Michael A Frohman

Publications of Michael A Frohman

• Visualizing mitochondrial lipids and fusion events in Mammalian cells.

  Authors: Huiyan Huang, Michael A Frohman

  Methods in cell biology. 01/2012; 108:131-45.

  Mitochondria are dynamic organelles that frequently undergo fusion and fission, the balance of which is critical for proper cellular functioning and viability. Most studies on mitochondrial fusionMitochondria are dynamic organelles that frequently undergo fusion and fission, the balance of which is critical for proper cellular functioning and viability. Most studies on mitochondrial fusion and fission mechanisms have focused on proteins thought to physically mediate the events. However, dynamic changes in membrane phospholipids also play roles in facilitating the fusion and fission events. This chapter will review the importance of lipids in mitochondrial dynamics and some of the methods that can be used to study the function of lipids in mitochondrial fusion and fission.

• Roles for the lipid-signaling enzyme MitoPLD in mitochondrial dynamics, piRNA biogenesis, and spermatogenesis.

  Authors: Qun Gao, Michael A Frohman

  BMB reports. 01/2012; 45(1):7-13.

  Phospholipase D (PLD), a superfamily of signaling enzymes that most commonly generate the lipid second messenger Phosphatidic Acid (PA), is found in diverse organisms from bacteria to man andPhospholipase D (PLD), a superfamily of signaling enzymes that most commonly generate the lipid second messenger Phosphatidic Acid (PA), is found in diverse organisms from bacteria to man and functions in multiple cellular pathways. A fascinating member of the family, MitoPLD, is anchored to the mitochondrial surface and has two reported roles. In the first role, MitoPLD-generated PA regulates mitochondrial shape through facilitating mitochondrial fusion. In the second role, MitoPLD performs a critical function in a pathway that creates a specialized form of RNAi required by developing spermatocytes to suppress transposon mobilization during meiosis. This spermatocyte-specific RNAi, known as piRNA, is generated in the nuage, an electron-dense accumulation of RNA templates and processing proteins that localize adjacent to mitochondria in a structure also called intermitochondrial cement. In this review, we summarize recent findings on these roles for MitoPLD functions, highlighting directions that need to be pursued to define the underlying mechanisms.

• A role for phospholipase d3 in myotube formation.

  Authors: Mary Osisami, Wahida Ali, Michael A Frohman

  PloS one. 01/2012; 7(3):e33341.

  Phospholipase D3 (PLD3) is a non-classical, poorly characterized member of the PLD superfamily of signaling enzymes. PLD3 is a type II glycoprotein associated with the endoplasmic reticulum, isPhospholipase D3 (PLD3) is a non-classical, poorly characterized member of the PLD superfamily of signaling enzymes. PLD3 is a type II glycoprotein associated with the endoplasmic reticulum, is expressed in a wide range of tissues and cells, and undergoes dramatic upregulation in neurons and muscle cells during differentiation. Using an in vitro skeletal muscle differentiation system, we define the ER-tethering mechanism and report that increased PLD3 expression enhances myotube formation, whereas a putatively dominant-negative PLD3 mutant isoform reduces myotube formation. ER stress, which also enhances myotube formation, is shown here to increase PLD3 expression levels. PLD3 protein was observed to localize to a restricted set of subcellular membrane sites in myotubes that may derive from or constitute a subdomain of the endoplasmic reticulum. These findings suggest that PLD3 plays a role in myogenesis during myotube formation, potentially in the events surrounding ER reorganization.

• Class III PI-3-kinase activates phospholipase D in an amino acid-sensing mTORC1 pathway.

  Authors: Mee-Sup Yoon, Guangwei Du, Jonathan M Backer, Michael A Frohman, Jie Chen

  The Journal of cell biology. 10/2011; 195(3):435-47.

  The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex, mTORC1, regulates cell growth in response to mitogenic signals and amino acid availability. Phospholipase D (PLD) and itsThe rapamycin-sensitive mammalian target of rapamycin (mTOR) complex, mTORC1, regulates cell growth in response to mitogenic signals and amino acid availability. Phospholipase D (PLD) and its product, phosphatidic acid, have been established as mediators of mitogenic activation of mTORC1. In this study, we identify a novel role for PLD1 in an amino acid-sensing pathway. We find that amino acids activate PLD1 and that PLD1 is indispensable for amino acid activation of mTORC1. Activation of PLD1 by amino acids requires the class III phosphatidylinositol 3-kinase hVps34, which stimulates PLD1 activity through a functional interaction between phosphatidylinositol 3-phosphate and the Phox homology (PX) domain of PLD1. Furthermore, amino acids stimulate PLD1 translocation to the lysosomal region where mTORC1 activation occurs in an hVps34-dependent manner, and this translocation is necessary for mTORC1 activation. The PX domain is required for PLD1 translocation, mTORC1 activation, and cell size regulation. Finally, we show that the hVps34-PLD1 pathway acts independently of, and in parallel to, the Rag pathway in regulating amino acid activation of mTORC1.

• Mammalian phospholipase D physiological and pathological roles.

  Authors: X Peng, M A Frohman

  Acta physiologica (Oxford, England). 03/2011; 204(2):219-26.

  Phospholipase D (PLD), a superfamily of signalling enzymes that most commonly generate the lipid second messenger phosphatidic acid, is found in diverse organisms from bacteria to humans andPhospholipase D (PLD), a superfamily of signalling enzymes that most commonly generate the lipid second messenger phosphatidic acid, is found in diverse organisms from bacteria to humans and functions in multiple cellular pathways. Since the early 1980s when mammalian PLD activities were first described, most of the important insights concerning PLD function have been gained from studies on cellular models. Reports on physiological and pathophysiological roles for members of the mammalian PLD superfamily are now starting to emerge from genetic models. In this review, we summarize recent findings on PLD functions in these model systems, highlighting newly appreciated connections of the superfamily to cancer, neuronal pathophysiology, cardiovascular topics, spermatogenesis and infectious diseases.

• piRNA-associated germline nuage formation and spermatogenesis require MitoPLD profusogenic mitochondrial-surface lipid signaling.

  Authors: Huiyan Huang, Qun Gao, Xiaoxue Peng, Seok-Yong Choi, Krishna Sarma, Hongmei Ren, Andrew J Morris, Michael A Frohman

  Developmental cell. 03/2011; 20(3):376-87.

  The mammalian Phospholipase D MitoPLD facilitates mitochondrial fusion by generating the signaling lipid phosphatidic acid (PA). The Drosophila MitoPLD homolog Zucchini (Zuc), a proposed cytoplasmicThe mammalian Phospholipase D MitoPLD facilitates mitochondrial fusion by generating the signaling lipid phosphatidic acid (PA). The Drosophila MitoPLD homolog Zucchini (Zuc), a proposed cytoplasmic nuclease, is required for piRNA generation, a critical event in germline development. We show that Zuc localizes to mitochondria and has MitoPLD-like activity. Conversely, MitoPLD(-/-) mice exhibit the meiotic arrest, DNA damage, and male sterility characteristic of mice lacking piRNAs. The primary function of MitoPLD seems to be the generation of mitochondrial-surface PA. This PA in turn recruits the phosphatase Lipin 1, which converts PA to diacylglycerol and promotes mitochondrial fission, suggesting a mechanism for mitochondrial morphology homeostasis. MitoPLD and Lipin 1 have opposing effects on mitochondria length and on intermitochondrial cement (nuage), a structure found between aggregated mitochondria that is implicated in piRNA generation. We propose that mitochondrial-surface PA generated by MitoPLD/Zuc recruits or activates nuage components critical for piRNA production.

• Phosphatidylinositol-4-phosphate-5-kinase alpha deficiency alters dynamics of glucose-stimulated insulin release to improve glucohomeostasis and decrease obesity in mice.

  Authors: Ping Huang, Oladapo Yeku, Haihong Zong, Phyllis Tsang, Wenjuan Su, Xiao Yu, Shuzhi Teng, Mary Osisami, Yasunori Kanaho, Jeffrey E Pessin, Michael A Frohman

  Diabetes. 02/2011; 60(2):454-63.

  Phosphatidylinositol-4-phosphate-5-kinase (PI4P5K) has been proposed to facilitate regulated exocytosis and specifically insulin secretion by generating phosphatidylinositol-4,5-bisphosphatePhosphatidylinositol-4-phosphate-5-kinase (PI4P5K) has been proposed to facilitate regulated exocytosis and specifically insulin secretion by generating phosphatidylinositol-4,5-bisphosphate (PIP(2)). We sought to examine the role of the α isoform of PI4P5K in glucohomeostasis and insulin secretion. The response of PI4P5Kα(-/-) mice to glucose challenge and a type 2-like diabetes-inducing high-fat diet was examined in vivo. Glucose-stimulated responses and PI4P5Kα(-/-) pancreatic islets and β-cells were characterized in culture. We show that PI4P5Kα(-/-) mice exhibit increased first-phase insulin release and improved glucose clearance, and resist high-fat diet-induced development of type 2-like diabetes and obesity. PI4P5Kα(-/-) pancreatic islets cultured in vitro exhibited decreased numbers of insulin granules docked at the plasma membrane and released less insulin under quiescent conditions, but then secreted similar amounts of insulin on glucose stimulation. Stimulation-dependent PIP(2) depletion occurred on the plasma membrane of the PI4P5Kα(-/-) pancreatic β-cells, accompanied by a near-total loss of cortical F-actin, which was already decreased in the PI4P5Kα(-/-) β-cells under resting conditions. Our findings suggest that PI4P5Kα plays a complex role in restricting insulin release from pancreatic β-cells through helping to maintain plasma membrane PIP(2) levels and integrity of the actin cytoskeleton under both basal and stimulatory conditions. The increased first-phase glucose-stimulated release of insulin observed on the normal diet may underlie the partial protection against the elevated serum glucose and obesity seen in type 2 diabetes-like model systems.

• Rapid amplification of cDNA ends (RACE).

  Authors: Oladapo Yeku, Michael A Frohman

  Methods in molecular biology (Clifton, N.J.). 01/2011; 703:107-22.

  Rapid Amplification of cDNA ends (RACE) provides an inexpensive and powerful tool to quickly obtain full-length cDNA when the sequence is only partially known. Starting with an mRNA mixture,Rapid Amplification of cDNA ends (RACE) provides an inexpensive and powerful tool to quickly obtain full-length cDNA when the sequence is only partially known. Starting with an mRNA mixture, gene-specific primers generated from the known regions of the gene and non-specific anchors, full-length sequences can be identified in as little as 3 days. RACE can also be used to identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5(') and 3(') cDNA ends using the "new RACE" technique.

• The phospholipase D1 pathway modulates macroautophagy.

  Authors: Claudia Dall'Armi, Andrés Hurtado-Lorenzo, Huasong Tian, Etienne Morel, Akiko Nezu, Robin B Chan, W Haung Yu, Kimberly S Robinson, Oladapo Yeku, Scott A Small, Karen Duff, Michael A Frohman, Markus R Wenk, Akitsugu Yamamoto, Gilbert Di Paolo

  Nature communications. 12/2010; 1:142.

  Although macroautophagy is known to be an essential degradative process whereby autophagosomes mediate the engulfment and delivery of cytoplasmic components into lysosomes, the lipid changesAlthough macroautophagy is known to be an essential degradative process whereby autophagosomes mediate the engulfment and delivery of cytoplasmic components into lysosomes, the lipid changes underlying autophagosomal membrane dynamics are undetermined. Here, we show that phospholipase D1 (PLD1), which is primarily associated with the endosomal system, partially relocalizes to the outer membrane of autophagosome-like structures upon nutrient starvation. The localization of PLD1, as well as the starvation-induced increase in PLD activity, are altered by wortmannin, a phosphatidylinositol 3-kinase inhibitor, suggesting PLD1 may act downstream of Vps34. Pharmacological inhibition of PLD and genetic ablation of PLD1 in mouse cells decreased the starvation-induced expansion of LC3-positive compartments, consistent with a role of PLD1 in the regulation of autophagy. Furthermore, inhibition of PLD results in higher levels of Tau and p62 aggregates in organotypic brain slices. Our in vitro and in vivo findings establish a role for PLD1 in autophagy.

• Mitochondria as integrators of signal transduction and energy production in cardiac physiology and disease.

  Authors: Michael A Frohman

  Journal of molecular medicine (Berlin, Germany). 10/2010; 88(10):967-70.

  Fascinating links are beginning to be discovered between mitochondrial function and cardiac physiology and disease in the context of diverse signaling mechanisms, energy production, and intersectionFascinating links are beginning to be discovered between mitochondrial function and cardiac physiology and disease in the context of diverse signaling mechanisms, energy production, and intersection with pathways producing reactive oxygen species. Proteins long known to drive mitochondrial fusion and fission are now reported to have emergent functions in intracellular calcium homeostasis, apoptosis, and vascular smooth muscle cell proliferation, all key issues in cardiac disease. Moreover, mitochondrial fusion has been demonstrated to be required for normal myofibril organization in skeletal muscle, and decreasing fission may confer protection against ischemic heart disease. These processes broaden the traditional role in energy production undertaken by mitochondria and provide new directions for potential therapeutic leads.

• A phosphatidic acid binding/nuclear localization motif determines lipin1 function in lipid metabolism and adipogenesis.

  Authors: Hongmei Ren, Lorenzo Federico, Huiyan Huang, Manjula Sunkara, Tracy Drennan, Michael A Frohman, Susan S Smyth, Andrew J Morris

  Molecular biology of the cell. 09/2010; 21(18):3171-81.

  Lipins are phosphatidic acid phosphatases with a pivotal role in regulation of triglyceride and glycerophospholipid metabolism. Lipin1 is also an amplifier of PGC-1α, a nuclear coactivator of PPAR-αLipins are phosphatidic acid phosphatases with a pivotal role in regulation of triglyceride and glycerophospholipid metabolism. Lipin1 is also an amplifier of PGC-1α, a nuclear coactivator of PPAR-α responsive gene transcription. Lipins do not contain recognized membrane-association domains, but interaction of these enzymes with cellular membranes is necessary for access to their phospholipid substrate. We identified a role for a conserved polybasic amino acid motif in an N-terminal domain previously implicated as a determinant of nuclear localization in selective binding of lipin1β to phosphatidic acid, using blot overlay assays and model bilayer membranes. Studies using lipin1β polybasic motif variants establish that this region is also critical for nuclear import and raise the possibility that nuclear/cytoplasmic shuttling of lipin1β is regulated by PA. We used pharmacological agents and lipin1β polybasic motif mutants to explore the role of PA-mediated membrane association and nuclear localization on lipin1β function in phospholipid metabolism and adipogenic differentiation. We identify a role for the lipin1 polybasic motif as both a lipid binding motif and a primary nuclear localization sequence. These two functions are necessary for full expression of the biological activity of the protein in intracellular lipid metabolism and transcriptional control of adipogenesis.

• Phospholipase D2-dependent inhibition of the nuclear hormone receptor PPARgamma by cyclic phosphatidic acid.

  Authors: Tamotsu Tsukahara, Ryoko Tsukahara, Yuko Fujiwara, Junming Yue, Yunhui Cheng, Huazhang Guo, Alyssa Bolen, Chunxiang Zhang, Louisa Balazs, Fabio Re, Guangwei Du, Michael A Frohman, Daniel L Baker, Abby L Parrill, Ayako Uchiyama, Tetsuyuki Kobayashi, Kimiko Murakami-Murofushi, Gabor Tigyi

  Molecular cell. 08/2010; 39(3):421-32.

  Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We findCyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists.

• A quantitative assay for mitochondrial fusion using Renilla luciferase complementation.

  Authors: Huiyan Huang, Seok-Yong Choi, Michael A Frohman

  Mitochondrion. 08/2010; 10(5):559-66.

  Mitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Large mitochondria produce energy more efficiently, whereas small mitochondria translocateMitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Large mitochondria produce energy more efficiently, whereas small mitochondria translocate better to subcellular sites where local production of ATP is acutely required. Mitochondrial fusion is currently assayed by fusing together cells expressing GFP or RFP in their mitochondria and then scoring the frequency of cells with yellow mitochondria (representing fused green and red mitochondria). However, this assay is labor-intensive and only semi-quantitative. We describe here a reporter system consisting of split fragments of Renilla luciferase and YFP fused to mitochondrial matrix-targeting sequences and to leucine zippers to trigger dimerization. The assay enables fusion to be quantitated both visually for individual cells and on a population level using chemiluminescence, laying the foundation for high throughput small molecule and RNAi screens for modulators of mitochondrial fusion. We use the assay to examine cytoskeletal roles in fusion progression.

• Phospholipase D2 mediates acute aldosterone secretion in response to angiotensin II in adrenal glomerulosa cells.

  Authors: Haixia Qin, Michael A Frohman, Wendy B Bollag

  Endocrinology. 03/2010; 151(5):2162-70.

  In primary bovine adrenal glomerulosa cells, the signaling enzyme phospholipase D (PLD) is suggested to mediate priming, the enhancement of aldosterone secretion after pretreatment with and removalIn primary bovine adrenal glomerulosa cells, the signaling enzyme phospholipase D (PLD) is suggested to mediate priming, the enhancement of aldosterone secretion after pretreatment with and removal of angiotensin II (AngII), via the formation of persistently elevated diacylglycerol (DAG). To further explore PLD's role in priming, glomerulosa cells were pretreated with an exogenous bacterial PLD. Using this approach, phosphatidic acid (PA) is generated on the outer, rather than the inner, leaflet of the plasma membrane. Although PA is not readily internalized, the PA is nonetheless rapidly hydrolyzed by cell-surface PA phosphatases to DAG, which efficiently flips to the inner leaflet and accesses the cell interior. Pretreatment with bacterial PLD resulted in priming upon subsequent AngII exposure, supporting a role of DAG in this process, because the increase in DAG persisted after exogenous PLD removal. To determine the PLD isoform mediating aldosterone secretion, and presumably priming, primary glomerulosa cells were infected with adenoviruses expressing GFP, PLD1, PLD2, or lipase-inactive mutants. Overexpressed PLD2 increased aldosterone secretion by approximately 3-fold over the GFP-infected control under basal conditions, with a significant enhancement to about 16-fold over the basal value upon AngII stimulation. PLD activity was also increased basally and upon stimulation with AngII. In contrast, PLD1 overexpression had little effect on aldosterone secretion, despite the fact that PLD activity was enhanced. In both cases, the lipase-inactive PLD mutants showed essentially no effect on PLD activity or aldosterone secretion. Our results suggest that PLD2 is the isoform that mediates aldosterone secretion and likely priming.

• Basis for the isoform-specific interaction of myosin phosphatase subunits protein phosphatase 1c beta and myosin phosphatase targeting subunit 1.

  Authors: Elizabeth Scotto-Lavino, Miguel Garcia-Diaz, Guangwei Du, Michael A Frohman

  The Journal of biological chemistry. 02/2010; 285(9):6419-24.

  Myosin II association with actin, which triggers contraction, is regulated by orchestrated waves of phosphorylation/dephosphorylation of the myosin regulatory light chain. Blocking myosin regulatoryMyosin II association with actin, which triggers contraction, is regulated by orchestrated waves of phosphorylation/dephosphorylation of the myosin regulatory light chain. Blocking myosin regulatory light chain phosphorylation with small molecule inhibitors alters the shape, adhesion, and migration of many types of smooth muscle and cancer cells. Dephosphorylation is mediated by myosin phosphatase (MP), a complex that consists of a catalytic subunit (protein phosphatase 1c, PP1c), a large subunit (myosin phosphatase targeting subunit, MYPT), and a small subunit of unknown function. MYPT functions by targeting PP1c onto its substrate, phosphorylated myosin II. Using RNA interference, we show here that stability of PP1c beta and MYPT1 is interdependent; knocking down one of the subunits decreases the expression level of the other. Associated changes in cell shape also occur, characterized by flattening and spreading accompanied by increased cortical actin, and cell numbers decrease secondary to apoptosis. Of the three highly conserved isoforms of PP1c, we show that MYPT1 binding is restricted to PP1c beta, and, using chimeric analysis and site-directed mutations, that the central region of PP1c beta confers the isoform-specific binding. This finding was unexpected because the MP crystal structure has been solved and was reported to identify the variable, C-terminal domain of PP1c beta as being the region key for isoform-specific interaction with MYPT1. These findings suggest a potential screening strategy for cardiovascular and cancer therapeutic agents based on destabilizing MP complex formation and function.

• Dependence of phospholipase D1 multi-monoubiquitination on its enzymatic activity and palmitoylation.

  Authors: Hao Yin, Yu Gui, Guangwei Du, Michael A Frohman, Xi-Long Zheng

  The Journal of biological chemistry. 02/2010; 285(18):13580-8.

  Phospholipase D (PLD) is an important lipase in many cellular processes, including vesicular trafficking, cell survival, and cell migration. In the present study, we show that PLD1, but not PLD2, isPhospholipase D (PLD) is an important lipase in many cellular processes, including vesicular trafficking, cell survival, and cell migration. In the present study, we show that PLD1, but not PLD2, is posttranslationally modified by multi-monoubiquitination. Intriguingly, suppression of lipase activity either by mutation of the HKD motif (PLD1 H896R, K898R, or D903A) or the phosphatidylinositol 4,5-bisphosphate binding motif (PLD1 R691G,R695G) or through use of PLD-selective inhibitors impaired the ubiquitination of PLD1, although stimulation of lipase activity by phorbol 12-myristate 13-acetate did not enhance its ubiquitination. A palmitoylation-deficient mutant PLD1 allele, which exhibits altered patterns of vesicular trafficking, had significantly lower levels of monoubiquitination. In addition, the expression of ubiquitin-fused PLD1 induced aberrantly enlarged vesicles partially co-localized with the Golgi complex but not with early endosomes. The altered localization was reduced by the K898R mutation, suggesting a role of multi-monoubiquitination in PLD1 subcellular localization. Surprisingly, the degradation of PLD1, but not of PLD1 K898R or PLD2, was blocked by inhibitors of proteasomes but not by inhibitors of lysosomes or other proteases, suggesting a role of the ubiquitination in proteasomal degradation of PLD1. In summary, our studies show that PLD1, but not PLD2, is multi-monoubiquitinated. The ubiquitination modification might represent a novel regulatory mechanism in PLD1 functioning, particularly in the context of subcellular trafficking between different membrane compartments.

• A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators.

  Authors: Astrid C Schauss, Huiyan Huang, Seok-Yong Choi, Liqun Xu, Sébastien Soubeyrand, Patricia Bilodeau, Rodolfo Zunino, Peter Rippstein, Michael A Frohman, Heidi M McBride

  BMC biology. 01/2010; 8:100.

  Mitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulateMitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulate mitochondrial morphology and the mechanisms that govern mitochondrial dynamics are poorly understood. However, recent evidence indicates that the core machinery that governs mitochondrial dynamics is linked within complex intracellular signalling cascades, including apoptotic pathways, cell cycle transitions and nuclear factor kappa B activation. Given the emerging importance of mitochondrial plasticity in cell signalling pathways and metabolism, it is essential that we develop tools to quantitatively analyse the processes of fission and fusion. In terms of mitochondrial fusion, the field currently relies upon on semi-quantitative assays which, even under optimal conditions, are labour-intensive, low-throughput and require complex imaging techniques. In order to overcome these technical limitations, we have developed a new, highly quantitative cell-free assay for mitochondrial fusion in mammalian cells. This assay system has allowed us to establish the energetic requirements for mitochondrial fusion. In addition, our data reveal a dependence on active protein phosphorylation for mitochondrial fusion, confirming emerging evidence that mitochondrial fusion is tightly integrated within the global cellular response to signaling events. Indeed, we have shown that cytosol derived from cells stimulated with different triggers either enhance or inhibit the cell-free fusion reaction. The adaptation of this system to high-throughput analysis will provide an unprecedented opportunity to identify and characterize novel regulatory factors. In addition, it provides a framework for a detailed mechanistic analysis of the process of mitochondrial fusion and the various axis of regulation that impinge upon this process in a wide range of cellular conditions.See Commentary: http://www.biomedcentral.com/1741-7007/8/99.

• Targeting phospholipase D with small-molecule inhibitors as a potential therapeutic approach for cancer metastasis.

  Authors: Wenjuan Su, Qin Chen, Michael A Frohman

  Future oncology (London, England). 11/2009; 5(9):1477-86.

  Phospholipase D (PLD)1 and PLD2, the classic mammalian members of the PLD superfamily, have been linked over the past three decades to immune cell function and to cell biological processes requiredPhospholipase D (PLD)1 and PLD2, the classic mammalian members of the PLD superfamily, have been linked over the past three decades to immune cell function and to cell biological processes required by cancer cells for metastasis. However, owing to the lack of effective small-molecule inhibitors, it has not been possible to validate these roles for the PLDs and to explore the possible utility of acute and chronic PLD inhibition in vivo. The first such inhibitors have recently been described and demonstrated to block neutrophil chemotaxis and invasion by breast cancer cells in culture, increasing the prospects for a new class of therapeutics for autoimmune disorders and several types of metastatic cancer.

• Lipid Signaling on the Mitochondrial Surface.

  Authors: Huiyan Huang, Michael A Frohman

  Biochimica et biophysica acta. 07/2009;

  Regulated production and elimination of the signaling lipids phosphatidic acid (PA), diacylglycerol (DAG), and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) creates a complex and interconnectedRegulated production and elimination of the signaling lipids phosphatidic acid (PA), diacylglycerol (DAG), and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) creates a complex and interconnected signaling network that modulates a wide variety of eukaryotic cell biological events. PA production at the plasma membrane and on trafficking membrane organelles by classical Phospholipase D (PLD) through the hydrolysis of phosphatidylcholine (PC) has been studied widely. In this chapter, we review a newly identified, non-canonical member of the PLD superfamily, MitoPLD, which localizes to the mitochondrial surface and plays a role in mitochondrial fusion via the hydrolysis of cardiolipin (CL) to generate PA. The role of PA in facilitating the mitochondrial fusion event carried out by proteins known as Mitofusins is intriguing in light of the role classic PLD-generated PA plays in facilitating SNARE-mediated fusion of secretory membrane vesicles into the plasma membrane. In addition, however, PA on the mitochondrial surface may also trigger a signaling cascade that elevates DAG, leading to downstream events that affect mitochondrial fission and energy production. PA production on the mitochondrial surface may also stimulate local production of PI4,5P(2) to facilitate mitochondrial fission and subcellular trafficking or facilitate Ca(2+) influx.

• Sequential Regulation of DOCK2 Dynamics by Two Phospholipids during Neutrophil Chemotaxis.

  Authors: Akihiko Nishikimi, Hideo Fukuhara, Wenjuan Su, Tsunaki Hongu, Shunsuke Takasuga, Hisashi Mihara, Qinhong Cao, Fumiyuki Sanematsu, Motomu Kanai, Hiroshi Hasegawa, Yoshihiko Tanaka, Masakatsu Shibasaki, Yasunori Kanaho, Takehiko Sasaki, Michael A Frohman, Yoshinori Fukui

  Science (New York, N.Y.). 04/2009;

  During chemotaxis activation of the small GTPase, Rac, is spatially regulated to organize the extension of membrane protrusions in the direction of migration. In neutrophils, Rac activation isDuring chemotaxis activation of the small GTPase, Rac, is spatially regulated to organize the extension of membrane protrusions in the direction of migration. In neutrophils, Rac activation is primarily mediated by DOCK2, an atypical guanine nucleotide exchange factor. Upon stimulation, we found that DOCK2 rapidly translocated to the plasma membrane in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner. However, subsequent accumulation of DOCK2 at the leading edge required phospholipase D-mediated synthesis of phosphatidic acid, which stabilized DOCK2 there via interaction with a polybasic amino acid cluster, resulting in increased local actin polymerization. When this interaction was blocked, neutrophils failed to form leading edges properly and exhibited defects in chemotaxis. Thus, intracellular DOCK2 dynamics are sequentially regulated by distinct phospholipids to localize Rac activation during neutrophil chemotaxis.