Small GTP-binding protein TC10 differentially regulates two distinct populations of filamentous actin in 3T3L1 adipocytes

Abstract

TC10 is a member of the Rho family of small GTP-binding proteins that has previously been implicated in the regulation of insulin-stimulated GLUT4 translocation in adipocytes. In a manner similar to Cdc42-stimulated actin-based motility, we have observed that constitutively active TC10 (TC10/Q75L) can induce actin comet tails in Xenopus oocyte extracts in vitro and extensive actin polymerization in the perinuclear region when expressed in 3T3L1 adipocytes. In contrast, expression of TC10/Q75L completely disrupted adipocyte cortical actin, which was specific for TC10, because expression of constitutively active Cdc42 was without effect. The effect of TC10/Q75L to disrupt cortical actin was abrogated after deletion of the amino terminal extension (DeltaN-TC10/Q75L), whereas this deletion retained the ability to induce perinuclear actin polymerization. In addition, alteration of perinuclear actin by expression of TC10/Q75L, a dominant-interfering TC10/T31N mutant or a mutant N-WASP protein (N-WASP/DeltaVCA) reduced the rate of VSV G protein trafficking to the plasma membrane. Furthermore, TC10 directly bound to Golgi COPI coat proteins through a dilysine motif in the carboxyl terminal domain consistent with a role for TC10 regulating actin polymerization on membrane transport vesicles. Together, these data demonstrate that TC10 can differentially regulate two types of filamentous actin in adipocytes dependent on distinct functional domains and its subcellular compartmentalization.

Figure 1

Activated TC10 induces actin polymerization through an N-WASP-dependent mechanism. CHO cells were transfected with either pcDNA3 (a), Cdc42/Q61L (b) or with TC10/Q75L (c–f). CHO cell extracts were prepared and mixed with Xenopus oocyte egg extracts plus rhodamine-actin as described under MATERIALS AND METHODS. The Xenopus oocyte extracts were also preincubated with 20 μM latruculin B (d), 1 μg/ml C. difficile toxin B (e), and 25 μg/ml N-WASP/ΔVCA dominant-interfering mutant (f) before the addition of the CHO cell extracts containing TC10/Q75L. These are representative confocal fluorescent microscopy low-magnification images taken at 10 min after the initiation of the actin polymerization assay (magnification 60×).

Figure 2

Time-lapse confocal microscopic observation of Cdc42/Q61L- and TC10/Q75L-induced actin comet tails. CHO cell extracts expressing Cdc42/Q61L (A) or TC10/Q75L (B) were mixed with Xenopus oocyte extracts plus rhodamine-actin and visualized every 22 s over a 2-min time period (a–e; magnification 100×, zoom ×2). This is a representative time-lapse image independently performed three times.

Figure 3

TC10 expression specifically disrupts cortical actin but induces actin polymerization in the perinuclear region in adipocytes. 3T3L1 adipocytes were microinjected with expression plasmids encoding for the myc-epitope–tagged Cdc42/Q61L cDNA (a, c, and e) or the TC10/Q75L cDNA (b, d, and f). The cells were allowed to recover for 24 h and then double labeled with a mAb directed against the myc-epitope tag (a and b) and with rhodamine-labeled phalloidin (c and d) as described under MATERIALS AND METHODS. The merged images are presented in e and f. This is a representative field obtained from three to five independent experiments. Quantitation of these data were obtained by counting 50–60 individual cells under each condition.

Figure 4

Expression of TC10/Q75L dirsupts cortical actin but increases perinuclear actin polymerization in adipocytes. (A) 3T3L1 adipocyte nuclei were microinjected with an expression plasmid encoding for YFP-actin plus empty vector (a–c) or TC10/Q75L (d–f) as described under MATERIALS AND METHODS. The living cells expressing YFP-actin were then continuously visualized by confocal fluorescent microscopy. The cells were also pretreated with 0.5 μg/ml C. difficile toxin B (b and e) or 20 μM latranculin B (c and f) followed by confocal fluorescent microscopy. The complete time-lapse image for toxin B-pretreated cell is provided in supplementary materials. (B) YFP-actin expressing cell was continuously visualized by confocal fluorescent microscopy and selected time images before and subsequent to the addition of latrunculin B (60 μM) as indicated in a–d. The complete time-lapse image is provided in supplementary materials. The time lapse images presented are representative images observed in four to seven individual cells examined under each condition.

Figure 5

TC10/Q75L expression induces perinuclear actin polymerization that is inhibited by a dominant-interfering N-WASP mutant. 3T3L1 nuclei were microinjected with expression plasmids encoding for YFP-actin plus either the empty vector (a), N-WASP/ΔVCA (b), TC10/Q75L (c), or N-WASP/ΔVCA plus TC10/Q75L (d). The cells were allowed to recover for 24 h and then visualized by time-lapse confocal fluorescent microscopy. These are individual representative YFP-actin time-lapse fluorescent images observed in four to seven independent cells examined.

Figure 6

Amino terminal domain of TC10/Q75L is responsible for the loss of adipocyte cortical actin. 3T3L1 adipocyte nuclei were microinjected with expression plasmids encoding for TC10/Q75L (a–c) and ΔN-TC10/Q75L (d–f). The cells were allowed to recovery for 24 h and then double labeled with a mAb directed against the myc-epitope tag (a and d) and with rhodamine-labeled phalloidin (b and e) as described under MATERIALS AND METHODS. The merged images are presented in c and f. This is a representative field obtained from three independent experiments. In each experimental condition a total of 53 to 77 individual cells were scored for the presence of cortical actin.

Figure 7

TC10/Q75L induces massive actin polymerization in the Golgi membrane region of adipocytes. 3T3L1 adipocytes were electroporated with expression plasmid encoding for the myc-tagged TC10/Q75L and the cells were allowed to recover for 24 h. The cells were then fixed and triple labeled with a rabbit polyclonal antibody directed against the myc-epitope tag (a and e), rhodamine-labeled phalloidin (b and f), and a mouse mAb directed against the p115 (c and g) as described under MATERIALS AND METHODS. The merged images are presented in d and h. These are representative fields obtained from three independent experiments.

Figure 8

Derangement of perinuclear actin inhibits VSV-G protein trafficking. (A) 3T3L1 adipocytes were coelectroporated with expression plasmids encoding for theVSV-G-ts045-GFP with either the empty vector (a and b) or expression plasmids encoding for TC10/Q75L (c and d), TC10/T31N (e and f), or N-WASP/ΔVCA (g and h) cDNA. The cells were incubated for 6 h at 40°C and then shifted to 32°C for 60 min. The cells were fixed and VSV-G-ts045-GFP was visualized by confocal microscopy. (B) The number of VSV-G-ts045-GFP-expressing cells displaying visually detectable plasma membrane fluorescence was counted from seven different fields (75–150 transfected cells/condition). This graph shows a result from two independent experiments from empty vector cotransfected adipocytes (●) or adipocytes coexpressing TC10/Q75L (□), TC10/T31N (▪), or N-WASP/ΔVCA (○) cDNAs.

Figure 9

TC10 interacts with coatomer through a carboxyl-terminal dilysine motif. (A) 3T3L1 adipocytes were electroporated with empty vector (lane 1) or expression plasmids encoding for the hemagglutinin-epitope–tagged TC10/Q75L (lane 2), Cdc42/Q61L (lane 3), empty vector (lane 4), TC10/WT (lane 5), or TC10/T31N (lane 6). The cells were allowed to recover for 24 h and then immunoprecipitated with the hemagglutinin antibody and immunoblotted with a β-COP antibody as described under MATERIALS AND METHODS. (B) 3T3L1 adipocytes were electroporated with empty vector (lane 1) or expression plasmids encoding for the hemigglutinin-epitope–tagged TC10/WT (lane 2), TC10/T31N (lane 3), TC10/Q75L (lane 4), or TC10/KK199,200SS (lane 5). The cells were allowed to recover for 24 h and then immunoprecipitated with the hemigglutinin antibody and immunoblotted with a γ-COP antibody as described under MATERIALS AND METHODS. These are representative immunoprecipitations independently performed three times.