Unveiling nanoscale optical signatures of cytokine-induced ß-cell dysfunction.

Pro-inflammatory cytokines contribute to ß-cell failure in both Type-1 and Type-2 Diabetes. Data collected so far allowed to dissect the genomic, transcriptomic, proteomic and biochemical landscape underlying cytokine-induced ß-cell progression through dysfunction. Yet, no report thus far complemented such molecular information with the direct optical nanoscopy of the ß-cell subcellular environment. Here we tackle this issue in Insulinoma 1E (INS-1E) ß-cells by label-free fluorescence lifetime imaging microscopy (FLIM) and fluorescence-based super resolution imaging by expansion microscopy (ExM). It is found that 24-h exposure to IL-1ß and IFN-γ is associated with a neat modification of the FLIM signature of cell autofluorescence due to the increase of either enzyme-bound NAD(P)H molecules and of oxidized lipid species. At the same time, ExM-based direct imaging unveils neat alteration of mitochondrial morphology (i.e. ~ 80% increase of mitochondrial circularity), marked degranulation (i.e. ~ 40% loss of insulin granules, with mis-localization of the surviving pool), appearance of F-actin-positive membrane blebs and an hitherto unknown extensive fragmentation of the microtubules network (e.g. ~ 37% reduction in the number of branches). Reported observations provide an optical-microscopy framework to interpret the amount of molecular information collected so far on ß-cell dysfunction and pave the way to future ex-vivo and in-vivo investigations.

Axonal plasticity in response to active forces generated through magnetic nano-pulling.

Mechanical force is crucial in guiding axon outgrowth before and after synapse formation. This process is referred to as "stretch growth." However, how neurons transduce mechanical input into signaling pathways remains poorly understood. Another open question is how stretch growth is coupled in time with the intercalated addition of new mass along the entire axon. Here, we demonstrate that active mechanical force generated by magnetic nano-pulling induces remodeling of the axonal cytoskeleton. Specifically, the increase in the axonal density of microtubules induced by nano-pulling leads to an accumulation of organelles and signaling vesicles, which, in turn, promotes local translation by increasing the probability of assembly of the "translation factories." Modulation of axonal transport and local translation sustains enhanced axon outgrowth and synapse maturation.

Manipulation of Axonal Outgrowth via Exogenous Low Forces.

Neurons are mechanosensitive cells. The role of mechanical force in the process of neurite initiation, elongation and sprouting; nerve fasciculation; and neuron maturation continues to attract considerable interest among scientists. Force is an endogenous signal that stimulates all these processes in vivo. The axon is able to sense force, generate force and, ultimately, transduce the force in a signal for growth. This opens up fascinating scenarios. How are forces generated and sensed in vivo? Which molecular mechanisms are responsible for this mechanotransduction signal? Can we exploit exogenously applied forces to mimic and control this process? How can these extremely low forces be generated in vivo in a non-invasive manner? Can these methodologies for force generation be used in regenerative therapies? This review addresses these questions, providing a general overview of current knowledge on the applications of exogenous forces to manipulate axonal outgrowth, with a special focus on forces whose magnitude is similar to those generated in vivo. We also review the principal methodologies for applying these forces, providing new inspiration and insights into the potential of this approach for future regenerative therapies.