Protein and lipid nanodomains are prevalent on the surface of mammalian cells. In particular, it ... more Protein and lipid nanodomains are prevalent on the surface of mammalian cells. In particular, it has been recently recognized that ion channels assemble into surface nanoclusters in the soma of cultured neurons. However, the interactions of these molecules with surface nanodomains display a considerable degree of heterogeneity. Here, we investigate this heterogeneity and develop statistical tools based on the recurrence of individual trajectories to identify subpopulations within ion channels in the neuronal surface. We specifically study the dynamics of the K^{+} channel Kv1.4 and the Na^{+} channel Nav1.6 on the surface of cultured hippocampal neurons at the single-molecule level. We find that both these molecules are expressed in two different forms with distinct kinetics with regards to surface interactions, emphasizing the complex proteomic landscape of the neuronal surface. Further, the tools presented in this work provide new methods for the analysis of membrane nanodomains, ...
Stochastic motion on the surface of living cells is critical to promote molecular encounters that... more Stochastic motion on the surface of living cells is critical to promote molecular encounters that are necessary for multiple cellular processes. Often the complexity of the cell membranes leads to anomalous diffusion, which under certain conditions it is accompanied by non-ergodic dynamics. Here, we unravel two manifestations of ergodicity breaking in the dynamics of membrane proteins in the somatic surface of hippocampal neurons. Three different tagged molecules are studied on the surface of the soma: the voltage-gated potassium and sodium channels Kv1.4 and Nav1.6 and the glycoprotein CD4. In these three molecules ergodicity breaking is unveiled by the confidence interval of the mean square displacement and by the dynamical functional estimator. Ergodicity breaking is found to take place due to transient confinement effects since the molecules alternate between free diffusion and confined motion.
A broad range of membrane proteins display anomalous diffusion on the cell surface. Different met... more A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized because of experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data confirm that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar meshwork. These results present a hierarchical nanoscale picture of the plasma membrane.
Voltage-gated sodium (Nav) channels are responsible for the depolarizing phase of the action pote... more Voltage-gated sodium (Nav) channels are responsible for the depolarizing phase of the action potential in most nerve cells, and Nav channel localization to the axon initial segment is vital to action potential initiation. Nav channels in the soma play a role in the transfer of axonal output information to the rest of the neuron and in synaptic plasticity, although little is known about Nav channel localization and dynamics within this neuronal compartment. This study uses single-particle tracking and photoactivation localization microscopy to analyze cell-surface Nav1.6 within the soma of cultured hippocampal neurons. Mean-square displacement analysis of individual trajectories indicated that half of the somatic Nav1.6 channels localized to stable nanoclusters ∼230 nm in diameter. Strikingly, these domains were stabilized at specific sites on the cell membrane for >30 min, notably via an ankyrin-independent mechanism, indicating that the means by which Nav1.6 nanoclusters are mai...
2010 Annual International Conference of the IEEE Engineering in Medicine and Biology, 2010
Kv2.1 are voltage gated potassium channels that form long-lived clusters on the surface of mammal... more 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.
Lipid-mediated membrane heterogeneity is proposed to be an important organizing principle in mamm... more Lipid-mediated membrane heterogeneity is proposed to be an important organizing principle in mammalian cells. Using single fluorescent particle tracking, we quantify diffusion parameters of a large panel of fluorescent fusion membrane proteins ranging in size, mode of membrane anchoring, and putative phase-association. These include palmitoylated or non-palmitoylated versions of three transmembrane proteins (truncated linker of activated T-cell, truncated haemagglutinin, and b 2 adrenergic receptor) as well as three proteins anchored with lipid moieties (GPI, palmitoyl and myristoyl, or geranylgeranyl). We present an analysis that utilizes Brownian simulations to aid in interpreting heterogeneity. With the exception of two of the palmitoylated transmembrane proteins, diffusion of all constructs is unconfined and consistent with Brownian motion at 37 C at the timescales investigated (20 ms-1 sec). We explore the contributions of lipid mixing to confinement by modulating cholesterol levels and through the use of biochemical perturbations that affect the temperature of the immiscibility phase transition in isolated plasma membrane vesicles. Among our findings is that diffusion and confinement are highly temperature sensitive. Furthermore, our results indicate a complicated size-dependence of diffusion suggesting that diffusion of small probes is particularly sensitive to dimerization when it occurs in either a biological context or due to labeling techniques. Overall, we explore the contribution of phase-mediated membrane heterogeneity to protein mobility while highlighting several factors that can complicate the interpretation of lateral diffusion data.
Cells are organized on length scales from Angstroms to microns. However, the mechanisms by which ... more Cells are organized on length scales from Angstroms to microns. However, the mechanisms by which Angstrom-scale molecular properties are translated to micron-scale macroscopic properties are not well understood. We have shown that interactions between multivalent proteins and multivalent ligands can cause liquid-liquid demixing phase transitions, resulting in formation of micron-sized liquid droplets in aqueous solution and micron-sized puncta on membranes. These transitions appear to occur concomitantly with sol-gel transitions to form large, dynamic polymers within the droplets/puncta. I will discuss how such transitions may control the spatial organization and biochemical activity of actin regulatory signaling pathways, and contribute to formation of PML nuclear bodies in the mammalian nucleus. Our data suggest a general mechanism by which cells may achieve micron-scale organization based on interactions between multivalent macromolecules.
While junctions between cortical endoplasmic reticulum (cER) and the plasma membrane (PM) are a s... more While junctions between cortical endoplasmic reticulum (cER) and the plasma membrane (PM) are a subtle but ubiquitous feature in mammalian cells very little is known about the functions and molecular interactions associated with neuronal ER/PM junctions. Here we report that Kv2.1, the primary delayed-rectifier K(+) channel in the mammalian brain, induces the formation of ER/PM junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, suggesting a function unrelated to membrane potential regulation. Accordingly, Kv2.1 clusters function as membrane trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER/PM junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 cluster...
Proceedings of the National Academy of Sciences, 2011
Diffusion in the plasma membrane of living cells is often found to display anomalous dynamics. Ho... more 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 implic...
Proceedings of the National Academy of Sciences, 2013
Significance Clathrin-mediated endocytosis is the primary pathway of cargo internalization in mam... more 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.
We describe a method for the analysis of the distribution of displacements, i.e., the propagators... more 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 obstacleinduced 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.
Voltage-gated K+(Kv) channels regulate membrane potential in many cell types. Although the channe... more 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 surf...
In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns... more 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 mamma... more 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.
study is to estimate the contribution of the nNavs in the late sodium current (I NaL) in ischemic... more study is to estimate the contribution of the nNavs in the late sodium current (I NaL) in ischemic condition in order to determine if an overexpression of the nNa v s can modify the APD and create an AS. Methods: Freshly isolated cardiomyocytes were placed under ischemic conditions for 45 minutes. I NaL currents were recorded with the patch clamp technique in whole cell configuration. Tetrodotoxin (a specific nNa v s blocker) and MTSEA (a specific Na v 1.5 blocker) were used to differentiate the two sodium channels isoforms. Results: In normal condition, nNa v s account for 11% of peak current. I NaL represents 0.3% of the peak current at a potential of À10 mV. Contribution of nNa v s (TTX sensitive) to I NaL was 36% 5 5%. Ischemia decreases the maximal current density from À73.3 nA/pF to À53.4 nA/pF. Surprisingly, nNa v s contribution was not modified (10% of the peak current). However, ischemia increases I NaL from 0.3% to 1.6% compared to the peak current. Conclusion: Ischemia increases by 5.3 times I NaL that can play a critical role in the duration of the action potential and facilitates the outcome of arrhythmias.
provide the first identification and characterization of the biophysical properties of the most p... more provide the first identification and characterization of the biophysical properties of the most prominently observed ion channel expressed within the inner and outer membrane of nuclei from adult skeletal muscle fibers. Excised insideout single channel recordings were obtained from individual nuclei acutely isolated from Flexor Digitorum Brevis (FDB) fibers of wild-type mice. The outer membrane of nuclei was readily accessible following isolation. For measurements of channels from the inner membrane, nuclei were treated with 1 % (w/v) sodium citrate in order to remove the outer membrane. We found that the predominant ion channel expressed in both the inner and outer nuclear membrane was a cationic channel that conducts monovalent ions with slight preference for potassium over sodium ions (a PK/PNa~1.22). A 10,000-fold difference in the concentration of free Ca2þ between the pipette and bath solutions did not affect the channel reversal potential in symmetric KCl (~0 mV), indicating that Ca2þ ions permeation is negligible. The maximum conductance of the channel in the outward direction was~162 pS. The mean open probability (PO) was~0.7 and voltage-independent between À50 mV to þ50 mV. We suggest that this novel monovalent cationic channel within the inner and outer membrane of skeletal muscle nuclei provides a countercurrent mechanism that minimizes voltage change across the nuclear membrane. This research was supported by NIH K01 award AR060831(to V.Y.) and NIH R01 grant AR44657 (to R.T.D).
Protein and lipid nanodomains are prevalent on the surface of mammalian cells. In particular, it ... more Protein and lipid nanodomains are prevalent on the surface of mammalian cells. In particular, it has been recently recognized that ion channels assemble into surface nanoclusters in the soma of cultured neurons. However, the interactions of these molecules with surface nanodomains display a considerable degree of heterogeneity. Here, we investigate this heterogeneity and develop statistical tools based on the recurrence of individual trajectories to identify subpopulations within ion channels in the neuronal surface. We specifically study the dynamics of the K^{+} channel Kv1.4 and the Na^{+} channel Nav1.6 on the surface of cultured hippocampal neurons at the single-molecule level. We find that both these molecules are expressed in two different forms with distinct kinetics with regards to surface interactions, emphasizing the complex proteomic landscape of the neuronal surface. Further, the tools presented in this work provide new methods for the analysis of membrane nanodomains, ...
Stochastic motion on the surface of living cells is critical to promote molecular encounters that... more Stochastic motion on the surface of living cells is critical to promote molecular encounters that are necessary for multiple cellular processes. Often the complexity of the cell membranes leads to anomalous diffusion, which under certain conditions it is accompanied by non-ergodic dynamics. Here, we unravel two manifestations of ergodicity breaking in the dynamics of membrane proteins in the somatic surface of hippocampal neurons. Three different tagged molecules are studied on the surface of the soma: the voltage-gated potassium and sodium channels Kv1.4 and Nav1.6 and the glycoprotein CD4. In these three molecules ergodicity breaking is unveiled by the confidence interval of the mean square displacement and by the dynamical functional estimator. Ergodicity breaking is found to take place due to transient confinement effects since the molecules alternate between free diffusion and confined motion.
A broad range of membrane proteins display anomalous diffusion on the cell surface. Different met... more A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized because of experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data confirm that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar meshwork. These results present a hierarchical nanoscale picture of the plasma membrane.
Voltage-gated sodium (Nav) channels are responsible for the depolarizing phase of the action pote... more Voltage-gated sodium (Nav) channels are responsible for the depolarizing phase of the action potential in most nerve cells, and Nav channel localization to the axon initial segment is vital to action potential initiation. Nav channels in the soma play a role in the transfer of axonal output information to the rest of the neuron and in synaptic plasticity, although little is known about Nav channel localization and dynamics within this neuronal compartment. This study uses single-particle tracking and photoactivation localization microscopy to analyze cell-surface Nav1.6 within the soma of cultured hippocampal neurons. Mean-square displacement analysis of individual trajectories indicated that half of the somatic Nav1.6 channels localized to stable nanoclusters ∼230 nm in diameter. Strikingly, these domains were stabilized at specific sites on the cell membrane for >30 min, notably via an ankyrin-independent mechanism, indicating that the means by which Nav1.6 nanoclusters are mai...
2010 Annual International Conference of the IEEE Engineering in Medicine and Biology, 2010
Kv2.1 are voltage gated potassium channels that form long-lived clusters on the surface of mammal... more 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.
Lipid-mediated membrane heterogeneity is proposed to be an important organizing principle in mamm... more Lipid-mediated membrane heterogeneity is proposed to be an important organizing principle in mammalian cells. Using single fluorescent particle tracking, we quantify diffusion parameters of a large panel of fluorescent fusion membrane proteins ranging in size, mode of membrane anchoring, and putative phase-association. These include palmitoylated or non-palmitoylated versions of three transmembrane proteins (truncated linker of activated T-cell, truncated haemagglutinin, and b 2 adrenergic receptor) as well as three proteins anchored with lipid moieties (GPI, palmitoyl and myristoyl, or geranylgeranyl). We present an analysis that utilizes Brownian simulations to aid in interpreting heterogeneity. With the exception of two of the palmitoylated transmembrane proteins, diffusion of all constructs is unconfined and consistent with Brownian motion at 37 C at the timescales investigated (20 ms-1 sec). We explore the contributions of lipid mixing to confinement by modulating cholesterol levels and through the use of biochemical perturbations that affect the temperature of the immiscibility phase transition in isolated plasma membrane vesicles. Among our findings is that diffusion and confinement are highly temperature sensitive. Furthermore, our results indicate a complicated size-dependence of diffusion suggesting that diffusion of small probes is particularly sensitive to dimerization when it occurs in either a biological context or due to labeling techniques. Overall, we explore the contribution of phase-mediated membrane heterogeneity to protein mobility while highlighting several factors that can complicate the interpretation of lateral diffusion data.
Cells are organized on length scales from Angstroms to microns. However, the mechanisms by which ... more Cells are organized on length scales from Angstroms to microns. However, the mechanisms by which Angstrom-scale molecular properties are translated to micron-scale macroscopic properties are not well understood. We have shown that interactions between multivalent proteins and multivalent ligands can cause liquid-liquid demixing phase transitions, resulting in formation of micron-sized liquid droplets in aqueous solution and micron-sized puncta on membranes. These transitions appear to occur concomitantly with sol-gel transitions to form large, dynamic polymers within the droplets/puncta. I will discuss how such transitions may control the spatial organization and biochemical activity of actin regulatory signaling pathways, and contribute to formation of PML nuclear bodies in the mammalian nucleus. Our data suggest a general mechanism by which cells may achieve micron-scale organization based on interactions between multivalent macromolecules.
While junctions between cortical endoplasmic reticulum (cER) and the plasma membrane (PM) are a s... more While junctions between cortical endoplasmic reticulum (cER) and the plasma membrane (PM) are a subtle but ubiquitous feature in mammalian cells very little is known about the functions and molecular interactions associated with neuronal ER/PM junctions. Here we report that Kv2.1, the primary delayed-rectifier K(+) channel in the mammalian brain, induces the formation of ER/PM junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, suggesting a function unrelated to membrane potential regulation. Accordingly, Kv2.1 clusters function as membrane trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER/PM junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 cluster...
Proceedings of the National Academy of Sciences, 2011
Diffusion in the plasma membrane of living cells is often found to display anomalous dynamics. Ho... more 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 implic...
Proceedings of the National Academy of Sciences, 2013
Significance Clathrin-mediated endocytosis is the primary pathway of cargo internalization in mam... more 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.
We describe a method for the analysis of the distribution of displacements, i.e., the propagators... more 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 obstacleinduced 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.
Voltage-gated K+(Kv) channels regulate membrane potential in many cell types. Although the channe... more 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 surf...
In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns... more 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 mamma... more 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.
study is to estimate the contribution of the nNavs in the late sodium current (I NaL) in ischemic... more study is to estimate the contribution of the nNavs in the late sodium current (I NaL) in ischemic condition in order to determine if an overexpression of the nNa v s can modify the APD and create an AS. Methods: Freshly isolated cardiomyocytes were placed under ischemic conditions for 45 minutes. I NaL currents were recorded with the patch clamp technique in whole cell configuration. Tetrodotoxin (a specific nNa v s blocker) and MTSEA (a specific Na v 1.5 blocker) were used to differentiate the two sodium channels isoforms. Results: In normal condition, nNa v s account for 11% of peak current. I NaL represents 0.3% of the peak current at a potential of À10 mV. Contribution of nNa v s (TTX sensitive) to I NaL was 36% 5 5%. Ischemia decreases the maximal current density from À73.3 nA/pF to À53.4 nA/pF. Surprisingly, nNa v s contribution was not modified (10% of the peak current). However, ischemia increases I NaL from 0.3% to 1.6% compared to the peak current. Conclusion: Ischemia increases by 5.3 times I NaL that can play a critical role in the duration of the action potential and facilitates the outcome of arrhythmias.
provide the first identification and characterization of the biophysical properties of the most p... more provide the first identification and characterization of the biophysical properties of the most prominently observed ion channel expressed within the inner and outer membrane of nuclei from adult skeletal muscle fibers. Excised insideout single channel recordings were obtained from individual nuclei acutely isolated from Flexor Digitorum Brevis (FDB) fibers of wild-type mice. The outer membrane of nuclei was readily accessible following isolation. For measurements of channels from the inner membrane, nuclei were treated with 1 % (w/v) sodium citrate in order to remove the outer membrane. We found that the predominant ion channel expressed in both the inner and outer nuclear membrane was a cationic channel that conducts monovalent ions with slight preference for potassium over sodium ions (a PK/PNa~1.22). A 10,000-fold difference in the concentration of free Ca2þ between the pipette and bath solutions did not affect the channel reversal potential in symmetric KCl (~0 mV), indicating that Ca2þ ions permeation is negligible. The maximum conductance of the channel in the outward direction was~162 pS. The mean open probability (PO) was~0.7 and voltage-independent between À50 mV to þ50 mV. We suggest that this novel monovalent cationic channel within the inner and outer membrane of skeletal muscle nuclei provides a countercurrent mechanism that minimizes voltage change across the nuclear membrane. This research was supported by NIH K01 award AR060831(to V.Y.) and NIH R01 grant AR44657 (to R.T.D).
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Papers by Michael Tamkun