Mitochondrial matrix Ca2+ as an intrinsic signal regulating mitochondrial motility in axons Karen T. Changa,1, Robert F. Niescierb, and Kyung-Tai Minb,1 aZilkha Neurogenetic Institute and Department of Cell and Neurobiology, University of Southern California, Los Angeles, CA 90033; and bDepartment of Biology, Indiana University, Bloomington, IN 47405 Edited by Ronald D. Vale, University of California, San Francisco, CA, and approved August 3, 2011 (received for review April 29, 2011) The proper distribution of mitochondria is particularly vital for a passive or active role in their own transport. Aside from the neurons because of their polarized structure and high energy endoplasmic reticulum, mitochondria are another main source 2+ 2+ demand. Mitochondria in axons constantly move in response to of Ca buffering within the cell. Following intracellular Ca 2+ physiological needs, but signals that regulate mitochondrial move- elevation, mitochondria rapidly take up and accumulate Ca 2+ ment are not well understood. Aside from producing ATP, Ca2+ buff- to maintain cellular Ca homeostasis (12). Studies also have shown that Ca2+ uptake into mitochondria occurs through the ering is another main function of mitochondria. Activities of many 2+ enzymes in mitochondria are also Ca2+-dependent, suggesting that mitochondrial Ca uniporter (12, 13), and that mitochondrial 2+ fl intramitochondrial Ca2+ concentration is important for mitochon- Ca in ux can increase ATP production by activating the TCA cycle, as well as enhance the activities of the electron transport- drial functions. Here, we report that mitochondrial motility in axons – is actively regulated by mitochondrial matrix Ca2+.Ca2+ entry chain enzymes and the ATP synthase complex (14 17). Never- theless, the relationship between mitochondrial energetic status through the mitochondrial Ca2+ uniporter modulates mitochondrial 2+ and mitochondrial movement remains controversial. A report transport, and mitochondrial Ca content correlates inversely with showed that anterogradely moving mitochondria have higher the speed of mitochondrial movement. Furthermore, the miro1 pro- 2+ membrane potential than those moving retrogradely (18), yet tein plays a role in Ca uptake into the mitochondria, which sub- another group found no difference between the membrane sequently affects mitochondrial movement. potentials of mitochondria moving in opposite directions or between the stationary and mobile mitochondria (19). The itochondria are dynamic organelles that constantly move parameters within mitochondria associated with changes in the Mwithin cells and undergo morphological changes in response pattern of mitochondrial motility thus remain unclear. Because to physiological needs (1–3). In neurons, mitochondria are abun- Ca2+ in mitochondria is associated with various mitochondrial dantly present throughout different subcellular compartments. functions, we hypothesized that mitochondrial Ca2+ may act as The machineries and signals that transport mitochondria from the a signal that allows mitochondria to actively regulate their own cell body (where they are synthesized) to the terminal need to be mobility. Here, we show Ca2+ influx through the mitochondrial carefully regulated because of the highly polarized structure and Ca2+ uniporter modulates mitochondrial transport, and that lengthy axon of a neuron (4–6). Defects in transport of mito- mitochondrial Ca2+ content correlated inversely with the speed chondria can cause deleterious effects on mitochondrial functions of mitochondrial movement. Furthermore, we demonstrate that in different parts of neurons, and hence affect neuronal survival the miro1 mutant modulates mitochondrial trafficking by alter- and function (2, 3). Recent efforts to investigate mitochondrial ing the amount of Ca2+ influx into the mitochondria in axons transportation have provided significant new information re- of hippocampal neurons. Taken together, our results imply that 2+ garding mitochondrial mobility, especially the mechanical com- mitochondrial matrix Ca is an intrinsic signal that actively ponents that modulate mitochondrial transport; however, it regulates mitochondrial transportation in neurons. remains unclear whether intrinsic signals inside of mitochondria also actively regulate mitochondrial movement. Results and Discussion Mitochondrial transport is mediated by interactions of the Visualization of Mitochondrial Ca2+ in Axons. Detecting small mitochondrial adaptor proteins to the kinesin and dynein changes in the level of Ca2+ within mitochondria of living neu- motors, as well as binding of the motor proteins to the cyto- rons have been challenging because of the lack of subcellular skeleton track (7, 8). It was posited that cytoplasmic Ca2+ level is specificity provided by synthetic Ca2+ indicators. To test the a key regulator of mitochondrial trafficking in axons and den- hypothesis that internal mitochondrial Ca2+ modulates mito- drites, and that intracellular Ca2+ influx impedes mitochondrial chondrial motility, we targeted a genetically encoded GFP-based movement by affecting the overall interactions between the mi- fluorescent Ca2+ indicator, Case12, to the mitochondria. This tochondrial adaptor, motor, and cytoskeleton track (9, 10). Two targeting was achieved by inserting a mitochondrial signal pep- different mechanisms for Ca2+-mediated stop in mitochondrial tide into the N terminus of the Case12 protein (mito-Case12). 2+ trafficking were proposed. Wang and Schwarz suggested that Case12 protein allows linear detection of Ca ion concentration 2+ Ca2+ binding to the EF hand motif of the mitochondrial adaptor within the physiological range and its binding to Ca is rapid 2+ protein miro1 recruits kinesin-1 motor and, hence, derails and reversible, thus making real-time detection of changes in Ca kinesin-1 from the microtubule track, thereby stopping mito- level within a cell possible (20, 21). Mito-Case12 colocalized with chondrial transportation in axons (10). Macaskill et al. proposed mito-RFP in transfected hippocampal neurons (Fig. 1A, Fig. S1 that following Ca2+ influx induced by glutamate or neuronal A and C), thus confirming that mito-Case12 is indeed targeted to activity, Ca2+ binding to the EF hand motif of the miro1 protein the mitochondria. Next, to test the ability of mito-Case12 to causes miro1 to dissociate from the kinesin-1 motor, and hence halts mitochondrial movement (11). Although the mechanisms 2+ proposed for this Ca -induced arrest in mitochondrial move- Author contributions: K.T.C. and K.-T.M. designed research; K.T.C. and R.F.N. performed ment are different, both groups agree that cytoplasmic Ca2+ research; K.T.C. and K.-T.M. analyzed data; and K.T.C. and K.-T.M. wrote the paper. elevation and its subsequent binding to the miro1 protein are The authors declare no conflict of interest. key events regulating mitochondrial motility in neurons. This article is a PNAS Direct Submission. 2+ Cytoplasmic Ca has been deemed important for mitochon- 1To whom correspondence may be addressed. E-mail: [email protected] or changkt@ drial motility in neurons; however, the relationship between usc.edu. 2+ mitochondrial Ca content and movement has not been es- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tablished. It also remains unclear whether mitochondria play 1073/pnas.1106862108/-/DCSupplemental. 15456e15461 | PNAS | September 13, 2011 | vol. 108 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1106862108 Downloaded by guest on September 24, 2021 respond to increases in Ca2+ content, we treated hippocampal stationary mitochondria are displayed as straight vertical lines as neurons containing mito-Case12 and mito-RFP with calcimycin, they are immobile over time, whereas the moving mitochondria aCa2+ ionophore (Fig. S1A). Following calcimycin treatment, are seen as diagonal lines. Interestingly, we noticed that compared the green fluorescence intensity of mito-Case12 increased sig- with stationary mitochondria, moving mitochondria tend to have nificantly within seconds in mitochondria, but the mito-RFP lower mito-Case12 fluorescence signal, and hence lower mito- signal remained constant (Fig. S1 A and B). Mito-Case12 signal chondrial Ca2+ content. To minimize for variability caused by is dependent on calcium influx into the cell, because calcimycin drifts in the focal plane as well as differences in size of mito- treatment in calcium-free extracellular solution failed to cause chondria, we normalized mito-Case12 labeling intensity to that of an increase in mito-Case12 signal (Fig. S1 C and D). To account mito-RFP intensity. Fig. 1 A and B and Fig. S2 show that average for the difference in the size of mitochondria and thus the mito-Case12:mito-RFP ratio is clearly lower in moving mito- amount of fluorescent protein inside of mitochondria, as well chondria (mean = 0.27 ± 0.01), but stationary mitochondria as change in focal plane that could occur over time, we exhibited higher ratio of mito-Case12:mito-RFP (mean = 0.39 ± also normalized mito-Case12 labeling intensity to that of mito- 0.01), confirming that mitochondria in motion tend to have lower RFP intensity. Similar to mito-Case12 signal, normalized mito- matrix Ca2+ content than stationary mitochondria. Case12:mito-RFP ratio increased following calcimycin treat- As mitochondria move in opposite directions (anterograde vs. ment. Together, our results suggest that (i) mito-Case12 is able retrograde) and with different speed, we next determined if the to detect Ca2+