Making a Division Apparatus on Mitochondria

Summary

Mitochondrial division apparatuses are generally thought to form by oligomerization of Drp1 at pre-determined sites on mitochondria. A recent study by Ji et al. [10] now shows that the Drp1 oligomers on mitochondria move, merge, and mature into a functional division apparatus.

Mitochondria are dynamic organelles: They grow, move, divide and fuse in cells in a highly regulated way [1]. Mitochondrial division is important for organelle function, distribution and turnover in human physiology and mutations in dynamin-related protein 1 (Drp1), a mechanochemical GTPase that drives constriction of mitochondria during division, cause human disorders that can severely affect the nervous system [2, 3]. Analysis of Drp1 knockout mice revealed the critical role that mitochondrial division plays in maintaining the functional integrity of the brain and heart [4]. Altered mitochondrial division via abnormal activation or inactivation due to misregulation of Drp1 has also been linked to age-related neurodegenerative disorders [5].

Drp1 is a cytosolic protein that is recruited to the surface of mitochondria via interactions with Mff (mitochondrial fission factor), Mid49/51 (mitochondrial dynamics proteins of 49 and 51 kDa) and Fis1 (mitochondrial fission 1 protein) [1]. Drp1 oligomerizes on mitochondria into ring-like structures that wrap around mitochondria to form a division apparatus. The dynamics of the assembly and disassembly of Drp1 on mitochondria appear to be regulated by actin and many actin-binding proteins such as INF2, myosin II, Spire, cortactin, cofilin and the Arp2/3 complex [6–9]. It would be intriguing to understand how Drp1 assembles into high-order oligomeric, force-generating helices around the large organelle, which is approximately 300 nm in diameter. It would also be fascinating to decipher where the assembly process begins and how the Drp1-binding proteins and the actin system help to create the dynamic protein apparatus on mitochondria.

A recent study by Ji et al. (2015) [10] investigated these questions, provided exciting new insights into the assembly process of the mitochondrial division apparatus. To investigate the mechanism of Drp1 oligomerization on mitochondria, these authors employed live imaging approaches of cells expressing green fluorescent protein (GFP)-Drp1 using confocal and super-resolution microscopy. To image Drp1 dynamics, the authors developed a cell system in which GFP-Drp1 was expressed at modest levels, then focused on the dynamics of oligomeric Drp1 by computationally removing the background GFP-Drp1 signal from the cytoplasm. The authors observed a wide variety of intensities for GFP-Drp1 punctae, corresponding to a variety of oligomeric states. These Drp1 oligomers move on mitochondria and, intriguingly, merge and become larger and more stationary prior to cutting the mitochondria. Interestingly, only a small fraction of these Drp1 oligomers engages in mitochondrial division. These observations suggest that the mitochondrial division apparatus grows via lateral merging of small Drp1 oligomers after they are recruited to the outer membrane from the cytosol (Figure 1). This oligomeric maturation model is critically different from a model in which Drp1 is assembled at pre-determined sites on the mitochondria via the incorporation of Drp1 from the cytosol.

Figure 1. Models for forming the mitochondrial division apparatus.

In the de novo assembly model, a mitochondrial division apparatus forms at pre-determined sites on the mitochondria by incorporating Drp1 from cytosol. In the oligomeric maturation model supported by Ji et al. [10], a mitochondrial division apparatus develops by laterally fusing small Drp1 oligomers after they translocate to the mitochondria from cytosol.

Ji et al. [10] demonstrate that actin filaments, myosin II and the endoplasmic reticulum (ER)-associated actin nucleation factor INF2 are involved in oligomeric maturation. To do this, they used genetic and pharmacological inhibition of actin polymerization, myosin II and INF2 in combination with calcium ionophore (ionomycin) treatment, which induces rapid mitochondrial division in a synchronized manner. Upon ionomycin treatment, the authors observed increased actin polymerization and oligomeric maturation of Drp1 on mitochondria, which colocalized even prior to mitochondrial division. Knockdown of INF2 blocks actin polymerization and the oligomeric maturation of Drp1; myosin II inhibition blocks the maturation process without inhibiting actin polymerization. Therefore, it appears that INF2 induces actin polymerization and that the myosin motor and actin filaments assemble Drp1 oligomers on mitochondria. How the actin system drives the maturation process is a fascinating and open question that should be addressed by future studies.

Oligomerization of Drp1 stimulates its GTPase activity, which is a critical biochemical event during mitochondrial division. Previous studies have shown that the major Drp1 receptor Mff stimulates GTP hydrolysis of Drp1, which suggests that Mff recruits and activates Drp1 on mitochondria [11]. Ji et al. have now shown that actin filaments interact with Drp1 in vitro and stimulate the GTPase activity of Drp1 together with Mff in a highly synergistic manner. These beautiful biochemical data suggest that Mff and actin filaments together drive the oligomeric maturation of Drp1.

An important future question is how ER-mitochondria contact sites play a role in the maturation process. Since INF2 is bound to the ER and regulates the actin polymerization necessary for the oligomeric maturation, Drp1 oligomers may continuously interact with the ER during the entire maturation process, allowing both the ER and Drp1 oligomers to co-migrate along the mitochondria. By contrast, Drp1 may begin to assemble independently of the ER; once Drp1 oligomers arrive at the contact site, the maturation process is enhanced and a functionally competent division apparatus appears at the organelle interface.

Finally, Drp1 is known to oligomerize and form a division apparatus. Previous studies have shown that the recruitment of Drp1 and the assembly of the division machine depend on the Drp1 receptor, the actin system and interactions with the ER [6, 7, 9, 12]. Thanks to the work of Ji et al. [10], we now have a better understanding about how these components orchestrate to build the membrane remodeling machinery on mitochondria. It is a stimulating possibility that the mitochondrial division apparatus examines the functional status of mitochondria to select where and when to sever mitochondria as the Drp1 oligomers move during maturation. It would be of great interest to test how Mff controls the oligomeric maturation in cells and the contribution of other Drp1 receptors such as Mid49/51 and Fis1 to this maturation process. These Drp1-binding proteins may act in distinct steps along the oligomeric maturation pathway.