Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs.

We present the first study of the 3D kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the early host response in a large lung volume using a combination of tissue imaging and transcriptomics. This approach allowed us to make a number of important findings: Spatially restricted antiviral response is shown, including the formation of monocytic macrophage clusters and upregulation of the major histocompatibility complex II in infected epithelial cells. The monocyte-derived macrophages are linked to SARS-CoV-2 clearance, and the appearance of these cells is associated with post-infection endothelial damage; thus, we shed light on the role of these cells in infected tissue.

IFN-γ-dependent interactions between tissue-intrinsic γδ T cells and tissue-infiltrating CD8 T cells limit allergic contact dermatitis.

Background: Elicitation of allergic contact dermatitis (ACD), an inflammatory type 4 hypersensitivity disease, induces skin infiltration by polyclonal effector CD8 αβ T cells and precursors of tissue-resident memory T (T) cells. Because T have long-term potential to contribute to body-surface immunoprotection and immunopathology, their local regulation needs a fuller understanding.

Objective: We sought to investigate how T-cell maturation might be influenced by innate-like T cells pre-existing within many epithelia.

Tissue optical clearing and 3D imaging of virus infections.

Imaging pathogens within 3D environment of biological tissues provides spatial information about their localization and interactions with the host. Technological advances in fluorescence microscopy and 3D image analysis now permit visualization and quantification of pathogens directly in large tissue volumes and in great detail. In recent years large volume imaging became an important tool in virology research helping to understand the properties of viruses and the host response to infection.

Ebola virus inclusion bodies are liquid organelles whose formation is facilitated by nucleoprotein oligomerization.

Viral RNA synthesis of several non-segmented, negative-sense RNA viruses (NNSVs) takes place in inclusion bodies (IBs) that show properties of liquid organelles, which are formed by liquid-liquid phase separation of scaffold proteins. It is believed that this is driven by intrinsically disordered regions (IDRs) and/or multiple copies of interaction domains, which for NNSVs are usually located in their nucleo - and phosphoproteins. In contrast to other NNSVs, the Ebola virus (EBOV) nucleoprotein NP alone is sufficient to form IBs without the need for a phosphoprotein, and to facilitate the recruitment of other viral proteins into these structures.

Evidence for Viral mRNA Export from Ebola Virus Inclusion Bodies by the Nuclear RNA Export Factor NXF1.

Many negative-sense RNA viruses, including the highly pathogenic Ebola virus (EBOV), use cytoplasmic inclusion bodies (IBs) for viral RNA synthesis. However, it remains unclear how viral mRNAs are exported from these IBs for subsequent translation. We recently demonstrated that the nuclear RNA export factor 1 (NXF1) is involved in a late step in viral protein expression, i.

Butyrophilin-like proteins display combinatorial diversity in selecting and maintaining signature intraepithelial γδ T cell compartments.

Butyrophilin-like (Btnl) genes are emerging as major epithelial determinants of tissue-associated γδ T cell compartments. Thus, the development of signature, murine TCRγδ intraepithelial lymphocytes (IEL) in gut and skin depends on Btnl family members, Btnl1 and Skint1, respectively. In seeking mechanisms underlying these profound effects, we now show that normal gut and skin γδ IEL development additionally requires Btnl6 and Skint2, respectively, and furthermore that different Btnl heteromers can seemingly shape different intestinal γδ IEL repertoires.

A wave of monocytes is recruited to replenish the long-term Langerhans cell network after immune injury.

A dense population of embryo-derived Langerhans cells (eLCs) is maintained within the sealed epidermis without contribution from circulating cells. When this network is perturbed by transient exposure to ultraviolet light, short-term LCs are temporarily reconstituted from an initial wave of monocytes but thought to be superseded by more permanent repopulation with undefined LC precursors. However, the extent to which this process is relevant to immunopathological processes that damage LC population integrity is not known.

Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays.

Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin-binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions.

Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin Subfragment 1.

Myosin head (myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain.

CDC42 switches IRSp53 from inhibition of actin growth to elongation by clustering of VASP.

Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth.

Semi-automated analysis of organelle movement and membrane content: understanding rab-motor complex transport function.

Organelle motility is an essential cellular function that is regulated by molecular motors, and their adaptors and activators. Here we established a new method that allows more direct investigation of the function of these peripheral membrane proteins in organelle motility than is possible by analysis of the organelle movement alone. This method uses multi-channel time-lapse microscopy to record the movement of organelles and associated fluorescent proteins, and automatic organelle tracking, to compare organelle movement parameters with the association of membrane proteins.

Response of rigor cross-bridges to stretch detected by fluorescence lifetime imaging microscopy of myosin essential light chain in skeletal muscle fibers.

We applied fluorescence lifetime imaging microscopy to map the microenvironment of the myosin essential light chain (ELC) in permeabilized skeletal muscle fibers. Four ELC mutants containing a single cysteine residue at different positions in the C-terminal half of the protein (ELC-127, ELC-142, ELC-160, and ELC-180) were generated by site-directed mutagenesis, labeled with 7-diethylamino-3-((((2-iodoacetamido)ethyl)amino)carbonyl)coumarin, and introduced into permeabilized rabbit psoas fibers. Binding to the myosin heavy chain was associated with a large conformational change in the ELC.

Natural killer cell signal integration balances synapse symmetry and migration.

Natural killer (NK) cells discern the health of other cells by recognising the balance of activating and inhibitory ligands expressed by each target cell. However, how the integration of activating and inhibitory signals relates to formation of the NK cell immune synapse remains a central question in our understanding of NK cell recognition. Here we report that ligation of LFA-1 on NK cells induced asymmetrical cell spreading and migration.

Direct visualisation and kinetic analysis of normal and nemaline myopathy actin polymerisation using total internal reflection microscopy.

Actin filaments were formed by elongation of pre-formed nuclei (short crosslinked actin-HMM complexes) that were attached to a microscope cover glass. By using TIRF illumination we could see actin filaments at high contrast despite the presence of 150 nM TRITC-phalloidin in the solution. Actin filaments showed rapid bending and translational movements due to Brownian motion but the presence of the methylcellulose polymer network constrained lateral movement away from the surface.

Morphoregulation by acetylcholinesterase in fibroblasts and astrocytes.

Acetylcholinesterase (AChE) terminates neurotransmission at cholinergic synapses by hydrolysing acetylcholine, but also has non-enzymatic morphoregulatory effects on neurons such as stimulation of neurite outgrowth. It is widely expressed outside the nervous system, but its function in non-neuronal cells is unclear. Here we have investigated the distribution and function of AChE in fibroblasts and astrocytes.

Rab27a and MyoVa are the primary Mlph interactors regulating melanosome transport in melanocytes.

Melanosome transport in melanocytes is a model system for the study of cytoskeletal regulation of intracellular transport. Melanophilin (Mlph) is a Rab27a- and myosin Va (MyoVa)-binding protein that regulates this process. Using yeast two-hybrid screening, we identified MT plus-end binding protein (EB1) as a melanocyte-expressed Mlph-interacting protein.

Rab27b regulates mast cell granule dynamics and secretion.

The Rab GTPase family regulates membrane domain organization and vesicular transport pathways. Recent studies indicate that one member of the family, Rab27a, regulates transport of lysosome-related organelles in specialized cells, such as melanosomes and lytic granules. Very little is known about the related isoform, Rab27b.

Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle.

Myosin head consists of a globular catalytic domain and a long alpha-helical regulatory domain. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. The proximal end of the regulatory domain contains the essential light chain 1 (LC1).