Irit Gottfried

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNA-mediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.

The DFNA15 Deafness Mutation Affects POU4F3 Protein Stability, Localization, and Transcriptional Activity

ABSTRACT A mutation in the POU4F3 gene (BRN-3.1, BRN3C) is responsible for DFNA15 (MIM 602459), autosomal-dominant nonsyndromic hearing loss. POU4F3 is a member of the POU family of transcription factors and is essential for inner-ear hair cell maintenance. To test the potential effects of the human POU4F3 mutation, we performed a series of experiments in cell culture to mimic the human mutation. Mutant POU4F3 loses most of its transcriptional activity and most of its ability to bind to DNA and does not function in a dominant-negative manner. Moreover, whereas wild-type POU4F3 is found exclusively in the nucleus, our studies demonstrate that the mutant protein is localized both to the nucleus and the cytoplasm. Two nuclear localization signals were identified; both are essential for proper nuclear entry of POU4F3 protein. We found that the mutant protein half-life is longer than that of the wild type. We propose that the combination of defects caused by the mutation on the function of the POU4F3 transcription factor eventually leads to hair cell morbidity in affected family H members.

Tomosyn Expression Pattern in the Mouse Hippocampus Suggests Both Presynaptic and Postsynaptic Functions

The protein tomosyn decreases synaptic transmission and release probability of vesicles, and is essential for modulating synaptic transmission in neurons. In this study, we provide a detailed description of the expression and localization patterns of tomosyn1 and tomosyn2 in the subareas of the mouse hippocampus. Using confocal and two-photon high-resolution microscopy we demonstrate that tomosyn colocalizes with several pre- and postsynaptic markers and is found mainly in glutamatergic synapses. Specifically, we show that tomosyn1 is differentially distributed in the mouse hippocampus and concentrated mainly in the hilus and mossy fibers. Surprisingly, we found that tomosyn2 is expressed in the subiculum, CA1 and CA2 pyramidal cell bodies, dendrites and spines, and colocalizes with PSD95, suggesting a postsynaptic role. These results suggest that in addition to the well-characterized presynaptic function of tomosyn in neurotransmitter release, tomosyn2 might have a postsynaptic function, and place tomosyn as a more general regulator of synaptic transmission and plasticity.

The Sla2p/HIP1/HIP1R family: similar structure, similar function in endocytosis?

HIP1 (huntingtin interacting protein 1) has two close relatives: HIP1R (HIP1-related) and yeast Sla2p. All three members of the family have a conserved domain structure, suggesting a common function. Over the past decade, a number of studies have characterized these proteins using a combination of biochemical, imaging, structural and genetic techniques. These studies provide valuable information on binding partners, structure and dynamics of HIP1/HIP1R/Sla2p. In general, all suggest a role in CME (clathrin-mediated endocytosis) for the three proteins, though some differences have emerged. In this mini-review we summarize the current views on the roles of these proteins, while emphasizing the unique attributes of each family member.

Munc13-1 translocates to the plasma membrane in a Doc2B- and calcium-dependent manner

Munc13-1 is a presynaptic protein activated by calcium, calmodulin, and diacylglycerols (DAG) that is known to enhance vesicle priming. Doc2B is another presynaptic protein that translocates to the plasma membrane (PM) upon elevation of internal calcium concentration ([Ca2+]i) to the submicromolar range, and increases both spontaneous and asynchronous release in a calcium-dependent manner. We speculated that Doc2B also recruits Munc13-1 to the PM since these two proteins have been shown to interact physiologically and this interaction is enhanced by Ca2+. However, this calcium-dependent co-translocation has never actually been shown. To examine this possibility, we expressed both proteins tagged to fluorescent proteins in PC12 cells and stimulated the cells to investigate the recruitment hypothesis using imaging techniques. We found that Munc13-1 does indeed translocate to the PM upon elevation in [Ca2+]i, but only when co-expressed with Doc2B. Interestingly, Munc13-1 co-translocates at a slower rate than Doc2B. Moreover, while Doc2B dislocates from the PM as soon as the [Ca2+]i returns to basal levels, Munc13-1 dislocates at a slower rate and a fraction of it accumulates on the PM. This accumulation is more pronounced under subsequent stimulations, suggesting that Munc13-1 accumulation builds up as some other factors accumulate at the PM. Munc13-1 co-translocation and accumulation was reduced when its mutant Munc13-1H567K, which is unable to bind DAG, was co-expressed with Doc2B, suggesting that Munc13-1 accumulation depends on DAG levels. These results suggest that Doc2B enables recruitment of Munc13-1 to the PM in a [Ca2+]i-dependent manner and offers another possible Munc13-1-regulatory mechanism that is both calcium- and Doc2B-dependent.

HIP1 exhibits an early recruitment and a late stage function in the maturation of coated pits

Huntingtin interacting protein 1 (HIP1) is an accessory protein of the clathrin-mediated endocytosis (CME) pathway, yet its precise role and the step at which it becomes involved are unclear. We employed live-cell imaging techniques to focus on the early steps of CME and characterize HIP1 dynamics. We show that HIP1 is highly colocalized with clathrin at the plasma membrane and shares similar dynamics with a subpopulation of clathrin assemblies. Employing transferrin receptor fused to pHluorin, we distinguished between open pits to which HIP1 localizes and newly internalized vesicles that are devoid of HIP1. Moreover, shRNA knockdown of clathrin compromised HIP1 membranal localization, unlike the reported behavior of Sla2p. HIP1 fragment, lacking its ANTH and Talin-like domains, inhibits internalization of transferrin, but retains colocalization with membranal clathrin assemblies. These data demonstrate HIP1’s role in pits maturation and formation of the coated vesicle, and its strong dependence on clathrin for membranal localization.

Rasosomes originate from the Golgi to dispense Ras signals

Ras proteins undergo an incompletely understood trafficking process in the cell. Rasosomes are protein nanoparticles of 80–100 nm diameter that carry lipidated Ras isoforms (H-Ras and N-Ras) as well as their effectors through the cytoplasm and near the plasma membrane (PM). In this study, we identified the subcellular origin of rasosomes and how they spread Ras proteins through the cell. We found no dependency of rasosome formation on galectins, or on the GDP-/GTP-bound state of Ras. We found that significantly more rasosomes are associated with forms of Ras that are localized to the Golgi, namely N-Ras or the singly palmitoylated H-Ras mutant (C181S). To explore the possibility that rasosome originate from the Golgi, we used photoactivatable (PA)-GFP-H-Ras mutants and showed that rasosomes bud from the Golgi in a two-step mechanism. Newly released rasosomes first move in an energy-dependent directed fashion and then convert to randomly diffusing rasosomes. Dual fluorescence time-lapse imaging revealed the appearance of dually labeled rasosomes, indicating a dynamic exchange of cytoplasmic and PM-associated Ras with rasosome-associated Ras. Finally, higher levels of rasosomes correlate with higher levels of ERK phosphorylation, a key marker of Ras downstream signaling. We suggest that H-Ras and N-Ras proteins exchange with rasosomes that can function as carriers of palmitoylated Ras and its signals.

Phosphatidylinositol (4, 5)-bisphosphate targets double C2 domain protein B to the plasma membrane

Double C2 domain protein B (DOC2B) is a high‐affinity Ca2+ sensor that translocates from the cytosol to the plasma membrane (PM) and promotes vesicle priming and fusion. However, the molecular mechanism underlying its translocation and targeting to the PM in living cells is not completely understood. DOC2B interacts in vitro with the PM components phosphatidylserine, phosphatidylinositol (4, 5)‐bisphosphate [PI(4, 5)P2] and target SNAREs (t‐SNAREs). Here, we show that PI(4, 5)P2 hydrolysis at the PM of living cells abolishes DOC2B translocation, whereas manipulations of t‐SNAREs and other phosphoinositides have no effect. Moreover, we were able to redirect DOC2B to intracellular membranes by synthesizing PI(4, 5)P2 in those membranes. Molecular dynamics simulations and mutagenesis in the calcium and PI(4, 5)P2‐binding sites strengthened our findings, demonstrating that both calcium and PI(4, 5)P2 are required for the DOC2B–PM association and revealing multiple PI(4, 5)P2–C2B interactions. In addition, we show that DOC2B translocation to the PM is ATP‐independent and occurs in a diffusion‐like manner. Our data suggest that the Ca2+‐triggered translocation of DOC2B is diffusion‐driven and aimed at PI(4, 5)P2‐containing membranes.

The Molecular Mechanisms Underlying Synaptic Transmission: A View of the Presynaptic Terminal

Abstract Synaptic transmission relies on spatially and temporally coordinated multistep processes that allow neuronal communication; activity-dependent changes in synaptic transmission underlie synaptic plasticity. These processes are coordinated by a large number of specific proteins whose dynamic interactions, expression, and regulation define the efficacy of transmission and the mode of synaptic plasticity. In this chapter, we discuss the molecular mechanisms of some of the basic processes associated with neurotransmission in the presynaptic terminal—vesicle docking, priming, and fusion—elaborate on the contribution of specific proteins to different modes of vesicle recycling, and discuss their nanoscale distribution in the synapses. We also describe the involvement of these proteins in synaptic plasticity and animal behavior, the expression ratios between specific proteins and the possible contribution of these ratios to various modes and kinetics of neurotransmitter release.Synaptic transmission relies on spatially and temporally coordinated multistep processes that allow neuronal communication; activity-dependent changes in synaptic transmission underlie synaptic plasticity. These processes are coordinated by a large number of specific proteins whose dynamic interactions, expression, and regulation define the efficacy of transmission and the mode of synaptic plasticity. In this chapter, we discuss the molecular mechanisms of some of the basic processes associated with neurotransmission in the presynaptic terminal—vesicle docking, priming, and fusion—elaborate on the contribution of specific proteins to different modes of vesicle recycling, and discuss their nanoscale distribution in the synapses. We also describe the involvement of these proteins in synaptic plasticity and animal behavior, the expression ratios between specific proteins and the possible contribution of these ratios to various modes and kinetics of neurotransmitter release.