Biology subjects

Parker, I.

Publications and source records attributed to Parker, I..

6 recordsLinked to original sources

bioRxiv2018

CellSpecks: A Software for Automated Detection and Analysis of Calcium Channels in Live Cells

To couple the fidelity of patch-clamp recording with a more high-throughput screening capability, we pioneered a novel approach to single channel recording that we named \"optical patch clamp\". By using highly-sensitive fluorescent Ca2+ indicator dyes in conjunction with total internal fluorescence microscopy techniques, we monitor Ca2+ flux through individual Ca2+-permeable channels. This approach provides information about channel gating analogous to patch-clamp recording at time resolution of ~ 2 ms, with the additional advantage of being massively parallel, providing simultaneous and independent recording from thousands of channels in native environment. However, manual analysis of the data generated by this technique presents severe challenges as a video recording can include many thousands of frames. To overcome this bottleneck, we developed an image processing and analysis framework called CellSpecks, capable of detecting and fully analyzing the kinetics of ion channels within a video sequence. By using a randomly generated synthetic data, we tested the ability of CellSpecks to rapidly and efficiently detect and analyze the activity of thousands of ion channels, including openings for a few milliseconds. Here, we report the use of CellSpecks for the analysis of experimental data acquired by imaging muscle nicotinic acetylcholine receptors and the Alzheimers disease-associated amyloid beta pores with multiconductance levels in the plasma membrane of Xenopus laevis oocytes. We show that CellSpecks can accurately and efficiently generate location maps, create raw and processed fluorescence time-traces, histograms of mean open times, mean close times, open probabilities, durations, and maximum amplitudes, and a channel chip showing the activity of all channels as a function of time. Although we specifically illustrate the application of CellSpecks for analyzing data from Ca2+ channels, it can be easily customized to analyze other spatially and temporally localized signals.

bioinformatics↗

bioRxiv2018

TraceSpecks: a software for automated idealization of noisy patch-clamp and imaging data

Experimental records of single molecules or ion channels from fluorescence microscopy and patch-clamp electrophysiology often include high-frequency noise and baseline fluctuations that are not generated by the system under investigation and have to be removed. More-over, multiple channels or conductance levels can be present at a time in the data that need to be quantified to accurately understand the behavior of the system. Manual procedures for removing these fluctuations and extracting conducting states or multiple channels are laborious, prone to subjective bias, and hinder the processing of often very large data-sets. We introduce a maximum likelihood formalism for separating signal from a noisy and drifting background such as fluorescence traces from imaging of elementary Ca2+ release events called puffs arising from clusters of channels and patch-clamp recordings of ion channels. Parameters such as the number of open channels or conducting states, noise level, and back-ground signal can all be optimized using the expectation-maximization (EM) algorithm. We implement our algorithm following the Baum-Welch approach to EM in the portable java language with a user-friendly graphical interface and test the algorithm on both synthetic and experimental data from patch-clamp electrophysiology of Ca2+ channels and fluorescence microscopy of a cluster of Ca2+ channels and Ca2+ channels with multiple conductance levels. The resulting software is accurate, fast, and provides detailed information usually not available through manual analysis. Options for visual inspection of the raw and processed data with key parameters, and exporting a range of statistics such as the mean open probabilities, mean open times, mean close times, and dwell time distributions for different number of channels open or conductance levels, amplitude distribution of all opening events, and number of transitions between different number of open channels or conducting levels in asci format with a single click are provided.

bioinformatics↗

bioRxiv2018

Piezo1 calcium flickers localize to hotspots of cellular traction forces

Piezo channels transduce mechanical stimuli into electrical and chemical signals, and in doing so, powerfully influence development, tissue homeostasis, and regeneration. While much is known about how Piezo1 responds to external forces, its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that actomyosin-based cellular traction forces generate spatially-restricted Ca2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched in regions proximal to force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca2+ flickers allow spatial segregation of mechanotransduction events, and that diffusion allows channel molecules to efficiently respond to transient, local mechanical stimuli.

biophysics↗

bioRxiv2017

Intermittent Ca2+ signals mediated by Orai1 regulate basal T cell motility

Ca2+ influx through Orai1 channels is crucial for several T cell functions, but a role in regulating basal cellular motility has not been described. Here we show that inhibition of Orai1 channel activity increases average cell velocities by reducing the frequency of pauses in human T cells migrating through confined spaces, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca2+ indicator, Salsa6f, which permits real-time monitoring of cytosolic Ca2+ along with cell motility, we show that spontaneous pauses during T cell motility in vitro and in vivo coincide with episodes of cytosolic Ca2+ signaling. Furthermore, lymph node T cells exhibited two types of spontaneous Ca2+ transients: short-duration \"sparkles\" and longer duration global signals. Our results demonstrate that spontaneous and self-peptide MHC-dependent activation of Orai1 ensures random walk behavior in T cells to optimize immune surveillance.

immunology↗

bioRxiv2017

T Cell Calcium Dynamics Visualized in a Ratiometric tdTomato-GCaMP6f Transgenic Reporter Mouse

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here we describe development and characterization of \"Salsa6f\", a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca2+, and a transgenic Ca2+ reporter mouse with Salsa6f floxed and targeted to the Rosa26 locus for expression in specific cell types. Using CD4-Cre-Salsa6f mice, we report normal development and function of T cells expressing Salsa6f and demonstrate Ca2+ signaling dynamics during T cell receptor engagement in naive T cells, helper Th17 T cells and regulatory T cells. Salsa6f expression also revealed functional expression of mechanosensitive Piezo1 channels in T cells. Transgenic expression of Salsa6f enables ratiometric imaging of Ca2+ signals in complex tissue environments found in vivo. Deep tissue two-photon imaging of T cells in the steady-state lymph node revealed a highly localized Ca2+ signaling behavior (\"sparkles\") as cells migrate.

cell biology↗

bioRxiv2017

Cell-intrinsic activation of Orai1 regulates human T cell motility

Ca2+ signaling through the store-operated Ca2+ channel, Orai1, is crucial for T cell function, but a role in regulating T cell motility in lymph nodes has not been previously reported. Tracking human T cells in immunodeficient mouse lymph nodes and in microfabricated PDMS channels, we show that inhibition of Orai1 channel activity with a dominant-negative Orai1-E106A construct increases average T cell velocities by reducing the frequency of pauses in motile T cells. Orai1-dependent motility arrest occurs spontaneously during confined motility in vitro, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca2+ indicator, Salsa6f, we show these spontaneous pauses during T cell motility in vitro coincide with episodes of spontaneous cytosolic Ca2+ signaling. Our results demonstrate that Orai1, activated in a cell-intrinsic manner, regulates T cell motility patterns that accompany immune surveillance.

cell biology↗