Live-cell Approaches Research Articles

Endosomal Transport to Lysosomes and the Trans-Golgi Network in Neurons and Other Cells: Visualizing Maturational Flux.

High-level microscopy enables the comprehensive study of dynamic intracellular processes. Here we describe a toolkit of combinatorial approaches for fixed cell imaging and live cell imaging to investigate the interactions along the trans-Golgi network (TGN)-endosome-lysosome transport axis, which underlie the maturation of endosomal compartments and degradative flux. For fixed cell approaches, we specifically highlight how choices of permeabilization conditions, antibody selection, and antibody multiplexing affect interpretation of results. For live cell approaches, we emphasize the use of sensors that read out pH and degradative capacity in combination with endosomal identity for elucidating dynamic compartment changes.

MTORC1 and mTORC2 Complexes Regulate the Untargeted Metabolomics and Amino Acid Metabolites Profile through Mitochondrial Bioenergetic Functions in Pancreatic Beta Cells.

Background: Pancreatic beta cells regulate bioenergetics efficiency and secret insulin in response to glucose and nutrient availability. The mechanistic Target of Rapamycin (mTOR) network orchestrates pancreatic progenitor cell growth and metabolism by nucleating two complexes, mTORC1 and mTORC2. Objective: To determine the impact of mTORC1/mTORC2 inhibition on amino acid metabolism in mouse pancreatic beta cells (Beta-TC-6 cells, ATCC-CRL-11506) using high-resolution metabolomics (HRM) and live-mitochondrial functions. Methods: Pancreatic beta TC-6 cells were incubated for 24 h with either: RapaLink-1 (RL); Torin-2 (T); rapamycin (R); metformin (M); a combination of RapaLink-1 and metformin (RLM); Torin-2 and metformin (TM); compared to the control. We applied high-resolution mass spectrometry (HRMS) LC-MS/MS untargeted metabolomics to compare the twenty natural amino acid profiles to the control. In addition, we quantified the bioenergetics dynamics and cellular metabolism by live-cell imaging and the MitoStress Test XF24 (Agilent, Seahorse). The real-time, live-cell approach simultaneously measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine cellular respiration and metabolism. Statistical significance was assessed using ANOVA on Ranks and post-hoc Welch t-Tests. Results: RapaLink-1, Torin-2, and rapamycin decreased L-aspartate levels compared to the control (p = 0.006). Metformin alone did not affect L-aspartate levels. However, L-asparagine levels decreased with all treatment groups compared to the control (p = 0.03). On the contrary, L-glutamate and glycine levels were reduced only by mTORC1/mTORC2 inhibitors RapaLink-1 and Torin-2, but not by rapamycin or metformin. The metabolic activity network model predicted that L-aspartate and AMP interact within the same activity network. Live-cell bioenergetics revealed that ATP production was significantly reduced in RapaLink-1 (122.23 ± 33.19), Torin-2 (72.37 ± 17.33) treated cells, compared to rapamycin (250.45 ± 9.41) and the vehicle control (274.23 ± 38.17), p < 0.01. However, non-mitochondrial oxygen consumption was not statistically different between RapaLink-1 (67.17 ± 3.52), Torin-2 (55.93 ± 8.76), or rapamycin (80.01 ± 4.36, p = 0.006). Conclusions: Dual mTORC1/mTORC2 inhibition by RapaLink-1 and Torin-2 differentially altered the amino acid profile and decreased mitochondrial respiration compared to rapamycin treatment which only blocks the FRB domain on mTOR. Third-generation mTOR inhibitors may alter the mitochondrial dynamics and reveal a bioenergetics profile that could be targeted to reduce mitochondrial stress.

Live-cell fluorescence imaging of microgametogenesis in the human malaria parasite Plasmodium falciparum.

Formation of gametes in the malaria parasite occurs in the midgut of the mosquito and is critical to onward parasite transmission. Transformation of the male gametocyte into microgametes, called microgametogenesis, is an explosive cellular event and one of the fastest eukaryotic DNA replication events known. The transformation of one microgametocyte into eight flagellated microgametes requires reorganisation of the parasite cytoskeleton, replication of the 22.9 Mb genome, axoneme formation and host erythrocyte egress, all of which occur simultaneously in <20 minutes. Whilst high-resolution imaging has been a powerful tool for defining stages of microgametogenesis, it has largely been limited to fixed parasite samples, given the speed of the process and parasite photosensitivity. Here, we have developed a live-cell fluorescence imaging workflow that captures the entirety of microgametogenesis. Using the most virulent human malaria parasite, Plasmodium falciparum, our live-cell approach captured early microgametogenesis with three-dimensional imaging through time (4D imaging) and microgamete release with two-dimensional (2D) fluorescence microscopy. To minimise the phototoxic impact to parasites, acquisition was alternated between 4D fluorescence, brightfield and 2D fluorescence microscopy. Combining live-cell dyes specific for DNA, tubulin and the host erythrocyte membrane, 4D and 2D imaging together enables definition of the positioning of newly replicated and segregated DNA. This combined approach also shows the microtubular cytoskeleton, location of newly formed basal bodies, elongation of axonemes and morphological changes to the erythrocyte membrane, the latter including potential echinocytosis of the erythrocyte membrane prior to microgamete egress. Extending the utility of this approach, the phenotypic effects of known transmission-blocking inhibitors on microgametogenesis were confirmed. Additionally, the effects of bortezomib, an untested proteasomal inhibitor, revealed a clear block of DNA replication, full axoneme nucleation and elongation. Thus, as well as defining a framework for broadly investigating microgametogenesis, these data demonstrate the utility of using live imaging to validate potential targets for transmission-blocking antimalarial drug development.

Live-Cell Synchrotron-Based FTIR Evaluation of Metabolic Compounds in Brain Glioblastoma Cell Lines after Riluzole Treatment.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor, characterized by short median survival and an almost 100% tumor-related mortality. The standard of care treatment for newly diagnosed GBM includes surgical resection followed by concomitant radiochemotherapy. The prevention of disease progression fails due to the poor therapeutic effect caused by the great molecular heterogeneity of this tumor. Previously, we exploited synchrotron radiation-based soft X-ray tomography and hard X-ray fluorescence for elemental microimaging of the shock-frozen GBM cells. The present study focuses instead on the biochemical profiling of live GBM cells and provides new insight into tumor heterogenicity. We studied bio-macromolecular changes by exploring the live-cell synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy in a set of three GBM cell lines, including the patient-derived glioblastoma cell line, before and after riluzole treatment, a medicament with potential anticancer properties. SR-FTIR microspectroscopy shows that GBM live cells of different origins recruit different organic compounds. The riluzole treatment of all GBM cell lines mainly affected carbohydrate metabolism and the DNA structure. Lipid structures and protein secondary conformation are affected as well by the riluzole treatment: cellular proteins assumed cross β-sheet conformation while parallel β-sheet conformation was less represented for all GBM cells. Moreover, we hope that a new live-cell approach for GBM simultaneous treatment and examination can be devised to target cancer cells more specifically, i.e., future therapies can develop more specific treatments according to the specific bio-macromolecular signature of each tumor type.

Mitochondrial Bioenergetics Profile With Different Mechanistic Target of Rapamycin Complexes (mTORC1/mTORC2) Inhibitors in Pancreatic Beta-Cell Lines (Beta-TC-6)

Abstract Objectives We investigated the impact of mTOR complexes inhibition with RapaLink-1, a third-generation bivalent mTORC1/mTORC2 inhibitor that links rapamycin with a mTOR-kinase inhibitor, or Torin-2 (ATP-competitive) on mitochondrial dynamics, bioenergetics, and extracellular flux. Methods We used insulin-secreting, glucose-responsive pancreatic beta cells derived from transgenic mice expressing SV40 (Beta-TC-6 cells, ATCC-11506). Cells were treated for 24 hours with either: RapaLink; Torin-2; rapamycin; metformin; a combination of metformin and RapaLink, or Torin-2; compared to vehicle control. Bioenergetic dynamics and cellular metabolism were quantified using MitoStress Test XF24 (Agilent, Seahorse). The real-time, live-cell approach simultaneously measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine cellular respiration and metabolism. Statistical significance was assessed by ANOVA followed by an unpaired t-test. Results Rapalink-1 and Torin-2 incubation decreased basal OCR compared to rapamycin alone or vehicle control (189.58 ± 48.58, 116.85 ± 26.55 versus 405.01 ± 21.57, 444.19 ± 58.59, p &amp;lt; 0.01) in Beta TC-6 pancreatic cell lines. ATP production was also significantly reduced in RapaLink-1 (122.23 ± 33.19), Torin-2 (72.37 ± 17.33) treated cells, compared to rapamycin (250.45 ± 9.41) and vehicle control (274.23 ± 38.17), p &amp;lt; 0.01. Similarly, proton leak decreased in RapaLink-1 (67.34 ± 15.40) and Torin-2 (44.48 ± 9.23) treated beta cells, compared to rapamycin (154.56 ± 12.53) or control (169.96 ± 20.52), p &amp;lt; 0.01. However, non-mitochondrial oxygen consumption was not statistically different between RapaLink (67.17 ± 3.52) Torin-2 (55.93 ± 8.76) or rapamycin (80.01 ± 4.36), but was less than the control group (108.80 ± 7.19), p = 0.006. Conclusions The combination of mTORC1 and mTORC2 inhibition by RapaLink-1 or Torin-2 decreased mitochondrial respiration compared to rapamycin treatment. Decreased OCR suggests reduced initial energy requirements, while decreased ATP production indicates decreased energy demands. Third-generation mTOR inhibitors may alter the mitochondrial dynamics and reveal a mitochondrial bioenergetics profile that could be targeted to reduce mitochondrial stress. Funding Sources City University of New York, GC Advanced Science Research Center Seed Grant Award

A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding

SummarySecreted chemokines are critical mediators of cellular communication that elicit intracellular signaling by binding membrane-bound receptors. Here we demonstrate the development and use of a sensitive real-time approach to quantify secretion and receptor binding of native chemokines in live cells to better understand their molecular interactions and function. CRISPR/Cas9 genome editing was used to tag the chemokine CXCL12 with the nanoluciferase fragment HiBiT. CXCL12 secretion was subsequently monitored and quantified by luminescence output. Binding of tagged CXCL12 to either chemokine receptors or membrane glycosaminoglycans could be monitored due to the steric constraints of nanoluciferase complementation. Furthermore, binding of native CXCL12-HiBiT to AlexaFluor488-tagged CXCR4 chemokine receptors could also be distinguished from glycosaminoglycan binding and pharmacologically analyzed using BRET. These live cell approaches combine the sensitivity of nanoluciferase with CRISPR/Cas9 genome editing to detect, quantify, and monitor binding of low levels of native secreted proteins in real time.

Treatment of atherosclerosis by macrophage-biomimetic nanoparticles via targeted pharmacotherapy and sequestration of proinflammatory cytokines

Vascular disease remains the leading cause of death and disability, the etiology of which often involves atherosclerosis. The current treatment of atherosclerosis by pharmacotherapy has limited therapeutic efficacy. Here we report a biomimetic drug delivery system derived from macrophage membrane coated ROS-responsive nanoparticles (NPs). The macrophage membrane not only avoids the clearance of NPs from the reticuloendothelial system, but also leads NPs to the inflammatory tissues, where the ROS-responsiveness of NPs enables specific payload release. Moreover, the macrophage membrane sequesters proinflammatory cytokines to suppress local inflammation. The synergistic effects of pharmacotherapy and inflammatory cytokines sequestration from such a biomimetic drug delivery system lead to improved therapeutic efficacy in atherosclerosis. Comparison to macrophage internalized with ROS-responsive NPs, as a live-cell based drug delivery system for treatment of atherosclerosis, suggests that cell membrane coated drug delivery approach is likely more suitable for dealing with an inflammatory disease than the live-cell approach.

Transendothelial Perforations and the Sphere of Influence of Single-Site Sonoporation

Acoustically driven gas bubble cavitation locally concentrates energy and can result in physical phenomena including sonoluminescence and erosion. In biomedicine, ultrasound-driven microbubbles transiently increase plasma membrane permeability (sonoporation) to promote drug/gene delivery. Despite its potential, little is known about cellular response in the aftermath of sonoporation. In the work described here, using a live-cell approach, we assessed the real-time interplay between transendothelial perforations (∼30–60 s) up to 650 µm2, calcium influx, breaching of the local cytoskeleton and sonoporation resealing upon F-actin recruitment to the perforation site (∼5–10 min). Through biophysical modeling, we established the critical role of membrane line tension in perforation resealing velocity (10–30 nm/s). Membrane budding/shedding post-sonoporation was observed on complete perforation closure, yet successful pore repair does not mark the end of sonoporation: protracted cell mobility from 8 µs of ultrasound is observed up to 4 h post-treatment. Taken holistically, we established the biophysical context of endothelial sonoporation repair with application in drug/gene delivery.

Live-cell monitoring of protein localization to membrane rafts using protein-fragment complementation.

The plasma membrane consists of a variety of discrete domains differing from the surrounding membrane in composition and properties. Selective partitioning of protein to these microdomains is essential for membrane functioning and integrity. Studying the nanoscale size and dynamic nature of the membrane microdomains requires advanced imaging approaches with a high spatiotemporal resolution and, consequently, expensive and specialized equipment, unavailable for most researchers and unsuited for large-scale studies. Thus, understanding of protein partitioning to the membrane microdomains in health and disease is still hampered by the lack of inexpensive live-cell approaches with an appropriate spatial resolution. Here, we have developed a novel approach based on Gaussia princeps luciferase protein-fragment complementation assay to quantitively investigate protein partitioning to cholesterol and sphingomyelin-rich domains, sometimes called ‘lipid rafts’, in intact living cells with a high-spatial resolution. In the assay, the reporter construct, carrying one half of the luciferase protein, is targeted to lipid microdomains through the fused acetylation motif from Src-family kinase Fyn. A protein of interest carries the second half of the luciferase protein. Together, this serves as a reversible real-time sensor of raft recruitment for the studied protein. We demonstrated that the assay can efficiently detect the dynamic alterations in raft localization of two disease-associated proteins: Akt and APP. Importantly, this method can be used in high-throughput screenings and other large-scale studies in living cells. This inexpensive, and easy to implement raft localization assay will benefit all researchers interested in protein partitioning in rafts.

Abstract 5266: Kinetic measurements of intracellular ATP levels in co-culture models using live-cell analysis

Abstract Differential metabolic requirements of tumor cells have gained recognition as attractive therapeutic targets. For example, clinical trials are currently underway to test the efficacy of the glutaminase-1 (GLS1) inhibitor CB-839 in a variety of indications, including triple-negative breast cancer (TNBC). Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that lack cell-specific data in complex co-culture models and provide limited kinetic information. Here we describe a live-cell approach to image and analyze intracellular ATP levels of tumor cells in mono- or co-culture over time using the IncuCyte® CytoATP Sensor. A panel of breast cancer cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated and cultured alone or with a monolayer of stromal cells. The effect of CB-839 treatment on tumor cell ATP levels was monitored over days and analyzed using the IncuCyte® S3 for Metabolism, which is equipped with a specialized optical module and image acquisition mode. In concordance with previous results, TNBC cell lines were more sensitive to GLS1 inhibition than their receptor-positive counterparts. Across all TNBC lines tested, CB-839 treatment resulted in a reduction in intracellular ATP that was sustained for the duration of the 3-day time course. In contrast, most receptor-positive cell lines displayed little to no effect of GLS1 inhibition. In some cases, a transient reduction followed by recovery within 48 hours was observed, highlighting the value of kinetic analysis. Co-culture with CCD-1086SK fibroblasts attenuated the effect of CB-839 in TNBC cell lines. This was not due to drug buffering, as the reduction in ATP induced by CB-839 treatment of MCF7 cells was unaffected by co-culture with stromal cells. Visualization tools included in the IncuCyte® ATP Analysis Software Module provide a quick, qualitative assessment of data across the assay plate and a view into the heterogeneity of responses to treatment. Measurements of proliferation, cell death, and mitochondrial membrane potential provided additional data supporting the observations generated via ATP readout. Overall, these data highlight the value of live-cell, kinetic analysis of metabolic and cell health measurements. Citation Format: Cicely L. Schramm, Michael L. Bowe, Laura A. Skerlos, Grigory S. Filonov, Yong X. Chen, Daniel M. Appledorn. Kinetic measurements of intracellular ATP levels in co-culture models using live-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5266.

Reorientation dynamics and structural interdependencies of actin, microtubules and intermediate filaments upon cyclic stretch application.

Any cell within a tissue is constantly confronted with a variety of mechanical stimuli. Sensing of these diverse stimuli plays an important role in cellular regulation. Besides shear stress, cells of the vascular endothelium are particularly exposed to a permanent cyclic straining originating from the interplay of outwards pushing blood pressure and inwards acting contraction by smooth musculature. Perpendicular alignment of cells as structural adaptation to this condition is a basic prerequisite in order to withstand deformation forces. Here, we combine live cell approaches with immunocytochemical analyses on single cell level to closely elucidate the mechanisms of cytoskeletal realignment to cyclic strain and consolidate orientation analyses of actin fibres, microtubules (MTs) and vimentin. We could show that strain-induced reorientation takes place for all cytoskeletal systems. However, all systems are characterized by their own, specific reorientation time course with actin filaments reorienting first followed by MTs and finally vimentin. Interestingly, in all cases, this reorientation was faster than cell body realignment which argues for an active adaptation mechanism for all cytoskeletal systems. Upon actin destabilization, already smallest alterations in actin kinetics massively hamper cell morphology under strain and therefore overall reorientation. Depolymerization of MTs just slightly influences actin reorientation velocity but strongly affects cell body reorientation.

TnI Structural Interface with the N-Terminal Lobe of TnC as a Determinant of Cardiac Contractility

The heterotrimeric cardiac troponin complex is a key regulator of contraction and plays an essential role in conferring Ca2+ sensitivity to the sarcomere. During ischemic injury, rapidly accumulating protons acidify the myoplasm, resulting in markedly reduced Ca2+ sensitivity of the sarcomere. Unlike the adult heart, sarcomeric Ca2+ sensitivity in fetal cardiac tissue is comparatively pH insensitive. Replacement of the adult cardiac troponin I (cTnI) isoform with the fetal troponin I (ssTnI) isoform renders adult cardiac contractile machinery relatively insensitive to acidification. Alignment and functional studies have determined histidine 132 of ssTnI to be the predominant source of this pH insensitivity. Substitution of histidine at the cognate position 164 in cTnI confers the same pH insensitivity to adult cardiac myocytes. An alanine at position 164 of cTnI is conserved in all mammals, with the exception of the platypus, which expresses a proline. Prolines are biophysically unique because of their innate conformational rigidity and helix-disrupting function. To provide deeper structure-function insight into the role of the TnC-TnI interface in determining contractility, we employed a live-cell approach alongside molecular dynamics simulations to ascertain the chemo-mechanical implications of the disrupted helix 4 of cTnI where position 164 exists. This important motif belongs to the critical switch region of cTnI. Substitution of a proline at position 164 of cTnI in adult rat cardiac myocytes causes increased contractility independent of alterations in the Ca2+ transient. Free-energy perturbation calculations of cTnC-Ca2+ binding indicate no difference in cTnC-Ca2+ affinity. Rather, we propose the enhanced contractility is derived from new salt bridge interactions between cTnI helix 4 and cTnC helix A, which are critical in determining pH sensitivity and contractility. Molecular dynamics simulations demonstrate that cTnI A164P structurally phenocopies ssTnI under baseline but not acidotic conditions. These findings highlight the evolutionarily directed role of the TnI-cTnC interface in determining cardiac contractility.

Redox Signaling from and to Peroxisomes: Progress, Challenges, and Prospects.

Peroxisomes are organelles that are best known for their role in cellular lipid and hydrogen peroxide (H2O2) metabolism. Emerging evidence suggests that these organelles serve as guardians and modulators of cellular redox balance, and that alterations in their redox metabolism may contribute to aging and the development of chronic diseases such as neurodegeneration, diabetes, and cancer. Recent Advances: H2O2 is an important signaling messenger that controls many cellular processes by modulating protein activity through cysteine oxidation. Somewhat surprisingly, the potential involvement of peroxisomes in H2O2-mediated signaling processes has been overlooked for a long time. However, recent advances in the development of live-cell approaches to monitor and modulate spatiotemporal fluxes in redox species at the subcellular level have opened up new avenues for research in redox biology and boosted interest in the concept of peroxisomes as redox signaling platforms. This review first introduces the reader to what is known about the role of peroxisomes in cellular H2O2 production and clearance, with a focus on mammalian cells. Next, it briefly describes the benefits and drawbacks of current strategies used to investigate the complex interplay between peroxisome metabolism and cellular redox state. Furthermore, it integrates and critically evaluates literature dealing with the interrelationship between peroxisomal redox metabolism, cell signaling, and human disease. As the precise molecular mechanisms underlying many of these associations are still poorly understood, a key focus for future research should be the identification of primary targets for peroxisome-derived H2O2.

Peroxisome Motility Measurement and Quantification Assay.

Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation with mitochondria and the endoplasmic reticulum (ER) is essential for the β-oxidation of fatty acids and the synthesis of myelin lipids and polyunsaturated fatty acids. A key assay to assess peroxisome motility in mammalian cells is the expression of a fluorescent fusion protein with a peroxisomal targeting signal (e.g., GFP-PTS1), which targets the peroxisomal matrix and allows live-cell imaging of peroxisomes. Here, we first present a protocol for the transfection of cultured mammalian cells with the peroxisomal marker EGFP-SKL to observe peroxisomes in living cells. This approach has revealed different motile behaviour of peroxisomes and novel insight into peroxisomal membrane dynamics (Rapp et al., 1996; Wiemer et al., 1997; Schrader et al., 2000). We then present a protocol which combines the live-cell approach with peroxisome motility measurements and quantification of peroxisome dynamics in mammalian cells. More recently, we used this approach to demonstrate that peroxisome motility and displacement is increased when a molecular tether, which associates peroxisomes with the ER, is lost (Costello et al., 2017b). Silencing of the peroxisomal acyl-CoA binding domain protein ACBD5, which interacts with ER-localised VAPB, increased peroxisome movement in skin fibroblasts, indicating that membrane contact sites can modulate organelle distribution and motility. The protocols described can be adapted to other cell types and organelles to measure and quantify organelle movement under different experimental conditions.

Abstract 4295: A 3D culture model for screening of cancer therapeutics

Abstract 3D cell culture models are currently of great interest to the bioscience community. This is most notable in the cancer biology area, where a growing body of evidence suggests that more relevant and translational observations can be made by testing compounds in a physiological relevant 3D model compared to a 2D model. In this study, we take a kinetic, live-cell imaging approach to measure the growth or shrinkage of non-adherent tumor spheroids using Ultra Low Attachment (ULA) 96-well and 384-well microtiter plates (Corning®). These round-bottomed, hydrogel coated plates facilitate the formation of spheroids by promoting self-adherence as opposed to adherence to the plate surface. In a single cell type, monoculture model, A549 lung epithelial carcinoma cells developed into single spheroid structures in each well of 96 and 384-well plates within 48-96 hours in culture. In both plate types, the resulting spheroid size, ranging from 330 µm - 600 µm, was proportional to the number of cells (1K - 5K cells per well). Post formation, spheroids remained untreated or were subjected to three test agents, staurosporine, oxaliplatin or cycloheximide. The increase or decrease in spheroid size was monitored over 3 - 10 days using the IncuCyte™ ZOOM Live Content Imaging system. Using the red fluorescent channel, spheroid growth was analyzed using fluorescent area and intensity metrics. Whereas staurosporine and oxaliplatin significantly decreased spheroid size at high concentrations, cycloheximide treatment resulted in a cytostatic response, effectively inhibiting spheroid growth in a concentration dependent manner, while not reducing total spheroid area at any tested concentration. Interestingly, in side-by-side comparisons of kinetic 2D and 3D assays, we observed a significant, 10-fold (one log) shift in the pharmacology of cycloheximide. In a 3D co-culture breast cancer model, this study also illustrates the effect of Lapatinib on SK-BR-3 cells grown in the presence of mammary derived stromal cells (fibroblasts). Together, this study describes a kinetic, live-cell approach to measuring spheroid size over time, illustrates a differential response of cycloheximide in a 3D system compared to identically treated cells grown in a 2D monolayer, and details a complex co-culture method for elucidating the effect of Lapatinib in a physiologically relevant 3D model of breast cancer. Citation Format: Kalpana Patel, Belinda O'Clair, Tim O'Callaghan, Daniel M. Appledorn, Derek Trezise. A 3D culture model for screening of cancer therapeutics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4295. doi:10.1158/1538-7445.AM2014-4295

High-Throughput Screening of T Cell Cytotoxic Events by Biomass Profiling

Adoptive immunotherapies against cancer, in which cytotoxic, CD8+ T cells engineered to express T cell receptors (TCRs) targeting cancer-associated antigens are transplanted into a patient, have shown dramatic promise in clinical trials. A major impediment to the widespread use of this technique for treatment of diverse cancers is the lack of a fast approach for the identification of TCRs from patient samples. In this talk, we present a method for high-throughput screening of T cell/target cell interactions by measurements of cell biomass. This live cell approach is label-free and allows cells to be recovered for downstream analysis. To ensure specificity, three parameters are tracked: target cell appearance, target cell mass loss during cell death, and T cell mass during and after the cytotoxic event (Figure panels a-c). Our results demonstrate, for the first time, the kinetics of T cell mass increase during activation. Finally, we will present an extension of this method to a microfabricated microwell format for the screening of patient samples and discovery of novel TCRs.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Live‐Cell Assays to Identify Regulators of ER‐to‐Golgi Trafficking

We applied fluorescence microscopy-based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)-to-Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi-to-ER relocalization of GalT-CFP (β1,4-galactosyltransferase I-cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash-out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER-to-Golgi trafficking. Nine of them interfered with the rate of BFA-induced redistribution of GalT-CFP from the Golgi complex to the ER, six of them interfered with GalT-CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash-out and six of them were positive effectors in both assays. Notably, our live-cell approach captures regulator function in ER-to-Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens.

The Role of Lipid Rafts in Virus Assembly. Identification and Characterization of Microdomain Partitioning Factors of the HIV-1 Glycoprotein gp41 using Flim-FRET and Fluorescence Anisotropy Microscopy

Recent experimental results indicate that host cell invasion as well as assembly and budding of the Human Immunodeficiency Virus (HIV) are highly cholesterol dependent. Supposably, cholesterol enriched plasma membrane microdomains, so called rafts, play an important role in different steps of the virus lifecycle. However, the exact function and molecular background of this sensitivity to bilayer composition remains unknown.We produced different variants of the HIV transmembrane protein gp41 labelled with a yellow fluorescent protein. Fluorescence lifetime imaging microscopy was used to report Forster Resonance Energy Transfer (FRET) between a raft marker labelled with a cyan fluorescent protein and gp41 chimeras in living cells. Since it is highly distance dependent, occurring FRET reflects a co-clustering of both fluorescent protein species in microdomains. By comparison of FRET efficiencies from different truncation and mutation variants of gp41, the Cholesterol Recognition Amino Acid Consensus (CRAC) was identified as main determinant of the protein's raft partitioning. Whereas localization and trafficking of the fusion proteins resembled reported wildtype behaviour, FACS experiments revealed a remarkable influence of CRAC mutations on plasma membrane perturbation properties of gp41. Furthermore, using fluorescence polarization anisotropy microscopy it could be shown, that wildtype gp41 oligomerization occurs at the plasma membrane. Oligomerization of CRAC mutants was found to be significantly impaired. This suggests a pooling function of lipid rafts not only for interactions with other viral components but also for assemblies of functional homo-oligomers.This study is to our knowledge the first live cell approach characterizing gp41 raft partitioning factors and relating lateral plasma membrane sorting to distinct protein functions and properties.The reported raft dependent oligomerization might be representative for general mechanisms of microdomain-facilitated protein interactions.

Defective mitochondrial translation differently affects the live cell dynamics of complex I subunits

Complex I (CI) of the oxidative phosphorylation system is assembled from 45 subunits encoded by both the mitochondrial and nuclear DNA. Defective mitochondrial translation is a major cause of mitochondrial disorders and proper understanding of its mechanisms and consequences is fundamental to rational treatment design. Here, we used a live cell approach to assess its consequences on CI assembly. The approach consisted of fluorescence recovery after photobleaching (FRAP) imaging of the effect of mitochondrial translation inhibition by chloramphenicol (CAP) on the dynamics of AcGFP1-tagged CI subunits NDUFV1, NDUFS3, NDUFA2 and NDUFB6 and assembly factor NDUFAF4. CAP increased the mobile fraction of the subunits, but not NDUFAF4, and decreased the amount of CI, demonstrating that CI is relatively immobile and does not associate with NDUFAF4. CAP increased the recovery kinetics of NDUFV1-AcGFP1 to the same value as obtained with AcGFP1 alone, indicative of the removal of unbound NDUFV1 from the mitochondrial matrix. Conversely, CAP decreased the mobility of NDUFS3-AcGFP1 and, to a lesser extent, NDUFB6-AcGFP1, suggestive of their enrichment in less mobile subassemblies. Little, if any, change in mobility of NDUFA2-AcGFP1 could be detected, suggesting that the dynamics of this accessory subunit of the matrix arm remains unaltered. Finally, CAP increased the mobility of NDUFAF4–AcGFP1, indicative of interaction with a more mobile membrane-bound subassembly. Our results show that the protein interactions of CI subunits and assembly factors are differently altered when mitochondrial translation is defective.

Self-organization phenomena during developing of cotton fibers

The observation of living cotton fibers is an important approach for describing, defining and refining some questions in the fields of cell biology, textile industry and agriculture. The goal of this review is to provide some recent examples that highlight the use of live-cell approaches in cotton plant fiber biology and to point to areas that should be productive for future experimentation.