Cell Biological Investigations Research Articles

Comprehensive Cell Biological Investigation of Cytochalasin B Derivatives with Distinct Activities on the Actin Network.

In search of a more comprehensive structure-activity relationship (SAR) regarding the inhibitory effect of cytochalasin B (2) on actin polymerization, a virtual docking of 2 onto monomeric actin was conducted. This led to the identification of potentially important functional groups of 2 (i.e., the NH group of the isoindolone core (N-2) and the hydroxy groups at C-7 and C-20) involved in interactions with the residual amino acids of the binding pocket of actin. Chemical modifications of 2 at positions C-7, N-2, and C-20 led to derivatives 3-6, which were analyzed for their bioactivities. Compounds 3-5 exhibited reduced or no cytotoxicity in murine L929 fibroblasts compared to that of 2. Moreover, short- and long-term treatments of human osteosarcoma cells (U-2OS) with 3-6 affected the actin network to a variable extent, partially accompanied by the induction of multinucleation. Derivatives displaying acetylation at C-20 and N-2 were subjected to slow intracellular conversion to highly cytotoxic 2. Together, this study highlights the importance of the hydroxy group at C-7 and the NH function at N-2 for the potency of 2 on the inhibition of actin polymerization.

Differential physical interactions of wild-type and S59L mutant CHCHD10 with mitochondrial proteases

Background CHCHD10 is a small mitochondrial protein with a C-terminal coiled-coil-helix–coiled-coil-helix domain. The N-terminal region of CHCHD10 is mostly intrinsically disordered. Therefore, CHCHD10 has no catalytic activity other than protein-protein interactions through the CHCH domain or intrinsically disordered region. The S59L mutation in CHCHD10 has been identified as a genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. However, the disease-causing mechanisms of CHCHD10S59L are not yet fully understood. Our previous study showed that CHCHD10S59L induces PINK1 and Parkin accumulation. PINK1 stabilization in the mitochondria is dependent on proteolytic processing by mitochondrial proteases. It has also been reported that mutant CHCHD10 disrupts protein homeostasis within mitochondria. Therefore, we hypothesized that CHCHD10S59L physically interacts with mitochondrial proteases during the disease pathogenesis. Methods To test this hypothesis, we performed co-immunoprecipitation using transiently transfected wild-type and mutant S59L CHCHD10 in HeLa cells. We employed a dithiobis (succinimidyl propionate) cross-linker to detect transient physical interactions. Results We expected that CHCHD10S59L may activate mitochondrial protease processing or expression and that it interacts with more diverse mitochondrial proteases than the wild type. In general, however, there was no difference in the expression and processing levels of mitochondrial proteases and the proteases that interact with wild-type and CHCHD10S59L. LonP1 degrading unfolded proteins in the mitochondrial matrix was the major proteases that can bind to wild-type CHCHD10 and CHCHD10S59L. LonP1 and HtrA2 were significantly less strongly bound to CHCHD10S59L. Conclusions This result can be interpreted in many different ways, including the regulatory role of CHCHD10 in mitochondrial proteases. However, more precise biochemical and cell biological investigations are required to determine the role of CHCHD10 in the activity of mitochondrial proteases, mechanism of PINK1 stabilization, and disruption of mitochondrial proteostasis.

A novel dual-release scaffold for fluorescent labels improves cyclic immunofluorescence

Cyclic immunofluorescence is a powerful method to generate high-content imaging datasets for investigating cell biology and developing therapies. A dual-release mechanism facilitates fast and efficient cleavage of fluorophores from target antigens.

Improved Methods for Bulk Cultivation and Fixation of Loxodes Ciliates for Fluorescence Microscopy

Loxodes is one of the best ecologically characterized ciliate genera with numerous intriguing physiological abilities, including gravity-sensing organelles and nitrate respiration. However, these cells have been considered challenging to cultivate in bulk, and are poorly preserved by conventional fixatives used for fluorescence microscopy. Here we describe methods to grow and harvest Loxodes cells in bulk with liquid soil extract medium, as well as a new fixative called ZFAE (zinc sulfate, formaldehyde, acetic acid, ethanol) that can fix Loxodes cells more effectively than buffered formaldehyde or methanol. We show that ZFAE is compatible with immunofluorescence and the nuclear stain DAPI. Loxodes is thus now amenable to long-term maintenance, large-scale growth, and modern cell biology investigations of monoclonal strains in laboratory conditions.

3d oxidized alginate-porcine liver acellular collagen droplets for tumor microenvironment mimicking

The traditional 2d culture has been proved inferior to reproduce the subtle interaction between cell-to-cell and cell-to-extracellular matrix (ECM) in tumor microenvironment (TME) and collagen in ECM contributes to various malignancies of tumors. Hence, the 3d model contained with collagen may overcome the shortcomings of 2d culture. In this study, the in vitro TME mimicking matrix was prepared by coupling porcine liver-derived collagen (COL) and the dialdehyde group of partially oxidized alginate (OA), namely OA-COL, and the 3d OA-COL droplets were polymerized by divalent calcium ions. In the 3d OA-COL droplets, cancer cells displayed vigorous proliferation, and the cells grew in clusters and formed a unique spindle like clone. Quantitative analysis proved that various gene transcription and protein expression were up-regulated for the cells in the 3d OA-COL droplets, including F-actin reassembling, focal adhesion, pseudopodia formation, and the proteins involved in epithelial-to-mesenchymal transition (EMT). The 3d OA-COL droplets induced the cells with strengthened polarity, invasiveness, higher IC50, and manifested stronger tumorigenicity in vivo. The fabricated 3d OA-COL droplets reproduced a variety of TME parameters, constructed an in vitro model similar to the TME in vivo, and it may facilitate many investigations in cell biology and tumor biology.

Multicolor expansion fluorescence emission difference microscopy to reveal potential organelle contacts

Biologists have long been interested in the submicroscopic structures of cells and interactions between organelles. However, due to resolution limitations, investigating cell biology using fluorescence microscopy remains a challenge. In recent decades, super-resolution microscopy has been developed as an essential solution. Recently, a new microscopic technique called expansion microscopy has been developed, which circumvents this resolution limit by increasing the size of the biological sample, thus allowing for super-resolution of the enlarged structure. In this study, we combined expansion microscopy with fluorescence emission difference microscopy (ExFED) and achieved a spatial resolution of approximately 40 nm in mammalian cells. Delicate subcellular and sub-organelle structures were investigated, and potential contacts were detected. The results indicated that ExFED is a simple and powerful super-resolution method for studying biological specimens.

Molecular evolutionary analysis of the SM and SNARE vesicle fusion machinery in ciliates shows concurrent expansions in late secretory machinery.

Protists in the phylum Ciliophora possess a complex membrane-trafficking system, including osmoregulatory Contractile Vacuoles and specialized secretory organelles. Molecular cell biological investigations in Tetrahymena thermophila have identified components of the protein machinery associated with the secretory organelles, mucocysts. The Qa-SNARE Syn7lp plays a role in mucocyst biogenesis as do subunits of the CORVET tethering complex (specifically Vps8). Indeed, Tetrahymena thermophila possesses expanded gene complements of several CORVET components, including Vps33 which is also a Sec1/Munc18 (SM) protein that binds Qa-SNAREs. Moreover, the Qa-SNAREs in Paramecium tetraurelia have been localized to various endomembrane organelles. Here, we use comparative genomics and phylogenetics to determine the evolutionary history of the SM and Qa-SNARE proteins across the Ciliophora. We identify that the last ciliate common ancestor possessed the four SM proteins and six Qa-SNAREs common to eukaryotes, including the uncommonly retained Syntaxin 17. We furthermore identify independent expansion of these protein families in several ciliate classes, including concurrent expansions of the SM protein-Qa SNARE partners Sec1:SynPM in the oligohymenophorean ciliates lineage, consistent with novel Contractile Vacuole specific innovations. Overall, these data are consistent with SM proteins and Qa-SNAREs being a common set of components for endomembrane modulation in the ciliates.

A Flexible Terahertz Metamaterial Biosensor for Cancer Cell Growth and Migration Detection.

Metamaterial biosensors have been extensively used to identify cell types and detect concentrations of tumor biomarkers. However, the methods for in situ and non-destruction measurement of cell migration, which plays a key role in tumor progression and metastasis, are highly desirable. Therefore, a flexible terahertz metamaterial biosensor based on parylene C substrate was proposed for label-free and non-destructive detection of breast cancer cell growth and migration. The maximum resonance peak frequency shift achieved 183.2 GHz when breast cancer cell MDA−MB−231 was cultured onto the surface of the metamaterial biosensor for 72 h. A designed polydimethylsiloxane (PDMS) barrier sheet was applied to detect the cell growth rate which was quantified as 14.9 µm/h. The experimental peak shift expressed a linear relationship with the covered area and a quadratic relationship with the distance, which was consistent with simulation results. Additionally, the cell migration indicated that the transform growth factor-β (TGF-β) promoted the cancer cell migration. The terahertz metamaterial biosensor shows great potential for the investigation of cell biology in the future.

Deep learning -- promises for 3D nuclear imaging: a guide for biologists.

ABSTRACTFor the past century, the nucleus has been the focus of extensive investigations in cell biology. However, many questions remain about how its shape and size are regulated during development, in different tissues, or during disease and aging. To track these changes, microscopy has long been the tool of choice. Image analysis has revolutionized this field of research by providing computational tools that can be used to translate qualitative images into quantitative parameters. Many tools have been designed to delimit objects in 2D and, eventually, in 3D in order to define their shapes, their number or their position in nuclear space. Today, the field is driven by deep-learning methods, most of which take advantage of convolutional neural networks. These techniques are remarkably adapted to biomedical images when trained using large datasets and powerful computer graphics cards. To promote these innovative and promising methods to cell biologists, this Review summarizes the main concepts and terminologies of deep learning. Special emphasis is placed on the availability of these methods. We highlight why the quality and characteristics of training image datasets are important and where to find them, as well as how to create, store and share image datasets. Finally, we describe deep-learning methods well-suited for 3D analysis of nuclei and classify them according to their level of usability for biologists. Out of more than 150 published methods, we identify fewer than 12 that biologists can use, and we explain why this is the case. Based on this experience, we propose best practices to share deep-learning methods with biologists.

Kinesin Motors in the Filamentous Basidiomycetes in Light of the Schizophyllum commune Genome.

Kinesins are essential motor molecules of the microtubule cytoskeleton. All eukaryotic organisms have several genes encoding kinesin proteins, which are necessary for various cell biological functions. During the vegetative growth of filamentous basidiomycetes, the apical cells of long leading hyphae have microtubules extending toward the tip. The reciprocal exchange and migration of nuclei between haploid hyphae at mating is also dependent on cytoskeletal structures, including the microtubules and their motor molecules. In dikaryotic hyphae, resulting from a compatible mating, the nuclear location, synchronous nuclear division, and extensive nuclear separation at telophase are microtubule-dependent processes that involve unidentified molecular motors. The genome of Schizophyllum commune is analyzed as an example of a species belonging to the Basidiomycota subclass, Agaricomycetes. In this subclass, the investigation of cell biology is restricted to a few species. Instead, the whole genome sequences of several species are now available. The analyses of the mating type genes and the genes necessary for fruiting body formation or wood degrading enzymes in several genomes of Agaricomycetes have shown that they are controlled by comparable systems. This supports the idea that the genes regulating the cell biological process in a model fungus, such as the genes encoding kinesin motor molecules, are also functional in other filamentous Agaricomycetes.

Collagen Sandwich Culture of Primary Hepatocytes for Image-Based Investigations.

Collagen sandwich culture of hepatocytes retains many in vivo-like properties and is used for many investigations in liver cell biology, and hepatic pharmacology and toxicology. This chapter describes the method of establishing collagen sandwich culture of hepatocytes in a glass bottom dish for image-based studies.

Proteomic Analysis of Marinesco-Sjogren Syndrome Fibroblasts Indicates Pro-Survival Metabolic Adaptation to SIL1 Loss.

Marinesco–Sjogren syndrome (MSS) is a rare multisystem pediatric disorder, caused by loss-of-function mutations in the gene encoding the endoplasmic reticulum cochaperone SIL1. SIL1 acts as a nucleotide exchange factor for BiP, which plays a central role in secretory protein folding. SIL1 mutant cells have reduced BiP-assisted protein folding, cannot fulfil their protein needs, and experience chronic activation of the unfolded protein response (UPR). Maladaptive UPR may explain the cerebellar and skeletal muscle degeneration responsible for the ataxia and muscle weakness typical of MSS. However, the cause of other more variable, clinical manifestations, such as mild to severe mental retardation, hypogonadism, short stature, and skeletal deformities, is less clear. To gain insights into the pathogenic mechanisms and/or adaptive responses to SIL1 loss, we carried out cell biological and proteomic investigations in skin fibroblasts derived from a young patient carrying the SIL1 R111X mutation. Despite fibroblasts not being overtly affected in MSS, we found morphological and biochemical changes indicative of UPR activation and altered cell metabolism. All the cell machineries involved in RNA splicing and translation were strongly downregulated, while protein degradation via lysosome-based structures was boosted, consistent with an attempt of the cell to reduce the workload of the endoplasmic reticulum and dispose of misfolded proteins. Cell metabolism was extensively affected as we observed a reduction in lipid synthesis, an increase in beta oxidation, and an enhancement of the tricarboxylic acid cycle, with upregulation of eight of its enzymes. Finally, the catabolic pathways of various amino acids, including valine, leucine, isoleucine, tryptophan, lysine, aspartate, and phenylalanine, were enhanced, while the biosynthetic pathways of arginine, serine, glycine, and cysteine were reduced. These results indicate that, in addition to UPR activation and increased protein degradation, MSS fibroblasts have profound metabolic alterations, which may help them cope with the absence of SIL1.

The molecular mechanism of RIG-I activation and signaling.

RIG‐I is our first line of defense against RNA viruses, serving as a pattern recognition receptor that identifies molecular features common among dsRNA and ssRNA viral pathogens. RIG‐I is maintained in an inactive conformation as it samples the cellular space for pathogenic RNAs. Upon encounter with the triphosphorylated terminus of blunt‐ended viral RNA duplexes, the receptor changes conformation and releases a pair of signaling domains (CARDs) that are selectively modified and interact with an adapter protein (MAVS), thereby triggering a signaling cascade that stimulates transcription of interferons. Here, we describe the structural determinants for specific RIG‐I activation by viral RNA, and we describe the strategies by which RIG‐I remains inactivated in the presence of host RNAs. From the initial RNA triggering event to the final stages of interferon expression, we describe the experimental evidence underpinning our working knowledge of RIG‐I signaling. We draw parallels with behavior of related proteins MDA5 and LGP2, describing evolutionary implications of their collective surveillance of the cell. We conclude by describing the cell biology and immunological investigations that will be needed to accurately describe the role of RIG‐I in innate immunity and to provide the necessary foundation for pharmacological manipulation of this important receptor.

Long-term culture of SH-SY5Y neuroblastoma cells in the absence of neurotrophins: A novel model of neuronal ageing

BackgroundStudying human ageing is of increasing importance due to the worldwide ageing population. However, it faces the challenge of lengthy experiments to produce an ageing phenotype. Often, to recreate the hallmarks of ageing requires complex empirical conditions that can confound data interpretation. Indeed, many studies use whole organisms with relatively short life spans, which may have little, or limited, relevance to human ageing. There has been extensive use of cell lines to study ageing in human somatic cells, but the modelling of human neuronal ageing is somewhat more complex in vitro. New MethodWe cultured the well-characterised SH-SY5Y human neural cell line to produce high purity cultures of cells differentiated to express a neuronal phenotype, and designed a protocol to maintain these cells in culture until they accumulated biomarkers of cellular ageing. ResultsOur data validate a novel and simple technique for the efficient differentiation and long-term maintenance of SH-SY5Y cells, expressing markers of neuronal differentiation and demonstrating electrical activity in culture. Over time in vitro, these cells progressively accumulate markers of ageing such as enhanced production of reactive oxygen species and accumulation of oxidative damage. Comparison to Existing MethodsIn comparison to existing techniques to model neuronal ageing our method is cost effective, requiring no specialist equipment or growth factors. ConclusionsWe demonstrate that SH-SY5Y cells, grown under these culture conditions, represent a simple model of neuronal ageing that is amenable to cell biological, biochemical and electrophysiological investigation.

A microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow.

Biological cells in vivo typically reside in a dynamic flowing microenvironment with extensive biomechanical and biochemical cues varying in time and space. These dynamic biomechanical and biochemical signals together act to regulate cellular behaviors and functions. Microfluidic technology is an important experimental platform for mimicking extracellular flowing microenvironment in vitro. However, most existing microfluidic chips for generating dynamic shear stress and biochemical signals require expensive, large peripheral pumps and external control systems, unsuitable for being placed inside cell incubators to conduct cell biology experiments. This study has developed a microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow. Further, based on the lumped-parameter and distributed-parameter models of multiscale fluid dynamics, the oscillatory flow field and the concentration field of biochemical factors has been simulated at the cell culture region within the designed microfluidic chip. Using the constructed experimental system, the feasibility of the designed microfluidic chip has been validated by simulating biochemical factors with red dye. The simulation results demonstrate that dynamic shear stress and biochemical signals with adjustable period and amplitude can be generated at the cell culture chamber within the microfluidic chip. The amplitudes of dynamic shear stress and biochemical signals is proportional to the pressure difference and inversely proportional to the flow resistance, while their periods are correlated positively with the flow capacity and the flow resistance. The experimental results reveal the feasibility of the designed microfluidic chip. Conclusively, the proposed microfluidic generator based on autonomously oscillatory flow can generate dynamic shear stress and biochemical signals without peripheral pumps and external control systems. In addition to reducing the experimental cost, due to the tiny volume, it is beneficial to be integrated into cell incubators for cell biology experiments. Thus, the proposed microfluidic chip provides a novel experimental platform for cell biology investigations.

First person – Maayan Barnea-Zohar and Sabina E. Winograd-Katz

ABSTRACT 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. Maayan Barnea-Zohar and Sabina E. Winograd-Katz are co-first authors on ‘ An SNX10-dependent mechanism downregulates fusion between mature osteoclasts’, published in JCS. Maayan is a research assistant in the lab of Ari Elson at the Weizmann Institute of Science, Rehovot, Israel, investigating cell–cell fusion in osteoclasts and how cell size is determined. Sabina is a research assistant and lab manager in the lab of Benjamin Geiger at the Weizmann Institute of Science, investigating cell biology and signaling.

Subcellular Specialization of 3D Mitochondrial Morphology and Mitochondria‐Lipid Droplet Interactivity is Associated with Proximity to Oxygen Supply in Muscle Cells

Advancements in imaging technology have considerably expanded our capacity to investigate cell biology, and our laboratory has recently employed high-resolution 3D microscopy to explore the subcellular structures and organelle-organelle interactions that support muscle metabolism. While this initial work has demonstrated that mitochondrial structure and mitochondria-organelle interactivity are regulated according to muscle cell type, it remains unclear how cellular metabolism is supported by the subcellular specialization of mitochondria. Certainly, several subcellular clusters of mitochondria have been identified within muscle cells, and these groups of mitochondria are often categorized according to their proximity to other cellular components (i.e. “intermyofibrillar” or “paravascular”). However, it is unknown how 3D mitochondrial morphology and organelle interactivity is altered to support the varying functional demands of different subcellular regions. Here, we combine 3D focused-ion beam scanning electron microscopy (FIB-SEM) with machine learning programs to quantify individual mitochondrial structure and organelle interactivity within large subcellular volumes (~8,000 µm3) (Fig. 1a-c). Using this high-throughput approach, we evaluate over 700 individual mitochondria per cell and leverage the statistical power of this large data set to identify 3D spatial relationships among multiple subcellular structures. We find that mitochondrial surface area to volume ratio (15.5±0.25 µm-1) is significantly correlated (R2= 0.39) to the minimal distance to capillary, but not distance to sarcolemma (R2= 0.02) (Fig. 1d). Further analysis revealed that mitochondria-organelle interactivity is also closely related to subcellular localization. Specifically, mitochondria within close proximity (<5 µm) to capillaries (paravascular) are ~3-fold more likely to make contact with lipid droplets compared to mitochondria located in other subcellular regions (Fig. 1e). Moreover, the paravascular mitochondria in contact with lipid droplets have 75% greater volume (1.4±0.07 µm3) compared to those not connected to lipid droplets (0.8±0.03 µm3). These preliminary analyses suggest that mitochondria in close proximity to the muscle oxygen supply, but not cell boundary, exhibit distinct 3D morphology and organelle interactivity patterns that may function to support metabolism and other cellular processes.

Overlapping roles of JIP3 and JIP4 in promoting axonal transport of lysosomes in human iPSC-derived neurons.

The dependence of neurons on microtubule-based motors for the movement of lysosomes over long distances raises questions about adaptations that allow neurons to meet these demands. Recently, JIP3/MAPK8IP3, a neuronally enriched putative adaptor between lysosomes and motors, was identified as a critical regulator of axonal lysosome abundance. In this study, we establish a human induced pluripotent stem cell (iPSC)-derived neuron model for the investigation of axonal lysosome transport and maturation and show that loss of JIP3 results in the accumulation of axonal lysosomes and the Alzheimer’s disease–related amyloid precursor protein (APP)-derived Aβ42 peptide. We furthermore reveal an overlapping role of the homologous JIP4 gene in lysosome axonal transport. These results establish a cellular model for investigating the relationship between lysosome axonal transport and amyloidogenic APP processing and more broadly demonstrate the utility of human iPSC–derived neurons for the investigation of neuronal cell biology and pathology.

An NKX2-1GFP and TP63tdTomato dual fluorescent reporter for the investigation of human lung basal cell biology

Basal cells are multipotent stem cells responsible for the repair and regeneration of all the epithelial cell types present in the proximal lung. In mice, the elusive origins of basal cells and their contribution to lung development were recently revealed by high-resolution, lineage tracing studies. It however remains unclear if human basal cells originate and participate in lung development in a similar fashion, particularly with mounting evidence for significant species-specific differences in this process. To address this outstanding question, in the last several years differentiation protocols incorporating human pluripotent stem cells (hPSC) have been developed to produce human basal cells in vitro with varying efficiencies. To facilitate this endeavour, we introduced tdTomato into the human TP63 gene, whose expression specifically labels basal cells, in the background of a previously described hPSC line harbouring an NKX2-1GFP reporter allele. The functionality and specificity of the NKX2-1GFP;TP63tdTomato hPSC line was validated by directed differentiation into lung progenitors as well as more specialised lung epithelial subtypes using an organoid platform. This dual fluorescent reporter hPSC line will be useful for tracking, isolating and expanding basal cells from heterogenous differentiation cultures for further study.

Activity‐Based NIR Enzyme Fluorescent Probes for the Diagnosis of Tumors and Image‐Guided Surgery

AbstractNear‐infrared (NIR) activatable fluorescent probes have been considered to be the effective edge tools for the investigation of cell biology and disease diagnosis because of their outstanding advantages. Related genes involved in tumor genesis and progression regulate the overexpression of certain enzymes. Owing to the distinctive characteristics of quick reaction time and favorable pharmacokinetics, enzyme‐reactive NIR optical probes have shown great potential in the diagnosis of tumorigenesis and in image‐guided intraoperative surgeries with high signal‐to‐noise ratios. In this review, we mainly summarize the latest advancements in enzyme‐reactive NIR fluorescent probes from design strategy to biomedical application. Specifically, some challenges and prospects in this field are presented at the end of the review, which will be beneficial to innovatively construct new multifunctional fluorescent probes and actively promote their clinical transformation in the future.