Several fluorescent proteins (FPs) are prone to forming low affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric under physiologic conditions. Membrane-associated FP-fusion proteins, by virtue of their constrained planar geometry achieve high effective concentrations. We exploited this propensity to develop an assay to measure FP tendencies to oligomerize in cells. FPs were fused on the cytoplasmic end of an endoplasmic reticulum (ER) signal anchor membrane protein (CytERM) and expressed in cells. Cells were scored based on the ability of CytERM to homo-oligomerize with proteins on apposing membranes and restructure the ER from a tubular network into organized smooth ER (OSER) whorl structures. The ratio of nuclear envelope and OSER structures mean fluorescent intensities for cells expressing EGFP or mGFP CytERM established standards for comparison of uncharacterized FPs. We tested three FPs and identified two as sufficiently monomeric while a third previously reported as monomeric was found to strongly oligomerize.

VAPB (vesicle-associated membrane protein-associated protein B) is an endoplasmic reticulum (ER)-resident tail-anchored adaptor protein involved in lipid transport. A dominantly inherited mutant, P56S-VAPB, causes a familial form of amyotrophic lateral sclerosis (ALS) and forms poorly characterized inclusion bodies in cultured cells. To provide a cell biological basis for the understanding of mutant VAPB pathogenicity, we investigated its biogenesis and the inclusions that it generates. Translocation assays in cell-free systems and in cultured mammalian cells were used to investigate P56S-VAPB membrane insertion, and the inclusions were characterized by confocal imaging and electron microscopy. We found that mutant VAPB inserts post-translationally into ER membranes in a manner indistinguishable from the wild-type protein but that it rapidly clusters to form inclusions that remain continuous with the rest of the ER. Inclusions were induced by the mutant also when it was expressed at levels comparable to the endogenous wild-type protein. Ultrastructural analysis revealed that the inclusions represent a novel form of organized smooth ER (OSER) consisting in a limited number of parallel cisternae (usually 2 or 3) interleaved by a approximately 30 nm-thick electron-dense cytosolic layer. Our results demonstrate that the ALS-linked VAPB mutant causes dramatic ER restructuring that may underlie its pathogenicity in motoneurons.

The proper function of neural circuits requires spatially and temporally balanced development of excitatory and inhibitory synapses. However, the molecular mechanisms coordinating excitatory and inhibitory synaptogenesis remain unknown. Here we demonstrate that SRGAP2A and its human-specific paralog SRGAP2C co-regulate the development of excitatory and inhibitory synapses in cortical pyramidal neurons in vivo. SRGAP2A promotes synaptic maturation, and ultimately the synaptic accumulation of AMPA and GABAA receptors, by interacting with key components of both excitatory and inhibitory postsynaptic scaffolds, Homer and Gephyrin. Furthermore, SRGAP2A limits the density of both types of synapses via its Rac1-GAP activity. SRGAP2C inhibits all identified functions of SRGAP2A, protracting the maturation and increasing the density of excitatory and inhibitory synapses. Our results uncover a molecular mechanism coordinating critical features of synaptic development and suggest that human-specific duplication of SRGAP2 might have contributed to the emergence of unique traits of human neurons while preserving the excitation/inhibition balance.

VAPB (vesicle-associated membrane protein-associated protein B) is a ubiquitously expressed, ER-resident tail-anchored protein that functions as adaptor for lipid-exchange proteins. Its mutant form, P56S-VAPB, is linked to a dominantly inherited form of amyotrophic lateral sclerosis (ALS8). P56S-VAPB forms intracellular inclusions, whose role in ALS pathogenesis has not yet been elucidated. We recently demonstrated that these inclusions are formed by profoundly remodelled stacked ER cisternae. Here, we used stable HeLa-TetOff cell lines inducibly expressing wild-type VAPB and P56S-VAPB, as well as microinjection protocols in non-transfected cells, to investigate the dynamics of inclusion generation and degradation. Shortly after synthesis, the mutant protein forms small, polyubiquitinated clusters, which then congregate in the juxtanuclear region independently of the integrity of the microtubule cytoskeleton. The rate of degradation of the aggregated mutant is higher than that of the wild-type protein, so that the inclusions are cleared only a few hours after cessation of P56S-VAPB synthesis. At variance with other inclusion bodies linked to neurodegenerative diseases, clearance of P56S-VAPB inclusions involves the proteasome, with no apparent participation of macro-autophagy. Transfection of a dominant-negative form of the AAA ATPase p97/VCP stabilizes mutant VAPB, suggesting a role for this ATPase in extracting the aggregated protein from the inclusions. Our results demonstrate that the structures induced by P56S-VAPB stand apart from other inclusion bodies, both in the mechanism of their genesis and of their clearance from the cell, with possible implications for the pathogenic mechanism of the mutant protein.

Highlights d GluD1 mediates the formation and specification of inhibitory synapses in the cortex d GluD1 forms trans-synaptic interactions via Cbln4, a protein secreted by SST + INs d GluD1 elicits agonist-dependent postsynaptic signaling via ARHGEF12 and PPP1R12A d GluD1 is a maverick iGluR operating through non-ionotropic mechanisms

Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABAB receptors (GABABRs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABABR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABABRs and extrasynaptic δ-subunit-containing GABAARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABABR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy.

The Golgi complex and ER are dynamically connected by anterograde and retrograde trafficking pathways. To what extent and by what mechanism outward-bound cargo proteins escape retrograde trafficking has been poorly investigated. Here, we analysed the behaviour of several membrane proteins at the ER/Golgi interface in live cells. When Golgi-to-plasma membrane transport was blocked, vesicular stomatitis virus glycoprotein (VSVG), which bears an ER export signal, accumulated in the Golgi, whereas an export signal-deleted version of VSVG attained a steady state determined by the balance of retrograde and anterograde traffic. A similar behaviour was displayed by EGF receptor and by a model tailanchored protein, whose retrograde traffic was slowed by addition of VSVG's export signal. Retrograde trafficking was energy-and Rab6-dependent, and Rab6 inhibition accelerated signal-deleted VSVG's transport to the cell surface. Our results extend the dynamic bi-directional relationship between the Golgi and ER to include surface-directed proteins, uncover an unanticipated role for export signals at the Golgi complex, and identify recycling as a novel factor that regulates cargo transport out of the early secretory pathway.

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