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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.

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.

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.

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.

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.