Neuronal synapses require precise regulation, particularly of membrane components. The composition and organization of synaptic membranes are dramatically remodeled during development, including accumulation of lipids associated with raft domains, and concomitant palmitoylation of PSD-95, suggesting recruitment of domains via scaffold lipidation.

The PKR-like endoplasmic reticulum kinase (PERK) arm of the Integrated Stress Response (ISR) is implicated in neurodegenerative disease, although the regulators and consequences of PERK activation following neuronal injury are poorly understood. Here we show that PERK signaling is a component of the mouse MAP kinase neuronal stress response controlled by the Dual Leucine Zipper Kinase (DLK) and contributes to DLK-mediated neurodegeneration. We find that DLK-activating insults ranging from nerve injury to neurotrophin deprivation result in both c-Jun N-terminal Kinase (JNK) signaling and the PERK- and ISR-dependent upregulation of the Activating Transcription Factor 4 (ATF4). Disruption of PERK signaling delays neurodegeneration without reducing JNK signaling. Furthermore, DLK is both sufficient for PERK activation and necessary for engaging the ISR subsequent to JNK-mediated retrograde injury signaling. These findings identify DLK as a central regulator of not only JNK but also PERK stress signaling in neurons, with both pathways contributing to neurodegeneration.DOI: http://dx.doi.org/10.7554/eLife.20725.001

bCryo-electron tomography (cryo-ET) has emerged as a leading technique for three-dimensional visualization of large macromolecular complexes and their conformational changes in their native cellular environment. However, the resolution and potential applications of cryo-ET are fundamentally limited by specimen thickness, preventing high-resolution in situ visualization of macromolecular structures in many bacteria (such as Escherichia coli and Salmonella enterica). Minicells, which were discovered nearly 50 years ago, have recently been exploited as model systems to visualize molecular machines in situ, due to their smaller size and other unique properties. In this review, we discuss strategies for producing minicells and highlight their use in the study of chemotactic signaling, protein secretion, and DNA translocation. In combination with powerful genetic tools and advanced imaging techniques, minicells provide a springboard for in-depth structural studies of bacterial macromolecular complexes in situ and therefore offer a unique approach for gaining novel structural insights into many important processes in microbiology.

Calcium-calmodulin-dependent protein kinase II (CaMKII) has been implicated in a wide variety of cellular processes, which include a critical regulatory role in actin cytoskeletal assembly. CaMKII is ubiquitous in cells, expressed as one of four isoforms referred to as α, β, γ, and δ. Characterization of the CaMKII-actin interaction has been mainly focused on the β isoform, which has been shown to bundle actin filaments and sequester actin monomers in an activity dependent manner. Much less is known about the interactions of other CaMKII isoforms with actin. In the present report, isoform specific interactions of CaMKII with actin are described and reveal that the δ isoform of CaMKII bundles F-actin filaments similarly to the β isoform while the γ isoform induces a novel layered structure to filaments. Using electron tomography, CaMKII holoenzymes are clearly identified in the complexes bridging the actin filaments, allowing direct visualization of the interactions between CaMKII isoforms and actin. In addition, we determined the isoform specificity of CaMKII-mediated inhibition of actin polymerization and discovered that all isoforms inhibit polymerization to varying degrees with β > γ ≈ δ > α (from most to least effective). Ca2+/CaM activation of all kinase isoforms produced a robust increase in actin polymerization that surpassed the rates of polymerization in the absence of kinase inhibition. These results indicate that diversity exists between the types of CaMKII-actin interactions mediated by the different isoforms and that CaMKII isoform composition differentially impacts the formation and maintenance of the actin cytoskeleton.

From injury to disease to aging, neurons, like all cells, may face various insults that can impact their function and survival. Although the consequences are substantially dictated by the type, context, and severity of insult, distressed neurons are far from passive. Activation of cellular stress responses aids in the preservation or restoration of nervous system function. However, stress responses themselves can further advance neuropathology and contribute significantly to neuronal dysfunction and neurodegeneration. Here we explore the recent advances in defining the cellular stress responses within neurodegenerative diseases and neuronal injury, and we emphasize axonal injury as a well-characterized model of neuronal insult. We highlight key findings and unanswered questions about neuronal stress response pathways, from the initial detection of cellular insults through the underlying mechanisms of the responses to their ultimate impact on the fates of distressed neurons.