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author:("fivlacz, Marc")
1.  STIM2 regulates PKA-dependent phosphorylation and trafficking of AMPARs 
Molecular Biology of the Cell  2015;26(6):1141-1159.
STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum and regulate store-operated Ca2+ entry. STIM2 mediates cAMP/PKA-dependent phosphorylation of the AMPA receptor subunit GluA1 in excitatory neurons. In addition, STIM2 promotes cAMP-dependent surface delivery of GluA1.
STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum (ER) and regulate store-operated Ca2+ entry (SOCE). The function of STIMs in the brain is only beginning to be explored, and the relevance of SOCE in nerve cells is being debated. Here we identify STIM2 as a central organizer of excitatory synapses. STIM2, but not its paralogue STIM1, influences the formation of dendritic spines and shapes basal synaptic transmission in excitatory neurons. We further demonstrate that STIM2 is essential for cAMP/PKA-dependent phosphorylation of the AMPA receptor (AMPAR) subunit GluA1. cAMP triggers rapid migration of STIM2 to ER–plasma membrane (PM) contact sites, enhances recruitment of GluA1 to these ER-PM junctions, and promotes localization of STIM2 in dendritic spines. Both biochemical and imaging data suggest that STIM2 regulates GluA1 phosphorylation by coupling PKA to the AMPAR in a SOCE-independent manner. Consistent with a central role of STIM2 in regulating AMPAR phosphorylation, STIM2 promotes cAMP-dependent surface delivery of GluA1 through combined effects on exocytosis and endocytosis. Collectively our results point to a unique mechanism of synaptic plasticity driven by dynamic assembly of a STIM2 signaling complex at ER-PM contact sites.
PMCID: PMC4357513  PMID: 25609091
2.  Impaired spatial memory and enhanced long-term potentiation in mice with forebrain-specific ablation of the Stim genes 
Recent findings point to a central role of the endoplasmic reticulum-resident STIM (Stromal Interaction Molecule) proteins in shaping the structure and function of excitatory synapses in the mammalian brain. The impact of the Stim genes on cognitive functions remains, however, poorly understood. To explore the function of the Stim genes in learning and memory, we generated three mouse strains with conditional deletion (cKO) of Stim1 and/or Stim2 in the forebrain. Stim1, Stim2, and double Stim1/Stim2 cKO mice show no obvious brain structural defects or locomotor impairment. Analysis of spatial reference memory in the Morris water maze revealed a mild learning delay in Stim1 cKO mice, while learning and memory in Stim2 cKO mice was indistinguishable from their control littermates. Deletion of both Stim genes in the forebrain resulted, however, in a pronounced impairment in spatial learning and memory reflecting a synergistic effect of the Stim genes on the underlying neural circuits. Notably, long-term potentiation (LTP) at CA3-CA1 hippocampal synapses was markedly enhanced in Stim1/Stim2 cKO mice and was associated with increased phosphorylation of the AMPA receptor subunit GluA1, the transcriptional regulator CREB and the L-type Voltage-dependent Ca2+ channel Cav1.2 on protein kinase A (PKA) sites. We conclude that STIM1 and STIM2 are key regulators of PKA signaling and synaptic plasticity in neural circuits encoding spatial memory. Our findings also reveal an inverse correlation between LTP and spatial learning/memory and suggest that abnormal enhancement of cAMP/PKA signaling and synaptic efficacy disrupts the formation of new memories.
PMCID: PMC4500926
excitatory synapse; AMPA receptor; endoplasmic reticulum; PKA; spatial memory; long-term potentiation; STIM1 and STIM2
3.  High-content imaging of presynaptic assembly 
Presynaptic assembly involves the specialization of a patch of axonal membrane into a complex structure that supports synaptic vesicle exocytosis and neurotransmitter release. In mammalian neurons, presynaptic assembly is widely studied in a co-culture assay, where a synaptogenic cue expressed at the surface of a heterologous cell induces presynaptic differentiation in a contacting axon. This assay has led to the discovery of numerous synaptogenic proteins, but has not been used to probe neuronal mechanisms regulating presynaptic induction. The identification of regulatory pathways that fine-tune presynaptic assembly is hindered by the lack of adequate tools to quantitatively image this process. Here, we introduce an image-processing algorithm that identifies presynaptic clusters in mammalian co-cultures and extracts a range of synapse-specific parameters. Using this software, we assessed the intrinsic variability of this synaptic induction assay and probed the effect of eight neuronal microRNAs on presynaptic assembly. Our analysis revealed a novel role for miR-27b in augmenting the density of presynaptic clusters. Our software is applicable to a wide range of synaptic induction protocols (including spontaneous synaptogenesis observed in neuron cultures) and is a valuable tool to determine the subtle impact of disease-associated genes on presynaptic assembly.
PMCID: PMC3939450  PMID: 24624059
synapse formation; presynaptic terminals; image processing; computer-assisted; synaptic vesicles; synaptogenic proteins; microRNA (miRNA); primary neuron culture
4.  The small GTPase HRas shapes local PI3K signals through positive feedback and regulates persistent membrane extension in migrating fibroblasts 
Molecular Biology of the Cell  2013;24(14):2228-2237.
An inducible activation approach is used to interrogate the positive feedback circuit underlying self-amplification of PI3K signals in fibroblasts. The results show that local positive feedback between PI3K and HRas drives asymmetric membrane extension and cell migration.
Self-amplification of phosphoinositide 3-kinase (PI3K) signaling is believed to regulate asymmetric membrane extension and cell migration, but the molecular organization of the underlying feedback circuit is elusive. Here we use an inducible approach to synthetically activate PI3K and interrogate the feedback circuitry governing self-enhancement of 3′-phosphoinositide (3-PI) signals in NIH3T3 fibroblasts. Synthetic activation of PI3K initially leads to uniform production of 3-PIs at the plasma membrane, followed by the appearance of asymmetric and highly amplified 3-PI signals. A detailed spatiotemporal analysis shows that local self-amplifying 3-PI signals drive rapid membrane extension with remarkable directional persistence and initiate a robust migratory response. This positive feedback loop is critically dependent on the small GTPase HRas. Silencing of HRas abrogates local amplification of 3-PI signals upon synthetic PI3K activation and results in short-lived protrusion events that do not support cell migration. Finally, our data indicate that this feedback circuit is likely to operate during platelet-derived growth factor–induced random cell migration. We conclude that positive feedback between PI3K and HRas is essential for fibroblasts to spontaneously self-organize and generate a productive migratory response in the absence of spatial cues.
PMCID: PMC3708728  PMID: 23676667
5.  Parkin Protects Against LRRK2 G2019S Mutant-induced Dopaminergic Neurodegeneration in Drosophila 
Mutations in the leucine-rich repeat kinas e 2 (LRRK2) gene are currently recognized as the most common genetic cause of parkinsonism. Among the large number of LRRK2 mutations identified to date, the G2019S variant is the most common. In Asia however, another LRRK2 variant, G2385R, appears to occur more frequently. To better understand the contribution of different LRRK2 variants towards disease pathogenesis, we generated transgenic Drosophila over-expressing various human LRRK2 alleles, including wild type, G2019S, Y1699C and G2385R LRRK2. We found that transgenic flies harboring G2019S, Y1699C or G2385R LRRK2 variant, but not the wild type protein, exhibit late-onset loss of DA neurons in selected clusters that is accompanied by locomotion deficits. Further, LRRK2 mutant flies also display reduced lifespan and increased sensitivity to rotenone, a mitochondrial complex I inhibitor. Importantly, co-expression of human parkin in LRRK2 G2019S-expressing flies provides significant protection against DA neurodegeneration that occurs with age or in response to rotenone. Taken together, our results suggest a potential link between LRRK2, parkin and mitochondria in the pathogenesis of LRRK2-related parkinsonism.
PMCID: PMC2771772  PMID: 19741132
Parkinson’s disease; LRRK2; parkin; rotenone; mitochondrial complex I; Drosophila
6.  Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking 
PLoS ONE  2007;2(9):e851.
Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics.
Methodology/Principal Findings
Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus.
These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation.
PMCID: PMC1952175  PMID: 17786222
7.  Spines and neurite branches function as geometric attractors that enhance protein kinase C action 
The Journal of Cell Biology  2005;170(7):1147-1158.
Ca2+ and diacylglycerol-regulated protein kinase Cs (PKCs; conventional PKC isoforms, such as PKCγ) are multifunctional signaling molecules that undergo reversible plasma membrane translocation as part of their mechanism of activation. In this article, we investigate PKCγ translocation in hippocampal neurons and show that electrical or glutamate stimulation leads to a striking enrichment of PKCγ in synaptic spines and dendritic branches. Translocation into spines and branches was delayed when compared with the soma plasma membrane, and PKCγ remained in these structures for a prolonged period after the response in the soma ceased. We have developed a quantitative model for the translocation process by measuring the rate at which PKCγ crossed the neck of spines, as well as cytosolic and membrane diffusion coefficients of PKCγ. Our study suggests that neurons make use of a high surface-to-volume ratio of spines and branches to create a geometric attraction process for PKC that imposes a delayed enhancement of PKC action at synapses and in peripheral processes.
PMCID: PMC2171530  PMID: 16186260
8.  Reversible intracellular translocation of KRas but not HRas in hippocampal neurons regulated by Ca2+/calmodulin 
The Journal of Cell Biology  2005;170(3):429-441.
The Ras/MAPK pathway regulates synaptic plasticity and cell survival in neurons of the central nervous system. Here, we show that KRas, but not HRas, acutely translocates from the plasma membrane (PM) to the Golgi complex and early/recycling endosomes in response to neuronal activity. Translocation is reversible and mediated by the polybasic-prenyl membrane targeting motif of KRas. We provide evidence that KRas translocation occurs through sequestration of the polybasic-prenyl motif by Ca2+/calmodulin (Ca2+/CaM) and subsequent release of KRas from the PM, in a process reminiscent of GDP dissociation inhibitor–mediated membrane recycling of Rab and Rho GTPases. KRas translocation was accompanied by partial intracellular redistribution of its activity. We conclude that the polybasic-prenyl motif acts as a Ca2+/CaM-regulated molecular switch that controls PM concentration of KRas and redistributes its activity to internal sites. Our data thus define a novel signaling mechanism that differentially regulates KRas and HRas localization and activity in neurons.
PMCID: PMC2171478  PMID: 16043511
9.  Sensitivity of Polarized Epithelial Cells to the Pore-Forming Toxin Aerolysin  
Infection and Immunity  2003;71(2):739-746.
Aerolysin is one of the major virulence factors produced by Aeromonas hydrophila, a human pathogen that produces deep wound infection and gastroenteritis. The toxin interacts with target mammalian cells by binding to the glycan core of glycosylphosphatidyl inositol (GPI)-anchored proteins and subsequently forms a pore in the plasma membrane. Since epithelial cells of the intestine are the primary targets of aerolysin, we investigated its effect on three types of polarized epithelial cells: Caco-2 cells, derived from human intestine; MDCK cells, a well-characterized cell line in terms of protein targeting; and FRT cells, an unusual cell line in that it targets its GPI-anchored proteins to the basolateral plasma membrane in contrast to other epithelial cells, which target them almost exclusively to the apical surface. Surprisingly, we found that all three cell types were sensitive to the toxin from both the apical and the basolateral sides. Apical sensitivity was always higher, even for FRT cells. In contrast, FRT cells were more sensitive from the basolateral than from the apical side to the related toxin Clostridium septicum alpha-toxin, which also binds to GPI-anchored proteins but lacks the lectin binding domain found in aerolysin. These observations are consistent with the notion that a shuttling mechanism involving low-affinity interactions with surface sugars allows aerolysin to gradually move toward the membrane surface, where it can finally encounter the glycan cores of GPI-anchored proteins.
PMCID: PMC145399  PMID: 12540553
10.  A Pore-forming Toxin Interacts with a GPI-anchored Protein and Causes Vacuolation of the Endoplasmic Reticulum  
The Journal of Cell Biology  1998;140(3):525-540.
In this paper, we have investigated the effects of the pore-forming toxin aerolysin, produced by Aeromonas hydrophila, on mammalian cells. Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid “rafts.” We show that the protoxin is then processed to its mature form by host cell proteases. We propose that the preferential association of the toxin with rafts, through binding to GPI-anchored proteins, is likely to increase the local toxin concentration and thereby promote oligomerization, a step that it is a prerequisite for channel formation. We show that channel formation does not lead to disruption of the plasma membrane but to the selective permeabilization to small ions such as potassium, which causes plasma membrane depolarization. Next we studied the consequences of channel formation on the organization and dynamics of intracellular membranes. Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments. Concomitantly we find that the COPI coat is released from biosynthetic membranes and that biosynthetic transport of newly synthesized transmembrane G protein of vesicular stomatitis virus is inhibited. Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.
PMCID: PMC2140172  PMID: 9456314

Results 1-10 (10)