A human dynamin-related protein controls the distribution of mitochondria

Abstract

Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.

Figure 1

Structure of dynamin-related protein and comparison with other dynamin family members. Boundaries of the Drp1 domains were as follows: the GTPase domain was from position 1–300 in the amino acid sequence, the middle domain was from position 301–508, the divergent domain was from position 509– 598, and the assembly domain was from position 599–699. Numbers above the individual domains show the percentage identity to the corresponding sequence of the nonneuronal isoform of Drp1.

Figure 2

Tissue-specific expression of human Drp1. A blot with 2 mg poly A+ RNA from each of the designated human tissues was hybridized with a radiolabeled Drp1 cDNA probe and processed for autoradiography. The mRNA sizes were estimated to be 2.8 and 4.8 kb, corresponding to the usage of different polyadenylation sites. Both mRNAs were ∼100 nt longer in brain (inset shows a lighter exposure).

Figure 3

(A) Immunoblot analysis of Drp1 in protein extracts from bovine brain, HeLa cells, and COS-7 cells, showing a prominent Drp1 band in brain. (B) Immunoblot analysis of untransfected COS-7 cells and COS-7 cells transfected with Drp1, showing overexpression of Drp1 in transfected cells. It is unclear whether the faint band in untransfected COS-7 cells represents a slightly larger splice variant of Drp1 or an unrelated protein that cross-reacts with the Drp1 antibody. (C) Indirect immunofluorescence of COS-7 cells with anti-Drp1 antibody. The brightly fluorescent cell on the right overexpresses Drp1, which indicates that this cell was a transfectant.

Figure 4

Absence of an effect of mutant Drp1 on the secretory pathway. The function of the secretory pathway was monitored by cotransfecting wild-type or mutant Drp1, together with a chimera containing VSV-G tsO45 fused to GFP (Presley et al., 1997). Overexpression of mutant or wild-type Drp1 was verified by double labeling with anti-Drp1 antibody (not shown). The VSV-G chimera was accumulated in the ER by growing transfected cells at 40°C. At 0 min the cells were transferred to the permissive temperature (32°C), allowing the VSV-G chimera to exit the ER. At 15 and 30 min, most of the label was concentrated in the Golgi, and at 60 min most of the label was transferred to the plasma membrane. There was no detectable difference between cells transfected with mutant or wild-type Drp1.

Figure 5

Absence of an effect of mutant Drp1 on the endocytic pathway. The function of the endocytic pathway was monitored by following LDL uptake. Cells transfected with the indicated Drp1 or dynamin constructs were incubated at 37°C in serum-free medium with Bodipy-LDL. After 25 min the cells were washed and fixed for staining with anti-Drp1 or anti-dynamin antibody. Cells that overexpress mutant or wild-type Drp1 or dynamin were identified by double labeling with anti-Drp1 or anti-dynamin antibody as indicated by the gray outlines. LDL does not appear in the lysosomes of 70% of cells transfected with mutant dynamin (Dyn1-K44A), demonstrating the validity of this approach. There was no detectable difference in the distribution of lysosomes in cells transfected with wild-type Drp1 (Drp1-wt), the K38A mutant (Drp1-K38A), or in untransfected cells, showing that mutant Drp1 does not affect the endocytic pathway.

Figure 6

Absence of an effect of dominant negative mutant Drp1 on organelles other than mitochondria. Cells that overexpress mutant or wild-type Drp1 were identified by double labeling with anti-Drp1 antibody as indicated by the gray outlines. The ER was detected with an anti-VSV–G antibody by staining cells that were cotransfected with Drp1 and VSV-G carrying an ER retention signal (KKTN at the COOH terminus). The inset shows that the reticulum extends to the cell periphery, both in cells transfected with wild-type (Drp1-wt) and with mutant (Drp1-K38A). COPII-coated vesicles which bud from the ER were detected with anti-sec13 antibody. COPI-coated vesicles, which bud from the Golgi, were detected with anti-β-COP antibody. Lysosomes were detected with anti-LAMP1 antibody. Endosomes detected by cotransfecting epitope-tagged rab5 and peroxisomes detected with anti-catalase antibody were also not affected (not shown).

Figure 7

Effect of Drp1 on mitochondrial morphology. (A) COS-7 cells transfected with wild-type Drp1, wild-type dynamin, or mutant dynamin all show normal mitochondrial morphology. The cells were transfected with Drp1-wt (the nonneuronal isoform; 1), B-Drp1-wt (the brain-specific isoform of Drp1; 2), Dyn1-wt (dynamin I wild-type; 3) or Dyn1-K44A (dynamin I K44A, which blocks endocytosis [van der Bliek et al., 1993]; 4). The cells were double-labeled with a mitochondrial antibody (anti-COXI) and either anti-Drp1 or anti-dynamin antibody. Outlines of cells that are overexpressing Drp1 or dynamin are superimposed on the mitochondrial staining. Untransfected cells were not outlined. (B) COS-7 cells transfected with mutant Drp1 show collapsed mitochondria. Two examples of each condition are shown. The following constructs were tested: Drp1-K38A (the K38A mutant of the non-neuronal isoform; 5 and 5′), and B-Drp1-K38A (the K38A mutant of the brain specific isoform; 6 and 6′).

Figure 8

EM analysis of COS-7 cells transfected with mutant or wild type Drp1. (A) Thin section of a wild-type cell. This cell is a typical example of the cells observed in transfection experiments with wild-type Drp1 in which untransfected cells were indistinguishable from transfected cells. Mitochondria (m) are scattered throughout the cytoplasm. The nucleus is visible in the upper part of the panel (n). (B) Thin sections of cells transfected with K38A mutant Drp1. Each of the three panels shows a cluster of mitochondria near the nucleus (n). 1 illustrates the larger diameter of the peripheral mitochondria (*) and the smaller diameter of the central mitochondria (x). 2 shows ring-like mitochondria (★), which are common in cells transfected with mutant Drp1. 3 shows an onion-like membranous structure (▴) adjacent to a mitochondrial cluster in a cell transfected with mutant Drp1.

Figure 8

EM analysis of COS-7 cells transfected with mutant or wild type Drp1. (A) Thin section of a wild-type cell. This cell is a typical example of the cells observed in transfection experiments with wild-type Drp1 in which untransfected cells were indistinguishable from transfected cells. Mitochondria (m) are scattered throughout the cytoplasm. The nucleus is visible in the upper part of the panel (n). (B) Thin sections of cells transfected with K38A mutant Drp1. Each of the three panels shows a cluster of mitochondria near the nucleus (n). 1 illustrates the larger diameter of the peripheral mitochondria (*) and the smaller diameter of the central mitochondria (x). 2 shows ring-like mitochondria (★), which are common in cells transfected with mutant Drp1. 3 shows an onion-like membranous structure (▴) adjacent to a mitochondrial cluster in a cell transfected with mutant Drp1.