Aubrey V. Weigel

Ergodic and nonergodic processes coexist in the plasma membrane as observed by single-molecule tracking

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.

Kv2.1 cell surface clusters are insertion platforms for ion channel delivery to the plasma membrane

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.

Quantifying the dynamic interactions between a clathrin-coated pit and cargo molecules

Significance Clathrin-mediated endocytosis is the primary pathway of cargo internalization in mammalian cells. However, little is known about the time-dependent interactions between the endocytic machinery and cargo molecules. Nevertheless, these interactions are known to regulate the maturation of a clathrin-coated pit. In this study, we attain a quantitative understanding of the interactions between clathrin-coated pits and cargo using a combination of imaging techniques, single-molecule tracking, and stochastic modeling. We observe that the binding times of cargo molecules are much shorter than the overall endocytic process, albeit they exhibit a very broad distribution. Our modeling explains the measured statistics of cargo captures and binding times. This work further identifies a mechanism for the large diversity in the dynamic behavior of clathrin structures. 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.

Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

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.

Size of Cell-Surface Kv2.1 Domains is Governed by Growth Fluctuations

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.

Anomalous diffusion of kv2.1 channels observed by single molecule tracking in live cells

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.

Single-Particle Tracking of Nav1.6 Demonstrates Different Mechanisms for Sodium Channel Anchoring within the AIS versus the Soma of Hippocampal Neurons

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.

Kv2.1 Cell Surface Clusters Promote Maturation of Clathrin-Coated Pits

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.

Rapid Cell Surface Kv2.1 Recycling Observed by Single Molecule Tracking

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 Single Potassium Channels in Live Mammalian Cells

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.