Live cell imaging

Fluorescence-based live-cell imaging (LCI) of lysosomal glycosidases is often hampered by unfavorable pH and redox conditions that reduce fluorescence output. Moreover, most lysosomal glycosidases are low-mass soluble proteins that do not allow for bulky fluorescent protein fusions. We selected α-galactosidase A (GALA) as a model lysosomal glycosidase involved in Anderson-Fabry disease (AFD) for the current LCI approach. Examination of the subcellular localization of AFD-causing mutants can reveal the mechanism underlying cellular trafficking deficits...

Human and mouse neutrophils are known to form tethers when rolling on selectins in vitro. Tethers are ∼0.2 μm thin, ∼5-10 μm-long structures behind rolling cells that can swing around to form slings that serve as self-adhesive substrates. Here, we developed a mouse intravital imaging method, where the neutrophil surface is labeled by injecting fluorescently labeled mAb to Ly-6G. Venules in the cremaster muscle of live mice were imaged at a high frame rate using a confocal microscope equipped with a fast resonant scanner...

Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca(2+) reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca(2+) levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca(2+) differential, aborting both cell migration and new branching...

Organic fluorophores have a wide range of biological uses and are usually needed to be prepared as water-soluble compounds or nanoparticles for applications in aqueous biosystems owing to their hydrophobic properties, which often is a complex, time-consuming and high-cost process. Here, the nanoparticle preparation of hydrophobic fluorophores and their application in cell imaging have been investigated. It was found: a) fetal bovine serum (FBS) shows an excellent dispersion effect on hydrophobic small-molecule organic compounds; b) a hydrophobic C6-unsubstituted tetrahydropyrimidine (Me-THP-Naph) can be prepared as nanosuspensions utilizing cell culture medium with 10% FBS and directly be used as a specific real-time imaging probe for the endoplasmic reticulum (ER), a dynamic organelle playing a crucial role in many cellular processes...

A dual-activatable, fluorogenic probe was developed to sense esterase activity with single-molecule resolution. Without enzymatic pre-activation, the diazoindanone-based probe has an electron poor core and, upon irradiation, undergoes Wolff rearrangement to give a ring-expanded xanthene core that is non-emissive. If the probe is pre-activated by carboxylesterases, the tricyclic core becomes electron-rich and the photoinduced Wolff rearrangement produces a highly emissive rhodol dye. Live-cell and solution studies confirmed the selectivity of the probe and revealed that the photoactivated dye does not diffuse away from the original location of activation because the intermediate ketene forms a covalent bond with surrounding macromolecules...

Cell replication is a fundamental attribute of growth and repair in multicellular organisms. Pancreatic beta-cells in adults rarely enter cell cycle, hindering the capacity for regeneration in diabetes. Efforts to drive beta-cells into cell cycle have so far largely focused on regulatory molecules such as cyclins and cyclin-dependent kinases (CDKs). Investigations in cancer biology have uncovered that adaptive changes in metabolism, the mitochondrial network, and cellular Ca(2+) are critical for permitting cells to progress through the cell cycle...

Mitochondria are cellular organelles with multifaceted tasks and thus composed of different sub-compartments. The inner mitochondrial membrane especially has a complex nano-architecture with cristae protruding into the matrix. Related to their function, the localization of mitochondrial membrane proteins is more or less restricted to specific sub-compartments. In contrast, it can be assumed that membrane proteins per se diffuse unimpeded through continuous membranes. Fluorescence recovery after photobleaching is a versatile technology used in mobility analyses to determine the mobile fraction of proteins, but it cannot provide data on subpopulations or on confined diffusion behavior...

Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed...

To form functional neural circuits, neurons migrate to their final destination and extend axons towards their targets. Whether and how these two processes are coordinated in vivo remains elusive. We use the zebrafish olfactory placode as a system to address the underlying mechanisms. Quantitative live imaging uncovers a choreography of directed cell movements that shapes the placode neuronal cluster: convergence of cells towards the centre of the placodal domain and lateral cell movements away from the brain...

The need for in vitro models that mimic the human brain to replace animal testing and allow high-throughput screening has driven scientists to develop new tools that reproduce tissue-like features on a chip. Three-dimensional (3D) in vitro cultures are emerging as an unmatched platform that preserves the complexity of cell-to-cell connections within a tissue, improves cell survival, and boosts neuronal differentiation. In this context, new and flexible imaging approaches are required to monitor the functional states of 3D networks...

Reported herein the synthesis, characterization and biologically important zinc ion binding propensity of a weakly fluorescent chemosensor, 4-methyl-2,6-bis((E)-(2-(4-phenylthiazol-2-yl)hydrazono)methyl)phenol (1). (1)H NMR spectroscopic titration experiment reveals the binding knack of 1 to the essential Zn(2+). The photo-physical studies of 1 exhibit an enhancement in the fluorescence by several folds upon binding with the zinc ions attributed to PET-off process, with a binding constant value of 5.22×10(3)M(-1)...

Transmission of polarity established early during cell lineage history is emerging as a key process guiding cell differentiation. Highly polarized neurons provide a fascinating model to study inheritance of polarity over cell generations and across morphological transitions. Neural crest cells (NCCs) migrate to the dorsal root ganglia to generate neurons directly or after cell divisions in situ. Using live imaging of vertebrate embryo slices, we found that bipolar NCC progenitors lose their polarity, retracting their processes to round for division, but generate neurons with bipolar morphology by emitting processes from the same locations as the progenitor...

Cell interaction with biomaterials is one of the keystones to developing medical devices for tissue engineering applications. Biomaterials are the scaffolds that give three-dimensional support to the cells, and are vectors that deliver the cells to the injured tissue requiring repair. Features of biomaterials can influence the behaviour of the cells and consequently the efficacy of the tissue-engineered product. The adhesion, distribution and motility of the seeded cells onto the scaffold represent key aspects, and must be evaluated in vitro during the product development, especially when the efficacy of a specific tissue-engineered product depends on viable and functional cell loading...

Single molecule imaging approaches like dSTORM and PALM resolve structures at 10-20 nm, and allow for unique insights into protein stoichiometry and spatial relationships. However, key obstacles remain in developing highly accurate quantitative single molecule approaches. The genomic tagging of PALM fluorophores through CRISPR-Cas9 offers an excellent opportunity for generating stable cell lines expressing a defined single molecule probe at endogenous levels, without the biological disruption and variability inherent to transfection...

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites...

Invasion of the extracellular matrix is a critical step in the colonization of metastatic tumors. The invasion process is thought to be driven by both chemokine signaling and interactions between invading cancer cells and physical components of the metastatic niche, including endothelial cells that line capillary walls and serve as a barrier to both diffusion and invasion of the underlying tissue. Transwell chambers, a tool for generating artificial chemokine gradients to induce cell migration, have facilitated recent work to investigate the chemokine contributions to matrix invasion...

Fluorescence photoswitching using nanomaterials has recently emerged as a promising approach for the imaging of biological targets. However, despite intensive research efforts during the last decade, practical microscopy of biological targets using photoswitchable nanoparticles in real time remains challenging. To address this problem, we have developed live macrophage cell imaging and single particle imaging methods, using photoswitchable fluorescent diarylethene-doped polymer nanoparticles (P-dots) under Xe lamp irradiation...

First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Dougall Norris is co-first author on 'An improved Akt reporter reveals intra- and intercellular heterogeneity and oscillations in signal transduction', published in Journal of Cell Science. Dougall is a PhD student in the Metabolic Systems Biology Group at The University of Sydney Charles Perkins Centre in Australia, where his project focuses on live-cell imaging, multiplexing fluorescent constructs and imaging modalities, coupled with powerful computational analysis...

Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1...

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice...