Diego Krapf

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.

Formation of nanopores in a SiN∕SiO2 membrane with an electron beam

An electron beam can drill nanopores in SiO2 or silicon nitride membranes and shrink a pore to a smaller diameter. Such nanopores are promising for single molecule detection. The pore formation in a 40?nm thick silicon nitride?SiO2 bilayer using an electron beam with a diameter of 8?nm (full width of half height) was investigated by electron energy loss spectroscopy with silicon nitride facing toward and away from the source. The O loss shows almost linear—independent of which layer faces the source, while N loss is quite complicated. After the formation of a pore, the membrane presents a wedge shape over a 70?nm radius around the nanopore.

Control of shape and material composition of solid-state nanopores.

Solid-state nanopores fabricated by a high-intensity electron beam in ceramic membranes can be fine-tuned on three-dimensional geometry and composition by choice of materials and beam sculpting conditions. For similar beam conditions, 8 nm diameter nanopores fabricated in membranes containing SiO(2) show large depletion areas (70 nm in radius) with small sidewall angles (55 degrees ), whereas those made in SiN membranes show small depletion areas (40 nm) with larger sidewall angles (75 degrees ). Three-dimensional electron tomograms of nanopores fabricated in a SiO(2)/SiN/SiO(2) membrane show a biconical shape with symmetric top and bottom and indicate a mixing of SiN and SiO(2) layers up to 30 nm from the edge of nanopore, with Si-rich particles throughout the membrane. Electron-energy-loss spectroscopy (EELS) reveals that the oxygen/nitrogen ratio near the pore depends on the beam sculpting conditions.

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.

Induction of stable ER-plasma-membrane junctions by Kv2.1 potassium channels.

ABSTRACT Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER–plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K+ channel in the mammalian brain, induces the formation of ER–plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is 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–plasma-membrane junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 clusters in neurons and subsequent retraction of the cER from the plasma membrane. We propose Kv2.1-induced ER–plasma-membrane junctions represent a new macromolecular plasma-membrane complex that is sensitive to excitotoxic insult and functions as a scaffolding site for both membrane trafficking and Ca2+ signaling.

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.

Mechanisms underlying anomalous diffusion in the plasma membrane.

The plasma membrane is a complex fluid where lipids and proteins undergo diffusive motion critical to biochemical reactions. Through quantitative imaging analyses such as single-particle tracking, it is observed that diffusion in the cell membrane is usually anomalous in the sense that the mean squared displacement is not linear with time. This chapter describes the different models that are employed to describe anomalous diffusion, paying special attention to the experimental evidence that supports these models in the plasma membrane. We review models based on anticorrelated displacements, such as fractional Brownian motion and obstructed diffusion, and nonstationary models such as continuous time random walks. We also emphasize evidence for the formation of distinct compartments that transiently form on the cell surface. Finally, we overview heterogeneous diffusion processes in the plasma membrane, which have recently attracted considerable interest.

The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm.

Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2−/− mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro. Highlighted article: The increase in tyrosine phosphorylation seen during sperm capacitation is mediated by the FER kinase, but this does not seem to be essential for fertilisation in vivo in mice.

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.

Infrared multispectral detection using Si/SixGe1−x quantum well infrared photodetectors

A modified p-type Si/SiGe quantum well infrared photodetector for multispectral infrared imaging applications is demonstrated. In order to improve the detector’s performances we have used a SiGe emitter and a low-temperature wet passivation process that give rise to a reduced dark current, even at relatively high bias voltages. Multispectral photoresponse at the long, mid and short wavelength infrared atmospheric windows was observed. The response peaks are assigned to the various classes of intervalence band transitions in the quantum wells and in the SiGe emitter layers.