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Companies are finally moving away from the traditional workplace setup that centers around a corporate headquarters where employees gather on a daily basis and all the leadership presides. The COVID-19 pandemic changed everything. When employees were forced to adjust to working away from the office, some began to discover that their productivity relied less on where they work, but how they work.
Now, employers are looking at innovative workplace models that incorporate the new lessons learned during the pandemic. These innovative models include improved versions of prior workplace trends. Hub and spoke, for example, is a model used by many companies that includes one central office hub and smaller spoke or satellite offices. The latest iteration of this model is part of a growing group of hybrid workplace models designed to offer a physical workplace for those who want one, while also appealing to increasing employee demands for flexibility.
According to a recent survey of workers in India, employees are receptive to such models. Fifty-eight percent of participants in the survey expressed an interest in working from a nearby satellite location like a branch office or coworking space provided by their employer.
These satellite locations do not have to be a step down from the main HQ as they sometimes currently are. They can be a vibrant part of a network with key leadership present and a dynamic mix of teams collaborating in various combinations. They can function like mini headquarters, or hubquarters, and this is only the beginning. The future of work calls for an even more decentralized workplace that prioritizes collaboration regardless of location. Let's take a look at this model and what comes next.
A different kind of hybrid
In a recent survey, 70 percent of respondents said that team management, collaboration, and networking are more effective in an office environment. This report points to the importance of having a physical base for better communication, collaboration, transparency, branding, and shared work culture among employees.
Despite the benefits of the in-office work experience, however, survey after survey indicates more employees want to work in a hybrid workplace where they have more freedom over how often they work in the office and how they collaborate. As a result, employers are increasingly embracing the idea in order to attract and retain top talent. In one recent survey the majority of executives said they expect that employees will be on-site between 21 and 80 percent of the time, or one to four days per week.
Under a basic hybrid model, employees work in the office on certain days and remotely on others, which can either be regulated or autonomous. This can create a number of challenges, but specifically, hybrid models can leave a large office headquarters looking like a ghost town. The new network model eliminates this problem by transforming that headquarters into one of many hubquarters. The former headquarters size may shrink, but the hubquarters will offset that loss of space.
Hubquarters don't have to be formal offices either. They can be any remote location where employees gather or work independently. Employers can provide smaller office spaces or hubs, such as coworking spaces, where employees can come together to work, or they can work in multiple locations outside of the offices.
It's important to note that this hubquarters network will look different in each market. Established hubs like Silicon Valley and New York will remain an important center and new hubs of talent will emerge driven by the desire for a shorter commute and in-person collaboration.
Benefits of hubquarters
During the pandemic, one of the top reasons cited by employees as to why they preferred to work remotely was that they no longer had a grueling commute. The new office network model offers employees multiple options to work in-person with colleagues as well as independently closer to home or at home.
Tech giants such as Amazon opened offices in 17 North American cities with plans to open six more, which closely resembles the new hubquarters network model. Last year, Google executives said they were considering adding new, smaller hub offices to give employees more flexibility around where they live. And Fujitsu recently announced plans to reduce its office footprint by 50 percent while giving employees the option to work from home, a hub, or satellite offices closer to where they live. These are all examples of a network designed to give employees freedom to work where they choose to or need to.
Cost is a major factor leading some companies to adopt a network model. Increasing real estate costs and decreasing space in city centres are leading many companies to reassess traditional workplace models. Maintaining a large headquarters that is half occupied on most days prohibits many companies from having smaller satellite offices. With the network model, multiple locations are more possible.
Transitioning to a network model can also help a company reduce its carbon footprint. In addition to reduced emissions resulting from shorter commutes, hubquarters take up less space and thus require less energy use from appliances, air conditioning, and heating.
Though the hubquarters network model offers increased flexibility for workers, if it remains hindered by tradition and leadership sticks close to one hubquarters, then employees who find they work better outside of the hub have fewer opportunities for collaboration with leadership.
Collaboration has proven key to business success. A recent survey found that satisfied employees in the U.S. are twice as likely as unsatisfied employees to say that collaboration makes them feel more productive. Additionally, more than half of all "happy" employees collaborate with five or more people on any given day.
Collaboration doesn't have to happen in an office to be successful, however. The survey also reported that nearly 70 percent of those who described themselves as satisfied with their job indicated that they collaborate with people outside of their office at least once or twice a week. This demonstrates that collaboration can happen anywhere and shouldn't be reserved for hubs.
If the pandemic showed employees they can work from anywhere, then leadership needs to be more proactive about meeting employees where they are. Moving forward, employees need a workplace that operates more like a central nervous system with leadership that traverses the entire system, moving from hub to hub, and serving as the catalyst to get synapses firing, literally.
Need help finding a workplace model that works for your company? Contact us now.


The names aren't unique (e.g. "tar" is the name of both a lute and a drum). These could be handled using disambiguation comments.
There are other sites with information about instruments, such as Wikipedia. We could use instrument-URL relationships to link to these.
Instruments (as credited in liner notes) do not form a nice tree (e.g. fue in Japanese refers to a variety of different Japanese flutes so doesn't fit nicely anywhere). Instead of a tree, we could use relationships to link to parent/child instruments. Then "shinobue" could be a child of both transverse flutes and fue, "shakuhachi" could be a child of both end-blown flutes and fue.
People can't easily edit descriptions, only relationship editors can do that. Annotations would let more people write instrument descriptions.
It's not possible to find out which artists/releases/recordings are using an instrument without directly searching the database. The entity page would display this information, like labels display releases from that label.


The global and local challenges lead us to utilize projects in European and local level in order to respond to every kind of social challenge.
Participation to the second Global Congress of Roma women in Spain 2016 -2017: Representatives of A.I.D. will attend the pre-congress meeting in order to give their input on the topics that should be pointed to the official Congress. During the official Congress, A.I.D. will be one of the main representatives of Greece.
Study Visit on Chios and Mytiline during November 2015. During the study visit, two members visited the islands in order to identify the needs of refugees and the situstion. As a result based on the needs, A.I.D. motivated its members to select clothes and food in order to be sent to the hot-spots.
Study visit in refugees hosting center in the region of Grevena on 1 December 2016. Three members of A.I.D. visited the region of Grevena in order to identify the number of children of refugees that are being hosted and their needs.


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Home » News » Walt Disney World News This Week
Walt Disney World News This Week
POSTED: Wed, 01/10/2018 - 06:06 | POSTED BY: Kristen Kirk
Hey! It's been a while since we've had a news round-up around here, so let's take on the flurry information that has been swirling the internet about what's going on down at the Walt Disney World Resort. We've got notes on refurbishments and new resort systems being tested, the winner of the Walt Disney World Marathon, a brand new runDisney event, and the introduction of the 2018 Disney Moms Panel. So keep on reading to discover all that's happening!
Nick Hilton, Winner Of The 25th Walt Disney World Marathon
Fans that love to book their trips around Disney dining deals should take note that the 2018 Summer Quick Service Meal Offer is booking now! This special package includes each guest of your party to one FREE quick service meal (breakfast, lunch, or dinner) per day of stay, when booked at select Walt Disney World Resort hotels.
Due to some refurbishments on Main Street, be aware that the First Aid station has been relocated to Tomorrowland, next to the Space Mountain entrance. Guests who anticipate needing care should take note that this will be the location through February 16, 2018 before returning to its regular location.
Fish and Chips from Yorkshire County Fish Shop
Rumor has it that guests staying Disney's Polynesian Village and Grand Floridian Resorts may be included in an upcoming test of a new Bluetooth room entry program that would function through the MyDisneyExperience App on their cell phones. This program that can already be found at hotels outside of the Walt Disney World Resort would be another great addition to the mobile technology forward trend we've seen in recent years.
Several Walt Disney World fan sites reported today that Walt Disney World has announced that it will be closing the Children's Activity Centers located at Animal Kingdom Lodge, Disney's Polynesian Village Resort, and Disney's Yacht and Beach Club Resorts as of July 31st, 2018. It is thought that the closures are due to a lack of interest in the programs, at this time it is unclear if the children's activity center at the Walt Disney World Dolphin will continue to operate.
Kids Activity Centers To Close In 2018
runDisney added a new event for 2018 - the Star Wars Virtual Half Marathon! Bring the magic of Disney to your run by participating in this race on your home turf. Registration is open for the Star Wars Virtual Half Marathon from now until February 28. Half Marathon must be completed by March 31st.
Speaking of runDisney, this weekend's Walt Disney World Marathon was won by an American, for the first time since 2004. Congratulations to Nick Hilton on his smashing accomplishment!
2018 Disney Moms Panel Members
Disney announced the new group of men and women that will round out the 2018 Disney Parks Mom's Panel. This great group of volunteers is on hand to answer your questions about planning your upcoming Disney vacation at DisneyParksMomsPanel.com
Children's Activity Centers
Mom's Panel
Virtual Half Marathon
Walt Disney World Maratho
Walt Disney World Marathon
About WDW For Grownups
We're a website dedicated to adults who visit Disney World because they love it, not just because their kids love it. Join in the discussion on our forums or find us here:
Who's Going to Walt Disney World (or Disneyland) ***2020***
Gourmet Cotton Candy Flavors at Goofy's Candy Co.
Wine and Dine Half Marathon Weekend Medals Are Out!
Disney Cruise Line Wins Top Ocean Cruise Line in Travel + Leisure World's Best Awards
First Look At Bo Peep In Toy Story Land
Early Shoppers at DisneyStore.com Have The Best Selection
Sunny For Springtime Minnie Mouse Rocks The Dots
Don't Miss The DisneyStore.com Twice Upon A Year Sale

redpajama_set_name: RedPajamaCommonCrawl


Extremely rare natural red Mogok Spinel loose gemstone for sale, available in our online gemstone shop, this red burmese spinel is completely natural and untreated it comes from the ground, it was genuine earth mined in Mogok, Myanmar, this nice gemstone comes with an elegant cushion faceted cut with good polish and well proportioned size that gives to this gemstone sparkle and fire. This red burmese Spinel weight over one carat is completely untreated and present a nice deep red color hue with medium dark tone and good saturation, this gemstone has an awesome transparent eye clean clarity without visible inclusions into the crystal. Considered a durable stone with its hardness of 8 on the Mohs scale, this valuable burmese spinel, offered at wholesale price is perfect for jewelry.
Dimensions: 5.7 X 5.6 X 3.7 mm.


Cloud atop his Pryor Mountain home in early July of 2013. In the process his mane flew up elegantly into the air! This museum quality, limited-edition print comes signed, numbered and matted. Matted size 12 x 14.


Music Studio 2 is available on the Apple App Store and the Google Play Store.
There is also a free Lite version for you to try some of the features. Please note that the Android version has a slightly reduced feature set compared to the iOS version.
Share option "Open with" changed to "Apps & more"


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Q: Formula based field in account instead of Roll-Up Summary Currently we are having (Opportunities(won)last 12 months) field which is of Roll-up summary data type , updating the count in this field on criteria when
Close date of opportunity >= (some date).
There is no way to give Value as TODAY or any inbuilt value. Instead of roll-up summary type can we use formula based field to count the closed won opportunity in account and update the field.
If possible could you please provide us the formula..
Thanks,
Chithra

A: Using a formula field to count related records is not possible.
You have a few options:

*

*Trigger to roll up in real time

*Batch job to perform rollup

*DLRS Package (Declarative Lookup Roll Up Summary)

Option 3 being the quickest route but be aware there were/are issues with some bulk operation with too many SOQL queries. Not sure if it is still there or not. For most cases it works well.
Or Option 4:

*

*Create a Checkbox field to indicate if in the last 12 months

*Create a WFR to update the checkbox field to true or false based on the formula field

*Create a batch job that sets the value of the checkbox daily

*Use the checkbox as the criteria of the rollup summary

Option 3 is still the fastest but those are your options AFAIK

redpajama_set_name: RedPajamaStackExchange


This W-2 scam has cropped up all across the country, targeting arts organizations and schools as well as businesses. The San Antonio Symphony, a school district in Illinois and an Indiana brewpub are among its other recent victims. The N.C. Department of Justice put out an alert about it earlier this month.
According to the alert, the state A.G.'s office had received reports of 18 such phishing breaches since the beginning of the year.
The emails typically appear to come from the organization's CEO or another top executive.
After the data leak, Macdonald said the symphony reported it to the Internal Revenue Service as well as the N.C. Department of Justice.


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Александер () — сільський муніципалітет в Канаді, у провінції Манітоба.

Населення
За даними перепису 2016 року, сільський муніципалітет нараховував 3333 жителів, показавши зростання на 11,7%, порівняно з 2011-м роком. Середня густина населення становила 2,1 осіб/км².

З офіційних мов обидвома одночасно володіли 510 жителів, тільки англійською — 2 815. Усього 270 осіб вважали рідною мовою не одну з офіційних, з них 20 — одну з корінних мов, а 50 — українську.

Працездатне населення становило 50,7% усього населення, рівень безробіття — 10,1% (13,1% серед чоловіків та 7,6% серед жінок). 82,8% були найманими працівниками, 15,8% — самозайнятими.

Середній дохід на особу становив $41 059 (медіана $34 608), при цьому для чоловіків — $48 869, а для жінок $33 144 (медіани — $42 470 та $27 904 відповідно).

30,8% мешканців мали закінчену шкільну освіту, не мали закінченої шкільної освіти — 22,6%, 46,8% мали післяшкільну освіту, з яких 22,3% мали диплом бакалавра, або вищий, 10 осіб мали вчений ступінь.

Населені пункти

До складу сільського муніципалітету входить містечко Паверв'ю-Пайн-Фоллс, а також хутори, інші малі населені пункти та розосереджені поселення.

Клімат
Середня річна температура становить 2°C, середня максимальна – 22,8°C, а середня мінімальна – -24,5°C. Середня річна кількість опадів – 501 мм.

Примітки

Населені пункти Манітоби

redpajama_set_name: RedPajamaWikipedia


The generation of cellular microtubules is initiated at specific sites such as the centrosome and the Golgi apparatus that contain nucleation complexes rich in γ-tubulin. The microtubule growing plus-ends are stabilized by plus-end tracking proteins (+TIPs), mainly EB1 and associated proteins. Myomegalin was identified as a centrosome/Golgi protein associated with cyclic nucleotide phosphodiesterase. We show here that Myomegalin exists as several isoforms. We characterize two of them. One isoform, CM-MMG, harbors a conserved domain (CM1), recently described as a nucleation activator, and is related to a family of γ-tubulin binding proteins, which includes Drosophila centrosomin. It localizes at the centrosome and at the cis-Golgi in an AKAP450-dependent manner. It recruits γ-tubulin nucleating complexes and promotes microtubule nucleation. The second isoform, EB-MMG, is devoid of CM1 domain and has a unique N-terminus with potential EB1-binding sites. It localizes at the cis-Golgi and can localize to microtubule plus-ends. EB-MMG binds EB1 and affects its loading on microtubules and microtubule growth. Depletion of Myomegalin by small interfering RNA delays microtubule growth from the centrosome and Golgi apparatus, and decreases directional migration of RPE1 cells. In conclusion, the Myomegalin gene encodes different isoforms that regulate microtubules. At least two of these have different roles, demonstrating a previously unknown mechanism to control microtubules in vertebrate cells.
The interphase microtubule (MT) cytoskeleton regulates cell morphology and motility, organelle transport and localization, and vesicular trafficking. Nascent MTs are formed from nucleation centers containing γ-tubulin ring complexes (γ-TuRCs). Subsequently, MTs grow by tubulin dimer polymerization at their plus-end (Kollman et al., 2011). The latter process is tightly regulated and requires plus-end stabilization by interacting proteins, the best characterized being end-binding (EB) proteins, mainly EB1 (Akhmanova and Steinmetz, 2008). The major MT nucleation center in vertebrate cells is the centrosome, also known as MTOC (microtubule organizing center), but other organelles such as the nucleus and the Golgi apparatus (GA) can concentrate MT-nucleating activities at their surface (Bartolini and Gundersen, 2006; Bornens, 2002; Chabin-Brion et al., 2001). The function of MTs is strongly linked to their capacity to bridge different cellular compartments at their minus- and plus-ends, such as the centrosome and the cell cortex, to organize directional movements and cell polarity.
In many eucaryotes, proteins related to Drosophila centrosomin bind and regulate subcellular localization of γ-tubulin, the major component of γ-TuRCs (Fong et al., 2008; Sawin et al., 2004; Zekert et al., 2010; Zhang and Megraw, 2007). Centrosomin regulates the recruitment of γ-tubulin to mitotic centrosomes, the formation of astral MTs and the proper orientation of mitotic spindles (Megraw et al., 2001). In fission yeast, Pcp1 and Mto1P are related proteins with similar functions that recruit γ-tubulin to spindle pole body (the equivalent of the centrosome in yeast) and non-spindle pole body associated MTOCs, respectively (Samejima et al., 2008; Sawin et al., 2004; Venkatram et al., 2004). In this protein family, Aspergillus nidulans AspB (Zekert et al., 2010) and mammalian CDK5RAP2/CEP215 (Fong et al., 2008) also associate with γ-tubulin to promote MT nucleation from cytoplasmic sites and centrosomes, respectively. All these proteins are large coiled-coil proteins with a small (around 60 amino acids long) N-terminal conserved domain known as the centrosomin motif 1 (CM1) (Samejima et al., 2008; Zhang and Megraw, 2007). Interestingly, the CM1 domain in centrosomin is required for γ-tubulin, D-TACC and Msps recruitment to centrosomes, but not of other centrosomal proteins such as Aurora-A and Map60 (Zhang and Megraw, 2007). In addition, a recent study has demonstrated that the CM1 domain of CDK5RAP2 is able to bind γ-TuRCs and enhance its ability to nucleate MTs (Choi et al., 2010). This motif was therefore named γ-TuNA (γ-TuRC-mediated nucleation activator).
Myomegalin/PDE4DIP is a CDK5RAP2 paralog in vertebrates. Myomegalin is highly expressed in muscle tissue and its product has been described as an interactor of phosphodiesterase 4D, an enzyme controling cAMP level (Taskén et al., 2001; Verde et al., 2001). In some mammalian cells, Myomegalin localizes to both the GA and the centrosome (Verde et al., 2001), but its function is currently unknown. Other proteins, such as CAP350 and AKAP450 (also known as AKAP9 or CG-NAP) localize at the centrosome and GA. CAP350 participates in MT anchoring at the centrosome and may stabilize MTs in the GA area to maintain its pericentrosomal structure (Hoppeler-Lebel et al., 2007). AKAP450, a γ-tubulin-interacting protein, in addition to its role as a kinase-anchoring scaffold protein at the centrosome, is also important for MT nucleation from the centrosome and GA, and for GA assembly (Hurtado et al., 2011; Takahashi et al., 1999; Takahashi et al., 2002). The GA and the centrosome cooperate in different cellular processes such as cell polarity, cell migration and ciliogenesis (Bisel et al., 2008; Follit et al., 2006; Hurtado et al., 2011; Magdalena et al., 2003; Marie et al., 2009; Sütterlin and Colanzi, 2010). Interestingly, activation of CDC42 at the GA regulates centrosome organization and function, and depends on the GA matrix protein GM130 (Kodani et al., 2009; Kodani and Sütterlin, 2008). Another GA matrix protein, GRASP65, also regulate centrosomes during mitosis (Sütterlin et al., 2005). In addition to the centrosome, the GA can be a potent MT-organizing organelle (Chabin-Brion et al., 2001; Efimov et al., 2007). Centrosome and GA-derived MTs cooperate for different functions such as proper ribbon formation and polarization during GA assembly (Vinogradova et al., 2012). The molecular machinery underlying the ability of the GA to organize MTs has begun to be identified (Efimov et al., 2007; Hurtado et al., 2011; Kim et al., 2007; Rivero et al., 2009). It includes AKAP450 and CLASP, a MT plus-end binding protein. AKAP450 is recruited to cis-Golgi membranes via GM130, and CLASP is anchored to the trans-Golgi membranes via GCC185.
In this study, we show that Myomegalin is a centrosome and cis-Golgi protein required for MT growth from the centrosome and GA. We also show that Myomegalin exists as several isoforms that participate by different means in MT regulation. We describe the role of two of them. The first isoform, CM-MMG, regulates MT nucleation via an interaction with the γ-TuRCs. The second isoform, EB-MMG, regulates MT dynamics via an interaction with the plus-end tracking protein (+TIP) EB1.
Immortalized human retinal pigment epithelial cells htert-RPE1 (thereafter referred as RPE1) were grown in DMEM/F12 (Life Technologies) supplemented with 10% fetal calf serum (heat inactivated 30 minutes at 56°C).
A rabbit polyclonal antibody (Ab#1) against Myomegalin was raised against the fragment 322–939 of clone IMAGE n° 4813248, fused to GST. The fusion protein was injected in rabbits and the serum was depleted in anti-GST immunoglobulins by affinity column. The specificity of this antibody was assessed by immunofluorescence (IF) and Western blotting (Fig. 1; supplementary material Fig. S1).
Fig. 1. The human Myomegalin gene encodes various isoforms.
(A) The Myomegalin gene is composed of at least 55 exons depicted as boxes. The 5′ and 3′ untranslated regions of exons are shown in gray and white, respectively. According to the Ensembl database, the alternative use of exons produces at least 8 protein isoforms depicted below the gene. Gaps in the representation of isoform 1 result from the use of alternative exon 29a and lack of exon 37 use. Dotted lines show the correspondence between initiating or terminating exons and the N and C-termini, respectively. The various isoforms are encoded by a first exon (gray/black), all the following full black exons, and a last exon (black/white). The two black boxes on isoforms at the N and C-termini depict the conserved CM1 and CM2 domains, respectively. As shown, the N-terminus of isoform 6 is encoded by a single specific exon. The positions of peptides used for antibody production and of sequences corresponding to siRNA are indicated. (B) Various Myomegalin isoforms are expressed in RPE1 cells. RPE1 cells were treated for 48 hours with the indicated siRNA and subsequently lysed in 1% Igepal-CA630-containing buffer. 50 µg of proteins from the lysates were separated by SDS-PAGE 4–12%, transferred onto nitrocellulose and immunoblotted with anti-Myomegalin antibodies Ab#1, Ab#2 or Ab#3. Bands indicated by arrows are identified according to their size and sensitivity to siRNA treatment.
Rabbit polyclonal antibody against Myomegalin (HPA008162, denoted Ab#3 in this study) was from Sigma and mouse monoclonal antibody against Myomegalin was from Abnova (M01, clone 2B5, denoted Ab#2 in this study). For Golgi compartment identification, GM130 antibody was from Abcam (rabbit monoclonal) or from BD Transduction Laboratories (mouse monoclonal), TGN46 from AbD serotec (sheep polyclonal). MTs were stained with a rat monoclonal YL1/2 antibody (Abcam). Anti-EB1 antibodies were from BD transduction Laboratories (mouse monoclonal), and Santa Cruz Biotech (rat monoclonal). Anti-γ-tubulin antibodies were from Sigma–Aldrich (mouse monoclonal GTU-88 and rabbit polyclonal T3559). Anti-GCP2 and NEDD1 were from Sigma–Aldrich and Novus Biological, respectively. Rabbit and mouse anti-AKAP450 were from Bethyl Laboratories and BD Biosciences, respectively. Mouse anti-c-Myc (9E10) was from Santa Cruz Biotechnology. Anti-GFP antibodies were purchased from Abcam (rabbit polyclonal) and Roche (mouse monoclonal).
For MT depolymerisation and/or Golgi dispersal, nocodazole (10 µM) or BFA (brefeldin-A) (5 µg/ml) was added to the culture medium for 2 hours. For dynein inhibition experiments, cells were transfected with a myc-p50 dynamitin coding expression plasmid.
Full-length Myomegalin cDNAs (KIAA0477/EB-MMG and KIAA0454/CM-MMG) were obtained from the Kazusa DNA Research Institute. The sequence coding for the full-length and truncated proteins were amplified by PCR with Gateway compatible primers using Platinum HiFi polymerase (Invitrogen) and transferred into pDONR/ZEO by Gateway® recombination. The sequences were subsequently transferred by Gateway® recombination into modified Gateway-compatible pRK5-Myc, pEGFP-C1 and pmCherry-C1 expression vectors. A pcDNA3/γ-tubulin coding vector was a gift from Alexey Khodjakov (Wadsworth Center, Albany, NY).
For plasmids, cells were transfected with Fugene6 (Roche Applied Science) according to manufacturer's protocol.
Single siRNA oligonucleotides or different combinations of mixed siRNA oligonucleotides against Myomegalin were transfected into cells using Oligofectamin (Life Technologies) according to the manufacturer's protocol. The sequences are the following: siRNA#1 (5′-CUAACGAGCUGGAGAAAUA-3′), siRNA#2 (5′-GAAGGGAAUAGUAAACUUA-3′), siRNA#3 (5′-AGAGCGAGAUCAUGACUUA-3′), siRNA#5 (5′-CAAGAAGAAUUGCAGAAUA-3′), siRNA#7 (5′-AGAGGAAGCCCUUGGAGAA-3′). In all the experiments, a second transfection was performed at 48 hours and experiments were conducted 72 hours after the first transfection. This protocol gave the best reduction of protein expression. To deplete GM130 or AKAP450 we used a mixture of two siRNA. The sequences are as described previously (Rivero et al., 2009). To deplete EB1 we used siRNA (5′-UUGCCUUGAAGAAAGUGAA-3′). A non-targeting siRNA or siRNA targeting GAPDH were used for controls. All siRNA were purchased from Dharmacon.
Cells were grown on coverslips, fixed in cold methanol (6–8 minutes at −20°C), saturated for 30 minutes in PBS 3% BSA and incubated with the appropriate mixture of primary antibodies for 60 minutes at room temperature. After washing in PBS 0.1% tween, they were incubated for another hour with the adequate secondary antibodies conjugated to cy2, cy3 or cy5 (Jackson laboratories) to which was added 1 µg/ml DAPI for DNA staining, rinsed and mounted in Prolong Gold anti-fade reagent (Life Technologies). Confocal images were acquired by capturing Z-series with 0.3 µm step size on a Zeiss LSM 510 laser scanning confocal microscope.
RPE1 cells were cultivated on coverslips and treated with appropriate siRNA for 72 hours before the addition of 1 µg/ml of nocodazole to the medium for 2 hours at 37°C followed by a 30-minute incubation on ice to depolymerize MTs. For repolymerization, coverslips were washed with medium and incubated 1, 3 or 5 minutes in warm medium. Before fixation, cells were permeabilized for 15 seconds in PHEM (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 4 mM MgSO4, pH 7.0) with 0.25% Triton and 320 mM sucrose. One volume of cold methanol was added and left for an additional 45 seconds before transferring cells to pure cold methanol for 4 minutes on ice. IF was then done as usual.
For time-lapse imaging, RPE1 EB1-GFP stable transfected cells were placed in 35 mm glass based dishes (Iwaki) and transfected as previously described with plasmid encoding mCherry-EB-MMG or mCherry-CM-MMG for 24 hours. Microscopy was carried out using a Zeiss LSM 510 laser scanning confocal microscope with a 63× oil lens. Images were taken every 2 seconds over 400-second periods. To determine EB1 velocity MT ends were tracked using the "track points" function of the MetaMorph software.
3×104 RPE1 cells were cultivated in 6 well plates and treated with appropriate siRNA as usual. The day before filming, cells were split in 6 well plates coated with collagen (25 µg/ml in 0.2% acetic acid) to be able to film individual cells. Phase contrast images were acquired every 2 minutes on a Zeiss Axiovert 200 microscope equipped with a thermocontroled chamber with a 10×/0.30 Zeiss objective for 4 hours. The velocity, the distance from start to end point – as a straight line – (D) and the total track distance (T) were measured in Image J with the manual tracking plugin. Directionality was calculated as the ratio D/T. Statistical analysis of the results was done with the GraphPad Prism software.
Myc and/or GFP-tagged constructs were transfected in RPE1 cells. Twenty four hours later, cells were washed in PBS and lysed in 1 ml of buffer containing 20 mM TRIS, 2 mM EDTA, 150 mM NaCl, 1% NP40 and a complete protease inhibitor cocktail (Roche). Lysates were collected, incubated on ice for 15 minutes and centrifuged for 30 minutes at 4°C at 13,000 g. Fifty µl were saved as input material. 10 µl anti-Myc conjugated sepharose beads (Santa Cruz Biotechnology) or anti-GFP conjugated sepharose beads (Abcam) were incubated for 2 hours at 4°C. Control immunoprecipitation was performed with lysates of untransfected cells. Beads were washed 3 times in lysis buffer, suspended in Laemmli sample buffer and boiled. Samples were electrophoresed on NuPAGE Novex Bis-Tris minigels (Life Technologies) and proteins were detected by Western blot.
The human Myomegalin gene (PDE4DIP) encompasses 224 kb on chromosome 1q12. In the Ensembl database it is predicted to encode 24 isoforms, 8 of which have a strong support from transcript sequences. These 8 isoforms originate from the use of common and alternative exons, leading to proteins with differences in their N-terminus, C-terminus and internal sequences (Fig. 1A). Seven of these isoforms have a conserved CM1 domain at their N-terminus, known to bind γ-TuRC (Choi et al., 2010). The three longer isoforms have a conserved CM2 domain at their C-terminus, characterized as a centrosome/Golgi targeting motif in CDK5RAP2 (Wang et al., 2010).
We first analyzed which isoforms are expressed in RPE1 cells using three different antibodies, and three siRNAs (Fig. 1B). Of the four major protein bands detected by Ab#2, two were consistent in apparent MW with isoforms 001/002/003 above 247 kDa and 004/005 around 120 kDa. The inhibitory effects of pooled or single siRNA confirmed this prediction, as only siRNA#3 inhibited the expression of the ∼120 kDa isoforms, while all 3 siRNA inhibited the large ones. Two other major bands around 80 and 30 kDa were also observed; they do not correspond to predicted isoforms, and were not inhibited by siRNA, possibly reflecting non-specific antibody reactivity.
As expected, Ab#3 detected protein products corresponding to the same large (above 240 kDa) and ∼120 kDa isoforms, albeit with a lower sensitivity than Ab#2. This lower sensitivity may account for the absence of residual large isoform in cells treated with siRNA#2, as seen with Ab#2. Interestingly, Ab#3 recognized a unique 140 kDa isoform not detected by Ab#2, and only inhibited by siRNA#3, which likely corresponds to isoform 006. This isoform lacks a CM1-containing N-terminus and has a unique C-terminus encoded by a unique alternative exon. A minor band corresponding to a protein product of 70 kDa is likely to be nonspecific for the same reasons as mentioned above for Ab#2. Ab#1 detected high molecular weight proteins very similar to Ab#2. Bands detected below 107 kDa are likely to be nonspecific as they do not correspond to any predicted Myomegalin products and are not inhibited by siRNAs.
RPE1 therefore express a complex pattern of different isoforms, which probably exert different functions within the cell.
We determined the subcellular localization of Myomegalin in RPE1 cells by IF. Ab#2 and Ab#3 antibodies were not used in these experiments because i) Ab#2 showed weak cross-reactivity with the human Myomegalin paralog CDK5RAP2 at the centrosome and ii) Ab#3 gave a strong centrosomal and cytoplasmic staining that was not specific as it was not inhibited by any Myomegalin-specific siRNA either used alone or pooled. A rabbit polyclonal antibody (Ab#1) raised against a CM1-containing fragment was the most reliable and specific reagent for IF experiments. Immunostaining with this antibody indicated that Myomegalin localizes at the centrosome, the GA and punctually in the cytoplasm (Fig. 2A). A similar staining has been described in Cos-7 cells and in FRTL-5 thyroid cells (Verde et al., 2001). However, we noticed that the GA staining in RPE1 cells was present in only 20 to 30% of proliferating cells as compared to 70 to 80% of resting cells. Consequently, subsequent experiments were done with overnight serum-starved cells. In these conditions, we observed that siRNA#1 and siRNA#3 were the most efficient at inhibiting Myomegalin expression at the GA but did not induce complete depletion at the centrosome (data not shown). The decrease in Myomegalin-specific staining by siRNA#2 was weak, as expected from Western blot experiments (data not shown; Fig. 1B). However, pooled siRNA (#1+#3) showed the strongest inhibitory effect and were used in subsequent experiments (supplementary material Fig. S1A,B). Of note, the GA showed enlargement and partial fragmentation upon Myomegalin depletion (supplementary material Fig. S1B). The IF results obtained with Ab#1 were confirmed with Ab#2, with the exception of a stronger centrosome staining probably due to the weak cross reactivity of the antibody with CDK5RAP2 (data not shown).
Fig. 2. Localization of Myomegalin at the centrosome and GA.
(A) Resting RPE1 cells were fixed in cold methanol and stained with rabbit anti-Myomegalin Ab#1 (Red) and mouse anti-GM130 (Green) (Upper panel) or mouse anti-γ-tubulin (Green) (Lower panel). DNA was stained with DAPI (Blue). Bars, 10 µm. (B) RPE1 cells were stained with mouse anti-GM130 (Green), rabbit anti-Myomegalin (Red) and sheep anti-TGN46 (Blue) (Upper images; bar, 5µm). A representative fluorescence density profile of the section indicated on the merge image is shown (Lower left). Pearson's correlation coefficients were calculated from triple labeling experiments. Values are the means ± s.d. of 36 measures (6 measures performed at random positions in 6 different cells) (Lower right).
Our results show that RPE1 cells express CM1-containing isoforms of Myomegalin at the GA and centrosomes. However, the localization of endogenous isoform 006 was not addressed in these experiments as it is not recognized by Ab#1.
To more precisely determine Myomegalin localization at the GA, RPE1 cells were labeled with anti-Myomegalin antibody together with antibodies directed against GM130/golgin95/GOLGA2 and TGN46/TGOLN2 to label the cis- and trans-regions of the GA, respectively. We observed an extensive colocalization of Myomegalin with GM130 at the cis-Golgi cisternae (Fig. 2B). TGN46-labeled structures were close but distinct from anti-Myomegalin staining (Fig. 2B).
We next studied the fate of Myomegalin during GA fragmentation. First, we examined the effect of a treatment with BFA or nocodazole. These drugs disrupt the structural integrity of the GA. They respectively block the traffic from the ER to the GA and depolymerize MTs, and cause Golgi fragmentation into small punctate remnants. After 30 minutes of BFA or 3 hours of nocodazole treatment, Myomegalin was found associated with GA remnants distributed throughout the cytoplasm, indicating that the localization of Myomegalin to the GA is independent of the integrity of this organelle. Like in intact GA, the cis-Golgi marker GM130 showed partial colocalization with Myomegalin (supplementary material Fig. S2A). Because nocodazole depolymerizes MTs, this result further indicated that Myomegalin is associated to the GA in a MT-independent manner. Second, we followed the fate of Myomegalin in cells overexpressing the p50 subunit of dynactin to inhibit dynein function, which causes fragmentation of the GA (Corthésy-Theulaz et al., 1992). Myomegalin association to GA ministacks persisted upon dynein inhibition (supplementary material Fig. S2A). Myomegalin thus localizes to the cis-Golgi independently of MTs and Golgi structure.
The human Myomegalin paralog CDK5RAP2 localizes to the GA in an AKAP450-dependent manner (Wang et al., 2010). Similarly, we could show that Myomegalin colocalizes with AKAP450 and depends on this protein for GA localization (supplementary material Fig. S2B,C). Interestingly, Myomegalin depletion altered localization of AKAP450 at the GA, which showed a more diffuse staining pattern in the cytoplasm and an impaired colocalization with GM130 compared to control cells. Myomegalin may thus control to some extent AKAP450 localization and/or stability (supplementary material Fig. S2D). As expected from the dependence of AKAP450 on GM130 for its presence at the GA (Rivero et al., 2009), GM130 depletion also prevented localization of Myomegalin on this organelle (supplementary material Fig. S3).
To better determine which isoforms are present at the GA and centrosome, we expressed full length and truncated GFP-tagged isoforms. We concentrated on isoforms 001 and 006 as representatives of two strikingly different forms of Myomegalin. We named isoform 001 CM-MMG because it has a CM1 and a CM2 domains and isoform 006 EB-MMG because, as detailed below, it has a unique tandem of putative EB1 binding motifs (Fig. 3A). At low expression levels, the two full length isoforms localized at the GA in close association with the cis-Golgi protein GM130 (Fig. 3B), but of the two only the CM-MMG isoform was detected at the centrosome. Both N and C-terminal CM-MMG truncated proteins localized at the centrosome but only the C-terminal part of CM-MMG localized at the GA. Strikingly, EB-MMG was addressed to the GA through its N-terminus, indicating that the two isoforms have two different, non-overlapping, Golgi-targeting regions (Fig. 3B,C).
Fig. 3. Different regions are required to address the CM-MMG and EB-MMG isoforms to the GA and/or centrosome.
(A) Schematic representation of the two Myomegalin isoforms and truncated proteins used in our study. Red boxes indicate coiled-coil regions and the unique sequence at the N-terminus of EB-MMG is shown in heavy gray, encompassing two putative EB1 binding motifs. A recapitulation of the localization experiments shown in panel B is presented on the right (Ct, centrosome). (B) The various GFP-tagged constructs were transfected in RPE1 cells. Cells were fixed by cold methanol at 24 hours post-transfection and labeled with mouse anti-GFP antibody (Green), rabbit anti-GM130 (Red) and rat-anti-FOR20 (Magenta) antibodies to label the cis-Golgi and centrosome, respectively. Centrosomes are indicated by arrows when colocalization is observed. Bars, 10 µm. (C) RPE1 cells were transfected with expression vectors coding for GPF-tagged CM-MMG_C and Myc-tagged EB-MMG_N. Cells were fixed and labeled with mouse anti-GFP (Green) and rabbit anti-Myc (Red) antibodies. DNA was stained with DAPI. Images show a single (left) and double (right) transfected cell. At the bottom is a magnification of the GA region of the double-transfected cell. Bar, 10 µm.
Human CDK5RAP2 and Myomegalin orthologs in various species, like Drosophila centrosomin, are known to associate with and to anchor γ-TuRCs MT nucleating complexes (Fong et al., 2008; Sawin et al., 2004; Zhang and Megraw, 2007). More importantly, the conserved CM1 domain not only binds γ-TuRCs directly but also stimulates its MT nucleation activity (Choi et al., 2010).
When overexpressed in RPE1 cells, four out of the six MMG constructs formed dense aggregates. In IF, aggregates formed by CM-MMG or its N-terminal part (CM-MMG_N), which has the CM1 domain, strongly recruited the γ-TuRCs components γ-tubulin (Fig. 4A), GCP2 (supplementary material Fig. S4B) and the associated protein NEDD1 (supplementary material Fig. S4A). In contrast, aggregates formed by EB-MMG_N, which is devoid of the CM1 domain, did not recruit γ-TuRCs (Fig. 4A). However, it was unique in its capacity to concentrate pericentrin (supplementary material Fig. S4C). The CM-containing Myomegalin isoforms are thus likely to bind γ-TuRCs through their CM1 domain, like other members of the same family. Surprisingly, we could not demonstrate specific binding between γ-tubulin and CM-MMG or CM-MMG_N. However, we found that a CM1-containing fragment, but not a fragment lacking the C-terminus of CM1 (Fig. 3A, CM1 and CM1ΔC), specifically associated with endogenous and ectopic GFP-tagged γ-tubulin (Fig. 4B).
Fig. 4. CM-MMG and γ-tubulin.
(A) Asynchronous RPE1 cells were transfected with GFP-tagged wild-type and truncated Myomegalin EB-MMG and CM-MMG coding constructs and fixed in cold methanol 24 hours post transfection. Cells were labeled with mouse anti-GFP (Green) and rabbit anti-γ-tubulin (Red). DNA was stained with DAPI (Blue). Bars, 10 µm. (B) RPE1 cells (Left panel) or RPE1 cells stably expressing GFP-γ-tubulin (Right panel) were transfected to express the Myc-tagged CM1 and CM1ΔC constructs (Fig. 3A). Cells were lysed 24 hours post-transfection and tagged proteins were precipitated with anti-Myc conjugated agarose beads. Immunoprecipitates were separated by SDS-PAGE, transferred onto nitrocellulose and immunoblotted with the indicated antibodies.
Next, we asked whether the aggregates of CM-MMG and CM-MMG_N associated with γ-TuRCs possess MT nucleation capacity. When we depolymerized MTs in transfected cells and subsequently allowed them to regrow, MT asters formed around nearly all CM-MMG and CM-MMG_N, but not control CM-MMG_C, aggregates (Fig. 5A). Strikingly, we observed that many CM-MMG_N associated asters also colocalized with anti-GM130 labeled GA remnants (Fig. 5B), suggesting that Myomegalin may participate in the regulation of MT production from the GA.
Fig. 5. CM-MMG promotes MT nucleation.
(A) Asynchronous RPE1 cells were transfected with GFP-tagged wild type CM-MMG and truncated forms and cells were subjected to a MT regrowth assay 24 hours post transfection. MTs were depolymerized with a high dose of nocodazole and a cold treatment. After the drug washout and transfer to 37°C, MTs were allowed to grow for 1 minute. Cells were fixed and labeled with mouse anti-α-tubulin (Red) and rabbit anti-γ-tubulin (Magenta). DNA was stained with DAPI (Blue). Bars, 10 µm. (B) RPE1 cells expressing GFP-CM-MMG were treated as in panel A and labeled with-α-tubulin (Red) and rabbit anti-GM130 (Magenta). The insert show a magnification of the indicated region of the merge image. Bar, 10 µm.
To test our hypothesis that Myomegalin regulates MT growth from the GA and/or the centrosome, MTs were depolymerized and allowed to regrow for various lengths of time in control and Myomegalin siRNA-treated RPE1 cells. α-tubulin staining after 1 minute of regrowth revealed numerous non-centrosomal MTs in addition to the centrosomal asters in control cells. These non-centrosomal MTs were associated with the dispersed Golgi remnants, as already described (Efimov et al., 2007; Rivero et al., 2009), to which Myomegalin was associated (Fig. 6A). Myomegalin siRNA-treated cells showed a striking difference with control cells in the formation of centrosomal and non-centrosomal MTs (Fig. 6B). After 1 minute regrowth, the aster size of Myomegalin siRNA-treated cells was diminished at the centrosome (1.35±0.39 µm versus 0.65±0.20 µm for control and Myomegalin siRNA, respectively) (Fig. 6C) and almost totally abrogated at the Golgi remnants. After 5 minutes, the difference at the centrosome was even higher with 7.85±2.60 µm and 1.61±0.71 µm for control and Myomegalin siRNA, respectively (Fig. 6C). However, a seemingly normal array of cytoplasmic MTs was recovered at later time points. Thus, MT regrowth is delayed in the absence of Myomegalin.
Fig. 6. Myomegalin is necessary for MT nucleation from the centrosome and GA.
(A) Control siRNA-transfected RPE1 cells were starved overnight and subjected to a MT regrowth assay. MTs were depolymerized with a high dose of nocodazole and a cold treatment. After the drug washout and transfer to 37°C, MTs were allowed to grow for 1 minute. Cells were then fixed and labeled with sheep anti-α-tubulin (Green), rabbit anti-Myomegalin Ab#1 (Red) and mouse anti-GM130 (Magenta) antibodies. DNA was stained with DAPI (Blue). Tubulin labeling was converted in negative grayscale to better visualize MT asters (Middle image) and a selected region was magnified (Rightmost images) to show Myomegalin (Upper image) and GM130 (Lower image) associated with asters. Bars, 10 µm. (B) Cells transfected with control or Myomegalin siRNA were treated as in panel A and MT growth was allowed for 1 and 5 minutes before fixation and staining as in panel A. Bars, 10 µm. (C) The sizes of centrosomal asters were measured in at least 30 cells for each experimental condition. The histogram displays the mean value and bars indicate SEM. P values compared with the control were calculated using unpaired Student's t-tests.
The unique sequence found at the N-terminus of Myomegalin isoform 006/EB-MMG is conserved in vertebrates. It is encoded by a single exon and does not show predicted domains. However, examination of the human sequence revealed three putative EB-1 binding motifs SxI/LP. These motifs are known to bind the end binding homology (EBH) domain of EB1 in +TIP binding partners such as CLASP, APC and CDK5RAP2 (Honnappa et al., 2009) (supplementary material Fig. S5). Validated binding motifs are found within basic serine/proline rich sequence regions. Site prediction using the Eukaryotic Linear Motif (ELM) resource confirmed that the second and third motifs of the 006/EB-MMG isoform are likely to constitute EB1 binding sites. The second motif is the best conserved in different species.
We found that EB-MMG associates with EB1 by co-immunoprecipitation in a cell line expressing GFP-EB1 and transfected with Myc-tagged full length or truncated constructs of EB-MMG. As expected, only EB-MMG and EB-MMG_N, which have the putative EB1 binding sites, but not EB-MMG_C, which does not, were able to bind EB1 (Fig. 6A). CM-MMG did not coprecipitate with EB1 (data not shown).
In RPE1 cells with moderate levels of overexpressed EB-MMG, the latter colocalized with EB1 at MT plus-ends (Fig. 7B), suggesting that EB-MMG may have a role in MT growth and dynamics. To test this hypothesis, we first analyzed the effect of overexpressing this isoform on the localization of EB1 in RPE1 cells. We observed a striking reduction of endogenous EB1 at MT plus ends in cells overexpressing EB-MMG but not CM-MMG (Fig. 7C,D; data not shown). We also looked at EB1 localization in RPE1 cells stably expressing ectopic GFP-EB1. In this cell line overexpressed EB1 labeled MTs along their entire length, as described previously by others (Askham et al., 2002; Ligon et al., 2003). Overexpressed EB-MMG strongly displaced EB1 from MTs to a diffuse cytoplasmic and nuclear localization identical to EB-MMG staining in these cells. This effect was not observed with CM-MMG (data not shown). These observations suggest that overexpressed EB-MMG may negatively regulate MT growth by trapping EB1 away from MT plus-ends. This was indeed the case in a MT regrowth assay where MT aster size was diminished by 50% in EB-MMG overexpressing cells compared to control cells (Fig. 6E). In addition, we used videomicroscopy to measure the velocity of GFP-EB1 comets in control and EB-MMG or CM-MMG overexpressing cells. This velocity was diminished by ∼30% in EB-MMG but not CM-MMG cells (Fig. 7F).
Fig. 7. EB-MMG isoform associates with EB1 and regulates MTs.
(A) RPE1 cells stably expressing GFP-EB1 were transfected to express Myc-tagged full length EB-MMG or EB-MMG N or C-terminal parts. Cells were lysed 24 hours after transfection and lysed in 1% Igepal-CA630-containing buffer. Myc-tagged proteins were isolated with anti-Myc-conjugated agarose beads. Immunoprecipitates and lysate fractions were separated by SDS-PAGE, transferred onto nitrocellulose membranes and immunoblotted with the indicated antibodies. (B) Asynchronous RPE1 cells expressing at a moderate level mCherry-EB-MMG were fixed with cold methanol 24 hours post transfection and labeled with mouse anti-EB1 (Green) and rat anti-mCherry (Red) antibodies. Magnified images of the framed region are shown to better visualize comets at MTs plus ends. Bar, 10 µm. (C) Asynchronous RPE1 cells (Upper panel) or RPE1 cells stably expressing GFP-EB1 at a high level (Lower panel) were transfected with a plasmid coding for Myc-EB-MMG and fixed with cold methanol 24 hours post transfection. Fixed cells were labeled with rabbit anti-Myc antibody (Red) and mouse anti-EB1 or mouse anti-GFP (Green) antibodies. Bar, 10 µm. (D) The number of EB1 comets was scored in 6 different cells in each condition (i.e. in cells expressing Myc-tagged Myomegalin constructs or not). Results are represented as the mean ± SEM. P values compared with the control were calculated using unpaired Student's t-tests. (E) We assayed MT regrowth in control and EB-MMG-overexpressing cells. The sizes of asters emanating from the centrosomes were measured at 1 minute. Results of a representative experiment out of three are shown as mean ± SEM (Control: n = 49, EB-MMG: n = 16). (F) We determined the velocity of GFP-EB1 comets at the plus-ends of growing MTs in control cells and EB-MMG or CM-MMG overexpressing cells. Results are the means ± s.d. of 60 to 160 measures performed on 11 control cells and 6 EB-MMG or CM-MMG overexpressing cells from two different experiments.
Finally, Myomegalin depletion by siRNA treatment did not alter EB1 localization at MTs plus-ends. In the reverse experiment, Myomegalin still localized at the GA and centrosome when EB1 was depleted (supplementary material Fig. S6).
Golgi-derived MTs are important for cell polarization and cell motility (Efimov et al., 2007; Miller et al., 2009; Nakano et al., 2010; Rivero et al., 2009). Because decreased Myomegalin expression affects MTs originating from the GA in RPE1 cells we wondered whether cell mobility was altered. We analyzed the individual movements of RPE1 cells seeded at low concentration (to prevent alteration of trajectories or speed by one cell encountering another one on its way). We tracked individual cells by time-lapse microscopy to analyze both the direction of the cell movement and the distance run by cells. We used two different siRNA pools targeting similar regions of Myomegalin mRNA, and thus likely to target the same Myomegalin isoforms (Fig. 1A). Control RPE1 cells filmed for 4 hours usually moved in a directionally persistent fashion. Cells treated with siRNA directed against Myomegalin ran shorter distances or were even immobilized, probably according to the level of the knock-down obtained with the siRNAs. The decrease in the distance run by cells was due to a reduced velocity but not to a change in migration directionality (measured by the ratio of the distance between start and finish point divided by the total track distance) (Fig. 8A,B). Values are probably underestimated as RNAi efficiency is not absolute (especially for the #1+#3 siRNA pool).
Fig. 8. Cell migration defects in Myomegalin-depleted cells.
Control and Myomegalin siRNA-treated cells (treated with two different pools) were plated on collagen-coated plates and imaged every 2 minutes for 4 hours. (A) Representative examples of cell trajectories in each condition. Bar, 50 µm. (B) Migration speed of control and Myomegalin siRNA treated cells and directionality of the migration. Directionality is the ratio of direct distance from start to end point divided by the total track distance. Results of a representative experiment out of three (control: n = 17, Myomegalin #1+#3 n = 18, Myomegalin #5+#7 n = 19). Histograms display the mean value and bars indicate SEM. P values compared with the control were calculated using unpaired Student's t-tests.
In conclusion, Myomegalin depletion affected cell motility but not migration directionality, which suggests that cell polarization was not affected. This is reminiscent of the role of AKAP450 in migration (Rivero et al., 2009). We propose that reduced MT polymerization (by reduction of MT nucleation and/or loss of EB1 from MT +ends) is responsible for the slower migration in cells with reduced Myomegalin expression.
The consensus coding sequences (CCDS) project has identified eight CCDS in the human Myomegalin/PDE4DIP gene, which are consistently annotated and of high quality. Most protein variability is found at the N and C-terminus. In both cases, a specific transcript is likely to be obtained from the initiation or termination of transcription downstream of alternative promoters and polyadenylation sites, respectively. Four putative promoters and three polyadenylation sites may thus account for part of the isoform diversity. Further complexity stems from alternative splicing events, such as those regulating the use of exons 29 and 37. Our study shows that a subset of the isoforms encoded by these CCDS is expressed in RPE1 cells. Centrosomin, the Drosophila ortholog of Myomegalin, acts during oogenesis and embryogenesis as a complex mix of isoforms to allow centrosome function (Eisman et al., 2009). We found centrosomin isoforms related to human CM-MMG (i.e. possessing a CM1 domain at their N-terminus) but none related to EB-MMG. Different isoforms can be present in a single cell or can be differentially regulated among various tissues or developmental stages to exert different functions (Bourdon et al., 2005; Pan et al., 2008; Wang et al., 2008). At least three Myomegalin isoforms are expressed in RPE1 cells, indicating that their functions are not exclusive but that they work together in independent or coordinated cellular processes, as we discuss below. There are rare cases in which two isoforms produced from one gene display alternative sequences at a similar location in the protein, conferring two different properties or function. Such exon switching is observed for example in the FGFR2 and calcitonin/CRGP genes, allowing the production of receptors binding different ligands and two different tissue-specific hormones sharing their N-terminus, respectively (Rosenfeld et al., 1983; Yan et al., 1993). The two different N-termini of Myomegalin (possessing or not a CM1 domain) confer to the protein the capacity to bind either γ-TuRCs (with CM1) or EB1 (without CM1). However, the mechanism responsible for the production of Myomegalin isoforms with or without a CM1 domain at the N-terminus does not involve alternative splicing but rather alternative promoters. Myomegalin orthologs from other species and vertebrate paralog CDK5RAP2 are related to the CM-MMG isoform. Interestingly, all vertebrate species have a Myomegalin gene that encodes the two isoforms with unique N-termini. It is therefore likely that the two different functions of Myomegalin, associated with the two types of isoforms, cannot work independently.
Myomegalin isoforms detected in RPE1 cells localize at the centrosome and the cis-Golgi. We found that, more specifically, the CM-MMG isoform (with conserved CM1 and CM2 domains) is present at both locations, whereas EB-MMG isoform is only detected at the cis-Golgi. The CM2 domain directs CDK5RAP2 to the centrosome and GA in a pericentrin and/or AKAP450-dependent manner (Wang et al., 2010). The preserved localization of the CM-MMG_C truncation mutant at the two organelles is consistent with a similar role of the CM2 domain in Myomegalin. The CM-MMG_N mutant, which is not present at the GA, is consistently localized at the centrosome (Fig. 3B; data not shown). This is possibly the result of an interaction of the Myomegalin CM1 domain with γ-TuRCs anchored at the centrosome through interactions with pericentrin, AKAP450 and/or CDK5RAP2 (Fong et al., 2008; Takahashi et al., 2002). Our study also underlines the central role of AKAP450 as a scaffold to recruit proteins important for MT nucleation at the GA, such as CDK5RAP2 and Myomegalin.
Surprisingly, EB-MMG is targeted to the GA through its N-terminus, suggesting a second, CM2-independent targeting mode. The colocalization of non-overlapping CM-MMG and EB-MMG truncated forms (CM-MMG_C and EB-MMG_N) at the GA (Fig. 3C) indicates that heterodimerization is an unlikely mechanism to explain this observation. Supporting this, CM-MMG_C, but not EB-MMG_N, localizes at the centrosome.
We have demonstrated that inhibiting Myomegalin expression leads to decreased MT growth from the centrosome and the GA during the recovery phase following nocodazole treatment. This is consistent with the localization of Myomegalin at these two organelles (this study; Verde et al., 2001), and with the recruitment of γ-TuRCs by Myomegalin in humans and related proteins in various species (Fong et al., 2008; Sawin et al., 2004; Terada et al., 2003; Veith et al., 2005). Nucleation at a specific site relies on γ-TuRC anchoring by large scaffolding proteins such as pericentrin, AKAP450 or CEP192. Recent studies suggest that it also depends on the nucleation-activating role of Myomegalin-related proteins such as CDK5RAP2 in humans and SPC110 in budding yeast (Choi et al., 2010; Kollman et al., 2011). Indeed, the two latter proteins are direct interactors of γ-TuRC and γ-TuSC (γ-tubulin small complex) respectively. They may help position γ-tubulin molecules at a distance compatible with tubulin protofilaments arrangement in the MT lattice (Kollman et al., 2011). Myomegalin does not seem to participate in γ-tubulin anchoring on interphase centrosomes (supplementary material Fig. S1B); it may instead play a nucleation-activating role at the centrosome and possibly at the GA. In contrast, CDK5RAP2 is required for both γ-TuRC anchoring and activation at the centrosome, because centrosomal γ-tubulin is greatly diminished following CDK5RAP2 depletion (Fong et al., 2008). It will be of interest to determine to what extent CDK5RAP2 and Myomegalin are redundant or complementary at the centrosome. Myomegalin may also play a role similar to other centrosomal proteins such as CEP70 and Centrobin, which regulate MT elongation and/or stabilization, but are not involved in centrosomal anchoring of γ-tubulin (Lee et al., 2010; Shi et al., 2011; Shi et al., 2012).
The molecular bases of the MT organizing activity of the GA only begin to be delineated. Clearly, it requires a GM130-AKAP450-γ-TuRC complex at the cis-Golgi and a GCC185-CLASP complex at the trans-Golgi (Efimov et al., 2007; Rivero et al., 2009). We found that aggregates of the overexpressed CM-MMG isoform promote MT nucleation at sites associated with the cis-Golgi protein GM130 and AKAP450, but not pericentrin. This suggests that a GM130-AKAP450-Myomegalin-γ-TuRC complex functions in MT nucleation at the cis-side of the GA. CDK5RAP2 was also found at the GA and may play a redundant or complementary role together with Myomegalin (Barr et al., 2010; Wang et al., 2010). Indeed, MT nucleation was delayed but not abolished following siRNA-mediated downregulation of Myomegalin. A defect in Golgi-associated MT organization impairs Golgi polarization and directional migration in RPE1 cells (Miller et al., 2009). The defect in cell migration that we observed after inhibiting Myomegalin expression suggests that the MT cytoskeleton and/or Golgi organization is not fully functional in the absence of Myomegalin.
We found that a specific Myomegalin isoform (EB-MMG) may regulate MT growth and organization through its direct interaction with the +TIP EB1 proteins. A number of +TIP protein bind MT ends indirectly through an interaction between the EBH C-terminal domain of EB1 and an SxI/LP motif in the +TIP (Honnappa et al., 2009). EB-MMG has tandem SxLP motifs, with the N-terminal one being highly conserved in vertebrate species. The sequence context of these motifs matches the basic serine/proline-rich consensus as defined in the Eukaryotic Linear Motif (ELM) resource for a functional EB1 binding motif. Such motifs are present in MT stabilizing (CLASP, APC) and destabilizing (MCAK) +TIPs. These motifs are likely to drive the binding of EB-MMG to EB1 we observed. Interestingly the interaction of overexpressed EB-MMG with EB1 resulted in a depletion of EB1 from MT plus-ends. MT dynamics was affected, at least using GFP-EB1 velocity measurements as a readout. To what extent this isoform was required for MT regulation in our MT regrowth assay is also not known, since we used siRNAs to downregulate most isoforms. Future experiments will require EB-MMG-specific antibodies and siRNA to confirm localization of the endogenous protein at the GA and possibly at plus-ends and to delineate its specific role in MT regulation. Similarly, we cannot exclude that depletion of the short CM1-containing isoforms 004 and 005 contributes to the phenotypes we observed. It is tempting to speculate that EB1, recruited at the GA in an EB-MMG-dependent manner, participates to the stabilization of MTs growing from the cis-Golgi, together with CLASP subsequently recruited at growing plus-ends at the trans-Golgi. EB-MMG at the Golgi surface may serve as a docking site for MTs plus-ends important for Golgi structure and/or positioning. In addition, it is possible that a cytoplasmic EB-MMG pool associates with EB1 to regulate EB1 availability or stability.
CDK5RAP2 exists as two isoforms resembling the CM-MMG isoform and also binds EB1 through an SxLP motif (Fong et al., 2009). The role of this interaction is still unclear because CDK5RAP2 inhibition only slightly affects MT dynamics and the SxLP motif in CDK5RAP2 is not conserved in all vertebrate species. We can hypothesize that in complex vertebrate organisms, MT nucleation and EB1-mediated stabilization activities must localize in very close proximity to ensure efficient and coordinated MT growth. This may be the case at the GA, but this will have to be substantiated by demonstrating the presence of EB1 (or its relative EB3) at the surface of this organelle. It is the case at the centrosome where γ-TuRC nucleation complexes are concentrated, EB1 is recruited by a CAP350-FOP complex (Yan et al., 2006). CDK5RAP2, but not Myomegalin, may recruit additional EB molecules at this site.
CDK5RAP2 alterations are associated with mitotic defects such as the formation of anastral spindles, impaired centrosomal attachment to spindle poles, abnormal mitotic spindle pole number and mitotic orientation (Barr et al., 2010; Barrera et al., 2010; Fong et al., 2008; Lizarraga et al., 2010). Mitotic Myomegalin-depleted RPE1 cells displayed normal levels of γ-tubulin, CDK5RAP2 and NEDD1 associated with spindle poles, and did not have an increased mitotic index suggestive of defects in mitosis (data not shown). CDK5RAP2 may compensate for the absence of Myomegalin in mitotic cells, but a detailed analysis of spindle structure/orientation and chromosome segregation in different cell type will be necessary to clarify the role of Myomegalin during mitosis.
In conclusion, our work establishes a role of Myomegalin in MT regulation. Future experiments are now required to clarify the specific functions exerted by the two Myomegalin isoforms at the cellular and molecular levels, and to identify the molecular complexes associated with these isoforms to perform their roles in a space and time-regulated manner. How these roles in MT regulation are integrated with the signaling activity of Myomegalin as an A-kinase anchoring and phosphodiesterase-associated protein will also be a matter of interest (Uys et al., 2011; Verde et al., 2001).
We thank A. Khodjakov for the kind gift of reagents and F. Gergely for comments on the manuscript. This work was supported by INSERM and Institut Paoli-Calmettes. F.S. was a recipient of a fellowship from the Ministère de la Recherche et de l'Enseignement Supérieur.
(2001). The Golgi complex is a microtubule-organizing organelle. Mol. Biol. Cell 12, 2047–2060.


IN APRIL 2015 we launched Beacon in the Dominican Republic in partnership with the local fire department, el Cuerpo de Bomberos Manzanillo, led by Colonel Ramon Soriano. Over the past year, the Colonel has taken charge of a group of local volunteers, provided training, structure and leadership, and has used our software, Beacon, to strengthen and expand their emergency services to neighboring communities. Last week they responded to their 100th request for emergency assistance, and the timing couldn't have been better. The 100th response came shortly after Cardinal Health and the Bindu Project donated together another $20,000 to our program, (their second round of support!), and just as we've been putting the finishing touches on our final report for the Google Impact grant ending this month — only a week before the ceremony we're having in the D.R. to celebrate the expansion of the program to new communities. (If you're interested in joining us, you're invited! — see details below).
Another interesting occurrence this month was the beginning of Marko's emergency medicine internship — at the same hospital that's now receiving patients transported via the EMS system he built. It's hard to say if you find a better example of local ownership than that; there's no doubt that the Mwanza program is in good hands and with a very bright future. The same can be said for Colonel Ramon's work in the D.R., and in both cases we're more excited than ever to be supporting these volunteers however we can to help them strengthen their own services. We're seeing better than ever that it doesn't take much to stand up a new quality program, get some big wins and lay the foundation to keep things moving forward.
To be sure, we're by no means trying to say that these programs are meeting the full demand for emergency care that exists across their communities — far from it — but, as we hope these initial statistics will start to show, the money we're spending is being well spent. Because of your support, local volunteers are providing life-saving emergency services and always eager for more, while the communities are now able to call for help when it's never been reliable. Marko told me that in Mwanza the fire department is getting more respect now because the community's seeing them out on the streets, responding to calls and providing help more frequently. This is inspiring for the firefighters, giving them more respect in the community and motivating them to give more at their job to the community. It's really gratifying to see. And it makes complete sense. I can't tell you how many times I've heard from the locals in different countries the same complaints: "The firefighters are lazy." "The Red Cross doesn't care." But it's not true! Those aren't the problems, those are symptoms of deeper root problems: the firefighters and community health workers simply haven't had the tools they need to do the job they're expected to do. That can be really, really self-defeating.
But now we know all of this can change. We've seen what it takes to get something started, and we're confident that once the stake has been planted it won't removed so long as the system is built using resources that are taken from within the community. Manzanillo and Mwanza are both proving how it can happen.
With these success in mind, and some preliminary data out now out for you to see, I'd like to you ask to please continue supporting us and the inspiring work our partner communities are doing by making a contribution at trekmedics.org/donate/ — every bit truly helps!
If you'd like to learn more about our work and how we approach EMS systems development in resource-limited communities, I had a great time sharing with our friends at DisasterPodcast.com — click here to listen to part 1 of the interview.


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Upgrade your Halloween decorating with AtmosFX digital decorations! Transform any any environment or surface into a frightfully entertaining experience using a digital projector, TV, or monitor. Bone Chillers includes digital decoration scenes appealing all ages, with a mix of scenes both scary or silly, in display mode options to suit a variety of different uses. Prepare for a chilling fright as skeletons rise from their graves to startle unsuspecting visitors with spine-tingling scares. Or, have a bone-rattling good time with an ensemble of family-friendly skeletons that dance, play music, and perform silly slapstick vignettes that are sure to leave you in stitches. AtmosFX DVDs are intended for private, home use. Commercial use of these decorations requires a special licensing agreement from AtmosFX.


text:
"The Natural" (PG) (1984) starring Robert Redford
"He lived for a dream that wouldn't die."
We rated this film 4 stars!!! Baseball movie lovers would agree. This is an excellent movie to watch and with a PG rating. It has so many great actors in this picture, Robert Redford, Robert Duvall, Glenn Close, Kim Basinger.
This film received 4 Academy Award Nominations:
Best Supporting Actress: Glenn Close
Best Music: Randy Newman
(The following information is from the DVD movie jacket and inserts:)
"This is a film filled with magic, wonder and awe." Los Angeles Times
Director Barry Lavinson and an all-star cast including Robert Redford, Glenn Close, Kim Basinger and Barbara Hershey deliver a "sport film which projects with understanding the mythical qualities that Americans associate with baseball." The Motion Picture Guide
Pulitzer Prize-winning author Bernard Malamud's first novel, The Natural, was published in 1952. An allegory in the guise of a baseball story, the book was considered unfilmable, too difficult to translate into a workable screenplay. In 1976 Malcolm Kahn and Rovert Dean decided to take a chance and acquired the film rights. A screenplay was written by Phil Dusenberry, but the project remained in limbo until 1979, when Columbia Pictures' story editor Roger Towne was asked to try his hand at adapting the fable. It would the firs to several drafts.
Over the next few years, many actors – including Jon Voight, Michael Douglas and Nick Nolte – expressed interest in the project, but no deal was ever consummated.
THE NATURAL finally came together with the formation of TriStar Pictures in the early '80s. The new studio sent Towne's third draft to Robert Redford, who had not made a movie since directing Ordinary People (1980). Impressed, the actor brought the script to Barry Levinson (Diner), who agreed to direct Redford in the film.
As the start date neared, Levinson and his crew began searching the country for a sutable ballpark that resembled a 1930's stadium. Eventually, the found what they were looking for in Buffalo, New York's War Memorial Stadium, built between 1935 and 1937 as a New Deal relief project, and the then-current home of the Buffalo Bisons. Beginning with the start of production on August 1, 1983, it also became home to the New York Knights.
Buffalo also became home to THE NATURAL. Originally, the filmmakers planned to shoot sonly the baseball sequences in the city, with the remainder of the picture to be made in Hollywood. Their plans soon changed.
Patrick Markey, production executive: "Buffalo was a city of great wealth in the 20's and early 30's, and much of its architecture is beautiful. We kept finding places that were perfect for our script."
Production designer Mel Bourne discovered the Masten Street Armory, a huge structure tha would not only house the film's production offices, but would also become the home to an editing room, wardrobe department, casting department and a sound stage that contained four sets: the knight's locker room, the Judge's office, Max Marcy's press box and Iris' apartment. Other locations included the Buffalo Psychiatric Center (Roy's hospital), the Grand Court of the Elliot Square Office Building (a Chicago hotel lobby) and the Parkside Candy Store (a Chicago ice cream pallor). The Hobbs farm and Iris' house were found in nearby Stafford New York. The carnival sequence, where Roy strikes out "The Slammer", was filmed in the South Dayton. Production in Buffalo was completed in mid-October; cast and crew returned to the Laird Studios in Culver City, California where the remaining scenes were shot.
THE NATURAL opened on May 11, 1984, grossed nearly $50 million and received four Academy Awards Nominations:
A mythic look at the American dream, THE NATURAL has been interpreted on many levels, from a sports film to a warning on the dangers of success. But perhaps it's truer meaning lies on a deeper, more existential plane of thought.
= = = = = = =
Comment by Roger Towne, the film's screenplay writer:
"Even the most gifted and most natural people are hurt by the capriciousness of fate. Yet they are often given a second chance to make up for the past. This is what The Natural is about."


text:
28th June 2018, 14:36 | Community
All-new Girls Hubs off to a flying start
The all-new Rugby Football League Girls Talent Hubs kicked off on Monday night, with 144 girls across u14s-u16s age groups involved.
The four-week skills programme sees sessions held with volunteer Rugby League coaches across three venues each Monday until July 16.
Sessions are held at Cowley College (St Helens), Tong (Bradford) and Featherstone, and players in attendance have the opportunity to develop their skills with the aim of creating future Women's Super League and England stars.
England internationals will visit the youngsters across the four-week process, which will end with a 9s Tournament to put their skills into action.
The sessions are aimed at improving Rugby League skills
Former England captain Natalie Gilmour MBE hands out tips at Featherstone
144 youngsters will be put through their paces over four weeks
Current England stars will be dropping in over the coming weeks
Phil Jones, National Player Development Manager at the Rugby Football League, said: "It's an incredibly exciting time for women's Rugby League in this country, and we are delighted to be able to offer more training and exposure to Rugby League to our most promising young players.
"The four-week programme aims to improve their skills as we look to prepare the next generation of talent for the Women's Super League and England team."
Lights, camera, action! Community Rugby League in the spotlight in 2021
Praise for Rugby League's pandemic response
therfl
Hull FC Community Foundation launch 'Connected By Rugby'


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Paris Gare du Nord: a long-awaited expansion
French national rail operator SNCF announced a massive expansion project at Paris Gare du Nord. Adele Berti looks at the plan, which will treble the station's capacity to accommodate an extra 100,000 passengers a day, just in time for the 2024 Olympic Games.
Gare du Nord station is one of Paris' most iconic symbols and a crucial connection point for rail travel in Europe, especially thanks to the Eurostar service connecting the French capital to the UK and the Netherlands.
Originally built in 1846, Gare du Nord has experienced increasing traffic over the past decades and is now the busiest railway station in Europe, able to accommodate around 700,000 people every day.
But the station has been struggling with capacity issues for a while. The flow of travellers is forecast to increase to unprecedented levels in the years leading up to and during the summer of the 2024 Olympic Games, when an extra 100,000 people are expected to use the station on a daily basis.
This led French national rail operator Société Nationale des Chemins de Fer Français (SNCF) to kick off a six-year-long project to revamp the station in time for the event. The proposed transformation of Gare du Nord will see the hub treble its size and acquire a new, more modern look that better fits its role for the European city of Paris.
Working in partnership with contractor Ceetrus, SNCF's Gares et Connexions division will draw its inspiration from London's St Pancras International and aims to turn Gare du Nord into a 'symbolic station of the 21st century', able to accommodate as many as 900,000 people every day.
According to the operator, the revamped station will feature an enlarged departure hall, an extended Eurostar terminal, major accessibility improvements and a new station front on Rue du Faubourg-Saint-Denis. The works are estimated to cost around €600m and should be completed by mid-2023.
A much needed expansion
Gare du Nord underwent its biggest renovation between 1861 and 1864, at a time when trains started becoming the main means of transport for the French population.
Works were carried out under the supervision of Baron Georges-Eugène Haussmann, who was also responsible for the urban renewal programme which gave Paris the appearance that has remained almost completely unchanged to the present day.
However, legend has it that a dispute between Haussmann and the designated architect Jacques Ignace Hittorff led to parts of the operation to be called off. After the station's iconic façade reached completion, Hittorff's plan to build a large avenue linking the station to the city centre was cancelled, and Gare du Nord was left with the poor access and lack of space that linger to these days.
Over 150 years later, the station will undergo its much awaited expansion. The public walkways will more than double, with the floor area planned to increase from 15,000m2 to more than 37,000m2. The overall surface of the station will be expanded from 36,000m2 to 110,000m2.
The Eurostar terminal will be larger, as SNCF aims to improve how border controls are handled in the aftermath of Brexit. Major accessibility improvements will lead to a redesigned bus station and more parking for bicycles.
Paying tribute to 19th century Paris
Early designs by two different groups of architects, Valode et Pistre and Walmotte, show that the new Gare du Nord will be a tribute to Haussman's era of urban redevelopment. This will be translated into a reinterpretation of the new departure terminal in 19th century style, featuring an 18m-wide gallery and a 300m-long passageway covered by a glazed roof.
The interior façade of the hall will also be restored to its original state, while the space inside the station will be destined for commercial, cultural and co-working activities.
This area will be called 'Transilien' as it provides access to the Transilien platform, as well as the Eurostar, TGV and RER.
Image courtesy of SNCF - Gares and connexions
Investing in the environment
The revamped Gare du Nord will bid to set new environmentally-friendly standards thanks to the introduction of 7,000m2 of public green areas, ecological energy production and 1,200 parking spaces for bikes.
SNCF is also planning to make space for solar panels spread over 3,200m2. The fourth and fifth floors of the station will be turned into a public garden offering spectacular views over Montmartre.
According to the operator, the entire renovation project will be carried out with particular attention to the neighbouring area. Within this framework, SNCF will put in place mechanisms to preserve air quality and install acoustic screens to keep noise pollution down to a minimum.
A sense of community for the neighbourhood
During a press conference held in July, rail bosses reiterated that the ultimate goal is to create a station inside the city and a city inside the station. In practical terms, this means that Gare du Nord will be used to create a sense of community for the neighbourhood, serving as a meeting point rather than a mere intersection between two destinations.
This concept will translate into building an area dedicated to sports, a move that will illustrate 'the role of urban sports in people's lives' and promote social activities.
Possibly the most interesting part of this plan is a 1km-long trail track – the first of its kind in the world – that allows for jogging on the station roof next to the public gardens.
In addition, SNCF is planning to build a basketball court, a golfing ground, and areas to practice paddle tennis, while dedicated cloakrooms and changing rooms will enable passengers to safely put their belongings away and shower.
About 27% of the new space will be destined for restaurants and 37% for boutique shops, in keeping with the operator's plan to integrate the station into the rest of the city. A theatre, a co-working space and a nursery will also be built in and around the station.
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