Guido M. Gaietta

Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors.

Regulation of AMPA receptor (AMPAR) trafficking is important for neural plasticity. Here we examined the trafficking and synthesis of the GluR1 and GluR2 subunits using ReAsH-EDT2 and FlAsH-EDT2 staining. Activity blockade of rat cultured neurons increased dendritic GluR1, but not GluR2, levels. Examination of transected dendrites revealed that both AMPAR subunits were synthesized in dendrites and that activity blockade enhanced dendritic synthesis of GluR1 but not GluR2. In contrast, acute pharmacological manipulations increased dendritic synthesis of both subunits. AMPARs synthesized in dendrites were inserted into synaptic plasma membranes and, after activity blockade, the electrophysiological properties of native synaptic AMPARs changed in the manner predicted by the imaging experiments. In addition to providing a novel mechanism for synaptic modifications, these results point out the advantages of using FlAsH-EDT2 and ReAsH-EDT2 for studying the trafficking of newly synthesized proteins in local cellular compartments such as dendrites.

A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells.

Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a well-established method to monitor protein-protein interactions or conformational changes of individual proteins. But protein functions can be perturbed by fusion of large tags such as CFP and YFP. Here we use G protein–coupled receptor (GPCR) activation in living cells as a model system to compare YFP with the small, membrane-permeant fluorescein derivative with two arsen-(III) substituents (fluorescein arsenical hairpin binder; FlAsH) targeted to a short tetracysteine sequence. Insertion of CFP and YFP into human adenosine A2A receptors allowed us to use FRET to monitor receptor activation but eliminated coupling to adenylyl cyclase. The CFP/FlAsH-tetracysteine system gave fivefold greater agonist-induced FRET signals, similar kinetics (time constant of 66–88 ms) and perfectly normal downstream signaling. Similar results were obtained for the mouse α2A-adrenergic receptor. Thus, FRET from CFP to FlAsH reports GPCR activation in living cells without disturbing receptor function and shows that the small size of the tetracysteine-biarsenical tag can be decisively advantageous.

The Arg451Cys-Neuroligin-3 Mutation Associated with Autism Reveals a Defect in Protein Processing

The neuroligins are a family of postsynaptic transmembrane proteins that associate with presynaptic partners, the β-neurexins. Neurexins and neuroligins play a critical role in initiating formation and differentiation of synaptic junctions. A recent study reported that a mutation of neuroligin-3 (NL3), an X-linked gene, was found in siblings with autistic spectrum disorder in which two affected brothers had a point mutation that substituted a Cys for Arg451. To characterize the mutation at the biochemical level, we analyzed expression and activity of the mutated protein. Mass spectrometry comparison of the disulfide bonding pattern between the native and the mutated proteins indicates the absence of aberrant disulfide bonding, suggesting that the secondary structure of the mutated protein is conserved. However, the mutation separately affects protein expression and activity. The Cys mutation causes defective neuroligin trafficking, leading to retention of the protein in the endoplasmic reticulum. This, in turn, decreases the delivery of NL3 to the cell surface. Also, the small fraction of protein that reaches the cell membrane lacks or has markedly diminished β-neurexin-1 (NX1β) binding activity. Other substitutions for Arg451 allow for normal cellular expression but diminished affinity for NX1β. Our findings reveal a cellular phenotype and loss of function for a congenital mutation associated with autistic spectrum disorders.

Pannexin1 Channels Contain a Glycosylation Site That Targets the Hexamer to the Plasma Membrane

Pannexins are newly discovered channel proteins expressed in many different tissues and abundantly in the vertebrate central nervous system. Based on membrane topology, folding and secondary structure prediction, pannexins are proposed to form gap junction-like structures. We show here that Pannexin1 forms a hexameric channel and reaches the cell surface but, unlike connexins, is N-glycosylated. Using site-directed mutagenesis we analyzed three putative N-linked glycosylation sites and examined the effects of each mutation on channel expression. We show for the first time that Pannexin1 is glycosylated at Asn-254 and that this residue is important for plasma membrane targeting. The glycosylation of Pannexin1 at its extracellular surface makes it unlikely that two oligomers could dock to form an intercellular channel. Ultrastructural analysis by electron microscopy confirmed that Pannexin1 junctional areas do not appear as canonical gap junctions. Rather, Pannexin1 channels are distributed throughout the plasma membrane. We propose that N-glycosylation of Pannexin1 could be a significant mechanism for regulating the trafficking of these membrane proteins to the cell surface in different tissues.

Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy

Combinations of molecular tags visible in light and electron microscopes become particularly advantageous in the analysis of dynamic cellular components like the Golgi apparatus. This organelle disassembles at the onset of mitosis and, after a sequence of poorly understood events, reassembles after cytokinesis. The precise location of Golgi membranes and resident proteins during mitosis remains unclear, partly due to limitations of molecular markers and the resolution of light microscopy. We generated a fusion consisting of the first 117 residues of α-mannosidase II tagged with a fluorescent protein and a tetracysteine motif. The mannosidase component guarantees docking into the Golgi membrane, with the tags exposed in the lumen. The fluorescent protein is optically visible without further treatment, whereas the tetracysteine tag can be reduced acutely with a membrane-permeant phosphine, labeled with ReAsH, monitored in the light microscope, and used to trigger the photoconversion of diaminobenzidine, allowing 4D optical recording on live cells and correlated ultrastructural analysis by electron microscopy. These methods reveal that Golgi reassembly is preceded by the formation of four colinear clusters at telophase, two per daughter cell. Within each daughter, the smaller cluster near the midbody gradually migrates to rejoin the major cluster on the far side of the nucleus and asymmetrically reconstitutes a single Golgi apparatus, first in one daughter cell and then in the other. Our studies provide previously undescribed insights into Golgi disassociation and reassembly during mitosis and offer a powerful approach to follow recombinant protein distribution in 4D imaging and correlated high-resolution analysis.

Fluorescent labeling of tetracysteine-tagged proteins in intact cells

In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder–ethanedithiol (FlAsH-EDT2). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg−1 of protein, such as G protein–coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT2 as described takes 2–3 h, depending on the number of samples to be processed.

Markers for correlated light and electron microscopy

Publisher Summary This chapter reviews whole cell, organelle, and protein labeling methods for correlated observations in the spatial domains of both light microscopy (LM) and electron microscopy (EM). The combination of EM tomographic reconstructions with labeling of specific components provides a powerful method for localizing proteins within their cellular components. Better fluorescent probes have been and are being developed that highlight a particular protein of interest, and better LM instrumentation allows to monitor these probes in three dimensions (3D) and real time (4D-imaging). In addition, developments in fluorescence LM provide great opportunities to determine protein colocalization, turnover, and function. New tools have been developed for correlated LM and EM—namely, the photooxidation of fluorescent probes and immuno-quantum dots (QD) labeling; these complement more established techniques such as peroxidase or colloidal gold (CG) labeling. A comparison of these methods has been achieved by using a standard structure, such as microtubules, which are immunolabeled for tubulin and then detected by photooxidation, QDs, CG, and enzymatic labeling.

The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodies

The C-terminus of the most abundant and best-studied gap-junction protein, connexin43, contains multiple phosphorylation sites and protein-binding domains that are involved in regulation of connexin trafficking and channel gating. It is well-documented that SDS/PAGE of NRK (normal rat kidney) cell lysates reveals at least three connexin43-specific bands (P0, P1 and P2). P1 and P2 are phosphorylated on multiple, unidentified serine residues and are found primarily in gap-junction plaques. In the present study we prepared monoclonal antibodies against a peptide representing the last 23 residues at the C-terminus of connexin43. Immunofluorescence studies showed that one antibody (designated CT1) bound primarily to connexin43 present in the Golgi apparatus, whereas the other antibody (designated IF1) labelled predominately connexin43 present in gap junctions. CT1 immunoprecipitates predominantly the P0 form whereas IF1 recognized all three bands. Peptide mapping, mutational analysis and protein-protein interaction experiments revealed that unphosphorylated Ser364 and/or Ser365 are critical for CT1 binding. The IF1 paratope binds to residues Pro375-Asp379 and requires Pro375 and Pro377. These proline residues are also necessary for ZO-1 interaction. These studies indicate that the conformation of Ser364/Ser365 is important for intracellular localization, whereas the tertiary structure of Pro375-Asp379 is essential in targeting and regulation of gap junctional connexin43.

Caulobacter PopZ forms a polar subdomain dictating sequential changes in pole composition and function

The bacterium Caulobacter crescentus has morphologically and functionally distinct cell poles that undergo sequential changes during the cell cycle. We show that the PopZ oligomeric network forms polar ribosome exclusion zones that change function during cell cycle progression. The parS/ParB chromosomal centromere is tethered to PopZ at one pole prior to the initiation of DNA replication. During polar maturation, the PopZ‐centromere tether is broken, and the PopZ zone at that pole then switches function to act as a recruitment factor for the ordered addition of multiple proteins that promote the transformation of the flagellated pole into a stalked pole. Stalked pole assembly, in turn, triggers the initiation of chromosome replication, which signals the formation of a new PopZ zone at the opposite cell pole, where it functions to anchor the newly duplicated centromere that has traversed the long axis of the cell. We propose that pole‐specific control of PopZ function co‐ordinates polar development and cell cycle progression by enabling independent assembly and tethering activities at the two cell poles.

Mutation of a conserved threonine in the third transmembrane helix of α- and β-connexins creates a dominant-negative closed gap junction channel

Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking, and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near the cytoplasmic end of the third transmembrane helix. This threonine is strictly conserved among members of the α- and β-connexin subgroups but not the γ-subgroup. In HeLa cells, connexin43 and connexin26 mutants are synthesized, traffic to the plasma membrane, and make gap junctions with the same overall appearance as wild type. We have isolated connexin26T135A gap junctions both from HeLa cells and baculovirus-infected insect Sf9 cells. By using cryoelectron microscopy and correlation averaging, difference images revealed a small but significant size change within the pore region and a slight rearrangement of the subunits between mutant and wild-type connexons expressed in Sf9 cells. Purified, detergent-solubilized mutant connexons contain both hexameric and partially disassembled structures, although wild-type connexons are almost all hexameric, suggesting that the three-dimensional mutant connexon is unstable. Mammalian cells expressing gap junction plaques composed of either connexin43T154A or connexin26T135A showed an absence of dye coupling. When expressed in Xenopus oocytes, these mutants, as well as a cysteine substitution mutant of connexin50 (connexin50T157C), failed to produce electrical coupling in homotypic and heteromeric pairings with wild type in a dominant-negative effect. This mutant may be useful as a tool for knocking down or knocking out connexin function in vitro or in vivo.