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How newly synthesized integral membrane proteins and secreted factors are sorted and trafficked to the appropriate location in different cell types remains an important problem in cell biology. One powerful approach for elucidating the trafficking route of a specific protein is to sequester it following synthesis in the endoplasmic reticulum and trigger its release with an externally applied cue. Combined with fluorescent probes, this approach can be used to directly visualize each trafficking step as cargo molecules progress through the different organelles of the secretory network. Here, we discuss design strategies and practical implementation of an inducible protein secretion system we recently developed (zapalog mediated ER trap: zapERtrap) that allows one to use light to initiate secretory trafficking from targeted cells or subcellular domains. We provide detailed protocols for experiments using this approach to visualize protein trafficking from the endoplasmic reticulum to the plasma membrane in fibroblast cell lines and primary cultured neurons.

Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.

SummaryFor normal synapse and circuit function, neurons must regulate the abundance and localization of transmembrane receptor, channel and adhesion proteins over vast cellular expanses, including remote sites in dendrites and axons. Whether the secretory network can support long-range trafficking of synaptic proteins synthesized in the cell body or precise trafficking of locally generated proteins at remote sites remains poorly characterized. We developed an approach for locally triggering secretory trafficking from specific subcellular domains to explore the rate, activity dependence and cargo-specificity of central and remote trafficking networks. Surprisingly, different postsynaptic proteins processed in the cell body were transported deep into dendrites, but with strikingly different kinetics, spatial distributions and activity dependencies. Proteins locally processed in dendrites were broadly dispersed prior to surface insertion, but could be directed locally to synapses. These results provide a novel interrogation of compartmentalized trafficking and reveal basic principles for protein targeting in complex cellular environments.

Fundamental cellular properties are determined by the repertoire and abundance of proteins displayed on the cell surface. As such, the trafficking mechanisms for establishing and maintaining the surface proteome must be tightly regulated for cells to respond appropriately to extracellular cues, yet plastic enough to adapt to ever-changing environments. Not only are the identity and abundance of surface proteins critical, but in many cases, their regulated spatial positioning within surface nanodomains can greatly impact their function. In the context of neuronal cell biology, surface levels and positioning of ion channels and neurotransmitter receptors play essential roles in establishing important properties, including cellular excitability and synaptic strength. Here we review our current understanding of the trafficking pathways that control the abundance and localization of proteins important for synaptic function and plasticity, as well as recent technological advances that are allowing the field to investigate protein trafficking with increasing spatiotemporal precision.

Summary Binding of two different CaM kinases, CaMKII and DAPK1, to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B near S1303 has been implicated in excitotoxic/ischemic neuronal cell death. The GluN2BΔCaMKII mutation (L1298A, R1300Q) is neuroprotective but abolishes only CaMKII but not DAPK1 binding. However, both kinases can additionally phosphorylate GluN2B S1303. Thus, we here tested S1303 phosphorylation for possible contribution to neuronal cell death. The GluN2BΔCaMKII mutation completely abolished phosphorylation by CaMKII and DAPK1, suggesting that the mutation could mediate neuroprotection by disrupting phosphorylation. However, S1303 phosphorylation was not increased by excitotoxic insults in hippocampal slices or by global cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation in vivo. In hippocampal cultures, S1303 phosphorylation was induced by chemical LTD but not LTP stimuli. These results indicate that the additional effect of the GluN2BΔCaMKII mutation on phosphorylation needs to be considered only in LTD but not in LTP or ischemia/excitotoxicity., Graphical abstract, Highlights • A neuroprotective GluN2B mutation blocked S1303 phosphorylation by CaMKII and DAPK1 • GluN2B S1303 is a better substrate for phosphorylation by CaMKII than by DAPK1 • Increased phospho-S1303 was detected after cLTD but not cLTP or excitotoxic stimuli • Increased phospho-S1303 was not detected after global cerebral ischemia in vivo, Biochemistry; Molecular biology; Neuroscience

The repertoire and abundance of proteins displayed on the surface of neuronal dendrites are tuned by regulated fusion of recycling endosomes (REs) with the dendritic plasma membrane. While this process is critical for neuronal function and plasticity, how synaptic activity drives RE fusion remains unexplored. We demonstrate a multistep fusion mechanism that requires Ca2+ from distinct sources. NMDA receptor Ca2+ initiates RE fusion with the plasma membrane, while L-type voltage-gated Ca2+ channels (L-VGCCs) regulate whether fused REs collapse into the membrane or reform without transferring their cargo to the cell surface. Accordingly, NMDA receptor activation triggered AMPA-type glutamate receptor trafficking to the dendritic surface in an L-VGCC-dependent manner. Conversely, potentiating L-VGCCs enhanced AMPA receptor surface expression only when NMDA receptors were also active. Thus L-VGCCs play a role in tuning activity-triggered surface expression of key synaptic proteins by gating the mode of RE fusion.

A DNA vaccine containing the glycoprotein (G) gene of the North American viral hemorrhagic septicemia virus (VHSV) genotype IVb was developed to evaluate the immune response of fish following vaccination and evaluate its efficacy in protecting a susceptible species, the Muskellunge Esox masquinongy, against VHSV-IVb challenge. Seven weeks (539 degree-days) following vaccination with 10 μg of either pVHSivb-G or a control plasmid, Muskellunge were challenged by immersion with 105 plaque-forming units (pfu)/mL of VHSV-IVb. Fish vaccinated with pVHSivb-G had a relative percent survival (RPS) of 45%. Vaccinated fish also had significantly lower mean viral titers in tissues (4.2 × 102 pfu/g) and viral prevalence (4%) than fish receiving the plasmid control vaccine (3.3 × 105 pfu/g; 82%). Neutralizing antibodies were detected 28 d (308 degree-days) postchallenge (11 weeks postvaccination) in 100% of Muskellunge vaccinated with pVHSivb-G compared with only 12% of plasmid-control-vaccinated Muskellunge, s...

Photosynthetic cells of most land plant lineages have numerous small chloroplasts even though most algae, and even the early diverging land plant group the hornworts, tend to have one or a few large chloroplasts. One constraint that small chloroplasts could improve is the resistance to CO2 diffusion from the atmosphere to the chloroplast stroma. We examined the mesophyll conductance (inverse of the diffusion resistance) of mutant Arabidopsis thaliana plants with one or only a few large chloroplasts per cell. The accumulation and replication of chloroplasts (arc) mutants of A. thaliana were studied by model fitting to gas exchange data and 13CO2 discrimination during carbon fixation. The two methods generally agreed, but the value of the CO2 compensation point of Rubisco (Γ *) used in the model had a large impact on the estimated photosynthetic parameters, including mesophyll conductance. We found that having only a few large chloroplasts per cell resulted in a 25–50 % reduction in the mesophyll conductance at ambient CO2.

Neurons face the challenge of regulating the abundance, distribution and repertoire of integral membrane proteins within their immense, architecturally complex dendritic arbors. While the endoplasmic reticulum (ER) supports dendritic translation, most dendrites lack the Golgi apparatus (GA), an essential organelle for conventional secretory trafficking. Thus, whether secretory cargo is locally trafficked in dendrites through a non-canonical pathway remains a fundamental question. Here we define the dendritic trafficking itinerary for key synaptic molecules in rat cortical neurons. Following ER exit, the AMPA-type glutamate receptor GluA1 and neuroligin 1 undergo spatially restricted entry into the dendritic secretory pathway and accumulate in recycling endosomes (REs) located in dendrites and spines before reaching the plasma membrane. Surprisingly, GluA1 surface delivery occurred even when GA function was disrupted. Thus, in addition to their canonical role in protein recycling, REs also mediate forward secretory trafficking in neuronal dendrites and spines through a specialized GA-independent trafficking network., eLife digest All cells must produce, sort and deliver molecular building blocks to the right places at the right time and in appropriate amounts. This is particularly important for neurons, which are the largest and most structurally complex cells in the body. A typical neuron consists of a cell body covered in branches called dendrites, plus a single cable-like structure known as an axon. Dendrites receive inputs from other neurons and relay the information to the cell body in the form of electrical signals. The cell body processes these electrical signals and the resulting signals then travel along the axon to terminals at the far-end. The axon terminals in turn pass the signals on to the dendrites of other neurons via junctions called synapses. For synapses to work correctly, the membranes surrounding the dendrites need to contain receptor proteins that can detect incoming signals. These proteins must be continually replenished, raising the question of how newly made receptor molecules are shuttled to the appropriate locations within the dendrites. A series of compartments called the Golgi complex play an important role in processing newly-made proteins in many different types of cells. As proteins pass through the Golgi, enzymes within the tunnel walls modify the proteins by adding or removing molecular groups. Therefore, it has been suggested that the route that the synapse receptor proteins take through the neuron to reach the dendrites always includes a visit to the Golgi. However, the Golgi complex in neurons is mostly confined to the cell body, raising the question of whether proteins that are locally produced within dendrites can make the journey to nearby synapses without visiting the Golgi complex. Bowen et al. used a microscope to follow the movements of synapse receptor proteins through neurons grown in a dish. The experiments show that proteins destined for the dendrites make a number of stops after leaving the cell body. However, some synaptic proteins reach the dendrites without passing through the Golgi at all, suggesting neurons are much less dependent on the Golgi to process newly-made proteins than other types of cells. Genetic mutations that prevent proteins from finding their way to their required destinations, or that disrupt the work of enzymes inside trafficking stations like the Golgi, cause numerous human diseases. Understanding how proteins travel to specific destinations inside healthy cells should also help reveal what happens when this process fails.

The repertoire and abundance of proteins displayed on the surface of neuronal dendrites are tuned by regulated fusion of recycling endosomes (REs) with the dendritic plasma membrane. While this process is critical for neuronal function and plasticity, how synaptic activity drives RE fusion remains unexplored. We demonstrate a multistep fusion mechanism that requires Ca(2+) from distinct sources. NMDA receptor Ca(2+) initiates RE fusion with the plasma membrane, while L-type voltage-gated Ca(2+) channels (L-VGCCs) regulate whether fused REs collapse into the membrane or reform without transferring their cargo to the cell surface. Accordingly, NMDA receptor activation triggered AMPA-type glutamate receptor trafficking to the dendritic surface in an L-VGCC-dependent manner. Conversely, potentiating L-VGCCs enhanced AMPA receptor surface expression only when NMDA receptors were also active. Thus L-VGCCs play a role in tuning activity-triggered surface expression of key synaptic proteins by gating the mode of RE fusion.

Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher affinity switch (0.8-47 μM) was less effective because more colocalization was seen in the dark. Additionally, with a point mutation in the LOV domain (N414L), we lengthened the reversion half-life of iLID. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light.