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Citizens form political action committee to fight proposed property tax rate increase in St. Johns
The new citizens political action committee is against any hike in the county.
Ponte Vedra Beach resident Adam Guillette is angry about the thought of paying more taxes to St. Johns County.
When he attended a town hall meeting June 22 at the Ponte Vedra Beach branch library that outlined a proposed property tax rate increase, and the reasons for it, he met others who shared his feelings.
At a County Commission meeting July 12, commissioners proposed raising the property tax rate about 0.75 percent.
This week, Guillette and other county citizens created a political action committee to defeat it.
Commissioner Jay Morris, who represents Ponte Vedra Beach, said he didn't have any idea how effective the grassroots group would be.
But Morris said he is going over the proposed budget line by line, and has some ideas about how the proposed tax increase could be reduced.
Guillette, chairman of the St. Johns County PAC, said the group was created "to give a voice to the overtaxed families of St. Johns County." On Tuesday he said there were about a dozen members, who are opposed to "reckless" spending by the county that "is twice as much for every man, woman, and child in the county as compared to 2000."
The county is citing declining property values amid the continuing economic downturn as the reason an increase is needed. Commissioners proposed an approximate 0.25 percent increase to pay for a federally mandated emergency radio system, an approximate 0.25 increase for county fire services and an approximate 0.25 percent increase for the general fund. Combined that would amount to about $12 million.
Combined, the increases would raise the property tax rate from the current 6.67 percent to 7.40 for the fiscal year 2011-12. The current rate is about $6.67 for every $1,000 of assessed property value, which means a homeowner with a house valued at $200,000 with a $50,000 homestead exemption pays about $1,000 in annual property taxes. At the new proposed rate, that homeowner would pay $1,100 in property taxes.
It's possible some home-owners would pay less in taxes if their property values have considerably declined, as might be the case with people who bought homes at the peak of the housing boom.
Since property values have been declining since 2008, the county budget decreased from $733 million in 2007 to $582 million in 2011, and is projected to drop to $545 million for 2012. Since property values continue to drop, the county would need to cut services if there is not a tax rate increase.
County Administrator Michael Wanchick held five town hall meetings around the county in June to discuss the budget, and Guillette said when he met other residents there who were upset by a possible tax increase, they decided to band together to oppose it.
Guillette said from 2000 to 2010 the county doubled its spending. That span included boom times and the economic bust.
"Their spending went down as property values went down," beginning in 2008, he said. But he said they should not have spent $16.5 million for the new county building on Lewis Speedway in St. Augustine.
"Instead of setting money aside for a rainy day, they wanted to spend it on their own comfort," he said.
Guillette also said the number of county employees increased "unnecessarily" during those years, with employees getting "lavish contracts and pension plans that could never be earned in the private sector."
There may be fewer employees now "through attrition, but no massive layoffs," he said. "During these troubled times, the County Commission should be focused on ways to cut spending and cut taxes, not add additional burdens to families."
He said he voted for Morris because he ran as a fiscal conservative, but that he feels betrayed because Morris and the other commissioners are acting like "liberals."
Morris said he has been a fiscal conservative all of his life, but that Guillette and the others need to look at the history of the county. Between 2000 and 2007 the population increased about 38 percent and spending did go up about 262 percent, he said. The 2007 budget reached an all-time high of $733 million, he said.
But since then, the county has lost $188 million in revenue because of property value decreases.
Morris said he wasn't a commissioner during the real estate boom years, because he was elected in November 2010. He said since the real estate bust, the county has done "an excellent job in cutting expenses." About 140 employees have been cut since 2007.
He said he would not have voted for the new county building, and "not one county commissioner would vote for that building today."
But he said the emergency radio system for police and firefighters is federally mandated, so the 0.25 percent increase is necessary for that. The fire department emergency medical system has been running on reserve, and will run out in 2013, so the 0.25 percent projected increase is also necessary for that. Closing down fire stations would increase people's insurance rates far more than what they'd pay in property taxes, he said.
For the general fund, the 0.25 percent is needed to keep libraries and ballparks open and keep the county's quality of life, Morris said.
But after going over that budget, "I believe we can cut some of the 0.25 from the general fund, reduce the millage rate somewhat down."
The next commission meeting is Tuesday, Aug. 2, and the new rate must be set by September.
Guillette said the PAC plans to meet with citizen groups around the county to get their support, including homeowners associations, Republican party members and tea party groups.
PAC member John Clegg of Ponte Vedra Beach said Wednesday that people are "calling us already. Which is quite surprising since we've only been going officially 24 hours."
He said there's a "groundswell."
"I personally know lots and lots of families who are really, really hurting in the recession," he said. Some have lost jobs, others fear they might lose theirs.
"When their revenues drop, they have to make cuts" to their household budget, he said, adding that the government should do that as well.
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Suburban train services in Mumbai are likely to be opened to all commuters from early January, Maharashtra Relief and Rehabilitation Minister Vijay Wadettiwar said on Tuesday.
Talking to reporters, Mr Wadettiwar said the state government’s correspondence with the Railway ministry in this regard was in final stages.
“We have been writing to the Railway ministry to open the suburban network to all commuters and it may be done from early January next year,” he said.
At present, only select categories of people, including staff of essential services and women, are allowed to board suburban trains.
The suburban network, called Mumbai’s lifeline, used to ferry over 70 lakh commuters daily before the coronavirus outbreak.
The vast rail network was closed in March-end after the coronavirus-induced lockdown was announced and was gradually opened for railway and essential services staffers.

domain: news


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Born in Reading and a graduate of the Newcastle College of Performing Arts the first instrument that Pete learned to play was the guitar. However in his early teens he discovered the bass guitar and subsequently the Double Bass and these have become his favoured instruments for live performance and recording.
At the Derby Folk Festival - image by permission © Mark Steele
His professional career started when he was still at school: he was spotted playing at the Henley Youth Festival and was asked to join the newly formed function band Flipside Jack as their bassist when still only 15. He enjoyed playing with the much acclaimed Flipside Jack for many years but his musical tastes have broadened considerably and he now moves effortlessly between Gypsy Jazz, Folk, Bluegrass, Blues and Rock.
Although he’s played several styles of Jazz his favourite by a considerable measure is Gypsy Jazz, always associated with the great guitarist Django Reinhardt. He's the bassist for several bands including The Nocturne Trio (recently rebranded as Lunascura), The Diminished Quartet, the Kourosh Kanani Trio, The London Django Collective, The Simon Moore Trio, the Johnny Kerry Quartet, Natalia Amy Douglas and Gonzalo Bergara (the Argentinian virtuoso guitarist, when Gonzalo tours in Europe) and many more. Throughout 2019 he’s also performed with Latchpen, The Belleville Hot Club and Café Manouche. He’s played venues in Scotland, Ireland, F rance, Italy, throughout England and his current home, London taking in The Edinburgh Fringe, The Cork Jazz Festival and Samois (the famous Gypsy Jazz Festival at Fontainbleau) on the way.
Folk music also features very highly in his musical interest and he has regularly played at venues from art centres to large festivals all over the country and abroad. He’s performed with many well known artists such as Jackie Oates, Megan Henwood, Martin and Eliza Carthy and bands such as Mawkin and Tyde, recording with some of them, but he is now the bassist for The Excess, Benji Kirkpatrick’s trio which first performed in 2018. Benji, well known for his performances with Bellowhead, Faustus and Steeleye Span, has formed his own band The Excess, a trio with Pete as the bassist. With two successful tours already behind them and their first album Gold Has Worn Away receiving much acclaim they are now an established band in the Folk World. A short UK tour was due to commence at the end of March but was cancelled owing to the current pandemic crisis. Later on in the year (August 31st) the band will be giving a virtual performance for the Shrewsbury Folk Festival which has been cancelled as an attended festival.
His interest in folk music is not limited to English Folk. He has performed and toured with the bluegrass band The Vanguards and the country style duet the Worry Dolls when they perform with their full band. He has recorded with both bands.
A diversion from his “normal” bass performances was his recent accompaniment to Ayenai – two dancers who perform modern dance - with the guitarist Andrea Todesco. Part of the Vault Festival the music was composed by Andrea with much input into the arrangement of the composition by Pete.
Pete was one of the support musicians at the Americana 2020 awards ceremony at the Troxy, east London on January 30th, a great occasion showcasing many new talents but also featuring stars such as Billy Bragg and Peter Donegan. (See the Blues page for a clip)
Although he started his career playing Rock and Roll and blues this takes less of his time now. However, he occasionally performs with Silvervoid who have released two EP’s the eponymous Silvervoid and Revive It. (Both are available to stream from Bandcamp.) The title track on the Revive It EP was selected by Classic Rock magazine in August 2019 as one of the best twelve tracks released by new bands.
For three days in March 2020 Pete should have been playing with the Album Project, a group of musicians that bring to life classic rock of times past, but this has been postponed because of the current crisis. It might still be performed in October but nothing is for sure in these uncertain times. This year the focus is 1973 when many memorable hits of that year will be performed; they can be seen at The Playhouse, Cheltenham; tickets will be available from their website if the performance goes ahead.
See the Discography page on this website for details and links for all the music mentioned above.

domain: artistic


To Conquer the Air: The Wright Brothers and the Great Race for Flight
To Conquer the Air: The Wright Brothers and the Great Race for Flight0 0 5 Forfatter: James Tobin
For years, Wilbur Wright and his younger brother, Orville, experimented in obscurity, supported only by their exceptional family. Meanwhile, the world watched as Samuel Langley, armed with a contract from the US War Department and all the resources of the Smithsonian Institution, sought to create the first manned flying machine. But while Langley saw flight as a problem of power, the Wrights saw a problem of balance. Thus their machines took two very different paths—Langley’s toward oblivion, the Wrights’ toward the heavens—though not before facing countless other obstacles. With a historian’s accuracy and a novelist’s eye, Tobin has captured an extraordinary moment in history. To Conquer the Air is itself a heroic achievement.
Mere info om e-bogen:Forlag: Free Press


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For common computers, benchmarks can signify a ceremony of passage of kinds into a new period of computing. As artificial intelligence and device mastering turn out to be more and a lot more ubiquitous, for instance, AI and ML benchmarks assistance every person realize and evaluate exactly how properly one particular neural internet performs in comparison to other units as perfectly as to reference architectures. Not shocking, then, that the rising discipline of quantum laptop or computer benchmarking will be supporting examination and improve upcoming-generation quantum processors, researchers say.
A quantum computer system with excellent more than enough complexity—for occasion, sufficient factors recognised as quantum bits or “qubits”—could theoretically reach a quantum gain wherever it can obtain the answers to problems no classical laptop could ever solve. In principle, a quantum laptop or computer with 300 qubits totally devoted to computing (not error correction) could conduct extra calculations in an quick than there are atoms in the noticeable universe.
Having said that, scientists at Sandia Countrywide Laboratories observe that it is currently complicated to properly predict a quantum processor’s capability—that is, the set of quantum plans it can run correctly. This is simply because the recent benchmarking programs made use of to analyze these equipment scale badly to quantum pcs with many qubits. Current quantum benchmarks are also not adaptable adequate to supply comprehensive seems on processor capabilities on quite a few various prospective purposes, they say.
“It truly is astonishingly tough to benchmark point out-of-the-art quantum pcs, simply because most benchmarks count on evaluating the success of a quantum computation with the suitable output, computed on a common laptop or computer,” states review lead writer Tim Proctor, a physicist at Sandia National Laboratories’ Quantum Efficiency Laboratory in Albuquerque, New Mexico. “That standard computation gets completely infeasible as the selection of qubits will increase, which was central to Google’s renowned ‘quantum supremacy’ demonstration.”
The new benchmarks depth a quantum computer’s efficiency on distinct tasks—compared to present benchmarks that generate just 1 variety.
In a new research, Proctor and his colleagues developed a novel approach for creating benchmarks for quantum personal computers that they phone circuit mirroring. This approach transforms any quantum software into an ensemble of intently linked benchmark applications dubbed “mirror circuits,” which every accomplish a established of calculations and then reverses it.
The mirror circuits are each at least as tricky to execute as the quantum application they are based mostly on. Having said that, not like several quantum systems, mirror circuits have simple, easy-to-predict results. As these types of, mirror circuits supply a way to confirm a quantum processor’s capabilities utilizing benchmarks identical to quantum packages the quantum desktops may truly operate, the scientists say.
“Quantum desktops are intricate, and quantifying their effectiveness is intricate too. Our techniques can be utilized to style and design benchmarks for finding out all kinds of facets of a quantum computer’s general performance,” Proctor claims. “In the long run, we feel that benchmarks created using our technique could be utilized to style and design a established of extensive tests for quantum computers. This would make it attainable to correctly compare quantum personal computers, and to uncover out which components is greatest for which endeavor.”
Applying circuit mirroring, the experts first made two people of benchmark programs—one that ran quantum processors by means of randomized sequences of functions, the other with very structured treatments. They subsequent executed these benchmarks on 12 publicly accessible quantum personal computers from IBM and Rigetti Computing to map out their capabilities.
Quantum benchmarking currently relies mostly on randomized courses. Nevertheless, Proctor notes these randomized benchmarks might not provide as perfectly as tests of the much more structured plans that quantum personal computers hire to implement quantum algorithms.
A competing typical, cycle benchmarking, is customized to each and every algorithm—and actions error fees for sets of quantum gates.
In truth, Proctor and his colleagues identified that how nicely quantum computer systems executed randomized mirror circuits did not forecast how very well they did with additional orderly mirror circuits. The effectiveness of some quantum processors depended greatly on the level of mirror circuit structure, though other individuals showed pretty much no indication of these kinds of a backlink.
“A specially nice aspect of our benchmarks is they’re intended to give a whole lot of detail—they explain to us about a quantum computer’s functionality on distinctive duties,” Proctor claims. “This contrasts to most current methods that, by style, explain a quantum computer’s general performance with 1 selection.”
A single potential flaw of benchmarks that utilize the reversal system used in mirror circuits is how they might fail to detect many essential glitches. “We make certain that our benchmarks are sensitive to all mistakes by inserting a random component in amongst the forward and reverse circuits, the two elements that comprise the bulk of a mirror circuit,” Proctor claims. “In the paper, we verify that our benchmarks are delicate to all mistakes.” The scientists comprehensive their findings Dec. 20 in the journal Character Physics.
Circuit mirroring faces a quantity of rivals in the arena of quantum benchmarking. For illustration, theoretical physicist Joseph Emerson at the College of Waterloo in Canada notes a process that he and his colleagues have made recognized as cycle benchmarking was demonstrated an efficient benchmarking technique on the quantum pcs at the Sophisticated Quantum Testbed at Lawrence Berkeley Countrywide Lab.
“Cycle benchmarking was shown to be preferable in the context of knowing application-precise performance—it is a scalable approach for benchmarking the general performance of the quantum components that is tailored to each and every precise algorithm or application of desire,” states Emerson, head of quantum strategy at Keysight Technologies.
Proctor notes that whereas cycle benchmarking steps mistake charges for sets of quantum gates—the quantum computing version of the logic gates that common desktops use to carry out computations—circuit mirroring actions a quantum computer’s effectiveness on full computations. As these types of, he implies they are basically various resources.
Circuit mirroring can theoretically be put together with quite a few other benchmarks, these kinds of as quantum quantity, to make scalable versions of these yardsticks, “which we assume will be definitely thrilling to the local community,” Proctor says. “We undoubtedly haven’t considered of all the appealing things you can do with our system, and we are fired up to see what the research group comes up with.”


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17 Feb 2010: Madhavan R, Gong ZL, Ma JJ, Chan AWS, Peng HB (2010) Correction: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction. PLOS ONE 5(2): 10.1371/annotation/7df19997-b7c7-40a2-9ed9-dc31233ce8f6. https://doi.org/10.1371/annotation/7df19997-b7c7-40a2-9ed9-dc31233ce8f6 View correction
Postsynaptic enrichment of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (NMJ) depends on the activation of the muscle receptor tyrosine MuSK by neural agrin. Agrin-stimulation of MuSK is known to initiate an intracellular signaling cascade that leads to the clustering of AChRs in an actin polymerization-dependent manner, but the molecular steps which link MuSK activation to AChR aggregation remain incompletely defined.
In this study we used biochemical, cell biological and molecular assays to investigate a possible role in AChR clustering of cortactin, a protein which is a tyrosine kinase substrate and a regulator of F-actin assembly and which has also been previously localized at AChR clustering sites. We report that cortactin was co-enriched at AChR clusters in situ with its target the Arp2/3 complex, which is a key stimulator of actin polymerization in cells. Cortactin was further preferentially tyrosine phosphorylated at AChR clustering sites and treatment of myotubes with agrin significantly enhanced the tyrosine phosphorylation of cortactin. Importantly, forced expression in myotubes of a tyrosine phosphorylation-defective cortactin mutant (but not wild-type cortactin) suppressed agrin-dependent AChR clustering, as did the reduction of endogenous cortactin levels using RNA interference, and introduction of the mutant cortactin into muscle cells potently inhibited synaptic AChR aggregation in response to innervation.
Citation: Madhavan R, Gong ZL, Ma JJ, Chan AWS, Peng HB (2009) The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction. PLoS ONE 4(12): e8478. https://doi.org/10.1371/journal.pone.0008478
Editor: Lin Mei, Medical College of Georgia, United States of America
Received: October 12, 2009; Accepted: December 3, 2009; Published: December 29, 2009
Copyright: © 2009 Madhavan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Research Grants Council (RGC) grants HKUST6401/05M and HKUST6417/06M, and Academic Outreach Endowment (AOE) grant grant/B-15/01 (http://www.ugc.edu.hk/eng/rgc/index.htm). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
At the vertebrate neuromuscular junction (NMJ) motor nerve-secreted acetylcholine (ACh) binds and opens postsynaptic ACh receptors (AChRs) to initiate excitation along the muscle membrane and cause contraction. One remarkable feature of the NMJ is its unfailing postsynaptic response to every nerve-stimulation, which is made possible by the selective enrichment of AChRs in muscle within a small membrane domain that apposes presynaptic “active zones” where synaptic vesicles dock and release ACh. Whereas ∼10,000 AChRs are present per µm2 of the synaptic muscle membrane, only ∼10 AChRs are found per µm2 of the extrasynaptic membrane . Thus, in the assembly of the NMJ, synaptic AChR clustering is a critical and perhaps also the most studied step .
During development AChRs form aggregates in embryonic muscle fibers even before motor innervation due to the activation of the muscle receptor tyrosine kinase MuSK , . This “pre-patterned” clustering of AChRs in the central regions of muscle fibers involves, in addition to MuSK, the transmembrane protein LRP4  and the adapter dok-7 which enhances MuSK signaling . Next, during innervation, a nerve-deposited heparan-sulfate proteoglycan named agrin  stimulates MuSK to promote AChR clustering and stabilization locally at synapses , . How agrin activates MuSK has remained unclear because agrin does not bind to MuSK , although recent studies suggest that agrin interacts with LRP4 and that LRP4 binds to MuSK and facilitates the aggregation and (trans)activation of MuSK , . Finally, as AChRs become accumulated at newly established synapses, the pre-patterned AChR clusters are disassembled, either by synaptogenic stimuli or by ACh , , , which helps the selective concentration of AChRs at NMJs.
How are AChRs concentrated in the postsynaptic membrane? It is thought that AChRs diffusing freely on the muscle surface become clustered at traps generated by synaptogenic stimuli at the NMJ ; this diffusion-mediated trapping of AChRs has recently been directly visualized through single-molecular tracking with quantum dots , . The clustering of AChRs is mediated by the protein rapsyn, which crosslinks and tethers AChRs to the cortical actin cytoskeleton , , , . F-actin and several proteins that bind to it are enriched at the NMJ and at AChR clusters in muscle cells, and inhibition of actin polymerization blocks the aggregation of AChRs in response to synaptogenic stimulation , . Furthermore, AChR clustering involves signaling by Rho-family GTPases , which regulate diverse actin polymerization-driven processes , p21-activated kinase 1, an effector of the GTPase Cdc42 , and geranylgeranyl transferase, an enzyme which enhances the membrane association and activation of GTPases . Conversely, inhibition of PI3 kinase signaling in myotubes reduces agrin-dependent activation of Rac and Cdc42 GTPases and impedes AChR clustering .
The above findings suggest that Rho GTPases influence AChR aggregation in multiple ways, but little is known about how the receptor clustering process is affected by other proteins that also regulate actin polymerization. The focus of this study is on one such protein – cortactin – which has been shown to localize at AChR clusters in cultured muscle cells , , but for which no functional role in NMJ formation has been described to date. Initially identified as a major src tyrosine kinase substrate in cells , , cortactin is today recognized as a modulator of numerous actin polymerization-dependent processes, ranging from cell motility to endocytosis to dendritic spine growth and synaptogenesis in central neurons . An important target of cortactin is the Arp2/3 protein complex, which binds to existing actin filaments and initiates new branch growth , ; association of cortactin with the Arp2/3 complex enhances Arp2/3-induced actin polymerization , . Intriguingly, phosphorylation by src allows cortactin to be linked by the adapter Nck1 to another Arp2/3-stimulator, N-WASP, and this tripartite complex composed of phospho-cortactin, Nck1 and N-WASP activates Arp2/3-dependent actin polymerization better than cortactin or N-WASP alone . In light of these recent findings related to cortactin and the importance of tyrosine kinase signaling and actin polymerization in AChR clustering, here we asked three specific questions: Is cortactin's tyrosine phosphorylation relevant in the context of AChR clustering in situ? Is cortactin's phosphorylation in muscle affected by agrin/MuSK signaling? Does cortactin signaling in any way regulate synaptic AChR aggregation? The results of our cell biological, biochemical and molecular assays presented below suggest that phosphorylation-dependent signaling by cortactin downstream from agrin/MuSK promotes AChR clustering at the NMJ.
Localization of Actin-Polymerization Regulators at AChR Clustering Sites
We previously showed that dynamic F-actin assembly occurs at sites of de novo AChR clustering and that green fluorescent protein (GFP)-tagged cortactin is recruited to such sites , . Because the Arp2/3 complex is a key cellular regulator of actin polymerization and a target of cortactin, we asked whether proteins of this complex are localized in muscle cells where AChR clustering is stimulated. Primary cultures of Xenopus embryonic muscle cells were labeled with rhodamine-α-bungarotoxin (R-BTX) to mark AChRs and then stimulated with polystyrene beads coated with heparan-binding growth associated molecule (HB-GAM); this procedure enables AChR clusters to be induced reliably and in a spatiotemporally controlled manner . Labeling of bead-treated cells with affinity-purified antibodies revealed that Arp2 and p34arc, two members of the Arp2/3 complex, were enriched at bead-muscle contacts where AChRs were focally clustered (Fig. 1, A–F), but numerous unrelated proteins were not (data not shown). The distribution of the Arp2/3 complex proteins relative to AChRs resembled that of cortactin , which can directly bind to the Arp2/3 complex , and when bead-treated muscle cells were labeled by the polyclonal anti-p34arc antibody and a monoclonal anti-cortactin antibody, p34arc and cortactin were found to colocalize at bead-muscle contacts (Fig. 1, G–I). However, in non-muscle cells which are found occasionally in primary muscle cultures, p34arc and cortactin were detected along the cell periphery but not at bead-contacts (panels J–K), suggesting that the antibodies against p34arc and cortactin recognized their targets at AChR clusters in muscle cells and did not simply mark all sites where beads contacted cells.
Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified polyclonal antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).
Association of Tyrosine-Phosphorylated Cortactin with AChR Clusters
Because recent work has shown that tyrosine phosphorylation of cortactin's src-target sites enhances the activation of Arp2/3-dependent actin polymerization , , we tested whether cortactin phosphorylation is relevant in the context of AChR clustering. For this the distribution of tyrosine-phosphorylated cortactin relative to AChR clusters was examined in Xenopus muscle cells by labeling (separately) with three different antibodies directed against cortactin phosphorylated on its major src-target sites (Y421, Y466 and Y482 in mouse cortactin). In quiescent muscle cells anti-Y482-phospho-cortactin antibody strongly labeled pre-patterned AChR clusters (Fig. 2, A–F) and also the edges of cells (where cortactin is known to localize) but only weakly labeled other regions of the cells. Y482-phosphorylated cortactin was detected reliably at pre-patterned AChR clusters (panel C), although sometimes it appeared more concentrated in certain regions of the clusters than others (panel F). Data from several muscle culture preparations showed phosphorylation of Y482-cortactin at >95% of pre-patterned clusters identified (Table S1, Supporting Documents).
Xenopus muscle cells were labeled with R-BTX and after fixation with an antibody that specifically recognizes Y482-phospho-cortactin (plus FITC-linked anti-rabbit antibodies) (A-F). In some cases muscle cells were first co-cultured for 1 d with spinal neurons and then labeled with R-BTX and anti-phospho-cortactin and secondary antibodies (G-I). In pure muscle cultures (A, D) large “pre-patterned” AChR clusters were present (B, E; arrows) and at these sites staining by anti-phospho-cortactin was significantly stronger than elsewhere in muscle cells (C, F; arrows). Labeling for phospho-cortactin was detected at almost all pre-patterned clusters examined (see Table S1), although within the clusters certain regions at times appeared to be more enriched in phospho-cortactin than others (as in F; arrow versus arrowhead). The anti-phospho-cortactin antibody also labeled muscle cell edges (C, F) where cortactin is known to be localized. In nerve-muscle co-cultures (G) AChRs were selectively concentrated at synaptic contacts (H; arrows) and these nerve-induced AChR clusters were also labeled by the anti-phospho-cortactin antibody (I; arrows).
Antibodies against Y421- and Y466-phospho-cortactin also labeled AChRs clusters and muscle cell edges, but with these two antibodies staining was somewhat weaker than with anti-Y482-phospho-cortactin (not shown). It is possible that these commercial antibodies have differing affinities for their target sites or that these sites are phosphorylated and/or accessible to different extents. Unlike these three antibodies, however, many control antibodies showed markedly different labeling of muscle cells. For example, a polyclonal antibody against tyrosine-phosphorylated AChR β-subunit labeled only AChR clusters, two different polyclonal antibodies against cadherin complex proteins strongly stained cell junctions but not AChR clusters, and other polyclonal antibodies either labeled muscle cells uniformly (such as an anti-phospho-Shp2 phosphatase antibody) or failed to label the cells (such as anti-synapsin) (R.M., A.W.S.C., H.B.P., unpublished observations).
Next we examined whether cortactin is phosphorylated at sites of new AChR cluster formation. In Xenopus nerve-muscle co-cultures, AChR clusters developed in muscle cells focally at sites where the cells were contacted by nerves and these clusters were strongly labeled by the anti-Y482-phospho-cortactin antibody (Fig. 2, G–I). Phospho-cortactin's localization closely matched that of postsynaptic AChR clusters, but labeling for phospho-cortactin was weak in presynaptic neurites (which were strongly labeled, as expected, by antibodies against neuronal markers such as synapsin; not shown). From several nerve-muscle co-culture preparations we found phospho-cortactin at >80% of nerve-induced AChR clusters (Table S1). Moreover, in muscle cultures stimulated with HB-GAM-beads, phospho-cortactin was found to be enriched at AChR clusters induced at bead-muscle contacts (Fig. 3, A–C), with pooled data showing that this was the case at ∼95% of such clusters (Table S1), and in agrin-treated muscle cells phospho-cortactin was accumulated at new AChR clusters (∼0.5–3 µm in diameter) generated across the muscle surface (Fig. 3, D–F). Interestingly, phospho-cortactin was also detected along myopodia (panel D–F) generated close to AChR clusters  and was often concentrated at myopodial tips (arrowheads in panel F). Taken together these results suggested that phosphorylation of cortactin's src-target tyrosines is enhanced at sites in muscle where synaptogenic stimuli such as agrin produce their known functional effects.
R-BTX-labeled Xenopus muscle cells were stimulated overnight with HB-GAM-beads (A-C) or neural agrin (D-F). In cells exposed to beads (A; asterisks) AChRs aggregated at bead-muscle contacts (B; arrows) and strong labeling was detected at these bead-induced AChR clusters for Y482-phospho-cortactin (C; arrows). Treatment of muscle cells with agrin (D) generated numerous small (∼0.5-3 µm) AChR clusters (D; arrows) and antibody labeling showed that phospho-cortactin was enriched at these clusters (E; arrows) and also along myopodia that formed near the AChR clusters (F; arrows and arrowheads).
Agrin-Dependent Tyrosine Phosphorylation of Cortactin
To extend the studies described above we tested whether agrin signaling directly affects cortactin's tyrosine phosphorylation through biochemical assays. For this C2 mouse myotubes were used because such assays cannot be easily performed on the limited material provided by Xenopus embryonic muscle primary cultures. Fully differentiated C2 myotubes were exposed to control or agrin-containing medium for 1 h and then cortactin was immuno-precipitated from them for analyses. Blotting of immuno-precipitates with anti-cortactin and anti-phosphotyrosine antibodies (Fig. 4A, top) showed that the anti-cortactin antibody (mAb4F11) captured cortactin from extracts but the control antibody did not (“IB:cort”, upper blot). Significantly, cortactin immuno-precipitated from extracts of agrin-treated myotubes was more strongly stained by the anti-phosphotyrosine antibody than cortactin captured from extracts of control myotubes (“IB: PY”, lower blot). From four such experiments we determined the band intensities of cortactin stained by anti-cortactin and anti-phosphotyrosine and then divided the latter values by the former to normalize for cortactin loading in each case. These results indicated that cortactin's tyrosine phosphorylation level in agrin-treated myotubes was more than twice that in control myotubes (Fig. 4A, bottom).
Cultured C2 mouse myotubes were exposed to medium without (-) or with added agrin (+) before preparing extracts for immuno-precipitation (A) with a monoclonal antibody against cortactin (IP: cort) or an unrelated protein (IP: ctl). When these samples were immuno-blotted for cortactin (IB: cort) and total phosphotyrosine (IB: PY; mAb4G10), cortactin was found to be captured only by the anti-cortactin antibody (upper lanes), and anti-phosphotyrosine staining showed that cortactin from extracts of agrin-treated cells was tyrosine phosphorylated significantly more than that captured from control extracts (lower lanes). This increase in cortactin phosphorylation was quantified from four experiments (A, graph) by measuring band densities, normalizing for cortactin loading (see Methods), and calculating the phosphotyrosine level change relative to control. B. To test whether the src-target sites in cortactin were phosphorylated in response to agrin-treatment, myotube extracts were blotted with antibodies against total cortactin and cortactin phosphorylated on Y421. Agrin-treatment did not alter the amount of cortactin present in extracts (upper lanes) but the staining of cortactin by the anti-Y421-phospho-cortactin antibody (IB: pCort) was enhanced by agrin-treatment more than two-fold, as shown by quantification from three experiments (B, graph). Positions of pre-stained MW markers (Bio-Rad) are indicated on the right side of blots, and in the graphs * represents P<0.02 in t-tests.
As discussed in the previous section, three major src-target sites have been identified in cortactin. To test whether agrin-treatment of muscle, which activates src signaling , triggers the phosphorylation of these specific sites, C2 myotube extracts were immuno-blotted with anti-cortactin and anti-Y421-phospho-cortactin antibodies. Both antibodies stained bands of cortactin's expected size and anti-cortactin staining showed equal protein loading (Fig. 4B); with the anti-phospho-cortactin antibody, however, cortactin was stained nearly three-times more strongly in extracts of agrin-treated myotubes than of control myotubes (Fig. 4B, bottom; data from three experiments). Similarly, a band corresponding to full-length cortactin was detected with the anti-Y466-phospho-cortactin antibody and this was enhanced by agrin as well (not shown), but staining by the anti-Y482-phospho-cortactin antibody in total extracts was weak and we have been unable to ascertain whether that staining is altered significantly by agrin-treatment. Nevertheless, these findings and our in situ labeling results together supported the conclusion that phosphorylation of cortactin's src-target sites is enhanced during agrin/MuSK signaling.
Involvement of Cortactin Signaling in Agrin/Nerve-Induced AChR Clustering
To investigate whether phosphorylation-dependent cortactin signaling is involved in the AChR clustering process, we expressed a dominant-negative, phosphorylation-defective mutant cortactin and wild-type cortactin in C2 myotubes and examined agrin-induction of AChR clusters. In the mutant cortactin (3YF-cortactin), the three major src phosphorylation sites – Y421, Y466 and Y482 – were eliminated , and both the mutant and the wild-type cortactin proteins were tagged with GFP. The GFP-tagged cortactin proteins (or GFP alone) were expressed in myotubes by mRNA transfection, which was carried out in parallel on myotubes grown on coverslips for analysis of AChR clustering and in culture dishes for biochemically confirming the expression of the exogenous proteins.
Myotubes growing on coverslips were incubated overnight with agrin and labeled with R-BTX (Fig. 5) and transfected cells were identified by green fluorescence. In these we found that compared to myotubes expressing GFP (panels A–C) or wild-type cortactin-GFP (panels G–I), those expressing the phospho-mutant cortactin had fewer and smaller AChR clusters (panels D–F). Immuno-blotting of extracts of myotubes transfected with mRNAs encoding the GFP-tagged cortactin proteins (but not GFP) demonstrated that anti-cortactin monoclonal antibody 4F11 stained endogenous cortactin plus the exogenous cortactin proteins, which were ∼25 kD larger (Fig. 5J). From five separate transfection experiments the numbers and lengths of AChR clusters in green fluorescent (transfected) myotubes were determined; for this the myotubes were examined in their entirety and all distinct clusters (such as those indicated by arrows in Fig. 5) were counted and measured. Data pooled from 200 or more (each) GFP-cells, wild-type cortactin-cells and phospho-mutant cortactin-cells showed that relative to control (GFP) myotubes, the phospho-mutant cortactin-expressing myotubes had nearly 60% fewer clusters (4.3/myotube instead of ∼10/myotube) which were nearly 30% smaller (∼19 µm long compared to ∼27 µm) (Fig. 5, K–L).
To examine the effect of exogenous cortactin proteins on AChR clustering, C2 myotubes were transfected with mRNAs encoding GFP (Ctl) or GFP-tagged phospho-mutant (3YF) cortactin or wild-type (WT) cortactin. After treating myotubes with agrin overnight, cells expressing exogenous proteins (A, D, G; asterisks) were identified by green fluorescence (B, E, H) and the AChR clusters present on the surface of these cells were examined by R-BTX-labeling (C, F, I; arrows). Forced expression of the phospho-mutant, but not wild-type, cortactin reduced the number and lengths of agrin-induced AChR clusters in myotubes. J. To biochemically confirm the expression of exogenous cortactin proteins in myotubes, extracts prepared from myotubes transfected with mRNAs encoding GFP, GFP-tagged WT and 3YF cortactin were immuno-blotted with anti-cortactin monoclonal antibody mAb4F11. Myotubes transfected with GFP mRNA (G) contained full-length endogenous cortactin (arrow on left), but those transfected with WT- and 3YF-cortactin mRNAs contained endogenous cortactin plus a protein (∼25 kD larger) corresponding to exogenous, GFP-tagged cortactin (asterisk). MW marker positions are indicated on the right. K-L. Myotubes transfected with GFP or GFP-tagged cortactin proteins were selected randomly and the numbers and lengths of the AChR clusters present on their surface were determined; data from five separate transfection experiments were pooled and normalized relative to values obtained from GFP-tranfected cells. Fewer (K) and smaller (L) AChR clusters were present in myotubes expressing phospho-mutant cortactin than in cells expressing GFP alone or WT-cortactin-GFP. Mean and SEM values are shown, *P<0.05.
The above results demonstrated that agrin-stimulated AChR clustering in myotubes was inhibited in the presence of exogenous phospho-mutant cortactin. Therefore, to directly test whether endogenous cortactin functions in the AChR cluster assembly process, we down-regulated its expression in myotubes using RNA interference (RNAi). C2 myoblasts were transfected with GFP cDNA mixed with a cocktail of mouse cortactin-specific small interfering RNAs (siRNAs) or siRNAs against unrelated proteins; cells were then allowed to differentiate for 4 d before they were treated overnight with agrin. When GFP-positive cells were examined after labeling with R-BTX, the results showed that AChR clustering in response to agrin was impaired in cortactin-siRNA-transfected myotubes compared to control-siRNA-transfected myotubes (Fig. 6, A–F). Immuno-blotting of total cell extracts from the same batch of myotubes (in each experiment; see Methods) confirmed that the cortactin-siRNAs down-regulated the expression of cortactin but did not produce any noticeable change in the expression of many unrelated proteins (Shp2, SIRPα1, p120 catenin (p120ctn), Arp2, Arp3, Nck1 and tubulin; not shown, except tubulin in panel G) and that control siRNAs (against Shp2, SIRPα1 and p120ctn; p120ctn-siRNA used as control in panel G) did not affect cortactin levels. AChR cluster numbers and lengths in control siRNA- and cortactin siRNA-transfected myotubes (150 or more each) were measured (see above) and data from four separate siRNA-transfection experiments revealed that the average values of these parameters were reduced by ∼40% in myotubes expressing lower than normal levels of cortactin (Fig. 6, H–I).
C2 myotubes generated from myoblasts transfected with control siRNAs (A-C) or a pool of siRNAs directed against mouse cortactin (D-F) (both mixed with a cDNA encoding GFP) were incubated overnight in differentiation medium containing agrin before labeling with R-BTX. Transfected myotubes (A, D; asterisks) were identified by green fluorescence (B, E), and the AChR clusters present on their surface (C, F; arrows) were counted and the lengths of these clusters were measured. G. To demonstrate that siRNAs against cortactin knocked down cortactin expression, in each experiment extracts were prepared from myotubes generated from myoblasts transfected in parallel and maintained under conditions identical to those used for examining agrin-induced AChR clustering. Extracts of cells transfected with GFP cDNA plus control (p120ctn) siRNA (Ctl; left lane), cortactin siRNA (middle lane) or GFP cDNA alone (right lane) were immuno-blotted with antibodies against cortactin (upper blot) or tubulin (lower blot). The cortactin siRNA suppressed the expression of cortactin without affecting unrelated proteins (such as tubulin, which is also shown here to demonstrate equal protein loading), and cortactin's expression was not affected by control siRNAs or by transfection procedures (where only GFP cDNA was used). From four transfection experiments AChR cluster data from control (Ctl) and mouse cortactin (msCort) siRNA-transfected myotubes were pooled and normalized relative to those obtained from cells transfected with the control siRNA. These results showed that agrin-induced AChR cluster numbers (H) and lengths (I) were significantly lower in myotubes expressing reduced levels of endogenous cortactin compared to those expressing normal levels of cortactin. Mean and SEM values are shown, *P<0.05.
Is cortactin signaling involved in AChR clustering at the NMJ in vivo? To answer this, we injected mRNAs encoding GFP or GFP-tagged wild-type and mutant cortactin proteins into Xenopus embryos with the goal of examining NMJs that can be observed (in a “chevron” pattern) in the developing tail muscle. These experiments, however, failed to reveal whether cortactin functions in AChR aggregation because the embryos injected with mutant cortactin mRNA died before reaching a stage (∼40 or later) when NMJs can be reliably identified by BTX-labeling. We therefore adopted an alternative approach for testing cortactin's function, an approach that has been successfully used by us  and by others  in which myotomal muscle cells and spinal neurons are isolated from early embryos (∼stage 20–22) and plated together. In these nerve-muscle co-cultures AChR clustering is triggered in response to innervation, and when nerve and muscle cells isolated from normal and mRNA-injected embryos are mixed together, nerve or muscle can be manipulated selectively with exogenous molecules (Methods). In this experimental system NMJ formation can be observed and quantified by R-BTX-labeling, which is used to monitor the focal aggregation of AChRs at nerve-muscle contacts . When we examined contacts between normal spinal neurons and muscle cells expressing GFP or the GFP-tagged cortactin proteins (Fig. 7), robust AChR clustering was detected at innervation sites in muscle cells expressing GFP (panels A–D) or GFP-tagged wild-type cortactin (E–H); in contrast, in cells expressing GFP-tagged phospho-mutant cortactin, often no synaptic AChR clustering was detected (I–J) or only weak and loosely organized AChR clusters were found (K–L). We also observed cases in which the same neurites contacted both mutant cortactin-expressing cells and normal cells present side-by-side, and here we found that AChR clustering was strongly induced in the normal cells but not mutant cells (M–N). In cells expressing GFP, wild-type and mutant cortactin we identified 168–225 nerve-contacts from multiple culture preparations and classified these innervation sites as those with or without AChR clusters; the quantified results showed that in mutant cortactin cells, taking into account even loosely organized AChR clusters (as in L), nerve-induction of AChR aggregation was only half as effective as that in GFP and wild-type cortactin cells (Fig. 7O).
Xenopus embryonic muscle cells expressing GFP (A-D) and GFP-tagged wild-type cortactin (WT-cort; E-H) and phospho-mutant cortactin (3YF-cort; I-N) were co-cultured with spinals neurons for 1 d and then labeled with R-BTX to visualize AChR clusters. Cells expressing the exogenous proteins fluoresced green and AChR clusters appeared red, as shown in this figure with 2-3 representative examples of nerves contacting muscle cells with GFP or GFP-tagged cortactin proteins. In the GFP and WT-cort muscle cells (A-H), AChRs were tightly clustered (arrows) along nerve-contacts identified (traced in white in colored panels) but this was not the case in 3YF-cort cells where nerves often induced no AChR clustering (J) or induced few clusters that were loosely organized (L; arrowheads). We found cases where the same neurites moved across normal muscle cells and 3YF-cells (M-N) and in such cases synaptic AChR clustering was robust in the normal cells (arrows) but not mutant cells (arrowheads). O. The percentages of nerve-contacts with AChR clusters were determined by examining several co-cultures with muscle cells expressing GFP or the GFP-tagged cortactin proteins (see Methods) and these values were normalized relative to numbers obtained from examining nerve-contacts on GFP-cells. In muscle cells expressing phospho-mutant cortactin, synaptic AChR clustering was almost halved. Nerve-muscle contacts examined: GFP cells, 168; WT-cort cells, 214; 3YF-cort cells, 225; mean and SEM shown, *P<0.0001.
In this study examining the molecular regulation of AChR aggregation at the NMJ we obtained these novel results: one, proteins of the Arp2/3 complex and cortactin (which can together promote actin polymerization) were co-distributed at AChR clustering sites (where F-actin assembly occurs). Two, AChR clusters were enriched in cortactin phosphorylated on its src-target tyrosine residues, whose phosphorylation enhances Arp2/3-mediated actin polymerization . Three, treatment of myotubes with agrin increased the phosphorylation of cortactin on tyrosine residues, including those known to be targeted by src. Four, forced expression in myotubes of a cortactin mutant lacking these (three major) src-target sites inhibited agrin-induced AChR clustering, as did the depletion of endogenous cortactin in myotubes using RNAi. And five, disruption of normal cortactin signaling in muscle cells by expression of the dominant-negative cortactin mutant inhibited the aggregation of AChRs at innervation sites. We propose that agrin/MuSK signaling acts on cortactin through src (and possibly other) tyrosine kinases, and that cortactin, on its own or with proteins such as Nck1, N-WASP and components of the Arp2/3 complex, promotes actin polymerization and AChR clustering at the NMJ (Fig. 8).
Activation of MuSK by agrin induces AChR clustering in an actin polymerization-dependent manner. This model depicts a possible way in which cortactin signaling might promote the AChR clustering process. Initiation of intracellular signaling by the activated MuSK complex could enhance cortactin's tyrosine phosphorylation through src family tyrosine kinases (SFKs) (and possibly other kinases such as abl), and cortactin, in turn, could increase actin polymerization. Alternatively, cortactin might trigger actin polymerization by activating the Arp2/3 complex, either on its own or in concert with WASP-related proteins (N-WASP, WIP, etc.) to which it could be linked by the adapter Nck. In parallel, via other signaling intermediates, MuSK could stimulate Rho-family GTPases and, through them, F-actin assembly. Such enhanced and dynamic actin polymerization at synaptic sites could generate a scaffold which “traps” AChRs through rapsyn.
Diverse cellular mechanisms collaborate to generate and maintain high-density AChR aggregates at the NMJ. Muscle-surface AChRs are selectively clustered in the synaptic region of muscle, the clustered AChRs are metabolically stabilized, and AChRs and many proteins that promote their synaptic accumulation are synthesized at high levels in the sub-synaptic domains of myotubes , , , . The first of these mechanisms – synaptic AChR clustering – has arguably been studied most extensively and current evidence supports the following (simplified) view of the molecular pathway involved: agrin secreted by nerve activates MuSK through the protein Lrp4 , , and MuSK, together with binding partners dok-7  and Tid1 , triggers downstream signaling through intracellular Ca2+, various kinases and phosphatases and Rho-family GTPases to direct AChR aggregation in an actin polymerization-dependent manner , .
New F-actin synthesis occurs at AChR clustering sites in muscle cells ,  and likely depends on the combined activity of Rho GTPases and their regulators and effectors that function in agrin/MuSK signaling , , , . Rho GTPases are potent stimulators of the Arp2/3 complex, which enhances actin polymerization in many types of cells . Arp2/3 complex-dependent actin polymerization is activated by proteins such as WASP/N-WASP and Scar/WAVE, and the binding of Rac and Cdc42 GTPases enables WASP/N-WASP and Scar/WAVE to stimulate the Arp2/3 complex , . Thus, our detection of Arp2/3 complex proteins at AChR clusters suggests that GTPase signaling accelerates Arp2/3-dependent F-actin assembly at synapses (Fig. 8).
In this study expression of either mutant cortactin or cortactin siRNAs in muscle cells inhibited agrin/nerve-dependent AChR aggregation partially rather than fully. Although this could have been due to incomplete disruption of normal cortactin function in the muscle cells, it could also be because of cortactin being one of many Arp2/3 complex/actin-regulators (such as the GTPases) at the synapse. Our observation that over-expression of wild-type cortactin in myotubes did not enhance AChR clustering is consistent with the latter view. It should be of interest to investigate in future studies the extent to which the inhibition of both cortactin and Rho-family GTPases blocks agrin-induced AChR clustering.
We have shown here that cortactin was colocalized with the Arp2/3 complex at AChR clustering sites. Notably, tyrosine phosphorylated-cortactin was enriched at AChR clusters and the phosphorylation of cortactin's src-target sites was enhanced by agrin treatment of muscle cells. Although cortactin was originally identified as a major src kinase-substrate in cells, the influence of phosphorylation on cortactin-dependent regulation of actin polymerization long remained unclear , . An elegant biochemical study carried out recently, however, has indicated a novel mechanism by which phosphorylation of cortactin's src-target sites can stimulate F-actin assembly . Using purified proteins this study showed that phosphorylation of cortactin by src enables the formation of a complex made up of cortactin and the SH2 domain-containing adapter Nck1, plus N-WASP or the WASP-interacting protein, WIP. Consistent with earlier work  cortactin stimulated Arp2/3-dependent polymerization of actin by its N-terminal acidic region; this activity was enhanced following src-phosphorylation of cortactin, which promoted Nck1-binding and through Nck1 the recruitment of WIP . After src-phosphorylation cortactin could also be linked by Nck1 to N-WASP, and this complex (through the “VCA” domain of N-WASP) more robustly stimulated F-actin assembly by Arp2/3 than cortactin or N-WASP alone. Importantly, a phospho-mutant (3YF) cortactin was used to demonstrate that src-dependent enhancement of cortactin's ability to stimulate Arp2/3 (through Nck1 and N-WASP or WIP) requires the Y421, Y466 and Y482 sites of cortactin . Forced expression of the same 3YF-mutant cortactin in myotubes, or the suppression of cortactin expression using RNAi, inhibited agrin-induced AChR clustering in this study. It is known that src kinases are rapidly activated by agrin-stimulation of MuSK and that they phosphorylate MuSK, AChRs and associated proteins . Moreover, like phospho-cortactin (this study), activated-src kinases are enriched at AChR clustering sites in situ . Thus src-cortactin signaling may be spatially restricted to the synaptic regions of muscle where intimate coupling of MuSK and src activation could lead to rapid tyrosine phosphorylation of cortactin; this, in turn, could locally enhance Arp2/3-dependent actin polymerization to generate “traps” that capture mobile AChRs.
Currently a role of cortactin at the NMJ in vivo cannot be directly tested because suitable models – viable animals with global or muscle-specific cortactin gene deletion – are unavailable. Moreover, through our own work we noted that Xenopus embryos which had been injected with the phospho-mutant cortactin mRNA failed to grow to a stage at which NMJs in the developing tail could be studied. We were, however, successful in using this dominant-negative approach to show that interference with phosphorylation-dependent cortactin signaling in muscle is sufficient for inhibiting nerve-induced AChR clustering, and by extension NMJ formation, in culture. Interestingly, others have studied mice lacking src kinase activity in muscle and have shown that while NMJs can develop in the muscles of these mice, the synaptic AChR aggregates found are less stable than their counterparts in the muscles of normal mice . One explanation for this observation is that src binds to AChRs and phosphorylates them, which enhances the cytoskeletal linkage of AChRs through rapsyn , , , . The results of this study raise the additional possibility that in the absence of normal src activity, sub-optimal signaling by cortactin leads to the assembly of synaptic scaffolds which hold AChRs poorly at NMJs.
Properly balanced src signaling is important for normal AChR clustering at the NMJ , but what all targets of src influence AChR clustering at the NMJ is incompletely understood. By uncovering a role of the src-substrate cortactin in AChR clustering this study suggests a previously unappreciated way by which src could regulate AChR aggregation at the NMJ. Interestingly, two other src-substrates we previously identified as targets in agrin/MuSK signaling produced effects distinct from those described here for cortactin: p120ctn promoted myopodial induction in response to agrin , whereas signal-regulatory protein α1 (SIRPα1) stimulated the tyrosine phosphatase Shp2 to limit MuSK-dependent AChR clustering  and to also facilitate the dispersal of pre-patterned AChR clusters by synaptogenic stimuli . Intriguingly, cortactin can participate in p120ctn signaling in epithelial cells  and can act together with the p120ctn-relative δ-catenin in generating filopodia-like dendritic protrusions in neurons . Moreover, cortactin itself also affects dendritic spine morphogenesis and remodeling, with the depletion of cortactin in neurons causing a reduction in spines , . Thus, cortactin-dependent actin polymerization triggered by src could potentially facilitate p120ctn-mediated myopodial assembly at the NMJ as well as synapse formation in central neurons. And, although it is not known which phosphatases dephosphorylate cortactin in muscle cells, the spread of phosphatase activity initiated by agrin/MuSK , ,  may bring about the dephosphorylation of extrasynaptic cortactin to maintain efficient actin polymerization by src-cortactin signaling selectively at developing NMJs where agrin stimulates MuSK. Future studies investigating the spatiotemporal control of cortactin's phosphorylation levels during neuronal synapse development and testing whether that phosphorylation affects cortactin's ability to influence the formation of structures such as dendritic spines should yield further insights into cortactin's functions at CNS synapses and elsewhere.
Agrin was obtained from the conditioned medium of HEK293 cells transfected with a plasmid encoding neural agrin . Recombinant heparin-binding growth-associated molecule (HB-GAM) was generously provided by Dr. Heikki Rauvala (University of Helsinki). Na-pervanadate, a potent tyrosine phosphatase inhibitor, was prepared by mixing 10 mM Na-orthovanadate with 1.7% hydrogen peroxide in a 50∶1 ratio just before use and diluting this solution as needed . These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).
Primary cultures of Xenopus myotomal muscle cells were prepared from stage 20–22 embryos as described previously  in accordance with HKUST's established animal handling and care procedures. Muscle cells were plated on glass coverslips coated with entactin-collagen-laminin substrate (Upstate Biotechnology) and used within one week. For some experiments neural tubes of stage 20–22 embryos were dissociated and spinal neurons were seeded on muscle cells plated 3–5 d earlier; these nerve-muscle co-cultures were examined 1 d later . C2 mouse myotube cultures were prepared by growing myoblasts (purchased from ATCC, Manassas, VA, USA) on glass coverslips or in culture dishes in DMEM containing 20% fetal bovine serum (growth medium) until confluence and then inducing differentiation by changing the medium to DMEM containing 2% horse serum (differentiation medium); fresh differentiation medium was added each day for 4–5 d .
Agrin-Treatment, Cell Extract Preparation, Immuno-Precipitation/Blotting
Differentiated C2 myotubes in culture dishes were incubated for 1 h in fresh differentiation medium without or with added agrin (plus 10 µM Na-pervanadate). After rinsing with cold phosphate buffered saline (PBS), extracts were prepared using a TX-100 buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% TX-100, 1 mM Na-pervanadate) , with 1 ml buffer being added to a 10 cm culture dish of cells. To clarified extracts anti-cortactin or control antibodies and Protein A/G agarose beads (Santa Cruz Biotechnology) were added, and after mixing for 2 h at room temp the beads were spun down, washed extensively with Tris-buffered saline containing 0.1% TX-100 and then mixed with SDS-electrophoresis sample buffer to elute immuno-precipitated proteins. Samples were electrophoresed, transferred to PVDF membranes and probed with primary antibodies and HRP-linked secondary antibodies for ECL-based detection.
Synthesis of mRNAs, Myotube Transfection
Wild-type and phospho-mutant mouse cortactin cDNAs were gifts from Drs. Tom Parsons (University of Virginia) and Xi Zhan (University of Maryland), respectively. In the phospho-mutant cortactin (3YF) construct, three src target sites – Y421, Y466 and Y482 – were changed to non-phosphorylatable Fs . Both wild-type and mutant cortactin proteins were tagged with green fluorescent protein (GFP) by inserting the cortactin cDNAs into pEGFP-N1 plasmid (Clonetech); the cortactin-GFP sequences were subcloned into pCS2+ vector  and then into pcDNA3.1. The cortactin-GFP encoding plasmids and pCS2+ vector with an insert encoding only GFP were linearized and used for mRNA preparation with the mMessage mMachine mRNA synthesis kit from Ambion, Inc. (Austin, TX, USA). The mRNAs were diluted with Opti-MEM I medium (GIBCO) before using Lipofectamine 2000 (Invitrogen) to transfect them into myotubes that had been maintained in differentiation medium for 4 d; 1 µg mRNA was used per 3.5 cm dish of cells . Myotubes were incubated in transfection solutions for 6 h and then transferred back to differentiation medium before use in experiments.
Cortactin Knockdown by RNA Interference
A pool of four small interfering RNAs (siRNAs) targeting mouse cortactin and several other siRNAs against unrelated proteins were purchased from Dharmacon RNA Technologies (Lafayette, CO, USA); the siRNAs (100 pmol) were mixed with GFP cDNA (1 µg) and used for transfecting C2 myoblasts (with Lipofectamine 2000) grown in 3.5 cm culture dishes or on glass coverslips placed within the dishes. Transfection was carried out (for 6 h) when cells were ∼60% confluent, and the cells were then put back in growth medium to allow them to reach confluence before transferring them to differentiation medium . After 4 d myotubes were treated overnight with agrin and used for examining total cortactin expression (by immuno-blotting extracts prepared from myotubes growing in dishes) as well as GFP fluorescence and AChR clustering (after R-BTX labeling of cells growing on coverslips placed in the same dishes).
Co-Culturing of Xenopus Neurons with Muscle Cells Expressing Exogenous Proteins
To express GFP-tagged wild-type and mutant cortactin (or GFP) selectively in muscle cells, mRNAs encoding these proteins were injected into one cell of 2-4-cell stage Xenopus embryos; myotomal muscle cells were cultured from the embryos after they had developed to stage 20–22 . Muscle cells were maintained for 3–5 d and then spinal neurons from normal (uninjected) embryos were seeded on them and allowed to spread for 1 d.
Labeling, Microscopy and Quantification of AChR Clustering
AChRs on Xenopus muscle cells were labeled with R-BTX either before exposing cells to control medium or medium containing agrin or HB-GAM beads, or after innervation in co-culture experiments. To localize Arp2/3 complex proteins, phospho-cortactin, etc., cells were fixed with cold ethanol, blocked with PBS containing bovine serum albumin, and labeled with primary and fluorescent-secondary antibodies. Cells were examined with a 63X, 1.4 n.a. lens using an Olympus IX70 inverted microscope, equipped with a Hamamatsu ORCAII cooled-CCD camera controlled by MetaMorph software (Universal Imaging, West Chester, PA, USA).
To analyze AChR clustering in C2 myotubes, cells were treated with agrin overnight, labeled with R-BTX, fixed with 4% paraformaldehyde and then mounted on slides. All transfection assays were carried out multiple times with duplicate or triplicate samples and AChR clusters were examined in (only) transfected (green fluorescent) myotubes. After selecting transfected myotubes randomly, all AChR clusters on those myotubes were counted and their lengths were measured. Average numbers of AChR clusters per myotube and the average lengths of clusters were calculated and normalized relative to values obtained for clusters from cells expressing GFP only.
In Xenopus nerve-muscle co-cultures, effects of exogenous proteins on synaptic AChR clustering was quantified as follows: in R-BTX-labeled live cultures, nerve-muscle pairs were randomly identified (in phase-contrast) in which nerves directly contacted muscle cells; then, if the muscle cell was found to be green fluorescent (indicating the expression of GFP- or GFP-tagged cortactin proteins), we determined (using the rhodamine filter) whether or not AChRs were concentrated focally at nerve-muscle contacts. Co-cultures were prepared using muscle cells from several separate batches of mRNA-injected embryos and neurons from normal embryos and in each preparation 3–6 coverslips were examined. The number of nerve-muscle contacts and the number of contacts with AChR clusters were determined and the latter values were divided by the former; pooled data were normalized using numbers obtained from co-cultures in which muscle cells only expressed GFP.
We thank Drs. Tom Parsons and Xi Zhan for wild-type and phospho-mutant cortactin constructs and Ms. Frances Chan for excellent technical help.
Conceived and designed the experiments: RM ZLG BP. Performed the experiments: RM ZLG JJM AWSC. Analyzed the data: RM ZLG BP. Contributed reagents/materials/analysis tools: RM ZLG AWSC. Wrote the paper: RM ZLG BP.
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domain: medical


text:
director Ray Kellogg
In the 1950’s, following perhaps the success of Them! (1954), an explosion of giant animal monster movies hit the theaters. While Jack Arnold’s Tarantula (1955) turned out pretty good and The Black Scorpion (1957) has its merits, not every filmmaker had real effects talents at their fingertips, not that that would stop them.
Ray Kellogg made a couple of serious lulu’s in this genre, scraping the bottom of the animal barrel even before the barrel was even that heavily tapped. I kind of have a soft spot for his hilariously bad The Killer Shrews (also 1959), but The Giant Gila Monster is a zebra of a different stripe. In this case, the gila monster is played by a Mexican beaded lizard, but of course gila monster has “monster” baked in to its name already.
Don Sullivan plays Chase Winstead, our movie’s hero. A garage/tow-truck owner, beau of the local French beauty Lisa (Lisa Simone), hot rod mechanical magician, doting brother to his crippled sister (saving all his cash to buy her leg braces), would be crooner (playing a mini-banjo), and shoot, he’s even got a book on reptiles to reference!
The Mexican beaded lizard crawls around on its belly, occasionally shown wrecking miniature trains and cars and a barn, but it would take a better editor than they had on hand to make it look like anything but a lizard of its regular size pretending that it’s a giant.
The film has a wonderful lack of explanation for its size, something about the salts in the area that might have allowed it to have grown so. And the monster’s end is tipped early on. Like Chekov’s gun in an opening act, you know that when someone shows up with nitroglycerin in an early scene that we’ve been shown the means of the eventual monster’s demise.
It’s bad, and somewhat boring at times, and most importantly the monster is just a lizard (with an occasional shot of a claw coming at the camera). It’s pretty laughable, though, which could make for some fun. I don’t know…is this the worst “giant animal” monster movie out there?

domain: entertainment


text:
Miami has 13,704 residents. It is located in Ottawa county.
For Oklahoma residents, the average tuition for all Miami clinical research schools for the 2009 - 2010 school year was $1,737.00. For non-residents, the average tuition for Miami clinical research schools was $5,558.00.
The cost, on average, for books and supplies needed for clinical research schools in Miami is $850.00. Students who live on campus while attending a clinical research school in Miami, can expect to pay an average of $3,858.00 for room and board. Students who live at home can expect average costs to be $2,666.00. Students have a choice of one clinical research college in Miami to attend. Clinical Research programs in Miami reportedly graduated 6 students in the 2008 - 2009 school year.
|Profession||Average Salary||Number Employed in City|
|Medical and Clinical Laboratory Technologists||$52,550.00||130|
|Medical and Clinical Laboratory Technicians||$32,360.00||130|


text:
Liberal theology doesn’t draw a crowd. For the Colson Center, I’m John Stonestreet with The Point.
A recent survey by LGBT advocacy group Church Clarity, found that none of the 100 largest churches in America affirm same-sex unions. These churches represent over a million Christians, and show that despite a media and political class that’s mostly accepted so-called same-sex “marriage,” the biggest churches in this country are not raising the rainbow flag.
Mark Tooley of the Institute on Religion and Democracy says that while this doesn’t mean all megachurches necessarily have solid theology, it does show that twisting Scripture to suit the sexual revolution doesn’t fill the pews. This is especially clear when you contrast megachurches and conservative denominations—many of which are growing—with dying, mainline, liberal Protestant churches.
Now, the news isn’t all good. The survey also found that while these megachurches weren’t affirming of homosexual relationships, many have not made their position public, or clear. That’s a pity, because Christ was clear about the definition of marriage. And His bride should be too.

domain: politics


We may be screaming about discrimination between female and male actors in Bollywood, but TV has predominantly been a heroine medium. The bahu, beti and biwi characterisation has been the most important aspect of our small screen binging. We take a look at the actresses who have added spark to the stories….read on:
When she entered our living rooms as a typical Bharatiya naari with a touch of modern element, we fell in love with her. Despite being around for over a decade, she has not let her lustre fade away. Also, she can give the younger actresses a run for their money. Any. Damn. Day.
Whether it was a village belle role, a modern woman’s avatar or the girl-next-door, this pretty lady has aced every role. She has also tried her hands at dancing, thanks to Jhalak Dikla Jaa. Apparently, the gifted actress is all set to play the most important role of her life…yes, she plans to settle down in January!
The Bong beauty is the fantasy of many a youngsters, while she is considered an icon by girls all over the country. Her striking good looks and acting chops make her one of the most popular faces of the small screen, and rightly so.
One of the few actresses to make unconventional look sexy, Kritika has constantly delivered power-packed performances and upped her stake in the industry. It also helps that she is such a good looking girl! Her evergreen beauty will take her a long way for sure.
The gorgeous actress made a huge impact as Madhubala and followed it up in a number of amazing ventures. She might have married the love of her life, but that hasn’t stopped her from making it big in the industry now. We definitely want to see more of her because she is a damn good actress.
Which actress is your favorite? C’mon, tell us now!


text:
CALL FOR SUBMISSIONS
ISSUE 7, DECEMBER 2023
Men Matters Online Journal (MMOJ) is interested in creative writing, literary and scholarly work that focus on issues related to men. It provides a space where male concerns and issues are explored, examined and discussed. We welcome creative explorations of—and critical inquiries into—gender, masculinity, sexuality, and identity from any historical, geographical, cultural, political, religious, and institutional context.
This is an open call for submissions; contributors are invited to submit any work that falls within MMOJ’s scope of interest. We welcome contributions from authors of all gender identities.
July 1st to October 31st 2023
Publication date: Last week of December 2023
Further submission guidelines can be found below.
Up to 5 poems. There is no minimum or maximum word limit but poems that run up to a maximum of 40 lines are preferred.
Fiction and Creative Non-Fiction:
A single work, maximum of 10,000 words.
A complete or a stand-alone extract of a play. A single work, maximum of 9,000 words.
Scholarly and Literary Essays:
Author Guidelines for Scholarly and Literary Essays
When preparing your citations and formatting your reference entries, please adhere to the conventions established by the Publication Manual of the American Psychological Association, Seventh Edition (2020). For more information about APA-7, please refer to https://apastyle.apa.org/blog.
Critical essays should not exceed 6,000 words (excluding footnotes and references). Please submit your manuscript as a Word Document. The text of your work should be in Garamond, 12 point size. Manuscripts should have 1.5 spacing on A4-sized paper with margins of 1.25” (right and left) and 1” (top and bottom).
Please use British spelling, except in cited texts or quotations.
The title page of your essay should include the following information:
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Do use the Document Inspector to find and remove hidden data and personal information in your manuscript. Please include the following information in a separate Word Document:
- Author name, institutional affiliation and email address
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Do let us know which publication or film you wish to review and proceed if the Editors show interest in it. This does not guarantee publication. Maximum word limit is 1,000 words.
Please follow APA-7 conventions for any quotations or citations: https://apastyle.apa.org/style-grammar-guidelines/citations/quotations
Do let us know about your planned interview and proceed if the Editors show interest in it. This does not guarantee publication. Maximum word limit is 3,000 words.
Language: English, original and translated works.
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Submit your work through email: email@example.com In the email cover letter please indicate that this is an original and unpublished work that is being submitted.
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Simultaneous submissions are welcome. It is your responsibility to inform us in time when the work submitted here is accepted somewhere else.
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domain: literature


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January 22, 2019
By Jill Serjeant and Lisa Richwine
LOS ANGELES (Reuters) – Netflix movie “Roma” and British historical romp “The Favourite” from Fox Searchlight led nominations for the Oscars on Tuesday in a history-making lineup that left the best picture race wide open.
The lists for the highest honors in the movie industry were packed with people of color, including nominees from Mexico, Greece and Poland, as well as films featuring the stories of women and gay and black people.
Mexican director Alfonso Cuaron’s semiautobiographical, black-and-white tale “Roma” scored a first best picture nod for streaming service Netflix Inc. It will also compete in the foreign language film category, where it is widely considered a shoo-in..
“Roma” and “The Favourite” shared a leading 10 nods, followed by Lady Gaga musical “A Star is Born” and scathing political comedy “Vice” with eight apiece.
Walt Disney Co’s “Black Panther,” which collected seven nods, became the first superhero movie in the 91-year history of the Academy Awards to win a best picture nomination.
“There is now no clear front-runner for best picture, making it an enormously suspenseful Oscar race,” Tom O’Neil, founder of awards website GoldDerby.com said in a telephone interview.
Shot entirely in Spanish and an indigenous Mexican language, “Roma” scored nominations across the board, including director, lead actress Yalitza Aparicio as a maid, supporting actress Marina de Tavira, screenplay, and multiple technical fields.
“It’s a movie centered on a domestic worker of indigenous origin who’s being celebrated. That’s what gives me the most pleasure,” Cuaron told Mexico City local radio program “Aristegui Noticias.”
Aparicio, 25, who had never acted professionally, said she was “humbled and honored.”
“As a daughter of a domestic worker and an indigenous woman myself, I am proud this movie will help those of us who feel invisible be seen,” she said in a statement.
The Oscars, chosen by the 8,000 members of the Academy of Motion Picture Arts and Sciences, will be handed out in Hollywood on Feb. 24.
“The Favourite,” set in the court of 18th-century British monarch Queen Anne, garnered nods for its British star, Olivia Colman, and supporting actresses Emma Stone and Rachel Weisz. Greek director Yorgos Lanthimos was also nominated for the film.
“Yorgos has such a unique vision. He is very witty, he takes a lot of risks,” Ceci Dempsey, one of the film’s producers, said in a telephone interview. “It’s a remarkable feeling to have a film with so many women in it get so many nominations.”
Warner Bros. “A Star is Born,” led by best actress contender Lady Gaga in her debut in a major movie, earned eight nods, including for Gaga’s hit song “Shallow.” But while Bradley Cooper will compete in the best actor category, he was snubbed in the director’s race.
Other snubs included the all-Asian cast romantic comedy “Crazy Rich Asians,” which got nothing, “Black Panther” director Ryan Coogler, and “If Beale Street Could Talk,” which was excluded from the best picture race but won a supporting actress nod for Regina King.
Crowd-pleaser “Bohemian Rhapsody” was nominated for five awards, including Egyptian-American Rami Malek for his role as Queen front man Freddie Mercury, who died of AIDS in 1991.
Gay and lesbian campaign group GLAAD said another three of the eight best picture nominees touched on gay issues, reflecting what it said was “a banner year for LGBTQ inclusion in film.”
Veteran actress Glenn Close, who has yet to win an Oscar, will compete for the seventh time, this year for her lead role in “The Wife.”
“Green Book” about the friendship that develops between a black pianist and his white driver in the 1960s, won nods for stars Viggo Mortensen and Mahershala Ali, but not for director Peter Farrelly. Nevertheless, the film remains a strong best picture contender after a win at the Producers Guild Awards on Saturday.
The Oscar ceremony looks set to take place without a host after the withdrawal in December of comedian Kevin Hart due to past homophobic comments he made on Twitter.
(Reporting by Jill Serjeant and Lisa Richwine; additional reporting by Daina Beth Solomon and Miguel Angel Gutierrez in Mexico City; editing by Jonathan Oatis)