2024
Date: Tuesday, October 15, 2024
Time and Location: 11 am EST, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Diego Presman (U Buenos Aires)
Title: “Insights on Glucocorticoid Receptor Activity Through Live Cell Imaging”
Summary: Eucaryotic transcription factors (TFs) must sort out many constraints imposed by the nuclear architecture to find their target sites and modulate transcription. A non-stoichiometric relationship between response elements and TFs is starting to emerge as a new layer in transcriptional regulation, wherein clustering of many molecules involved in transcription ‘condense’ around enhancers to facilitate RNA polymerase II activity. Whether these transcriptional condensates positively or negatively affect transcription remains controversial. In this seminar, I will focus on the glucocorticoid receptor (GR), a ligand regulated TF involved in many physiological and pathological processes. Specifically, I will discuss how its quaternary structure and its ability to form condensates correlates with its transcriptional activity.
Date: Tuesday, September 17, 2024
Time and Location: 11 am EST, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Harshad Vishwasrao (NIH\NIBIB)
Title: “The latest aims of AIM (Advanced Imaging and Microscopy)”
Summary: AIM is a trans-NIH shared resource that houses, operates, disseminates, and improves next-generation and prototype optical imaging systems. AIM also provides support for advanced image processing, including applications of high-performance computing and artificial intelligence. Microscopy and image processing are fields of active research and development, with new technologies constantly emerging to enable ever better interrogation of biological systems. However, these emerging technologies typically require years to become easily accessible as user-friendly commercial products. There is a need for early adopters to make such technologies rapidly accessible to the broader research community. To fill this need, AIM monitors developments in microscopy and advanced image processing, identifies useful technologies, and makes them rapidly available to biologists at the NIH before they become easily and broadly accessible. This provides NIH researchers with unique tools for biological measurement and insight that are not available elsewhere.
Date: Tuesday, June 11, 2024
Time and Location: 11 am EST, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Simona Patange (NIST)
Title: “Evaluation of Fluid Force Microscopy (FluidFM) for genome editing in single cells”
Summary: Genome editing is a rapidly evolving biotechnology with the potential to transform many sectors of industry. To introduce genome editing biomolecules into cells, one must use a reagent delivery method that could either manipulate cells in bulk or at the single-cell level. In this talk, I present our results using the ‘FluidFM OMNIUM,’ a single-cell manipulation technology with applications for genome editing. I give an overview of the U.S. NIST Genome Editing Program and present our data on the OMNIUM measurement capabilities, including the ability to perform targeted single-cell injections, single-cell isolations, and 1-2 picoliter cytoplasmic extractions. Our research with FluidFM technology builds upon guidance from our recent publication (Patange and Maragh 2022) in which we summarize technologies related to research applications where it is advantageous to have controlled reagent delivery and high-resolution data on the distribution of genomic and phenotypic outcomes of individual cells within a population.
Reference: Patange S, Maragh S. Fire Burn and Cauldron Bubble: What Is in Your Genome Editing Brew? ACS Biochemistry 2022, Article ASAP. https://doi.org/10.1021/acs.biochem.2c00431
Date: Tuesday, May 21, 2024
Time and Location: 11 am EST, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Fernando Stefani (CIBION, Buenos Aires)
Title: “Fluorescence nanoscopy with sub-10 nm resolution”
Summary: Super-resolution fluorescence microscopy, also known as fluorescence nanoscopy, represented a breakthrough for bioimaging as it delivers sub-diffraction resolution using far-field microscopes. Although they do not face any fundamental limit, the resolution of the first generation of methods was bound by the limited photostability of fluorophores under ambient conditions to about 10-30 nm resolution. This has motivated the development of a second generation of fluorescence nanoscopy methods that aim to surpass sub-10 nm resolutions, thus providing true molecular resolution. In this talk, I will present the latest efforts of our lab to address this challenge trough four different approaches: pulsed-interleaved MINFLUX1, SIMPLER2, STED-FRET3, and RASTMIN4.
1. Masullo, L. A. et al. Pulsed Interleaved MINFLUX. Nano Lett. 21, 840–846 (2021).
2. Szalai, A. M. et al. Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules. Nat. Commun. 12, 517 (2021).
3. Szalai, A. M. et al. Super-resolution Imaging of Energy Transfer by Intensity-Based STED-FRET. Nano Lett. 21, 2296–2303 (2021).
4. Masullo, L. A. et al. An alternative to MINFLUX that enables nanometer resolution in a confocal microscope. Light Sci. Appl. 11, 199 (2022).
Date: Tuesday, April 16, 2024
Time and Location: 11 am EST, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. C-H Bear Huang (Johns Hopkins Med Sch)
Title: “Decoding Cellular Conversations: Unveiling Complex Signaling Networks with Multiplexed Biosensor Barcoding”
Summary: The survival of cells depends on their ability to properly respond to signals from other cells and the environment, mediated by a complex network of signaling molecules. The feedback loops in signaling networks give rise to dynamic spatiotemporal patterns that serve crucial functional roles. Our research is focused on how the dynamic behavior of the RTK/Ras/PI3K/ERK signaling network regulates cellular processes such as migration, proliferation, and metabolism. By employing a new method for highly multiplexed tracking of signaling activities in live cells, we provide insights into the regulatory mechanisms of signaling networks and the effects of oncogenic mutations. Our methodology and findings have implications for elucidating the structure and function of signaling networks in cancer and other diseases.
Date: Tuesday, February 20, 2024
Time and Location: 11 am EST, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Anthony Asmar (NIST)
Title: “Development of the Wide Scale Digital Optical Microscopy (WSDOM) laboratory for high throughput single cell tracking and analysis of iPSCs”
2023
Date: Tuesday, December 12, 2023
Time and Location: 11 am EST, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Martin Pauli (U Wuerzburg, Germany)
Title: “Freezing Life: SMLM Nanotopology in high pressure frozen physiological sample”.
Summary: Presynaptic active zones (AZs) are key structures for mediating and regulating neurotransmitter release. Although the key molecular components of AZs have been known for many years, their nanotopology and molecular dynamics remain elusive. Using single-molecule localization microscopy (SMLM) we could show that synaptic homeostasis involves subtle changes in AZ- nanotopology.
SMLM techniques are based on Immunofluorescence labelling and mostly used in aldehyde-fixed tissue. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure. We established a protocol that adopted HPF/Fs techniques to fit the requirements SMLM. We could demonstrate that presynaptic AZs are smaller in HPF samples showing a well-preserved structure, that allowed the analysis of subclusters.
Date: Tuesday, November 14, 2023
Time and Location: 11 am EST, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Sebastian Streichan (U California SB)
Title: “Physics of living matter: how the embryo gets its shape”.
Summary: Organ architecture is often composed of multiple concentric tissue layers. Morphogenesis folds these organs into a specific shape that is required for proper function. Genetic signals that determine cell fate have been uncovered – yet the dynamic interplay of tissue layers giving rise to specific form remains elusive. We combine multi-layer analysis of cellular dynamics on evolving surfaces with physical modeling to obtain testable quantitative descriptions of how genetic patterning controls physics giving rise to shape. I will discuss two examples: (I) Quantitative analysis of visceral organogenesis in D. melanogaster reveals how a hox code in the mesoderm triggers a dynamic molecular mechanism to control physical processes in the adjacent endoderm layer. (II) A chip-based culture system enables self-organization of micro patterned stem cells into precise three-dimensional cell-fate patterns and form. This system recreates aspects of neural tube folding and indicates basal interactions between non-neural and neural ectoderm are required for tube closure.
Date: Tuesday, October 17, 2023
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Sandra Vidak (NIH/NCI)
Title: “The role of molecular chaperones in a premature ageing disease”.
Summary: Human aging is the biggest risk factor for many diseases including cancer, cardiovascular and neurodegenerative diseases. Since several human premature aging syndromes are characterized by features resembling normal aging, they provide important insights into the molecular mechanisms underlying human aging. One such disease is Hutchinson-Gilford Progeria Syndrome (HGPS), an extremely rare premature aging disorder reflecting several aspects of normal aging. Classical HGPS is caused by a de novo heterozygous mutation in the LMNA gene encoding A-type lamins, major structural components of the cell nucleus. The HGPS-linked LMNA mutation leads to the expression of a mutant lamin A termed progerin, which causes numerous nuclear and cellular defects, including post-transcriptional reduction of select cellular proteins, pointing to an effect of the disease-causing progerin protein on protein homeostasis. To test this hypothesis, we have analyzed the levels and localization of several major cellular chaperones by high-throughput imaging. In inducible GFP-progerin and in patient-derived HGPS fibroblasts several endoplasmic reticulum (ER) chaperones localize at the nuclear periphery where they co-localize with the nuclear protein progerin. This re-localization is accompanied by the activation of an adaptive response to ER stress in vitro and in vivo, including an increase in various ER chaperones and transcriptional activation of Unfolded Protein Response (UPR). Interestingly, induction of ER stress is dependent on the inner nuclear membrane protein SUN2 and its ability to cluster in the nuclear membrane. Our observations suggest that the presence of nuclear protein aggregates can be sensed and signaled to the ER lumen via immobilization and clustering of an inner nuclear membrane protein. These results identify a novel mechanism of communication between the nucleus and the ER, and they provide insight into the molecular disease mechanisms of HGPS.
Date: Tuesday, September 19, 2023
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Ben Donovan (NIH/NCI)
Title: “Real-Time Visualization of Spliceosome Assembly in Live Cells”.
Summary: Among the first steps of spliceosome assembly is 3’ splice site (3’SS) recognition by the U2AF heterodimer. However, U2AF binds pervasively throughout the entire pre-mRNA. A long intron, for example, may contain nearly a hundred U2AF binding sites. Considering this, we sought to uncover the characteristics of U2AF binding that lead to productive spliceosome assembly at the correct location. Surprisingly, in vitro high throughput binding assays indicate that 3’SSs contain low-affinity U2AF binding sites, indicating that sequence alone does not impart the specificity required for accurate splice site selection. Despite this narrow distribution of RNA binding affinities, we observe a broad distribution of U2AF dwell times in live-cell single-molecule tracking assays which we show, through a combination of complementary in vitro and in vivo single-molecule assays, reflects a wide range of processes, from initial binding site sampling to involvement in spliceosome assembly. Importantly, these experiments establish an approach to uncover the kinetic regulation of splice site selection (from the E- to the A-complex) on endogenous pre-mRNAs and suggest a model where specificity is refined as the spliceosome progresses towards the A-complex. Therefore, we sought to identify additional factors that may interact with U2AF to influence the specificity and kinetics of spliceosome assembly. U2AF IP-MS identifies DDX42, an RNA helicase that competes with the helicase DDX46 to bind the SF3B1 component of the U2 snRNP. Interestingly in orbital tracking assays, where U2AF binding and pre-mRNA splicing are observed simultaneously, DDX42 knockdown stabilizes U2AF binding while DDX46 knockdown destabilizes binding. Together, these results provide new insight into how U2AF binding is interrogated during spliceosome assembly to ensure highly specific 3’SS recognition.
Date: Tuesday, June 20, 2023
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speakers: Alexander Peterson, PhD and Edward Kwee, PhD (NIST)
Title: “Quantifying viral vector heterogeneity through biophysical and infectivity assay imaging”
Summary: Characterization of viruses is critical for the development of gene delivery therapies and pandemic preparedness. We will present two applications of imaging to characterize gene delivery particle heterogeneity and quantify SARS-CoV-2 serum neutralization.First, we introduce a novel light scattering technique, interferometric dark-field total internal reflection microscopy to measure mass, size, and concentration of individual gene delivery particles to provide a multi-attribute comprehensive characterization of critical particle metrics to help characterize and enable a deeper understanding of their heterogeneity. We observe that different manufactured viral vector particles exhibited distinct mass profiles, suggesting potential differences in their structural composition or cargo content. Additionally, we observed a potential correlation between viral vector mass and infectivity, with heavier particles displaying enhanced infectivity compared to lighter counterparts.Second, we developed a cell based pseudovirus neutralization assay that measured neutralization by live cell imaging and flow cytometry. For live cell imaging, brightfield and fluorescence microscopy was performed on serum samples incubated with pseudotype particles expressing the SARS-CoV-2 spike protein and target cells. Image processing and analysis enabled quantification of infection and neutralization dynamics. The pseudovirus neutralization assay demonstrated reliable performance for detecting varying degrees of neutralization against SARS-CoV-2 in patient serum samples, with good agreement between live cell imaging and flow cytometry readouts.Together, these imaging approaches can provide a holistic approach to characterize virus heterogeneity with single particle characterization and infectivity quantification.
Date: Tuesday, May 16, 2023
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Steve Presse (Arizona State U)
Title: “Bayesian methods for FISH and single photon smFRET analysis”.
Summary: Biological processes are directed by molecular actors conspiring, often in small numbers, to achieve tasks relevant to life. Yet these actors are not directly observable. Within the fluorescence paradigm, multiple layers of stochasticity lie between what we care about (molecular actor behavior) and what we observe (detector counts). These layers include, but are not limited to, optical aberrations, detector noise, and photo-physics. As a direct consequence, we have been limited as a community to superresolving the positions of static molecules and obtaining snapshots of otherwise dynamical events. Here we present a probabilistic framework starting from single spot confocal and generalizing to widefield to demonstrate how we can leverage the statistics of the stochasticity to achieve superresolved tracking in crowded environments.
Date: Tuesday, April 18, 2023
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Greta Babakhanova (NIST)
Title: “Measuring cell viability with imaging-based methods and in 3D scaffolds”.
Summary: In the field of tissue engineering, 3D scaffolds and cells are often combined to yield constructs that are used as therapeutics to repair or restore tissue function in patients. Viable cells are often required to achieve the intended mechanism of action for the therapy, where the live cells may build new tissue or may release factors that induce tissue regeneration. Thus, there is a need to reliably measure cell viability in 3D scaffolds as a quality attribute of a tissue-engineered medical product. We developed a noninvasive, label-free, 3D optical coherence tomography (OCT) method to rapidly (2.5 min) image large sample volumes (1 mm3) to assess cell viability and distribution within scaffolds. OCT imaging was assessed using a model scaffold-cell system consisting of a polysaccharide-based hydrogel seeded with human Jurkat cells. Four test systems were used: hydrogel seeded with live cells, hydrogel seeded with heat-shocked or fixed dead cells and hydrogel without any cells. Time series OCT images demonstrated changes in the time-dependent speckle patterns due to refractive index (RI) variations within live cells that were not observed for pure hydrogel samples or hydrogels with dead cells. The changes in speckle patterns were used to generate live-cell contrast by image subtraction. In this way, objects with large changes in RI were binned as live cells. Using this approach, on average, OCT imaging measurements counted 326 ± 52 live cells per 0.288 mm3 for hydrogels that were seeded with 288 live cells (as determined by the acridine orange-propidium iodide cell counting method prior to seeding cells in gels). Considering the substantial uncertainties in fabricating the scaffold-cell constructs, such as the error from pipetting and counting cells, a 13% difference in the live-cell count is reasonable. Additionally, the 3D distribution of live cells was mapped within a hydrogel scaffold to assess the uniformity of their distribution across the volume. Our results demonstrate a real-time, noninvasive method to rapidly assess the spatial distribution of live cells within a 3D scaffold that could be useful for assessing tissue-engineered medical products.
Date: Tuesday, March 21, 2023
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Kaustubh Wagh (NIH/NCI)
Title: “Single-molecule tracking reveals dynamic switching between two low-mobility states for chromatin and chromatin-bound transcriptional regulators”.
Summary: How transcription factors (TFs) navigate the complex nuclear environment to assemble the transcriptional machinery at specific genomic loci remains elusive. Using single-molecule tracking, coupled with machine learning, we examined the mobility of multiple transcriptional regulators. We show that H2B and ten different transcriptional regulators display the same two low-mobility states. This suggests that, on our imaging timescales, we can study the dynamics of transcriptional regulators when they are bound to chromatin. Ligand activation results in a dramatic increase in the proportion of steroid receptors in the lowest mobility state. Mutational analysis revealed that only chromatin interactions in the lowest mobility state require an intact DNA-binding domain as well as oligomerization domains. Importantly, these states are not spatially separated as previously believed but in fact, individual molecules can dynamically switch between them. Together, our results identify two unique and distinct low-mobility states of chromatin-bound transcriptional regulators that appear to represent common pathways for transcription activation in mammalian cells.
2022
Date: Tuesday, February 21, 2022
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Yihan Wan (Westlake U, Hangzhou, China)
Title: “Single cell tracking of orchestrated transcription dynamics during stem cell fate decision”.
Summary: Transcription is a sporadic and stochastic process. Our previous study proved that the abundance and identity of mRNA molecules in a single cell are dynamic features. However, the stringent regulation in development and the stochasticity of gene expression seems to be a paradox. How do the sporadic transcription and stochastic splicing dynamics coordinate with lineage commitment? Here, I will present our preliminary data to elucidate the nascent RNA dynamics during human embryonic stem cell fate decisions. Through Fucci-assisted cell cycle tracking, barcoding mediated genome-wide nascent RNA dynamic tracking, we aim to reveal the real-time dynamics of global gene expression during the lineage commitment process.
Date: Tuesday, January 17, 2022
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Andrew Moore (HHMI/Janelia)
Title: “Vimentin intermediate filaments (IFs) play a role in clustering and stabilizing matrices of peripheral ER tubules”.
Summary: The endoplasmic reticulum (ER) is a continuous membrane-bound organelle found in all eukaryotes. In cultured cells, the ER adopts an elaborate net-like organization characterized by densely stacked sheets in the perinuclear region, sparsely interconnected tubules in the cell periphery, and a complex mixture of sheets and tubule matrices therebetween. Whilst the factors controlling the sheet/tubule balance have been extensively examined, the mechanisms by which these geometries are spatially segregated within the cytoplasm are not well understood. In this talk, I will outline our efforts to investigate the mechanism of ER positioning in cultured cells and focus on an unexpected role for vimentin intermediate filaments (IFs) in clustering and stabilizing matrices of peripheral ER tubules.
Date: Tuesday, December 13, 2022
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Roberto Weigert (NIH/NCI)
Title: “Imaging tumor initiation and progression in live animals at cellular and subcellular resolution”.
Summary: Intravital Microscopy is a powerful tool to image the dynamics of a variety of biological processes in live animals across scales, ranging from tissue, individual cells, and subcellular compartments.
Here, we will show how this technology can be applied to study at an unprecedented level of resolution the initiation and progression of tumor lesions within the same animal. The combination of 1) genetically engineered mice expressing a variety of fluorescently tagged molecules which highlight selected cell populations or subcellular compartments, and 2) the ability of non-linear microscopy to excite endogenous molecules such as NADH and FAD, or collagen I via second harmonic generation provide an unique way to follow the transformation of epithelial cells during tumor progression, their metabolic reprogramming, and the role of immune cells and the tumor microenvironment. Finally, examples of potential applications for the use of non-linear microcopy in diagnostics will be presented.
Date: Tuesday, November 8, 2022
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Edouard Bertrand (IGH, CNRS, Montpellier U, France)
Title: “Single molecule imaging reveals a new mechanism for RNA transport and local translation”.
Summary: Local translation allows for a spatial control of gene expression. Here, we performed RNA localization screen using high throughput smFISH and discovered mRNAs locally translated at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, the Golgi apparatus, the nuclear envelope and centrosomes. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. We also discovered that several mRNAs accumulated in foci distinct from P-bodies, which served as specialized translation sites, i.e. translation factories. Most interestingly, we found a conserved family of mRNAs that localize to centrosomes in both human and drosophila cells. These mRNAs localize to centrosomes at different stages of the cell cycle and some also localize to cilia in quiescent cells. Drug treatments and reporter analyses revealed that mRNA localization required translation of the nascent protein in cis. Moreover, using ASPM and NUMA1 as models, single mRNA and polysome imaging revealed active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. These data identify a conserved family of centrosomal mRNAs, which localize by a novel mechanism involving active polysome transport mediated by nascent proteins.
Date: Tuesday, October 18, 2022
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Jiji Chen (NIH\NIBIB)
Title: “Deep learning to denoise and enhance resolution for super resolution imaging”.
Summary: To obtain quality data in bioimaging, one needs to compromise between spatial resolution, temporal resolution, and signal to noise. It is difficult to optimize one factor without affecting the other two. To overcome these barriers, traditional methods including spatial filtering, wavelet thresholding, deconvolution, etc. have been used but typically cannot recover images to the desired level. Deep learning-based methods have shown very promising growth in image processing, analysis, and resolution enhancement. This talk will describe recent work we have done to develop a 3D residual channel attention network (3D RCAN) that enables simultaneous denoising and deconvolution of image volumes that is competitive with other state-of-the-art neural networks. 3D-RCAN allows us to acquire tens of thousands of images without apparent photobleaching, enhance confocal images to STED resolution, and iSIM images to expansion microscopy resolution. We have also integrated the 3D RCAN deep learning pipeline with a new 4 beam 3D SIM, enabling 120 nm isotropic resolution without using the reflected beam. 3D-RCAN and the 4beam 3D SIM are now available at the trans-NIH Advanced Imaging and Microscopy (AIM) Resource. AIM provides support for general image computation including software/pipeline development, large data visualization, high-performance computing, and AI assisted image segmentation/denoising.
Date: Tuesday, May 17, 2022
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Carsen Stringer (HHMI, Janelia)
Title: “Cellpose 2.0 : How to train your own model”.
Summary: Generalist models for cellular segmentation, like Cellpose, provide good out-of-the-box results for many types of images. However, such models do not allow users to adapt the segmentation style to their specific needs and may perform sub-optimally for test images that are very different from the training images. Here we introduce Cellpose 2.0, a new package which includes an ensemble of diverse pretrained models as well as a human-in-the-loop pipeline for quickly prototyping new specialist models. We show that specialist models pretrained on the Cellpose dataset can achieve state-of-the-art segmentation on new image categories with very little user-provided training data. Models trained on 500-1000 segmented regions-of-interest (ROIs) performed nearly as well as models trained on entire datasets with up to 200,000 ROIs. A human-in-the-loop approach further reduced the required user annotations to 100-200 ROIs, while maintaining state-of-the-art segmentation performance. This approach enables a new generation of specialist segmentation models that can be trained on new image types with only 1-2 hours of user effort. We provide software tools including an annotation GUI, a model zoo and a human-in-the-loop pipeline to facilitate the adoption of Cellpose 2.0.
Date: Tuesday, April 19, 2022
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Michael W. Halter (NIST)
Title: “Quantitative live imaging of pluripotent stem cells to probe gene regulatory networks”.
Summary: Induced pluripotent stem cell (iPSC) populations are complex, dynamic and heterogeneous. Individual cells within a population are constantly changing all while maintaining the capacity to differentiate into numerous possible cell types. Time lapse imaging of live pluripotent stem cells can be used to follow the large numbers of individual cells over time, but the analysis of images necessary to segment and track single cells is challenging. Image analysis based on deep learning algorithms is dramatically improving the capability for obtaining dynamic information from single cells. Our work focuses on using fluorescent protein reporters to monitor the dynamics of gene expression, but the label-free image analysis pipelines are potentially very general. This talk will discuss 1) measuring a modeling gene expression dynamic in single cells, 2) measurement assurance strategies for single cell dynamics from time lapse images, and 3) the development of high speed imaging systems that can generate training and testing data for the analytical pipelines will be discussed.
Date: Tuesday, March 15, 2022
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. David J. Nesbitt (U. Colorado, Boulder)
Title: “Kinetics and Thermodynamics of Nucleic Acid Folding: “Raising the bar” for Real Time Studies at the Single Molecule Level”.
Summary: The ability to look with laser microscopy at single biomolecules has led to a revolution in research opportunities for chemistry, physics and molecular biology. This talk will present three “vignettes” with the common theme of confocal microscopy, fluorescence resonance energy transfer (FRET), and single photon counting methods for single molecule kinetics and thermodynamics of conformational RNA folding into biocompetent structures. (1) Exquisite temperature control in single molecule “nanobathtubs” is used to permit systematic deconstruction of free energies landscapes (DG0) into enthalpic (DH0) and entropic (-TDS0) components, as well as begin to elucidate properties of transition state barriers (e.g., DH‡, DS‡) for folding/unfolding. (2) the effect of molecular “crowding” on RNA/DNA loop/stem formation and hybridization at the single molecule level, to explore conditions relevant to in vivo crowding in the cellular cytoplasm. (3) recent extensions of these methods into the kinetics and thermodynamics of folding/unfolding at high hydrostatic pressures (Pext = 1-4000bar), which allows one to interrogate the impact of sequence, mono/divalent cations, ligands, osmolytes, etc. on stabilities and free volumes (DV0, DV‡) for nucleic acid folding. A unifying goal in these vignettes is the development of simple physical pictures to help us interpret, explain, and potentially control the underlying biophysics of nucleic acid folding at the single molecule level.
Date: Tuesday, February 15, 2022
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Stephen A Boppart (U Illinois, Urbana Champaign)
Title: “Imaging for Biomarkers of Cancer using Simultaneous Label-free Auto-fluorescence Multi-harmonic (SLAM) Microscopy”.
Summary: Innovations in biomedical imaging have historically led to discoveries in the life sciences and new detection and diagnostic technologies in medicine and surgery. Label-free intravital optical imaging and imaging of fresh, unstained, resected tissue specimens, offers a wealth of new biomarkers for revealing the true colors of cancer and diagnosing disease. Using new optical source technology and nonlinear optics to generate new excitation wavelengths and manipulate the light stimulus in new ways, Simultaneous Label-free Auto-fluorescence Multi-harmonic (SLAM) microscopy can achieve fast simultaneous visualization of the rich intrinsic molecular and metabolic features within tissues. Quantitative machine/deep learning analyses of these multi-dimensional datasets can be used to identify selective biomarkers for cancer. Specifically, tumor-associated extracellular vesicles (EVs) were analyzed via their optical signatures and spatial distributions. Analysis showed that EVs from the tumor microenvironment have unique optical signatures, in comparison to those from healthy subjects. The clinical demonstration of these optical biomedical imaging technologies offers new paradigms for point-of-procedure diagnosis and guidance.
Date: Tuesday, January 18, 2022
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Matthew L. Ferguson (Boise State U)
Title: “Real Time Single Molecule Live Cell Measurement of Gene Activation by 3D Orbital Tracking Fluorescence Cross Correlation Spectroscopy”.
Summary: Mechanisms of transcription and translation take the information encoded in the genome and make it “work” in cells, through the production of proteins defined by nucleic acid coding regions. This involves the coordination of many multi-subunit complexes about which most knowledge is inferred from ensemble and/or in vitro assays, giving a detailed but static picture. How these macromolecular machines coordinate in living cells remains unknown but recent advances in the application of fluctuation analysis to time resolved multi-color fluorescence imaging can now give an unprecedented level of dynamic in vivo information. My talk will describe recent applications of 3D Orbital Tracking and Fluorescence Cross Correlation Spectroscopy to the study of transcriptional activation in living cells and outline possible future applications. Orbital tracking provides faster sampling and longer measurements than traditional microscopy, while minimizing photobleaching. Using these methods, we can begin to understand and model the living genome.
2021
Date: Tuesday, December 14, 2021
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Boyang Hua (Johns Hopkins MI)
Title: “Monitoring co-transcriptional RNA folding in real time with smFRET”.
Summary: We develop an RNA polymerase-free assay to capture the “essence” of co-transcriptional RNA folding process. Combining this assay with the powerful single-molecule FRET techniques, we observe how individual ribozymes and riboswitches choose their fold and make regulatory decisions in real time. The lessons learnt from these observations help us tease out the effects of different rate parameters in the kinetically controlled folding regime.
Date: Tuesday, November 9, 2021
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Jadranka Lonkarek (NIH/NCI)
Title: “Combining various super resolution approaches to decipher centrosome organization”.
Summary: Centrosomes are small, ~500 nm in diameter, multifunctional membrane-less organelles, that are critically important for development, signaling and cell- and organ- homeostasis, and proper chromosome segregation during mitosis. Centrosomes were first described by Edouard Van Beneden and named and linked to chromosome segregation by Theodor Boveri around 1870. In the 1960-1980s, electron microscopy studies have unraveled the remarkable nine-fold symmetrical ultrastructure of a centriole — a microtubular cylindrical structure that resides within a centrosome and organizes it. About 15 years ago, proteomics and genomic screens identified hundreds of centriole and centrosome core proteins and revealed the evolutionary highly conserved nature of the centriole assembly pathway across multiple organisms. Nowadays, super resolution microscopy approaches and improvements in cryo-tomography are enabling us to build a nanoscale-detailed picture of the centriole and centrosome architecture. In this seminar, I will present our efforts to combine various super resolution methods to decipher intra-centrosomal organization of a human centrosome and to understand how centrosomal dynamic localization of various centrosomal proteins affects centriole and centrosome cycle and function.
Date: Tuesday, October 19, 2021
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Krishna Mudumbi (Yale Cancer Biol Inst)
Title: “EGFR dimerization and phosphorylation by single-molecule microscopy”.
Summary: Epidermal growth factor receptor (EGFR) is a single-pass transmembrane protein and a member of the receptor tyrosine kinase family of proteins that is critically involved in the regulation many cellular pathways. EGFR is comprised of five major domains: the extracellular ligand binding domain, transmembrane domain, intracellular juxtamembrane domain, kinase domain, and the tyrosine residue containing C-tail. Ligand binding induces dimerization of EGFR, which is believed to be the key step in receptor activation and signaling. Previous work has shown that receptor mobility can be used to determine monomeric and dimeric states of the receptor – with a reduction in diffusivity as a readout for dimerization. However, it is unclear how other cellular factors, such as phosphorylation and adaptor protein recruitment, may contribute to this change in receptor mobility. Furthermore, each of the five major domains of EGFR have been previously shown to play a role in the dimerization of the receptor, but the relative contribution of each element to EGFR dimerization is unknown. Using several single-molecule microscopy techniques, we monitored the contribution of phosphorylation, adaptor protein recruitment, and of each individual domain of EGFR to its dimerization kinetics in live cells, giving us valuable insight into the mechanisms underlying receptor dimerization.
Date: Tuesday, September 14, 2021
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. David Alejandro Garcia Grisales (NIH/NCI, U. Maryland)
Title: “The Role of Transcription Factor Dynamics in Gene Expression: Does Time Matter?”.
Summary: Transcription factors (TFs) regulate gene expression by binding to specific consensus motifs within the local chromatin context. The mechanisms by which TFs navigate the nuclear environment as they search for binding sites remain unclear. Here, we used single-molecule tracking and machine-learning based classification to directly measure the nuclear mobility of the glucocorticoid receptor (GR) in live cells. We revealed two distinct and dynamic low-mobility populations. One accounts for specific binding to chromatin, while the other represents a confinement state that requires an intrinsically disordered region (IDR), implicated in liquid-liquid condensate subdomains. Further analysis showed that the dwell times of both subpopulations follow a power-law distribution, consistent with a broad distribution of affinities on the GR cistrome and interactome. Altogether, our data link IDRs with a confinement state that is functionally distinct from specific chromatin binding and modulates the transcriptional output by increasing the local concentration of TFs at specific sites.
Date: Tuesday, June 15, 2021
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Marina Feric (NIH/NCI)
Title: “The mitochondrial genome as a transcriptional condensate”.
Summary: Mitochondria contain their own genome (mitochondrial DNA, mtDNA). mtDNA is not soluble in the mitochondrial matrix, but instead, is packaged by proteins to form membrane-less nucleoprotein structures called mitochondrial nucleoids. We have recently found that phase separation is the primary physical mechanism for assembly and size-control of mitochondrial nucleoids. The major mt-nucleoid architectural protein TFAM spontaneously phase separates on its own via weak, multivalent interactions into droplets with slow internal dynamics in vitro. Indeed, the mitochondrial transcriptional components, including mtDNA, the polymerase POLRMT, and transcription factor TFB2M, further partition with TFAM to form highly heterogenous, viscoelastic droplets in vitro, which recapitulate the dynamics and behavior of mt-nucleoids in vivo. Transcription can be targeted to these mitochondrial condensates in vitro, leading to significant structural changes associated with localized RNA production, yet with dampened kinetics compared to when all components are fully solubilized. The mitochondrial transcriptional machinery thus serves as a model system for studying the biophysics and kinetics of transcriptional condensates. Our results point to phase separation as an evolutionarily conserved mechanism of genome organization and function.
Date: Tuesday, April 20, 2021
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Vu Nguyen (Johns Hopkins U)
Title: “Spatio-temporal coordination of transcription preinitiation complex assembly in live cells”.
Summary: Transcription initiation by RNA polymerase II (Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus where thousands of promoters are broadly distributed in chromatin, it is unclear how ten individual components converge on any target to establish the PIC. Here, we use live-cell, single-molecule tracking in S. cerevisiae to document spatially constrained exploration of the nucleoplasm by PIC components, which is guided by the large coactivator complexes Mediator and TFIID. On chromatin, Mediator and TFIID/TBP instruct assembly of a short-lived PIC, which occurs infrequently but efficiently at an average promoter where initiation-coupled disassembly may occur within a few seconds. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Collectively, our study provides a global spatio-temporal model for transcription initiation in live cells.
Date: Tuesday, March 16, 2021
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. Varun Sood (NIH/NCI)
Title: “Identification of chromatin determinants of gene bursting”.
Summary: Stochastic bursting is a fundamental and an evolutionarily conserved feature of transcription, yet it is unclear how chromatin modulates the size and frequency of transcriptional bursts. Lack of high throughput models for testing the direct effects has limited a comprehensive understanding. We have performed comprehensive imaging-based screens of libraries of more than five hundred direct inhibitors of chromatin modifying enzymes for a set of twelve endogenous genes that have a wide range of bursting. We screened in two steps. First, we adapted nascent RNA FISH to estimate transcription site frequencies and used it as a parameter to assay acute changes in bursting in a high throughput imaging setup. Next, we validated hits by assaying changes in bursting kinetics using MS2 reporter cell lines. We discovered four broad categories of modulators of gene bursting: Inhibitors of histone deacetylase (HDAC-i), bromodomain containing proteins, demethylases and Janus kinases. Amongst these, HDAC-i showed the strongest impact on bursting that resulted from a 10-30% decrease in the mean Off-time. This decrease was restricted to the longer Off-times while the shorter Off-times were unaffected. On the other hand, changes in On-time were gene specific. HDAC-i also decreased the burst size for several genes across three cell lines and drastically increased the heterogeneity of burst sizes for MYC, EGFR and P53 in MCF7 cells. At the chromatin level, the HDAC-i primarily increased the gene body acetylation of histone 3 lysine 27. However, these changes were quantitatively different between genes with increased vs decreased bursting. Our results suggest that chromatin can regulate both size and frequency of bursting. Modulation of bursting occurs by changing the fraction of long Off-times. Finally, the gene specific regulation of bursting is influenced by both chromatin context and signaling.
Date: Tuesday, February 16, 2021
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. James McNally (Helmholtz Center, Berlin)
Title: “Cryo soft X-ray tomography: a tool for unbiased detection of ultrastructural changes in perturbed cells”.
Summary: Soft X-ray tomography enables 3D nanoscale imaging of intact, unstained biological cells in their near native state, subject only to cryo-preservation. This talk will discuss the underlying principles of the technique and illustrate some of its applications. Soft X-ray tomography can visualize most cytoplasmic organelles at resolutions approaching 30 nm without staining or chemical fixation. Cells are instead cryo-preserved, and cellular structure is detected based on the natural contrast of organic matter in this soft X-ray energy range. Furthermore, correlative fluorescence and X-ray microscopy can also be performed on the same sample under cryo conditions. These capabilities are ideally suited for initial, unbiased detection of changes in cell ultrastructure induced by any sort of perturbation: unbiased because no a priori knowledge is required about which cellular components should be stained (in contrast to fluorescence microscopy), and because the entire 3D cell volume is visible and so rare or localized changes are easily found (in contrast to electron microscopy). We illustrate how these capabilities have enabled the detection of structural changes induced by nanoparticle uptake into cells, which we found induces a cytoplasmic reorganization that had not been seen before by traditional microscopy analyses. These changes in cytoplasmic organelle content after nanoparticle uptake may reflect a more common cellular response to nanoparticles, which are now widely used in commercial products, as well as in medicine, including the mRNA-based Covid 19 vaccines.
Kepsutlu, B., Wycisk, V., Achazi, K., Kapishnikov, S., Pérez-Berná, A.J., Guttmann, P., Cossmer, A., Pereiro, E., Ewers, H., Ballauff, M., Schneider, G. and McNally, J.G. (2021) Cells undergo major changes in the quantity of cytoplasmic organelles after uptake of gold nanoparticles with biologically relevant surface coatings. ACS Nano 14:2248-2264.
Müller, W.G., Heymann J.B., Nagashima, K., Guttmann, P., Werner, S., Rehbein, S., Schneider, G., McNally, J.G. (2011) Towards an atlas of mammalian cell ulstrastructure by cryo soft X-ray tomography. J. Struct. Biol. 177:179-192.
Schneider, G., Guttmann, P., Heim, S., Rehbein, S., Mueller, F., Nagashima, K., Heymann, J.B., Müller, W.G., McNally, J.G. (2010) Three-dimensional cellular ultrastructure resolved by X-ray microscopy. Nat. Meth. 7:985-987.
Date: Tuesday, January 19, 2021
Time and Location: 11 am, ZOOM (INVITATION BY LMIG LIST SERVER)
Speaker: Dr. James Zhe Liu (HHMI, Janelia)
Title: “Organizing mechanism of the accessible genome”.
Summary: To image active chromatin at nanometer scale in situ, we developed 3D ATAC-PALM that integrates the assay for transposase-accessible chromatin (ATAC), PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA–FISH, RNA–FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we identify the BET family scaffold protein BRD2 as a key factor responsible for compartmentalization of the accessible genome. Specifically, BRD2 mixes and compacts active compartments in the absence of Cohesin. This activity is independent of transcription but requires BRD2 to recognize acetylated nucleosomes through its double bromodomain. We also show that BRD2 safeguards compartmental boundaries by preventing intermingling between active and inactive chromatin. Notably, genome organization mediated by BRD2 is antagonized on one hand by Cohesin and on the other by the BET homolog protein BRD4, both of which inhibit BRD2 binding to chromatin. Polymer simulation of the data supports a BRD2-Cohesin ‘tug-of-war’ model of nuclear topology, where genome compartmentalization results from a competition between loop extrusion and chromatin state-specific affinity interactions.