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Cells contain hundreds of organelles and macromolecular assemblies. Obtaining a complete understanding of their intricate organization requires the nanometre-level, three-dimensional reconstruction of whole cells, which is only feasible with robust and scalable automatic methods. Here, to support the development of such methods, we annotated up to 35 different cellular organelle classes—ranging from endoplasmic reticulum to microtubules to ribosomes—in diverse sample volumes from multiple cell types imaged at a near-isotropic resolution of 4 nm per voxel with focused ion beam scanning electron microscopy (FIB-SEM)1. We trained deep learning architectures to segment these structures in 4 nm and 8 nm per voxel FIB-SEM volumes, validated their performance and showed that automatic reconstructions can be used to directly quantify previously inaccessible metrics including spatial interactions between cellular components. We also show that such reconstructions can be used to automatically register light and electron microscopy images for correlative studies. We have created an open data and open-source web repository, ‘OpenOrganelle’, to share the data, computer code and trained models, which will enable scientists everywhere to query and further improve automatic reconstruction of these datasets. Focused ion beam scanning electron microscopy (FIB-SEM) combined with deep-learning-based segmentation is used to produce three-dimensional reconstructions of complete cells and tissues, in which up to 35 different organelle classes are annotated.

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues., Cells in many tissues fuse into syncytia acquiring new functions. By investigating whether physical remodelling promotes differentiation, here, the authors show that plasma membrane diminution post-fusion causes transient nutrient stress that inhibits YAP1 activity and may reduce proliferation-promoting transcription.

Arrays of actin filaments (F-actin) near the apical surface of epithelial cells (medioapical arrays) contribute to apical constriction and morphogenesis throughout phylogeny. Here, superresolution approaches (grazing incidence structured illumination, GI-SIM, and lattice light sheet, LLSM) microscopy resolve individual, fluorescently labeled F-actin and bipolar myosin filaments that drive amnioserosa cell shape changes during dorsal closure in

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity, and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal activation. In exploring the basis of these N-glycosylation alterations, we discovered that they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed during neuronal excitation were in close association with endoplasmic reticulum (ER) exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite’s satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction, and disease.

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of “fluorogenic” compounds—ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone–zwitterion equilibrium constant (KL–Z) is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore—Janelia Fluor 526 (JF526)—with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF526 is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF526 constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging., We developed a general rubric for creating fluorogenic stains, resulting in the novel fluorophore JF526. This dye can be used in numerous imaging modalities including super-resolution microscopy.

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal excitation. In exploring the basis of these N-glycosylation alterations, we discovered they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed with neuronal excitation were in close association with ER exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway, or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite’s satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction and disease.

Cells contain hundreds of different organelle and macromolecular assemblies intricately organized relative to each other to meet any cellular demands. Obtaining a complete understanding of their organization is challenging and requires nanometer-level, threedimensional reconstruction of whole cells. Even then, the immense size of datasets and large number of structures to be characterized requires generalizable, automatic methods. To meet this challenge, we developed an analysis pipeline for comprehensively reconstructing and analyzing the cellular organelles in entire cells imaged by focused ion beam scanning electron microscopy (FIB-SEM) at a near-isotropic size of 4 or 8 nm per voxel. The pipeline involved deep learning architectures trained on diverse samples for automatic reconstruction of 35 different cellular organelle classes - ranging from endoplasmic reticulum to microtubules to ribosomes - from multiple cell types.Automatic reconstructions were used to directly quantify various previously inaccessible metrics about these structures, including their spatial interactions. We show that automatic organelle reconstructions can also be used to automatically register light and electron microscopy images for correlative studies. We created an open data and open source web repository, OpenOrganelle, to share the data, computer code, and trained models, enabling scientists everywhere to query and further reconstruct the datasets.

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometer resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations inasmuch as they only visualize a single slice or a relatively small volume of the cell, respectively. Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) demonstrated the ability to image cellular samples at 4-nm isotropic voxels with rather limited imageable volume. Here, we present 3D EM images of whole cells and tissues with two orders of magnitude increases in imageable volume at 4-nm voxels. Such data with a combined fine resolution scale and large sample size do not currently exist, and are enabled by the advances in higher precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning. More importantly, we have generated a volume EM atlas encompassing ten diverse datasets of whole cells and tissues, from cancer cells to immune cells, and from mouse pancreatic islets to Drosophila neural tissues. These open-access data (via OpenOrganelle) represent a foundation to nucleate a new field of high-resolution whole-cell volume EM and subsequent analyses, and invite biologists to explore this new paradigm and pose fundamentally new questions.

How nicotine exposure produces long-lasting changes that remodel neural circuits with addiction is unknown. Here, we report that long-term nicotine exposure alters the trafficking of α4β2-type nicotinic acetylcholine receptors (α4β2Rs) by dispersing and redistributing the Golgi apparatus. In cultured neurons, dispersed Golgi membranes were distributed throughout somata, dendrites and axons. Small, mobile vesicles in dendrites and axons lacked standard Golgi markers and were identified by other Golgi enzymes that modify glycans. Nicotine exposure increased levels of dispersed Golgi membranes, which required α4β2R expression. Similar nicotine-induced changes occurred in vivo at dopaminergic neurons at mouse nucleus accumbens terminals, consistent with these events contributing to nicotine’s addictive effects. Characterization in vitro demonstrated that dispersal was reversible, that dispersed Golgi membranes were functional, and that membranes were heterogenous in size, with smaller vesicles emerging from larger “ministacks”, similar to Golgi dispersal induced by nocadazole. Protocols that increased cultured neuronal synaptic excitability also increased Golgi dispersal, without the requirement of α4β2R expression. Our findings reveal novel activity- and nicotine-dependent changes in neuronal intracellular morphology. These changes regulate levels and location of dispersed Golgi membranes at dendrites and axons, which function in local trafficking at subdomains.

At the center of the secretory pathway, the Golgi complex ensures correct processing and sorting of cargos toward their final destination. Cargos are diverse in topology, function and destination. A remarkable feature of the Golgi complex is its ability to sort and process these diverse cargos destined for secretion, the cell surface, the lysosome, or retained within the secretory pathway. Just as these cargos are diverse so also are their sorting requirements and thus, their trafficking route. There is no one-size-fits-all sorting scheme in the Golgi. We propose a coexistence of models to reconcile these diverse needs. We review examples of differential sorting mediated by proteins and lipids. Additionally, we highlight recent technological developments that have potential to uncover new modes of transport.

SummaryDeveloping cells divide and differentiate, and in many tissues, such as bone, muscle, and placenta, cells fuse acquiring specialized functions. While it is known that fused-cells are differentiated, it is unclear what mechanisms trigger the programmatic-change, and whether cell-fusion alone drives differentiation. To address this, we employed a fusogen-mediated cell-fusion system involving undifferentiated cells in tissue culture. RNA-seq analysis revealed cell-fusion initiates a dramatic transcriptional change towards differentiation. Dissecting the mechanisms causing this reprogramming, we observed that after cell-fusion plasma-membrane surface area decreases through increased endocytosis. Consequently, glucose-transporters are internalized, and cytoplasmic-glucose and ATP transiently decrease. This low-energetic state activates AMPK, which inhibits YAP1, causing cell-cycle arrest. Impairing either endocytosis or AMPK prevents YAP1 inhibition and cell-cycle arrest after fusion. Together these data suggest that cell-fusion-induced differentiation does not need to rely on extrinsic-cues; rather the plasma-membrane diminishment forced by the geometric-transformations of cell-fusion cause transient cell-starvation that induces differentiation.

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.

GRAPHICAL ABSTACT HIGHLIGHTS Hyperactive neurons generate and release excess peroxidated fatty acids (FAs) Astrocytes endocytose toxic FAs as lipoprotein particles, delivering them to lipid droplets Lipid droplets trigger upregulation of genes involved in metabolism and detoxification Astrocytes detoxify and consume FAs by mitochondrial oxidation in response to neural activity SUMMARY 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, peroxidated fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by lipoprotein 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. Together, our findings reveal that FA metabolism between neurons and astrocytes is coupled in an activity-dependent manner to protect neurons from FA toxicity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

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 endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle that plays crucial roles in numerous cellular functions. We used emerging superresolution imaging technologies to clarify the morphology and dynamics of the peripheral ER, which contacts and modulates most other intracellular organelles. Peripheral components of the ER have classically been described as comprising both tubules and flat sheets. We show that this system consists almost exclusively of tubules at varying densities, including structures that we term ER matrices. Conventional optical imaging technologies had led to misidentification of these structures as sheets because of the dense clustering of tubular junctions and a previously uncharacterized rapid form of ER motion. The existence of ER matrices explains previous confounding evidence that had indicated the occurrence of ER "sheet" proliferation after overexpression of tubular junction-forming proteins.

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.

We describe a method for the analysis of the distribution of displacements, i.e., the propagators, of single-particle tracking measurements for the case of obstructed subdiffusion in two-dimensional membranes. The propagator for the percolation cluster is compared with a two-component mobility model against Monte Carlo simulations. To account for diffusion in the presence of obstacle concentrations below the percolation threshold, a propagator that includes the transient motion in finite percolation clusters and hopping between obstacle-induced compartments is derived. Finally, these models are shown to be effective in the analysis of Kv2.1 channel diffusive measurements in the membrane of living mammalian cells.

Diffusion in the plasma membrane of living cells is often found to display anomalous dynamics. However, the mechanism underlying this diffusion pattern remains highly controversial. Here, we study the physical mechanism underlying Kv2.1 potassium channel anomalous dynamics using single-molecule tracking. Our analysis includes both time series of individual trajectories and ensemble averages. We show that an ergodic and a nonergodic process coexist in the plasma membrane. The ergodic process resembles a fractal structure with its origin in macromolecular crowding in the cell membrane. The nonergodic process is found to be regulated by transient binding to the actin cytoskeleton and can be accurately modeled by a continuous-time random walk. When the cell is treated with drugs that inhibit actin polymerization, the diffusion pattern of Kv2.1 channels recovers ergodicity. However, the fractal structure that induces anomalous diffusion remains unaltered. These results have direct implications on the regulation of membrane receptor trafficking and signaling.

Experimental single-particle tracking (SPT) is extensively used to study the dynamics of membrane proteins and lipids in living cells. Most studies show that molecular motion in the plasma membrane, as well as in the cytoplasm and the nucleus, undergoes anomalous subdiffusion. SPT data is usually analyzed in terms of the temporal mean square displacement because averaging along individual trajectories is more readily available than ensemble averages. However, in some intriguing physical phenomena ergodicity is broken and thus temporal averages do not converge to the ensemble measurements. Here we measured more than 1,000 Kv2.1 potassium channel trajectories in live human embryonic kidney (HEK) cells, analyzed their ergodic and non-ergodic properties and uncovered the physical mechanism underlying their anomalous diffusion pattern.We have labeled Kv2.1 channels with quantum dots (QDs) in HEK cells, imaged the cell basal membrane with total internal fluorescence microscopy and analyzed the individual channel trajectories. We found that the distributions of the two types of averages are clearly different. The temporal average yielded a much broader MSD than the ensemble-average. Nevertheless, the diffusion pattern of these channels follows anomalous subdiffusion in the lag time, i.e. the MSD is sublinear, both for the temporal and the ensemble averages. Our data reveal that two processes simultaneously coexist and only one of them is ergodic. The ergodicity breaking is found to be maintained by a set of anchoring points associated to the actin cytoskeleton. Control experiments indicate these effects are not induced by the quantum dots. These results are accurately modeled by a continuous time random walk on a fractal structure. When the actin cytoskeleton is disrupted, ergodicity is recovered. These experimental observations have direct biological implications in the dynamics of membrane proteins.

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.

Kv2.1 are voltage gated potassium channels that form long-lived clusters on the surface of mammalian cells. We have used single molecule tracking to study the interesting dynamics of these channels in live HEK cells. Both the channels inside the clusters and non-clustering channels are found to follow anomalous subdiffusion. The effect of actin cytoskeleton on the diffusion properties of the channels is also investigated in the presence of cytochalasin D, a F-actin binding drug that blocks actin polymerization.

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.

Single molecule tracking in concert with mean square displacement and cumulative distribution function analysis is used to study Kv2.1 ion channel dynamics. Results show the channels are confined to clusters and they undergo anomalous subdiffusion.

The successful identification and detection at the single molecule level of Antigen 85b, an antigen released by tuberculosis, was accomplished using a fluorescence-based immunoassay. This work enables a method for the diagnosis of latent tuberculosis.

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.

Clathrin mediated endocytosis is the major route of cargo internalization in mammalian cells. The assembly of clathrin coated pits (CCPs) is a multistep process that includes nucleation of a clathrin coat and growth by recruitment of clathrin molecules, adaptors and cargo. This process is terminated either nonproductively (the pit breaks up) or productively (a vesicle forms and is internalized). Even though the association of cargo to CCPs is crucial in the regulation of endocytosis, the study of this interaction in vivo remains challenging. Here we study the recruitment of cargo by characterizing the interactions of clathrin coated pits with Kv2.1, a potassium channel that is effectively internalized via clathrin mediated endocytosis.TIRF-based single-particle tracking reveals that Kv2.1 displays a confined subdiffusion type of motion on the cell surface and frequent stalls occur during individual trajectories. Multicolor imaging indicates that these stalls are caused by stable CCPs that capture Kv2.1 channels. By monitoring the residence time of Kv2.1 within a CCP, we are able to study the binding strength as a function of the age of a pit, e.g., the time since coat initiation. Due to the dynamic growth of CCPs, the interaction between the pit and cargo is not Poissonian. A kinetic model that takes into account the coat assembly via the recruitment of adaptor proteins leads to nonstationary and nonergodic Kv2.1 dynamics. This model accurately predicts the statistics of binding energies between pit and cargo.

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.

Voltage-gated sodium channels (Nav) are densely accumulated at the axon initial segment (AIS) of neurons where they are responsible for action potential initiation. The dense clustering of channels at the AIS involves ankyrinG binding, however the details of trafficking these channels to the AIS remains elusive. Furthermore, it is unclear what percentage of AIS channels is actually conducting. Since the large sodium channel cDNAs are difficult to manipulate and suffer from rearrangements in E. coli, the most elegant trafficking work to date has utilized chimeric proteins containing the sodium channel ankyrin-binding motif fused to other membrane proteins. To fully address trafficking in real time, an appropriately tagged full-length and functional channel is required. Therefore, we developed a Nav1.6 tagged with either GFP or Dendra2 fluorescent-proteins on the C-terminus and an extracellular biotin-acceptor-domain (BAD). The BAD allows for visualization and single molecule tracking of quantum-dot-bound sodium channels on the neuronal surface. This modified Nav1.6 demonstrated wild-type activity when expressed in hippocampal neurons. The tagged channel efficiently trafficked to the cell surface and was localized at the AIS as indicated by both confocal and TIRF microscopy. Alexafluor 594-conjugated-streptavidin binding indicated the surface-density of channels at the AIS was approximately 60 times greater than on the soma, comparable to endogenous Nav1.6 channels. Fluorescence recovery after photobleaching (FRAP) and single particle tracking showed that channels at the AIS had recovery time constants of greater than 2 hours and were confined to 60nm +/- 20nm compartments. In summary, we constructed a sodium channel with fluorescent protein and extracellular biotin reporters that has both wild-type trafficking and biophysical properties. This construct will permit the examination of sodium channel turnover, trafficking, diffusion and location-dependent function in neuronal cells.

We study the insertion and retrieval of voltage-gated potassium channels, Kv2.1, at the single molecule level. Kv2.1 channels are labeled with quantum dots (QDs) at an extracellular domain. We observe QDs being internalized by the cell and new QD-tagged channels being inserted into the membrane. Because labeling occurs solely on the cell surface, only recycled channels that were previously in the plasma membrane can carry emerging QDs. Controls with both GFP and QD labels indicate that newly arriving QDs are indeed Kv2.1 channels. Channels that are in the plasma membrane from the beginning of the experiment can be either recycled or newly synthesized channels, as we cannot separate between these two in this measurement. The residence time distribution of channels that are on the cell surface from the beginning of our measurements has a median of 119 s, whereas for recycled channels the median is only 81 s, a 32% reduction (n = 334). In both instances it is surprising how short the residence time is on the cell surface of these channels. We propose that rapid channel turnover, via recycling pathways, helps the cell to maintain specialized regions in the membrane, which are entropically unfavorable. We investigate the role of actin in Kv2.1 trafficking using actin polymerization inhibitors. Upon the application of 5 μM cytochalasin D and 80 μM swinholide A, we observe that the residence times of both newly synthesized and recycled proteins are significantly reduced. In cells treated with actin inhibitors, channels are no longer sequestered into specific microdomains. Thus, channel recycling may function as an important factor in membrane compartmentalization and may be enhanced by stimuli that disrupt this organization.

The Nav1.6 isoform is one of the major Nav channels of the central nervous system. In addition to its AIS localization, this isoform is found at a lower density throughout the somatodendritic region of the neuron, where it is thought to contribute to the back-propagation of the action potential. Due to the low number of somatodendritic Nav channels, immunocytochemistry is not sensitive enough to detect these channels and instead electron microscopy is required to visualize them. Thus, the dynamics and diffusive behavior of these channels have never been previously observed in real time. To visualize this behavior, we have utilized a Nav1.6 construct tagged with the photoswitchable fluorophore, Dendra2 (Nav1.6-Dendra2). This construct was transfected into cultured rat hippocampal neurons and imaged via TIRF microscopy. We combined single-particle tracking with photoactivated localization microscopy (sptPALM) such that we tracked only a small subset of Nav1.6-Dendra2 molecules at any given time. A steady-state density of active fluorophores was maintained via a low-power activation laser and the trajectory of each molecule was determined using an automated detection and tracking algorithm. We typically obtained statistics of tens of thousands of trajectories in each cell with high spatial and temporal resolution. Then these trajectories were used to obtain a diffusivity map across the neuronal surface using a Bayesian inference scheme. Consistent with previous observations, this method showed that AIS Nav1.6 channels are stably anchored, presumably to AnkyrinG. In contrast, Nav1.6 channels in the somatodendritic region showed both diffusive behavior and periods of transient confinement within specific membrane regions, thus creating small membrane clusters. Interestingly, we found large, micron-size regions of membrane completely devoid of Nav1.6, which implies that the channel is physically excluded from these domains.

Tracking individual potassium channels in the plasma membrane reveals complex dynamics involving anomalous diffusion. Theoretical models show that anomalous subdiffusion can be caused by several different processes. In particular, transient binding events, modeled by a continuous time random walk (CTRW), may not only induce anomalous subdiffusion but also weak ergodicity breaking, that is, the ensemble and time averages do not coincide. We studied the physical mechanism underlying Kv2.1 and Kv1.4 potassium channel anomalous dynamics by performing time series analysis of extensive single molecule tracking in the membrane of live mammalian cells. We find ample evidence showing that the ensemble and temporal distributions are different. Our data reveal that two anomalous subdiffusion processes simultaneously coexist and only one of them is ergodic. Weak ergodicity breaking is found to be maintained by immobilization events as long as 60 seconds. In the presence of either actin or chlathrin inhibitors, ergodicity is recovered. In order to elucidate the effects of clathrin endocytosis on Kv2.1 trafficking and diffusion, we have performed simultaneous total internal reflection fluorescence imaging of quantum-dot-tagged Kv2.1 and RFP-tagged clathrin. Our results show that Kv2.1 channels are frequently recycled from and to the plasma membrane. Retrieval of Kv2.1 from the membrane is found to be attained via a clathrin-mediated endocytic pathway. These endocytic/insertion processes are needed to maintain a nonergodic CTRW. Furthermore, the Kv2.1 stalling events colocalize with clathrin clusters. These results suggest that abortive endocytic events are responsible for the observed channel immobilization in the plasma membrane.

Cortical actin is a complex meshwork essential for the dynamic organization and localization of plasma membrane proteins. In order to characterize dynamics of the cytoskeleton of living mammalian cells with resolution beyond the diffraction limit, we used photoactivated localization microscopy (PALM). We transfected ND7/23 and HEK 293 cells to express Dendra2 labeled actin. The cells were imaged in a custom-built total internal reflection microscope maintaining the cells under physiological conditions for prolonged periods of time. Dynamic PALM images of the actin cytoskeleton were reconstructed in a 5-second sliding time window. A resolution of 50 nm was achieved.By combining dynamic super-resolution actin images and single particle tracking in the plasma membrane, we studied the actin cytoskeleton's role in the organization of Kv2.1 channels into segregated microdomains. Kv2.1 forms stable clusters in hippocampal neurons and transfected HEK cells, but the mechanism by which the clusters are formed is not well understood. By labeling Kv2.1 channels with quantum dots which are spectrally well separated from Dendra2, two-color images were obtained which revealed complex interactions between the actin cytoskeleton and Kv2.1 channels. This work demonstrates that PALM combined with single particle tracking is an effective technique to probe the dynamic interactions between cortical actin and membrane proteins.

Voltage-gated sodium channels are responsible for the initiation of action potentials in excitable cells. These channels are highly concentrated at the axon initial segment (AIS) of neurons due to their interactions with ankyrin-G. This interaction is mediated by a 9 amino acid sequence, termed the Ankyrin Binding Motif (ABM) present on the II-III linker. In order to study the dynamics of sodium channels in living neurons in real time, we created a fluorescently labeled Nav1.6 protein with an extracellular tag (biotin acceptor domain). We used single-particle tracking of channels labeled with streptavidin conjugated quantum dots (QDs) and/or Alexa594 to directly compare the mobility of Nav1.6 channels localized to the AIS and somatodendritic compartments of 8DIV hippocampal neurons. We observed two populations of Nav1.6 channels, a small mobile population and a much larger immobile population. The mobile channels on the soma had a diffusion coefficient of 0.016 ± 0.008 μm2/s. To determine the role of ankyrin-G binding in the diffusion of the full-length sodium channel, we deleted the ABM from the Nav1.6 construct. As expected, this mutant channel did not concentrate at the AIS and instead was localized throughout the soma and processes, based on both GFP fluorescence and labeling of surface channels using streptavidin conjugated-Alexa594. Single-particle tracking of the mutant channels revealed that the majority of these channels (∼80%) are also immobile in the plasma membrane of the soma and dendrites. This suggests that although binding to ankyrin-G is necessary and sufficient for Nav1.6 to localize to the AIS, a different mechanism is responsible for the localization and membrane dynamics in the somatodendritic region of hippocampal neurons.