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Lipid droplets (LDs) and peroxisomes form a functional alliance to maintain lipid and energy homeostasis. Chang et al. provide mechanistic insights by describing a tethering complex that comprises LD-localized M1 Spastin and peroxisomal ABCD1 at LD–peroxisome contact sites and supports fatty acid trafficking., Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two interrelated mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD–peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1- and CHMP1B-dependent modifications in LD membrane morphology. Furthermore, LD-to-peroxisome FA trafficking mediated by M1 Spastin is required to relieve LDs of lipid peroxidation. M1 Spastin’s dual roles in tethering LDs to peroxisomes and in recruiting ESCRT-III components to LD–peroxisome contact sites for FA trafficking may underlie the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes.

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial β-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

This study investigates the hypothesis that trafficking of membrane proteins occurs at plasma membrane (PM) domains adjacent to underlying cortical endoplasmic reticulum (cER). The authors observe exocytosis of transferrin receptor and vesicular stomatitis virus G-protein to occur preferentially (>80%) at cER-enriched PM domains. They also report a preferential (>80%) localization of clathrin-coated pits at these domains., In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

The Kv2.1 voltage-gated potassium channel forms stable clusters on the surface of different mammalian cells. Even though these cell-surface structures have been observed for almost a decade, little is known about the mechanism by which cells maintain them. We measure the distribution of domain sizes to study the kinetics of their growth. Using a Fokker-Planck formalism, we find no evidence for a feedback mechanism present to maintain specific domain radii. Instead, the size of Kv2.1 clusters is consistent with a model where domain size is established by fluctuations in the trafficking machinery. These results are further validated using likelihood and Akaike weights to select the best model for the kinetics of domain growth consistent with our experimental data.

Kv2.1 surface clusters in transfected HEK cells and hippocampal neurons are shown to be trafficking platforms involved in potassium channel movement to and from the cell surface. This work is the first to define stable cell surface sites for ion channel delivery and retrieval at the cell surface., Voltage-gated K+ (Kv) channels regulate membrane potential in many cell types. Although the channel surface density and location must be well controlled, little is known about Kv channel delivery and retrieval on the cell surface. The Kv2.1 channel localizes to micron-sized clusters in neurons and transfected human embryonic kidney (HEK) cells, where it is nonconducting. Because Kv2.1 is postulated to be involved in soluble N-ethylmaleimide–sensitive factor attachment protein receptor–mediated membrane fusion, we examined the hypothesis that these surface clusters are specialized platforms involved in membrane protein trafficking. Total internal reflection–based fluorescence recovery after photobleaching studies and quantum dot imaging of single Kv2.1 channels revealed that Kv2.1-containing vesicles deliver cargo at the Kv2.1 surface clusters in both transfected HEK cells and hippocampal neurons. More than 85% of cytoplasmic and recycling Kv2.1 channels was delivered to the cell surface at the cluster perimeter in both cell types. At least 85% of recycling Kv1.4, which, unlike Kv2.1, has a homogeneous surface distribution, is also delivered here. Actin depolymerization resulted in Kv2.1 exocytosis at cluster-free surface membrane. These results indicate that one nonconducting function of Kv2.1 is to form microdomains involved in membrane protein trafficking. This study is the first to identify stable cell surface platforms involved in ion channel trafficking.

Clathrin-mediated endocytosis takes place through the recruitment of cargo molecules into a growing clathrin-coated pit (CCP). Despite the importance of this process to all mammalian cells, little is yet known about the interaction dynamics between cargo and CCPs. These interactions are difficult to study because CCPs display a large degree of lifetime heterogeneity and the interactions with cargo molecules are time dependent. We use single-molecule total internal reflection fluorescence microscopy, in combination with automatic detection and tracking algorithms, to directly visualize the recruitment of individual voltage-gated potassium channels into forming CCPs in living cells. We observe association and dissociation of individual channels with a CCP and, occasionally, their internalization. Contrary to widespread ideas, cargo often escapes from a pit before abortive CCP termination or endocytic vesicle production. Thus, the binding times of cargo molecules associating to CCPs are much shorter than the overall endocytic process. By measuring tens of thousands of capturing events, we build the distribution of capture times and the times that cargo remains confined to a CCP. An analytical stochastic model is developed and compared with the measured distributions. Due to the dynamic nature of the pit, the model is non-Markovian and it displays long-tail power law statistics. The measured distributions and model predictions are in excellent agreement over more than five orders of magnitude. Our findings identify one source of the large heterogeneities in CCP dynamics and provide a mechanism for the anomalous diffusion of proteins in the plasma membrane.

Neuronal Kv2.1 potassium channels localize into micron-sized clusters which are regulated by extracellular glutamate and intracellular Ca2+ levels. The physical mechanisms underlying the formation and maintenance of these unique structures are largely unknown. We are investigating the dynamics of clustered Kv2.1 channels using high resolution total internal reflection fluorescence microscopy (TIRFM) to track single molecules with 8 nm accuracy.Transfected human embryonic kidney (HEK) cells expressing biotinylated and GFP-tagged Kv2.1 channels are detected with streptavidin-conjugated red quantum dots (QD). While the red QDs enable tracking of individual channels, GFP fluorescence provides characteristics of clusters as an ensemble. The channel dynamics inside Kv2.1 clusters are analyzed in the membrane of live cells in terms of their mean square displacement (MSD) and cumulative distribution function (CDF).In our current model, the actin cytoskeleton plays a dominant role in Kv2.1 cluster formation and maintenance. To test this model we are studying the effects of depolymerization agents such as Cytochalasin and Swinholide A on the individual channel dynamics.Clustered channels remain confined within the cluster perimeter throughout the entire imaging time, up to 25 minutes. MSD analysis indicates similar diffusion constants for clustered and non-clustered channels, D = 0.013 ± 0.017 μm2/s and 0.014 ± 0.011 μm2/s respectively. The CDF of all analyzed trajectories (n=900) deviates from a monoexponential, indicating a discrepancy with Brownian diffusion. Instead, the data can be accurately fit to a double exponential.Our results show a bimodal distribution of channels (clustered and non-clustered) and indicate that both populations experience anomalous subdiffusion. The double exponential term of the CDF suggests two stochastic processes which have slow and fast mobility respectively. Single molecule tracking with simultaneous channel cluster imaging is shown to be an effective way to study the mechanisms underlying clustering phenomena.

The Kv2.1 delayed-rectifier K+ channel regulates electrical activity in neurons and plays a non-conducting role in SNARE-mediated protein fusion in neuroendocrine cells. Kv2.1 is unusual among voltage-gated K+ channels in that it localizes to micron-sized clusters on the cell-surface of neurons and transfected HEK cells. Within these clusters Kv2.1 is non-conducting. Here we demonstrate that these surface structures are specialized platforms involved in trafficking of Kv channels to and from the cell surface. TIRF-based studies indicated that GFP-Kv2.1 containing vesicles tether directly to and deliver cargo in a discrete fashion to the Kv2.1 surface clusters in both transfected HEK and cultured hippocampal neurons. Qdot-based single molecule experiments indicated that the delivery and surface retrieval of Kv2.1 occurs at the perimeter of the surface clusters. Overall, 85±8.4% of newly synthesized channels in HEK cells and 84.9±10.4% in hippocampal neurons were inserted at the cluster perimeter even though the Kv2.1 clusters represent only 21.4 ±3.8% of the basal cell surface. When 132 continuously recycling Kv2.1 channels in HEK cells were examined, 96.2% were also inserted at the cluster perimeter. The actin depolymerizing agents swinholide A and cytochalasin D reduced the cluster localized insertion to 5 and 0% of control, respectively. Unlike Kv2.1, the Kv1.4 K+ channel has a homogeneous cell-surface expression in transfected cells. Demonstrating that Kv2.1 clusters represent cell-surface platforms for more than just Kv2.1 insertion and retrieval, the non-clustering Kv1.4 K+ channel also inserted into the HEK cell plasma membrane at the Kv2.1 cluster perimeter. Kv1.4 endocytosis also occurred at this region. Together, these results indicate that a non-conducting function of Kv2.1 is to form specialized cell-surface microdomains which are involved in ion channel trafficking. These domains may also be involved in additional cellular processes.

The voltage-gated potassium channel Kv2.1 localizes to stable, micro-domains on the cell surface where it plays a non-conducting role. These surface structures are specialized platforms involved in trafficking of Kv channels to and from the cell surface in hippocampal neurons and transfected HEK cells [Deutsch et al., MBoC 15, pp 2917-29 (2012)]. Internalization of Kv2.1 occurs through clathrin-mediated endocytosis and clathrin-coated pits (CCP) localizes adjacent to these micro-domains. This study examines the relationship between Kv2.1 clusters and CCP maturation.TIRF-microscopy was used to study GFP-tagged-clathrin light chain CCPs in live HEK293 cells. HEK cells do not express endogenous Kv2.1, making them a suitable model system in which to investigate the role of Kv2.1 in clathrin-mediated endocytosis. We tracked individual CCPs and measured their lifetimes. This analysis is obtained from the appearance and disappearance of GFP fluorescence within the evanescent field illumination. We compare the dynamics of CCPs in control cells transfected with GFP-CLC and cells co-transfected with Kv2.1 or a Kv2.1 mutant lacking the last 318 amino acids of the C-terminus (ΔC-Kv2.1) necessary for cluster formation.In control cells, CCPs had a mean lifetime of 12.6±0.3 s (mean±sem). The lifetime of CCPs in cells co-expressing Kv2.1 and GFP-CLC was reduced by 50%. When GFP-CLC was co-transfected with the non-clustering ΔC-Kv2.1, the lifetime of CCPs increased by 17%. ΔC-Kv2.1 also decreased the rate of channel endocytosis by 12%.These data reveal that Kv2.1, specifically the C-terminal tail, has a direct effect on CCP lifetimes and thereby maturation. Cells expressing clustering Kv2.1 exhibit more rapidly maturing CCPs as seen by the rate of Kv2.1 internalization. Non-clustering Kv2.1 increases CCP lifetimes, and complete loss of Kv2.1 results in even longer lifetimes, i.e. slower maturation. These results indicate that Kv2.1 cluster formation remodels clathrin-mediated endocytosis.

Outside of striated muscle, endoplasmic reticulum/plasma membrane (ER/PM) junctions are best known for their role in store-operated Ca2+ influx via the Stim/Orai complex. Adding to their functional significance we demonstrate here that these microdomains also operate as trafficking hubs for membrane protein transport to and from the cell surface. We first monitored exocytosis in HEK cells using TIRF microscopy and the transferrin receptor fused to a pH-sensitive GFP variant, superecliptic pHluorin (TfR-SEP). ER/PM junctions were defined by the fluorescent ER markers DsRed2-ER or ER-Tracker Green within the TIR illumination field which typically accounted for 10-20% of the cell footprint. Exocytic delivery of TfR-SEP was detected as the transient appearance and subsequent diffusion of bright puncta on the PM. Greater than 80% of the TfR-SEP delivery events occurred adjacent to ER/PM junctions indicating these microdomains are preferred sites for TfR exocytosis. The temperature-sensitive VSV-G protein mutant (YFP-VSV-G-ts045) was delivered to the cell surface at these microdomains following temperature-dependent release from the ER, demonstrating that ER/PM junctions are trafficking hubs for nascent membrane proteins. Automated tracking of Qdot-labeled GFP-Kv1.4-loopBAD channels indicated that this ion channel also trafficked both to and from the cell surface at ER/PM junctions. To highlight sites of endocytosis we expressed RFP clathrin light chain (RFP-CLC) which formed dynamic puncta. TfR-SEP formed similar puncta which co-localized with RFP-CLC when expressed together. Endocytosis of both types of puncta was observed frequently suggesting these puncta are endocytic sites. Almost 90% of both types of puncta were present within 0.3 μm of the ER perimeter. Together, these data indicate that both exo- and endocytosis preferentially occur adjacent to ER/PM junctions. This suggests that ER/PM junctions are trafficking hubs and are playing a role more central to basic cell biology than previously appreciated.