You know it's a good day when you head out to an old building that has been bought and renovated and they just, "want the stuff outta here". Musical words to my ears. I couldn't resist taking a peek at all they had to offer. Of course, this vintage Art Deco waterfall hope chest caught my eye and I had to bring it home.
To say I'm excited for the young couple I got the chest from is an understatement. They purchased a downtown building in my city that has seen many days and ugly updates. However, they are renovating it back to the original glory it once was. We're talking uncovering the pressed tin ceiling hiding behind a drop ceiling, old wood floorboards revealed under carpet, and so much more. I wanted to do a happy dance! It makes my heart happy when others see past the obvious to the beauty beyond. Count me first in line to see it in the after.
I came home with many treasures from this place and will show them all to you in time. Today, I'm focusing on this waterfall hope chest. It had wear to the top with missing veneer, water rings, dents, scratches, etc. Nothing too hard to handle.
The interior was immaculate and has a beautiful cedar lining. I just love the smell of cedar, and wood in general. Do you ever pick up raw wood just to smell it. Stop and try it sometime – bliss, I tell ya.
The lock was missing part of its components, but that's fine with me because I have to remove it for resell anyway due to recalls, etc. To get this makeover started, I first grabbed some veneer remnants from my stash that I salvaged off of past projects. It's hard to find good veneer nowadays so I keep all I can. My stash scrap was adhered to other layers of veneer, so I soaked it in water and separated the layers.
While doing that, I chiseled out the damaged section on the chest top to make it square to receive a repair patch. After many attempts, I finally got an exact piece to fit and it looks good as new.
Next up, I removed the lock set and filled it with a piece of dowel using wood glue . I cut it to length, used wood filler to fill in any gaps.
Then, I used my orbital sander and my Corner Cat to knock down the worn finish on the top and on the sides to prepare it for the next steps.
Before starting any finish work, I decided to add a few decorative strips to the front to add dimension. I simply cut them to length, and attach with wood glue and few brads using my brad nailer . I love that thing! No compressor needed, easy to use, and so fast. BAM!
Once prepped, the top got a deep new color that I've loved to use in the past with an Espresso Stain . It give the wood such richness and I like it better than the orangey feel the wood had prior.
I applied the stain, let rest for 5 minutes and then wiped off with a lint-free cloth. I let the stain cure for about 30 minutes, and then applied two coats Dixie Belle Satin topcoat using my Palm Pro, making sure to let the coats cure for 2 hours in between.
For the base of the waterfall hope chest, I chose these two complimentary colors: Putty and Dropcloth. You can see how I used these colors in my faux brick treatment in my kitchen, and how Dropcloth is one of my favorite colors in these posts.
Dropcloth would be the main color with an accent of Putty. Again, using my Palm Pro, I give the cabinet two coats letting cure 2 hours in between.
Now comes the fun part…adding dimension. Woot! I wanted to create a point of interest on the front of this piece, so I purchased this wooden medallion applique . Isn't it lovely?
I used my Dixie Belle paints in Dropcloth, Putty, Sage, Spanish Moss, and even more Espresso stain to give it depth and character. The added bonus was I topped it off with these beautiful gilding waxes in Bronze and Warm Gold.
I'll show you what it looks like in a bit, but first, I had even more fun. If that's possible, right? I like to play in the dirt, but this kinda dirt is a blast! Dixie Belle offers this product called Dixie Dirt in various colors, so that you can use it to create texture on your project. Why dirt you ask? Well, you have seen before how I use glazing, waxes, and even different paints to add dimension to ornate pieces. This Dixie Dirt is so easy to use and makes it fun.
First, I apply a coat of wax using a brush or lint-free cloth. I chose to use Clear Wax, but you can use any of their waxes (white, black, grunge gray, or brown). While the wax is still tacky, I use my Square Brush to just pick up a hint of Dirt in Earth color. Doesn't that sound funny?!
Tap off any excess because less is more when using this product. I apply the Dirt to the places I want to highlight and shade by brushing it on. I hit the carved area and am aiming to shadow the panels.
For the center panel that I painted in a different color, I also use the Dirt to make an oval where that wooden applique I showed you earlier is going to go. I daresay that this is easier to use than glazing and gives such a fun effect.
Now the base of the waterfall hope chest is ready for my last step to add my wooden applique. With wood glue and my nailer, I just pop it on and I'm done! Hooray!
Here's an upclose peek of that applique with the painted details and gilded waxes highlighted with the Dixie Dirt.
The new look has the aged feel I love. mmm…My favorite part is the outcome of the top. You can hardly even see the repair piece I patched with. High five for having a veneer stash, right?
The interior cedar lining didn't need anything, so I left it in its raw beauty. Those built-in trays are super handy for holding your treasures. I still have my hope chest that my parents gifted to me on my high school graduation day that also happened to be my 18th birthday. It is crammed full of my treasures – I love it.
What a fun project this was and I hope you learned a few tricks too. For me, it's all about making your home your own. There is no need to buy high end furniture from box stores when there are so many quality built vintage and antique pieces that just need a little love.
Normally about now I would be telling you this chest is available in my shop, but it sold before I could even list it. I'm so excited that it's on its way out west to a very special young lady as a gift from her wonderful mama. Such a blessing to be a part of it all.
Because Dixie Belle Paint Company and I want you to be able to get your DIY on and create your own home story, we're giving away THREE PRIZE PACKAGES to three of you. Wahoo!! Giveaway is closed.
Easy entry – just complete as many steps in the widget as you like (daily even). Open to U.S. & Canada residents 18 years of age and older. Winner will be notified via email, so be sure to check your spam!
Do pin and share this waterfall hope chest makeover to inspire other to give DIY a try. You never know who needs that spark of inspiration to get them on their own journey.
Up next, my little stash of wooden thrifted items get new life. Any guesses? Take a peek at their new life HERE in the reveal post.
My pleasure and thank you for the kind words, Lori.
Well, you just made my day! Thank you so much, Payton. I just love Dixie Belle and I'm not just saying that. I truly do!
Absolutely gorgeous! Too bad it sold already because that's a piece I could have totally gotten into! I'm so curious about the Dirt…I need to try that stuff!
You're so fun, Christina! Yes, time to take a breath every now and then. 🙂 Fingers crossed for ya!
Wow, what a difference. Love the pretty applique.
I just love these kinds of pieces. Thanks!
That is beautiful. I have a buffet i would love to paint in a two tone and thosr 2 colors look great together.
Thank you, Norma. Love buffets!
What a lovely transformation. Like me; I also love them old furniture pieces. This hope / cedar chest is gorgeous.
I have so many pieces that need painting that don't know where to begin. Gardening season now so busy with that. I can only paint in my spare time of which I have little of.
Always been wanting to try the Dixie Belle paints but with my tight budget I just haven't been able to afford it.
Thank you, Colleen. Our garden season has yet to start, but I'm planning. I hope you win!
Love the transformation! Who knew Dixie Belle could make dirt look so good! If I have to choose what to paint first it would be either my dining table or the TV stand. They both need some paint love.
So pretty! Love the applique!
It's great to see how you bring back things people see as junk or too far gone to be of use. Thanks for the inspiration!
I have a couple of chairs I'd like to paint.
You are a super talented artist. My favorite of all the pieces I've seen!
How wonderful to hear, Jewell! Thank you for making my day.
You have outdone yourself with this one! Just beautiful!
Very nice. A keepsake for some lucky young gal.
I love the different paint colors and stains you used – they tie it all together and highlight what was already there instead of being distracting.
Congrats on your third place win in the Zibra furniture makeover contest! I really enjoy your posts. I have several pieces of furniture that I would love to experiment on, so the Dixie Belle products would be a great start.
Sweet! And congrats on winning 3rd on the Zibra "One Of A Kind" Furniture Refinishing Contest!
First, Congratulations on the Zibra contest! So happy you made the top 3 !! Love the cedar chest. I have an old one I bought years ago at a thrift store, and I love it. I can't imagine painting it though. I grew up with parents who refinished antiques and I always remember my dad cursing those painted pieces. haha! Although these days I am getting into some of the painted furniture. Some things are just too gorgeous ! Since I have a plethora of little tables, that's probably what I'd use the paint for. Thank you for the chance, Larissa!
This is the most beautiful hope chest I have ever seen. You are sooo talented. Thank you for sharing with all of us. I love the dirt and applique.
Thank you so much, Jolinda.
Stunning! How did you apply stain on the applique without getting it all over??!! And congratulations on your 3rd place finish for the Zibra blog!!!!
you truly honored this piece with the way you finished it. My heart smiled when I saw the reveal picture. That chest is beautiful.
Thank you as some always chastise for painting wood. They just don't get what I do.
You always come up with the greatest ideas! Really good job on this piece. So much out there is paint-distress-seal-done. You take your projects to the next level with such beauty. All those extra touches take time and patience and CONFIDENCE! to accomplish. I have my grandmother's Lane waterfall cedar chest. It needs work but can't paint it because of the beautiful veneer. Would love to find a fixer-upper chest to do something like you have done. Great job!
Thank you for getting what I do. I am not one of those for sure and to be lumped in with them drives me crazy. Hugs, Carol.
That is so pretty! You do fantastic work!
Oh my! I am so in love with your work! Your talent defies all boundries girl!!! The hope chest is a piece of beauty now, not just an old piece in the attic. Love it!
Thank you for the kind words, Kathy.
You did such a fantastic job from fixing the veneer and your color combination. Definitely a unique piece of furniture.
I'm delighted to hear it. Thanks!
I couldn't figure out where you going with the colors and the pieces you were adding to the chest but when you were finished with it, it was beautiful! You have a very good eye when it comes to seeing how a project will turn out. That is truly a gift! Another job well done!
Thanks for the love, Renee.
very pretty and I was glad to learn how to fix veneer. Great Job! Congrats on 3rd place in the Zibra Brush contest!
It can be done, thank goodness. haha. Thanks!
So gorgeous! And I am with you on loving the smell of wood, I have to take a stroll through the wood aisle when I go to Home Depot and my husband thinks I'm nuts! I'm still contemplating painting that swan table!
You are completely sane. haha!
Larissa…..you have the patience of Job! (-: I actually have an old chest that we used as a "toy chest" when my kids were small. I'd spiff it up!
haha…not the first time I've been told that. Thanks so much, Constance!
Another gorgeous piece! You are so talented. Love the applique and the DB dirt sets it off. I have several pieces that need repaired and painted. Maybe you could do a giveaway where we win you helping us redo a piece…ha ha. Congrats on the Zibra win. I voted for you!
Thanks! You're too funny, Debra! Thank you for the love.
I really need to try Dixie dirt one of these days!
My favorite part is the medallion!
I hope you find one, Robin. They're so useful in the home.
I have a dresser that could use some love, that is what I would use the Dixie Bell paints on!
I'm delighted that she's going to be surprised. Yay for fun!
I Love it!!! Pinned it for inspiration when I finally get my hands on a hope chest!
Beautiful and congratulations on your win on the zebra website.
Beautiful chest! I'm sad it is already sold!
This turned out beautiful!! My mother had one exactly like it! …. My brother had refinished it for her…and now it belongs to him…I've wished already, I would have liked her Waterfall bedroom suite …alas, it was sold, becouse nobody wanted it. Btw, does this comment enter me into the giveaway?
Thanks! Bummer on the bedroom set. I know what you mean. You leave a comment and then fill out the widget saying you did. Make sense?
I'm so glad to see this! I have a buffet that I planned to do in drop cloth and the top in espresso gel stain. I'm trying to,decide if I should use black wax.
Ooh…I love buffets!! Just brought another one home. Thanks!
Larissa, I have visited the same building and had a great time going through all their treasures and chatting with the couple. What a neat place with all those offices and apartments upstairs! I probably walked right past that hope chest and didn't give it a second look. You are an inspiration! Every time I watch you transform something, I wish I had the nerve to tackle similar projects. Maybe part of my problem is that I'm lacking the right tools. Yay for giveaways! Maybe you could offer a class?!?!?! Sign me up!
Sweet to hear, Jeanette! I'm so excited for them. Like my motto, #toolsoverjewels! haha.
That came out lovely, Larissa! I've never tried this brand of paint. Have heard great things & would love to!
Thank you, Lisa. I was hooked on first dip after trying dozens of paints over the years.
Wonderful makeover. I just found a shop near me that sells Dixie Belle paints and I will have to visit. You always amaze me with your creative finishes.
Hope you have fun hunting! Thank you for the encouraging words.
Love the waterfall chest makeover. Very pretty. I am about to paint my childhood French provincial bedroom set and considering using Dixie Belle paint for the first time. I would love too win some.
Thank you! That would be sweet.
I love what you did with the Art Deco Waterfall Chest, so inventive!
Love it so much! If I win I'll use DB for more jewelry boxes.
This turned out so beautiful. Thanks for showing us a lot of new and different Dixie Belle products. I appreciate that you are so willing to share how products work.
It makes it fun to teach others so they can do their own too.
Looking forward to getting settled in new home and redoing pieces for coastal cottage look. This chest is beautiful.
That is such a cool chest and getting it free is a big bonus! Congrats on the Zibra contest!
It wasn't free, but a good price that I couldn't pass up, but mostly wanted to see it revived. Thanks, Lisa!
The chest looks beautiful. The applique is a nice touch.
Beautiful, as always. Love the applique on the painted body of the chest combined with the stained top.
I had to cover those two faces on the front in the veneer…haha…just couldn't work around that design. Thanks!
This turned out so beautiful. And congratulations on winning a place in the Zibra contest. I am so happy for you.
You're always so good to me, Debra. Thank you so much.
Larissa that is absolutely gorgeous! I love the detail work you did on the floral medallion! That is one fine veneer patch too. I'm with you on picking up wood and smelling it, such wonderful scents.
How sweet of you to say, Rachel. Thank you! You are my kinda gal.
Love the chest! If I win I'd try to make my old hope chest look that great.
The chest turned out beautiful! I feel the need to make a Goodwill trip and find something to paint. I've never used this paint before and would love to try it!
The need is real…haha… Can't wait for you to try it!
i have a similar one that belonged to my mother-in-law. Unfortunately the inside is not in very good shape. She had painted the outside and later covered it with woodgrain contact paper. I love what you did with yours.
You have an eclectic style that I love. This cedar chest came out beautiful. Love your blog! Would love to try the Dixie Belle products!
It's totally fun to switch up style as the pieces come in my path. Thanks for hanging around with me, Liz!
I'd love to try this on an old wood jewelry box I have.
Wow, what a gorgeous chest. I am so inspired by your work! There are several things around here that could use a creative, elegant redo like this chest that you brought to new life, the first one would be a wardrobe-type piece that needs to be a linen storage cabinet. I think Dixie Belle would be just the right product to transform that piece. Thank you for the give-a-way opportunity for some Dixie Belle products!!! Really glad I discovered your blog today.
I'm so happy to have you here, Gina! That project sounds like the perfect project piece. Hope you win!
I have a beautiful antique buffet I'd love to try this on.
Those are so wonderful to have.
Your so talented, that girl is sure lucky to have that chest! All your pieces are so beautiful.
Thank you, Mary. I am so delighted for her and her mom.
Yay! You're so fun, Debbie!
The first thing that I will paint is a secretary desk that I found on the roadside….it will be beautiful! I just need the paint…… Thanks for the opportunity to win!
Score!! So excited for you, Dale!
Wow! What a beautiful chest!
Thank you for the smile, Kathleen.
Awesome project ! I have the same hope chest…it was my mom's.Don't know if I can bring myself to re-do it 😕 However..I do have a dresser I need to re-do , and I love Dixie Belle paints & products ! I will keep following to see your inspired transformations !!
This is beautiful! I love all of the different layers! Grest job on the repair… I'm savine this for future reference!!
Love the makeover on the hope chest!
How fun! Thank you, Kayla.
This turned out great!! Love how you really made it look "fancy" with the added decorative item but kept the age looking older with the techniques!!
I love the detail you used on the applique! Absolutely beautiful piece!
I'm delighted you like it, Connie. Thank you!
So beautiful; gorgeous detailing! At one point I assumed that the cedar made the furniture orange (growing up) because all of it was that orange-y look. When I found out I could have my beloved cedar lined furniture and give it deep, rich stain I was ecstatic.
Wahoo! Great feeling, isn't it?
You are amazing…Beautiful job done with lot of creativity.I have a very old bedroom set which i am going to paint for my 15 years old daughter with Dixie Belle paint.
That turned out absolutely gorgeous! You are so talented and I love that you share all of your pieces with us. I need to look into a brad nailer for myself, we have one bt is heavy and does need the compresser.
It's my pleasure to do so because I love seeing other's tackle their own pieces. Makes my day! This nailer is a dream without the hose pulling on my wrist.
Beautiful! The first thing I painted with Dixie Belle was a corner hutch. It was so beautiful, I made over my entire kitchen! I can't stop paining!
I love this makeover! so glad you left the inside in its pristine condition. ❤ I'm currently on the hunt for a pair of nightstands to re-do, that would be my choice of painting next.
Thanks so much, Roxie! I honestly was hesitant at first too because I thought it would be like all the other paints I tried, but I was wrong. So glad I tried it!
You're so awesome, Marti! Thank you! I need a twin for sure. haha.
So glad you won, you deserved it……………and I voted for you!!!! I have entered the contest and would absolutely love to win……I have just found Dixie Belle Paint and so happy that I have. Love Love Love it and would love to win the goodies!!! Thanks for such a great giveaway!!!! Looking forward to more of your post and trying more of the paint!! It is so easy to work with and has a beautiful finish!!!
I'm happy to hear it! I use DB all the time, so you're sure to see it again. woot!
Congratulations on the win ! Nice job on the cedar chest !
You did an amazing job on this hope chest! I had one just like it that was my mother in laws but gave it to my sister in law when we moved. I sure wish I had it back! The appliqué is beautiful!
They are such treasures to me. Thank you for the kind words, Candy.
Great find. I am amazed at the condition of the interior. As usual, you created a beautiful transformation.
Yes, other than the broken lock, all was well. Thanks!
I forgot the #2. In addition to how impressed I am with you work, I would paint my two very old, very sturdy kitchen chairs.
Love it and that applique. I love that you have a veneer stash too!
I soooooo admire your creativity. I haven't used Dixie Bell paint before but would love to try it. I have 2 French Provincial library tables I would paint first. I'm a neutral color girl so probably would use Dropcloth. Keep up the good work. I'm inspired by you!
I love it all!!! I have a few pieces I am planning to redo. One is an old headboard, small half moon table.
I learn something in every post. Love the transformation of this chest. It's beautiful!
I love the little wall cabinet! The saying is perfect.
I always enjoy your interesting transitions! Thanks for sharing.
Part 2 – I need to paint a drop side coffee table. I am thinking a stripe would be good.
I plan on painting a book case and a jewelry armoire. I appreciate all of the tutorials, blogs, etc. that you and other talented people share. I learn so much and am confident that with everyone's helpful tips and tricks, I will end up with beautiful pieces.
That's what it's all about. Yay!
What a transformation! It is gorgeous!
Another beautiful transformation, you're so talented!
You are so sweet to say so, Sian. Thank you!
Nice job. I'd paint my office desk.
It is beautiful! I have my mother's Hope Chest. It looks just like the one that you have. I would love to paint it too.
I think I'm ready for some color! I love the Dixie Belle Cotton and Fluff, but it's time to branch out. I have a little foot stool that's begging for a makeover.
Go on, girl! Get some color and have at it. So fun!
Beautiful! The detail you did on the medallion is gorgeous!
I'm so happy to hear it, Cathy. Thanks!
Always enjoy your blog and learn so much from you. Thanks for the give away.
Chest turned out absolutely beautiful !! I have a marble top dresser I'm ready to get painted. You are an inspiration !
Sounds like a perfect project piece. Have fun!
Thank you for sharing your work in amazing.
OH, MY! That is lovely! When I first looked at the finished project it took my breath away. I hadn't really been able to picture it from the photos of you working on it, so it was a complete surprise. It's transformation is just amazing. How did you know what you wanted to do with it? Had you seen something similar or did the idea just pop into your mind? It sure turned out to be a unique piece.
Thank you for the smile, Naomi. I just try to keep them true to their era and give them new life.
Sorry I forgot to tell you what my first Dixie Bell paint project would be. I think it would be a little end table with drop down leaves on each side that I got second-hand and have in my living room. Not sure yet which Dixie Belle color I will choose. Probably a shade of green or turquoise.
It's me again! I can't tell which of the photos of this project is the is the pinable image. None of them do anything when I click on them. Help!
You can pin any of them, but the long image with text near the bottom is the pin image. You can use the pin button on my site or if you have it in your chrome extensions that works too.
What a treasure you have. Enjoy!


Julia Abeyta was born on January 28, 1888. She died in April 1977 at 89 years old. We know that Julia Abeyta had been residing in San Francisco, San Francisco County, California.
Julia Abeyta's biography is built and maintained by people like you. Create an online profile of Julia so that her life is remembered forever. If any factual information is incorrect, please edit Julia's biography.
This genealogy profile is dedicated to the life and ancestry of Julia Abeyta and her immediate Abeyta family. Add to Julia Abeyta's genealogy page to share your memories & historical research with her family and other genealogy hobbyists.
Do you know the final resting place - gravesite in a cemetery or location of cremation - of Julia Abeyta? Add burial and funeral information.
Julia Abeyta lived 23 years longer than the average Abeyta family member when she died at the age of 89.
It is unknown if Julia Abeyta is a military veteran.
1888 - In the year that Julia Abeyta was born, Irishman John Robert Gregg published a pamphlet in the U.S., teaching his first version of shorthand - Gregg shorthand. When he improved on the first version and published it 5 years later, Gregg shorthand became popular.
1904 - By the time she was 16 years old, the Russo-Japanese war began. The Russian Empire and the Japanese Empire began fighting over the territories of Manchuria and Korea. Russia wanted a warm water port on the Pacific Ocean while Japan feared growing encroachment from Russia into Asia. So the Japan fleet launched a surprise attack on the Russian Navy and a one year war began. President Roosevelt of the United States brokered peace between the two nations. It was the first time in the modern era that an Asian power showed its dominance over a European power.
1952 - Julia was 64 years old when on July 2, Dr. Jonas E. Salk tested the first dead-virus polio vaccine on 43 children. The worst epidemic of polio had broken out that year - in the U.S. there were 58,000 cases reported. Of these, 3,145 people had died and 21,269 were left with mild to disabling paralysis.
1971 - Julia was 83 years old when in March, Intel shipped the first microprocessor to Busicom, a Japanese manufacturer of calculators. The microprocessor has since allowed computers to become smaller and faster, leading to smaller and more versatile handheld devices, home computers, and supercomputers.
1977 - In the year of Julia Abeyta's passing, on January 20th, Jimmy Carter became the 39th President of the United States. Running against incumbent Gerald Ford, he won 50.1% of the popular vote to Ford's 48.0%. He was elected to only one term.
This obit of Julia Abeyta is updated by the community. Edit this biography to contribute to her obituary. Include details such as cemetery, burial, newspaper obituary and grave or marker inscription if available.
Julia Abeyta passed away in April 1977 at 89 years old. No cause of death has been entered. She was born on January 28, 1888. We have no information about Julia's family or relationships. We know that Julia Abeyta had been residing in San Francisco, San Francisco County, California.
What do you remember about Julia Abeyta? Share your memories of special moments and stories you have heard about her. Or just leave a comment to show the world that Julia is remembered.


text:
Lululemon has a 'long international runway' that will get it to $1 billion in sales
Kimberly Chin
Lululemon plans to reach $US1 billion in international sales by 2020.
Analysts at Oppenheimer believe that company is well on its way to getting there thanks to its strong fundamentals.
Although former CEO and chairman Laurent Potdevin abruptly resigned over allegations of fostering a "toxic" work culture in February, the analysts believe that the company's fundamentals are still strong.
You can view Lululemon's stock price in real time here.
Lululemon has set its sights on reaching $US1 billion in international sales by 2020, and its well on its way to getting there, analysts at Oppenheimer say.
According to its third-quarter results, the athleisure retailer's sales in Asia doubled in the third quarter, while in Europe they were up 40%. The company reported total quarterly sales of $US619 million, up 14% from the same quarter last year, and sales for the first three quarters ended October 29 was $US1.72 billion. That "long international runway," could help reach the $US1 billion target, Oppenheimer analysts Anna Andreeva and Samantha Lanman wrote in a note to clients.
Online penetration in China alone is expected to grow 40% to 50% by 2020, the analysts said. This should offset slower sales growth in Europe. The company's ambitious 2020 sales target is part of a larger plan to grow the company's total sales to $US4 billion. Lululemon expects $US1 billion of that figure to come from international sales, $US1 billion from online sales, and another $US1 billion in sales of men's apparel, its former CEO and chairman Laurent Potdevin said in an investor call in December.
Although Potdevin abruptly resigned over allegations of fostering a "toxic" work culture, the analysts believe that the company's fundamentals are still strong.
They raised their price to $US88 per share, from $US75, and gave the company an "Outperform" rating.
Lululemon's stock was trading at $US79.50 per share on Friday, unchanged for the year. The company is expected to report fourth-quarter 2017 earnings results on March 27.
You can view Market Insider's earnings calender here.
Read more about why Lululemon's management woes will end up hurting it in the long run.
athleisure clusterstock laurent potdevin lululemon stock price moneygame-us retail

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: How to determine which trigger executed background task in Windows 8? I've got a problem with background task in WinRT application. I register task with two triggers
var builder = new BackgroundTaskBuilder
        {
            Name = "MyTask",
            TaskEntryPoint = "Task.MyTask"
        };
        builder.SetTrigger(new TimeTrigger(60, false));
        builder.Register();

Then i do almost the same:
builder = new BackgroundTaskBuilder
        {
            Name = "MyTask",
            TaskEntryPoint = "Task.MyTask"
        };
        builder.SetTrigger(new SystemTrigger(SystemTriggerType.InternetAvailable, false));
        builder.Register();

and in my background task logic i want to determine, which trigger executed background task. taskInstance.TriggerDetails == null
this is always true, when task starts working.
please, help. Thanks in advance!

redpajama_set_name: RedPajamaStackExchange


Warning! This interaction will be hotfixed with the weekly reset on Jan 9. Mutilate should again be your main generator in all Single Target situations.
With enough Mastery or Poison Knives relics, dropping Mutilate and using Fan of Knives as the main generator actually becomes an increase. If you see variations including "FoK" for Combinations or Relics, those are profiles using Fan of Knives instead of Mutilate.
To sim yourself and check whether using FoK as the main builder for Assassination is an increase, you can copy and paste this code after your /simc output. E.g. on Raidbots, open "Advanced", insert your /simc Addon output and then, right after, the following code.
If you click on "Copy", the input will be automatically copied to your clipboard.


text:
Tag Archives: win
Beyond- Free Speak
May 4, 2014 Black Ops, Dragon, et abductions, Maker, military abductions, Uncategorizeddanger, fear, games, gods, stablization, winmaxillya
In the Old Days, we're the days of the Gods, or the Godalarians, a race of Beginners, who traverse all Universes, realms, experiences, expressions, effects and life.
Each Godalarian, has a speciality, which is unique to them, but they, are beyond, in all the stitches, sprines, and dangum, or to put it more up to date- talents, abilities and skills.
No Godalarian, could fight another, if they tried, they were knocked clear across the Universe, and made to make their way back- slowly.
The Godalarians were who the humans of old, called Gods, but Godalarians, never claimed to be Gods, but only wanted to help the humans, on their path to their Daily, or here and now.
The Godalarians have self-authority, over themselves, no other can tell them what to do, but they can choose to follow another, and all of them, chose to listen to the Silverness of purity and love, made available through the expression of music, and chimes.
Later bells, would be added, but all of them simply gave you an impression of the twinkle in the eye of what is not seen, but can be touched, but never known, this is the True, WalkerSilence.
The other Godalarians, called him or her or it, King of the Godalarian.
Humans called him/her/it- King of the Gods.
He never made a decision, but simply showed them what he saw, and they did what they felt, from the truth, he would show them.
Some Godalarians, started called him WisEsen, which later, became Wizard.
In fun, the Godalarians, tried to defeat him in any kind of combat they could think of, they always lost.
He/she/it never gloated, or accepted adulation, but always helped everyone, who did this, to find the truth that laid within them.
Together they developed Free Speak, the non-language of the Beyond Experiencers.
The Humans, saw Free Speak, as only for the gods, and the only talk, must be the ones, who come to rule them, so they limited their speech, and lowered their ability to- do.
In this limited speech, came distortions, and new anger at themselves, which led to war, killing, butchery, and so on.
They, then, made the Godalarians, Gods, and the one who led them, was Odin/Zeus/etc., and he was the mightiest of all that lived.
Those who were less limited, even more than the humans; the Dark, rose by stealth, death, fear, lies, deceits, mindclouding, and so on. They called themselves, the Dark, and became the opposite of the Gods, and the humans, and rose in power and destruction, they were war, it was and is all they care about.
They were thrown down, by the humans, with the help of the Gods, and the light of self-search, which made the Dark run away from the battle fields, and combat, and hide in the darker realms, but even there, the Truth ruled supreme, by not ruling, but being.
It is now, when the duality set in, the Truth, and the Game, which allowed all kinds of truth, to confuse those who would play the game.
To help guide those players, within the game, music came into being, and the purer the music, the greater the level jumping in the game.
The Game led to the stablization of the Universes, Realms, Crafteds, and so on.
Beyond: Free Speech- we are the Truth of each; we win our life and our purity of love and freedom, day by day, here and now.


Who doesn't love cakes and donuts?!? I know we do! Tastykakes sent some delicious goodies our way to try out, and we were not disappointed. My favorite of the bunch is the Sweetie Kakes, because I'm partial to chocolate cake..haha. I love how they spread the icing across the cakes in a sheet. It's super cute, and I think the icing is much softer than other leading brands! They sent some coconut cakes, but since we aren't a coconut eating household, my dad gladly took those off of our hands…haha. The donuts, however, were a huge hit with my teenage boys and hubby! If you are looking to make a goody basket for Valentine's Day, these are the perfect additions!
Fancy Panz is the most awesome thing ever! You know how, when you want to take a dish somewhere, but those foil pans are flimsy? Well, you won't have to worry about that anymore! This is the most amazing foil pan carrier! Not only does it hold your pan, but it comes with a plastic serving spoon, so no taking your good ones 🙂 I love that it's easy to wash, and that it has a lid. Have you ever used those flimsy lids that come with a foil pan? Oy!
Disposable pans are so handy, but so unattractive. So Jane Casey created a travel pan that dresses up the typical use-and-toss variety and makes them easier to take on the go. Fancy Panz are Made in the USA to fit over standard size disposable pans, so your dish (hot or cold) stays neatly contained and looks perfectly presentable. It's made from dishwasher-friendly, BPA-free plastic and also comes with an insert to fit a half lasagna pan perfectly. You can serve straight out of the Fancy Panz or through the removable lid.
Use Code MAJENICA10 for 10% off your order!
The jewelry from VolanaKate has real whiskers in it!! Real. Whiskers. That is the best thing I have ever heard! Now, before you get upset, please know that the whiskers used are not plucked from unsuspecting kitties and puppies. These are naturally fallen whiskers. I promise, they do fall naturally. We collect all of the whiskers that we find from our kitties. In fact, just yesterday I found another one. It's said that they bring good luck.
Now, something that is really neat about this shop is that they will even allow you to send your own whiskers in to be used in your jewelry! I don't know of any other place that will do that! When I got these studs, I was so excited! I am a crazy cat lady, so I had to have them! They are very well made, and will last me a long while. I will definitely be ordering in the future!
Oomo earbuds are not just another cheap earbud. They are super comfy to wear and the sound quality is outstanding! I like wearing earbuds when I'm watching movies and videos on my iPad, because I feel that I get the best sound experience. These earbuds from Oomo are my new favorite earbuds! It's like having surround sound in your ears.
3 cavity, 5 channel Acoustic structure separates the sound frequencies to provide clarity and 3D Virtual 5.1 Surround Sound. You will hear every aspect of the output produced, as all the sounds are distributed in distinct channels. Fidelity is unbelievable. It has the frequency response of 8Hz-20KHz. Infrasonic frequencies develop in the back cavity allowing you to hear crystal clear bass. Using a viscoelastic material inside the mechanical structure, frequencies are refined to produce clear output. The material absorbs unwanted feedback inside the structure providing the optimal acoustic environment.
This Tourma blanket is one of the softest blankets I own. The material is very comforting, and relaxing. Generally, I have to fight my cats for it, because they love to curl up and nap on it! It washes very easy, and doesn't lose it's softness at all. Mine has been washed a few times and still looks like new.
Tourmaline is a therapeutic mineral that has been used for millennia in Eastern medicine, as well as around the world. When must people work hard, they like to unwind. Snuggling up with Tourma®Blanket can help you to do just that, but in a healthy and therapeutic manner.
My Tourma scarf keeps me nice and toasty when I'm outside! Thank goodness, because it's been in the subzero temps here in Indiana! It's nicely sized, so that anyone could wear it comfortably, and stay warm. It's the same material as the Tourma blanket, which is amazingly soft and snuggly.
This is a fun gift for the music lover in your life. You can find fun music facts about any date! This is one of those great gifts that will provide entertainment for a long time. On This Date In Music is a book with a daily story about a significant event that happened in the world of music on each date. Stories range from Edison patenting the phonograph in the late 1800's to Pharrell Williams' incredible ten week run at #1 in 2014 with his uplifting song "Happy." Each story is told in approximately 800 words aimed at being concise and interesting. This is the perfect compendium for any music lover and can be used as a reference book or for a daily dose of compelling tales of tunes we all love and bands we all follow.
This is a beautiful necklace! At first I didn't think there was any way that there were 100 different ways to say I Love You inside of it! Boy was I wrong! I held it up to the light, and sure enough, there they were! It is such a neat, and unique gift. It's very nice quality, and comes in either rose gold or silver plated. The price is budget friendly, and your loved one is sure to be thrilled!
Objects With Purpose offers candles, roll on perfumes, and gift baskets. What makes their candles unique is that they are not only vegan, but they also can be used as multiple items. They are candles, massage oil (when lit), body lotion and can be used as perfume when rubbed on the wrists! This is amazing, because you are purchasing one product, but getting many different uses, making this a very unique gift. They sent me the scent "Crisp Ocean Air, Salt & Citrus" and I love it! It has the most amazing smell to it, like I'm standing by the ocean, which is my favorite place to be 🙂 They also sent 3 of the small tins for me to try out, and I am really impressed with the range of scents they have available. You will have no problem finding a scent for the loved one in your life!
This orchid is beautiful! I love how they took great care to make sure it arrived safely. They use clips and rods to ensure that the orchids stand tall. The wooden base that comes with it is super darling! The best part is that you don't have to worry about the amount of water you have to give it because it literally only takes 3 ice cubes per week!
Just Add Ice® orchids are not only beautiful and easy-to-grow; they are also produced with the most innovative orchid-growing technologies available today. That includes our state-of-the-art grading system to determine orchid quality, the only one of its kind in North America. The computerized system in our growing facility maximizes space efficiency, reduces labor expenses and guarantees quality. We are constantly testing new ways to ship our plants in small packages directly to homes.
Stella Rosa Black Lux is a wonderful wine. It has a great flavor, and isn't too sweet. The presentation is beautiful, and would make a great wine to share with your Valentine! It's budget friendly, but tastes like the expensive stuff.
More sparkles than the twinkle in his eyes. More romance than the red on her lips. Darker than your richest fantasy. Stella Rosa Imperiale Black Lux is the desirable sparkling red blend, a sweet bold nectar plucked from the same grapes of your favorite Stella Rosa Black, and perfect for any special occasion. Created by recent winner of the American Winery of the Year award – from the bottle to the seductive taste, this wine is the epitome of Valentines Day.
These sticks are pretty convenient! Not only are they convenient, but they are also tasty. Some mornings, I just don't want to brew coffee, yet I want to drink coffee. These Nescafe sticks are the perfect alternative to having to brew coffee. They are also great to take on the go, and slip easily into your pocketbook or backpack.
NESCAFÉ can't stop bringing more excitement to your coffee experience. Introducing another People's Favorite Nescafe Latte Mocha, a 3-in-1 specialty coffee that gives your taste buds surprises to your everyday boring joe.
Just like other Nescafe 3-in-1 coffee, this Latte Mocha comes ready, made with coffee, creamer and sugar for the ultimate coffee brewing convenience. It is milky, chocoliciously creamy and frothy.
The uniqueness of this latte is that it has no harshness and bitterness, like regular coffee. That's the NESCAFÉ way to impress and delight your taste buds. Even non-coffee drinkers can't resist the unique taste of this Latte Mocha.
Domino Sugar has long been a staple in my pantry. The quality has always been stellar, and it makes my recipes delicious!
Valentine's day is synonymous for love, roses, baked goods, candies, and truffles. Both Domino Light and Dark Brown Sugars create a flavor that's deep, rich and real, while being ideal for baking full-flavored or savory recipes. Domino Brown Sugars are ideal for baking of all kinds— especially cookies, shortbread, candies, fudge, chocolate cakes, brownies and crumble toppings. What better way to show your friends, family, significant others, etc. that you love them? Let Domino Sugar sweeten up your Valentine's Day this year.
This is a wonderful journal for a couple to complete. It really brings back some memories, that's for sure! Each page is something different, and keeps it interesting. It is a nice, hard back book, with a closure strap. The pages are a good quality, and the ink doesn't bleed.
Designed to be filled out by one partner and given to the other, or to be explored as a couple, the prompts and questions in this guided journal offer up insight and fun as you write your love story in your own words!
Measures 6-1/4" wide x 8-1/4" high.
Pretty red journal design features raised embossing, glossy heart design highlights, and gold foil accents.


Talin is a cytoskeletal protein involved in constructing and regulating focal adhesions in animal cells. The cellular slime mold Dictyostelium discoideum has two talin homologues, talA and talB, and earlier studies have characterized the single knockout mutants. talA− cells show reduced adhesion to the substrates and slightly impaired cytokinesis leading to a high proportion of multinucleated cells in the vegetative stage, while the development is normal. In contrast, talB− cells are characterized by reduced motility in the developmental stage, and they are arrested at the tight-mound stage. Here, we created and analyzed a double mutant with a disruption of both talA and talB. Defects in adhesion to the substrates, cytokinesis, and development were more severe in cells with a disruption of both talA and talB. The talA− talB− cells failed to attach to the substrates in the vegetative stage, exhibited a higher proportion of multinucleated cells than talA− cells, and showed more-reduced motility during the development and an earlier developmental arrest than talB− cells at the loose-mound stage. Moreover, overexpression of either talA or talB compensated for the loss of the other talin, respectively. The analysis of talA− talB− cells also revealed that talin was required for the formation of paxillin-rich adhesion sites and that there was another adhesion mechanism which is independent of talin in the developmental stage. This is the first study demonstrating overlapping functions of two talin homologues, and our data further indicate the importance of talin.
Formation and regulation of focal adhesions are crucial for cell locomotion, since cells need to adhere to substrates in order to exert traction forces. Talin is a large cytoskeletal protein which assembles at focal adhesions in mammalian cells (8). It is thought to link actin filaments to the plasma membrane at focal adhesions, since it has a FERM domain that interacts with the plasma membrane at the N terminus and an I/LWEQ module, which is an actin binding domain conserved in certain proteins, including talin and yeast Sla2, at the C terminus (22). This view is supported by the findings of previous studies that showed that inhibitions of talin's function or a disruption of a talin gene compromise the assembly and stabilization of focal adhesions (4, 27, 29). Several other studies have attempted to clarify not only the structural but also the regulatory role of talin. For instance, talin nucleates and organizes actin filaments and is involved in the processing and activation of integrins (1, 13, 34). Talin binds and regulates type Iγ phosphatidylinositol phosphate kinase at synapses and focal adhesions (9, 19, 21, 24). Furthermore, talin is implicated in cytokinesis through a so-far-unknown mechanism (3, 14, 26).
It is intriguing that a second talin has been discovered for certain organisms, considering the wide spectrum of functions of talin (30). The cellular slime mold Dictyostelium discoideum was the first organism for which two different talin homologues, talin A and talin B, which are encoded by talA and talB, respectively, have been identified. Dictyostelium is an established model system to study the mechanisms of cell movement. It possesses a large number of cytoskeletal proteins that are also present in mammalian cells, and the amoeboid cells move actively, in a manner similar to that of leukocytes (11). The availability of various genetic approaches allowed for identifying genes involved in diverse processes and revealing the dynamics and functions of the gene products. When deprived of a food supply, the amoeboid cells aggregate and build up a hemispherical structure called a "mound." A small protrusion forming a "tip" subsequently emerges from the top of the mound, which then can elongate to form a cylindrical structure known as a "slug" that finally transforms into a fruiting body consisting of a stalk tube and a spore sorus.
There are clear phenotypic differences between talA− and talB− strains that appear to suggest distinct roles for the two talins. Talin A's main role appears to be in the vegetative stage. talA− cells show much weaker adhesion to the substrates and bacterial surfaces than wild-type cells, resulting in a distinct phagocytosis defect, and improper cytokinesis, resulting in multinucleated cells in suspension cultures during the vegetative stage. Although severe defects are observed during the growth phase, development in talA− cells proceeds normally (26). In contrast to talA− cells, cells lacking talin B are defective in development, although the cells do not exhibit any obvious deficiencies during the vegetative stage (35). talB− cells never form a tip on the mound, probably due to weaker motile forces of the differentiated cells within the mounds (36).
Recent studies suggested that the two mammalian talins also have discernible functions. Talin 1 was suggested to have a function in myoblasts, while talin 2 is induced during striated muscle differentiation. In addition, talin 1 and talin 2 mRNAs show different distributions in adult mouse tissues (31). Different binding partners have been described for mouse talin 1 and talin 2 (30). Disruption of the tln1 gene encoding talin 1 in mice leads to embryonic death due to an incomplete gastrulation, indicating that talin 2, encoded by tln2, is not able to compensate for the loss of talin 1 (23). Similarly, talin 2 is not able to replace talin 1 in the formation of the minimal linkage between fibronectin and the cytoskeleton in mouse differentiated embryonic stem cells (16), although the expression of tln2 was not examined in these two experiments. Furthermore, alternative variants of a talin gene in the nonvertebrate chordate Ciona intestinalis are suggested to have different roles (32). However, the analysis of a double mutant with a disruption of both talin genes and the rescue of a mutant lacking one talin gene by the overexpression of the other talin gene, which would enable us to clearly demonstrate the unique and overlapping functions of two talins, have never been attempted. Here, we applied our analysis to the two talins in Dictyostelium discoideum and provided evidence that the functions of the two talins were largely redundant. The more-severe phenotypic defects of the double mutant further confirm the important role of talin.
Cloning of expression constructs.To create the green fluorescent protein (GFP)-paxillin construct, full-length paxB paxillin cDNA (5) was amplified from a Dictyostlium cDNA library by PCR using the primer set 5′-GGATCCAATGTCAAATAAAAATCCATTAAATAATAGTA-3′ and 5′-GAGCTCTTATTTTCTTTGTTGTACAAGTGT-3′. The PCR product was fused to GFP cDNA by subcloning into GFP/pBIG vector (25) as a BamHI/SacI fragment.
To generate the FLAG-tagged full-length talin B construct, three parts of talB cDNA (4 to 1164, 2988 to 7275, and 6234 to 7845, numbered from the first nucleotide of the coding sequence) were obtained from a Dictyostlium cDNA library by PCR or from total RNA by reverse transcription (RT)-PCR using three primer sets: 5′-CGAGCTCTCACTTACACTCAAGATTCAAATCGT-3′ and 5′-GGAATTCACTCTCAACGTTGGCAATATCAG-3′, 5′-CGTTCAAGCCTTAGAAGCCG-3′ and 5′-GATGATTTGGGCGTTTGATAAG-3′, and 5′-GGCTGCTTATGATAGTGCAACC-3′ and 5′-GCTCTAGATTAGAATAAACCAAGTTTAGTTTTTATATTATTTC-3′, respectively. The fragment 4 to 1164 was subcloned into the pCRBluntIITOPO vector as a SacI/EcoRI fragment (ptalB-head). A KpnI/KpnI fragment of the talB gene was acquired from the plasmid containing the talB knockout construct, which was obtained using XbaI from the genome of the talB− mutant (35). The fragment was inserted into ptalB-head that was digested with KpnI to extend the talB cDNA fragment (ptalB-Nhalf). The fragment 6234 to 7845 was subcloned into HincII/XbaI-digested pBluescriptIISK+ after digestion with the same enzymes (ptalB-tail). A KpnI/EcoRI fragment of ptalB-tail was replaced by a KpnI/EcoRI fragment of the fragment 2988 to 7275 to obtain a longer talB cDNA fragment (ptalB-Chalf). A SacI/SacI fragment and a SacI/XbaI fragment from ptalB-Nhalf and ptalB-Chalf, respectively, were ligated to the SacI/XbaI-digested pTX-FLAG (20) construct to complete the expression construct of FLAG-tagged full-length talin B.
To create the talin A-GFP construct, which drives the expression of talin A fused with GFP at the C terminus, the full-length talA gene was amplified by PCR using the primer set 5′-GATCGGATCCAATGTCAATTTCATTAAAAATTAATATTGTTGG-3′ and 5′-ATCGGATCCATTTTTATTATAATTTTGTTTTCTTG-3′. The PCR product was subcloned into pA15GFP vector (kindly provided by Rex Chisholm, Northwestern University) as a BamHI/BamHI fragment.
Strains and conditions for growth and development.Strains used in the present study were Dictyostelium Ax2 as a wild-type strain, HG1664 as a talA− strain (26), HKT104 as a talB− strain (36), and a strain with a disruption of both talA and talB (a talA− talB− strain). All the strains were grown axenically in HL5 axenic medium (0.5% yeast extract, 0.5% peptone, 1% glucose, 8.82 mM KH2PO4, 2.46 mM Na2HPO4 [pH 6.3]) at 22°C in plastic petri dishes (33). FLAG-tagged talin B and talin A-GFP constructs were introduced separately into talA−, talB−, and talA− talB− strains. GFP-actin and GFP-paxillin constructs were introduced into wild-type and talA− talB− strains separately. The expression of all the tagged proteins was driven by actin 15 promoter. Those transformants were selected in HL5 axenic medium containing 10 μg/ml G418. Cells were allowed to develop at 22°C on 1% nonnutrient agar buffered with KK2 phosphate (16.5 mM KH2PO4 and 3.8 mM K2HPO4, pH 6.2) or on a lawn of Klebsiella aerogenes made on SM/5 agar plates (33).
Analysis of translational regulations.Wild-type cells growing exponentially in HL5 shaken cultures (2 × 106 to 5 × 106 cells/ml) were harvested and washed once in KK2 phosphate buffer. The cells were resuspended in KK2 phosphate buffer at a density of 5 × 104 cells/μl, and the cell suspension was spotted as 10-μl aliquots on mixed cellulose ester filters (Advantec). The membrane filters were placed on filter papers (Advantec) soaked with KK2 phosphate buffer and were kept at 22°C to allow cells to develop. Cells (107) were taken every 3 h after the onset of development to be lysed in 100 μl of sample buffer (75 mM Tris-HCl [pH 6.8], 3% sodium dodecyl sulfate [SDS], 9% β-mercaptoethanol, 15% glycerol, and 0.075% bromophenol blue). For Western blotting analysis, 15 μl of the lysates was fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) (SuperSep 5 to 20%; WAKO) and transferred to polyvinylidene difluoride membranes. Talin A and talin B were visualized by chemiluminescence assay (Immobilon Western; Millipore) using their specific antibodies as in previous studies (18, 36) and horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (Jackson ImmunoResearch). To confirm the equal loading amounts, 3 μl of lysates was subjected to SDS-PAGE, and the gel was stained with Coomassie blue.
Analysis of gene expression by RT-PCR.talB− and talA− talB− cells were allowed to develop on membrane filters as described for sample preparation for Western blotting analysis. After 36 h, total RNA was extracted from the mounds by using an RNeasy Mini kit (Qiagen). RT-PCR was performed using total RNA, a Titanium one-step RT-PCR kit (BD Biosciences), and specific primer sets to amplify the genes.
Generation of a talA− talB− double mutant.For the generation of a talA− talB− double mutant, 8 × 106 blasticidin-resistant talA− cells were transformed by electroporation with the disruption construct of talB carrying a hygromycin resistance cassette (36). The resultant cells were diluted in HL5 axenic medium containing 10 μg/ml blasticidin S (Funakoshi, Japan) and 50 μg/ml hygromycin B (Wako), and the aliquots were seeded into six 96-well plates. In addition, autoclaved bacteria were included in the selection medium to raise the efficiency of transformation (17). For the preparation of autoclaved bacteria, K. aerogenes grown in YT medium (2% tryptone, 1% yeast extract, 1% NaCl) at 37°C overnight were collected, washed twice with KK2 phosphate buffer, and concentrated 100× in KK2 phosphate buffer. The bacteria slurry was autoclaved and added to HL5 axenic medium at a 100-fold dilution. Cells that were resistant to both blasticidin S and hygromycin B appeared in a small number of wells after the selection, and those clones were transferred to a plate with a lawn of K. aerogenes to observe the morphogenesis during development. Disruption of the talB gene by homologous recombination was confirmed by genomic PCR using primers 5′-CGAAACACACAACAACAAAATACAC-3′ and 5′-GACCACCTTTAACAATTTCAATTGCC-3′.
Adhesion assays.For the adhesion assay, 106 cells in 1 ml HL5 axenic medium were plated into plastic petri dishes (35-mm nontreated dish; Iwaki). Cells were allowed to settle and adhere to the bottom surfaces for 20 min, and then the dishes were agitated by using an orbital shaker at speeds ranging from 0 to 200 rpm for 30 min. Subsequently, the media containing detached cells were taken, and the fractions of detached cells were determined by counting.
For the adhesion assay in KK2 phosphate buffer, 106 cells grown in HL5 axenic medium were collected by using a microcentrifuge (MX-300; Tomy) at 10,000 rpm for 5 seconds and were suspended in 1 ml KK2 phosphate buffer or HL5 axenic medium for a control. The cell suspension was immediately transferred into 35-mm nontreated dishes. The fraction of nonadherent cells was determined by counting the cells contained in the supernatant removed from the dishes after 5 min. To assay the inhibition of adhesion in HL5 axenic medium, 106 talA− talB− cells grown axenically were allowed to adhere to the bottom surfaces of 35-mm nontreated dishes in 1 ml KK2 phosphate buffer for 20 min. After the fraction of adherent cells was determined by counting the nonadherent cells in the buffer removed from the dishes, the adherent cells were incubated in 1 ml HL5 axenic medium for 3 h. The fraction of detached cells was subsequently determined by counting the number of cells contained in the removed medium. The control experiment was performed by the same procedure except with KK2 phosphate buffer for the 3-h incubation instead of HL5 axenic medium.
To assay adhesion to the agar surface, 1 ml of a cell suspension grown in HL5 axenic medium to a density of 106 cells/ml was placed on a 0.5% nonnutrient agar plate. After 30 min, the medium containing nonadherent cells was removed by slanting the dish to determine the proportion of nonadherent cells.
Recording cell movement and fluorescence microscopy.Vegetative cells were harvested and resuspended in KK2 phosphate buffer. An aliquot of the cell suspension was placed in a glass-bottom dish (35-mm; Iwaki) and overlaid with a thin piece of a sheet made of 1% nonnutrient agar. The agar sheet was produced by pouring 3 ml of melted agar of KK2 phosphate buffer into a 6-cm plastic dish (Falcon). After 4 h, phase-contrast images of the cells were captured every 3 min for 90 min using NIH Image software version 1.62 and an inverted microscope (IX50; Olympus, Japan) equipped with a charge-coupled-device camera (C5985; Hamamatsu Photonics, Japan) and a time-lapse system (Argus-20; Hamamatsu Photonics). The cell centroids were tracked using ImageJ software version 1.32, and the mean velocity of cell migration was determined by averaging the displacements during 3-min intervals. Fluorescent signals of GFP-actin and GFP-paxillin in the transformants were observed using a confocal microscope (CSU10; Yokogawa, Japan).
Analysis of development.Vegetative cells (2 × 106) were collected by centrifugation at 2,000 rpm for 3 min and suspended in 100 μl KK2 phosphate buffer. Aliquots (10 μl) of the cell suspensions were spotted on 1% nonnutrient agar to allow the cells to develop. Pictures of the spots were taken at certain time points by using a stereo microscope (MZ7.5; Leica) equipped with a charge-coupled-device camera (FX380; Olympus) to monitor the developmental processes. Different spots were photographed at the onset and after 12 h of development.
Cytokinesis assay.Cells (3 × 105) were grown axenically on 0.5% nonnutrient agar submerged in 3 ml HL5 axenic medium in 6-cm plastic petri dishes to a cell density of approximately 2 × 106 cells/ml, and nonadherent cells were collected by centrifugation. The harvested cells were fixed with 1% formaldehyde in ethanol at −20°C. After the cells were washed three times with phosphate-buffered saline (PBS; 137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4), they were incubated in PBS containing 1 μg/ml DAPI (4′,6′-diamidino-2-phenylindole) for 10 min. Subsequently, cells were washed three times with PBS, and the number of nuclei was counted using an IX70 fluorescence microscope.
Developmental time courses of talin A and talin B expressions.The defects of talA− and talB− cells are seen only in the vegetative and developmental stages, respectively. We first analyzed the translational regulations of talin A and talin B during development by Western blotting analysis in order to examine whether they are expressed mainly in the stages where each mutant exhibits the impairments. Both proteins were present in the vegetative stage and throughout development, although the patterns were different for the two stages. Talin A was maximally expressed in the vegetative stage, and considerable accumulations occurred at the mound and slug stages (Fig. 1). In contrast, the expression of talin B reached the maximum between mound and slug stages (Fig. 1). Therefore, each talin exhibited plausible maximal accumulations at times when it was suggested to play a more important role based on the mutant analysis, but considerable levels of expression were also detected in other periods for both talins.
Time courses of talin A and talin B expression. Wild-type cells developing on membrane filters were harvested every 3 h and were lysed in SDS sample buffer. Equal amounts of lysates were separated by three individual SDS-PAGEs. One was used for the staining with Coomassie blue (CBB) to confirm the equal amount of loading, and the other two were used for Western blotting analysis to examine the time courses of talin A and talin B expression, which was detected using each specific antibody. Only a portion of the gel is shown for Coomassie blue staining (top).
Generation of a talA− talB− double mutant.In order to obtain a Dictyostelium double mutant lacking both talin A and talin B, we eliminated talin B in talA− cells (26). talA− cells were transformed with a disruption construct targeted against talB (Fig. 2A) (36). Transformed cells, which were potential candidates of talA− talB− double-mutant clones, emerged following selection. We transferred the transformant clones to bacterial lawns to observe their developmental phenotype. Only 12 out of 142 clones from two independent transformations were arrested at the mound stage, and the remainder formed normal fruiting bodies. Since the inactivation of talB in wild-type cells resulted in a developmental arrest at the tight-mound stage (35), we selected 4 of the 12 clones that were arrested at the mound stage and tested them for disruption of talB. Genomic PCR analysis confirmed that the talB genes of all four clones were replaced by the knockout construct due to homologous recombination (Fig. 2B). In contrast, talB genes in two resistant clones that formed fruiting bodies were not disrupted, suggesting that the constructs were integrated into the genome nonhomologously (Fig. 2B).
Disruption of the talB gene in talA− cells. (A) Schematic representation of the talB gene disruption with the disruption construct (36). The sites of the first codon of talB and the positions of the primers used to confirm the disruption are indicated by an arrow and arrowheads, respectively. (B) Confirmation of talB gene disruption by PCR using genomic DNA as the template. The bands obtained with the indicated primers (arrowheads in panel A) were approximately 4 kb in size in the parent talA− strain and also in the two clones of random integrants (RI-1 and 2) whose talB genes were not disrupted. In contrast, the bands obtained from genomic DNA of the talB− strain and the two clones of the talA− talB− strain were 0.8 kb larger in size due to the replacement of a 1.1-kb fragment of talB gene by the 1.9-kb hygromycin cassette.
talA− talB− double-mutant cells are severely affected in adhesion to substrates.Phenotypic differences in comparison to control strains became obvious when talA− talB− cells were grown axenically in plastic petri dishes. Unlike wild-type and both talA− and talB− cells, most of the talA− talB− cells were spherical in shape and unable to attach to the substrates. Furthermore, many of the talA− talB− cells were significantly larger than wild-type and both talA− and talB− cells (Fig. 3A and B and data not shown).
Attachment of wild-type (WT) and talin mutant cells to a solid substrate. (A, B, and D) Phase-contrast micrographs of talA− (A), talA− talB− (B), and talA− talB− cells expressing FLAG-tagged talin B (talB OE in talA−/talB−) (D) grown in HL5 axenic medium in plastic petri dishes. talA− talB− cells are more spherical and enlarged than talA− and talB OE in talA− talB− cells. The scale bars represent 20 μm. (C) Fractions of detached cells in HL5 axenic medium examined after 30 min of shaking the dishes. (E) Fractions of adherent cells examined 5 min after substituting KK2 phosphate buffer for HL5 axenic medium or maintained in HL5 axenic medium. (F) Fractions of cells that remained attached in KK2 phosphate buffer after 30 min of shaking the dishes at a speed of 75 rpm. (G) Fractions of detached talA− talB− cells, which had been induced to adhere to the substrates in KK2 phosphate buffer, after the incubation in HL5 axenic medium or KK2 phosphate buffer for 3 h. All data are means ± standard deviations (SD; n = 3).
In order to investigate wild-type, talA− and talB− single-mutant, and talA− talB − double-mutant cell adhesion to the surfaces of plastic petri dishes, we counted the number of detached cells after shaking the dishes at different speeds for 30 min. More than 60% of wild-type and both talA− and talB− cells remained attached at 100 rpm, whereas talA− talB− cells, even without shaking, were nonadherent (Fig. 3C). This result indicates that both talin A and talin B are involved in adhesion to the substrates in the vegetative stage and that expression of either talin gene is sufficient for an adhesion strength comparable to that of wild-type cells in this assay. The number of nonattached talA− talB− cells was smaller in the absence than in the presence of shaking. This was not a consequence of a few cells being able to adhere in the absence of shaking but instead occurred because it was difficult to remove all detached cells from the bottoms of the dishes under these conditions. Expression of FLAG-tagged talin B in talA− talB− cells restored their ability to adhere to the surfaces of plastic dishes (Fig. 3D). This result confirms that the defect in attachment to the substrates is due to the talA− talB− double mutation.
The talA− talB− mutant formed mounds, which suggests that talA− talB− cells are able to attach to the substrates for translocation after starvation. Therefore, we examined adhesion of talA− talB− cells to the substrates in the absence of nutrient medium. We collected both wild-type and talA− talB− cells from HL5 axenic medium, suspended the cells in KK2 phosphate buffer at the same cell density, and plated the cell suspensions immediately in plastic petri dishes. Nonadherent cells were counted after 5 min. Approximately 57% of talA− talB− cells were able to attach to the substrate under these conditions, although a higher fraction (72%) of wild-type cells adhered to the surfaces (Fig. 3E). When we used HL5 axenic medium instead of KK2 phosphate buffer as a control in this assay, almost no cells attached to the bottom surfaces of the dishes (Fig. 3E). The weak adhesion ability of talA− talB− cells compared to wild-type cells in KK2 phosphate buffer became more obvious when the shaking adhesion assay was carried out with KK2 phosphate buffer (Fig. 3F). Only 40% of talA− talB− cells remained attached to the substrates after shaking, in contrast to 82% of wild-type cells. These results demonstrate that, unlike results with the nutrient medium, talA− talB− cells are able to adhere to the substrates in the nonnutrient KK2 buffer, although more weakly than wild-type cells.
Subsequently, we examined whether the addition of HL5 axenic medium inhibited the attachment of talA− talB− cells that became adherent to the substrates in KK2 phosphate buffer. talA− talB− cells, which had been allowed to attach to the substrates in KK2 phosphate buffer in advance, were incubated in HL5 axenic medium for 3 h, and the fraction of detached cells was determined. Approximately 80% of talA− talB− cells detached in this assay, while only 20% of them detached when they were incubated in KK2 phosphate buffer for 3 h instead of in HL5 axenic medium (Fig. 3G). The inactivation of adhesion ability by HL5 axenic medium was not as fast as the activation of adhesion by KK2 phosphate buffer, since most of the talA− talB− cells remained attached 5 min after the buffer was replaced by HL5 axenic medium.
Overexpression of FLAG-tagged talin B compensates for the adhesion defect in the talA− mutant.We were unable to find any significant differences in the adhesion levels between wild-type and talA− cells in our assay described above (Fig. 3C), even though talA− cells have been reported to be impaired in adhesion to the substrates during the vegetative stage (26). Therefore, another, more sensitive assay was developed in order to compare adhesiveness between wild-type and talA− cells. When talA− cells were allowed to adhere to the surfaces of agar submerged in HL5 axenic medium, approximately 80% of talA− cells did not attach to the agar surfaces after 30 min of settling. In contrast, almost all wild-type and talB− cells attached, demonstrating the weaker adhesion ability of talA− cells (Fig. 4B). However, overexpression of FLAG-tagged talin B in talA− cells raised the fraction of attached cells by 67% (Fig. 4A and B).
Overexpression of FLAG-tagged talin B rescued the adhesion defect in the talA− mutant. (A) Vegetative wild-type (WT) cells, talA− mutant cells, and the transformant expressing FLAG-tagged talin B (talB OE in talA−) were lysed in SDS sample buffer. Equal amounts of lysates were separated by two individual SDS-PAGEs. One was used for the staining with Coomassie blue (CBB) to confirm the equal loading amounts, and the other was used to examine the expression of internal talin B or FLAG-tagged talin B by Western blotting analysis using anti-talin B antibody. The signal in the transformant was so strong that it did not appear as a band with this exposure time. Only a portion of the gel is shown for staining of Coomassie blue. (B) Fractions of cells adherent to agar surfaces under submerged conditions in HL5 axenic medium after 30 min of settling. All data are means ± SD (n = 3).
Cytokinesis of talA− talB− cells.Since a considerable proportion of talA− talB− cells grown submerged in HL5 axenic medium in plastic petri dishes were significantly larger than wild-type cells, we stained the nuclei of these cells with DAPI and found that the enlarged cells were multinucleated (Fig. 5A). The deficiency of talin A causes impairment in substrate-independent cytokinesis, which is consistent with the multinucleation of talA− talB− cells, although talA− cells are able to divide almost normally when they adhere to the substrates (26). In order to compare the numbers of nuclei in talA− and talA− talB− cells grown in plastic petri dishes with HL5 axenic medium under static detached conditions, we incubated both strains on nonnutrient agar submerged in HL5 axenic medium, which significantly inhibited the substrate adhesion of talA− cells as described above, and we counted the number of nuclei of the detached cells. talA− cells seemed to exhibit significant multinucleation, as expected, though we were not able to compare it with that of wild-type cells, because almost all wild-type cells adhered to the agar surface in this assay. More importantly, talA− talB− cells showed more-severe impairment in cytokinesis than talA− cells under these conditions (Fig. 5B).
Cytokinesis defect of talA− talB− cells. (A) Fluorescence images of DAPI-stained talA− talB− cells grown in HL5 axenic medium in a petri dish. (B) Comparison of the number of nuclei per cell between talA− and talA− talB− cells cultivated under detached and static conditions. All data are means ± SD (n = 3).
The talA− talB− double mutant is arrested at the early mound stage.The talB− mutant is arrested at the tight-mound stage, when the two cell types, prestalk cells and prespore cells, have already differentiated and their mutual sorting has occurred within the mound (35). We observed that the mound of talA− talB− mutant was flatter and more loosely packed than that of the talB− mutant (Fig. 6A and B). The flat and loose hemispherical structure is generally called a "loose mound" and occurs following completion of cell aggregation before cell sorting occurs within the mound. When allowed to develop on nonnutrient agar containing neutral red, which specifically stains prestalk cells, the red staining clearly concentrated at the top of the mound in the talB− mutant, as reported previously (Fig. 6C) (35). However, stained cells remained at the periphery of the mound and did not noticeably accumulate at the top of the mound in the talA− talB− mutant, suggesting that cell sorting has not been completed yet (Fig. 6D). In order to confirm the earlier developmental block of the talA− talB− mutant, we compared the expression levels of a prestalk-specific gene, ecmA, and prespore-specific genes SP60 and D19, the expression of all of which increases during tight-mound formation, between the mounds formed by the talB− and talA− talB− mutants (10, 15, 28). RT-PCR analysis demonstrated that all the three genes were less expressed in the mounds of the talA− talB− mutant than in the talB− mounds (Fig. 6E). Therefore, not only cell sorting but also differentiation was impaired in the mounds of the talA− talB− mutant, further implying that there is an earlier developmental arrest for the talA− talB− mutant.
Development in the talA− talB− double mutant is arrested at an earlier stage than in the talB− single mutant. (A and B) Top views of the plaques on bacterial lawns of talB− (A) and talA− talB− (B) strains. Cells inside the edges of the plaques started morphogenesis due to starvation. (C and D) The mounds of talB− (C) and talA− talB− (D) mutants stained with neutral red. (E) RT-PCR analysis of the marker genes in the development of talB− and talA− talB− mutants. The expression levels of a prestalk-specific gene, ecmA, and two prespore-specific genes, SP60 and D19, were analyzed at the mound stage in talB− and talA− talB− mutants by RT-PCR using primer sets to amplify each gene fragment. Ig7 is a constitutively expressed gene used as the control. (F and G) The processes of mound formation in wild-type (F) and talA− talB− (G) strains. Slugs formed at 15 h in wild-type cells are magnified in the bottom right corner of panel F. (H) Fruiting bodies formed by talA− talB− cells expressing FLAG-tagged talin B. The scale bars represent 1 mm (A, B, and F), 100 μm (C and D), and 20 μm (H).
We also monitored carefully the processes of mound formation of wild-type and talA− talB− cells on nonnutrient agar by taking pictures at certain time points. In wild-type cells, aggregation domains became visible on lawns of cells at about 8 h; these domains resulted in the rough surfaces of the lawns, and streams of cells were clearly visible at about 12 h when elongated cells were moving toward the aggregation centers to form mounds. Many of the wild-type mounds formed slugs at 15 h (Fig. 6F). In contrast, the lawns of talA− talB− cells gradually separated into mounds without forming discernible streams, and the formation of the loose-mound structures was completed at approximately 36 h (Fig. 6G).
Expression of FLAG-tagged talin B in talA− talB− cells rescued the developmental defect, confirming that disruption of talB caused the developmental phenotype of the talA− talB− double mutant (Fig. 6H).
Overexpression of talin A-GFP rescued the developmental block of the talB− mutant.The fact that developmental arrest of the talA− talB− mutant occurred earlier than that of the talB− mutant suggests that talin A is also involved in the developmental process. Therefore, we transformed talB− cells with the talin A-GFP expression construct, which was able to rescue the defects of adhesion to bacterial surfaces and of cytokinesis in talA− cells (see Fig. S1 in the supplemental material), to examine whether the overexpression of talin A-GFP compensates for the loss of talin B. Western blotting analysis confirmed the overexpression of talin A-GFP in the transformant (Fig. 7A). On nonnutrient agar, the talin A-GFP-expressing talB− cells formed mounds, many of which proceeded further to form aberrant fruiting body-like structures (Fig. 7B). The representative terminal structures had a tiny cell mass on top of a stalk extending from a thick column or formed an irregularly shaped bolus on a column (Fig. 7C and D). This result implies that the overexpression of talin A, at least in part, compensates for the loss of talin B during development. Interestingly, we also found that the expression of talin A was upregulated in talB− cells (Fig. 7A), while the expression of talin B was not significantly increased in talA− cells (Fig. 4A).
Developmental defects of a talB− mutant partially rescued by the overexpression of talin A-GFP. (A) Mounds formed by the wild type (WT), the talB− mutant, and the transformant expressing talin A-GFP (talA OE in talB−) were lysed in SDS sample buffer. Equal amounts of lysates were separated by two individual SDS-PAGEs. One was used for the staining with Coomassie blue (CBB) to show the loading amounts, and the other was used to examine the expression of internal talin A or talin A-GFP by Western blotting analysis using anti-talin A antibody. Overexpression of talin A-GFP in the transformant was confirmed, and the expression of talin A was found to be upregulated in talB− cells. Only a portion of the gel is shown for staining of Coomassie blue. (B) Top view of the final structures of talA OE in talB− developed on nonnutrient agar. (C and D) Two representative final structures of talA OE in talB− developed on nonnutrient agar. The scale bars represent 1 mm (B), and 40 μm (C and D).
Movement of talA− talB− cells at the early developmental stage.Individual talB− cells migrate less effectively than wild-type cells during development, which would explain the developmental block at the tight-mound stage (36). We expected that the mound formation without distinct streams and earlier developmental arrest of talA− talB− mutant might have resulted from more seriously impaired motility in the early stage of development; therefore, we compared the cell locomotion of wild-type, talA−, talB−, and talA− talB− cells at 4 h after onset of starvation in KK2 phosphate buffer. The starved talA− talB− cells were unable to move on glass surfaces (data not shown). Since we found that placing a thin agar sheet over wild-type cells in this stage enhanced locomotion, we decided to examine the motility of mutant cells in more detail under these agar overlay conditions. Both talA− and talB− cells migrated as actively as wild-type cells under these conditions (Fig. 8A and Table 1). In contrast, talA− talB− cells extended lamellipodia actively but did not show displacements, even when the motility of wild-type cells was enhanced (Fig. 8A and B).
Random movements in wild-type (WT), talA−, talB−, and talA− talB− cells starved for 4 h between a glass surface and an overlaid agar sheet. (A) Four representative traces of the random movements of each strain. Cell images were recorded every 3 min for 90 min. 1 pixel corresponds to 1.1 μm. (B) Panels show sequential recordings of two representative talA− talB− cells over a period of 12 min. The cells extended lamellipodia actively (asterisks). Time (in minutes) is indicated at the upper right of each panel. The scale bar represents 20 μm.
As talA− talB− cells failed to translocate under the experimental conditions described above, we investigated whether the cells form any adhesive structures. For this purpose, GFP-paxillin and GFP-actin constructs were introduced into wild-type and talA− talB− cells separately, since both molecules are known to accumulate at adhesion sites (2, 5, 38). Confocal microscopic observations identified a dot-like fluorescence of GFP-paxillin on the substrate-attached surfaces in transformants derived from wild-type cells. Dot-like fluorescent signals were not observed in the upper sections above the surface, suggesting that the fluorescent dots corresponded to adhesion sites (Fig. 9A) (see Fig. S2 in the supplemental material). However, the dot-like signals of GFP-paxillin were not observed on the substrate-attached surfaces of the transformants derived from the talA− talB− mutant (Fig. 9B). In contrast, GFP-actin dots were observed on the substrate-attached surfaces of both wild-type and talA− talB− cells (Fig. 9C and D).
Adhesion sites in wild-type (WT) and talA− talB− cells visualized by fluorescence microscopy. Fluorescent images of GFP-paxillin (A and B) and GFP-actin (C and D) at the substrate-attached surfaces in the wild-type (A and C) and talA− talB− (B and D) transformants. The fluorescent signals of GFP-paxillin and GFP-actin were observed using confocal microscopy. Adhesion sites revealed by GFP-paxillin were exhibited not only in wild-type cells forming clumps but also in single cells (see Fig. S2 in the supplemental material). The scale bars represent 5 μm.
The redundant and specific properties of two talins have attracted much interest since second talins were identified recently for various organisms (30). However, only limited information has been obtained regarding this issue. The functions of both talin A and talin B in Dictyostelium have been comprehensively investigated by analyses of each single mutant. These studies favored the notions that talin A is important for the vegetative stage, whereas talin B is required for proceeding through development, and that each talin has unique functions (26, 35, 36). These ideas are consistent with the time courses of the expression levels of talin A and talin B. The accumulation of talin A was maximal in the vegetative stage, although the transcript of talA gene was reported to be constantly expressed (18). In contrast, the accumulation of talin B reached the peak between the mound and slug stages, which roughly correlates with the kinetics of the expression of talB mRNA (35). In this study, however, talA− talB− cells exhibited more-severe defects than talA− and talB− cells in adhesion to the substrates and cytokinesis of vegetative cells, and in developmental arrest. These findings indicate that talin A and talin B also work in the developmental and vegetative stages, respectively, despite the fact that each single mutant did not show any detectable alterations in these stages. Furthermore, overexpression of each tagged-talin in the other talin disruptants largely rescued their impaired functions. Hence, this is the first report that demonstrates that two talin homologues in an organism have overlapping functions. We also found that the expression of talin A was upregulated in talB− cells, suggesting that there is cross talk between the two talins. The mechanism of regulation should be addressed in the future.
Although the distinct functions of these two mammalian talins have been emphasized, it is conceivable that they are also able to compensate for the loss of each other. Several cell lines lacking talin 1 do not show serious adhesion defects despite the fact that talin is a major molecule for activating an adhesion receptor, integrin (12, 29). In those cells, talin 2 may mediate the adhesion of cells lacking talin 1, as either talin A or talin B is sufficient for the substrate attachment in Dictyostelium vegetative cells. Further research into the elimination or knockdown of both talins in mammalian cells would be required to reveal the full extent of the defects caused by the loss of talin and the complete spectrum of talin's functions.
The stage-specific impairments of talA− and talB− cells could be caused by a reduction of the total amount of talin molecules if the properties of the two talins are completely redundant. In contrast to this model, overexpression of each tagged talin was unable to fully correct the defects of the mutant lacking the other talin, suggesting that talin A and talin B have specific functions. However, we cannot exclude the possibilities that a certain proportion of transformed cells did not express sufficient levels of the tagged talins and that this caused the defects manifested by the population of transformed cells or that the tagged talins were not fully functional for some unknown reasons.
talA− talB− cells completely lost the ability to attach to the substrates in the vegetative stage, suggesting that no other molecule is able to replace talin in this stage. Vertebrate talin generally mediates adhesion by activating integrin (6, 34). Five Dictyostelium molecules have been reported to have several features that are common with vertebrate integrin β, which include binding activity to talin (7). We speculate that Dictyostelium talin mediates adhesion through these molecules. In contrast to the vegetative stage, talA− talB− cells gained adhesion ability within 5 min when they were transferred to phosphate buffer. Thus, there is another adhesion mechanism, which is blocked in HL5 medium but active in phosphate buffer. Dot-like fluorescence of GFP-actin at the substrate-attached surfaces of talA− talB− cells sandwiched between a glass surface and an agar sheet of phosphate buffer also implies that they are capable of forming adhesion structures in the absence of HL5 medium, although it is not yet proven whether all of the GFP-actin dots are involved in substrate adhesion. Certain mutants generated by chemical mutagenesis were reported to be defective in attachment to a plastic surface in HL5 medium but not in phosphate buffer (37). The study suggested that adhesion molecules were altered in the mutants, though the genes responsible for this defect have not been identified. We suggest that the genes mutagenized in those strains were related to talin functions, though they cannot be talin itself, since a single knockout of one of the two talin genes did not elicit severe defects in substrate adhesion. Further studies need to be done to characterize the culture medium-sensitive, talin-independent adhesion.
Both talins are also important after starvation in Dictyostelium. The adhesion strength of talA− talB− cells was lower than that of wild-type cells even in phosphate buffer, and the cell motility was impaired in the early developmental stage in talA− talB− cells. In addition, talin is required for the formation of adhesion structures containing paxillin. A similar function of talin has been reported for mammalian cells. Although undifferentiated embryonic stem cells from talin 1-deficient mice are able to adhere to fibronectin, the adhesion structures do not contain paxillin. Also, talin 1-deficient mouse fibroblast-like cells are delayed in the initiation of the formation of paxillin-containing focal adhesions (12). Actin-rich dots and paxillin-rich dots were proposed to be distinct structures in Dictyostelium because their appearances and dynamics are different (5). Our study also showed that the dimensions of the dots enriched with GFP-paxillin were relatively smaller than the ones enriched with GFP-actin, and the dots of GFP-paxillin were localized in the peripheral region of the substrate-attached surfaces, while the dots of GFP-actin were evenly distributed. talA− talB− cells on nonnutrient agar were able to aggregate and develop until reaching the loose-mound stage. Therefore, they should be able to adhere to and obtain traction forces from the substrates. We speculate that the adhesion system represented by GFP-actin dots enable talA− talB− cells to develop until the loose-mound stage is reached. In addition, cell-cell adhesion might help talA− talB− cells to aggregate, because talA− talB− cells gradually packed into mounds rather than forming streams composed of actively moving cells, which appeared to be mediated by cell-cell adhesion. The paxillin-containing adhesion sites would be important for proper cell adhesions and transmission of motile forces in development.
We thank Kei Inouye, Department of Botany, Kyoto University, Japan, for his valuable comments on the manuscript.
This study was supported in part by Japan Society for the Promotion of Science and Special Postdoctoral Researchers Program of RIKEN.
↵▿ Published ahead of print on 28 March 2008.
↵‡ Supplemental material for this article may be found at http://ec.asm.org/.
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text:
@implementation NSOperationQueue (BaseKit)

////////////////////////////////////////////////////////////////////////////////////////////////////
- (BOOL)isEmpty {
    return self.operationCount == 0;
}

@end

redpajama_set_name: RedPajamaGithub