QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 30 September 2008
doi: 10.1242/jcs.032904

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tan, F. J. Articles by Hill, R. B. Search for Related Content PubMed PubMed Citation Articles by Tan, F. J. Articles by Hill, R. B. Social Bookmarking                         What's this?

CED-9 and mitochondrial homeostasis in C. elegans muscle

¹ Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA
² Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA
³ Integrated Imaging Center, Johns Hopkins University, Baltimore, MD 21218, USA
⁴ Departments of Pathology and Genetics, Stanford University SOM, Stanford, CA 94305, USA

View larger version (50K): [in this window] [in a new window]   Fig. 1. Analysis of mitochondria in wild-type, loss-of-function ced-9(n2812lf); ced-3(n717lf) and gain-of-function ced-9(n1950gf) animals. (A-C) Mitochondrial morphology in wild-type, loss-of-function ced-9(n2812lf); ced-3(n717lf) and gain-of-function ced-9(n1950sd) genetic backgrounds was examined by creating multiple independently generated transgenic lines that expressed GFP targeted to the mitochondrial matrix (mitoGFP). Wide-field epifluorescene images are of body wall muscle cells from young adult animals. n, nucleus. Bar, 10 µm. (D) The average mitochondrial length in a given muscle cell was estimated using ImageJ (see Materials and Methods), and plotted as an individual point [wild-type n=16, ced-9(n2812lf); ced-3(n717lf)n=19, ced-9(n1950gf) n=14]; the mean value is indicated by a horizontal bar. (E) Mitochondrial ultrastructure in wild-type, ced-9(n2812lf); ced-3(n717lf) and ced-9(n1950sd) animals was examined in both body wall muscle and hypodermis by transmission electron microscopy. m, mitochondria. Bar, 500 nm.

View larger version (86K): [in this window] [in a new window]   Fig. 2. Mitochondrial fission and fusion occur in the absence of CED-9. (A-F) Wild-type and dominant negative forms of DRP-1 were expressed using a myo-3 promoter in wild-type ced-9(+) and ced-9(n2812lf); ced-3(n717lf) animals. Similar mitochondrial morphologies were observed in both genetic backgrounds when body wall muscle cells were labeled with mitoGFP. n, nucleus. Bar, 10 µm. (G,H) Wild-type DRP-1 expressed using a myo-3 promoter in ced-9(n1950gf) animals resulted in a similar phenotype. (I) Increased DRP-1 expression induced mitochondrial fragmentation in wild-type, ced-9(n2812lf); ced-3(n717lf), and ced-9(n1950sd) backgrounds. Each point represents the proportion of cells with fragmented mitochondria from an independently generated transgenic line; the mean value is indicated by a horizontal bar, and error bars represent the standard error. The differences between wild-type and ced-9(n2812lf); ced-3(n717lf) backgrounds is significant, z=2.42, P<0.05; raw data is included as Table S1 in supplementary material. (J) Inactivating DRP-1 with dominant negative DRP-1(K40A) resulted in interconnected mitochondria in both wild-type and ced-9(n2812lf); ced-3(n717lf) backgrounds.

View larger version (59K): [in this window] [in a new window]   Fig. 3. Increased CED-9 expression alters mitochondrial morphology. (A,B) Increased CED-9 expression resulted in dilated, interconnected mitochondria. This effect is similar to inactivation of mitochondrial fission by expression of dominant negative DRP-1(K40A) (Fig. 2C). However, we note that DRP-1(K40A)-induced mitochondria are more rounded, and only a fraction of the cells possess thin tubules interconnecting various mitochondria. (C) Removing the N-terminal 67 residues of CED-9 did not abolish the ability of CED-9 to alter mitochondria. However, removing either (D) a core structural helix or (E) the C-terminal transmembrane domain prevented CED-9 from altering mitochondria. Increased CED-9 expression also altered mitochondrial outer membrane morphology as judged by mitochondria labeled with TOM70::GFP, a mitochondrial outer membrane tethered GFP (Labrousse et al., 1999). (F) In otherwise wild-type animals, the mitochondria had a fairly stereotypical tubular morphology. (G) By contrast, transgenic animals with increased CED-9 expression displayed altered outer membrane morphologies. n, nucleus. Bar, 10 µm. (H) Mitochondrial morphology in the body wall muscle of adult animals with increased CED-9 expression was further examined by transmission electron microscopy. Two animals were found that possessed markedly distinct mitochondrial morphologies; shown are two cells from one of the animals. Bar, 500 nm.

View larger version (43K): [in this window] [in a new window]   Fig. 4. CED-9-altered mitochondria can be partially suppressed by co-expression of DRP-1. (A) Co-expression of DRP-1 and CED-9 from the same transgene resulted in three classes of mitochondrial morphology: tubular, fragmented and interconnected (percentages represent the mean value of three independently generated transgenic lines; raw data is presented in C and Table S1 in supplementary material). These three phenotypes suggest that increased DRP-1 expression is able to partially suppress the effects of CED-9 on mitochondria, or vice-versa. n, nucleus. Bar, 10 µm. (B) Structure of CED-9 (yellow surface) bound to a portion of the BH3 containing protein EGL-1 (blue ribbon) (1TY4.pdb). In the gain-of-function ced-9(n1950sd) allele, glycine 169, which resides in the CED-9 BH3 binding pocket, is mutated to glutamate (G169E). This mutation has been reported to decrease EGL-1 binding to CED-9 (del Peso et al., 2000). (C) Co-expression of DRP-1 and CED-9(G169E) from the same transgene also resulted in three classes of mitochondrial morphology. (D) Each point represents the proportion of cells with interconnected mitochondria from an independently generated transgenic line; the mean value is indicated by a horizontal bar, and error bars represent the standard error. No mean was calculated for the CED-9(G169E) construct because of the apparent bimodal distribution. This variation among lines expressing CED-9(G169E) was examined by western blot analysis (Fig. S4 in supplementary material). The difference in proportions between co-expression of DRP-1 with CED-9 and co-expression of DRP-1 with CED-9(G169E) is significant, z=2.59, P<0.01. (E) The GTPase activity of 2.5 µM human DRP1 was assayed using a continuous assay coupled to a regenerative system. (F) The amount of GTP hydrolyzed by 2.5 µM human DRP1 with 12.5 µM CED-9 after 40 minutes (296±23 µM) was significantly higher than the amount of GTP hydrolyzed by 2.5 µM human DRP1 alone after 40 minutes (221±17 µM), t(4)=4.56, P<0.025.

View larger version (5K): [in this window] [in a new window]   Fig. 5. A model for CED-9 function in mitochondrial homeostasis. In C. elegans, CED-9 does not appear to be essential for mitochondrial homeostasis. Mitochondrial fusion, which probably involves the mitofusin FZO-1, can occur in the absence of CED-9. Similarly, mitochondrial fission, which involves DRP-1 can also occur in the absence of CED-9. This and other studies (Delivani et al., 2006; Jagasia et al., 2005), however, suggest that CED-9 may play a role in potentiating the activities of both DRP-1 and FZO-1. Additionally, CED-9 may itself be regulated by the mitochondrial fission protein DRP-1. If true, regulation of CED-9 by DRP-1 would provide a mechanism for cross-regulation between the mitochondrial fission and fusion machineries. Such a pathway would allow DRP-1 to promote mitochondrial fission both directly, and through downregulation of FZO-1 by way of inhibiting CED-9.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter