Subcellular Dynamics Research Articles

Intravital imaging with two-photon microscopy reveals cellular dynamics in the ischeamia-reperfused rat heart

Recent advances in intravital microscopy have provided insight into dynamic biological events at the cellular level in both healthy and pathological tissue. However, real-time in vivo cellular imaging of the beating heart has not been fully established, mainly due to the difficulty of obtaining clear images through cycles of cardiac and respiratory motion. Here we report the successful recording of clear in vivo moving images of the beating rat heart by two-photon microscopy facilitated by cardiothoracic surgery and a novel cardiac stabiliser. Subcellular dynamics of the major cardiac components including the myocardium and its subcellular structures (i.e., nuclei and myofibrils) and mitochondrial distribution in cardiac myocytes were visualised for 4–5 h in green fluorescent protein-expressing transgenic Lewis rats at 15 frames/s. We also observed ischaemia/reperfusion (I/R) injury-induced suppression of the contraction/relaxation cycle and the consequent increase in cell permeability and leukocyte accumulation in cardiac tissue. I/R injury was induced in other transgenic mouse lines to further clarify the biological events in cardiac tissue. This imaging system can serve as an alternative modality for real time monitoring in animal models and cardiological drug screening, and can contribute to the development of more effective treatments for cardiac diseases.

Exposure to naphthenic acids and the acid extractable organic fraction from oil sands process-affected water alters the subcellular structure and dynamics of plant cells

Oil sands surface mining generates vast quantities of oil sands process-affected water (OSPW) as a by-product of bitumen extraction. The acid extractable organic (AEO) fraction of OSPW contains several contaminants, including naphthenic acids (NAs). While responses of living organisms to NA and AEO exposure have been described at the developmental, physiological, metabolic and gene expression levels, the effects of these compounds at the cellular and subcellular level are limited. Using live cell fluorescence microscopy and a suite of fluorescent marker proteins, we studied the intracellular responses of the plant cell cytoskeleton and several membrane-bound organelles to NA and AEO treatments. A rapid disassembly of cortical microtubules and a decrease in dynamics associated with actin filaments was observed in response to these treatments. Concomitantly, the integrity and dynamics of mitochondria, peroxisomes, Golgi stacks, and endoplasmic reticulum were also altered. AEO treatments were the most toxic to cells and resulted in the accumulation reactive oxygen species. This study provides foundational evidence for intracellular responses to NA and AEO exposure using two evolutionarily diverse model plant cell types. This cellular assay could be used to identify the most toxic components of AEO sub-fractions, and assist in determining the effectiveness of OSPW remediation efforts.

Mechanical Regulation of Photosynthesis

Photosynthesis is regulated in response to many diverse stimuli including light intensity and wavelength, nutrient availability, and temperature to prevent formation of reactive oxygen species and cell death. However, it has remained a challenge to investigate this regulation in the context of a living photosynthetic cell due to the heterogeneity inherent to batch culture analysis. To overcome this obstacle, we utilized long-term, quantitative time-lapse fluorescence microscopy to visualize the growth and subcellular dynamics of individual wildtype and mutant cyanobacterial cells over multi-generational lineages. Our results demonstrate that in addition to known abiotic regulators of photosynthesis, mechanical perturbations lead to attenuation of photosynthesis and can play a major role in governing cellular fitness and productivity in cyanobacteria. These results provide insight into how photosynthetic organisms navigate the physical environment and a new perspective on source-sink regulation of photosynthesis that incorporates conversion of light into mechanical energy.

A Proteomic Map to Navigate Subcellular Reorganization in Fatty Liver Disease

In a tour de force in this issue of Developmental Cell, Krahmer etal. leverage proteomic platforms to reveal subcellular proteome dynamics during development of nonalcoholic fatty liver disease. Their findings uncover a remarkable degree of subcellular reorganization, alterations in protein localization, and remodeling of organelle contact sites.

Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein.

Alpha-synuclein (aSyn) is a central player in Parkinson's disease (PD) but the precise molecular mechanisms underlying its pathogenicity remain unclear. It has recently been suggested that nuclear aSyn may modulate gene expression, possibly via interactions with DNA. However, the biological behavior of aSyn in the nucleus and the factors affecting its transcriptional role are not known. Here, we investigated the mechanisms underlying aSyn-mediated transcription deregulation by assessing its effects in the nucleus and the impact of phosphorylation in these dynamics. We found that aSyn induced severe transcriptional deregulation, including the downregulation of important cell cycle-related genes. Importantly, transcriptional deregulation was concomitant with reduced binding of aSyn to DNA. By forcing the nuclear presence of aSyn in the nucleus (aSyn-NLS), we found the accumulation of high molecular weight aSyn species altered gene expression and reduced toxicity when compared with the wild-type or exclusively cytosolic protein. Interestingly, nuclear localization of aSyn, and the effect on gene expression and cytotoxicity, was also modulated by phosphorylation on serine 129. Thus, we hypothesize that the role of aSyn on gene expression and, ultimately, toxicity, may be modulated by the phosphorylation status and nuclear presence of different aSyn species. Our findings shed new light onto the subcellular dynamics of aSyn and unveil an intricate interplay between subcellular location, phosphorylation and toxicity, opening novel avenues for the design of future strategies for therapeutic intervention in PD and other synucleinopathies.

HCPro-mediated transmission by aphids of purified virions does not require its silencing suppression function and correlates with its ability to coat cell microtubules in loss-of-function mutant studies

Native and amino acid (aa) substitution mutants of HCPro from potato virus Y (PVY) were transiently expressed in Nicotiana benthamiana leaves. Properties of those HCPro variants with regard to silencing suppression activities, mediation of viral transmission by aphids, and subcellular localization dynamics, were determined. One mutant failed to suppress silencing in agropatch assays, but could efficiently mediate the transmission by aphids of purified virions. This mutant also retained the ability to translocate to microtubules (MTs) in stressed cells. By contrast, another single aa substitution mutant displayed native-like silencing suppression activity in agropatch assays, but could not mediate transmission of PVY virions by aphids, and could not relocate to MTs. Our data show that silencing suppression by HCPro is not required in the aphid-mediated transmission of purified virions. In addition, since the same single aa alteration compromised both, viral transmission and coating of MTs, those two properties could be functionally related.

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

RGB‐Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells

AbstractAdenosine triphosphate (ATP) provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to our understanding of the mechanisms underlying cellular energy metabolism. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling molecules. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with additional fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca2+ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metabolism.

RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells.

Adenosine triphosphate (ATP) provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to our understanding of the mechanisms underlying cellular energy metabolism. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling molecules. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with additional fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca2+ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metabolism.

A Proposed Chaperone of the Bacterial Type VI Secretion System Functions To Constrain a Self-Identity Protein.

The bacterium Proteus mirabilis can communicate identity through the secretion of the self-identity protein IdsD via the type VI secretion (T6S) system. IdsD secretion is essential for self-versus-nonself recognition behaviors in these populations. Here we provide an answer to the unresolved question of how the activity of a T6S substrate, such as IdsD, is regulated before secretion. We demonstrate that IdsD is found in clusters that form independently of the T6S machinery and activity. We show that the IdsC protein, which is a member of the proposed DUF4123 chaperone family, is essential for the maintenance of these clusters and of the IdsD protein itself. We provide evidence that amino acid disruptions in IdsC are sufficient to disrupt IdsD secretion but not IdsD localization into subcellular clusters, strongly supporting the notion that IdsC functions in at least two different ways: maintaining IdsD levels and secreting IdsD. We propose that IdsC, and likely other DUF4123-containing proteins, functions to regulate T6S substrates in the donor cell both by maintaining protein levels and by mediating secretion at the T6S machinery.IMPORTANCE Understanding the subcellular dynamics of self-identity proteins is crucial for developing models of self-versus-nonself recognition. We directly addressed how a bacterium restricts self-identity information before cell-cell exchange. We resolved two conflicting models for type VI secretion (T6S) substrate regulation by focusing on the self-identity protein IdsD. One model is that a cognate immunity protein binds the substrate, inhibiting activity before transport. Another model proposes that DUF4123 proteins act as chaperones in the donor cell, but no detailed molecular mechanism was previously known. We resolve this discrepancy and propose a model wherein a chaperone couples IdsD sequestration with its localization. Such a molecular mechanism restricts the communication of identity, and possibly other T6S substrates, in producing cells.

Engineering the Synaptic Kinetic Process into Memristive Device

AbstractThe important synaptic functionalities, such as long‐term memory, short‐term memory, and experience‐dependent learning, are extensively emulated by the emerging memristive devices, which provide a main step toward brain inspired computing. The majority of the electronic synapses based on memristors usually focus on the emulation of the physiological behaviors at the cellular level. However, the research on the subcellular synaptic dynamic properties of electronic synapses is rarely reported. In this article, Ag/Ta2O5/Pt‐based memristors are utilized to emulate both the long‐term memory and short‐term facilitation of biological synapse. Under pulse‐train measurements, the device exhibits short‐term facilitation depending on varying the electrical pulse intensity, duration, and frequency. Based on the experimental results, one‐to‐one correspondence is established between the filament dynamics in our device and the subcellular synaptic dynamics during short‐term facilitation. By fitting the experimental results into the biological model, the dynamic displacements of the rear and head ends of the Ag filament are drawn analogy to the neurotransmitter and Ca2+ dynamics. The dynamic process is explained by simulations of the nanoparticle dynamics in which the Ag filament is simplified to a 1D “stick.” This research enables the emulation of synaptic function with improved biofidelity and broad applications to neuromorphic computing.

Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms.

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.

Carotenoid dynamics and lipid droplet containing astaxanthin in response to light in the green alga Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis accumulates large amounts of the red ketocarotenoid astaxanthin to protect against environmental stresses. Haematococcus cells that accumulate astaxanthin in the central part (green-red cyst cells) respond rapidly to intense light by distributing astaxanthin diffusively to the peripheral part of the cell within 10 min after irradiation. This response is reversible: when astaxanthin-diffused cells were placed in the dark, astaxanthin was redistributed to the center of the cell. Although Haematococcus possesses several pigments other that astaxanthin, the subcellular distribution and content of each pigment remain unknown. Here, we analyzed the subcellular dynamics and localization of major pigments such as astaxanthin, β-carotene, lutein, and chlorophylls under light irradiation using time-lapse and label-free hyperspectral imaging analysis. Fluorescence microscopy and freeze-fracture transmission electron microscopy showed that, preceding/following exposure to light, astaxanthin colocalized with lipid droplets, which moved from the center to the periphery through pathways in a chloroplast. This study revealed that photoresponse dynamics differed between astaxanthin and other pigments (chlorophylls, lutein, and β-carotene), and that only astaxanthin freely migrates from the center to the periphery of the cell through a large, spherical, cytoplasm-encapsulating chloroplast as a lipid droplet. We consider this to be the Haematococcus light-protection mechanism.

Revealing Subcellular Redox Dynamics with Multiplex Imaging of Compartment‐Specific Redox Probes

Reactive oxygen species (ROS), redox enzymes, and redox buffers are central to the propagation of redox signals and oxidative stress between cells in the tissue environment and between organelles in the cellular environment. For example, hydrogen peroxide generated in mitochondria can alter the thiol oxidation states on proteins to alter their function, and subsequently redox active biomolecules such as glutathione can act as reducing equivalents in the face of oxidative stress. In some cases, it has become apparent that ROS mediate physiologically important signaling across compartments, such as in mitonuclear retrograde signaling. However, it has remained technically challenging to quantitatively correlate compartment‐specific redox dynamics with subcellular resolution within live single cells. In order to address this challenge, we are developing genetically‐encoded biosensors that enable the visualization of redox dynamics simultaneously within multiple compartments within the same cell. Our approach utilizes Förster‐type resonance energy transfer in a spectral relay strategy to extend the fluorescence emission of the redox‐sensitive green fluorescent protein (roGFP) into orange and red emission wavelengths. Furthermore, we are developing multiplexed, multiphoton imaging methods to study redox coupling between organelles and compartment‐specific dynamics in thick neural tissues.Support or Funding InformationWe gratefully acknowledge support from the Ralph W. and Grace M. Showalter Trust, the Purdue Research Foundation, and from National Institutes of Health grants R21 NS092010 and R21 EY026425.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy.

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing.

LITE microscopy: Tilted light-sheet excitation of model organisms offers high resolution and low photobleaching.

Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.

Subcellular Localization and Dynamics of the Bcl-2 Family of Proteins.

Bcl-2 family proteins are recognized as major regulators of the mitochondrial pathway of apoptosis. They control the mitochondrial outer membrane permeabilization (MOMP) by directly localizing to this organelle. Further investigations demonstrated that Bcl-2 related proteins are also found in other intracellular compartments such as the endoplasmic reticulum, the Golgi apparatus, the nucleus and the peroxisomes. At the level of these organelles, Bcl-2 family proteins not only regulate MOMP in a remote fashion but also participate in major cellular processes including calcium homeostasis, cell cycle control and cell migration. With the advances of live cell imaging techniques and the generation of fluorescent recombinant proteins, it became clear that the distribution of Bcl-2 proteins inside the cell is a dynamic process which is profoundly affected by changes in the cellular microenvironment. Here, we describe the current knowledge related to the subcellular distribution of the Bcl-2 family of proteins and further emphasize on the emerging concept that this highly dynamic process is critical for cell fate determination.

Vertical Light Sheet Enhanced Side-View Imaging for AFM Cell Mechanics Studies

The ability to measure dynamic structural changes within a cell under applied load is essential for developing more accurate models of cell mechanics and mechanotransduction. Atomic force microscopy is a powerful tool for evaluating cell mechanics, but the dominant applied forces and sample strains are in the vertical direction, perpendicular to the imaging plane of standard fluorescence imaging. Here we report on a combined sideways imaging and vertical light sheet illumination system integrated with AFM. Our system enables high frame rate, low background imaging of subcellular structural dynamics in the vertical plane synchronized with AFM force data. Using our system for cell compression measurements, we correlated stiffening features in the force indentation data with onset of nuclear deformation revealed in the imaging data. In adhesion studies we were able to correlate detailed features in the force data during adhesive release events with strain at the membrane and within the nucleus.

High-Resolution Imaging of STIM/Orai Subcellular Localization Using Array Confocal Laser Scanning Microscopy.

The expression of chimeras that consist of a fluorescent protein (FP) conjugated with a protein of interest provides the ability to visualize, track, and quantify the subcellular localization and dynamics of specific proteins in biological samples. Array confocal laser scanning microscopy is an eminently suitable technique for live-cell imaging of FP-tagged fusion proteins. Here, we describe real-time monitoring of the subcellular dynamics of the stromal-interacting molecule 1 (STIM1) and Orai1, the key protagonists of store-operated Ca2+ entry (SOCE) under resting conditions, and upon Ca2+ mobilization from the endoplasmic reticulum (ER).