ER-plasma membrane junctions: Why and how do we study them?

Chang, Chi‐Lun; Chen, Yu‐Ju; Liou, Jen

doi:10.1016/j.bbamcr.2017.05.018

Cited by 67 publications

(65 citation statements)

References 173 publications

(218 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…DsRed-Kv2.1 S586A and DsRed-Kv2.1 P404W were generated using QuikChange PCR-based mutagenesis using DsRed-Kv2.1 (Bishop et al, 2018) as a template. GFP-Kv2.1 S586A and GFP-Kv2.1 P404W were similarly generated using GFP-Kv2.1 (Antonucci et al, 2001) as a template. The generation of rat Kv2.1 truncation, internal deletion, and point mutants, as well as Kv1.5N-Kv2.1C chimeras, has been described previously (Lim et al, 2000;.…”

Section: Methodsmentioning

confidence: 99%

Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum–Plasma Membrane Junctions in Mammalian Brain Neurons

et al. 2018

View full text Add to dashboard Cite

Membrane contacts between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are ubiquitous in eukaryotic cells and are platforms for lipid and calcium signaling and homeostasis. Recent studies have revealed proteins crucial to the formation and function of ER-PM junctions in non-neuronal cells, but little is known of the ER-PM junctions prominent in aspiny regions of mammalian brain neurons. The Kv2.1 voltage-gated potassium channel is abundantly clustered at ER-PM junctions in brain neurons and is the first PM protein that functions to organize ER-PM junctions. However, the molecular mechanism whereby Kv2.1 localizes to and remodels these junctions is unknown. We used affinity immunopurification and mass spectrometry-based proteomics on brain samples from male and female WT and Kv2.1 KO mice and identified the resident ER vesicle-associated membrane protein-associated proteins isoforms A and B (VAPA and VAPB) as prominent Kv2.1-associated proteins. Coexpression with Kv2.1 or its paralog Kv2.2 was sufficient to recruit VAPs to ER-PM junctions. Multiplex immunolabeling revealed colocalization of Kv2.1 and Kv2.2 with endogenous VAPs at ER-PM junctions in brain neurons from male and female mice and in cultured rat hippocampal neurons, and KO of VAPA in mammalian cells reduces Kv2.1 clustering. The association of VAPA with Kv2.1 relies on a "two phenylalanines in an acidic tract" (FFAT) binding domain on VAPA and a noncanonical phosphorylation-dependent FFAT motif comprising the Kv2-specific clustering or PRC motif. These results suggest that Kv2.1 localizes to and organizes neuronal ER-PM junctions through an interaction with VAPs. Our study identified the endoplasmic reticulum (ER) proteins vesicle-associated membrane protein-associated proteins isoforms A and B (VAPA and VAPB) as proteins copurifying with the plasma membrane (PM) Kv2.1 ion channel. We found that expression of Kv2.1 recruits VAPs to ER-PM junctions, specialized membrane contact sites crucial to distinct aspects of cell function. We found endogenous VAPs at Kv2.1-mediated ER-PM junctions in brain neurons and other mammalian cells and that knocking out VAPA expression disrupts Kv2.1 clustering. We identified domains of VAPs and Kv2.1 necessary and sufficient for their association at ER-PM junctions. Our study suggests that Kv2.1 expression in the PM can affect ER-PM junctions via its phosphorylation-dependent association to ER-localized VAPA and VAPB.

show abstract

Section: Methodsmentioning

confidence: 99%

Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum–Plasma Membrane Junctions in Mammalian Brain Neurons

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Membrane contact sites are cellular domains where two organelles interact to facilitate molecular function and promote functional integration. As a network, the ER reaches to the farthest corners of the cell and interacts with many organelles including the plasma membrane . In fact, the ER is the largest intracellular organelle and contains the majority of cellular lipids.…”

Section: Lysosomal Positioning Regulated By Other Organellesmentioning

confidence: 99%

“…As a network, the ER reaches to the farthest corners of the cell and interacts with many organelles including the plasma membrane. 30,31 In fact, the ER is the largest intracellular organelle and contains the majority of cellular lipids. There is growing evidence that the ER forms membrane contact sites with almost all organelles and the plasma membrane.…”

Section: Lysosomal Positioning Regulated By Other Organellesmentioning

confidence: 99%

Mechanisms of lysosomal positioning and movement

Cabukusta

Neefjes

2018

Traffic

213

177

View full text Add to dashboard Cite

Lysosomes are highly dynamic organelles that can move rapidly throughout the cell. They distribute in a rather immobile pool located around the microtubule‐organizing center in a “cloud,” and a highly dynamic pool in the cell periphery. Their spatiotemporal characteristics allow them to carry out multiple biological functions, such as cargo degradation, antigen presentation and plasma membrane repair. Therefore, it is not surprising that lysosomal dysfunction underlies various diseases, including cancer, neurodegenerative and autoimmune diseases. In most of these biological events, the involvement of lysosomes is dependent on their ability to move throughout the cytoplasm, to find and fuse to the correct compartments to receive and deliver substrates for further handling. These dynamics are orchestrated by motor proteins moving along cytoskeletal components. The complexity of the mechanisms responsible for controlling lysosomal transport has recently been appreciated and has yielded novel insights into interorganellar communication, as well as lipid‐protein interplay. In this review, we discuss the current understanding of the mechanisms of lysosomal transport and the molecular machineries that control this mobility.

show abstract

“…ER is the organelle where most steps of cholesterol synthesis take place, but cholesterol is rapidly transferred from the ER to other organelles via vesicular trafficking and nonvesicular pathways (4). There are reports showing existence of endoplasmic reticulum (ER)-plasma membrane (PM) junctions as contact sites between the ER and the PM (30,31). These membrane contact sites (MCSs) are domains where two membranes come to close proximity, typically less than 30 nm, that favor exchange between the two organelles.…”

Section: Discussionmentioning

confidence: 99%

Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders

Králová

Jurášek

Krčová

et al. 2018

Preprint

View full text Add to dashboard Cite

The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analyzed in detail and showed an intensive labeling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 labeling in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.Significance statementCholesterol is a vital steroid molecule with many important functions in animal cells. Although its dysregulation is associated with an expanding list of clinically important pathologies, the study of its role is limited by a lack of reliable tools for live intracellular monitoring. This study demonstrates the applicability of a novel class of heterocyclic sterol probes. These probes exhibit fast cellular uptake with effective fluorescence labeling of sterol species in a variety of living cells, without a need for artificial carriers. When applied to Niemann-Pick disease type C1 cells, they identified massive accumulation of cholesterol in the endosome/lysosome compartment. Thus, several probes from the same series can also be used for visualizing lysosomal storage disorders and sterol transporting pathologies.

show abstract

ER-plasma membrane junctions: Why and how do we study them?

Cited by 67 publications

References 173 publications

Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum–Plasma Membrane Junctions in Mammalian Brain Neurons

Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum–Plasma Membrane Junctions in Mammalian Brain Neurons

Mechanisms of lysosomal positioning and movement

Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders

Contact Info

Product

Resources

About