Mouse lines with photo‐activatable mitochondria to study mitochondrial dynamics

Many pathological states involve dysregulation of mitochondrial fusion, fission, or transport. These dynamic events are usually studied in cells lines because of the challenges in tracking mitochondria in tissues. To investigate mitochondrial dynamics in tissues and disease models, we generated two mouse lines withphoto‐activatable mitochondria (PhAM). In the PhAM ^floxed line, a mitochondrially localized version of the photo‐convertible fluorescent protein Dendra2 (mito‐Dendra2) is targeted to the ubiquitously expressed Rosa26 locus, along with an upstream loxP‐flanked termination signal. Expression of Cre in PhAM ^floxed cells results in bright mito‐Dendra2 fluorescence without adverse effects on mitochondrial morphology. When crossed with Cre drivers, the PhAM ^floxed line expresses mito‐Dendra2 in specific cell types, allowing mitochondria to be tracked even in tissues that have high cell density. In a second line (PhAM ^excised), the expression of mito‐Dendra2 is ubiquitous, allowing mitochondria to be analyzed in a wide range of live and fixed tissues. By using photo‐conversion techniques, we directly measured mitochondrial fusion events in cultured cells as well as tissues such as skeletal muscle. These mouse lines facilitate analysis of mitochondrial dynamics in a wide spectrum of primary cells and tissues, and can be used to examine mitochondria in developmental transitions and disease states. © genesis 1–11, 2012. © 2012 Wiley Periodicals, Inc.     

p57KIP2 control of actin cytoskeleton dynamics is responsible for its mitochondrial pro-apoptotic effect

p57 (Kip2, cyclin-dependent kinase inhibitor 1C), often found downregulated in cancer, is reported to hold tumor suppressor properties. Originally described as a cyclin-dependent kinase (cdk) inhibitor, p57^KIP2 has since been shown to influence other cellular processes, beyond cell cycle regulation, including cell death and cell migration. Inhibition of cell migration by p57^KIP2 is attributed to the stabilization of the actin cytoskeleton through the activation of LIM domain kinase-1 (LIMK-1). Furthermore, p57^KIP2 is able to enhance mitochondrial-mediated apoptosis. Here, we report that the cell death promoting effect of p57^KIP2 is linked to its effect on the actin cytoskeleton. Indeed, whereas Jasplakinolide, an actin cytoskeleton-stabilizing agent, mimicked p57^KIP2''s pro-apoptotic effect, destabilizing the actin cytoskeleton with cytochalsin D reversed p57^KIP2''s pro-apoptotic function. Conversely, LIMK-1, the enzyme mediating p57^KIP2''s effect on the actin cytoskeleton, was required for p57^KIP2''s death promoting effect. Finally, p57^KIP2-mediated stabilization of the actin cytoskeleton was associated with the displacement of hexokinase-1, an inhibitor of the mitochondrial voltage-dependent anion channel, from the mitochondria, providing a possible mechanism for the promotion of the mitochondrial apoptotic cell death pathway. Altogether, our findings link together two tumor suppressor properties of p57^KIP2, by showing that the promotion of cell death by p57^KIP2 requires its actin cytoskeleton stabilization function.     

Bacterial tail anchors can target to the mitochondrial outer membrane

Background

During the generation and evolution of the eukaryotic cell, a proteobacterial endosymbiont was re-fashioned into the mitochondrion, an organelle that appears to have been present in the ancestor of all present-day eukaryotes. Mitochondria harbor proteomes derived from coding information located both inside and outside the organelle, and the rate-limiting step toward the formation of eukaryotic cells may have been development of an import apparatus allowing protein entry to mitochondria. Currently, a widely conserved translocon allows proteins to pass from the cytosol into mitochondria, but how proteins encoded outside of mitochondria were first directed to these organelles at the dawn of eukaryogenesis is not clear. Because several proteins targeted by a carboxyl-terminal tail anchor (TA) appear to have the ability to insert spontaneously into the mitochondrial outer membrane (OM), it is possible that self-inserting, tail-anchored polypeptides obtained from bacteria might have formed the first gate allowing proteins to access mitochondria from the cytosol.

Results

Here, we tested whether bacterial TAs are capable of targeting to mitochondria. In a survey of proteins encoded by the proteobacterium Escherichia coli, predicted TA sequences were directed to specific subcellular locations within the yeast Saccharomyces cerevisiae. Importantly, TAs obtained from DUF883 family members ElaB and YqjD were abundantly localized to and inserted at the mitochondrial OM.

Conclusions

Our results support the notion that eukaryotic cells are able to utilize membrane-targeting signals present in bacterial proteins obtained by lateral gene transfer, and our findings make plausible a model in which mitochondrial protein translocation was first driven by tail-anchored proteins.

Reviewers

This article was reviewed by Michael Ryan and Thomas Simmen.

     

Regulation of actin dynamics by annexin 2

Annexin 2 is a ubiquitous Ca(2+)-binding protein that is essential for actin-dependent vesicle transport. Here, we show that in spontaneously motile cells annexin 2 is concentrated in dynamic actin-rich protrusions, and that depletion of annexin 2 using siRNA leads to the accumulation of stress fibres and loss of protrusive and retractile activity. Cells co-expressing annexin 2-CFP and actin-YFP exhibit Ca(2+)-dependent fluorescense resonance energy transfer throughout the cytoplasm and in membrane ruffles and protrusions, suggesting that annexin 2 may directly interact with actin. This notion was supported by biochemical studies, in which we show that annexin 2 reduces the polymerisation rate of actin monomers in a dose-dependent manner. By measuring actin polymerisation rates in the presence of barbed-end and pointed-end cappers, we further demonstrate that annexin 2 specifically inhibits filament elongation at the barbed ends. These results show that annexin 2 has an essential role in maintaining the plasticity of the dynamic membrane-associated actin cytoskeleton, and that its activity in this context may be at least partly explained through direct interactions with polymerised and monomeric actin.     

Heterogeneity of mitochondrial matrix free Ca2+: resolution of Ca2+ dynamics in individual mitochondria in situ

The role of mitochondria inCa²⁺ homeostasis is controversial.We employed the Ca²⁺-sensitive dyerhod 2 with novel, high temporal and spatial resolution imaging toevaluate changes in the matrix freeCa²⁺ concentration of individualmitochondria([Ca²⁺]_m)in agonist-stimulated, primary cultured aortic myocytes. Stimulation with 10 µM serotonin (5-HT) evoked modest cytosolicCa²⁺ transients[cytosolic freeCa²⁺ concentration([Ca²⁺]_cyt)<500 nM; measured with fura 2] and triggered contractions inshort-term cultured myocytes. However, 5-HT triggered a large mitochondrial rhod 2 signal (indicating pronounced elevation of [Ca²⁺]_m)in only 4% of cells. This revealed heterogeneity in the responses ofindividual mitochondria, all of which stained with MitoTracker GreenFM. In contrast, stimulation with 100 µM ATP evoked large cytosolicCa²⁺ transients (>1,000 nM) andinduced pronounced, reversible elevation of[Ca²⁺]_m(measured as rhod 2 fluorescence) in 60% of cells. This mitochondrial Ca²⁺ uptake usually lagged behindthe cytosolic Ca²⁺ transient peakby 3-5 s, and[Ca²⁺]_mdeclined more slowly than did bulk[Ca²⁺]_cyt.The uptake delay may prevent mitochondria from interfering with rapidsignaling events while enhancing the mitochondrial response to large,long-duration elevations of[Ca²⁺]_cyt.The responses of arterial myocytes to modest physiological stimulationdo not, however, depend on such marked changes in [Ca²⁺]_m.     

From mitochondrial dynamics to arrhythmias

The reactive oxygen species (ROS)-dependent mitochondrial oscillator described in cardiac cells exhibits at least two modes of function under physiological conditions or in response to metabolic and oxidative stress. Both modes depend upon network behavior of mitochondria. Under physiological conditions cardiac mitochondria behave as a network of coupled oscillators with a broad range of frequencies. ROS weakly couples mitochondria under normal conditions but becomes a strong coupling messenger when, under oxidative stress, the mitochondrial network attains criticality. Mitochondrial criticality is achieved when a threshold of ROS is overcome and a certain density of mitochondria forms a cluster that spans the whole cell. Under these conditions, the slightest perturbation triggers a cell-wide collapse of the mitochondrial membrane potential, Δψ_m, visualized as a depolarization wave throughout the cell which is followed by whole cell synchronized oscillations in Δψ_m, NADH, ROS, and GSH. This dynamic behavior scales from the mitochondrion to the cell by driving cellular excitability and the whole heart into catastrophic arrhythmias. A network collapse of Δψ_m under criticality leads to: (i) energetic failure, (ii) temporal and regional alterations in action potential (AP), (iii) development of zones of impaired conduction in the myocardium, and, ultimately, (iv) a fatal ventricular arrhythmia.     

Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy

Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release.     

Coupling actin dynamics and membrane dynamics during endocytosis.

A convergence of cellular, genetic and biochemical studies supports the hypothesis that the actin cytoskeleton is coupled to endocytic processes, but the roles played by actin filaments during endocytosis are not yet clear. Recent studies have identified several proteins that may functionally link the endocytic machinery with actin filament dynamics. Three of these proteins, Abp1p, Pan1p and cortactin, are activators of actin assembly nucleated by the Arp2/3 complex, a key regulator of actin assembly in vivo. Two others, intersectin and syndapin, bind N-WASp, a potent activator of actin assembly via the Arp2/3 complex. All of these proteins also bind components of the endocytic machinery, and thus, could coordinately regulate actin assembly and trafficking events. Hip1R, an F-actin-binding protein that associates with clathrin-coated vesicles, may physically link endocytic vesicles to actin filaments. The GTPase dynamin is implicated in modulating actin filaments at specialized actin-rich structures of the cell cortex, suggesting that dynamin may regulate the organization of cortical actin filaments as well as regulate actin dynamics during endocytosis. Finally, myosin VI may generate actin-dependent forces for membrane invagination or vesicle movement during the early stages of endocytosis.     

RhoA-mediated MLC2 regulates actin dynamics for cytokinesis in meiosis

During oocyte meiosis, the bipolar spindle forms in the central cytoplasm and then migrates to the cortex. Subsequently, the oocyte extrudes the polar body through two successive asymmetric divisions, which are regulated primarily by actin filaments. Myosin light chain2 (MLC2) phosphorylation plays pivotal roles in smooth muscle contraction, stress fiber formation, cell motility and cytokinesis. However, whether MLC2 phosphorylation participates in the oocyte polarization and asymmetric division has not been clarified. The present study investigated the expression and functions of MLC2 during mouse oocyte meiosis. Our result showed that p-MLC2 was localized in the oocyte cortex, with a thickened cap above the chromosomes. Meanwhile, p-MLC2 was also localized in the poles of spindle. Disruption of MLC2 activity by MLC2 knock down (K_D) caused the failure of polar body extrusion. Immunofluorescent staining showed that a large proportion of oocytes arrested in telophase stage and failed to undergo cytokinesis after culturing for 12 hours. In the meantime, actin filament staining at oocyte membrane and cytoplasm were reduced in MLC2 K_D oocytes. Finally, we found that the phosphorylation of MLC2 protein levels was decreased after disruption of RhoA activity. Above all, our data indicated that the RhoA-mediated MLC2 regulates the actin organization for cytokinesis during mouse oocyte maturation.     

In vivo visualization of actin dynamics and actin interactions by BiFC

The method of bimolecular fluorescence complementation (BiFC) enables selective visualization of protein interactions. While BiFC complex formation under in vitro conditions is considered to be essentially irreversible, there are hints that under in vivo conditions BiFC complex formation can be reversible. In the present study we used the BiFC method to visualize in vivo actin cytoskeleton dynamics. We demonstrate that in living cells formation of actin/actin BiFC complexes is reversible. Furthermore, we show heterologous binding between actin and protein kinase C delta (PKCdelta). Treatment with phorbol esters caused translocation of actin/PKCdelta complexes from the cytosol to the plasma membrane independent of an intact actin cytoskeleton. Our experiments demonstrate that the BiFC method might be a useful tool to investigate participation of the actin cytoskeleton in regulation of cell function.     

p57Kip2 regulates actin dynamics by binding and translocating LIM-kinase 1 to the nucleus

p57Kip2 is the only cyclin-dependent kinase (Cdk) inhibitor shown to be essential for mouse embryogenesis. The fact suggests that p57 has a specific role that cannot be compensated by other Cdk inhibitors. LIM-kinase 1 (LIMK-1) is a downstream effector of the Rho family of GTPases that phosphorylates and inactivates an actin depolymerization factor, cofilin, to induce the formation of actin fiber. Here we demonstrate that p57 regulates actin dynamics by binding and translocating LIMK-1 from the cytoplasm into the nucleus, which in turn results in a reorganization of actin fiber. The central region of p57, a unique feature among the Cdk inhibitors, and the N-terminal region of LIMK-1, which contains the LIM domains were essential for the interaction. Expression of p57, but not p27Kip1 or a p57 mutant, with a deletion in the central region was shown to induce marked reorganization of actin filament and a translocation of LIMK-1. Our findings indicate p57 may act as a key regulator in embryogenesis by bearing two distinct functions, the regulation of cell cycle through binding to Cdks and the regulation of actin dynamics through binding to LIMK-1, both of which should be important in developmental procedure.     

Fusion of mitochondria in mammalian cells is dependent on the mitochondrial inner membrane potential and independent of microtubules or actin

Mitochondrial fusion is a poorly characterized process which has mainly been studied in yeast and Drosophila but is thought to occur in all eukaryotes. Until now, there was only indirect evidence to support such a process in mammalian cells. In this study, using a cell fusion system, we found that mitochondrial fusion occurs rapidly in mammalian cells and is completed in less than 24 h. We report that the fusion of mitochondria requires an intact mitochondrial inner membrane potential but is independent of a functional cytoskeleton.