Endoplasmic reticulum morphology regulation by RTN4 modulates neuronal regeneration by curbing luminal transport.

Cell functions rely on intracellular transport systems distributing bioactive molecules with high spatiotemporal accuracy. The endoplasmic reticulum (ER) tubular network constitutes a system for delivering luminal solutes, including Ca2+, across the cell periphery. How the ER structure enables this nanofluidic transport system is unclear. Here, we show that ER membrane-localized reticulon 4 (RTN4/Nogo) is sufficient to impose neurite outgrowth inhibition in human cortical neurons while acting as an ER morphoregulator. Improving ER transport visualization methodologies combined with optogenetic Ca2+ dynamics imaging and in silico modeling, we observed that ER luminal transport is modulated by ER tubule narrowing and dilation, proportional to the amount of RTN4. Excess RTN4 limited ER luminal transport and Ca2+ release, while RTN4 elimination reversed the effects. The described morphoregulatory effect of RTN4 defines the capacity of the ER for peripheral Ca2+ delivery for physiological releases and thus may constitute a mechanism for controlling the (re)generation of neurites.

Palbociclib releases the latent differentiation capacity of neuroblastoma cells.

Neuroblastoma is the most common extracranial solid tumor in infants, arising from developmentally stalled neural crest-derived cells. Driving tumor differentiation is a promising therapeutic approach for this devastating disease. Here, we show that the CDK4/6 inhibitor palbociclib not only inhibits proliferation but induces extensive neuronal differentiation of adrenergic neuroblastoma cells. Palbociclib-mediated differentiation is manifested by extensive phenotypic and transcriptional changes accompanied by the establishment of an epigenetic program driving expression of mature neuronal features. In vivo palbociclib significantly inhibits tumor growth in mouse neuroblastoma models. Furthermore, dual treatment with retinoic acid resets the oncogenic adrenergic core regulatory circuit of neuroblastoma cells, further suppresses proliferation, and can enhance differentiation, altering gene expression in ways that significantly correlate with improved patient survival. We therefore identify palbociclib as a therapeutic approach to dramatically enhance neuroblastoma differentiation efficacy that could be used in combination with retinoic acid to improve patient outcomes.

Architecture of cell-cell junctions in situ reveals a mechanism for bacterial biofilm inhibition.

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam-milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.