Pontus Aspenström

Miro1 Is a Calcium Sensor for Glutamate Receptor-Dependent Localization of Mitochondria at Synapses

Summary Energy use, mainly to reverse ion movements in neurons, is a fundamental constraint on brain information processing. Trafficking of mitochondria to locations in neurons where there are large ion fluxes is essential for powering neural function. Mitochondrial trafficking is regulated by Ca2+ entry through ionotropic glutamate receptors, but the underlying mechanism is unknown. We show that the protein Miro1 links mitochondria to KIF5 motor proteins, allowing mitochondria to move along microtubules. This linkage is inhibited by micromolar levels of Ca2+ binding to Miro1. With the EF hand domains of Miro1 mutated to prevent Ca2+ binding, Miro1 could still facilitate mitochondrial motility, but mitochondrial stopping induced by glutamate or neuronal activity was blocked. Activating neuronal NMDA receptors with exogenous or synaptically released glutamate led to Miro1 positioning mitochondria at the postsynaptic side of synapses. Thus, Miro1 is a key determinant of how energy supply is matched to energy usage in neurons.

Effectors for the Rho GTPases.

The Rho GTPases are simple enzymes with complex roles in regulating cell morphology, gene transcription, cell cycle progression, apoptosis and tumour progression. The picture has been further complicated by the steady rise in the number of known Rho GTPases as well as in the number of known regulators and target proteins of these GTPases. Recent implications of Rho effectors in human disease, however, might give important clues to how specificity is achieved in cell signalling pathways employing Rho GTPases.

Bidirectional Ca2+-dependent control of mitochondrial dynamics by the Miro GTPase

Calcium oscillations suppress mitochondrial movements along the microtubules to support on-demand distribution of mitochondria. To activate this mechanism, Ca2+ targets a yet unidentified cytoplasmic factor that does not seem to be a microtubular motor or a kinase/phosphatase. Here, we have studied the dependence of mitochondrial dynamics on the Miro GTPases that reside in the mitochondria and contain two EF-hand Ca2+-binding domains, in H9c2 cells and primary neurons. At resting cytoplasmic [Ca2+] ([Ca2+]c), movements of the mitochondria were enhanced by Miro overexpression irrespective of the presence of the EF-hands. The Ca2+-induced arrest of mitochondrial motility was also promoted by Miro overexpression and was suppressed when either the Miro were depleted or their EF-hand was mutated. Miro also enhanced the fusion state of the mitochondria at resting [Ca2+]c but promoted mitochondrial fragmentation at high [Ca2+]c. These effects of Miro on mitochondrial morphology seem to involve Drp1 suppression and activation, respectively. In primary neurons, Miro also caused an increase in dendritic mitochondrial mass and enhanced mitochondrial calcium signaling. Thus, Miro proteins serve as a [Ca2+]c-sensitive switch and bifunctional regulator for both the motility and fusion-fission dynamics of the mitochondria.

Rho GTPases have diverse effects on the organization of the actin filament system.

The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1-Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1-Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.

A Cdc42 target protein with homology to the non-kinase domain of FER has a potential role in regulating the actin cytoskeleton

BACKGROUND Members of the Rho family of small GTPases have been shown to have a diverse role in cell signalling events. They were originally identified as proteins that, by regulating the assembly of the actin cytoskeleton, are important determinants of cell morphology, and have recently been shown to be involved in transcriptional activation by the JNK/SAPK signalling pathway. In order to understand the mechanisms underlying the effects of Rho GTPases on these processes, the yeast two-hybrid system has been used to identify proteins that bind to an activated mutant of Cdc42, a Rho-family member. RESULTS A cDNA encoding a previously unidentified Cdc42 target protein, CIP4, which is 545 amino-acids long and contains an SH3 domain at its carboxyl terminus, was cloned from a human B-cell library. The amino terminus of CIP4 bears resemblance to the non-kinase domain of the FER and Fes/Fps family of tyrosine kinases. In addition, similarities to a number of proteins with roles in regulating the actin cytoskeleton were noticed. CIP4 binds to activated Cdc42 in vitro and in vivo and overexpression of CIP4 in Swiss 3T3 fibroblasts reduces the amount of stress fibres in these cells. Moreover, coexpression of activated Cdc42 and CIP4 leads to clustering of CIP4 to a large number of foci at the dorsal side of the cells. CONCLUSIONS CIP4 is a downstream target of activated GTP-bound Cdc42, and is similar in sequence to proteins involved in signalling and cytoskeletal control. Together, these findings suggest that CIP4 may act as a link between Cdc42 signalling and regulation of the actin cytoskeleton.

Tyrosine phosphorylation of the Wiskott-Aldrich syndrome protein by Lyn and Btk is regulated by CDC42.

The Wiskott‐Aldrich syndrome (WAS) is a rare immunodeficiency disease affecting mainly platelets and lymphocytes. Here, we show that the WAS gene product, WASp, is tyrosine phosphorylated upon aggregation of the high affinity IgE receptor (FcϵRI) at the surface of RBL‐2H3 rat tumor mast cells. Lyn and the Brutons tyrosine kinase (Btk), two protein tyrosine kinases involved in FcϵRI‐signaling phosphorylate WASp and interact with WASp in vivo. Interestingly, expression of a GTPase defective mutant form of CDC42, that interacts with WASp, is accompanied by a substantial increase in WASp tyrosine phosphorylation. This study suggests that activated CDC42 recruits WASp to the plasma membrane where it becomes phosphorylated by Lyn and Btk. We conclude that WASp represents a connection between protein tyrosine kinase signaling pathways and CDC42 function in cytoskeleton and cell growth regulation in hematopoietic cells.

A novel GTPase-activating protein for Rho interacts with a PDZ domain of the protein-tyrosine phosphatase PTPL1.

PTPL1 is an intracellular protein-tyrosine phosphatase that contains five PDZ domains. Here, we present the cloning of a novel 150-kDa protein, the four most C-terminal amino acid residues of which specifically interact with the fourth PDZ domain of PTPL1. The molecule contains a GTPase-activating protein (GAP) domain, a cysteine-rich, putative Zn2+- and diacylglycerol-binding domain, and a region of sequence homology to the product of the Caenorhabditis elegans geneZK669.1a. The GAP domain is active on Rho, Rac, and Cdc42in vitro but with a clear preference for Rho; we refer to the molecule as PTPL1-associated RhoGAP 1, PARG1. Rho is inactivated by GAPs, and protein-tyrosine phosphorylation has been implicated in Rho signaling. Therefore, a complex between PTPL1 and PARG1 may function as a powerful negative regulator of Rho signaling, acting both on Rho itself and on tyrosine phosphorylated components in the Rho signal transduction pathway.

Interaction between Smad7 and β-Catenin: Importance for Transforming Growth Factor β-Induced Apoptosis

ABSTRACT Members of the transforming growth factor β (TGF-β) and Wnt/wingless superfamilies regulate cell fate during development and tissue maintenance. Here we report that Smad7 interacts with β-catenin and lymphoid enhancer binding factor 1/T-cell-specific factor (LEF1/TCF), transcriptional regulators in Wnt signaling, in a TGF-β-dependent manner. Smad7 was found to be required for TGF-β1-induced accumulation of β-catenin and LEF1 in human prostate cancer (PC-3U) cells as well as in human keratinocytes (HaCaT cells). Moreover, when the endogenous Smad7 was repressed by specific small interfering RNA, TGF-β-induced increase of activated p38, Akt phosphorylated on Ser473, glycogen synthase kinase 3β phosphorylated on Ser9 was prevented, as well as the TGF-β-induced association between β-catenin and LEF1. Notably, the observed physical association of Smad7 and β-catenin was found to be important for TGF-β-induced apoptosis, since suppression of β-catenin expression by small interfering RNA decreased the apoptotic response to TGF-β.

Isolation and characterisation of DOCK8, a member of the DOCK180‐related regulators of cell morphology

In a yeast two‐hybrid system screen for Cdc42‐interacting proteins, we identified a protein with similarity to the CrkII‐binding protein DOCK180. A cDNA clone of this protein, designated DOCK8, encoded a gene‐product of 1701 amino acid residues with a molecular mass of 190 kDa. Immunofluorescence staining showed that transiently transfected HA‐tagged DOCK8, as well as endogenous DOCK8, was present at the cell edges in areas undergoing lamellipodia formation. Transient transfection of a C‐terminal fragment of DOCK8 resulted in the formation of vesicular structures. Interestingly, these vesicles also contained filamentous actin. These data suggest an involvement of DOCK8 in processes that affect the organisation of filamentous actin.

12/15-lipoxygenase–derived lipid peroxides control receptor tyrosine kinase signaling through oxidation of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are regulated through reversible oxidation of the active-site cysteine. Previous studies have implied soluble reactive oxygen species (ROS), like H2O2, as the mediators of PTP oxidation. The potential role(s) of peroxidized lipids in PTP oxidation have not been described. This study demonstrates that increases in cellular lipid peroxides, induced by disruption of glutathione peroxidase 4, induce cellular PTP oxidation and reduce the activity of PDGF receptor targeting PTPs. These effects were accompanied by site-selective increased PDGF β-receptor phosphorylation, sensitive to 12/15-lipoxygenase (12/15-LOX) inhibitors, and increased PDGF-induced cytoskeletal rearrangements. Importantly, the 12/15-LOX–derived 15-OOH-eicosatetraenoic acid lipid peroxide was much more effective than H2O2 in induction of in vitro PTP oxidation. Our study thus establishes that lipid peroxides are previously unrecognized inducers of oxidation of PTPs. This identifies a pathway for control of receptor tyrosine kinase signaling, which might also be involved in the etiology of diseases associated with increased lipid peroxidation.