Mitochondria have recently attracted attention because of their role in apoptosis (Kluck et al., 1997
). There is also a huge body of literature on the role of mitochondria in oxidative phosphorylation, mitochondrial DNA, the transcription/translation machinery, and protein import (Tyler, 1992
). However, little or nothing is known about factors contributing to mitochondrial distribution, although this poses fascinating biological problems. It is not widely appreciated that mitochondria exist as an extensive tubular network with projections that move, break, and reseal in response to local environmental changes (Bereiter-Hahn and Voth, 1994
; Berger and Yaffe, 1996
; Hermann and Shaw, 1998
). Our results suggest that Drp1 is specifically required to establish this morphology.
The following observations indicate that mitochondria are the principle target of Drp1 function: (a
) The collapse of the mitochondrial network is induced by mutant Drp1, but not by mutant dynamin; (b
) the morphologies of ER, Golgi, endosomes, lysosomes, and peroxisomes are not visibly altered by mutant Drp1; (c
) the vesicular transport functions of the endocytic and secretory pathways were unaltered by mutant Drp1. The observed effect of Drp1 on mitochondrial morphology is consistent with recent studies in yeast and C
. Yeast dnm1 mutants have collapsed mitochondria, suggesting that yeast Dnm1p and human Drp1 are functional equivalents (see the accompanying article by Otsuga et al.). Mitochondria are also collapsed in C
expressing mutant isoforms of C
Drp1, although further experiments are required to ensure that this effect is specific (our unpublished results). Functional equivalence is also likely considering the high degree of sequence identity between C
and human Drp1 (62% identity, which is remarkably close to the 61% identity between C
dyn-1 and human dynamin; Clark et al., 1997
). Thus, it seems likely that the mitochondrial function of Drp1 is conserved throughout eukaryotic evolution.
Our localization results are in agreement with previous reports showing that Drp1 is mostly cytosolic (Imoto et al., 1998
; Kamimoto et al., 1998
; Shin et al., 1997
; Yoon et al., 1998
). The large fraction of cytosolic protein suggests that Drp1 cycles between the cytosol and a target organelle. A similar cycling occurs with dynamin, which cycles to and from the plasma membrane where it functions in endocytosis (Scaife and Margolis, 1990
). In addition, Yoon et al. (1998)
observed punctate staining of Drp1 interspersed with punctate ER staining, which led them to suggest that Drp1 is associated with ER. However, it seems unlikely that Drp1 also has an ER-associated function since mutant Drp1 had no effect on ER morphology or on VSV-G transport in our experiments, yet it dramatically changed mitochondrial morphology under the same conditions. Instead, the punctate staining detected by Yoon et al. (1998)
may correspond to mitochondria since they are often in close apposition to ER (Bereiter-Hahn and Voth, 1994
; Cascarano et al., 1995
; Tyler, 1992
Recently, Imoto et al. (1998)
reported transfecting dominant interfering mutant Drp1 and measuring its effect on the secretion of cotransfected luciferase, which they used as a reporter. They found a 50% reduction in secreted luciferase at high concentrations of transfected Drp1 DNA. Since no other cellular functions were tested, it is not possible to assess whether the observed inhibition of secretion was specific. It seems more likely that the inhibitory effect was an indirect consequence of mitochondrial collapse. Cells transfected with high concentrations of mutant Drp1 could have a range of secondary defects because the disruption of mitochondrial morphology might eventually affect oxidative phosphorylation and other vital mitochondrial functions. However, in our comparisons of different cellular functions, the first readily observed defect is the mitochondrial collapse from which we conclude that mitochondria are the principal target of Drp1.
How might Drp1 affect mitochondrial distribution? We envision two alternative explanations. First, Drp1 may help drag mitochondria to the cell periphery. Such a role for Drp1 would complement the well-established role of kinesin in transporting mitochondria along microtubules (Morris and Hollenbeck, 1995
). The importance of kinesin was demonstrated by injecting anti-kinesin antibodies, which caused mitochondria to retract from the cell periphery (Rodionov et al., 1993
). In addition, some kinesin light chains are specifically associated with mitochondria, where they might serve as adapters to connect mitochondria to a conventional kinesin (Khodjakov et al., 1998
). Neurons also possess a specialized kinesin, KIF1B, responsible for axonal transport of mitochondria (Nangaku et al., 1994
). KIF1B, like Drp1, is expressed at high levels in the brain, but is different from resident mitochondrial proteins, which are usually expressed at high levels in muscles, but not in the brain (Tiranti et al., 1997
). The difference in expression between resident mitochondrial proteins and KIF1B or Drp1 indicates that mitochondrial distribution is more taxing in neurons than in other cell types, regardless of their energy requirements. It is not clear how Drp1 might contribute to mitochondrial motility, if that proves to be its primary function.
Alternatively, Drp1 might affect mitochondrial distribution by helping to pinch off mitochondrial fragments, analogous to the role of dynamin in pinching off clathrin-coated vesicles. To understand how a defect in pinching off might lead to mitochondrial tubule clustering, we propose that normal subcellular distributions require shorter fragments or the separation of branched structures. Two lines of reasoning make a role in pinching seem more likely than a role in transport. First, the structural similarity between dynamin and Drp1 suggests that Drp1 might form a multimeric complex similar to the dynamin spiral. Second, the cytosolic localization of Drp1 is consistent with a transient role such as the scission of mitochondrial tubules. The lengthened mitochondria resulting from mutant Drp1 might then be too taxing for further distribution throughout the cell. Nothing is known about mitochondrial scission, but it must occur frequently.
The widening of mitochondrial tubules towards the periphery of the clusters in cells transfected with mutant Drp1 may simply help to accommodate the displacement of excess internal matrix from within the cluster of mitochondrial tubules. Mitochondrial clustering may have also induced the odd morphological transformations observed in some of the transfected cells. Club-, cup-, ring-, and onion-shaped mitochondria occur occasionally in normal cell types or under certain adverse conditions (De Robertis and Sabatini, 1958
; Ghadially, 1997
), which suggests that these abnormalities may be a secondary response to other changes in mitochondrial function. Interestingly, mutations in the Drosophila fuzzy onions
gene that affects mitochondrial fusion also induce odd cup or ring shapes, supporting the view that these transformations are not directly linked to a single protein function (Hales and Fuller, 1997
Although it is not yet known how Drp1 might interact with mitochondria, genetic screens conducted with yeast revealed a series of other proteins that affect mitochondrial morphology (Berger and Yaffe, 1996
; Hermann et al., 1997
; Shepard and Yaffe, 1997
). Some of these might interact with Drp1. The order of binding interactions may resemble the recruitment steps needed to form a dynamin spiral at the neck of a budding clathrin coated vesicle (Schmid, 1997
). The nature of these interactions will become clear in future studies.
In conclusion, we propose that Drp1 establishes mitochondrial morphology through a role in the distribution of mitochondrial tubules throughout the cytoplasm. Tubule formation and scission contribute to the dynamic nature of mitochondria (Bereiter-Hahn and Voth, 1994
; Hermann and Shaw, 1998
). Mitochondria can change shape during cell division and during differentiation, for example forming elaborate networks in muscle cells (Tyler, 1992
). Mitochondria also rapidly respond to local changes in the intracellular environment, sending out projections that break and reseal elsewhere within seconds (Bereiter-Hahn and Voth, 1994
). Our results suggest that Drp1 is a key factor controlling these morphological changes.