Gunther G, Malacrida L, Jameson DM, Gratton E, Sánchez SA.
LAURDAN since Weber: The quest for visualizing membrane heterogeneity.
Acc Chem Res. 2021; 54(4): 976-987.Conspectus Any chemist studying the interaction of molecules with lipid assemblies will eventually be confronted by the topic of membrane bilayer heterogeneity and may ultimately encounter the heterogeneity of natural membranes. In artificial bilayers, heterogeneity is defined by phase segregation that can be in the nano- and micrometer range. In biological bilayers, heterogeneity is considered in the context of small (10-200 nm) sterol and sphingolipid-enriched heterogeneous and highly dynamic domains. Several techniques can be used to assess membrane heterogeneity in living systems. Our approach is to use a fluorescent reporter molecule immersed in the bilayer, which, by changes in its spectroscopic properties, senses physical-chemistry aspects of the membrane. This dye in combination with microscopy and fluctuation techniques can give information about membrane heterogeneity at different temporal and spatial levels: going from average fluidity to number and diffusion coefficient of nanodomains. LAURDAN (6-dodecanoyl-2-(dimethylamino) naphthalene), is a fluorescent probe designed and synthesized ... [truncated at 150 words]
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Single cell physiological characterization in living tissue.
65th Annual Meeting of the Biophysical Society. Online. February 22-26.
Biophys J. 2021; 120(3, Suppl 1): 180a, 875-Pos.The omics approach can provide single cell genomics, proteomic and lipidomics and other biological important parameters and at the level of single cell for some of these methods. However the information obtained is at one time and on a fixed tissue. Even if we had this information it is unclear what we can learn about the interactions of the different compartments in a single cell and even less in a living tissue which involves the interaction among cells. Fluorescence and tissue fluorescence has the potential to offer a comprehensive understanding of the relationship between cellular compartments in the context of a tissue. In this abstract we show new fluorescence technologies that have the potential to reach our goals. To meet this goal, we have constructed a scanning fluorescence microscope that con provide gene expression, proteomics and other parameters in living cell on selected genes and proteins. More important, on the ... [truncated at 150 words]
Scipioni L, Rossetta A, Tedeschi G, Gratton E.
Highly multiplexed determination of the cell state by phasor spectral FLIM.
65th Annual Meeting of the Biophysical Society. Online. February 22-26.
Biophys J. 2021; 120(3, Suppl 1): 356a-357a, 1721-Pos.Many high throughput “-omics” techniques (genomics, transcriptomics, proteomics, metabolomics) have been developed to characterize the state of a cell. However, such methods either require highly specific expertise and instrumentation or they’re not applicable to single cells, let alone over multiple time points. We aim to propose a novel, fluorescence-based, high throughput method that allows for the acquisition of multiple physiological parameters over extended periods of time for the characterization of the physiological state of a cell. Environment-sensitive probes can report on important physiological parameters including mitochondrial activity (membrane potential), lipid metabolism (lipid droplet concentration), transcriptional activity (chromatin compaction) and cellular homeostasis (intracellular pH), to name a few. Despite being well characterized, many technical challenges (spectral overlap, sequential acquisition, need for lifetime information) prevent their combination and therefore the acquisition of multiple physiological parameters at the same time. One way to solve this issue is the simultaneous acquisition of spectral and ... [truncated at 150 words]
Ranjit S, Henriksen K, Dvornikov A, Delsante M, Rosenberg A, Levi M, Gratton E.
Quantification of chronic renal parenchymal injury using phasor approach to autofluorescence lifetime imaging.
65th Annual Meeting of the Biophysical Society. Online. February 22-26.
Biophys J. 2021; 120(3, Suppl 1): 357a, 1725-Pos.Diabetic kidney disease is the leading cause of chronic kidney disease. Tubulointerstitial fibrosis, in addition to the well characterized glomerular alterations including mesangial expansion, podocyte injury, and glomerulosclerosis, is an important component of diabetic kidney injury. Any chronic kidney injury often includes tubulointerstitial fibrosis as a critical component. Sensitive and quantitative identification of tubulointerstitial fibrosis is critical for the assessment of long-term prognosis of kidney disease. Phasor approach to fluorescence lifetime imaging, commonly known as phasor-FLIM from autofluorescence measurements is employed here to understand tissue heterogeneity and calculate changes in the tissue autofluorescence lifetime signatures due to diabetic kidney disease. FLIM imaging was performed on cryostat sections of snap-frozen biopsy material of patients with diabetic nephropathy. There was an overall increase in phase lifetime (τphase) with increased disease severity. To understand the heterogeneity and small differences in these biopsy samples multiparametric phasor analysis was employed. Multiparametric phasor analysis is a ... [truncated at 150 words]
Malacrida LS, Kamaid A, Gratton E, Hedde PN.
Hyperspectral imaging in a single plane illumination microscope by spectral phasor analysis with sine-cosine transmission filters.
65th Annual Meeting of the Biophysical Society. Online. February 22-26.
Biophys J. 2021; 120(3, Suppl 1): 358a, 1727-Pos.The combination of hyperspectral imaging and single plane illumination microscopy (SPIM) is a powerful approach to address large samples in a multidimensional fashion. We have developed an ultrafast phasor-based hyperspectral imaging method using sine/cosine interference filters on the side-SPIM microscope. This instrumentation is 10-100 times faster than regular hyperspectral imaging in scanning confocal, while maintaining resolution and achieving high photon recovery. We have tested this new method in Spectrum of Fates (SoFa) zebrafish lines, in which the expression of different fluorescent proteins is driven from promoters of a set of transcription factors differentially expressed in the developing retina: Crx:gapCFP, Ptf1a:cytGFP, and Atoh7:gapRFP. Thus, SoFa is an attractive model with several cell types co-expressing more than one FP, so no simple “single color” pattern was expected. SoFa zebrafish embryos were fixed at 72 hours post fertilization, nucleic acids stained with SYTOX Orange (SO) to label the nucleus and imaged by 4D ... [truncated at 150 words]
Vallmitjana A, Torrado B, Gratton E.
Automatic image segmentation based on phasor space clustering.
65th Annual Meeting of the Biophysical Society. Online. February 22-26.
Biophys J. 2021; 120(3, Suppl 1): 360a, 1739-Pos.We present our latest developments in automatic image segmentation for measurements on spectral and lifetime fluorescence imaging. The algorithm operates on the basis of the phasor approach and makes use of a well-established machine learning method for clustering N-dimensional data points, in our case the pixel data points on the phasor space. The technique is based on Gaussian mixture models solved by the use of the expectation-maximization algorithm. We show the method is the best candidate out of the many machine learning clustering techniques available due to the nature of the phasor data and also due to the spreading of points in the phasor space for the cases of low pixel photon counts. In order to quantify the power of the method we performed simulations with synthetic phasor data and we tested the method by measuring its performance against a series of established clustering techniques. We also show results on ... [truncated at 150 words]
Cinco R, Dragan M, Gratton E, Welch H, Timpson P, Dai X, Digman MA.
Spatiotemporal dynamics of RAC1 signaling during wound healing in Forster resonance energy transfer biosensor mice.
65th Annual Meeting of the Biophysical Society. Online. February 22-26.
Biophys J. 2021; 120(3, Suppl 1): 105a, 506-Plat.The advent of Förster Resonance Energy Transfer (FRET) biosensor transgenic animals presents the unique opportunity to spatially specify and quantify the level of active signal transduction within individual cells of a tissue with subcellular resolution. The type of information gleaned by these biosensors are key for understanding the dynamic role of signaling intermediates in regulating cellular plasticity during developmental and regenerative processes. The modified Raichu-Rac1 biosensors transgenically encoded in mice FRET when bound to GTP and report active signal transduction. We hypothesize Rac1 signal transduction regulates the proliferative and migratory functions of keratinocytes during wound repair and that functional specificity of Rac1 is achieved through a combination of subcellular targeting and signal transduction level thresholds. To explore these hypotheses, we imaged the wound repair geography at the proliferative, mixed and migratory zones at 4 days post wounding to quantify the subcellular signal transduction levels of Rac1 in keratinocytes using the ... [truncated at 150 words]
Torrado B, Dvornikov A, Gratton E.
Hyperspectral analysis of fluorescence images in scattering media using optical filters.
65th Annual Meeting of the Biophysical Society. Online. February 22-26.
Biophys J. 2021; 120(3, Suppl 1): 106a, 509-Plat.We present the simple fluorescence imaging technique that allows hyperspectral analysis of images even obtained in highly scattering media. This technique can be applied to any fluorescence microscope equipped with a single detector or camera. The conventional hyperspectral imaging usually utilizes spectrometers embedded in a microscope, which are designed for use with collimated light and thus not efficient for imaging in scattering samples, such as tissues. Also, this is an expensive addition to the microscope. In the proposed method, we utilize optical filters which have certain transmittance characteristics in the spectral range of interest. The images are collected through these filters, either in sequence or parallel, and then the spectral phasor coordinates can be calculated to perform hyperspectral analysis. Several types of filters can be used in this method, including filters with sin-cos and linear transmittance profiles. The sin-cos filters allow recovering information on central wavelength and width of fluorophore ... [truncated at 150 words]
Ranjit S, Lanzanò L, Libby AE, Gratton E, Levi M.
Advances in fluorescence microscopy techniques to study kidney function.
Nat Rev Nephrol. 2021; 17(2): 128-144.Fluorescence microscopy, in particular immunofluorescence microscopy, has been used extensively for the assessment of kidney function and pathology for both research and diagnostic purposes. The development of confocal microscopy in the 1950s enabled imaging of live cells and intravital imaging of the kidney; however, confocal microscopy is limited by its maximal spatial resolution and depth. More recent advances in fluorescence microscopy techniques have enabled increasingly detailed assessment of kidney structure and provided extraordinary insights into kidney function. For example, nanoscale precise imaging by rapid beam oscillation (nSPIRO) is a super-resolution microscopy technique that was originally developed for functional imaging of kidney microvilli and enables detection of dynamic physiological events in the kidney. A variety of techniques such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) enable assessment of interaction between proteins. The emergence of other super-resolution techniques, including super-resolution stimulated emission depletion ... [truncated at 150 words]
Sameni S, Zhang R, Digman MA.
The phasor FLIM method reveals a link between a change in energy metabolism and mHtt protein spread in healthy mammalian cells when co-cultured with Huntington diseased cells.
Methods Appl Fluoresc. 2021; 9(1), 015005.Huntington Disease (HD) is a late-onset autosomal neurodegenerative disease characterized by the aggregations of mutant Huntingtin proteins (mHTT). A glutamine stretch (PolyQ) at the N-terminal of the Huntingtin protein is generated by the abnormal expansion of CAG trinucleotide repeats in exon 1 of the HTT gene. While the resulting polyQ aggregates are the predominate feature of HD , the intercellular spread of the expanded protein and the effect upon this transfer inside healthy cells have not yet fully understood. Here, we have employed the phasor Fluorescence Lifetime Imaging Microscopy (FLIM) method to measure NADH fluorescence lifetime change after the internalization of the PolyQ protein. Based on our analysis, we have found a significant decrease in the fraction of bound NADH in both cytoplasmic and nucleus regions when cells are co-cultured or when healthy cells uptake the supernatant containing polyQ proteins and aggregates. Overall, our FLIM study combined with confocal fluorescence ... [truncated at 150 words]
Greco CM, Cervantes M, Fustin JM, Ito K, Ceglia N, Samad M, Shi J, Koronowski KB, Forne I, Ranjit S, Gaucher J, Kinouchi K, Kojima R, Gratton E, Li W, Baldi P, Imhof A, Okamura H, Sassone-Corsi P.
S-adenosyl-l-homocysteine hydrolase links methionine metabolism to the circadian clock and chromatin remodeling.
Sci Adv. 2020; 6(51), eabc5629. PMCID: PMC7744083Circadian gene expression driven by transcription activators CLOCK and BMAL1 is intimately associated with dynamic chromatin remodeling. However, how cellular metabolism directs circadian chromatin remodeling is virtually unexplored. We report that the S-adenosylhomocysteine (SAH) hydrolyzing enzyme adenosylhomocysteinase (AHCY) cyclically associates to CLOCK-BMAL1 at chromatin sites and promotes circadian transcriptional activity. SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases, and timely hydrolysis of SAH by AHCY is critical to sustain methylation reactions. We show that AHCY is essential for cyclic H3K4 trimethylation, genome-wide recruitment of BMAL1 to chromatin, and subsequent circadian transcription. Depletion or targeted pharmacological inhibition of AHCY in mammalian cells markedly decreases the amplitude of circadian gene expression. In mice, pharmacological inhibition of AHCY in the hypothalamus alters circadian locomotor activity and rhythmic transcription within the suprachiasmatic nucleus. These results reveal a previously unappreciated connection between cellular metabolism, chromatin dynamics, and circadian regulation.
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System and method for hyperspectral imaging in highly scattering media by the spectral phasor approach using two filters.
Patent US20200378830A1, 03 Dec 2020.An imaging system comprises an excitation light source, a directing element positioned to direct light from the excitation light source toward a sample, a detector configured to measure incoming light from the sample, a filter cavity positioned between the sample and the detector, a first filter configured to be inserted into the filter cavity, a sine filter configured to be inserted into the filter cavity, and a processing unit communicatively connected to the detector, configured to receive image data from the detector to form an image. Methods of constructing a hyperspectral image of a sample are also described.
Vallmitjana A, Torrado B, Dvornikov A, Ranjit S, Gratton E.
Blind resolution of lifetime components in individual pixels of fluorescence lifetime images using the phasor approach.
J Phys Chem B. 2020; 124(45): 10126-10137.The phasor approach is used in fluorescence lifetime imaging microscopy for several purposes, notably to calculate the metabolic index of single cells and tissues. An important feature of the phasor approach is that it is a fit-free method allowing immediate and easy to interpret analysis of images. In a recent paper, we showed that three or four intensity fractions of exponential components can be resolved in each pixel of an image by the phasor approach using simple algebra, provided the component phasors are known. This method only makes use of the rule of linear combination of phasors rather than fits. Without prior knowledge of the components and their single exponential decay times, resolution of components and fractions is much more challenging. Blind decomposition has been carried out only for cuvette experiments wherein the statistics in terms of the number of photons collected is very good. In this paper, we show ... [truncated at 150 words]
Makaremi S, Rose M, Ranjit S, Digman MA, Bowdish DME, Moran-Mirabal JM.
Lateral diffusion of CD14 and TLR2 in macrophage plasma membrane assessed by raster image correlation spectroscopy and single particle tracking.
Sci Rep. 2020; 10(1): 19375. PMCID: PMC7652837The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; consequently, several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. The membrane-anchored receptor cluster of differentiation 14 (CD14) and the transmembrane toll-like receptor 2 (TLR2) are important receptors in the plasma membrane of macrophages that activate the intracellular signaling cascade in response to pathogenic stimuli. The aim of the present work was to compare the diffusion coefficients of CD14 and TLR2 on the apical and basal membranes of macrophages using two fluorescence-based methods: raster image correlation spectroscopy (RICS) and single particle tracking (SPT). In the basal membrane, the diffusion coefficients obtained from SPT and RICS were found to be comparable and revealed significantly faster diffusion of CD14 compared with TLR2. In addition, RICS showed that the diffusion of both receptors was ... [truncated at 150 words]
Ranjit S, Henriksen K, Dvornikov A, Delsante M, Rosenberg A, Levi M, Gratton E.
Phasor approach to autofluorescence lifetime imaging FLIM can be a qantitative biomarker of chronic renal Parenchymal injury.
Kidney Int. 2020; 98(5): 1341-1346. PMCID: PMC7606347Diabetic kidney disease continues to be the leading cause of chronic kidney disease, often advancing to end stage kidney disease. In addition to the well characterized glomerular alterations including mesangial expansion, podocyte injury, and glomerulosclerosis, tubulointerstitial fibrosis is also an important component of diabetic kidney injury. Similarly, tubulointerstitial fibrosis is a critical component of any chronic kidney injury. Therefore, sensitive and quantitative identification of tubulointerstitial fibrosis is critical for the assessment of long-term prognosis of kidney disease. Here, we employed phasor approach to fluorescence lifetime imaging, commonly known as FLIM, to understand tissue heterogeneity and calculate changes in the tissue autofluorescence lifetime signatures due to diabetic kidney disease. FLIM imaging was performed on cryostat sections of snap-frozen biopsy material of patients with diabetic nephropathy. There was an overall increase in phase lifetime (τphase) with increased disease severity. Multicomponent phasor analysis shows the distinctive differences between the different disease states. Thus, ... [truncated at 150 words]
Chen YC, Sood C, Marin M, Aaron J, Gratton E, Salaita K, Melikyan GB.
Super-resolution fluorescence imaging reveals that Serine incorporator protein 5 inhibits human immunodeficiency virus fusion by disrupting envelope glycoprotein clusters.
ACS Nano. 2020; 14(9): 10929-10943.Serine incorporator protein 5 (SERINC5) is the host anti-retroviral factor that reduces HIV-1 infectivity by incorporating into virions and inhibiting the envelope glycoprotein (Env) mediated virus fusion with target cells. We and others have shown that SERINC5 incorporation into virions alters the Env structure and sensitizes the virus to broadly neutralizing antibodies targeting cryptic Env epitopes. We have also found that SERINC5 accelerates the loss of Env function over time compared to control viruses. However, the exact mechanism by which SERINC5 inhibits HIV-1 fusion is not understood. Here, we utilized 2D and 3D super-resolution microscopy to examine the effect of SERINC5 on the distribution of Env glycoproteins on single HIV-1 particles. We find that, in agreement with a previous report, Env glycoproteins form clusters on the surface of mature virions. Importantly, incorporation of SERINC5, but not SERINC2, which lacks antiviral activity, disrupted Env clusters without affecting the overall Env content. ... [truncated at 150 words]
Hedde PN, Abram TJ, Jain A, Nakajima R, de Assis RR, Pearce T, Jasinskas A, Toosky MN, Khan S, Felgner PL, Gratton E, Zhao W.
A modular microarray imaging system for highly specific COVID-19 antibody testing.
Lab Chip. 2020; 20(18): 3302-3309. PMCID: PMC7263497To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100× more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months ... [truncated at 150 words]
Harris CR, Millman KJ, van der Walt SJ, Gommers R, Virtanen P, Cournapeau D, Wieser E, Taylor J, Berg S, Smith NJ, Kern R, Picus M, Hoyer S, van Kerkwijk MH, Brett M, Haldane A, Río JFd, Wiebe M, Peterson P, Gérard-Marchant P, Sheppard K, Reddy T, Weckesser W, Abbasi H, Gohlke C, Oliphant TE.
Array programming with NumPy.
Nature. 2020; 585(7825): 357-362. PMCID: PMC7759461Array programming provides a powerful, compact and expressive syntax for accessing, manipulating and operating on data in vectors, matrices and higher-dimensional arrays. NumPy is the primary array programming library for the Python language. It has an essential role in research analysis pipelines in fields as diverse as physics, chemistry, astronomy, geoscience, biology, psychology, materials science, engineering, finance and economics. For example, in astronomy, NumPy was an important part of the software stack used in the discovery of gravitational waves1 and in the first imaging of a black hole2. Here we review how a few fundamental array concepts lead to a simple and powerful programming paradigm for organizing, exploring and analysing scientific data. NumPy is the foundation upon which the scientific Python ecosystem is constructed. It is so pervasive that several projects, targeting audiences with specialized needs, have developed their own NumPy-like interfaces and array objects. Owing to its central position ... [truncated at 150 words]
Palczewska G, Boguslawski J, Stremplewsk P, Kornaszewski L, Zhang J, Dong Z, Liang XX, Gratton E, Vogel A, Wojtkowski M, Palczewski K.
Noninvasive two-photon optical biopsy of retinal fluorophores.
Proc Natl Acad Sci USA. 2020; 117(36): 22532-22543. PMCID: PMC7486747High-resolution imaging techniques capable of detecting identifiable endogenous fluorophores in the eye along with genetic testing will dramatically improve diagnostic capabilities in the ophthalmology clinic and accelerate the development of new treatments for blinding diseases. Two-photon excitation (TPE)-based imaging overcomes the filtering of ultraviolet light by the lens of the human eye and thus can be utilized to discover defects in vitamin A metabolism during the regeneration of the visual pigments required for the detection of light. Combining TPE with fluorescence lifetime imaging (FLIM) and spectral analyses offers the potential of detecting diseases of the retina at earlier stages before irreversible structural damage has occurred. The main barriers to realizing the benefits of TPE for imaging the human retina arise from concerns about the high light exposure typically needed for informative TPE imaging and the requirement to correlate the ensuing data with different states of health and disease. To overcome ... [truncated at 150 words]
Li W, Hu J, Sh B, Palomba F, Digman MA, Gratton E, Jiang H.
Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis.
Nat Cell Biol. 2020; 22(8): 960-972. PMCID: PMC7425812It remains unknown if biophysical or material properties of biomolecular condensates regulate cancer. Here we show that AKAP95, a nuclear protein that regulates transcription and RNA splicing, plays an important role in tumorigenesis by supporting cancer cell growth and suppressing oncogene-induced senescence. AKAP95 forms phase-separated and liquid-like condensates in vitro and in nucleus. Mutations of key residues to different amino acids perturb AKAP95 condensation in opposite directions. Importantly, the activity of AKAP95 in splice regulation is abolished by disruption of condensation, significantly impaired by hardening of condensates, and regained by substituting its condensation-mediating region with other condensation-mediating regions from irrelevant proteins. Moreover, the abilities of AKAP95 in regulating gene expression and supporting tumorigenesis require AKAP95 to form condensates with proper liquidity and dynamicity. These results link phase separation to tumorigenesis and uncover an important role of appropriate biophysical properties of protein condensates in gene regulation and cancer.
Hedde PN, Bouzin M, Abram TJ, Chen X, Toosky MN, Vu T, Li Y, Zhao W, Gratton E.
Rapid isolation of rare targets from large fluid volumes.
Sci Rep. 2020; 10(1), 12458. PMCID: PMC7385493apidly isolating rare targets from larger, clinically relevant fluid volumes remains an unresolved problem in biomedicine and diagnosis. Here, we describe how 3D particle sorting can enrich targets at ultralow concentrations over 100-fold within minutes not possible with conventional approaches. Current clinical devices based on biochemical extraction and microfluidic solutions typically require high concentrations and/or can only process sub-milliliter volumes in time. In a proof-of-concept application, we isolated bacteria from whole blood as demanded for rapid sepsis diagnosis where minimal numbers of bacteria need to be found in a 1–10 mL blood sample. After sample encapsulation in droplets and target enrichment with the 3D particle sorter within a few minutes, downstream analyses were able to identify bacteria and test for antibiotic susceptibility, information which is critical for successful treatment of bloodstream infections.
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Fluorescence lifetime imaging microscopy and LAURDAN spectral imaging for dynamically investigating osteoclast differentiation.
PhD in Biomedical Engineering, University of California, Irvine, 2020.
Advisor: Michelle A Digman
Osteoclasts, the multinucleated bone-resorbing cells, are involved in the destructive breakdown of bones in many diseases such as osteoporosis and rheumatoid arthritis. Designing an efficient and specific therapeutic strategy to these diseases would depend on understanding osteoclasts differentiation. Although gene expression quantification and biochemical techniques have been used extensively to study osteoclast differentiation, they lack the capability to dynamically examine live osteoclasts at the single-cell level. In this thesis, we explored the practicality of the two minimally invasive microscopy techniques, NAD(P)H Fluorescence Lifetime Imaging and LAURDAN spectral imaging, in observing cellular metabolic profiles and membrane dynamics respectively during osteoclast differentiation. In addition to establishing the practicality of these two imaging platforms, our report offered a deeper understanding regarding the roles of metabolism and membrane dynamics in osteoclasts differentiation and pathogenesis of osteoclasts-associated diseases.
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Fluorescence based adaptive optics and multidimensional fluorescence microscopy.
PhD in Biomedical Engineering, University of California, Irvine, 2020.
Advisor: Enrico Gratton
Studying the structure and interactions of molecules and cells in their native environments has always been a challenge in the life sciences. When an organism of interest is physically out of reach or invisible to the naked eye, scientists have historically turned to reductionist experiments that first isolate the organism from a complex environment and then study the bulk signal from a large population of cells in tissue or in bacterial colonies. However, the structure, function, and interactions of a population of cells can vary at a subpopulation and even single-cell level. Non-invasive tools that can study a population in relevant conditions, such as in tissue, capable of resolving single cells in a large region of interest are necessary to improve treatment of disease and understanding of physiological phenomena.
Microscopy – in particular multiphoton fluorescence microscopy – holds enormous potential to discover new science by elucidating biological properties with subcellular resolution. ... [truncated at 150 words]
Cervantes M, Forné I, Ranjit S, Gratton E, Imhof A, Sassone-Corsi P.
BMAL1 associates with NOP58 in the nucleolus and contributes to pre-rRNA processing.
iScience. 2020; 23(6), 101151. PMCID: PMC7256328The transcription factor BMAL1 is a core element of the circadian clock that contributes to cyclic control of genes transcribed by RNA polymerase II. By using biochemical cellular fractionation and immunofluorescence analyses we reveal a previously uncharacterized nucleolar localization for BMAL1. We used an unbiased approach to determine the BMAL1 interactome by mass spectrometry and identified NOP58 as a prominent nucleolar interactor. NOP58, a core component of the box C/D small nucleolar ribonucleoprotein complex, associates with Snord118 to control specific pre-ribosomal RNA (pre-rRNA) processing steps. These results suggest a non-canonical role of BMAL1 in ribosomal RNA regulation. Indeed, we show that BMAL1 controls NOP58-associated Snord118 nucleolar levels and cleavage of unique pre-rRNA intermediates. Our findings identify an unsuspected function of BMAL1 in the nucleolus that appears distinct from its canonical role in the circadian clock system.
Vallmitjana A, Dvornikov A, Torrado B, Jameson DM, Ranjit S, Gratton E.
Resolution of 4 components in the same pixel in FLIM images using the phasor approach.
Methods Appl Fluoresc. 2020; 8(3), 035001.In several cellular systems, the phasor FLIM approach has shown the existence of more than 2 components in the same pixel, a typical example being free and bound NADH. In order to properly quantify the concentrations and the spatial distributions of fluorescence components associated with different molecular species we developed a general method to resolve 3 and 4 components in the same pixel using the phasor approach. The method is based on the law of linear combination of components valid after transformation of the decay curves to phasors for each pixel in the image. In principle, the linear combination rule is valid for an arbitrary number of components. For 3 components we use only the phasor position for the first harmonic, which has a small error, while for 4 components we need the phasor location at higher harmonics that have intrinsically more noise. As a result of the noise in ... [truncated at 150 words]
Takahashi S, Luo Y, Ranjit S, Xie C, Libby AE, Orlicky DJ, Dvornikov A, Wang XX, Myakala K, Jones BA, Bhasin K, Wang D, McManaman JL, Krausz KW, Gratton E, Ir D, Robertson CE, Frank DN, Gonzalez FJ, Levi M.
Bile acid sequestration reverses liver injury and prevents progression of NASH in western diet-fed mice.
J Biol Chem. 2020; 295(14): 4733-4747. PMCID: PMC7135973Non-alcoholic fatty liver disease (NAFLD) is a rapidly rising problem in the 21st century and is a leading cause of chronic liver disease that can lead to end-stage liver diseases, including cirrhosis and hepatocellular cancer. Despite this rising epidemic, no pharmacological treatment has yet been established to treat this disease. The rapidly increasing prevalence of NAFLD and its aggressive form, nonalcoholic steatohepatitis (NASH), requires novel therapeutic approaches to prevent disease progression. Alterations in microbiome dynamics and dysbiosis play an important role in liver disease, and these may represent targetable pathways to treat liver disorders. Improving microbiome properties or restoring normal bile acid metabolism may prevent or slow the progression of liver diseases such as NASH. Importantly, aberrant systemic circulation of bile acids can greatly disrupt metabolic homeostasis. Bile acid sequestrants (BAS) are orally administered polymers that bind bile acids in the intestine forming nonabsorbable complexes. BAS interrupts intestinal ... [truncated at 150 words]
Planes N, Vanderheyden PPML, Gratton E, Caballero-George C.
Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells.
Microsc Res Tech. 2020; 83(4): 381-392.The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G-protein-coupled receptors namely Angiotensin II type 1 (AT1 ) and Endothelin 1 type A (ETA ) receptors and their corresponding receptor-ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT1 and ETA receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local ... [truncated at 150 words]
Pascua SM, McGahey GE, Ma N, Wang JJ, Digman MA.
Caffeine and Cisplatin effectively targets the metabolism of a triple-negative breast cancer cell line assessed via Phasor-FLIM.
Int J Mol Sci. 2020; 21(7), E2443. PMCID: PMC7177700Triple-negative tumor cells, a malignant subtype of breast cancer, lack a biologically targeted therapy. Given its DNA repair inhibiting properties, caffeine has been shown to enhance the effectiveness of specific tumor chemotherapies. In this work, we have investigated the effects of caffeine, cisplatin, and a combination of the two as potential treatments in energy metabolism for three cell lines, triple-negative breast cancer (MDA-MB-231), estrogen-receptor lacking breast cancer (MCF7) and breast epithelial cells (MCF10A) using a sensitive label-free approach, phasor-fluorescence lifetime imaging microscopy (phasor-FLIM). We found that solely using caffeine to treat MDA-MB-231 shifts their metabolism towards respiratory-chain phosphorylation with a lower ratio of free to bound NADH, and a similar trend is seen in MCF7. However, MDA-MB-231 cells shifted to a higher ratio of free to bound NADH when cisplatin was added. The combination of cisplatin and caffeine together reduced the survival rate for MDA-MD231 and shifted their energy metabolism ... [truncated at 150 words]
Haensel D, Jin S, Sun P, Cinco R, Dragan M, Nguyen Q, Cang Z, Gong Y, Vu R, MacLean AL, Kessenbrock K, Gratton E, Nie Q, Dai X.
Defining epidermal basal cell states during skin homeostasis and wound healing using single-cell transcriptomics.
Cell Rep. 2020; 30(11): 3932-3947.e6. PMCID: PMC7218802Our knowledge of transcriptional heterogeneities in epithelial stem and progenitor cell compartments is limited. Epidermal basal cells sustain cutaneous tissue maintenance and drive wound healing. Previous studies have probed basal cell heterogeneity in stem and progenitor potential, but a comprehensive dissection of basal cell dynamics during differentiation is lacking. Using single-cell RNA sequencing coupled with RNAScope and fluorescence lifetime imaging, we identify three non-proliferative and one proliferative basal cell state in homeostatic skin that differ in metabolic preference and become spatially partitioned during wound re-epithelialization. Pseudotemporal trajectory and RNA velocity analyses predict a quasi-linear differentiation hierarchy where basal cells progress from Col17a1Hi/Trp63Hi state to early-response state, proliferate at the juncture of these two states, or become growth arrested before differentiating into spinous cells. Wound healing induces plasticity manifested by dynamic basal-spinous interconversions at multiple basal transcriptional states. Our study provides a systematic view of epidermal cellular dynamics, supporting a revised ... [truncated at 150 words]
Liang Z, Lou J, Scipioni L, Gratton E, Hinde E.
Quantifying nuclear wide chromatin compaction by phasor analysis of histone Förster resonance energy transfer (FRET) in frequency domain fluorescence lifetime imaging microscopy (FLIM) data.
Data Brief. 2020; 30, 105401. PMCID: PMC7152662The nanometer spacing between nucleosomes throughout global chromatin organisation modulates local DNA template access, and through continuous dynamic rearrangements, regulates genome function [1]. However, given that nucleosome packaging occurs on a spatial scale well below the diffraction limit, real time observation of chromatin structure in live cells by optical microscopy has proved technically difficult, despite recent advances in live cell super resolution imaging [2]. One alternative solution to quantify chromatin structure in a living cell at the level of nucleosome proximity is to measure and spatially map Förster resonance energy transfer (FRET) between fluorescently labelled histones - the core protein of a nucleosome [3]. In recent work we established that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) is a robust method for the detection of histone FRET which can quantify nuclear wide chromatin compaction in the presence of cellular autofluorescence [4]. Here we share FLIM data recording histone ... [truncated at 150 words]
Perinbam K, Chacko JV, Kannan A, Digman MA, Siryaporn A.
A shift in central metabolism accompanies virulence activation in Pseudomonas aeruginosa.
mBio. 2020; 11(2), e02730-18. PMCID: PMC7064766The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total ... [truncated at 150 words]
Davis RT, Blake K, Ma D, Gabra MBI, Hernandez GA, Phung AT, Yang Y, Maurer D, Lefebvre AEYT, Alshetaiwi H, Xiao Z, Liu J, Locasale JW, Digman MA, Mjolsness E, Kong M, Werb Z, Lawson DA.
Transcriptional diversity and bioenergetic shift in human breast cancer metastasis revealed by single-cell RNA sequencing.
Nat Cell Biol. 2020; 22(3): 310-320.Although metastasis remains the cause of most cancer-related mortality, mechanisms governing seeding in distal tissues are poorly understood. Here, we establish a robust method for the identification of global transcriptomic changes in rare metastatic cells during seeding using single-cell RNA sequencing and patient-derived-xenograft models of breast cancer. We find that both primary tumours and micrometastases display transcriptional heterogeneity but micrometastases harbour a distinct transcriptome program conserved across patient-derived-xenograft models that is highly predictive of poor survival of patients. Pathway analysis revealed mitochondrial oxidative phosphorylation as the top pathway upregulated in micrometastases, in contrast to higher levels of glycolytic enzymes in primary tumour cells, which we corroborated by flow cytometric and metabolomic analyses. Pharmacological inhibition of oxidative phosphorylation dramatically attenuated metastatic seeding in the lungs, which demonstrates the functional importance of oxidative phosphorylation in metastasis and highlights its potential as a therapeutic target to prevent metastatic spread in patients with breast ... [truncated at 150 words]
