mutant or GFP-INF2–non-CAAX treated with 1 μM LatB for the indicated amount of time. The cells were then fixed and stained with TRITC phalloidin. Arrow indicates one such actin aggregate where INF2 colocalizes. Scale bar, 20 μm.

FIGURE 7:

INF2 enriches around the Golgi apparatus. (A) U2OS cells were stained with anti-INF2 (green) and golgin-97 (a marker for the trans-Golgi network; red). Top, one confocal z-section. Bottom, the maximum-intensity projection of multiple confocal z-sections. Arrow represents INF2 enrichment around the Golgi. Scale bar, 20 μm. (B) U2OS cells treated for 0 or 30 min with 2.5 μg/ml brefeldin A. Arrow represents INF2 enrichment around the Golgi The cells were then fixed and stained with anti-INF2 (green) and anti–golgin-97 (red) antibodies. Scale bar, 20 μm.

FIGURE 8:

INF2 depletion causes dispersal of the Golgi apparatus in U2OS cells. (A) INF2-depleted U2OS cells stained with anti-INF2 (green) and DAPI (blue). The stars indicate cells that have been knocked down. Scale bar, 20 μm. (B) Western blot showing INF2 depletion using two different siRNAs. Tubulin was used as a loading control. (C) Representative images of compact, spread, and dispersed Golgi. GM130 (green) was used as the Golgi marker. DAPI is in blue. Scale bar, 20 μm. (D) Quantification of Golgi dispersion. The data represent the average of three independent experiments (n > 200 cells). Error bars, SE. (E) Quantification of Golgi morphology upon INF2 depletion and LatB treatment. C, control siRNA; S, INF2 siRNA. Cells were treated with 0, 1, or 2 μM LatB for 1 h prior to fixation.

FIGURE 9:

Peri-Golgi actin patches are reduced and dispersed in INF2 knockdown. (A) Cells transfected with control (top) or INF2 (bottom) siRNAs were fixed and stained with anti-GM130 (green) and TRITC-phalloidin (red). Maximum-intensity projections of three consecutive 0.2-μm sections taken through the middle of Golgi region are shown. Arrows indicate actin patches found in close proximity to Golgi stacks. Scale bar, 10 μm. Quantification of peri-Golgi actin patches reveals an approximate 30% decrease in patch number upon INF2 knockdown (12.7 patches/Golgi in control siRNA [217 patches in 17 cells] vs. 9.0 in INF2 siRNA [190 patches in 21 cells]). Quantification is the result of two separate experiments that showed similar 30% decrease for INF2 siRNA. (B) Colocalization of INF2 with actin patches. Single confocal slice of U2OS stained with anti-INF2 (green), TRITC-phalloidin (red), and anti–golgin-97 (blue). Arrow indicates a patch that colocalizes with INF2, and arrowhead indicates a patch that does not colocalize with INF2. Scale bar, 5 μm.