imaging-subcellular-structures-in-the-living-zebrafish-embryo

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Technische Universität München

Autoantibody-induced cellular signaling mechanisms contribute to the pathogenesis of autoimmune blistering skin disease pemphigus vulgaris (PV). Recently, it was proposed that epidermal growth factor receptor (EGFR) might be involved in PV signaling pathways. In this study, we investigated the role of EGFR by comparing the effects of epidermal growth factor (EGF) and PV-IgG on the immortalized human keratinocyte cell line HaCaT, and primary normal human keratinocytes. In contrast to EGF treatment, PV-IgG neither caused the canonical activation of EGFR via phosphorylation at tyrosine (Y)1173 followed by internalization of EGFR nor the phosphorylation of the EGFR at the c-Src-dependent site Y845. Nevertheless, both PV-IgG and EGF led to cell dissociation and cytokeratin retraction in keratinocyte monolayers. Moreover, the effects of EGF were blocked by inhibition of EGFR and c-Src whereas the effects of PV-IgG were independent of both signaling pathways. Similarly, laser tweezer experiments revealed that impaired bead binding of epidermal cadherins desmoglein (Dsg) 3 and Dsg 1 in response to PV-IgG was not affected by inhibition of either EGFR or c-Src. In contrast, EGF treatment did not interfere with Dsg bead binding. Taken together, our study indicates that the loss of Dsg-mediated adhesion and keratinocyte dissociation in pemphigus is independent of EGFR. Moreover, the mechanisms by which both EGF and PV-IgG lead to keratinocyte dissociation and cytokeratin retraction appear to be different.

Pemphigus vulgaris IgG cause loss of desmoglein-mediated adhesion and keratinocyte dissociation independent of epidermal growth factor receptor.

The release of transmitter-filled vesicles from presynaptic terminals is a key step of neurotransmission. Prior to release, synaptic vesicles get clustered at a specialized patch of the presynaptic membrane, here referred to as the active zone. So far, mainly biochemical regulations at the active zone were regarded as decisive for synaptic vesicle clustering and release. However, using biophysical approaches, a recent paper [Siechen, et al. (2009). Proc. Natl. Acad. Sci. U.S.A. 106, 12611-12616] indicated also that the micromechanical regulations within axon and terminal could be crucial for proper vesicle clustering. The authors demonstrated that the synaptic vesicle accumulations vanished after axotomy but were restored after the application of physical tension. Furthermore, axons seem to be under an intrinsic tension, which could be perceived and tuned by an axon-internal tension sensing mechanism. Therefore, mechanical force could steer vesicle clustering and consequently synapse function. Here, we review this interdisciplinary study of Siechen, et al. [Proc. Natl. Acad. Sci. U.S.A. 106, 12611-12616 (2009)] and discuss the significance of cellular mechanics on synaptic function.

Relax? Don't do it!-Linking presynaptic vesicle clustering with mechanical tension.

Using a descanned, laser-induced guide star and direct wavefront sensing, we demonstrate adaptive correction of complex optical aberrations at high numerical aperture (NA) and a 14-ms update rate. This correction permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens and to recover diffraction-limited imaging over large volumes (>240 mm per side). We applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain.

Rapid adaptive optical recovery of optimal resolution over large volumes.

Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.

An assay to image neuronal microtubule dynamics in mice.

CentrinFish permit the visualization of centrosome dynamics in a cellular context in vivo.

To characterize phenotypes of T cells that accumulated in multiple sclerosis (MS) lesions, to compare the lesional T-cell receptor (TCR) repertoire of T-cell subsets to peripheral blood, and to identify paired α and β chains from single CD8(+) T cells from an index patient who we followed for 18 years.

Article	Year
Pemphigus vulgaris IgG cause loss of desmoglein-mediated adhesion and keratinocyte dissociation independent of epidermal growth factor receptor. The American journal of pathology\| PubMed ID: 19147829	2009
Relax? Don't do it!-Linking presynaptic vesicle clustering with mechanical tension. HFSP journal\| PubMed ID: 20514128	2009
Rapid adaptive optical recovery of optimal resolution over large volumes. Nature methods\| PubMed ID: 24727653	2014
An assay to image neuronal microtubule dynamics in mice. Nature communications\| PubMed ID: 25219969	2014
CentrinFish permit the visualization of centrosome dynamics in a cellular context in vivo. Zebrafish\| PubMed ID: 25387243	2014
αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell-related features. Neurology® neuroimmunology & neuroinflammation\| PubMed ID: 25977934	2015

Peter Engerer

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