The envelope glycoproteins (Env) of HIV-1 mediate cell entry through fusion of the viral envelope with a target cell membrane. Intramembrane mobility and clustering of Env trimers at the viral budding site are essential for its function. Previous live-cell and super-resolution microscopy studies were limited by lack of a functional fluorescent Env derivative, requiring antibody labeling for detection. Introduction of a bio-orthogonal amino acid by genetic code expansion, combined with click chemistry, offers novel possibilities for site-specific, minimally invasive labeling. Using this approach, we established efficient incorporation of non-canonical amino acids within HIV-1 Env in mammalian cells. The engineered protein retained plasma membrane localization, glycosylation, virion incorporation, and fusogenic activity, and could be rapidly and specifically labeled with synthetic dyes. This strategy allowed us to revisit Env dynamics and nanoscale distribution at the plasma membrane close to its native state, applying fluorescence recovery after photo bleaching and STED nanoscopy, respectively.

A key component allowing a neuron to function properly within its dynamic environment is the axon initial segment (AIS), the site of action potential generation. In visual cortex, AIS of pyramidal neurons undergo periods of activity-dependent structural plasticity during development. However, it remains unknown how AIS morphology is organized during development for downstream cells in the visual pathway (retinal ganglion cells; RGCs) and whether AIS retain the ability to dynamically adjust to changes in network state. Here, we investigated the maturation of AIS in RGCs during mouse retinal development, and tested putative activity-dependent mechanisms by applying visual deprivation with a focus on the AIS-specific cisternal organelle (CO), a presumed Ca2+-store. Whole-mount retinae from wildtype and Thy1-GFP transgenic mice were processed for multi-channel immunofluorescence using antibodies against AIS scaffolding proteins ankyrin-G, βIV-spectrin and the CO marker synaptopodin (synpo). Confocal microscopy in combination with morphometrical analysis of AIS length and position as well as synpo cluster size was performed. Data indicated that a subset of RGC AIS contains synpo clusters and that these show significant dynamic regulation in size during development as well as after visual deprivation. Using super resolution microscopy, we addressed the subcellular localization of synpo in RGC axons. Similar to cortical neurons, RGCs show a periodic distribution of AIS scaffolding proteins. A previously reported scaffold-deficient nanodomain correlating with synpo localization is not evident in all RGC AIS. In summary, our work demonstrates a dynamic regulation of both the AIS and synpo in RGCs during retinal development and after visual deprivation, providing first evidence that the AIS and CO in RGCs can undergo structural plasticity in response to changes in network activity.

Patients with Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) positive lung cancer are sensitive to ALK-kinase inhibitors. TAE684 is a potent second generation ALK inhibitor that overcomes Crizotinib resistance. Radiotherapy is an integral therapeutic component of locally advanced lung cancer. Therefore, we sought to investigate the effects of combined radiotherapy and ALK-inhibition via TAE684 in ALK-positive vs. wild type lung cancer cells.

Mechanical signaling plays an important role in cell physiology and pathology. Many cell types, including neurons and glial cells, respond to the mechanical properties of their environment. Yet, for spinal cord tissue, data on tissue stiffness are sparse. To investigate the regional and direction-dependent mechanical properties of spinal cord tissue at a spatial resolution relevant to individual cells, we conducted atomic force microscopy (AFM) indentation and tensile measurements on acutely isolated mouse spinal cord tissue sectioned along the three major anatomical planes, and correlated local mechanical properties with the underlying cellular structures. Stiffness maps revealed that gray matter is significantly stiffer than white matter irrespective of directionality (transverse, coronal, and sagittal planes) and force direction (compression or tension) (K(g) = ∼ 130 P(a) vs. K(w) = ∼ 70 Pa); both matters stiffened with increasing strain. When all data were pooled for each plane, gray matter behaved like an isotropic material under compression; however, subregions of the gray matter were rather heterogeneous and anisotropic. For example, in sagittal sections the dorsal horn was significantly stiffer than the ventral horn. In contrast, white matter behaved transversely isotropic, with the elastic stiffness along the craniocaudal (i.e., longitudinal) axis being lower than perpendicular to it. The stiffness distributions we found under compression strongly correlated with the orientation of axons, the areas of cell nuclei, and cellular in plane proximity. Based on these morphological parameters, we developed a phenomenological model to estimate local mechanical properties of central nervous system (CNS) tissue. Our study may thus ultimately help predicting local tissue stiffness, and hence cell behavior in response to mechanical signaling under physiological and pathological conditions, purely based on histological data.