Cellular inertia

It has been experimentally reported that chemotactic cells exhibit cellular memory, that is, a tendency to maintain the migration direction despite changes in the chemoattractant gradient. In this study, we analyzed a phenomenological model assuming the presence of cellular inertia, as well as a response time in motility, resulting in the reproduction of the cellular memory observed in the previous experiments. According to the analysis, the cellular motion is described by the superposition of multiple oscillative functions induced by the multiplication of the oscillative polarity and motility. The cellular intertia generates cellular memory by regulating phase differences between those oscillative functions. By applying the theory to the experimental data, the cellular inertia was estimated at $$m=3-6$$ m = 3 - 6 min. In addition, physiological parameters, such as response time in motility and intracellular processing speed, were also evaluated. The agreement between the experiemental data and theory suggests the possibility of the presence of the response time in motility, which has never been biologically verified and should be explored in the future.

Molecular Mechanisms of Borrelia burgdorferi Phagocytosis and Intracellular Processing by Human Macrophages

Lyme disease is the most common vector-borne illness in North America and Europe. Its causative agents are spirochetes of the Borrelia burgdorferi sensu latu complex. Infection with borreliae can manifest in different tissues, most commonly in the skin and joints, but in severe cases also in the nervous systems and the heart. The immune response of the host is a crucial factor for preventing the development or progression of Lyme disease. Macrophages are part of the innate immune system and thus one of the first cells to encounter infecting borreliae. As professional phagocytes, they are capable of recognition, uptake, intracellular processing and final elimination of borreliae. This sequence of events involves the initial capture and internalization by actin-rich cellular protrusions, filopodia and coiling pseudopods. Uptake into phagosomes is followed by compaction of the elongated spirochetes and degradation in mature phagolysosomes. In this review, we discuss the current knowledge about the processes and molecular mechanisms involved in recognition, capturing, uptake and intracellular processing of Borrelia by human macrophages. Moreover, we highlight interactions between macrophages and other cells of the immune system during these processes and point out open questions in the intracellular processing of borreliae, which include potential escape strategies of Borrelia.

First person – Matthias Klose

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. Matthias Klose is first author on ‘FIB-SEM-based analysis of Borrelia intracellular processing by human macrophages’, published in JCS. Matthias is a postdoc in the lab of Professor Stefan Linder at Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Eppendorf, Hamburg, Germany, investigating mechanisms that drive uptake and intracellular processing of Borrelia burgdorferi in primary human macrophages.

In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli

Recombinant proteins are generally fused with solubility enhancer tags to improve target protein folding and solubility. However, the fusion protein strategy usually requires the use of expensive proteases to perform in vitro proteolysis and additional chromatography steps to obtain tag-free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co-expression of a site-specific protease, are useful for simplifying the recombinant protein purification process, for screening molecules that fail to remain soluble after tag removal, and to promote higher yields of soluble target protein. Herein, we review controlled intracellular processing (CIP) systems, tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular protein processing regarding system design features, significant advantages and limitations of the various strategies.

FIB/SEM-based analysis of Borrelia intracellular processing by human macrophages

Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, joints and nervous system. Successful internalization and intracellular processing of borreliae by immune cells like macrophages is decisive for the outcome of a respective infection. Here, we use for the first time focused ion beam scanning electron microscopy tomography (FIB/SEM tomography) to visualize the interaction of borreliae with primary human macrophages with high resolution. We report that interaction between macrophages and the elongated and highly motile borreliae can lead to formation of membrane tunnels that extend deeper into the host cytoplasm than the actual phagosome, most probably as a result of partial extrication of captured borreliae. We also show that membrane tubulation at borreliae-containing phagosomes, a process suggested earlier as a mechanism leading to phagosome compaction, but hard to visualize in live cell imaging, is apparently a frequent phenomenon. Finally, we demonstrate that the endoplasmic reticulum (ER) forms multiple STIM1-positive contact sites with both membrane tunnels and phagosome tubulations, confirming the important role of the ER during uptake and intracellular processing of borreliae.

Multispectral Fingerprinting Resolves Dynamics of Nanomaterial Trafficking in Primary Endothelial Cells

Intracellular vesicle trafficking involves a complex series of biological pathways used to sort, recycle, and degrade extracellular components, including engineered nanomaterials which gain cellular entry via active endocytic processes. A recent emphasis on routes of nanomaterial uptake has established key physicochemical properties which direct certain mechanisms, yet relatively few studies have identified their effect on intracellular trafficking processes past entry and initial subcellular localization. Here, we developed and applied an approach where single-walled carbon nanotubes (SWCNTs) play a dual role - that of an engineered nanomaterial (ENM) undergoing intracellular processing, in addition to functioning as the signal transduction element reporting these events in individual cells with single organelle resolution. We used the unique optical properties exhibited by non-covalent hybrids of single-stranded DNA and SWCNTs (DNA-SWCNTs) to report the progression of intracellular processing events via two orthogonal hyperspectral imaging approaches of near-infrared (NIR) fluorescence and resonance Raman scattering. A positive correlation between fluorescence and G-band intensities was uncovered within single cells, while exciton energy transfer and eventual aggregation of DNA-SWCNTs were observed to scale with increasing time after internalization. These were confirmed to be consequences of intracellular processes using pharmacological inhibitors of endosomal maturation, which suppressed spectral changes through two distinct mechanisms. An analysis pipeline was developed to colocalize and deconvolute the fluorescence and Raman spectra of subcellular regions of interest (ROIs), allowing for single-chirality component spectra to be obtained with sub-micron spatial resolution. This approach uncovered a complex relationship between DNA-SWCNT concentration, fluorescence intensity, environmental transformations, and irreversible aggregation resulting from the temporal evolution of trafficking events. Finally, a spectral clustering analysis was applied to delineate the dynamic sequence of processes into four distinct populations, allowing stages of the intracellular trafficking process to be identified by the multispectral fingerprint of encapsulated DNA-SWCNTs.

Kinetic analysis of the intracellular processing of siRNAs by confocal microscopy

Here, we describe a method for tracking intracellular processing of small interfering RNA (siRNA) containing complexes using automated microscopy controls and image acquisition to minimize user effort and time. This technique uses fluorescence colocalization to monitor dual-labeled fluorescent siRNAs delivered by silica nanoparticles in different intracellular locations, including the early/late endosomes, fast/slow recycling endosomes, lysosomes and the endoplasmic reticulum. Combining the temporal association of siRNAs with each intracellular location, we reconstructed the intracellular pathways used in siRNA processing, and demonstrate how these pathways vary based on the chemical composition of the delivery vehicle.