RASSF4: Regulator of plasma membrane PI(4,5)P2.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a negatively charged phospholipid that plays a major role in recruiting and regulating proteins at the plasma membrane-cytosol interface. In this issue, Chen et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201606047) demonstrate that RAS association domain family 4 (RASSF4) positively influences PI(4,5)P2 synthesis through ARF6-dependent regulation of PIP5K

Niemann-Pick Type C Disease Reveals a Link between Lysosomal Cholesterol and PtdIns(4,5)P2 That Regulates Neuronal Excitability.

There is increasing evidence that the lysosome is involved in the pathogenesis of a variety of neurodegenerative disorders. Thus, mechanisms that link lysosome dysfunction to the disruption of neuronal homeostasis offer opportunities to understand the molecular underpinnings of neurodegeneration and potentially identify specific therapeutic targets. Here, using a monogenic neurodegenerative disorder, NPC1 disease, we demonstrate that reduced cholesterol efflux from lysosomes aberrantly modifies neuronal firing patterns. The molecular mechanism linking alterations in lysosomal cholesterol egress to intrinsic tuning of neuronal excitability is a transcriptionally mediated upregulation of the ABCA1 transporter, whose PtdIns(4,5)P2-floppase activity decreases plasma membrane PtdIns(4,5)P2. The consequence of reduced PtdIns(4,5)P2 is a parallel decrease in a key regulator of neuronal excitability, the voltage-gated KCNQ2/3 potassium channel, which leads to hyperexcitability in NPC1 disease neurons. Thus, cholesterol efflux from lysosomes regulates PtdIns(4,5)P2 to shape the electrical and functional identity of the plasma membrane of neurons in health and disease

β-adrenergic-mediated dynamic augmentation of sarcolemmal CaV 1.2 clustering and co-operativity in ventricular myocytes.

Key pointsPrevailing dogma holds that activation of the β-adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L-type CaV 1.2 channel activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. CaV 1.2 channel clusters decorate T-tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca2+ -dependent co-operative gating behaviour mediated by physical interactions between adjacent channel C-terminal tails. We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A-dependent augmentation of CaV 1.2 channel abundance along cardiomyocyte T-tubules, resulting in the appearance of channel 'super-clusters', and enhanced channel co-operativity that amplifies Ca2+ influx. On the basis of these data, we suggest a new model in which a sub-sarcolemmal pool of pre-synthesized CaV 1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation-contraction coupling.AbstractVoltage-dependent L-type CaV 1.2 channels play an indispensable role in cardiac excitation-contraction coupling. Activation of the β-adrenergic receptor (βAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced CaV 1.2 activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. CaV 1.2 channels exhibit a clustered distribution along the T-tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca2+ -dependent co-operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C-terminal tails. This amplifies Ca2+ influx and augments myocyte Ca2+ transient and contraction amplitudes. We investigated whether βAR signalling could alter CaV 1.2 channel clustering to facilitate co-operative channel interactions and elevate Ca2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the βAR agonist, isoproterenol (ISO), promotes enhanced CaV 1.2-CaV 1.2 physical interactions. Super-resolution nanoscopy and dynamic channel tracking indicate that these interactions are expedited by enhanced spatial proximity between channels, resulting in the appearance of CaV 1.2 'super-clusters' along the z-lines of ISO-stimulated cardiomyocytes. The mechanism that leads to super-cluster formation involves rapid, dynamic augmentation of sarcolemmal CaV 1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt co-operative gating behaviour of CaV 1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of βAR-mediated regulation of CaV 1.2 channels in the heart and support the novel concept that a pre-synthesized pool of sub-sarcolemmal CaV 1.2 channel-containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma to tune excitation-contraction coupling to meet metabolic and/or haemodynamic demands

NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes

Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann-Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4-phosphate (PtdIns4P) countertransport cycle between Golgi-endoplasmic reticulum (ER), as well as lysosome-ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4-kinases-PI4KIIα and PI4KIIIβ-which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease

IP3R-driven increases in mitochondrial Ca2+ promote neuronal death in NPC disease

Ca2+ is the most ubiquitous second messenger in neurons whose spatial and temporal elevations are tightly controlled to initiate and orchestrate diverse intracellular signaling cascades. Numerous neuropathologies result from mutations or alterations in Ca2+ handling proteins; thus, elucidating molecular pathways that shape Ca2+ signaling is imperative. Here, we report that loss-of-function, knockout, or neurodegenerative disease-causing mutations in the lysosomal cholesterol transporter, Niemann-Pick Type C1 (NPC1), initiate a damaging signaling cascade that alters the expression and nanoscale distribution of IP3R type 1 (IP3R1) in endoplasmic reticulum membranes. These alterations detrimentally increase Gq-protein coupled receptor-stimulated Ca2+ release and spontaneous IP3R1 Ca2+ activity, leading to mitochondrial Ca2+ cytotoxicity. Mechanistically, we find that SREBP-dependent increases in Presenilin 1 (PS1) underlie functional and expressional changes in IP3R1. Accordingly, expression of PS1 mutants recapitulate, while PS1 knockout abrogates Ca2+ phenotypes. These data present a signaling axis that links the NPC1 lysosomal cholesterol transporter to the damaging redistribution and activity of IP3R1 that precipitates cell death in NPC1 disease and suggests that NPC1 is a nanostructural disease

Biophysical physiology of phosphoinositide rapid dynamics and regulation in living cells

Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future

Phosphoinositide transport and metabolism at membrane contact sites.

Phosphoinositides are a family of signaling lipids that play a profound role in regulating protein function at the membrane-cytosol interface of all cellular membranes. Underscoring their importance, mutations or alterations in phosphoinositide metabolizing enzymes lead to host of developmental, neurodegenerative, and metabolic disorders that are devastating for human health. In addition to lipid enzymes, phosphoinositide metabolism is regulated and controlled at membrane contact sites (MCS). Regions of close opposition typically between the ER and other cellular membranes, MCS are non-vesicular lipid transport portals that engage in extensive communication to influence organelle homeostasis. This review focuses on lipid transport, specifically phosphoinositide lipid transport and metabolism at MCS