Vimar Is a Novel Regulator of Mitochondrial Fission through Miro

abstract: ...As fundamental processes in mitochondrial dynamics, mitochondrial fusion, fission and transport are regulated by several core components, including Miro. As an atypical Rho-like small GTPase with high molecular mass, the exchange of GDP/GTP in Miro may require assistance from a guanine nucleotide exchange factor (GEF). However, the GEF for Miro has not been identified. While studying mitochondrial morphology in Drosophila , we incidentally observed that the loss of vimar , a gene encoding an atypical GEF, enhanced mitochondrial fission under normal physiological conditions. Because Vimar could co-immunoprecipitate with Miro in vitro , we speculated that Vimar might be the GEF of Miro. In support of this hypothesis, a loss-of-function (LOF) vimar mutant rescued mitochondrial enlargement induced by a gain-of-function (GOF) Miro transgene; whereas a GOF vimar transgene enhanced Miro function. In addition, vimar lost its effect under the expression of a constitutively GTP-bound or GDP-bound Miro mutant background. These results indicate a genetic dependence of vimar on Miro. Moreover, we found that mitochondrial fission played a functional role in high-calcium induced necrosis, and a LOF vimar mutant rescued the mitochondrial fission defect and cell death. This result can also be explained by vimar's function through Miro, because Miros effect on mitochondrial morphology is altered upon binding with calcium. In addition, a PINK1 mutant, which induced mitochondrial enlargement and had been considered as a Drosophila model of Parkinsons disease (PD), caused fly muscle defects, and the loss of vimar could rescue these defects. Furthermore, we found that the mammalian homolog of Vimar, RAP1GDS1, played a similar role in regulating mitochondrial morphology, suggesting a functional conservation of this GEF member. The Miro/Vimar complex may be a promising drug target for diseases in which mitochondrial fission and fusion are dysfunctional.

... homolog of Vimar, RAP1GDS1, played a similar role in regulating mitochondrial morphology, suggesting a functional conservation of this GEF member. The Miro/Vimar complex may be a promising drug target for diseases in which mitochondrial fission and fusion are dysfunctional. Author Summary Mitochondrial dynamics including fusion, fission and transport are essential for energy supply in eukaryotic cells; and defects in mitochondrial dynamics often result in premature aging and diseases such as

... of vimar promoted mitochondrial shortening; and this function was mediated through Miro. As a GEF, Vimar partially localized on mitochondria and could physically interact with Miro. In the pathophysiological conditions, including a Pink1 mutant to model PD and a calcium-overload induced stress to model neuronal necrosis in Drosophila , loss of vimar suppressed both aberrant mitochondrial fusion and fragmentation in PD and necrosis, respectively. As the mammalian homolog of Vimar, RAP1GDS1 function was similar to Vimar. Therefore, Vimar/ RAP1GDS1 may be a great drug target to deal with diseases caused by defective mitochondrial dynamics. This work is supported by the Chinese Ministry of Science and Technology (Grant No. 2013CB530700; http://www.most.gov.cn/ ) to LL and the National Science Foundation (Grant No. 81325007; http://www.nsfc.gov.cn/ ) for Distinguished Young Scholars to XJ. The funders had no role in study design, data collection and analysis, decision to publish, or

... not regulate apoptosis. The mammalian homolog of vimar (RAP1GDS1) plays a similar role in mitochondrial morphology and cell death A protein sequence comparison showed that Drosophila vimar shares great similarity with the mammalian protein RAP1GDS1 ( S6 Fig ); however, it is not clear whether vimar is a functional homolog of RAP1GDS1 [ 51 ]. Here, we further investigated the role of RAP1GDS1 in mitochondrial morphology. First, we used a lentivirus to transfect a RAP1GDS1 shRNA into HEK293T cells and established a stable cell line. As expected, the protein level of the RAP1GDS1 was significantly reduced in the shRNA line ( S7A Fig ). Then, this shRNA line was transiently transfected with a mitochondrial reporter, mitoDsRed. We found that the mitochondrial length showed a trend of reduction in the RAP1GDS1 shRNA cells ( Fig 5A ). Next, we studied the effect of RAP1GDS1 on necrosis. Necrotic cell death was induced by a calcium ionophore (A23187), which causes calcium overloading and necrosis [ 52 ]. As expected, the calcium ionophore induced mitochondrial fragmentation, and the RAP1GDS1 shRNA rescued the mitochondrial defect ( Fig 5B ). To quantify the cell death, we measured cellular ATP level and performed propidium iodide (PI) staining [ 53 ]. The result showed that RAP1GDS1 shRNA rescued necrosis in both assays ( Fig 5C and 5D ). Moreover, we tested the RAP1GDS1 shRNA in another human cell line, the SH-SY5Y neuroblastoma cells. Similar to the HEK293T cells, the RAP1GDS1 shRNA protected the SH-SY5Y cells from calcium overload ( S7B and S7C Fig ). In addition, we examined the effect of a Miro-1 siRNA on calcium ionophore induced necrosis. The result showed that it also rescued the cell death ( Fig 5E and 5F , and the Miro-1 siRNA effect is shown in S7D Fig ). Furthermore, the HA-tagged Miro1 and the Flag-tagged RAP1GDS1 could co-immunoprecipitate in vitro ( Fig 5G ). Together, these results indicate that the function of the Miro1/RAP1GDS1 complex in regulating mitochondrial

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Gene: RAP1GDS1 - RAP1, GTP-GDP dissociation stimulator 1 - (NCBI)

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