Evidence that dynamin-2 functions as a signal-transducing GTPase

Abstract

The role of dynamin GTPases in the regulation of receptor-mediated endocytosis is well established. Here, we present new evidence that the ubiquitously expressed isoform dynamin-2 (dyn2) can also function in a signal transduction pathway(s). A </=5-fold increase of dyn2 relative to endogenous levels activates the transcription factor p53 and induces apoptosis, as demonstrated by reduced cell proliferation, DNA fragmentation, and caspase-3 activation. Dyn2-triggered apoptosis occurs only in dividing cells and is p53 dependent. A mutant defective in GTP binding does not trigger apoptosis, indicating that increased levels of dyn2.GTP, rather than protein levels per se, are required to transduce signals that activate p53. A truncated dyn2 lacking the COOH-terminal proline/arginine-rich domain (PRD), which interacts with many SH3 domain-containing partners implicated in both endocytosis and signal transduction, triggers apoptosis even more potently than the wild-type. This observation provides additional support for the importance of the NH(2)-terminal GTPase domain for the apoptotic phenotype. All described effects are dyn2-specific because >200-fold overexpression of dyn1, the 70% identical neuronal isoform, has no effect. Our data suggest that dyn2 can act as a signal transducing GTPase affecting transcriptional regulation.

Figure 1

Dynamin-2 specifically induces cell toxicity in tTA HeLa cells. (A) Phase contrast micrographs of tTA-HeLa cells infected with an equivalent moi of adenovirus expressing either dyn1^wt, dyn2^wt, or dyn2^K44A under control of a tetracycline-regulatable promoter, as described in Materials and Methods. After infection, cells were incubated for 36 h in media containing the indicated concentration of tetracycline. (B) Western blot analysis using the pan-dynamin antibody 748 showing endogenous dynamin-2 (detected in control lanes and in lanes where exogenous expression is suppressed in the presence of 1 μg/ml tet) and tetracycline-dependent expression levels of exogenous dyn1^wt, dyn2^wt, or dyn2^K44A. (C–E) Immunofluorescence images of dynamin staining in uninfected tTA HeLa cells (C) or tTA HeLa cells infected with adenovirus encoding dyn1^wt (D) or dyn2^wt (E) and cultured for 24 h in the absence of tetracycline. Bar, 20 μm.

Figure 2

At low levels of expression, dynamin-2 specifically induces cell death. (A) Equal numbers of tTA-HeLa cells were infected with 10 moi of adenovirus expressing either dyn1^wt(□), dyn2^wt (○), or tTA (•). Cells were harvested at the indicated times postinfection and analyzed using Trypan blue exclusion to quantify cell viability. (B) Equal numbers of tTA-HeLa cells were infected with a high moi (500) which was chosen to detect some toxicity of adenoviruses encoding dyn1^wt, and incubated in the presence of varying concentrations of tetracycline to regulate expression of dyn1^wt (□) and dyn2^wt (○). Cells were harvested 36 h postinfection and analyzed for viability by Trypan blue exclusion. The insert in B shows Western blot analysis of corresponding levels of dyn1 and dyn2 expression under these conditions detected using the pan-dynamin polyclonal antibody 748. Dyn1^wt at an moi 500 in the absence of tetracycline is expressed ∼100-fold over endogenous dyn2 as quantified by densitometric measurements of Western blots.

Figure 2

At low levels of expression, dynamin-2 specifically induces cell death. (A) Equal numbers of tTA-HeLa cells were infected with 10 moi of adenovirus expressing either dyn1^wt(□), dyn2^wt (○), or tTA (•). Cells were harvested at the indicated times postinfection and analyzed using Trypan blue exclusion to quantify cell viability. (B) Equal numbers of tTA-HeLa cells were infected with a high moi (500) which was chosen to detect some toxicity of adenoviruses encoding dyn1^wt, and incubated in the presence of varying concentrations of tetracycline to regulate expression of dyn1^wt (□) and dyn2^wt (○). Cells were harvested 36 h postinfection and analyzed for viability by Trypan blue exclusion. The insert in B shows Western blot analysis of corresponding levels of dyn1 and dyn2 expression under these conditions detected using the pan-dynamin polyclonal antibody 748. Dyn1^wt at an moi 500 in the absence of tetracycline is expressed ∼100-fold over endogenous dyn2 as quantified by densitometric measurements of Western blots.

Figure 3

Dynamin-2–induced apoptosis detected by loss of membrane integrity. Subconfluent monolayers of tTA-HeLa cells were either uninfected or infected with adenovirus encoding either dyn1^wt, dyn2^wt, or dyn2^K44A and incubated 24 h postinfection with the fluorescent DNA dyes, YO-PRO-1 (1 μM), propidium iodine (PI, 5 μg/ml) and Hoechst 33342 (10 μg/ml) for 10 min at 37°C as described in Materials and Methods. The immunofluorescence images shown are representative fields of adenovirally infected tTA-HeLa cells expressing dyn1^wt (A) or dyn2^wt (B) taken 24 h postinfection. Early apoptotic cells within the population with compromised plasma membrane integrity are labeled with YO-PRO-1^® (green fluorescence). Cells at later stages of apoptosis and dead cells are labeled with the membrane impermeant dye, PI (red fluorescence). Hoechst, a cell permeant DNA dye (blue fluorescence), labels all cells in the population. Quantification of the number of early apoptotic cells (i.e., YO-PRO-1 positive), expressed as the percentage of viable cells (i.e., PI negative), at various times postinfection is shown in C. 100 cells were counted for each condition.

Figure 3

Dynamin-2–induced apoptosis detected by loss of membrane integrity. Subconfluent monolayers of tTA-HeLa cells were either uninfected or infected with adenovirus encoding either dyn1^wt, dyn2^wt, or dyn2^K44A and incubated 24 h postinfection with the fluorescent DNA dyes, YO-PRO-1 (1 μM), propidium iodine (PI, 5 μg/ml) and Hoechst 33342 (10 μg/ml) for 10 min at 37°C as described in Materials and Methods. The immunofluorescence images shown are representative fields of adenovirally infected tTA-HeLa cells expressing dyn1^wt (A) or dyn2^wt (B) taken 24 h postinfection. Early apoptotic cells within the population with compromised plasma membrane integrity are labeled with YO-PRO-1^® (green fluorescence). Cells at later stages of apoptosis and dead cells are labeled with the membrane impermeant dye, PI (red fluorescence). Hoechst, a cell permeant DNA dye (blue fluorescence), labels all cells in the population. Quantification of the number of early apoptotic cells (i.e., YO-PRO-1 positive), expressed as the percentage of viable cells (i.e., PI negative), at various times postinfection is shown in C. 100 cells were counted for each condition.

Figure 4

Dynamin-2–induced apoptosis detected by increased caspase-3 activity. tTA-HeLa cells infected with adenovirus encoding dyn1^wt or dyn2^wt or the dynamin mutants dyn2^K44A and dyn2^ΔPRD. Cells were harvested at the indicated times postinfection and viable cells were assayed for caspase-3 activity using the fluorescent substrate Ac-DEVD-AMC as described in Materials and Methods. For comparison of independent experiments, caspase-3 activity is plotted in arbitrary units setting dyn2^wt at 36 h postinfection as 100 after substracting the background of tTA-infected cells. Representative data of three independent experiments are shown.

Figure 5

Nondividing cells are resistant to dynamin-2–induced apoptosis. At 8 d post differentiation, primary monocyte-derived macrophage cultures were coinfected with 100 moi each of Ad-Trans and either Ad-Dyn1^wt (A) or Ad-Dyn2^wt (B) recombinant adenoviruses. These infection conditions were required to obtain maximum efficiency of ∼60% of cells expressing HA-dynamin as detected immunofluorescence using 12CA5 (C). Bars, 20 μm.

Figure 6

Dynamin-2 increases nuclear staining for p53. 24 h after adenoviral infection tTA Hela cells expressing dyn1^wt (A) or dyn2^wt (B) were processed for immunostaining with p53-specific antibodies (green). Nuclei (red) were stained with Hoechst. p53 redistribution to the nucleus was observed in >90% of dynamin-2–expressing cells. Bar, 20 μm.

Figure 7

Dynamin-2–induced apoptosis is p53 dependent. (A) p53+/+ and p53−/− mouse embryo fibroblasts were infected with the indicated adenoviruses and incubated for 24 h before staining with the fluorescent DNA dye YO-PRO-1 (1 μM). Quantification of the number of early apoptotic cells (i.e., YO-PRO-1 positive), expressed as the percentage of viable cells (i.e., PI negative) is shown at 24 h postinfection. 100 cells were counted for each condition. (B) p53^ts cells were grown at 37 or 32°C for 24 h and then infected with the indicated adenoviruses and incubated for a further 24 h at the same temperature. MTT conversion assays were used to measure cell proliferation and viability. The results are expressed as percent of uninfected control ± SD (n = 3).

Figure 7

Dynamin-2–induced apoptosis is p53 dependent. (A) p53+/+ and p53−/− mouse embryo fibroblasts were infected with the indicated adenoviruses and incubated for 24 h before staining with the fluorescent DNA dye YO-PRO-1 (1 μM). Quantification of the number of early apoptotic cells (i.e., YO-PRO-1 positive), expressed as the percentage of viable cells (i.e., PI negative) is shown at 24 h postinfection. 100 cells were counted for each condition. (B) p53^ts cells were grown at 37 or 32°C for 24 h and then infected with the indicated adenoviruses and incubated for a further 24 h at the same temperature. MTT conversion assays were used to measure cell proliferation and viability. The results are expressed as percent of uninfected control ± SD (n = 3).