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.Circadian 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.
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]
Ranjit S, Lanzanò L, Libby AE, Gratton E, Levi M.
Advances in fluorescence microscopy techniques to study kidney function.
Nat Rev Nephrol. 2020; [Online ahead of print].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]
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.Array 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]
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. 2020; [Epub ahead of print].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]
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]
Titlow J, Robertson F, Järvelin A, Ish-Horowicz D, Smith C, Gratton E, Davis I.
Syncrip/hnRNP Q is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation.
J Cell Biol. 2020; 219(3), e201903135. PMCID: PMC7055005Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. Here, we show that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator ... [truncated at 150 words]
Tetreau G, Banneville AS, Andreeva EA, Brewster AS, Hunter MS, Sierra RG, Teulon JM, Young ID, Burke N, Grünewald TA, Beaudouin J, Snigireva I, Fernandez-Luna MT, Burt A, Park HW, Signor L, Bafna JA, Sadir R, Fenel D, Boeri-Erba E, Bacia M, Zala N, Laporte F, Després L, Weik M, Boutet S, Rosenthal M, Coquelle N, Burghammer M, Cascio D, Sawaya MR, Winterhalter M, Gratton E, Gutsche I, Federici B, Pellequer JL, Sauter NK, Colletier JP.
Serial femtosecond crystallography on in vivo-grown crystals drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade.
Nat Commun. 2020; 11(1): 1153. PMCID: PMC7052140Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.
Abram TJ, Cherukury H, Ou CY, Vu T, Toledano M, Li Y, Grunwald JT, Toosky MN, Tifrea DF, Slepenkin A, Chong J, Kong L, Pozo DVD, La KT, Labanieh L, Zimak J, Shen B, Huang SS, Gratton E, Peterson EM, Zhao W.
Rapid bacterial detection and antibiotic susceptibility testing in whole blood using one-step, high throughput blood digital PCR.
Lab Chip. 2020; 20(3): 477-489. PMCID: PMC7250044Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates ... [truncated at 150 words]
Palomba F, Scipioni L, Gratton E, Digman MA.
Water dynamics of a model protein phase separation via fluorescence lifetime and spectral analysis of ACDAN.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 308a, 1509-Pos.Cells can organize many of their biochemical reactions in membrane-less compartments. These can be achieved by self-assembly of proteins leading to protein phase separations (PPS). The mechanism of de-mixing can be physiological (inner centromere) and/or pathological (stress granules). PPS organize cellular regions via confinement and exclusion of the molecules affecting their dynamics within and surrounding the compartment. PPS, also called coacervates, can express different types of phase, from liquid- to solid-like. To characterize that, investigations commonly starts with the definition of the phase separation diagram of the purified protein that drive the water/protein liquid demixing. In our opinion, to better understand these complex phases, the characterization of the water dynamics is fundamental. In this contest, we proposed the study of the water dipolar relaxation measuring the emission spectra and fluorescence lifetime at optical resolution of ACDAN (6-acetyl-2-dimethylaminonaphthalene, solvofluorochromic dye) that colocalize on a model PPS. Indeed, we implement the method ... [truncated at 150 words]
Torrado B, Scipioni L, Gratton E, Badano JL, Malacrida LS, Irigoín F.
Primary cilium submicron organization and dynamics.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 437a, 2144-Pos.The aim of this work is to contribute to the characterization of the biophysical properties of the ciliary microenvironment in order to understand central processes of the organelle's biology such as protein import, movement and crowding organization. First, we analyzed the movement of a small soluble protein (EGFP) to establish its pattern of diffusion in different regions of the ciliary compartment (body and distal tip). Using advanced fluorescence fluctuation correlation spectroscopy (Comprehensive correlation analysis) with the Zeiss Airyscan Detector, we characterized the organization and dynamic fingerprint of these subcompartments. Preliminary results showed that both ciliary body and distal tip have permeable microdomains explaining why EGFP presents a pattern of diffusion compatible with dynamic partition rather than free diffusion. However, at the ciliary body diffusion is more confined than at the ciliary tip. Measurements on cytoplasm and nucleus were made to compare with cilia crowding organization. Moreover, to study ciliary structural ... [truncated at 150 words]
Hedde PN, Staaf E, Singh SB, Johansson S, Gratton E.
Pair correlation analysis reveals barriers to natural killer cell receptor motion at the synapse.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 246a, 1211-Pos.In living systems, barriers and obstacles to biomolecular dynamics, including receptor motion, critically regulate many mechanisms. Conventional methods including raster image correlation spectroscopy (RICS) and image mean square displacement (iMSD) analysis focus on measuring the average, isotropic motion of molecules in a larger region of interest [1]. Instead, the two-dimensional pair correlation function (2D-pCF) approach can visualize and quantify the paths taken by molecules with high spatial resolution to reveal spatial heterogeneities in motion as caused by barriers and obstacles that break the symmetry [2]. We used this method to study the anisotropy in the motion of natural killer (NK) cell receptors at the immune synapse. In cultured human HLA null 721.221 cells, we observed more barriers to motion for inhibitory receptor HLA-Cw4-YFP coexpressed with KIR3DL1 compared to inhibitory receptor HLA-B51-YFP coexpressed with KIR2DL1. Further, in a mouse model for NK cell education, we found the dynamic spatial organization of ... [truncated at 150 words]
Oneto M, Scipioni L, Sarmento M, Cainero I, Cerutti E, Pelicci S, Furia L, Pelicci PG, Dellino GI, Bianchini P, Faretta M, Gratton E, Diaspro A, Lanzano L.
Nanoscale distribution of nuclear sites analyzed by superresolution STED image cross-correlation spectroscopy.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 20a, 93-Plat.Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, superresolution microscopy has gained considerable interest as it can be used to probe the spatial organization of functional sites in intact single cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a pre-segmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet.
Hsu C, Gratton E, Anders R, Rosenberg A, Levi M, Ranjit S.
Image correlation microscopy approach to study collagen accumulation for distinguishing recurrence in liver cancer patients.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 309a-310a, 1514-Pos.Collagen alignment, also known as tumor associated collagen signature, has been used to predict worse prognosis in breast cancer. It has been shown in breast cancer studies that when collagen is aligned perpendicular to the tumor, it allows for cancer invasion. Cirrhosis is a risk factor for liver cancer development. Thus, we aim to test the hypothesis that there is a similar collagen signature in patients who recurred with liver cancer after liver transplant when compared to those who did not recur. In this study, we have used image correlation techniques to identify the changes in collagen structures around different cancer samples collected from liver cancer patients. Different properties of the image correlation function, including the anisotropic shape of the correlation function, the shape parameters of the ellipsoid describing the correlation function and orientation of the ellipses were used to create overlapping maps of image correlation properties with the SHG ... [truncated at 150 words]
Vallmitjana A, Vorontsova I, Torrado B, Schilling TF, Hall JE, Gratton E, Malacrida LS.
Measuring the spatial distribution of dipolar relaxation in live Zebrafish eye lenses during development.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 308a, 1508-Pos.This work presents image processing techniques we developed to analyze spectral images of in vivo zebrafish eye lenses using ACDAN (6-acetyl-2-dimethylaminonaphthalene) as a probe for intracellular dipolar relaxation (DR). Using these techniques we analyzed the development of regional DR in zebrafish lenses, as water homeostasis is crucial for normal development of structure and optical properties of the lens. We imaged lenses between two and nine days post-fertilization encompassing stages ranging from immature embryonic to functional emmetropic lenses at larval stages. ACDAN fluorescence was collected using a 32-channel spectral detector at anterior, equatorial and posterior planes of the ocular lens in anesthetized zebrafish incubated in ACDAN. We used the spectral phasor transformation to obtain a continuum of values within an arbitrarily-set interval in order to measure DR at each pixel of the images. From these images we interpolated radial DR profiles in all spatial directions from which a mean radial profile ... [truncated at 150 words]
Tedeschi G, Scipioni L, Abbott G, Digman MA.
Dynamic characterization of KCNQ1 and its regulatory subunits revealed by fluorescence fluctuation techniques.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 262a, 1285-Pos.KCNQ1 is the α subunit of a voltage-gated potassium channel expressed on the membrane of a variety of cells, including cardiac myocytes, where it has a key role in ventricular repolarization. KCNQ1 is known to interact with and be regulated by single-transmembrane domain ancillary subunits encoded by the family of KCNE genes, such as KCNE1 and KCNE2. Mutations in these genes are associated with life-threatening cardiac arrhythmias, including long QT syndrome.
