Visualizing bleb mass dynamics in single cells using quantitative phase microscopy.

Understanding biological responses to directed energy (DE) is critical to ensure the safety of personnel within the Department of Defense. At the Air Force Research Laboratory, we have developed or adapted advanced optical imaging systems that quantify biophysical responses to DE. One notable cellular response to DE exposure is the formation of blebs, or semi-spherical protrusions of the plasma membrane in living cells. In this work, we demonstrate the capacity of quantitative phase imaging (QPI) to both visualize and quantify the formation of membrane blebs following DE exposure. QPI is an interferometric imaging tool that uses optical path length as a label-free contrast mechanism and is sensitive to the non-aqueous mass density, or dry mass, of living cells. Blebs from both CHO-K1 and U937 cells were generated after exposure to a series of 600 ns, 21.2 kV/cm electric pulses. These blebs were visualized in real time, and their dry mass relative to the rest of the cell body was quantified as a function of time. It is our hope that this system will lead to an improved understanding of both DE-induced and apoptotic blebbing.

Visualization of Dynamic Sub-microsecond Changes in Membrane Potential.

Direct observation of rapid membrane potential changes is critical to understand how complex neurological systems function. This knowledge is especially important when stimulation is achieved through an external stimulus meant to mimic a naturally occurring process. To enable exploration of this dynamic space, we developed an all-optical method for observing rapid changes in membrane potential at temporal resolutions of ∼25 ns. By applying a single 600-ns electric pulse, we observed sub-microsecond, continuous membrane charging and discharging dynamics. Close agreement between the acquired results and an analytical membrane-charging model validates the utility of this technique. This tool will deepen our understanding of the role of membrane potential dynamics in the regulation of many biological and chemical processes within living systems.

Filament displacement image analytics tool for use in investigating dynamics of dense microtubule networks.

The fate and motion of cells is influenced by a variety of physical characteristics of their microenvironments. Traditionally, mechanobiology focuses on external mechanical phenomena such as cell movement and environmental sensing. However, cells are inherently dynamic, where internal waves and internal oscillations are a hallmark of living cells observed under a microscope. We propose that these internal mechanical rhythms provide valuable information about cell health. Therefore, it is valuable to capture the rhythms inside cells and quantify how drugs or physical interventions affect a cell's internal dynamics. One of the key dynamical entities inside cells is the microtubule network. Typically, microtubule dynamics are measured by end-protein tracking. In contrast, this paper introduces an easy-to-implement approach to measure the lateral motion of the microtubule filaments embedded within dense networks with (at least) confocal resolution image sequences. Our tool couples the computer vision algorithm Optical Flow with an anisotropic, rotating Laplacian of Gaussian filtering to characterize the lateral motion of dense microtubule networks. We then showcase additional image analytics used to understand the effect of microtubule orientation and regional location on lateral motion. We argue that our tool and these additional metrics provide a fuller picture of the active forcing environment within cells.

Comparing the segmentation of quantitative phase images of neurons using convolutional neural networks trained on simulated and augmented imagery.

Significance: Quantitative phase imaging (QPI) can visualize cellular morphology and measure dry mass. Automated segmentation of QPI imagery is desirable for tracking neuron growth. Convolutional neural networks (CNNs) have provided state-of-the-art results for image segmentation. Improving the amount and robustness of training data is often crucial to improving CNN output on novel samples, but acquiring enough labeled data can be labor intensive. Data augmentation and simulation can be used to address this, but it is unclear whether low-complexity data can result in useful network generalization. Aim: We trained CNNs on abstract images of neurons and on augmented images of real neurons. We then benchmarked the resulting models against human labeling. Approach: We used a stochastic simulation of neuron growth to guide abstract QPI image and label generation. We then tested the segmentation performance of networks trained on augmented data and networks trained on simulated data against manual labeling established via consensus of three human labelers. Results: We show that training on augmented real data resulted in a model that achieved the best Dice coefficients in our group of CNNs. The largest percent difference in dry mass estimation with respect to the ground truth was driven by segmentation errors of cell debris and phase noise. The error in dry mass when considering the cell body alone was similar between the CNNs. Neurite pixels only accounted for ∼6% of the total image space, making them a difficult feature to learn. Future efforts should consider methods for improving neurite segmentation quality. Conclusions: Augmented data outperformed the simulated abstract data for this testing set. The quality of segmentation of neurites was the key difference in performance between the models. Notably, even humans performed poorly when segmenting neurites. Further work is needed to improve the segmentation quality of neurites.

Rapid and precise tracking of water influx and efflux across cell membranes induced by a pulsed electric field.

Quantitative measurements of water content within a single cell are notoriously difficult. In this work, we introduce a single-shot optical method for tracking the intracellular water content, by mass and volume, of a single cell at video rate. We utilize quantitative phase imaging and a priori knowledge of a spherical cellular geometry, leveraging a two-component mixture model to compute the intracellular water content. We apply this technique to study CHO-K1 cells responding to a pulsed electric field, which induces membrane permeabilization and rapid water influx or efflux depending upon the osmotic environment. The effects of mercury and gadolinium on water uptake in Jurkat cells following electropermeabilization are also examined.

Comprehensive single-shot biophysical cytometry using simultaneous quantitative phase imaging and Brillouin spectroscopy.

Single-cell analysis, or cytometry, is a ubiquitous tool in the biomedical sciences. Whereas most cytometers use fluorescent probes to ascertain the presence or absence of targeted molecules, biophysical parameters such as the cell density, refractive index, and viscosity are difficult to obtain. In this work, we combine two complementary techniques-quantitative phase imaging and Brillouin spectroscopy-into a label-free image cytometry platform capable of measuring more than a dozen biophysical properties of individual cells simultaneously. Using a geometric simplification linked to freshly plated cells, we can acquire the cellular diameter, volume, refractive index, mass density, non-aqueous mass, fluid volume, dry volume, the fractional water content of cells, both by mass and by volume, the Brillouin shift, Brillouin linewidth, longitudinal modulus, longitudinal viscosity, the loss modulus, and the loss tangent, all from a single acquisition, and with no assumptions of underlying parameters. Our methods are validated across three cell populations, including a control population of CHO-K1 cells, cells exposed to tubulin-disrupting nocodazole, and cells under hypoosmotic shock. Our system will unlock new avenues of research in biophysics, cell biology, and medicine.

Intrinsic properties of primary hippocampal neurons contribute to PIP2 depletion during nsEP-induced physiological response.

High-energy, short-duration electric pulses (EPs) are known to be effective in neuromodulation, but the biological mechanisms underlying this effect remain unclear. Recently, we discovered that nanosecond electric pulses (nsEPs) could initiate the phosphatidylinositol4,5-bisphosphate (PIP2) depletion in non-excitable cells identical to agonist-induced activation of the Gq11 coupled receptors. PIP2 is the precursor for multiple intracellular second messengers critically involved in the regulation of intracellular Ca2+ homeostasis and plasma membrane (PM) ion channels responsible for the control of neuronal excitability. In this paper we demonstrate a novel finding that five day in vitro (DIV5) primary hippocampal neurons (PHNs) undergo significantly higher PIP2 depletion after 7.5 kV/cm 600 ns EP exposure than DIV1 PHNs and day 1-5 (D1-D5) non-excitable Chinese hamster ovarian cells with muscarinic receptor 1 (CHO-hM1). Despite the age of development, the stronger 15 kV/cm 600 ns or longer 7.5 kV/cm 12 µs EP initiated profound PIP2 depletion in all cells studied, outlining damage of the cellular PM and electroporation. Therefore, the intrinsic properties of PHNs in concert with nanoporation explain the stronger neuronal response to nsEP at lower intensity exposures. PIP2 reduction in neurons could be a primary biological mechanism responsible for the stimulation or inhibition of neuronal tissues.

Structural rearrangements in loop F of the GABA receptor signal ligand binding, not channel activation.

Structure-function studies of the Cys loop family of ionotropic neurotransmitter receptors (GABA, nACh, 5-HT(3), and glycine receptors) have resulted in a six-loop (A-F) model of the agonist-binding site. Key amino acids have been identified in these loops that associate with, and stabilize, bound ligand. The next step is to identify the structural rearrangements that couple agonist binding to channel opening. Loop F has been proposed to move upon receptor activation, although it is not known whether this movement is along the conformational pathway for channel opening. We test this hypothesis in the GABA receptor using simultaneous electrophysiology and site-directed fluorescence spectroscopy. The latter method reveals structural rearrangements by reporting changes in hydrophobicity around an environmentally sensitive fluorophore attached to defined positions of loop F. Using a series of ligands that span the range from full activation to full antagonism, we show there is no correlation between the rearrangements in loop F and channel opening. Based on these data and agonist docking simulations into a structural model of the GABA binding site, we propose that loop F is not along the pathway for channel opening, but rather is a component of the structural machinery that locks ligand into the agonist-binding site.

Stoichiometric pore mutations of the GABAAR reveal a pattern of hydrogen bonding with picrotoxin.

Picrotoxin (PTX) is a noncompetitive antagonist of many ligand-gated ion channels, with a site of action believed to be within the ion-conducting pore. In the A-type gamma-aminobutyric acid receptor, a threonine residue in the second transmembrane domain is of particular importance for the binding of, and ultimate inhibition by, PTX. To better understand the relationship between this residue and the PTX molecule, we mutated this threonine residue to serine, valine, and tyrosine to change the structural and biochemical characteristics at this location. The known subunit stoichiometry of the A-type gamma-aminobutyric acid receptor allowed us to create receptors with anywhere from zero to five mutations. With an increasing number of mutated subunits, each amino acid substitution revealed a unique pattern of changes in PTX sensitivity, ultimately encompassing sensitivity shifts over several orders of magnitude. The electrophysiological data on PTX-mediated block, and supporting modeling and docking studies, provide evidence that an interaction between the PTX molecule and three adjacent uncharged polar amino acids at this position of the pore are crucial for PTX-mediated inhibition.

Phosphorylation of the recombinant rho1 GABA receptor.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. While a growing body of literature indicates that postsynaptic GABA receptors are regulated by phosphorylation, there is discrepancy as to the specific effects of phosphorylation on GABA receptor function. Here, we have identified phosphorylation sites on the human rho1 GABA receptor for six protein kinases widely expressed in the brain: protein kinase C (PKC); cAMP-dependent protein kinase (PKA); calmodulin-dependent kinase (CaMKII); casein kinase (CKII); mitogen-activated protein kinase (MAPK); and cGMP-dependent protein kinase (PKG). We demonstrate that in nearly all cases, the consensus sites and actual phosphorylation sites do not agree supporting the risk of relying on a sequence analysis to identify potential phosphorylation sites. In addition, of the six kinases examined, only CKII phosphorylated the human rho2 subunit. Site-directed mutagenesis of the phosphorylation sites, or activation/inhibition of select kinase pathways, did not alter the receptor sensitivity or maximal GABA-activated current of the rho1 GABA receptor expressed in Xenopus laevis oocytes suggesting phosphorylation of rho1 does not directly alter receptor properties. This study is a first and necessary step towards elucidating the regulation of rho1 GABA receptors by phosphorylation.

Function and modulation of delta-containing GABA(A) receptors.

alphabetadelta-Containing GABA(A) receptors are (1) localized to extra- and perisynaptic membranes, (2) exhibit a high sensitivity to GABA, (3) show little desensitization, and (4) are believed to be one of the primary mediators of tonic inhibition in the central nervous system. This type of signaling appears to play a key role in controlling cell excitability. This review article briefly summarizes recent knowledge on tonic GABA-mediated inhibition. We will also consider the mechanism of action of many clinically important drugs such as anxiolytics, anticonvulsants, and sedative/hypnotics and their effects on delta-containing GABA receptor activation. We will conclude that alphabetadelta-containing GABA(A) receptors exhibit a relatively low efficacy that can be potentiated by endogenous modulators and anxiolytic agents. This scenario enables these particular GABA receptor combinations, upon neurosteroid exposure for example, to impart a profound effect on excitability in the central nervous system.

Stoichiometry of a pore mutation that abolishes picrotoxin-mediated antagonism of the GABAA receptor.

Picrotoxin, a potent antagonist of the inhibitory central nervous system GABAA and glycine receptors, is believed to interact with residues that line the central ion pore. These pore-lining residues are in the second transmembrane domain (TM2) of each of the five constituent subunits. One of these amino acids, a threonine at the 6' location, when mutated to phenylalanine, abolishes picrotoxin sensitivity. It has been suggested that this threonine, via hydrogen bonding, directly interacts with the picrotoxin molecule. We previously demonstrated that this mutation, in the alpha, beta or gamma subunit, can impart picrotoxin resistance to the GABA receptor. Since the functional pentameric GABA receptor contains two alpha subunits, two beta subunits and one gamma subunit, it is not clear how many alpha and beta subunits must carry this mutation to impart the resistant phenotype. In this study, by coexpression of mutant alpha or beta subunits with their wild-type counterparts in various defined ratios, we demonstrate that any single subunit carrying the 6' mutation imparts picrotoxin resistance. Implications of this finding in terms of the mechanism of antagonism are considered.

Mapping the rho1 GABA(C) receptor agonist binding pocket. Constructing a complete model.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The GABA receptor type C (GABA(C)) is a ligand-gated ion channel with pharmacological properties distinct from the GABA(A) receptor. To date, only three binding domains in the recombinant rho1 GABA(C) receptor have been recognized among six potential regions. In this report, using the substituted cysteine accessibility method, we scanned three potential regions previously unexplored in the rho1 GABA(C) receptor, corresponding to the binding loops A, E, and F in the structural model for ligand-gated ion channels. The cysteine accessibility scanning and agonist/antagonist protection tests have resulted in the identification of residues in loops A and E, but not F, involved in forming the GABA(C) receptor agonist binding pocket. Three of these newly identified residues are in a novel region corresponding to the extended stretch of loop E. In addition, the cysteine accessibility pattern suggests that part of loop A and part of loop E have a beta-strand structure, whereas loop F is a random coil. Finally, when all of the identified ligand binding residues are mapped onto a three-dimensional homology model of the amino-terminal domain of the rho1 GABA(C) receptor, they are facing toward the putative binding pocket. Combined with previous findings, a complete model of the GABA(C) receptor binding pocket was proposed and discussed in comparison with the GABA(A) receptor binding pocket.