CiteULike: lechristophe's library [402 articles]

[Surface mobility of postsynaptic AMPARs tunes synaptic transmission.]

D Choquet — 2008-05-13T18:18:04-00:00

Médecine sciences : M/S, Vol. 24, No. 5. (May 2008), pp. 548-550.

An essential role for cortactin in the modulation of the potassium channel Kv1.2.

MR Williams — 2008-05-13T18:16:19-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, No. 44. (30 October 2007), pp. 17412-17417.

Ion channels are key determinants of membrane excitability. The actin cytoskeleton has a central role in morphology, migration, intracellular transport, and signaling. In this article, we show that the actin-binding protein cortactin regulates the potassium channel Kv1.2 and thereby provides a direct link between actin dynamics and membrane excitability. In previous reports, we showed that the tyrosine phosphorylation-mediated suppression of Kv1.2 ionic current occurs by endocytosis of the channel protein. Pull-down assays using recombinant-purified cortactin and Kv1.2 demonstrated that their interaction is direct and reduced by tyrosine phosphorylation of Kv1.2. This finding suggests a link between cortactin and Kv1.2 endocytosis. Here, we confirm that relationship and identify the molecular mechanisms involved. We use FRET to demonstrate that Kv1.2 and cortactin interact in vivo. By manipulating the cortactin-binding site within Kv1.2, we confirm that cortactin proximity influences channel function. We used flow cytometry in conjunction with cortactin gene replacement to identify C-terminal tyrosines, the fourth repeat actin-binding domain, and the N-terminal Arp2/3-binding region, as critical to Kv1.2 regulation. Surprisingly, cortactin's dynamin-binding Src homology 3 domain is not required for Kv1.2 endocytosis, despite that process being dynamin-dependent. These findings predict that cortactin-mediated actin remodeling in excitable cells is not only important for cell structure, but may directly impact membrane excitability.

Interdomain cytoplasmic interactions govern the intracellular trafficking, gating, and modulation of the Kv2.1 channel.

DP Mohapatra — 2008-05-13T12:24:28-00:00

The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 28, No. 19. (7 May 2008), pp. 4982-4994.

Voltage-gated potassium (Kv) channels comprise four transmembrane alpha subunits, often associated with cytoplasmic beta subunits that impact channel expression and function. Here, we show that cell surface expression, voltage-dependent activation gating, and phosphorylation-dependent modulation of Kv2.1 are regulated by cytoplasmic N/C interaction within the alpha subunit. Kv2.1 surface expression is greatly reduced by C-terminal truncation. Tailless Kv2.1 channels exhibit altered voltage-dependent gating properties and lack the bulk of the phosphorylation-dependent modulation of channel gating. Remarkably, the soluble C terminus of Kv2.1 associates with tailless channels and rescues their expression, function, and phosphorylation-dependent modulation. Soluble N and C termini of Kv2.1 can also interact directly. We also show that the N/C-terminal interaction in Kv2.1 is governed by a 34 aa motif in the juxtamembrane cytoplasmic C terminus, and a 17 aa motif located in the N terminus at a position equivalent to the beta subunit binding site in other Kv channels. Deletion of either motif disrupts N/C-terminal interaction and surface expression, function, and phosphorylation-dependent modulation of Kv2.1 channels. These findings provide novel insights into intrinsic mechanisms for the regulation of Kv2.1 trafficking, gating, and phosphorylation-dependent modulation through cytoplasmic N/C-terminal interaction, which resembles alpha/beta subunit interaction in other Kv channels.

K+ Channels at the Axon Initial Segment Dampen Near-Threshold Excitability of Neocortical Fast-Spiking GABAergic Interneurons

Ethan Goldberg — 2008-05-13T10:24:23-00:00

Neuron, Vol. 58, No. 3. (8 May 2008), pp. 387-400.

Summary Fast-spiking cells (FS cells) are a prominent subtype of neocortical GABAergic interneurons with important functional roles. Multiple FS cell properties are coordinated for rapid response. Here, we describe an FS cell feature that serves to gate the powerful inhibition produced by FS cell activity. We show that FS cells in layer 2/3 barrel cortex possess a dampening mechanism mediated by Kv1.1-containing potassium channels localized to the axon initial segment. These channels powerfully regulate action potential threshold and allow FS cells to respond preferentially to large inputs that are fast enough to "outrun" Kv1 activation. In addition, Kv1.1 channel blockade converts the delay-type discharge pattern of FS cells to one of continuous fast spiking without influencing the high-frequency firing that defines FS cells. Thus, Kv1 channels provide a key counterbalance to the established rapid-response characteristics of FS cells, regulating excitability through a unique combination of electrophysiological properties and discrete subcellular localization.

Surface mobility of postsynaptic AMPARs tunes synaptic transmission.

M Heine — 2008-04-13T06:35:31-00:00

Science (New York, N.Y.), Vol. 320, No. 5873. (11 April 2008), pp. 201-205.

AMPA glutamate receptors (AMPARs) mediate fast excitatory synaptic transmission. Upon fast consecutive synaptic stimulation, transmission can be depressed. Recuperation from fast synaptic depression has been attributed solely to recovery of transmitter release and/or AMPAR desensitization. We show that AMPAR lateral diffusion, observed in both intact hippocampi and cultured neurons, allows fast exchange of desensitized receptors with naïve functional ones within or near the postsynaptic density. Recovery from depression in the tens of millisecond time range can be explained in part by this fast receptor exchange. Preventing AMPAR surface movements through cross-linking, endogenous clustering, or calcium rise all slow recovery from depression. Physiological regulation of postsynaptic receptor mobility affects the fidelity of synaptic transmission by shaping the frequency dependence of synaptic responses.

Single-molecule diffusion study of activated EGFR implicates its endocytic pathway.

Z Xiao — 2008-05-12T15:32:56-00:00

Biochemical and biophysical research communications, Vol. 369, No. 2. (2 May 2008), pp. 730-734.

In this work, we have imaged the lateral diffusion of activated epidermal growth factor receptor (EGFR) on cell membrane for studying its internalization pathway. After EGF activation, the mobility of individual EGFR molecules was measured and compared with that in the cells disrupted of clathrin-coated pits and caveolae, the two endocytosis-competent membrane microdomains. The results implicated that activated EGFR molecules associated with clathrin-coated pits but not caveolae at low doses of EGF, whereas they were located in these two domains at high EGF doses. It provided supporting evidence for the occurrence of both clathrin-dependent and caveolae-dependent EGFR endocytosis.

Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism

Tanya Daigle — 2008-04-14T04:41:21-00:00

Journal of Neurochemistry, Vol. 0, No. 0. (0), pp. ???-???.

Abstract Agonists stimulate cannabinoid 1 receptor (CB1R) internalization. Previous work suggests that the extreme carboxy-terminus of the receptor regulates this internalization - likely through the phosphorylation of serines and threonines clustered within this region. While truncation of the carboxy-terminus (V460Z CB1) and consequent removal of these putative phosphorylation sites prevents endocytosis in AtT20 cells, the residues necessary for CB1R internalization remain elusive. To determine the structural requirements for internalization, we evaluated endocytosis of carboxy-terminal mutant CB1Rs stably expressed in HEK293 cells. In contrast to AtT20 cells, V460Z CB1R expressed in HEK293 cells internalized to the same extent and with similar kinetics as the wild-type receptor. However, mutation of serine and/or threonine residues within the extreme carboxy-terminal attenuated internalization when these receptors were expressed in HEK293 cells. These results establish that the extreme carboxy-terminal phosphorylation sites are not required for internalization of truncated receptors, but are required for internalization of full-length receptors in HEK293 cells. Analysis of beta-arrestin-2 recruitment to mutant CB1R suggests that putative carboxy-terminal phosphorylation sites mediate beta-arrestin-2 translocation. This study indicates that the local cellular environment affects the structural determinants of CB1R internalization. Additionally, phosphorylation likely regulates the internalization of (full-length) CB1Rs.

Mechanisms of neurodegenerative diseases: insights from live cell imaging.

C Weissmann — 2008-05-12T15:24:26-00:00

Journal of neuroscience research, Vol. 86, No. 3. (15 February 2008), pp. 504-511.

Pathologic alterations in protein dynamics such as changes in protein degradation, accumulation of misfolded proteins, and deficits in cellular transport mechanisms are a common feature of most if not all neurodegenerative diseases. Live cell imaging studies promise to contribute to a better understanding of the molecular mechanisms underlying these diseases by visualizing the turnover, accumulation, and transport of proteins in a living cellular context in real time. In this review, we discuss recent work in which different live cell imaging approaches are applied in cellular models of amyotrophic lateral sclerosis, polyQ diseases, and tauopathies as paradigmatic examples of diseases with different types of alterations in protein dynamics. It becomes evident that live cell imaging studies provide new insights into different aspects of protein dynamics, such as the understanding that aggregates are not as static as concluded from previous studies but exhibit a remarkable molecular exchange and that the dynamicity state of the neuronal cytoskeleton might have a critical role in neuronal degeneration. It can be anticipated that live cell imaging studies will lead to a more dynamic view of protein turnover and aggregation, which may aid in identifying drugs that specifically interfere with disease-related changes.

Endocytosis as a Mechanism for Tyrosine Kinase-dependent Suppression of a Voltage-gated Potassium Channel

Edmund Nesti — 2008-05-12T15:21:40-00:00

Mol. Biol. Cell, Vol. 15, No. 9. (1 September 2004), pp. 4073-4088.

The voltage-gated potassium channel Kv1.2 undergoes tyrosine phosphorylation-dependent suppression of its ionic current. However, little is known about the physical mechanism behind that process. We have found that the Kv1.2 alpha-subunit protein undergoes endocytosis in response to the same stimuli that evoke suppression of Kv1.2 ionic current. The process is tyrosine phosphorylation-dependent because the same tyrosine to phenylalanine mutation in the N-terminus of Kv1.2 that confers resistance to channel suppression (Y132F) also confers resistance to channel endocytosis. Overexpression of a dominant negative form of dynamin blocked stimulus-induced Kv1.2 endocytosis and also blocked suppression of Kv1.2 ionic current. These data indicate that endocytosis of Kv1.2 from the cell surface is a key mechanism for channel suppression by tyrosine kinases. 10.1091/mbc.E03-11-0788

Contrast, resolution, pixelation, dynamic range and signal-to-noise ratio: fundamental limits to resolution in fluorescence light microscopy

Stelzer — 2008-04-30T10:00:52-00:00

Journal of Microscopy, Vol. 189, No. 1. (1998), pp. 15-24.

In a perfect optical system numerical aperture and wavelength determine resolution. In a real optical system, however, the number of photons collected from a specimen determines the contrast and this limits the resolution. Contrast is affected by the number of picture elements per unit area, the number of photons and the aberrations present in every optical system. The concept of contrast vs. distance functions is used to compare the resolution achievable in confocal and wide-field fluorescence microscopes and the effect of a further reduction of the observable volume. In conclusio: (a) real optical systems will never be able to achieve the theoretical resolution, (b) wide-field fluorescence microscopy will often provide a better resolution than confocal fluorescence microscopy, (c) decreasing the observed volume does not necessarily increase the resolution and (d) using multiple fluorophores can improve the accuracy with which distances are measured. Some numbers for typical situations are provided.

Role of Septin cytoskeleton in spine morphogenesis and dendrite development in neurons.

T Tada — 2008-04-30T09:53:48-00:00

Current biology : CB, Vol. 17, No. 20. (23 October 2007), pp. 1752-1758.

Septins are GTP-binding proteins that polymerize into heteromeric filaments and form microscopic bundles or ring structures in vitro and in vivo. Because of these properties and their ability to associate with membrane, F-actin, and microtubules, septins have been generally regarded as cytoskeletal components [1, 2]. Septins are known to play roles in cytokinesis, in membrane trafficking, and as structural scaffolds; however, their function in neurons is poorly understood. Many members of the septin family, including Septin 7 (Sept7), were found by mass-spectrometry analysis of postsynaptic density (PSD) fractions of the brain [3, 4], suggesting a possible postsynaptic function of septins in neurons. We report that Sept7 is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons--a distribution reminiscent of septin localization in the bud neck of budding yeast. Overexpression of Sept7 increased dendrite branching and the density of dendritic protrusions, whereas RNA interference (RNAi)-mediated knockdown of Sept7 led to reduced dendrite arborization and a greater proportion of immature protrusions. These data suggest that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.

Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells

Jason Mercer — 2008-04-25T16:18:21-00:00

Science, Vol. 320, No. 5875. (25 April 2008), pp. 531-535.

Viruses employ many different strategies to enter host cells. Vaccinia virus, a prototype poxvirus, enters cells in a pH-dependent fashion. Live cell imaging showed that fluorescent virus particles associated with and moved along filopodia to the cell body, where they were internalized after inducing the extrusion of large transient membrane blebs. p21-activated kinase 1 (PAK1) was activated by the virus, and the endocytic process had the general characteristics of macropinocytosis. The induction of blebs, the endocytic event, and infection were all critically dependent on the presence of exposed phosphatidylserine in the viral membrane, which suggests that vaccinia virus uses apoptotic mimicry to enter cells. 10.1126/science.1155164

Drosophila Ankyrin 2 Is Required for Synaptic Stability

Iris Koch — 2008-04-24T14:24:05-00:00

Neuron, Vol. 58, No. 2. (24 April 2008), pp. 210-222.

Summary Synaptic connections are stabilized through transsynaptic adhesion complexes that are anchored in the underlying cytoskeleton. The Drosophila neuromuscular junction (NMJs) serves as a model system to unravel genes required for the structural remodeling of synapses. In a mutagenesis screen for regulators of synaptic stability, we recovered mutations in Drosophila ankyrin 2 (ank2) affecting two giant Ank2 isoforms that are specifically expressed in the nervous system and associate with the presynaptic membrane cytoskeleton. ank2 mutant larvae show severe deficits in the stability of NMJs, resulting in a reduction in overall terminal size, withdrawal of synaptic boutons, and disassembly of presynaptic active zones. In addition, lack of Ank2 leads to disintegration of the synaptic microtubule cytoskeleton. Microtubules and microtubule-associated proteins fail to extend into distant boutons. Interestingly, Ank2 functions downstream of spectrin in the anchorage of synaptic microtubules, providing the cytoskeletal scaffold that is essential for synaptic stability.

A Presynaptic Giant Ankyrin Stabilizes the NMJ through Regulation of Presynaptic Microtubules and Transsynaptic Cell Adhesion

Jan Pielage — 2008-04-24T14:22:25-00:00

Neuron, Vol. 58, No. 2. (24 April 2008), pp. 195-209.

Summary In a forward genetic screen for mutations that destabilize the neuromuscular junction, we identified a novel long isoform of Drosophila ankyrin2 (ank2-L). We demonstrate that loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. We provide genetic evidence that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that we demonstrate can directly bind and organize synaptic microtubules. We propose that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal.

Imaging of Rab5 activity identifies essential regulators for phagosome maturation

Masahiro Kitano — 2008-04-03T16:42:31-00:00

Nature (02 April 2008)

Mechanisms of voltage-gated ion channel regulation: from gene expression to localization.

D Schulz — 2008-04-24T08:09:07-00:00

Cellular and molecular life sciences : CMLS (14 April 2008)

The ion channel milieu present in a neuron in large part determines the inherent excitability of a given cell and is responsible for the translation of sensory transduction and synaptic input to axonal output. Intrinsic excitability is a dynamic process subject to multiple levels of regulation from channel gene expression to post-translational modifications that influence channel activity. The goal of this review is to provide an overview of some of the mechanisms by which channels can be modified in order to influence neuronal output. We focus on four levels of regulation: channel gene transcription, alternative splicing of channel transcripts, post-translational modifications that alter channel kinetics (phosphorylation), and subcellular localization and trafficking of channel proteins.

The diffusion of molecules in axonal plasma membranes: the sites of insertion of new membrane molecules and their distribution along the axon surface.

R Khanin — 2008-04-23T09:01:56-00:00

Journal of theoretical biology, Vol. 193, No. 3. (7 August 1998), pp. 371-382.

The neuronal cell surface consists of two domains, the somatodendritic and axonal plasma membranes. Each domain serves different functions, and has a different complement of membrane molecules. Since membrane molecules are able to diffuse in the plane of the plasma membrane lipid bilayer, with diffusion coefficients ranging from 10-8 cm 2 s-1 for lipids to 10-10 cm 2 s-1 for proteins, mechanisms must exist to prevent as intermixing of membrane molecules from each domain by diffusion. Presented here is a theoretical analysis of the distribution of axonal molecules in both growing and non-growing axons based on two models for the insertion of these molecules into the axonal membrane, namely insertion exclusively at the distal end of the axon, or insertion with equal probability all along the axon. In all cases, assuming that the molecules have a finite half-life in the axonal membrane, compositional differences between the axonal and somatodendritic membranes can be obtained that are similar to those observed in other polarized cells, even in the absence of a physical barrier to prevent the intermixing of membrane molecules. Moreover, our analyses demonstrate that the diffusion of membrane molecules in the plane of the axonal lipid bilayer is a sufficiently slow process to preclude the possibility that membrane molecules are inserted into axonal membranes at a site remote from their final location, and then move to their final locations for diffusion. Thus, in long axons, for membrane molecules that are localized all along the length of the axon, mechanisms must exist for their insertion in the axonal membrane at sites all along the axon, and not just at the distal end.

The Subspine Organization of Actin Fibers Regulates the Structure and Plasticity of Dendritic Spines

Naoki Honkura — 2008-04-14T16:24:42-00:00

Neuron, Vol. 57, No. 5. (13 March 2008), pp. 719-729.

Summary Synapse function and plasticity depend on the physical structure of dendritic spines as determined by the actin cytoskeleton. We have investigated the organization of filamentous (F-) actin within individual spines on CA1 pyramidal neurons in rat hippocampal slices. Using two-photon photoactivation of green fluorescent protein fused to [beta]-actin, we found that a dynamic pool of F-actin at the tip of the spine quickly treadmilled to generate an expansive force. The size of a stable F-actin pool at the base of the spine depended on spine volume. Repeated two-photon uncaging of glutamate formed a third pool of F-actin and enlarged the spine. The spine often released this "enlargement pool" into the dendritic shaft, but the pool had to be physically confined by a spine neck for the enlargement to be long-lasting. Ca2+/calmodulin-dependent protein kinase II regulated this confinement. Thus, spines have an elaborate mechanical nature that is regulated by actin fibers.

Flotillin-dependent clustering of the amyloid precursor protein regulates its endocytosis and amyloidogenic processing in neurons.

A Schneider — 2008-04-23T08:17:58-00:00

The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 28, No. 11. (12 March 2008), pp. 2874-2882.

The flotillins/reggie proteins are associated with noncaveolar membrane microdomains and have been implicated in the regulation of a clathrin- and caveolin-independent endocytosis pathway. Endocytosis is required for the amyloidogenic processing of the amyloid precursor protein (APP) and thus to initiate the release of the neurotoxic beta-amyloid peptide (Abeta), the major component of extracellular plaques found in the brains of Alzheimer's disease patients. Here, we report that small interference RNA-mediated downregulation of flotillin-2 impairs the endocytosis of APP, in both neuroblastoma cells and primary cultures of hippocampal neurons, and reduces the production of Abeta. Similar to tetanus neurotoxin endocytosis, but unlike the internalization of transferrin, clathrin-dependent endocytosis of APP requires cholesterol and adaptor protein-2 but is independent of epsin1 function. Moreover, on a nanoscale resolution using stimulated emission depletion microscopy and by Förster resonance energy transfer with fluorescence lifetime imaging microscopy, we provide evidence that flotillin-2 promotes the clustering of APP at the cell surface. We show that the interaction of flotillin-2 with APP is dependent on cholesterol and that clustering of APP enhances its endocytosis rate. Together, our data suggest that cholesterol/flotillin-dependent clustering of APP may stimulate the internalization into a specialized clathrin-dependent endocytosis pathway to promote amyloidogenic processing.

Culturing hippocampal neurons.

S Kaech — 2008-04-21T14:16:21-00:00

Nature protocols, Vol. 1, No. 5. (2006), pp. 2406-2415.

We provide protocols for preparing low-density dissociated-cell cultures of hippocampal neurons from embryonic rats or mice. The neurons are cultured on polylysine-treated coverslips, which are suspended above an astrocyte feeder layer and maintained in serum-free medium. When cultured according to this protocol, hippocampal neurons become appropriately polarized, develop extensive axonal and dendritic arbors and form numerous, functional synaptic connections with one another. Hippocampal cultures have been used widely for visualizing the subcellular localization of endogenous or expressed proteins, for imaging protein trafficking and for defining the molecular mechanisms underlying the development of neuronal polarity, dendritic growth and synapse formation. Preparation of glial feeder cultures must begin 2 weeks in advance, and it takes 5 d to prepare coverslips as a substrate for neuronal growth. Dissecting the hippocampus and plating hippocampal neurons takes 2-3 h.

Casein kinase 2-dependent serine phosphorylation of MuSK regulates acetylcholine receptor aggregation at the neuromuscular junction

Tatiana Cheusova — 2006-07-07T02:37:46-00:00

Genes Dev., Vol. 20, No. 13. (1 July 2006), pp. 1800-1816.

The release of Agrin by motoneurons activates the muscle-specific receptor tyrosine kinase (MuSK) as the main organizer of subsynaptic specializations at the neuromuscular junction. MuSK downstream signaling is largely undefined. Here we show that protein kinase CK2 interacts and colocalizes with MuSK at post-synaptic specializations. We observed CK2-mediated phosphorylation of serine residues within the kinase insert (KI) of MuSK. Inhibition or knockdown of CK2, or exchange of phosphorylatable serines by alanines within the KI of MuSK, impaired acetylcholine receptor (AChR) clustering, whereas their substitution by residues that imitate constitutive phosphorylation led to aggregation of AChRs even in the presence of CK2 inhibitors. Impairment of AChR cluster formation after replacement of MuSK KI with KIs of other receptor tyrosine kinases correlates with potential CK2-dependent serine phosphorylation within KIs. MuSK activity was unchanged but AChR stability decreased in the presence of CK2 inhibitors. Muscle-specific CK2[beta] knockout mice develop a myasthenic phenotype due to impaired muscle endplate structure and function. This is the first description of a regulatory cross-talk between MuSK and CK2 and of a role for the KI of the receptor tyrosine kinase MuSK for the development of subsynaptic specializations. 10.1101/gad.375206

Polarized distribution of ion channels within microdomains of the axon initial segment.

A Van Wart — 2007-09-25T13:32:43-00:00

J Comp Neurol, Vol. 500, No. 2. (10 January 2007), pp. 339-352.

Voltage-gated sodium (Na(v)) channels accumulate at the axon initial segment (IS), where their high density supports spike initiation. Maintenance of this high density of Na(v) channels involves a macromolecular complex that includes the cytoskeletal linker protein ankyrin-G, the only protein known to bind Na(v) channels and localize them at the IS. We found previously that Na(v)1.6 is the predominant Na(v) channel isoform at IS of adult rodent retinal ganglion cells. However, here we report that Na(v)1.6 immunostaining is consistently reduced or absent in short regions of the IS proximal to the soma, although both ankyrin-G and pan-Na(v) antibodies stain this region. We show that this proximal IS subregion is a unique axonal microdomain, containing an accumulation of Na(v)1.1 channels that are spatially segregated from the Na(v)1.6 channels of the distal IS. Additionally, we find that axonal K(v)1.2 potassium channels are present within the distal IS, but are also excluded from the Na(v)1.1-enriched proximal IS microdomain. Because ankyrin-G was prominent in both proximal and distal subcompartments of the IS, where it colocalized with either Na(v)1.1 or Na(v)1.6, respectively, mechanisms other than association with ankyrin-G must mediate differential targeting of Na(v) channel subtypes to achieve the spatial precision observed within the IS. This precise arrangement of ion channels within the axon initial segment is likely an important determinant of the firing properties of ganglion cells and other mammalian neurons.

Pathway selection to the axon depends on multiple targeting signals in NgCAM.

Chan Choo Yap — 2008-04-21T11:44:23-00:00

Journal of cell science (14 April 2008)

Similar to most differentiated cells, both neurons and epithelial cells elaborate distinct plasma membrane domains that contain different membrane proteins. We have previously shown that the axonal cell-adhesion molecule L1/NgCAM accumulates on the axonal surface by an indirect transcytotic pathway via somatodendritic endosomes. MDCK epithelial cells similarly traffic NgCAM to the apical surface by transcytosis. In this study, we map the signals in NgCAM required for routing via the multi-step transcytotic pathway. We identify both a previously mapped tyrosine-based signal as a sufficient somatodendritic targeting signal, as well as a novel axonal targeting signal in the cytoplasmic tail of NgCAM. The axonal signal is glycine and serine rich, but only the glycine residues are required for activity. The somatodendritic signal is cis-dominant and needs to be inactivated in order for the axonal signal to be executed. Additionally, we show that the axonal cytoplasmic signal promotes apical targeting in MDCK cells. Transcytosis of NgCAM to the axon thus requires the sequential regulated execution of multiple targeting signals.

No barrier to diffusion between cell soma and neurite membranes in sympathetic neurons for a GPI-anchored glycoprotein.

MR Howard — 2008-04-21T11:42:30-00:00

Molecular and cellular neurosciences, Vol. 24, No. 2. (October 2003), pp. 296-306.

As neurons extend their axons, it is thought that newly synthesised membrane components travel in vesicles along the axon, fuse with the growth cone membrane, and diffuse back along the axonal membrane. However, it is difficult to explain how axons continue to be populated with membrane proteins as they extend in length. To investigate this problem, we have used a CEPU-green fluorescent protein (GFP) chimeric protein to study the site of insertion of new glycosyl phosphatidyl inositol (GPI)-anchored glycoproteins and their subsequent behaviour in chick dorsal root ganglia (DRG) neurons. Infection of cultures grown for 24 h revealed rapid expression of CEPU-GFP over the whole surface of the neuron, more rapidly than could be accounted for by diffusion from the growth cone, and fluorescence intensity was uniform along the length of the neurite. Photobleaching experiments of neurite membrane revealed that recovery of fluorescence was due to diffusion from adjacent membranes and there was no evidence for membrane flow in either direction. Photobleaching of membrane adjacent to the cell body also showed rapid recovery, with chimera diffusing both from cell body membrane and the distal neurite membrane into the bleached area. These results suggest there is no barrier to diffusion between the cell body and neurite membrane in DRG and sympathetic neurons cultured for 1 or 2 days in vitro. We propose that the neurite is populated by newly synthesised chimera by diffusion from both regions. This situation may also occur in neurons in the early stages of extending axons in vivo prior to polarisation and the development of the dendritic field.

Heterogeneity of parvalbumin-containing neurons in the mouse main olfactory bulb, with special reference to short-axon cells and betaIV-spectrin positive dendritic segments.

T Kosaka — 2008-04-21T11:38:45-00:00

Neuroscience research, Vol. 60, No. 1. (January 2008), pp. 56-72.

The structural features of parvalbumin-positive neurons were studied in the mouse main olfactory bulb (MOB). Parvalbumin-positive neurons were heterogeneous, including numerous medium-sized interneurons in the external plexiform layer (EPL), some few large short-axon cells and a few periglomerular cells. Their overall distribution pattern and structural features resembled those of the rat MOB. However, large short-axon cells were frequently encountered in the internal plexiform and granule cell layers, which were rare in the rat MOB. In addition a few large short-axon cells were also encountered throughout the EPL. These short-axon cells extended their axons mainly in the EPL, usually making columnar axonal fields. Most parvalbumin-positive cells except periglomerular cells were confirmed to be glutamic acid decarboxylase positive. We examined the immuno-localization of the markers for the axon initial segments (AISs), betaIV-spectrin and sodium channels, to determine whether or not heterogeneous parvalbumin-positive neurons have axons. We confirmed their localization on the AISs of the large short-axon cells and periglomerular cells. However, these markers were encountered on some patch-like segments on the dendritic processes instead of the thin axon-like processes of the medium-sized EPL interneurons. The present study revealed the diversity of parvalbumin-positive neurons in the mouse MOB and their particular structural properties hitherto unknown.

Voltage-gated ion channels in the axon initial segment of human cortical pyramidal cells and their relationship with chandelier cells.

MC Inda — 2008-04-21T08:46:33-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, No. 8. (21 February 2006), pp. 2920-2925.

The axon initial segment (AIS) of pyramidal cells is a critical region for the generation of action potentials and for the control of pyramidal cell activity. Here we show that Na+ and K+ voltage-gated channels, together with other molecules involved in the localization of ion channels, are distributed asymmetrically in the AIS of pyramidal cells situated in the human temporal neocortex. There is a high density of Na+ channels distributed along the length of the AIS together with the associated proteins spectrin betaIV and ankyrin G. In contrast, Kv1.2 channels are associated with the adhesion molecule Caspr2, and they are mostly localized to the distal region of the AIS. In general, the distal region of the AIS is targeted by the GABAergic axon terminals of chandelier cells, whereas the proximal region is innervated, mostly by other types of GABAergic interneurons. We suggest that this molecular segregation and the consequent regional specialization of the GABAergic input to the AIS of pyramidal cells may have important functional implications for the control of pyramidal cell activity.

Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice

Daniel Huber — 2007-12-20T05:53:40-00:00

Nature (19 December 2007)

Endosomal trafficking of AMPA-type glutamate receptors

H Hirling — 2008-04-17T08:12:05-00:00

Neuroscience, Vol. In Press, Corrected Proof

Many different forms of synaptic plasticity have been shown to ultimately modulate the number of AMPA-type glutamate receptors at the synapse. This trafficking involves lateral movements between synaptic and extrasynaptic sites at the neuron surface, as well as vesicular transport between the plasma membrane and intracellular compartments. Several new studies have shed light on the location and regulation of AMPA-type receptor (AMPAR) endocytosis, their intracellular sorting to divergent pathways at the level of endosomes, and the mechanism and sites of receptor recycling. This review summarizes this recent data on the trafficking along the endocytic pathway, and follows the path of internalized AMPAR from endocytosis up to sites of recycling.

Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.

MH Kole — 2008-01-20T23:09:07-00:00

Neuron, Vol. 55, No. 4. (16 August 2007), pp. 633-647.

Action potentials are binary signals that transmit information via their rate and temporal pattern. In this context, the axon is thought of as a transmission line, devoid of a role in neuronal computation. Here, we show a highly localized role of axonal Kv1 potassium channels in shaping the action potential waveform in the axon initial segment (AIS) of layer 5 pyramidal neurons independent of the soma. Cell-attached recordings revealed a 10-fold increase in Kv1 channel density over the first 50 microm of the AIS. Inactivation of AIS and proximal axonal Kv1 channels, as occurs during slow subthreshold somatodendritic depolarizations, led to a distance-dependent broadening of axonal action potentials, as well as an increase in synaptic strength at proximal axonal terminals. Thus, Kv1 channels are strategically positioned to integrate slow subthreshold signals, providing control of the presynaptic action potential waveform and synaptic coupling in local cortical circuits.

Action potential generation requires a high sodium channel density in the axon initial segment

Maarten Kole — 2008-01-31T11:58:16-00:00

Nature Neuroscience, Vol. 11, No. 2. (20 January 2008), pp. 178-186.

Tracking the ends: a dynamic protein network controls the fate of microtubule tips

Anna Akhmanova — 2008-03-21T04:35:37-00:00

Nature Reviews Molecular Cell Biology, Vol. 9, No. 4. (05 March 2008), pp. 309-322.

Genetic dissection of neural circuits.

L Luo — 2008-03-20T12:34:55-00:00

Neuron, Vol. 57, No. 5. (13 March 2008), pp. 634-660.

Understanding the principles of information processing in neural circuits requires systematic characterization of the participating cell types and their connections, and the ability to measure and perturb their activity. Genetic approaches promise to bring experimental access to complex neural systems, including genetic stalwarts such as the fly and mouse, but also to nongenetic systems such as primates. Together with anatomical and physiological methods, cell-type-specific expression of protein markers and sensors and transducers will be critical to construct circuit diagrams and to measure the activity of genetically defined neurons. Inactivation and activation of genetically defined cell types will establish causal relationships between activity in specific groups of neurons, circuit function, and animal behavior. Genetic analysis thus promises to reveal the logic of the neural circuits in complex brains that guide behaviors. Here we review progress in the genetic analysis of neural circuits and discuss directions for future research and development.

Type-1 cannabinoid receptors colocalize with caveolin-1 in neuronal cells.

M Bari — 2008-04-01T10:20:38-00:00

Neuropharmacology, Vol. 54, No. 1. (January 2008), pp. 45-50.

Type-1 (CB1) and type-2 (CB2) cannabinoid receptors belong to the rhodopsin family of G protein-coupled receptors, and are activated by endogenous lipids termed "endocannabinoids". Recent reports have demonstrated that CB1R, unlike CB2R and other receptors and metabolic enzymes of endocannabinoids, functions in the context of lipid rafts, i.e. plasma membrane microdomains which may be important in modulating signal transduction. Here, we present novel data based on cell subfractionation, immunoprecipitation and confocal microscopy studies, that show that in C6 cells CB1R co-localizes almost entirely with caveolin-1. We also show that trafficking of CB1R in response to the raft disruptor methyl-beta-cyclodextrin (MCD) is superimposable on that of caveolin-1, and that MCD treatment increases the accessibility of CB1R to its specific antibodies. These findings may be relevant for the manifold CB1R-dependent activities of endocannabinoids, like the regulation of apoptosis and of neurodegenerative diseases.

Lateral diffusion of the GABAB receptor is regulated by the GABAB2 C terminus.

AM Pooler — 2008-04-01T10:17:47-00:00

J Biol Chem, Vol. 282, No. 35. (31 August 2007), pp. 25349-25356.

GABAB (gamma-aminobutyric acid, type B) is a heterodimeric G-protein-coupled receptor. The GABAB1 subunit, which contains an endoplasmic reticulum retention sequence, is only transported to the cell surface when it is associated with the GABAB2 subunit. Fluorescence recovery after photobleaching studies in transfected COS-7 cells and hippocampal neurons revealed that GABAB2 diffuses slowly within the plasma membrane whether expressed alone or with the GABAB1 subunit. Treatment of cells with brefeldin A revealed that GABAB2 moves freely within the endoplasmic reticulum, suggesting that slow movement of GABAB2 is a result of its plasma membrane insertion. Disruption of the cytoskeleton did not affect the mobility of GABAB2, indicating that its restricted diffusion is not due to direct interactions with actin or tubulin. To determine whether the C terminus of GABAB2 regulates its diffusion, this region of the subunit was attached to the lymphocyte membrane protein, CD2, which then exhibited a slower rate of lateral diffusion. Furthermore, co-expression of a cytoplasmically expressed soluble form of the GABAB2 C terminus increased movement of the GABAB2 subunit. We constructed forms of GABAB2 with various C-terminal truncations. Truncation of GABAB2 after residue 862, but not residue 886, caused a dramatic increase in its mobility, suggesting that the region between these two residues is critical for restricting GABAB2 diffusion. Finally, we investigated whether activation of GABAB might modulate its movement. Treatment of COS-7 cells with the GABAB receptor agonist baclofen significantly increased its mobile fraction. These data show that the restricted movement of GABAB at the cell surface is regulated by a region within its C terminus.

Local presentation of L1 and N-cadherin in multicomponent, microscale patterns differentially direct neuron function in vitro.

P Shi — 2008-04-01T10:12:21-00:00

Dev Neurobiol, Vol. 67, No. 13. (November 2007), pp. 1765-1776.

The ability to pattern multiple bioactive cues on a surface is valuable for understanding how neurons interact with their complex extracellular environment. In this report, we introduce a set of methods for creating such surfaces, with the goals of understanding how developing neurons integrate multiple biologically relevant signals and as a tool for studying interactions between multiple neurons. Multiple microcontact printing steps are combined on a single surface to produce an array of polylysine nodes, interconnected by lines of proteins based on the extracellular domains of L1 or N-cadherin. Surprisingly, the N-cadherin protein could also be directly printed onto surfaces while retaining its biological activity. Rat hippocampal neurons selectively attached to the polylysine nodes, differentially extending axonal and dendritic processes along the patterns of L1 and N-cadherin, thus demonstrating control over neuron attachment and outgrowth. Combining these three biomolecules on a single surface revealed a highly complex pattern of protein recognition. Dendrites extended exclusively on N-cadherin patterns, while axons exhibited a very high degree of selectivity on L1 patterns, preferentially at distances greater than 55 mum from the cell body. At shorter distances, axonal processes recognized both L1 and N-cadherin, revealing a new aspect of neuron polarity and axon specification. This onset of L1 selectivity correlated with the establishment of intracellular L1 polarity, suggesting a functional outcome of the process of neuron polarization that has implications in development of neural tissues and creation of in vitro neuron networks.

Actin filament disruption blocks cerebellar granule neurons at the unipolar stage of differentiation in vitro.

JF Zmuda — 2008-04-01T10:10:46-00:00

J Neurobiol, Vol. 43, No. 4. (15 June 2000), pp. 313-328.

Cerebellar granule neurons developing in vitro initially extend a single axon, with the Golgi apparatus and centrosome positioned at the base of this axon and then begin the transition to a bipolar morphology by rotating the Golgi-centrosome to the opposite pole of the cell and extending a secondary axon; granule cells reach a mature, complex morphology by extending multiple, short dendrites by 5-6 days in vitro. (Zmuda and Rivas, 1998. Cell Motil Cytoskel 41:18-38). To test the effects of actin depolymerization on this characteristic pattern of granule cell axonogenesis, cultured granule cells were treated with either cytochalasin D (CD) or latrunculin A (Lat A) to depolymerize filamentous actin. Although actin depolymerization did not inhibit initial axon extension, it prevented the cells from proceeding on to the transitional, bipolar, or complex stages of differentiation, effectively blocking the cells at the unipolar stage of differentiation. Although the Golgi apparatus resided at the base of the axon in nontreated unipolar cells, or at the opposite pole of the cell body in nontreated transitional cells, the Golgi was randomly localized within the cytoplasm of cells that had been treated with either CD or Lat A. These results show that the transition from the unipolar to the bipolar stage and on to more mature stages of granule cell differentiation is dependent on an intact actin cytoskeleton and suggest that the characteristic pattern of granule cell differentiation may be dependent on the repositioning of the Golgi-centrosome during morphological development.

Integrating molecular and network biology to decode endocytosis

Eva Schmid — 2007-08-23T14:27:13-00:00

Nature, Vol. 448, No. 7156. (2007), pp. 883-888.

Kinesin-5 regulates the growth of the axon by acting as a brake on its microtubule array.

KA Myers — 2007-11-09T17:57:14-00:00

J Cell Biol, Vol. 178, No. 6. (10 September 2007), pp. 1081-1091.

Kinesin-5 is a homotetrameric motor protein that interacts with adjacent microtubules in the mitotic spindle. Kinesin-5 is also highly expressed in developing postmitotic neurons. Axons of cultured neurons experimentally depleted of kinesin-5 grow up to five times longer than controls and display more branches. The faster growth rates are accompanied by a doubling of the frequency of transport of short microtubules, suggesting a major role for kinesin-5 in the balance of motor-driven forces on the axonal microtubule array. Live-cell imaging reveals that the effects on axonal length of kinesin-5 depletion are caused partly by a lower propensity of the axon and newly forming branches to undergo bouts of retraction. Overexpression of wild-type kinesin-5, but not a rigor mutant of kinesin-5, has the inverse effect on axonal length. These results indicate that kinesin-5 imposes restrictions on the growth of the axon and does so at least in part by generating forces on the axonal microtubule array.

Highlighting protein kinase CK2 movement in living cells.

N Theis-Febvre — 2008-04-01T09:58:13-00:00

Mol Cell Biochem, Vol. 274, No. 1-2. (June 2005), pp. 15-22.

Protein kinase CK2 has traditionally been described as a stable heterotetrameric complex (alpha2beta2) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol 23: 975-987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio-temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell.

Visualization of AP-1 NF-kappaB ternary complexes in living cells by using a BiFC-based FRET

John Shyu — 2008-01-03T08:10:19-00:00

Proceedings of the National Academy of Sciences (2 January 2008), 0705181105.

Proteinprotein interactions are essential for maintaining cell structure and for executing almost all cellular processes. Determination of where and how each protein interacts with its partners provides significant insight into proteins' cellular roles. Although several assays, such as FRET and bimolecular fluorescence complementation (BiFC), have been developed and widely used for visualization and identification of protein interactions in living cells, there is no simple and convenient assay to visualize and identify multiple protein complexes in living cells. Because many signaling molecules often function as ternary complexes, availability of an assay for visualization and identification of ternary complexes will significantly expand the repertoire of protein interaction studies in living cells. By using the FosJunnuclear factor of activated T cells (NFAT) ternary complex as a model and the fluorescent proteins Cerulean and Venus, two mutant proteins of CFP and YFP with better folding and less environment sensitivity, as a donor and acceptor, respectively, we have combined a Venus-based BiFC system with Cerulean to develop a BiFC-based FRET (BiFC-FRET) assay for visualization of ternary complexes in living cells with a conventional three-filter FRET setup. We also have applied the BiFC-FRET to identify a ternary complex formed between FosJun heterodimers and the NF-kappaB subunit, p65. This finding reveals a cross-talk between AP-1 and NF-kappaB. Thus, the BiFC-FRET represents a convenient assay for identification and visualization of ternary complexes in living cells. 10.1073/pnas.0705181105

Real-time imaging of myosin II regulatory light-chain phosphorylation using a new protein biosensor.

A Yamada — 2008-03-21T14:44:01-00:00

Biochem J, Vol. 385, No. Pt 2. (15 January 2005), pp. 589-594.

Phosphorylation of the RMLC (regulatory myosin light chain) regulates the activity of myosin II, which is critically involved in the motility of both muscle and non-muscle cells. There are both Ca2+-dependent and -independent pathways for RMLC phosphorylation in smooth-muscle cells, and the latter pathway is often involved in an abnormal contractility in pathological states such as asthma and hypertension. Therefore pharmacological interventions of RMLC phosphorylation may have a therapeutic value. In the present study, we developed a new genetically encoded biosensor, termed CRCit (ECFP-RMLC-Citrine, where ECFP is enhanced cyan fluorescent protein), that detects RMLC phosphorylation using fluorescence resonance energy transfer between two variants of the green fluorescent protein fused to both the N- and C-termini of RMLC. When expressed in primary cultured vascular smooth-muscle cells, CRCit detected the Ca2+-dependent RMLC phosphorylation with a high spatiotemporal resolution. Furthermore, we could specifically assay the agonist-induced Ca2+-independent phosphorylation of RMLC when Ca2+ signalling in cells expressing CRCit was suppressed. Thus CRCit may also be used for the high throughput screening of compounds that inhibit abnormal smooth-muscle contraction.

Fast 4D Microscopy.

JR De Mey — 2008-03-21T14:39:55-00:00

Methods Cell Biol, Vol. 85 (2008), pp. 83-112.

Many cellular processes involve fast movements of weakly labeled cellular structures in all directions, which should be recorded in 3D time-lapse microscopy (4D microscopy). This chapter introduces fast 4D imaging, which is used for sampling the cell's volume by collecting focal planes in time-lapse mode as rapidly as possible, without perturbing the sample by strong illumination. The final images should contain sufficient contrast allowing for the isolation of structures of interest by segmentation and the analysis of their intracellular movements by tracking. Because they are the most sensitive, systems using wide-field microscopy and deconvolution techniques are discussed in greater depth. We discuss important points to consider, including system components and multifunctionality, spatial resolution and sampling conditions, and mechanical and optical stability and how to test for it. We consider image formation using high numerical aperture optics and discuss the influence of optical blur and noise on image formation of living cells. Spherical aberrations, their consequences for axial image quality, and their impact on the success of deconvolution of low intensity image stacks are explained in detail. Simple protocols for acquiring and treating point spread functions (PSFs) and live cells are provided. A compromise for counteracting spherical aberration involving the use of a kit of immersion oils for PSF and cell acquisition is illustrated. Recommendations for evaluating acquisition conditions and deconvolution parameters are given. Finally, we discuss future developments based on the use of adaptive optics which will push back many of today's limits.

Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells

Jin-Yu Fan — 2008-01-15T09:48:13-00:00

Biochemical and Biophysical Research Communications, Vol. 367, No. 1. (29 February 2008), pp. 47-53.

Bimolecular fluorescence complementation (BiFC) is a recently developed technique for detection of protein-protein interactions in living cells. In this study, a new red BiFC system was developed by splitting mCherry, a mutant monomeric red fluorescent protein, into two fragments between amino acids 159-160 and was verified using a pair of interacting proteins, SV40 large T antigen (LTag), and human p53 protein. By combined use of the mCherry-based red BiFC system with a Venus-based yellow BiFC system, the interaction between LTag and p53 as well as the interaction between sp100 and promyelocytic leukemia protein (PML), were detected simultaneously in Vero cells. The brilliant redness, short maturation time, and the long excitation and emission wavelengths (587/610 nm) of mCherry make the new BiFC system an excellent candidate for analyzing protein-protein interactions in living cells and for studying multiple protein-protein interactions when coupled with other BiFC systems.

Bimolecular fluorescence complementation: visualization of molecular interactions in living cells.

TK Kerppola — 2008-03-21T14:33:58-00:00

Methods Cell Biol, Vol. 85 (2008), pp. 431-470.

A variety of experimental methods have been developed for the analysis of protein interactions. The majority of these methods either require disruption of the cells to detect molecular interactions or rely on indirect detection of the protein interaction. The bimolecular fluorescence complementation (BiFC) assay provides a direct approach for the visualization of molecular interactions in living cells and organisms. The BiFC approach is based on the facilitated association between two fragments of a fluorescent protein when the fragments are brought together by an interaction between proteins fused to the fragments. The BiFC approach has been used for visualization of interactions among a variety of structurally diverse interaction partners in many different cell types. It enables detection of transient complexes as well as complexes formed by a subpopulation of the interaction partners. It is essential to include negative controls in each experiment in which the interface between the interaction partners has been mutated or deleted. The BiFC assay has been adapted for simultaneous visualization of multiple protein complexes in the same cell and the competition for shared interaction partners. A ubiquitin-mediated fluorescence complementation assay has also been developed for visualization of the covalent modification of proteins by ubiquitin family peptides. These fluorescence complementation assays have a great potential to illuminate a variety of biological interactions in the future.

Recent advances using green and red fluorescent protein variants.

A Müller-Taubenberger — 2008-03-21T14:32:16-00:00

Appl Microbiol Biotechnol, Vol. 77, No. 1. (November 2007), pp. 1-12.

Fluorescent proteins have proven to be excellent tools for live-cell imaging. In addition to green fluorescent protein (GFP) and its variants, recent progress has led to the development of monomeric red fluorescent proteins (mRFPs) that show improved properties with respect to maturation, brightness, and the monomeric state. This review considers green and red spectral variants, their paired use for live-cell imaging in vivo, in vitro, and in fluorescence resonance energy transfer (FRET) studies, in addition to other recent "two-color" advances including photoswitching and bimolecular fluorescence complementation (BiFC). It will be seen that green and red fluorescent proteins now exist with nearly ideal properties for dual-color microscopy and FRET.

Visualization of molecular interactions by fluorescence complementation

Tom Kerppola — 2006-04-22T08:01:49-00:00

Nature Reviews Molecular Cell Biology, Vol. 7, No. 6. (19 April 2006), pp. 449-456.

The visualization of protein complexes in living cells enables the examination of protein interactions in their normal environment and the determination of their subcellular localization. The bimolecular fluorescence complementation assay has been used to visualize interactions among multiple proteins in many cell types and organisms. Modified forms of this assay have been used to visualize the competition between alternative interaction partners and the covalent modification of proteins by ubiquitin-family peptides.

Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.

TK Kerppola — 2008-03-21T14:28:33-00:00

Nat Protoc, Vol. 1, No. 3. (2006), pp. 1278-1286.

Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the discoveries that two non-fluorescent fragments of a fluorescent protein can form a fluorescent complex and that the association of the fragments can be facilitated when they are fused to two proteins that interact with each other. BiFC must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. It is not necessary for the interaction partners to juxtapose the fragments within a specific distance of each other because they can associate when they are tethered to a complex with flexible linkers. It is also not necessary for the interaction partners to form a complex with a long half-life or a high occupancy since the fragments can associate in a transient complex and un-associated fusion proteins do not interfere with detection of the complex. Many interactions can be visualized when the fusion proteins are expressed at levels comparable to their endogenous counterparts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different subcellular locations and in different cell types and organisms. It is technically straightforward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents.

Fluorescent protein FRET: the good, the bad and the ugly

David Piston — 2007-09-17T10:26:58-00:00

Trends in Biochemical Sciences, Vol. 32, No. 9. (September 2007), pp. 407-414.

Dynamic protein interactions play a significant part in many cellular processes. A technique that shows considerable promise in elucidating such interactions is Forster resonance energy transfer (FRET). When combined with multiple, colored fluorescent proteins, FRET permits high spatial resolution assays of protein-protein interactions in living cells. Because FRET signals are usually small, however, their measurement requires careful interpretation and several control experiments. Nevertheless, the use of FRET in cell biological experiments has exploded over the past few years. Here we describe the physical basis of FRET and the fluorescent proteins appropriate for these experiments. We also review the approaches that can be used to measure FRET, with particular emphasis on the potential artifacts associated with each approach.

Organization of ion channels in the myelinated nerve fiber.

SG Waxman — 2008-03-21T14:21:01-00:00

Science, Vol. 228, No. 4707. (28 June 1985), pp. 1502-1507.

The functional organization of the mammalian myelinated nerve fiber is complex and elegant. In contrast to nonmyelinated axons, whose membranes have a relatively uniform structure, the mammalian myelinated axon exhibits a high degree of regional specialization that extends to the location of voltage-dependent ion channels within the axon membrane. Sodium and potassium channels are segregated into complementary membrane domains, with a distribution reflecting that of the overlying Schwann or glial cells. This complexity of organization has important implications for physiology and pathophysiology, particularly with respect to the development of myelinated fibers.

Whatever happened to the 'microtrabecular concept'?

J Heuser — 2008-03-21T14:15:35-00:00

Biol Cell, Vol. 94, No. 9. (December 2002), pp. 561-596.

Keith Porter culminated his stellar career as the founding father of biological electron microscopy by acquiring, in the late 1970s, a high-voltage electron microscope (HVEM). With this magnificent instrument he examined whole-mounts of cultured cells, and perceived within them a structured cytoplasmic matrix he named the "microtrabecular lattice". Over the next decade Porter published a series of studies, together with a team of outstanding young colleagues, which elaborated his broader "microtrabecular concept." This concept posited that microtrabeculae were real physical entities that represented the fundamental organization the cytoplasm, and that they were the physical basis of cytoplasmic motility and of cell-shape determination. The present review presents Porter's original images of microtrabeculae, after conversion to a more interpretable "digital-anaglyph" form, and discusses the rise and fall of the microtrabecular concept. Further, it explains how the HVEM images of microtrabeculae finally came to be considered as an artifact of the preparative methods Porter used to prepare whole cells for HVEM. Still, Keith's "microtrabecular concept" foretold of our current appreciation of the complexity and pervasiveness of the cytoskeleton, which has now been found by more modern methods of EM to actually be the fundamental organizing principle of the cytoplasmic matrix. During the impending eclipse of Porter's microtrabecular concept in the late 1980s, many of Keith's colleagues fondly described the cell as being filled, not with protoplasm, but with "Porterplasm." Despite the fact that Keith's view was clouded by the methods of his time, it would be fitting and apt to retain this name, still today, for the ordered matrix of cytoskeletal macromolecules that exists in the living cell. In the end, the story of what happened to Porter's microtrabecular concept should be an object lesson in scientific hubris that should humble and inform all of us in cell biology, even today--particularly when we begin to think that our most recent methods and observations are achieving "the last word".