Impaired dynamic interaction of axonal endoplasmic reticulum and ribosomes contributes to defective stimulus-response in spinal muscular atrophy

Background:Axonal degeneration and defects in neuromuscular neurotransmission represent a pathological hall-mark in spinal muscular atrophy(SMA)and other forms of motoneuron disease.These pathological changes do not only base on altered axonal and presynaptic architecture,but also on alterations in dynamic movements of organelles and subcellular structures that are not necessarily reflected by static histopathological changes.The dynamic inter-play between the axonal endoplasmic reticulum(ER)and ribosomes is essential for stimulus-induced local translation in motor axons and presynaptic terminals.However,it remains enigmatic whether the ER and ribosome crosstalk is impaired in the presynaptic compartment of motoneurons with Smn(survival of motor neuron)deficiency that could contribute to axonopathy and presynaptic dysfunction in SMA.Methods:Using super-resolution microscopy,proximity ligation assay(PLA)and live imaging of cultured motoneu-rons from a mouse model of SMA,we investigated the dynamics of the axonal ER and ribosome distribution and activation.Results:We observed that the dynamic remodeling of ER was impaired in axon terminals of Smn-deficient motoneu-rons.In addition,in axon terminals of Smn-deficient motoneurons,ribosomes failed to respond to the brain-derived neurotrophic factor stimulation,and did not undergo rapid association with the axonal ER in response to extracellular stimuli.Conclusions:These findings implicate impaired dynamic interplay between the ribosomes and ER in axon terminals of motoneurons as a contributor to the pathophysiology of SMA and possibly also other motoneuron diseases.

Characterization of an infectious pancreatic necrosis virus from rainbow trout fry(Onhorhynchus mykiss) in West Ukraine

Dear Editor,Aquatic birnaviruses are a group within the family Birnaviridae that comprise isolates from fish and shellfish from both fresh-and seawater(Woo and Bruno,1999).Members of the family Birnaviridae are icosahedral viruses approximately 60 nm in diameter composed of five polypeptides and two segments of double-stranded RNA(Dixon et al.,2008).In this report,an aquatic birnavirus was isolated from rainbow trout fry,Onhorhynchus myki-

A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants

Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolu- tion fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluores- cent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ＇positively switchable＇ PADRON C-s with the ＇negatively switchable＇ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localiza- tion analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 （Arabidopsis thaliana glycine-rich RNA-binding protein 7） in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each pho- toactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7：rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants, Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells.

A Trojan Horse for live-cell super-resolution microscopy

New peptide vehicles enable the efficient live-cell labeling of intracellular organelles with cell-impermeable fluorescent probes by simple coincubation,paving the way for refined multicolor super-resolution fluorescence imaging.

Variant signaling topology at the cancer cell–T-cell interface induced by a two-component T-cell engager

Bispecific T-cell-engaging antibodies(BiTEs)convey antigenic information from cancer to the T-lymphocyte-associated CD3 complex,thus redirecting immune effector cells to induce target cell lysis.Since antigenic information from transformed tissues is rarely unambiguous,we recently introduced novel T-cell-engaging antibody fragments called hemibodies for the combinatorial,ultraprecise targeting of cancer.1 Here,we report the unprecedented enhancement of the plasticity of immunological synapse(IS)formation and variant signaling topology at the cancer cell–T-cell interface induced by hemibodies compared to that induced by BiTEs.

Sharpening emitter localization in front of a tuned mirror

Single-molecule localization microscopy(SMLM)aims for maximized precision and a high signal-to-noise ratio1.Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2–4.Here,we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two.The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength,as experimentally demonstrated for dual-color SMLM in cells.