Quantitative imaging of protein networks and genome structure in single human cells

Jan Ellenberg

Cell Division and Nuclear Organization, European Molecular Biology Laboratory

Abstract:

The rapid development of new imaging technologies allows unprecedented insights into the molecular

machinery inside living cells and organisms. For the first time, light and electron microscopy have

molecular sensitivity and resolving power in situ, and, used together, can connect the scales of atomic

structural detail and molecular dynamics with the whole living cell. Aided by machine learning driven

image analysis powered by open sharing of image data, this provides unprecedented opportunities for

new insights into the molecular mechanisms that drive life’s core functions at the scale of the cell.

I will present the progress we have made to study the dynamic protein network, assembly of individual

protein complexes and genome re-folding that are essential for one of life’s most fundamental functions,

cell division. Our work has studied cell division in human cells and early mammalian embryos using

advanced imaging methods, including light-sheet, quantitative fluorescence correlation spectroscopy

(FCS)-calibrated, super-resolution and correlative light and electron microscopy. The quantitative

molecular data that these new technologies deliver, allow us to better understand how the molecular

machinery functions in space and time to ensure faithful cell division and prevent errors, that underlie

congenital disease, infertility and cancer.

The exciting opportunities for open access to such cutting-edge imaging technologies provided by the

EMBL Imaging Centre and support in image data archiving and sharing provided by the EMBL Bioimage

Archive will also be discussed.

Multi-cellular dynamics measurement and their manipulation

Hiroaki Wake 

Division of Multicellular Circuit Dynamics, NIPS/Department of Anatomy and Molecular Cell Biology

Nagoya University Graduate School of Medicine

Abstract:

The interaction of neuron and glia is essential for functional neuronal circuits. We have been especially focused on microglia, a sole immune cell in CNS. In addition to the pathological function of microglia, recent developments in molecular probes and optical imaging in vivo have revealed that microglia are highly motile cell in the healthy brain, extending and retracting their process that extend from a largely stationary cell soma. We reveal their physiological and pathological function on synapse and vessels. In this session, we will show microglial role for cross modal plasticity and show how they can contribute on sensory discrimination by their effect on neural circuit function. In addition, we will introduce our recent developed holographic microscope that can precisely measure and manipulate neuron and glial cell activities in a spatiotemporal manner in living mice to ultimately affect on behavior output. Furthermore, we will show our successful evaluation method to analyze the functional neuronal connectivity to integrate the transcriptome

information.

High-speed fluorescence microscopy and beyond: a new approach to collecting big data from life

Hideharu Mikami

Laboratory of Information Biophotonics,  Research Institute for Electronic Science, Hokkaido University.

Abstract:

Fluorescence microscopy is a crucial tool in biomedical research, as it provides insights into the morphology of complex organisms. In recent decades, the spatial resolution of fluorescence microscopy has greatly improved by the emergence of super-resolution techniques. However, its temporal resolution (imaging speed) has been overlooked and limits its usage. To overcome this limitation, researchers have been developing high-speed fluorescence microscopy techniques. High-speed imaging allows for the study of rapid biological processes, as well as the generation of large amounts of data for single-cell analysis, 3D imaging of tissues, and real-time recording of living organisms. As a result, high-speed fluorescence microscopy can serve as a big-data generator in life sciences. For instance, an ultrafast confocal fluorescence microscope has been developed that can acquire images at a remarkable speed (2D frame rate of 16,000 frames/s, 3D volume rate of 104 volumes/s, and cellular image acquisition throughput of >10,000 cells/s), generating approximately 10 TB of data in an hour. To analyze such large volumes of data, automated methods such as machine learning are needed. This presentation will cover recently developed high-speed fluorescence microscopy techniques, as well as data analysis using machine learning. Finally, the future of high-speed fluorescence imaging, which will involve the integration of photonics and information technologies, will be discussed.

Elucidating the neural mechanisms of subjective experience through cognitive biases and neuroimaging

Makiko Yamada

Nat. Inst. for Quantum Sci. and Tech.

Abstract: 

Subjective experience encompasses the internal sensations and perceptions unique to an individual, representing a personal reality that is inherently private and inaccessible to others. This presentation will discuss our research into the molecular and neural mechanisms that give rise to these subjective experiences. A key focus of our approach is the use of perceptual illusions and cognitive biases as tools to objectively quantify subjective experience. By measuring the extent of these distortions, we aim to provide an objective assessment of subjective phenomena. We will present findings that integrate these assessments with functional brain activity observed through fMRI and molecular imaging via positron emission tomography (PET), with particular emphasis on the role of the dopaminergic system. Our research offers insights into the neural underpinnings of subjective experience, advancing our understanding of the brain mechanisms involved.

Label-retention expansion microscopy and its applications in nuclear organization

Xiaoyu Shi                                                                                                                                                        Dept Developmental & Cell Biology, Chemistry, and Biomedical Engineering, University of California, Irvine

Abstract:

Between genotype and phenotype lies a complex landscape of mechanisms of action, where structure-function relationships are vital for understanding these mechanisms. There is ongoing debate regarding whether direct manipulation of structure-function relationships can facilitate phenotypic rescue, or whether gene editing is invariably required. Our research supports the former possibility. In this study, we present a case study on nuclear deformation and illustrate a method for rescuing its influence on ribosome biogenesis by artificially reshaping the nuclei in live cells.

Both cancer and progeria cells exhibit two common structural and functional characteristics: a deformed nuclear envelope and elevated ribosome biogenesis. The existence of a direct relationship between these two features, however, is unknown. We uncovered their strong correlation by employing Label-retention expansion microscopy (LR-ExM). With the super-resolution provided by LR-ExM, we identified spatial contacts between the nucleolus and nuclear envelope invaginations in nearly every examined breast cancer cell. Surprisingly, only contacts with high-curvature invaginations were associated with increased rRNA levels in the nucleolus, whereas contacts with low-curvature indentations appeared benign. Informed by this insight, we utilized nanopillars to transform high-curvature nuclear invaginations into low-curvature indentations by growing breast cancer cells on the nanopillars with low curvatures. Remarkably, this led to a significant reduction of rRNA levels in nucleoli to those comparable with normal breast cells. This structural intervention in ribosome biogenesis applies to breast cancer, progeria, and even wild-type cells, suggesting a universal aspect of nuclear-nucleolus interaction in cell biology.