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- 6 Wand Candy Premium Silicone Wand Massager Attachments. Included are the Rabbit Thriller, Naughty Triple Thriller, Triple Play Thriller, VibaCup, Straight-Up Thriller and G-Curve Thriller.
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subset: commoncrawl


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'Dark Knight' Alleged Shooter James Holmes May Not Have Acted Alone
A moviegoer at the midnight premiere of 'The Dark Knight Rises' claims he saw someone prop open the emergency exit door that the gunman entered through.
Although police initially thought 'Dark Knight' alleged shooter James Holmes acted alone, a witness told CBS that the shooter may have had an accomplice.
"As I was sitting down to get my seat, I noticed that a person came up to the front row, the front right, sat down, and as credits were going, it seemed like he got a phone call," the witness told CBS. "So he went out toward the emergency exit doorway, which I thought was unusual to take a phone call. And it seemed like he probably pried it open, or probably did not let it latch all the way."
The witness believes the person who took the phone call may have propped open the emergency exit door, making it easy for the shooter to enter the theater.
"As soon as the movie started, somebody came in, all black, gas mask, armour, and threw a gas can into the audience, and it went off, and then there were gunshots that took place," the witness said.
Although the witness' claim could point to an accomplice in the 'Dark Knight' shootings, police are still focusing on James as the only suspect.
"All the evidence we have, every single indicator, is that this was all Mr. Holmes's activity," police chief Daniel Oates told CBS. "He wasn't particularly aided by anyone else."
More James Holmes News:


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Organizing events are never been so easier with the event websites. User from around the world can make their reservations on your site and can pay from their place itself. No more waiting in a long queue. These are the best event website templates you can use to build a user attracting event website.
When you come to an event website templates the biggest dilemma you will come across is, whether to choose a single page website template or a multi-page website template. Well, both the types have their own advantages. In the one page website templates only the most important information is provided for the users, so the users don't have to wander around the site to find the correct information. With the multi-page website templates, you have plenty of pages and spaces to explain in detail about the features of the event.
The second most important thing is the online ticket reservation feature. Some of the event website templates listed here support PayPal for receiving amount and the users can reserve their tickets. But for more convenient usage it is better to opt for a WordPress event themes. Because in WordPress you have plenty of customization and functionality extending features. To give you more flexible choice we have mentioned all types of event website templates on this list. Select the one that meets your needs.
If you are an event organizer, a website is an absolute must. There, you can tell all about the upcoming conference, forum or any other gathering. From introducing speakers and sharing schedule to other useful information that will persuade potential attendee to buy a ticket, make it all visible on your page.
To establish an event website, a template like Divi will do the trick. With very little work and barely any time needed, you can have an outcome that will spark their interest. To quicken things up, all you need to do is to choose a predefined demo and go from there.
What's more, Divi also comes with its own page builder, giving you a chance to customize it to your needs and branding regulations to a T. Doing the editing happens without coding; indeed, Divi offers visual coding which gives real-time results.
One more versatile and easily editable tool for building event and conference websites is Jevelin. It is easy to use, thanks to the WPBakery page builder. In other words, working with Jevelin asks for no programming knowledge. With that in mind, even if you are making your first page, you can still successfully do it with the practical Jevelin.
Moreover, there are also ready-made samples available for you to put them into play. Even if using Jevelin out of the box, the final product is a guaranteed masterpiece. But you know already that the drag and drop page building method gives you all the rights to make corrections and improvements accordingly.
Outstanding performance, search engine optimization, responsive layout, compatibility with browsers and retina-friendliness, Jevelin rocks it all and then some. It is an all-in-one item which brings to fruition top-notch event pages just like that.
Spker takes things to an entirely new degree with its outstanding look and performance. This event website template is packed with four demos, each creative, modern and attention-grabbing. Although these layouts are highly adaptive and ready to use as is, with Elementor page builder, you can customize them according to your meticulous taste precisely. After all, all it takes is some dragging and dropping and you are ready to roll. With that in mind, why wouldn't you want to brand Spker to your business regulations and make it unique?
Other goodness comes in the form of three schedule layouts, speaker profiles, map for attendees, call to actions, pricing plans and official sponsors section to name a few. You can also start a blog and keep everyone up to date, announce additional information and even share other compelling articles on the topic of the conference.
Important gatherings like conventions always look so formal, and Eventium is going to provide your attendees with that vibe. This event website template has a modern, neat and tidy look suitable for conferences, meetings, exhibitions, seminars, gigs, webinars and similar activities. In the package, Eventium provides all the essentials, divided into multiple sections with many layouts to choose from. There is no need to be building anything from scratch anymore.
Eventium gives you access to 22 HTML files which all are super simple to modify and edit due to their orderly structure. The template is also based on the famous Bootstrap Framework what gives it the much-needed flexibility and responsiveness. On top of that, Eventium is retina ready, comes with Sass files and entirely documented. Each Eventium user also receives pro support so you will never feel lonely.
Make captivating memories possible with a website that highlights their potential and attracts new folks. Emeet is an event website template that will do just that for you. It is a well-designed, super optimized and high performing HTML template perfect for meetups, conferences, congresses, seminars and similar pages. Basically, Emeet is so easy to customize you can use it for a broad range of purposes. Moreover, all layouts that Emeet has to offer look stunning on either desktop, tablet or smartphone.
Emeet is put together using the latest and greatest technologies and rocks a pixel perfect design. It has 18 different homes, ten internal pages and all sorts of enticing effects. Speaking of which, animated typewriter and scrolling parallax effects are just a few of the cool extras of the amazing Emeet.
Harmoni is a tool that has all in store for building websites for event management companies. There is loads of predefined content at your disposal, from four sophisticated front to several other inner pages. Pick what works best for you and start from there. In fact, you can use Harmoni exactly how it comes out of the box. Still, if you truly want to make it your own, feel free to perform different tweaks and customize Harmoni to your likings.
List and grid views for events, an entire event booking page, six color presets, boxed and full-width layouts, you name it, Harmoni delivers the necessary and then some. Countdown timer, large slideshow, call-to-action buttons, sticky navigation and additional PSD files, all this is what you will find in the Harmoni kit.
Building a suitable website to promote social events, DJ, night clubs, parties, dance clubs and so on requires a flexible event website template. Anondho is suitable for the features it has to build such a stunning and attractive platform. This will provide an excellent experience for your visitors, getting all the necessary information about the forthcoming event. Using Anondho as an event website template ensures an active online presence that makes the platform appropriate for all kinds of devices.
Anondho is easy to use, and you can change the settings without the need to write a single line of code. It's the Elementor page builder that does the magic for you. With Anondho, you will also enjoy 24/7 support to get your website running smoothly when in need of help. Get the Anondho theme today and promote your business effectively.
People will prefer an event platform that has excellent features and can showcase the works of the speakers and organizers. Booth offers you the opportunity to promote a responsive website that will encourage potential attendees to take action. It is easy to build and requires no coding, so both the inexperienced and experienced can use this event website template to its full potential.
Booth comes with nine beautiful home page demos, all easily editable and adjustable to make them match your branding directions. Still, they work fantastically well out of the box, too. Moreover, the tool includes several shortcodes that make it easy to integrate new features to the website. In short, with Booth you have all the necessary amenities you need to bring into fruition a first-class event page. It enables you to build and promote your event, conference, forum, social gathering, you name it, professionally.
Event organizers that require to place more information on their platform should try Voelas WordPress theme. This is one of the best event website templates that will help you to organize schedules, speakers, pricing, blog post, news and other features, all in one place. With Voelas, you are giving your visitors transparency and more information that will improve their user experience. Not just that, but the likelihood of buying the tickets increases.
Voelas allows visitors to do more while promoting your business and make a profit. It provides the options of booking tickets, seeking sponsors, signing up for newsletters and other call to action formats. Some more specialties of Voelas include Elementor, Slider Revolution, testimonials and Bootstrap Framework. The layout is also 100% mobile-ready, cross-browser compatible and in tune with modern retina screens. In conclusion, with Voelas, you are ready for a difference that will produce great results.
For events and all sorts of exhibitions, you better check out Blazen event website template. This is a feature-packed site canvas that brings to the table a wide specter of options and numerous possibilities. The time has come to create the exact page that you want for your forthcoming project and spark the interest of even more potential attendees. Instead of doing things from the ground up, do yourself a favor and let a template help you out.
Blazen is flexible, mobile and retina ready, compatible with all modern web browsers and sports a working AJAX contact form. In addition, Blazen has two home pages and all the must-have inner sections, like about, gallery, speakers, blog and FAQ. Push your event's details in a stunning way and show the world what they can expect from the meeting.
Bands, musicians, concerts and gigs, MP9 is the ideal event website template. It requires very little work to craft your very own website, get online and stand out from the crowd. MP9 has a beautiful dark layout which just happens to be responsive and retina ready. However, that is something all templates should sport to treat the modern mobile web user right. As far as the technical part of the tool goes, MP9 is built using all the up-to-the-minute technologies, as well as follows all modern web trends.
In total, MP9 has fourteen HTML files, is simple to adjust and does not shy away from any modifications you would like to perform. That said, enrich it with your personal touch and do your thing like pros do it. Whether you are the one performing music or you are the one organizing a party, MP9 template is ready for both of you.
If you can spare a few minutes of your time to publish a flawless event website, then Wiscon is within your reach. It is an event website template known for its functional and friendly features, having its specialization on the field. Providing the fundamentals in web building, it is no wonder why this template has been favored by organizers, companies, planners and administrators. With two preset homes, you can quickly decide how you will present your conference or event on the web.
Of course, you do not need to end the customization process here.
Feel free to upgrade and improve the main look of Wiscon to suit your needs precisely. Insert texts, titles, photos, logos, files, forms and any other content you would like to push. In short, modify Wiscon to your heart's content. What's more, you also gain access to professional support which lets you start your website confidently.
Get a kickstart with your website by trying out the Fizcon template. Completeness and versatility are two characteristics that put this event website template on top of its competitors. In fact, Fizcon is way more generous than any conference template out there. Designing a website has never been this easy – select a home page demo that you prefer, and you can even modify it according to your concepts as well. For your information, Fizcon rocks four homes and twelve additional internal sections.
Entice your attendees' anticipation further with the program section, containing pertinent information like event date, location and speakers. Fizcon has a clean, modern and minimalistic look that will spark everyone interest and encourage them to take action. Other features are transition and parallax effects, smooth scrolling, working contact form and a free lifetime worth of updates.
Launching events can be fun, no matter how colossal or private they are. However, certain complexities make it a bit challenging, like spreading the news to everyone. Well, Maxbeat has the perfect solution to this issue. This event website template lets you personalize layouts, add spectacular features, integrate media forms, build amazing pages and more. Indeed, a website is one of the best mediums to market your events, conferences, forums and meetups.
Many beginners and accomplished developers use Maxbeat, essentially because it's easy and fast to learn, yet the results are always breathtaking. You can get features such as contact form, blogs, sliders and pricing plans. There are also three pre-built index page demos and ten additional layouts for a fast completion of your online presence. Maxbeat enables potential attendees to find you effortlessly.
Is it really possible to put simplicity and elegance in the same sentence? Well, for Gen8, it is! It is an event website template that features some truly impressive material for you to use in crafting the best web space for your needs. Gen8 features two one-page layouts and all the other additional features and assets to cover every section of your page. Created with Bootstrap 4, Gen8 is clean, stylish and contemporary. Gen8 creates a smooth browsing experience with zero distractions.
Gen8 is full commented with an option to style the layout with SCSS or CSS for your convenience. In addition, it is fully responsive and compatible with pretty much any browser imaginable. For an added touch, loads of attention-grabbing sections come part of the kit which you rarely see in the classic event websites.
If you are after a cool, nifty and fun event website template, chances are, you need nothing else than Eventex. This tool is enhanced with great characteristics that will help you impress and amaze all your site visitors and turn them into potential attendees. While it keeps things super clean and minimal, Eventex still shows all the needed via its segments that an event or similar page requires. That said, Eventex has five homes and twelve inner pages to choose from for a fast and straightforward page establishment.
Some of the great features of Eventex are countdown timer, parallax effect, smooth scrolling, event schedule, pricing tables and a working contact form with Google Maps. There are also social media icons and a newsletter subscription form to use to capture guests' emails for future email promotions. Get things rocking on the web with Eventex.
Many people underestimate visuals when it comes to event pages, not knowing that this indirectly stimulates them to visit the gathering in the first place. Do not be that person when setting up a website. With EventTalk, your page will be more alive and interactive and those willing to take part in the event will appreciate it even more. EventTalk is ideal for any individual event, as well as event management companies who are dealing with loads of different seminars, conferences and other whatnots.
EventTalk is a beginner-friendly tool which is easy to use and modify according to your needs. It comes with loads of specialized features and pages that will help you out tremendously. From four different homes and four event schedule pages to about, shop, sponsors, speaker details and schedule sections to name a few. EventTalk includes all the mandatory and a whole lot more.
Event Pro
Choosing a well-organized template enables you to unleash your creative genius in designing a quality website, even if you do not have any talent for doing so. All of a sudden, you become a pro with close to none prior experience. This is what the Event Pro tool promises. It is an event website template suitable for companies that plan meetings, as well as manage events, no matter how big or small they are. Even if it is just a single seminar, Event Pro is a great option to spread the word out, too.
Event Pro is constructed with the latest technologies, like HTML5, CSS3, Bootstrap, jQuery and LESS. It has four front and ten internal pages and additional eight predefined stylesheets. With access to Event Pro, you can get your hands on many pre-built sections and pages to play around with. All you have to do is plan what you need, select a design, input everything and boom! The template itself will do the magic for you.
Couple Heart
Wedding invitation cards are getting outdated and so old-school, not to mention, they cost a fortune. Do you want to wow your guests and still retain a sacred matrimonial vibe? Do things effectively with Couple Heart. This is the event website template that works best with wedding events, whether you are the planner or the bride or groom. Couple Heart comes with all the essential features to effectively spread out the message to your friends and family.
In the bundle, Couple Heart has more than 150 HTML pages of which 20 are homes so you will surely find a design that matches you. It is also coupled (no pun intended) with gorgeous shop pages, eleven headers, five footers and all sorts of other goodies in case you are turning this website into a business.
Wedding Invitation
For couple events and celebrations, Wedding Invitation is the event website template you should look into. It has a ton of stuff at your disposal, so you do not need to do anything from the ground up. All you can do to improve the tool is to modify it and make it follow your regulations to the T. That said, Wedding Invitation is totally customizable and easily adaptable to suit your needs perfectly. The only limitation there is is your imagination.
Wedding Invitation template is packed with eleven different wedding pages, dark and light layouts, five preset color skins and Revolution Slider. With a small amount of time and effort invested, you can do remarkable things using Wedding Invitation. The web design is also mobile-ready and cross-browser compatible to work smoothly at all times.
Utouch is a large website template which you can use for multiple purposes, including event websites. Whether you are a startup or any other business who is continuously up to something, hosting different events, meetings and forums, you should better further investigate Utouch. The tool has a contemporary and delightful look that will definitely attract everyone who will ever come across your web space. Make it impressive and they will be intrigued to learn more about your content.
In total, Utouch comes with 34 HTML pages, from tempting front sections to all the requisite internal components. Sliders, galleries, SVG icons, sticky header with four hover effects, drop-down menu and footer parallax feature are some of the assets Utouch is equipped with. If you need something different or even if you already run a business but you feel a refreshment is needed, pick Utouch.
Paper is a multi-purpose website template which has enough pre-built material at your service to craft the exact page that you want. From numerous homes and inner pages, Paper also comes with an admin panel from where you can have complete control over your online project. Paper is a modern, clean, neat and optimized page canvas that is exceptionally simple to use for a quick site launch. The performance is top-notch across all devices and platforms for satisfied visitors.
Mega and off-canvas menus, portfolio and blog pages, shortcodes, parallax effect, pricing tables, you name it, all this and a ton more gives you the freedom to construct the right page for your upcoming event. With Paper, you are always one step ahead of the competition, always making sure all your users and guests get the exact same thing that they are looking for.
Event Organizer
Event Organizer is a fresh new modern looking event website template. This template is an apt choice for the event organizing companies. It includes all the necessary features and options you need to create a professional website.
Trendy color gradients are used as the primary color scheme of this template. Event Organizer website template includes five homepage variations, each one of them is unique from each other. This template uses HTML5, CSS3, and Bootstrap framework, along with these codes this template also supports Javascript to help you get well-animated web elements. This template uses custom icons that fits overall design style of the template.
What's Your Choice?
These are the best event website templates you can use for your event website. To give you more flexible choice we have listed both the feature-packed website templates and simple website templates. Based on your needs select the best event website template for your site. If you are still not able to come to a conclusion of which is the best template for your site, take your time to see our other theme and template collections for a better idea.


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URL: www.teamstrykercomic.com
Creator/s: Andrew Grieve
Run: 2/12-current
Schedule: Mondays
Section/s: "Space Commies from Outer Space"
Website: The explosion design's awesome, and it manages to make the comic seem exciting even though it's a fairly simple graphic. The large, detailed title also helps to make the comic look bold and impressive, and the oranges and reds match the coloring in the artwork and layout. ComicPress sites are usually pretty plain, so this is a great example of how they don't have to be just a generic template. It's too bad the creator's name isn't centered, though, as it's unclear what he was going for by putting it off to the left.
There's enough bonus content that the site would be impressive all-around if it wasn't for an extremely annoying "feature," which is that every comic page is followed by its penciled and inked versions. This means that the reader has to click the "Next" button three times just to keep reading the story, and it's annoying enough that I don't know if I would've kept going if I wasn't doing a review. I guess it's a way to get more ad revenue, or to encourage readers to buy the print versions, but it mainly just seems like a dumb gimmick that's probably preventing this webcomic from being more popular.
Writing: Most of the superhero webcomics that have been reviewed on this blog are parodies, and the idea seems to be to present an alternative to mainstream American publishing. However, there isn't actually a competition between print comics and webcomics, as webcomics are just so much better. So, I'll go ahead and make it official: webcomics won, print comics lost. From now on, the latter only belongs in museums and in the hands of antique collectors.
The death of print comics was caused by the medium becoming a circlejerk. It's messed up that you have its creators and fans giving its characters cutesy nicknames and taking its stuff so seriously when the Internet's the place for modern, high-quality comics. Yeah, the print stuff has amazing artwork, but the concepts and the way they're executed are unambitious and plagued by low expectations. With webcomics, though, there are so many of them and they cost nothing to read, so something has to be really special and competently done in order to get noticed at all. In addition, since it's so difficult, time-consuming, and unrewarding to be a webcartoonist, these individuals have to be super-passionate and force themselves to push their limits. Obviously, there are a lot of duds, as well as webcomics that are popular despite sucking and/or relying on fan-service, but the Internet's the main publisher now. And while I've seen articles that are, like, "Comics... on the Internet?!" it's clear that those writers are hopelessly out of touch. Webcomics have only really been around for about fifteen years, but it shows just how pathetic the print industry is that they were able to be dominated so quickly. Also, superhero movies are as big as ever, so the genre's still appealing, and it's just a matter of movie studios reacting to higher expectations.
Team Stryker's another webcomic that parodies superhero comics, and particularly their nationalistic aspect. In "Space Commies from Outer Space," it's making fun of the idea of some dumb meatheads promoting American values, and it doesn't work because it's tied to outdated contexts. The Cold War and superhero comics are both historical subjects, and they hold this webcomic back from being modern enough to be noteworthy. The jokes about drinking vodka and stuff are quirky enough to be a little entertaining, but they're not substantial enough to do an entire issue about them, and especially not multiple issues. It's also problematic that each of the four team members goes through their own communist transformation, making the gag quickly become repetitive. I get that it's anachronistic and all, but there's room for contemporary social commentary that could've made this a way better comic.
Another big problem with the writing is that it's just super-lazy. None of the meathead protagonists are memorable, and the only ones with a bit of personality are Casanova, who quips about being a ladies' man despite there being no women in the comic, and the "random" character, V-Scope, who appears to be mentally handicapped. And the villains are generic, nameless Martians, so there's no characterization there either. The webcomic might be trying to make fun of bland superheroes, but in that case there should really be more exaggeration so that it's actually goofy rather than just dumb. The other lazy part is that the grammar and spelling get a lot worse as the story progresses, which suggests that the creator hasn't been bothering to read his own webcomic. There's "comminism" instead of "communism," "Regan" instead of "Reagan," and "Uranius" instead of "Uranus," but lately, common words have started being misspelled too. For example, in the second issue, there's "gret" instead of "great," "lizzard" instead of "lizard," and "rile" instead of "rifle," in addition to other mistakes. Also, on the page where "great" is misspelled, there's an obvious screw-up where some of the dialogue's missing the speech bubble.
Art: Action's handled really well, with dynamic poses, detailed armor and muscles, and slick coloring, and it's impressive that the creator's been able to post high-quality action sequences week after week. The scenes are also pretty complex, as there are always multiple characters interacting, and there are plenty of busy panels with Martians everywhere. The pages get sort of tedious after a while, though, as all of the characters have a permanent "angry badass" expression, and the backgrounds are almost all just empty moon terrain. I mean, the creator does the best he can with the setting, but there are too many pages spent in such a boring location. Another problem is perspectives, as the creator struggles to draw characters properly when viewed from above(1, 2, 3).
Splash pages and double splash pages are generally ineffective since they waste opportunities for character and plot development. The first chapter uses double splash pages twice (1, 2), and while the artwork looks great, the dialogue's so tiny that it's barely readable. Fortunately, the creator provides links to larger versions in the comments sections, but it's an awkward setup just to add something that doesn't really benefit the comic anyways.
Finally, Comic Sans is just an ugly font, and while it has the word "comic" in its name, it doesn't belong in comics (or anywhere, really). The font used for the Martians' dialogue is better, though, and the special fonts used to introduce characters are a nice touch.
Overall: Team Stryker has some pretty good action scenes and shows some solid design skills, but all aspects of the writing are handled in a really apathetic way. It's like the comic's supposed to just be cool drawings of meatheads punching each other, and while it's only a parody, it's a dumb parody of dumb, old print comics. A much better idea would be to do a parody of webcomics, as that's something that hasn't been done as much before, and it would work better since it'd be more targeted towards webcomics readers. And while it might seem unfair to amateurs that they have to deal with higher expectations than the pros, it's ultimately helpful since it pushes them to make better comics.
|Scores (out of 5)
|Recommended sentence


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On November 11 in Jerusalem one of the world's best known scholars of the New Testament died, Dominican Father Jerome Murphy-O'Connor, 78. Father Jerry, as he was known, was certainly the best known priest in Jerusalem. He lived with poor health in recent years.
A tall man with a big personality was certainly a force to be reckoned with on all planes. He was certainly a provocative thinker, particularly on Saint Paul, was one who pushed the boundaries; but he was a man of trust in Divine Mystery. Some may say he was a modernist scholar; a keen interest was the real humanity of Jesus, especially as Jesus approached the crucifixion. Hence, you may not agree with all things that he said, but one would hope that you'd do your own research and draw your own conclusions, but you can't dismiss out of hand professional and honest work. I certainly think history will show us that JMC was a on to something.
His last book was, Keys to Jerusalem: Collected Essays (Oxford, 2012).
I met Father Jerry at University of Notre Dame several years ago while he was there doing some teaching and lecturing in NT studies.
Several articles ought to be read:
- The Franciscan Media Center produced this video clip of Father Murphy-O'Connor's funeral.
- Catholic Herald's "Church mourns renowned Bible scholar Fr Jerome Murphy O'Connor."
- The Telegraph obit, "Father Jerome Murphy O'Connor – Obituary"
- Brian B. Pinter has an appreciative essay of Father Jerome in America, "Jerome Murphy-O'Connor, O.P.: A Life well Lived."
- And a 2012 interview given to the Catholic Herald, "Jesus had a brief nervous breakdown" on what is speculated happened to Jesus in Gethsemani.
May God be merciful to Father Jerome. May Our Lady and Saint Dominic guide Father to the Beatific Vision.


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During development, morphogenesis involves migration and changes in the shape of epithelial sheets, both of which require coordination of cell adhesion. Thus, while modulation of integrin-mediated adhesion to the ECM regulates epithelial motility, cell-cell adhesion via cadherins controls the remodelling of epithelial sheets. We have used the Drosophila wing epithelium to demonstrate that cell-ECM interactions mediated by integrins also regulate the changes in cell shape that underly epithelial morphogenesis. We show that integrins control the transitions from columnar to cuboidal cell shape underlying wing formation, and we demonstrate that eliminating the ECM has the same effect on cell shape as inhibiting integrin function. Furthermore, lack of integrin activity also induces detachment of the basal lamina and failure to assemble the basal matrix. Hence, we propose that integrins control epithelial cell shape by mediating adherence of these cells to the ECM. Finally, we show that the ECM has an instructive rather than a structural role, because inhibition of Raf reverses the cell shape changes caused by perturbing integrins.
One fundamental question in developmental biology is how the zygote acquires the complex shape of the adult organism. Although embryonic tissues are shaped in a specific manner in different species, the fundamental process always involves the rearrangement of cell layers and the movement of cells from one location to another. This is mainly accomplished by changing the shape of individual cells or of groups of cells. In many cases, changes in cell shape are necessary for cells to move to distant locations where they aggregate to form tissues. In other cases, groups of cells undergo such changes in a coordinated manner, shaping and orientating tissues. Although, in the past few years a great deal of progress has been made in understanding how cell movement is regulated in animals during morphogenesis, little is known about how the changes in cell shape associated with epithelial morphogenesis are controlled (reviewed in Schock and Perrimon, 2002).
The developing Drosophila wing provides an excellent system to investigate the molecular mechanisms that control the changes in cell shape associated with rearrangement of epithelial sheets during organogenesis. Wing formation is a relatively simple process involving the unfolding and flattening of an epithelial sac, the wing imaginal disc. Indeed, this process has already been described in detail elsewhere and, hence, we will just summarise some relevant aspects here (Fristrom and Fristrom, 1993). The formation of the wing starts during early embryonic development when about 30 cells are allocated to form the wing imaginal disc. These cells will ultimately give rise to the adult wing and most of the thorax. During larval life, the number of cells in the disc increases to about 50,000 (Garcia-Bellido and Merrian, 1971) and in this proliferative phase, the imaginal disc forms an epithelial sac comprised of a folded columnar epithelium on one side and a squamous epithelium (the peripodial membrane) on the other. At the onset of metamorphosis, some 2 hours after puparium formation, local changes in cell shape promote the transformation of the single-layered columnar epithelium to a flattened bi-layer. More specifically, wing margin cells shorten, while cells on either side of this margin expand their apical surfaces and become wedge-shaped. These cell shape changes appear to drive the bending of the wing pouch along the wing margin (Fristrom and Fristrom, 1993) (Fig. 1). Subsequently, the rest of the dorsal and ventral wing surfaces become apposed, tight contacts are formed between the intervein regions of the wing, and the cells adopt a more cuboidal shape. During the remainder of pupal development, an additional round of division and further flattening of the epithelial layers accompanies the increase in the surface area of the wing.
Although the molecular mechanisms that regulate the changes in cell shape underlying wing morphogenesis remain elusive, it has long been thought that the extracellular matrix (ECM) participates in this process. The trypsin digestion of imaginal discs in vitro produces the partial dissociation of cell contacts at the basal surface, and this leads to cell flattening and partial evagination (Poodry and Schneiderman, 1971). Even though it was not originally clear how trypsin digestion might accelerate this process, it was proposed that specific proteolytic cleavage of ECM components triggers the cellular events that normally mediate the subsequent changes in cell shape.
The integrin family of cell surface receptors mediates most interactions between cells and the ECM. Each integrin is a heterodimer of a α and β subunit that extends across the membrane. The extracellular domain of integrins recognises and binds to ECM proteins, whereas the intracellular domain associates with cytoskeletal elements. Integrins are fundamental for the cell to adhere to the ECM and for the transmission of signals from the ECM to the interior of the cell, signals that influence a multitude of cellular activities. During epithelial morphogenesis, integrins have been shown to be involved in cell migration and recent experiments in Xenopus embryos have shown that they are also required for cell intercalation that drives epiboly at gastrulation (Marsden and DeSimone, 2001). The role of integrins during Drosophila wing morphogenesis has been studied extensively (reviewed in Brower, 2003; Brown et al., 2000). Two integrins are expressed in the Drosophila wing, the αPS1βPS (PS1) on the dorsal side and αPS2βPS (PS2) in the ventral domain. Eliminating integrin activity produces a loss of adhesion between the two surfaces, generating a blister in the adult appendage. Thus, it was proposed that during pupal stages integrins hold the ventral and dorsal wing epithelia together.
We have identified new roles for integrins during epithelial morphogenesis using the Drosophila wing disc epithelium as a model system. Here, we show that integrins play an essential role in regulating the changes in cell shape underlying epithelial rearrangements during organogenesis. Specifically, we have found that eliminating integrin function in the wing disc epithelium results in a precocious change from a columnar to a cuboidal cell shape. In addition, we show that eliminating ECM components without affecting the distribution of integrins results in similar changes in cell morphology. These results suggest that these activities depend on the interactions between integrins and the ECM. In addition, we found that abrogating integrin function also leads to a failure to correctly distribute and/or assemble the laminin-A-containing matrix. Finally, we identify the Raf pathway as a potential regulator of these activities.
Expression of dominant-negative chimeric integrins results in changes in cell shape and disorganisation of the ECM
To determine whether integrins influence the changes in cell shape that underlie wing morphogenesis, we eliminated integrin function in large areas of the wing disc by expressing chimeric integrins that have been shown to act as dominant negative both in cultured cells and during animal development. These chimeras contain the β integrin cytoplasmic domain fused to an extracellular reporter domain, and have been shown to inhibit endogenous integrin activity that mediates cell spreading, cell migration, and matrix assembly in cell culture experiments (LaFlamme et al., 1994; Lukashev et al., 1994). During Xenopus development, expression of these chimeric integrins blocks radial intercalation in the blastocoel roof (Marsden and DeSimone, 2001). Similar chimeras have been generated that can be used in Drosophila, and these contain the integrin βPS subunit cytoplasmic domain fused to the extracellular and transmembrane domains of mutant forms of the Torso receptor tyrosine kinase. These chimeras localise to the sites of endogenous integrins (Tanentzapf et al., 2006). There, they can act both as activated integrins and as dominant negatives. Whereas on one hand, they can substitute for the endogenous integrin and regulate integrin target genes in the Drosophila midgut (Martin-Bermudo and Brown, 1999) they can, on the other hand, also inhibit cell adhesion, matrix assembly and cell migration mediated by the endogenous integrins (Narasimha and Brown, 2004; Tanentzapf et al., 2006). Since the key feature of this chimera appears to be the dimerisation of the βPS cytoplasmic tail, we refer to it henceforth as diβ.
We used the UAS-GAL4 system (Brand and Perrimon, 1993) to drive expression of diβ in the wing. The expression of diβ in the whole wing pouch by using the GAL4 line CY2 (Queenan et al., 1997) produced a blister in the adult wing. Whereas this phenotype strongly resembles that of loss of integrin function, suggesting that expression of diβ in the wing was causing a dominant-negative effect (Fig. 2B), wing blistering may also result from a gain of integrin function (Baker et al., 2002). To confirm whether diβ was indeed inhibiting integrin function, we analysed whether the wing blister observed upon diβ overexpression was sensitive to mutations in the βPS subunit. If diβ was behaving as a dominant negative chimera, then reducing the endogenous integrin activity should enhance the effects of diβ overexpression. Since null mutants of the gene encoding the βPS integrin subunit (myospheroid, mys) are lethal, we used two hypomorphic homozygous viable alleles, mysB43 (Fig. 2A) (Jannuzi et al., 2004) and mysnj42 (Wilcox et al., 1989). In control flies, overexpression of diβ in the wing produced small wing blisters in both wings in 100% of adult females (Fig. 2B). Under these conditions, 55% of males displayed small wing blisters in both wings, 21% showed small wing blisters in one wing and no blisters were observed in 24% of the flies. In either a mysB43 or a mysnj42 background, there was a dramatic enhancement of wing blistering in adult females and males, all of which displayed big blisters in both wings (Fig. 2C,D and data not shown). These results indicated that overexpression of diβ did indeed produce a dominant-negative effect in the wing.
We then assessed whether there was a critical period for the generation of blisters. The blister phenotype increased with temperature due to sensitivity of the GAL4 system. Thus, the animals were given a pulse of 29°C during either the prepupa or pupal period and wing blistering was assayed when the flies eclosed. In control experiments, flies were grown at 18°C continuously and blistering was observed in 2.9% of the cases. When cultures were given a pulse of 29°C during the prepupal period, and then shifted back to 18°C until hatching, blistering was seen in 52% of the cases. However, if the shift to 29°C was done during the pupal-period blistering was observed in 32% of the cases. Continuous culture at 29°C resulted in blistering in 100% of the flies (in all cases analysed n>100). These experiments suggested an early integrin function sensitive to diβ overexpression.
We next decided to investigate this early integrin function by analysing the effects produced by diβ overexpression. To directly compare affected and unaffected cells in the same wing disc we expressed diβ in subsets of cells using either the engrailed-GAL4 (en-GAL4), which drives expression in all cells of the posterior compartment (see inset in Fig. 3F), or Flp-out GAL4 to drive expression in clonal patches of cells throughout the wing disc (de Celis and Bray, 1997) (Fig. 3D,I). In both cases, the expression of diβ altered disc morphology producing additional folds (Fig. 3F-H, and data not shown). These additional folds could be due to an increase in proliferation or changes in cell shape. A change from a columnar to cuboidal morphology would expand the surface area of the wing disc and, given the constraint of the peripodial membrane, may result in the formation of extra folds. To study the morphological changes that arose upon diβ overexpression, affected wing discs were sectioned and stained with methylene and Toluidine Blue. Whereas cells in the wing pouch of late third-instar wild-type discs were elongated and columnar (Fig. 3C and anterior wild type side of wing disc in Fig. 3H), expression of diβ in the disc caused a reduction in cell height (compare posterior diβ+ cells with anterior wild-type ones in Fig. 3H). This reduction in cell height was accompanied with an enlargement of the apical surface (Fig. 3D,I) as revealed with an apical marker, the anti-phosphotyrosine antibody (Muller and Wieschaus, 1996). Moreover, staining with an antibody against phosphorylated histone revealed that cell proliferation was not affected (see supplementary material Fig. S1). Thus, diβ overexpression appeared to cause cells to lose their columnar morphology and become cuboidal, suggesting that integrins are required to maintain the columnar state.
Wing epithelial cells are surrounded by a thin amorphous basal lamina that contains type IV collagen, laminin and sulphated proteoglycans (Fessler and Fessler, 1989). Integrins have been shown to be required for the distribution of laminin A at the amnioserosa-yolk interface (Narasimha and Brown, 2004), while this is not the case for the rest of the embryo (Prokop et al., 1998). Thus, we decided to investigate whether integrin function in the wing imaginal disc also involved proper assembly of the basal matrix by examining lamininA distribution. In wild-type wing cells, laminin A localised to focal adhesion-like structures at the basal surface of the wing imaginal disc (as seen in the anterior en-GAL4/UAS-diβ wing discs, Fig. 3E,E'). This distribution is lost in posterior diβ+ cells (Fig. 3E,E'). A second form of basal matrix has been observed between the basal lamina and the basal surface of the imaginal disc, forming a loose fibrous network (Brower et al., 1987). Using an antibody that binds to a component of this matrix we found that in the mutant posterior domain this basal matrix was also affected, adopting a less fibrous appearance when in contact with these cells (Fig. 3J).
To analyse the consequences of diβ overexpression on subsequent morphogenetic processes, we analysed wing discs shortly after the onset of metamorphosis (4 hours after puparium formation). By this stage, the wild-type wing disc has folded along the wing margin, whose cells have already shortened. Additionally, projections emanate from the basal surfaces of the dorsal and ventral epithelia that, except for a small gap where the marginal vein forms, have become apposed (Fig. 4A,A'). However, although folding occurred in wing discs that express diβ in the whole wing pouch, using the GAL4 line 638 (Barrio and de Celis, 2004), the basal surfaces remained smooth, lacked projections and did not contact the apposed surface (compare Fig. 4B,B' with A,A'). In addition, folding did not always take place at the wing margin cells (Fig. 4A,B). During normal wing morphogenesis local changes in cell shape at the wing margin ensure that folding always takes place along the middle of the wing disc (see above). This process is affected in wing discs that uniformly express diβ, leading to a mismatch between dorsal and ventral surfaces (arrows in Fig. 4A,B). This is probably due to the fact that all the cells of the disc have now reduced their height. In fact, while in late third-instar wild-type wing discs wing margin cells can be morphologically distinguished from the rest by scanning microscopy (Fig. 4C), in diβ-expressing wing discs this was not the case (Fig. 4D) – although they maintain their identity (data not shown). In summary, eliminating integrin function in the wing disc by overexpressing the chimeric integrin diβ produces premature changes in cell shape from a columnar to cuboidal form and disruption of basal matrix assembly.
Eliminating integrin function in wing disc cells causes changes in cell shape and defects in basal matrix assembly
To confirm that the experiments with diβ have revealed a normal function of integrins in epithelial morphogenesis, we tested whether genetic loss of integrins would give the same defects. We used a mutation in the gene encoding the βPS subunit, which eliminates all integrin heterodimers expressed in the wing. We generated large numbers of cells that lack the βPS subunit in the posterior compartment by inducing clones of homozygous mutant cells with engrailed-GAL4 driving UAS-Flp (Fig. 5). Cells lacking the βPS subunit formed ectopic folds in mid- to third-instar wing discs, very similar to those obtained upon diβ overexpression (compare the posterior compartments in Fig. 5B and Fig. 3F). Similarly, the basal matrix on the mutant side of the wing disc looked less fibrous than that associated with the anterior wild-type cells, again reminiscent of the effects caused by diβ overexpression (compare Fig. 5D and Fig. 3J). Furthermore, sections of these chimeric mutant wing discs show that the mutant βPS cells have a reduced height when compared with their wild-type counterparts, as seen for diβ overexpressing cells (Fig. 5E-G). Thus, removing integrins recapitulates the phenotype obtained upon diβ expression.
Taken together, these experiments have identified new roles for integrins during epithelial wing morphogenesis. Integrins are required to maintain the columnar shape of epithelial wing cells and to mediate the correct assembly of a laminin-containing basal lamina. These functions are fundamental for the proper folding of the wing epithelial sheet and necessary to establish and maintain contact between the two opposing wing surfaces. It is possible that two of these events are connected, such that alterations in the basal lamina lead to changes in cell shape. Alternatively, integrins might regulate these two processes independently. To determine to what extent these activities depend on the matrix, we examined whether disruption of the ECM caused similar defects.
Ectopic expression of metalloproteinases mimics integrin loss of function
Evidence that ECM components participate in maintaining the wing epithelial cells in a columnar state was first presented some time ago (Poodry and Schneiderman, 1971). Trypsin digestion of imaginal discs in vitro provoked a precocious change from a columnar to cuboidal morphology in most cells, similar to the phenotype we observed upon disrupting integrin activity. Although the basis of this effect is poorly understood it may be that trypsin proteolysis cleaves specific extracellular proteins. We are now able to test this directly by expressing matrix metalloproteinases (MMPs) to cleave the ECM within the developing animal. The MMPs can cleave virtually all ECM components (Matrisian and Brinckerhoff, 2002), and two such MMPs exist in Drosophila, Mmp1 and Mmp2 (Page-McCaw et al., 2003). Indeed, these MMPs have been shown to be required for tissue remodelling but not for embryonic development. Only Mmp2 appears to be expressed in wing discs and, thus, we analysed the consequences of its overexpression in the wing disc. Since all GAL4 lines we had so far used in this study were lethal when combined with UAS-Mmp2, we then used the MS1096 GAL4 line that is mainly active in the wing pouch (Capdevila and Guerrero, 1994). Expression of Mmp2 in the wing disc resulted in the formation of extra folds in late third-instar wing discs (Fig. 6A,A',B,B'), Scanning microscopy show that in some cases, these extra folds seemed to extend to form a pouch (C). Indeed, most imaginal disc cells had adopted a cuboidal morphology rather than maintaining their normal columnar shape (Fig. 6F). In addition, the expression of UAS-Mmp2 in the wing discs produced blisters in the adult (Fig. 6G) – consistent with results showing that mutations in Timp, an inhibitor of MMPs, produce a blistering phenotype (Godenschwege et al., 2000). All these effects resemble those observed upon disrupting integrin activity and suggest that in these processes, integrins mediate cell adhesion to the ECM. However, the overproduction of metalloproteinases could also eliminate cell surface proteins – including integrins. Prior to pupariation integrins cluster into focal adhesion-like structures at the basal surface of the wing imaginal disc (Fig. 6D) (Brown et al., 2002). When βPS expression was analysed in wing discs, Mmp2 overexpression did not grossly alter their distribution (Fig. 6E).
In summary, disrupting the interactions between cells and the ECM, either by perturbing integrin activity or by cleaving ECM components, induces cells to prematurely adopt a cuboidal morphology. Hence, integrin-ECM interactions appear to be necessary to maintain the columnar cell shape. One way this could occur is simply structural, in that cells might need to hold on to the basement membrane to maintain their elongated shape. Alternatively, the contact with the ECM may signal to the cytoskeleton to maintain an elongated shape – independent of the adhesion. To distinguish between them, we examined the mechanisms of action of diβ.
The chimeric integrin diβ competes with cytoplasmic components required for integrin localisation and adhesion
We first examined whether diβ expression affected the distribution of endogenous integrins and found that integrins failed to localise into basal focal adhesion-like structures upon diβ overexpression (Fig. 7A,B). Furthermore, xz confocal optical sections of wing disc cells expressing diβ revealed that the βPS protein was still present on the lateral and apical surfaces (Fig. 7B). Although it seems as if there is less βPS protein in diβ+ cells, this could be due to the fact that mislocalised protein is less stable. In addition, analysis by in situ hybridisation showed that mRNA levels of βPS, αPS1 or αPS2 did not change (supplementary material Fig. S2 and data not shown). Hence, diβ affects the correct distribution of endogenous integrins. We had already proposed that diβ affects endogenous integrin activity by competing for cytoplasmic elements required for integrin localisation and adhesion. The cytoplasmic protein Talin is required for integrin clustering into focal adhesion like structures in the wing (Brown et al., 2002), and we found that diβ was able to recruit Talin (Fig. 7C-E). In fact, it seemed to interact with Talin more efficiently than the endogenous integrins, because more Talin appeared to be recruited to the sites of diβ expression. This is in agreement with our recent findings, showing that muscles overexpressing diβ do not contain integrins at their ends but can still recruit talin (Tanentzapf et al., 2006). These results suggest that diβ inhibits integrin function by binding and competing Talin away from the endogenous integrins. To test this we analysed whether overexpression of talin could rescue the bubble phenotype caused by diβ, and used either an UAS-talin construct (Tanentzapf et al., 2006) or an EP insertion line that inserts a GAL4-dependent UAS promoter upstream of the talin transcription unit, the EP line P{EPgy2}ergic53EY09736. However, because we were unable to rescue the bubble phenotype (see supplementary material Fig. S3), sequestration of talin cannot be the only cause of the diβ dominant-negative phenotype.
The effects of diβ might be mediated by sequestering other cytoplasmic proteins or, alternatively, its dominant-negative activity could arise from its ability to recruit cytoplasmic factors that negatively regulate integrin function. In a screen for suppressors of integrin activation, a MAP kinase pathway linked to the effector kinase Raf-1, was seen to regulate integrin activation (Hughes et al., 1997). Since clustering of integrins can activate the classic MAP kinase pathway, it was proposed that a MAP kinase dependent negative feedback loop might regulate integrins. In this scenario, the trans-dominant inhibition produced by diβ could in part be due to the activation of an integrin suppression pathway dependent on Raf.
Inhibition of integrin function by diβ requires a Raf-dependent pathway.
We tested whether Raf participates in the trans-dominant inhibition exerted by diβ, by coexpressing in the whole wing pouch (using the GAL4 line 638) a dominant-negative form of Raf (RafDN) (Martin-Blanco et al., 1999). Overexpression of RafDN almost completely suppressed the wing blister phenotype induced by diβ expression, levels fell from 98% to 2% (Fig. 8A-C,G). These results were confirmed using other GAL4 lines that also drive expression in the wing pouch, such as 69B and 179 (Brand and Perrimon, 1993) (Fig. 8G). In addition, coexpression of diβ with other GAL4-regulated constructs, such as UAS-β-gal or UAS-src-GFP, did not rescue the wing blister phenotype, demonstrating that suppression was not simply due to the titration of GAL4 (data not shown). To further test the involvement of Raf in the suppression of integrin activation by diβ we decided to analyse the ability of overexpression of RafDN to rescue the blistering effect due to genetic loss of integrin function. For this purpose, we used ectopically expressed RafDN in the posterior compartment of wings mutant clones for the βPS integrin subunit have been induced (see Materials and Methods). We found that overexpression of RafDN could not rescue the blistering phenotype (data not shown).
We next tested the ability of RafDN to suppress the effects of diβ on cell shape. Longitudinal sections of discs coexpressing diβ and RafDN in the whole wing pouch revealed that inhibition of the Raf pathway was able to suppress the change in cell shape by diβ (Fig. 8D-F). The effects of RafDN on the redistribution of endogenous integrins caused by diβ and on the integrity of the ECM were also evaluated. We were unable to directly compare wild-type cells and those coexpressing diβ and RafDN using the engrailed-GAL4 line because the expression of RafDN unexpectedly blocked the transcriptional stimulation of this construct. We therefore used the ptc-GAL4 line, which drives expression in a narrow band of cells along the dorso-ventral boundary (see inset in Fig. 9A), and the Flp-out GAL4 line (data not shown). Coexpression of RafDN with diβ resulted in a substantial redistribution of the endogenous integrin to focal contacts (compare Fig. 9A with 9B). In addition, the integrity of the basal matrix was also restored (compare Fig. 9D with 9C). Our results indicate that a Raf pathway is required for diβ to exert its dominant-negative effect on endogenous integrins. To further support our hypothesis, that the trans-dominant inhibition produced by diβ could in part be due to the activation of MAP kinase dependent negative feedback loop to regulate integrins, we analysed whether MAPK activity was enhanced upon diβ overexpression. In a wild-type third-instar larval wing disc MAPK activation is specifically detected in all veins and in the wing margin (Fig. 9E). In wing discs that express diβ using the engrailed-GAL4 line this pattern is altered and MAPK activation is detected uniformly in the posterior compartment (Fig. 9F).
The generation of form in early animal development involves key cellular process such as epithelial morphogenesis. The reorganisation of cell shape is commonly associated with epithelial morphogenesis, which requires a precise and coordinated remodelling of the cytoskeleton and the adhesive properties of cells. In view of the two predominant cell adhesion systems, there is considerable evidence indicating that interactions between cells and the ECM modulate the shape of cells in culture (Watt, 1986). Here, we have used the Drosophila wing to show that the regulation of cell shape by integrins also plays an important role during epithelial organ morphogenesis. Furthermore, we show that this integrin function relies on interactions with a matrix whose assembly also depends on integrin activity. Finally, we provide evidence that the Raf kinase may act as a putative intracellular regulator of this integrin activity.
Integrins are required for the maintenance of columnar cell shape in the Drosophila wing epithelium
Integrins are thought to perform two distinct functions in the wing: an early regulatory one in which integrins signal to make cells competent for re-apposition and a later, more traditional one, where integrins mediate adhesion (Brabant et al., 1996). Here, we have unravelled a new early function for integrins in maintaining the columnar cell shape of wing epithelial cells. We propose that maintenance of this columnar shape is necessary to achieve proper contact and recognition of cells on opposing surfaces during folding. Thus, interfering with this activity results in cells adopting a cuboidal shape, which prevents them from establishing appropriate dorso-ventral connections. These early connections are probably necessary for re-apposition and final adhesion between the dorsal and ventral epithelia. Indeed, disruption of integrin function by diβ overexpression during the initial apposition period results in the formation of wing blisters in the adult.
The simplest hypothesis as to how integrins maintain the columnar shape of cells is that they keep the wing disc cells firmly attached to the basal matrix. However, recent evidence supports the idea that integrins also play a role in mediating adhesion between the lateral surfaces of cells during the process of dorsal closure in the embryo (Narasimha and Brown, 2004). In the wing, integrins seem to be distributed basolaterally when cells are in close contact, such as during apposition and adhesion. By contrast, integrins are absent form the lateral cell surfaces and become restricted to basal junctions when cells diminish their basal contact, i.e. during the expansion period (Brabant et al., 1996; Fristrom et al., 1993). It therefore seems reasonable to propose that, integrins can maintain the columnar shape by mediating basolateral contact between adjacent cells.
Cell culture studies have shown that integrins and members of the Rho family of GTPases function in a coordinated manner to regulate the morphological changes that accompany cell spreading and migration upon binding to the ECM (reviewed in Schwartz and Shattil, 2000). In the Drosophila wing, the Rho GTPase Dcdc42 localises predominantly to the basal and apical regions of epithelial columnar cells. Furthermore, expression of a dominant-negative form of Dcdc42 results in a shortening of epithelial cells in the third instar larvae and produces a wing blister in the adult (Eaton et al., 1995). Therefore, it is possible that integrins and Rho GTPases also interact to regulate the changes in cell shape underlying epithelial morphogenesis during development.
Maintenance of the columnar state through integrins is also required for folding along the wing margin. During normal wing morphogenesis this is mainly accomplished by local changes in cell shape of the wing margin cells, involving a reduction in height. This ensures that folding only occurs at the middle of the wing disc, thereby allowing an alignment match between dorsal and ventral cells. We show here that, when integrin function is disrupted in most of the wing pouch, folding does not always occur along the wing margin, probably because all cells now adopt a similar shape. In fact, wing margin cells cannot be distinguished morphologically from the rest, although they do maintain their identity. This results in a mismatch between dorsal and ventral cells that might also be important for later differentiation processes. In summary, we show here that cell-ECM interactions mediated by integrins are required for the temporal and, most likely, the spatial regulation of the changes in cell shape that accompany the folding of epithelial sheets during organogenesis.
Integrin adhesion versus signalling
It still remains unclear to what extent signalling contributes to the activity of integrins during development. One of the main problems is how to distinguish between direct integrin signalling and the indirect effects caused by a lack of integrin adhesion. In Drosophila, the differentiation of some but not all cell types depends directly on integrin signalling downstream of diβ (Martin-Bermudo, 2000; Martin-Bermudo and Brown, 1999). The results presented here support the idea that the regulation of cell morphology by integrins depends more on integrin adhesion to the ECM than on signalling. Indeed, the diβ chimera does not prevent the changes in cell shape by interfering with integrin activity. However, we cannot rule out the possibility that the signal pathway activated by diβ does not fully mimic integrin signalling. In fact, we recently demonstrated in muscle that the chimeric diβ integrin is not capable of recruiting certain proteins associated with sites of integrin activity, such as ILK, paxillin, PINCH and tensin (Tanentzapf et al., 2006). Hence, it remains possible that the regulation of cell shape requires integrin signals that are only triggered when a complete integrin complex is assembled.
We have also generated additional evidence to support the idea that the interactions between integrins and the matrix affect cell shape. We found that the elimination of ECM components by overexpressing metalloproteinases provoked changes in cell morphology that strongly resemble those observed when integrin activity is disrupted. Moreover, overexpression of metalloproteinases does not affect the normal distribution of endogenous integrins, which can still cluster in focal-adhesion like structures. Hence, integrins alone are insufficient to regulate changes in cell shape but, rather, they must interact with ECM components. This is in agreement with findings that, in most cases, a threshold of both clustering and binding to integrins must be reached before fully functional focal adhesion complexes are formed (Miyamoto et al., 1995).
The interactions of cells with the ECM have long been proposed to involve `dynamic reciprocity', whereby a cell response to its ECM affects the composition of the new matrix it secretes, which in turn alters the ensuing response of the cell (Bissell et al., 1982). Here, we show that disrupting integrin function leads to changes in the basal matrix containing laminin. As such, it seems reasonable to consider a model by which the main function of integrins in regulating cell shape during wing development is the correct assembly and/or attachment to the ECM. An organised ECM can then in turn modulate the activity of the integrins themselves and/or other receptors to regulate cell morphology.
Raf as regulator of integrin function in the wing
Overexpression of chimeras containing the cytoplasmic domains of the β1 or β3 subunits reduces integrin affinity. By contrast, chimeras containing a mutated β3 cytoplasmic domain with defective inside-out signalling, reduce the ability of the β3 cytoplasmic domain to block activation (Chen et al., 1994). These results suggest that there are limiting factors that bind to the cytoplasmic domains of integrins and which regulate ligand binding affinity. Modulation of these factors could be a way of regulating integrin activity. Here, we show that diβ recruits the cytoplasmic protein Talin, opening the possibility that diβ exerts its dominant-negative effect by competing for Talin. However, this does not seem to be the case because overexpression of Talin does not rescue the diβ phenotype, contradicting data from CHO cells showing that competition for Talin underlies the trans-dominant inhibition exerted by isolated β tails (Calderwood et al., 2004). Nevertheless, we recently demonstrated complementation between mutations in different motifs of the βPS cytoplasmic domain that eliminate the dominant-negative activity of diβ (Tanentzapf et al., 2006). This suggests that, if the dominant negative activity of diβ were due to competition for cytoplasmic components, in our system this would involve the recruitment of at least two cytoplasmic proteins.
Alternatively, diβ could initiate a signalling cascade leading to the activation of a Raf-dependent integrin-suppressing pathway. Integrin-mediated adhesion to the ECM can trigger clustering and increase tyrosine phosphorylation of a number of intracellular proteins, including focal adhesion kinase (FAK), Raf, Ras, and MAPKs (reviewed in Boudreau and Jons, 1999). However, it has been shown that activation of MAPK suppresses high-affinity ligand binding in integrins (Hughes et al., 1997). Thus, a model has been proposed in which MAPK regulates integrin function through a negative feedback loop. Here, we show that a dominant-negative form of Raf suppresses the capacity of diβ to inhibit integrin function. Furthermore, we demonstrate that overexpression of diβ enhances Raf activity. Therefore, we propose that the trans-dominant inhibition exerted by diβ could result from the activation of a Raf-dependent signal transduction pathway that inhibits or modifies integrin-ECM interactions.
We also believe that the negative regulation exerted by the Raf pathway could be part of a negative feedback loop that regulates integrin function during normal development. If this were the case, the expression of RafDN would be expected to constitutively activate integrin signalling and, therefore. provoke changes in cell morphology. But, we have not observed any of these effects upon expression of RafDN in the wing disc. However, since integrins can activate other pathways that are Raf independent, affecting one of these pathways might not produce a dramatic effect because the other pathways may compensate this deficiency. In this context, our results suggest that the chimeric diβ integrin is not able to activate intracellular signals other than those associated to the Raf pathway – probably be due to the failure of diβ to assemble a complete integrin complex (see above).
The regulation of cell shape through cell-ECM interactions has been shown to have a dramatic influence on cell proliferation, patterning, differentiation, cell migration, cell branching and matrix production during development. Here, we show that these interactions also play a crucial role in regulating the changes in cell shape that drive epithelial morphogenesis underlying the formation of organs and tissues. We must now identify the molecular mechanisms by which these cell-ECM interactions influence the cytoskeleton and regulate cell shape during morphogenesis. The easily detectable wing blister phenotype caused by expression of diβ in the wing provides us with the foundation to screen for mutations in genes required to modulate these integrin-ECM interactions.
Materials and Methods
Drosophila stocks
The mutant integrin allele mysXG43 has been described by (Bunch et al., 1992), and the two viable alleles mysb43 (Jannuzi et al., 2004) and mysnj42 (Wilcox et al., 1989) were kindly provided by D. Brower (University of Arizona, AZ). The 638-GAL4 line is a homozygous insertion on the X chromosome (Barrio and de Celis, 2004), and the en-GAL4 and ptc-GAL4 lines are insertions of the GAL4 enhancer-trap construct pGawB (Bloomington) in the engrailed and patched genes, respectively, The chimeric integrin UAS-diβ has been described previously in (Martin-Bermudo and Brown, 1999). UAS-RafDN is a dominant-negative mutation in which the catalytic kinase domain has been inactivated (Martin-Blanco et al., 1999). UAS-Mmp2 was used to drive ectopic expression of the metalloproteinase 2 (Page-McCaw et al., 2003). UAS-talin, a full-length talin construct, has been described previously (Tanentzapf et al., 2006). The EP line P {EPgy2} ergic53EY09736 inserted upstream of the rhea (talin) locus was obtained from Bloomington Stock Centre.
Generation of mys mutant clones and diβ expressing clones
Mitotic recombination was induced using the FRT/FLP system (Chou and Perrimon, 1992). Larvae of the genotype y w mysXG43 FRT101/y w Ubq::GFP FRT101; HsFlp38 were subjected to heat shock for 1 hour at 37°C, allowed to develop and then dissected. To generate large βPS– clones in the posterior compartment, the FRT/FLP system was combined with the GAL4 system (Brand and Perrimon, 1993). Larvae of the genotype y w mysXG43 FRT101/y w Ubq::GFP FRT101; enGAL4/UAS-Flp38 were generated by crossing females of the genotype y w mysXG43 FRT101/FM6; UAS-Flp38 to y w Ubq::GFP FRT101; enGAL4/CyO males. Integrin mutant clones were identified by the absence of staining with either a monoclonal antibody specific for βPS or an antibody to β-galactosidase. To test the ability of RafDN to suppress the wing blistering due to genetic loss of βPS larvae from the cross between y w mysXG43 FRT101/FM6; UAS-RafDN/TM6 and hsGFhsFlp1.22 FRT101; enGAL4 were subjected to heat shock as described above. Wing blistering was compared between the female progeny with somatic mys wing clones (ywmysXG43FRT101/hsGFPhsFlp1.22FRT101; enGAL4/+; TM6/+) and females with somatic mys wing clones overexpressing RafDN (ywmysXG43FRT101/hsGFPhsFlp1.22FRT101; enGAL4/+; UAS-RafDN/+). To produce diβ expressing clones, larvae from the cross between UAS-diβ and ywhsFlp1.22; abx/Ubx [f+] GAL4-lacZ/CyO (de Celis and Bray, 1997) were subjected to the same heat shock treatment. Larval discs were dissected out and stained with an antibody against either β-galactosidase or the myc epitope to distinguish the diβ+ cells.
Immunostaining and histological sections
Imaginal discs were fixed in 4% PFA in PBS for 1 hour on ice and antibody staining was performed according to standard procedures. The primary antibodies used were: mouse monoclonal anti-βPS integrin CF6G11 (1:10; Developmental Hybridoma Bank), mouse monoclonal anti-talin (1:5) (Brown et al., 2002), rabbit anti-GFP (1:500, Molecular Probes), mouse monoclonal anti-phosphotyrosine clone pY20 (1:500, Zymed), rabbit anti-lamininA (1:250) (Gutzeit et al., 1991) and rabbit anti-β-galactosidase (1:5000, Capell); the monoclonal CN6D10 binds to a component of the basal matrix (1:500) (Brower et al., 1987), mouse monoclonal anti-myc (1:500 Oncogene Research Products) and anti-active MAPK antibody (1:200 Sigma). Alexa-Fluor-conjugated secondary antibodies were from Molecular Probes and were used at a dilution of 1:200. Actin was visualised with Rhodamine-phalloidin (Sigma) as described before (Eaton et al., 1995), and imaginal discs were sectioned and stained with Toluidine Blue as described before (Eaton et al., 1995). Images were collected using a confocal microscope Leica TCS-SP2-AOBS and scanning electron microscopy was performed using a Zeiss DSM 950 microscope at the Microscopy Unit of the University of Granada.
We thank D. Martín, for his assistance with the confocal microscope. We also thank the microscopy unit from the University of Granada (Spain), especially C. Hernández, for their assistance with the sections and the scanning electron microscopy. We thank A. González-Reyes, S. Gregory and I. Palacios for critically reading the manuscript, and M Sefton for improvements to the manuscript. This work was supported by grants awarded to M.D.M.B.: a Project Grant 054613 from the Wellcome Trust, a Royal Society University Research Fellowship (516002.K5510), a project grant (BMC2001-2298) from the Spanish Ministery of Science and Technology and a European Molecular Biology Organization Young Investigator Award. P.D.G. and M.D.M.B. performed experiments and analysed data. M.D.M.B. conceived, designed and wrote the paper. N.H.B. advised on the latter.
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/6/1061/DC1
- Accepted January 11, 2007.
- © The Company of Biologists Limited 2007


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California landlords need to be aware of the different ways in which a tenant can be legally compelled to leave the property. While there are five options when giving an eviction notice, there are three in which the tenant is simply being asked to leave or "quit" the property. Those that will not be discussed here provide alternatives for the tenant to remain. This post relates to the tenant moving out when asked to do so with no option to stay. There are three alternatives: a 3-day notice to quit; a 30-day or 60-day notice to quit; and a 90-day notice to quit.
A 3-day notice to quit is used when there are problems with a tenant who has created a nuisance, is using the property for illegal activity, has threatened the health and safety of other tenants or the public, has done damage that significantly reduces the property value, and moves in other tenants without the landlord's approval. In this notice, the following must be included in writing: the full name of the tenant, details of what the tenant did to warrant the 3-day notice with dates of infractions, and instructions that the tenant must move out once the 3 days have passed.
With a 30-day or 60-day notice to quit, the landlord can end a tenant's stay if it has been a month-to-month lease and the tenant has lived at the property for less than one year. The landlord will use a 60-day notice if the tenant has lived there for one year or more. This notice is required to be in writing, have the tenant's full name, have the property address, and say that the month-to-month agreement will be terminated in 30 days or in 60 days, depending on the situation.
The landlord must give a 90-notice if the residence is Section 8 or subsidized housing. The landlord is required to provide an explanation as to why the tenant is being asked to leave. There must also be just cause for the decision. It is perfectly legal for a landlord to request that a tenant leave with these notices. However, the law must be followed. If there is a problem with a tenant who refuses to adhere to the rental agreement or other issue, a landlord should speak to an attorney who is experienced with the eviction process.
Source: courts.ca.gov, "Eviction Notices," accessed on March 21, 2017


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Homework Helper
Help your child take control of his homework load. Your child may not realize it memorizing the periodic table at 2 a.m., but homework is a good thing. It helps your child:
• practice what was learned during the day;
• establish study habits that will be critical in college;
• prepare for classes;
• get a sense of progress.
Help your child take control of his homework load. Your child may not realize it memorizing the periodic table at 2 a.m., but homework is a good thing. It helps your child:
• practice what was learned during the day;
• establish study habits that will be critical in college;
• prepare for classes;
• get a sense of progress.
Homework tips:
How can you help your child get the most out of homework? Here are 10 ideas to get you started.
Set the mood
Help your child create a good study area with all the resources she needs (for example, a dictionary). If you don't have a quiet place at home, she should try the school or local library.
Know where to begin
Your child should make a prioritized list of everything she needs to do, so she can't use "I don't know where to start" as an excuse. It's important not to over-schedule. Without some flexibility, your child will set herself up to fail.
Study at the same time every day
Even if your child doesn't have homework, she can use the time to review notes. If homework is something your child accepts as part of her day, she'll approach it with less dread. Plus, she will become a pro at using time productively.
Keep things in perspective
Your child should know how much weight each assignment or test carries, and use her time accordingly.
Get more involved
Does your child ever feel like she can't stay awake to read something, let alone process it? To keep her mind from wandering, your child may want to take notes, underline sections, discuss topics with others, or relate her homework to what she is studying in another class.
Organize the information
People process information in different ways. Some people like to draw pictures or charts to digest information, other people like to read out loud or make detailed outlines. Your child should try to find the best methods that work for her. She should ask her teacher for recommendations if she's experiencing any difficulty.
Take advantage of any free time
If your child has a study hall, or a long bus ride, she can use the time to review notes, prepare for an upcoming class, or start homework.
Studying with a friend
Unless it's too distracting, your child may want to get together with friends and classmates to quiz herself, compare notes, and predict test questions. To you, this may seem like mostly a social time, but it can be very beneficial to your child to prepare for an assignment as part of a group.
Celebrate your child's achievements
Reward your child for hitting milestones, or doing something well. You can provide treats or small rewards for your child while she is working on a big assignment. Your appreciation of your child's accomplishments in school is still very important to her, even though she may not always show it.
Communication is key
Keeping the lines of communication open will help to broaden your understanding of what teachers and counselors expect of your child and may help you to think of new ways to be supportive while still giving your child the independence that she's craving. It will also help you to understand how much time your child needs to allot for her homework, time that might take away from her participation in family activities or helping out around the house.
If your child has concerns about the amount or type of homework she has, she may want to talk to her teacher, adviser, or counselor. Encourage your child to ask for help if she needs it.
Creating a dream study space for your child
It's very important to think about helping your child with at-home assignments. Here are our favorite ways to assemble the perfect homework spot.
Get a CLUE
One of the most important keys to your child's school success is found right at home: Your kid's study space.
But all too often, a home study spot is given short shrift — with little thought to how and where it's set up — undermining how well your child can do his homework. Time to get a CLUE by asking yourself these questions:
Calm — Are there distractions (such as animals, TV, and music) that keep your kid from concentrating?
Light — How is the light? Low lighting may make your child sleepy and unfocused.
Uncluttered — Who wants to work at a messy desk?
Easy — Is it easy for your child to find and organize his stuff?
Now that you know the questions to ask, here are seven ways to create the best study area for your child.
How does your child work best?
Sure, that fancy desk and chair you're thinking of getting for your child's bedroom seems like a good idea. But before rushing out to buy all the bells and whistles for a catalog-beautiful "dream" space, remember the golden rule of designing the perfect area: create it specifically with your child's personality and study habits in mind.
If he works best around people, set him up in the dining room, kitchen, or living room (for kids who sometimes prefer lying down to work, the couch doubles as a great alternate work space). If he's easily distracted by clutter and noise, set up his desk in a quiet, secluded space.
Study that study space
Now that you've chosen the location, study the space to make sure your child is working under the best conditions. Consider his age and size to ensure he's not sitting at a giant chair and desk for his height, especially if your child is working at an adult-size computer and straining his head to look up — a winning recipe for neck, shoulder, and back pain. (If you have a young child working at a computer, consider investing in a kid-size keyboard and mouse to accommodate smaller hands.)
The overall work surface should be waist height. When your child sits down — ideally at a chair that has a back and arm rests — his elbows should rest on the table without hunching, bent at an angle of about 90 degrees or more. If it's not high enough, add a pillow or folded towel to raise the seat. His feet should be resting flat on the floor and not dangling. If they are, put a foot rest or box underneath.
To shoot for ergonomic perfection, for a typical first grader, the chair should be around 12 inches (30 cm) high, the table at about 18 inches (45 cm); by seventh grade, the chair should be around 14 inches (35 cm) high and the table 24 inches (60 cm) high. After middle school, aim for a 16-inch-high (40 cm) chair and 25-inch-high (63 cm) desk for girls, an 18-inch-high (45 cm) chair and 27-inch-high (68 cm) desk for boys.
Finally, how's the lighting? Consider getting a desk lamp for task lighting. Squinting strains the eyes and tires the mind.
Banish distractions
Is the TV on? Is an older sibling blasting music, or are two younger ones squabbling? Is the dog barking and the bird squawking?
Some kids require peace and quiet to focus. If you notice that your child gets easily distracted, or that you're frequently asking family members to pipe down, think about relocating her study space — or moving the pets. Turn off the TV. Turn off or ignore all phones (cell and land line; she's not allowed to answer hers).
Parents might even consider creating a "Homework time" sign so that everyone remembers to be quiet as church mice between, say, 5 and 5:30 p.m. One thing to remember: some kids do better with music in the background. But try to choose something without vocals, which can be distracting.
Stay close
Especially for younger children who need to build up their homework skills, try to stay close by to be available for questions or guidance. (But resist giving the answer!)
If your child is working in a more remote spot in the house, you can still let her know that you're there if she needs you. This lets her know you care about and value her schoolwork.
Stock up on supplies
"Ugh! I can't find the dictionary!" "Who took the glue stick?" To avoid these stress-filled moments — and needlessly wasting time hunting for missing items — duplicate a school supply list at home.
Store everything in a central area. Shoe boxes do nicely to store pencils, pens, rulers, and scissors. Consider labeling big-ticket items like staplers, scissors, and rulers with a sticker that reads "Return to homework box". That way, everything is easy to find, and nothing goes missing when your child needs it most!
Store backup materials in one spot
These days parents regularly get everything from weekly homework lists to semester calendars for big projects from the teacher. Keep your stash of homework paperwork close by so you can verify what is due when. This is especially helpful for parents of younger kids who are still mastering the fine art of keeping track of their homework.
Keep the mess at bay
Finally, your child's workspace doesn't need to be military-precision clean, but encourage her to put things away after finishing her assignments (again, so supplies and important papers don't go missing), and keep the area in order for the next day's homework.


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Stationary and Office Supplies
Industry data
There are approximately 580 active stores selling stationary and office supplies in Israel. There are a number of major chains in the industry: Office depot operates 27 stores, Kravitz operates 40 stores, and Arta opertes approximately 15 stores.
Industry Sub-division
Existing types of businesses are:
- Stores selling stationary and office supplies- these stores are usually located at city centers, malls, and commercial or trade areas.
- Diverse stores selling stationary alongside many other products such as textbooks, toys, gifts, etc. these stores are usually located in small neighborhoods and are based on private clientele.
Market Characteristics
- High seasonality is reflected in significant disparity in sales especially at stores that also sell textbooks and school supplies. At stores targeted for the business segments, an increase in corporate customer sales is usually evident towards the end of December.
- High chain activity in the industry by corporations such as Office Depot, Grafitti, Kravitz, etc.
Customers: Defining the Target Market and its Size, Market segmenting
Target market includes the general population, segmented according to the following criteria:
- Business customers- In recent year corporations are increasingly purchasing office supplies in bulk from a single vendor.
- Private customers purchase equipment and supplies for personal use.
- Large retail chains (such as Office Depot, Graffiti, and Kravitz).
- Stationary and office supply stores targeted to the private or business sector.
- Large supermarkets offer school supplies especially around "back to school" season.
- Internet- large chains and vendors specializing in the business sector offer various purchasing options via the internet.
There are many active vendors in the industry, selling a range of products, quality, and prices. Vendors in the industry are local manufacturers and importers, although in recent years import has increased from cheap countries in South Asia and Turkey.
Marketing resources
Marketing resources may be divided according to type of customer.
Private Sector:
Great importance is credited to store's location, correct use of promotional sales, advertisement in local or national press, local advertisement by using flyers. Stores selling brand names sometimes benefit from manufacturer advertisement on additional media. Franchise stores benefit from cooperative chain marketing.
Business Sector:
Use of catalogs, internet sites for online orders, direct marketing, or email.
Factors Affecting Success:
- Location of the store and density of competitors in the area
- Uniqueness and selection
- For the corporate market, incorporation of additional products such as furniture or office kitchenette supplies
Human Resources:
Employees in the industry are usually non-professionals, although it is preferable that store manager and sales representatives have previous experience.
Required Equipment:
- Furniture- including shelves (in the store and storage area), display shelves.
- Cash register (or computer to include inventory management), telephone and fax.
Required Investment
- Store remodeling- separating the store into a selling area, display area, and storage area
- Initial inventory- initial inventory investment is high, as the aspiration is to present a wide variety and a variety of models for each product sold.
Industry revenue and profitability
Yearly revenue per working business and typical profit percentages depend on the store's characteristics, location, reputation, price levels, and product quality. Percentages of raw revenue:
- At stationary and office supply stores selling mainly to large corporations and institutions: 20-25%
- At stationary and office supply stores selling mainly to private customers: 31-35%.
- At small stationary stores- 34-38%
Typical Terms of Payment
- Private customer sales are conducted in cash, check, or credit cards. Occasionally, business owners allow the customer to pay with a postponed check or in installments.
- Business customers- formal credit terms are EOM+30 to EOM+60 but in actuality, due to considerable competition, customer credit terms are better.
Business insurance (for content and inventory), third party insurance, employer's liability insurance, and loss of income insurance. In addition to the above, an insurance agent should be consulted in regards to the need for additional insurance.
Search Company By Name


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A Breach Could Have Significant Impact on Your Business
- Type: First Data Perspectives
- Tags: Fraud & Risk Management, Small Business
This is the third in a series of four brief summaries about what small business owners need to know about data breach risk and liability.
There are many ways that your business could be impacted by a payment card data breach.
- Substantial fees and fines that could potentially total in the tens of thousands of dollars or more.
- Loss of customer confidence and trust.
- Damage to your brand and good business reputation, especially as word travels quickly through social media and review sites like Yelp.
- Considerable time that you and other employees will have to devote to dealing with and recovering from the breach event.
- Loss of payment card privileges, meaning your business will not be permitted to accept debit and credit card payments if the card associations refuse to do business with you.
- Costs to upgrade or replace your POS system and associated security measures.
It's not inconceivable that a breach could put you out of business, for a short time or for good.
You can read more in Payment Card Data Breaches: What You Need to Know About Your Risk and Liability.