Dynamic remodeling of the actin cytoskeleton by FMNL1γ is required for structural maintenance of the Golgi complex

Abstract

Formin-like 1 (FMNL1) is a member of the formin family of actin nucleators, and is one of the few formins for which in vitro activities have been well characterized. However, the functional roles of this mammalian formin remain ill-defined. In particular, it is unclear how the unique in vitro biochemical properties of FMNL1 relate to its regulation of cellular processes. Here, we demonstrate that FMNL1 depletion caused a dramatic increase in cellular F-actin content, which resulted in Golgi complex fragmentation. Moreover, increased F-actin and maintenance of Golgi structure were distinctly regulated by the gamma isoform of FMNL1, which required binding to actin. Importantly, in addition to Golgi fragmentation, increased F-actin content in the absence of FMNL1 also led to cation-independent mannose 6-phosphate receptor dispersal, lysosomal enlargement and missorting of cathepsin D. Taken together, our data support a model in which FMNL1 regulates cellular F-actin levels required to maintain structural integrity of the Golgi complex and lysosomes.

Fig. 1.

Expression of diaphanous and FMNL formins in a panel of cancer cell lines. Clarified cell lysates were prepared from Jurkat, HeLa and pancreatic cancer cell lines. Equivalent amounts of protein were separated by SDS-PAGE and immunoblotted using (A) anti-FMNL1, (B) anti-FMNL2, (C) anti-FMNL3 and (D) anti-Dia1 and -Dia2. α-tubulin was used as loading control.

Fig. 2.

Depletion of FMNL1 increases F-actin content. (A) HeLa and Jurkat T cells were transfected with suppression vector shFMNL1–GFP or a control vector (shControl–GFP). Cell lysates were immunoblotted for FMNL1 and α-tubulin 72 hours after transfection. (B) HeLa and Jurkat T cells were transfected with shControl–GFP or shFMNL1–GFP vectors and stained with phalloidin. All cells are GFP positive (not shown). Images are a maximum intensity projection from single z-stacks. Scale bars: 10 μm. (C) Jurkat T cells were transfected with shControl (gray line), shFMNL1 (black line) vectors, labeled with FITC-conjugated phalloidin and analyzed by flow cytometry. (D) Graphical representation of the mean fluorescence intensity of the FITC histograms in C.

Fig. 3.

FMNL1 localizes throughout the cytoplasm and is enriched at a punctate structure near the MTOC. The subcellular localization of FMNL1 was studied in HeLa cells using immunofluorescence. (A) HeLa cells were immunostained for FMNL1. F-actin was stained with TRITC-phalloidin. (B) HeLa cells were labeled for FMNL1 and α-tubulin. Scale bar: 5 μm.

Fig. 4.

FMNL1 localizes to the Golgi complex. HeLa cells were immunostained for FMNL1 and Golgi markers. (A) FMNL1 colocalizes with the trans-Golgi marker Golgin97 (G97). (B) FMNL1 colocalizes with the cis-Golgi marker GM130 and is dispersed by treatment with brefeldin A (+BFA). (C,D) Immunofluorescence of Jurkat cells co-stained with FMNL1 and the Golgi markers G97 (C) or GM130 (D). Nuclei were stained with Hoechst 33341 (blue). Scale bars: 5 μm.

Fig. 5.

FMNL1 depletion causes fragmentation of the Golgi complex. (A) HeLa cells were transfected with shControl–GFP and shFMNL1–GFP vectors. After 72 hours, cells were immunolabeled with anti-G97 antibody and stained with Hoechst. (B) Quantification of Golgi dispersal in HeLa cells transfected with the indicated shRNA knockdown vectors. Over 100 GFP-positive cells were analyzed and scored on the basis of their Golgi phenotype. The graph shows the average percentage of cells with a fragmented Golgi. Bars represent means + s.d. from three independent experiments. ***P<0.0001 (t-test).

Fig. 6.

FMNL1γ and its ability to bind actin barbed ends specifically regulate Golgi structure integrity. (A,B) HeLa cells were transfected with the indicated suppression–re-expression vectors and scored for Golgi fragmentation as described in Fig. 5. (A) Over 50 YFP-positive cells were scored for the fragmented Golgi phenotype. Bars represent means ± s.d. from three independent experiments; **P<0.001, ***P<0.0001 compared with a control. (B) Over 100 YFP-positive cells from C were scored for the fragmented Golgi phenotype. (C) HeLa cells were transfected with the indicated HA–YFP control or HA–YFP FMNL1 suppression–re-expression vectors. After 72 hours, cells were immunostained with anti-G97. Transfected cells are YFP positive. Bars represent means ± s.d. from three independent experiments; *P<0.05, t-test.

Fig. 7.

FMNL1γ rescues the increase in F-actin observed in FMNL1-depleted cells. HeLa cells were transfected with vectors expressing shFMNL1 and coexpressing (A) HA–YFP–FMNL1α, (B) HA–YFP–FMNL1β, (C) HA–YFP–FMNL1γ, (D) HA–YFP FMNL1ΔFH2 or (E) FMNL1γ defective for actin barbed-end binding mutants HA–YFP–FMNL1γK871D. After 72 hours, cells were stained with TRITC-phalloidin and Hoechst. Transfected cells are YFP positive. Scale bars: 10 μm.

Fig. 8.

FMNL1 depletion increases F-actin at the Golgi in an FMNL1γ-dependent manner. HeLa cells were transfected with vectors expressing (A) shControl–HA–YFP, (B) shFMNL1–HA–YFP, (C) shFMNL1–HA–YFP–FMNL1α, (D) shFMNL1–HA–YFP–FMNL1β, (E) shFMNL1–HA–YFP–FMNL1γ, (F) shFMNL1–HA–YFP–FMNL1γK871D. After 72 hours, cells were stained with TRITC-phalloidin, and G97 was detected with a Cy5 secondary antibody but is represented in green. Cells are YFP positive (not shown). Images are a maximum intensity projection from single z-stacks. Scale bars: 10 μm.