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Black Ink: Literary Legends on the Peril, Power, and Pleasure of Reading and Writing is one of the best anthologies I have read in a long, long time. Not only is it well-written, interesting, and important, it's also extremely relevant to my field of study, my profession, and my passions.
A collection of 25 essays written between the early 1800s and the early 2000s, this anthology of Black writers' perspectives on reading and writing is a lament for the struggles of those excluded from the literary world and a celebration of the power of that world.
The early essays move readers through the literary journeys of some of the most influential Black authors at the turn of the 20th century. Kept from reading and writing by practice and law, Blacks often learned in secret, protesting their restrictions and then using their newfound power to tell the world the truth about slavery and racism. This section on the Peril of Reading reveals the revolutionary power of words to communicate truth and effect change by excerpting works from The Narrative of the Life of Frederick Douglas, Up From Slavery, The Souls of Black Folk, and Twelve Years a Slave. Writing is activism.
The middle section, on the Power of Reading, contains 13 essays from writers such as Zora Neale Hurston, James Baldwin, Martin Luther King Jr., and Toni Morrison, to name a few. This is a much broader cross-section of authors and topics than part 1 and attempting to effectively paraphrase them in their entirety is just not possible, nor would it communicate the breadth of force these essays possess.
I was particularly interested in Hurston's "Books and Things" as I had just finished teaching her novel Their Eyes Were Watching God. One of the topics the students discovered in researched discussion was Hurston's contention that books by Black authors did not have to be about the Race Problem and that Hurston received quite a bit of flack for that belief and her practice of it. This essay touches on that theme and I was quite pleased to be able to share it with my students – despite the fact we had moved on to our next novel by that time. 🙂
The third section focuses on the Pleasure of Reading and Writing, featuring authors such as Junot Diaz, Roxane Gay, and Ta-Nehisi Coates. The final selection is an interview with Barack Obama on his relationship with reading. My favorite essay here is "The Danger of a Single Story" by Chimamanda Ngozi Adichie who so beautifully argues the necessity of multiplicity and diversity in stories.
I teach American Literature since 1865 and while I missed the opportunity this time around, I will definitely be using these essays – possibly this entire book – the next time this class is on the schedule and I am fortunate enough to teach it.
Finally, I want to share with you this section from Henry Louis Gates Jr.'s essay:
"What makes these books special – "classic" – however, is something else. Each text has the uncanny capacity to take the seemingly mundane details of the day-to-day African American experience of its time and transmute those details and the characters' actions into something that transcends its ostensible subject's time and place, its specificity. These texts reveal the human universal through the African American particular: all true art, all classics, do this; this is what 'art' is, a revelation of that which makes each of us sublimely human, rendered in the minute details of the actions and thoughts and feelings of a compelling character embedded in a time and place."
Nuff said.


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- You certainly can, and some people do. This approach has advantages and disadvantages. When you pre-pay for your funeral, you get to personalize it to your taste. You can grill different funeral directors until you find one you love. You can pick out the perfect casket and the choicest plot in the cemetery. Another advantage of pre-paying is that it will most likely prompt you to talk your choices over with loved ones, leaving them more confident that they know your wishes.
- Different states have different guidelines governing funeral pre-payment. The government wants to protect people from writing checks to unscrupulous folks who will take the money and run, or charge your family extra on top of what you pre-pay. Before you pre-pay, check your state guidelines for how the money will be held until your death. Make sure you know what you're paying for. Most importantly, are you locking in the rate for your funeral, or is there a chance your family would face an up-charge when the time comes? If you do any pre-planning or pre-payment, it's important to tell a loved one what you did and where you keep the documentation.
- The disadvantage of pre-payment is that it's less flexible than burial insurance. Will you or your family be able to get a refund if your funeral plans change? Will you lose the money you pre-paid if you move to a different area? What happens if the funeral parlor goes out of business? With final expense insurance, your surviving relatives will receive a payout they can spend anywhere. If you go with final expense insurance instead of pre-payment, you have less control, but your family has more flexibility. The choice is yours.
- If you want to leave a clear indication of your wishes without necessarily locking in the funeral home, you could always combine burial insurance with a document stating your preferences. Kept wherever you keep your will (you have a will, right?), this document would cover issues like burial vs. cremation, open vs. closed casket. etc.
- Find out about Social Security Death Benefits

subset: commoncrawl


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WHER Chat: Gender Gaps in STEM Across the World
According to the Institute for Women's Policy Research (IWPR), median annual earnings for women in STEM fields are $64,000 versus $78,000 for men, and women are only three in 10 of STEM workers. There is a discernible gender gap certain high-tech jobs in the United States. For instance, women accounted for less than 20 percent of those employed in these positions in 2017. In particular, women made up 18.7 percent of software developers, applicants and systems software positions; 4.2 percent of computer network architects; and 8.9 percent of aerospace engineers.


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Do you ever feel like your business has lost its focus? Remember when you first started out and you thought as long as you took care of what was at the heart of your organization everything would turnout okay? That was kind of naïve thinking wasn't it, or was it?
The truth is, businesses are complex and there are quite a few moving parts that need attention in order to keep them running smoothly. And a good way to keep your business humming along is to outsource functions that are not central to your core service. This will allow you to focus on what you do best.
So what is outsourcing? It is a task, job, operation, or process that could be done by someone within your company, but is contracted out to a third-party for an extended period of time.
I know outsourcing has gotten a lot of media attention lately—and it's not all good. But the kind of outsourcing that's been getting the negative press refers to jobs that are being sent oversees to countries like Mexico, India or China. This type of outsourcing is often called offshoring. I'm not talking about sending work overseas; I'm thinking more like…down the road.
There are many reasons why a company may choose to outsource one or more of their business functions, but saving either time or money usually heads the list. Another big reason why managers get motivated to outsource involves a lack of resources. This could be in the form of materials or employees. It can sometimes be difficult to find the right people—with the right knowledge—to perform certain tasks within your operation.
To be a successful manager you don't have to know all the answers, but you do need to be smart enough to surround yourself (and your business) with those who do. Outsourcing can sometimes be the best way to provide expertise to a very necessary but peripheral business function. Let's take accounts receivable management and debt collection as a couple of examples. Getting paid the money owed to your organization may not be the heart of your business, but it sure is going to be a vital contributing factor to its success.
I guess you could make the argument that you really don't need to outsource these services as long as you can hire "the experts" yourself. And you would be correct, if you don't mind dealing with the added cost and time associated with salaries, ongoing training, benefits, employment taxes, additional work space, office supplies, computers, and advanced telephone systems—all things a third-party vendor already has in place—that and more.
Imagine if there was an organization that was created for no other reason than to improve your bottom-line. That's right; an entire company that spends all of their time, money and energy collecting money owed to you. Wouldn't that be great? We're talking about amassing knowledge; through extensive and ongoing training to ensure compliance and great customer service for consumers and clients, and the technological infrastructure; complete with unrivaled data security, industry-tested Customer Relationship Management systems, skiptracing software, and advance telephone systems with predictive dialers and recording capabilities for operating efficiency and quality control—all working cohesively to improve your bottom-line.
That's what you could get if you outsourced your accounts receivables to a reputable third-party accounts receivable management, collection agency. If you are concerned you don't have money in your budget to outsource your accounts receivable, don't worry, many third-party collection agencies get paid strictly on a contingency basis, which means they get paid on a percentage of what they collect. Get your focus back on what you do best and let a third-party collection agency focus on what they do best, collecting money for you.
A. Alliance Collection Agency, Inc. is a full service, licensed accounts receivable management and debt collection agency providing highly effective, customized one on one management and recovery solutions for our business partners. Founded in northern Illinois in 2005, we have been proudly improving the bottom-line on behalf of our business partners in and around Chicagoland for over 10 years.


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Tag: Metallica
LIVE, IN PHOTOS: Metallica Rocks S&M2 with the San Francisco Symphony
The band celebrated the twentieth anniversary of "S&M" in SF this weekend.
WATCH: Jack Black Rocks the Chris Cornell Tribute Concert
The comedian, musician, and FLOOD 9 cover star regaled the crowd with Soundgarden and Metallica hits.
LIVE, IN PHOTOS: Metallica at The Masonic
The iconic metal group went unplugged—or at least slightly de-electrified—for a benefit show this past weekend.
Homegrown's Alright with Texas at Austin City Limits 2018
Amidst heavyweight legends like Paul McCartney and Metallica at this year's ACL, plenty of fresh-faced acts showcased their love for Texas, and a charity group helped to celebrate music as the best medicine.
LIVE, IN PHOTOS: Outside Lands (8/11–13/2017)
Belle and Sebastian, Fleet Foxes, Kaytranada, and more play in the Bay.
Metallica, "Hardwired…to Self-Destruct"
Historically, metal's biggest act has suffered the most when they try something new. "Hardwired…To Self-Destruct" finds them slogging their way back to basics.
Lollapalooza 2015: No Calm Before the Storm
Evacuations, lightning, and humidity couldn't keep the crowds or the good music away from Grant Park.
Festival Season Is Officially Here as Lollapalooza Announces 2015 Lineup
…and it's already sold out.
More Awards, More Nominations: The Grammys Announce Their List for 2015 [UPDATED]
The Academy Of Recording Arts And Sciences are making a whole morning event to announce their list of nominations for their 57th annual awards show, the…


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Availability: In stock
We stock a range of these different pressure gauges.
These are quite easy to install into a tank and depending on the thickness of the tank wall all you need to do to fit a Gauge like this is drill a hole in the tank about 8mm in diameter then insert the gauge and secure with screws.
We have several different models available in different pressure ratings:
KL14984 - Mini Gauge 0-23psiKL14991 - Mini Gauge 0-30psiKL15004 - Mini Gauge 0-60psiKL15011 - Mini Gauge 0-150psi
These gauges are designed to fit into either our inline regulator or our blowtie units. These can be used for water or gas.
SpecificationsPressure: 0-60psi (0-4bar)Graduations: PSI + BarMaterial: Brass/Plastic/Copper CapillaryGauge Diameter: 20mmProbe Diameter: 7.8mm (designed to fit 8mm hole)Accuracy: These gauges are accurate to 10% of the whole pressure range. (ie the 0-30psi Gauge is accurate to +/- 3psi). For more accurate gauges we recommend the larger 50mm gauges that we sell.
WARNING:Do not exceed gauge pressure. If you exceed gauge pressure it will damage the capillary in the gauge and prevent the gauge from working correctly. NEVER exceed the maximum gauge display pressure range.
Not tested or approved for pure oxygen.
Please ensure that your shipping information is 100% correct before submitting your order.
Once an order has been placed KegLand cannot change/add to an order or change the shipping information.
Any such changes will fall onto the customer to contact the shipping provider and attempt redirection.
For larger items we do recommend that you use a business address as your primary shipping choice. This limits un-deliverable items as it will be delivered within business hours ensuring a recipient can sign for the package.
If you require extra items, a secondary order will have to be placed with additional shipping costs.
Please note that some items are coming from multiple warehouse locations and may not be delivered at the same time.
Full Terms & Conditions.


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The aim of the 2nd Annual AICAR conference is to provide a platform to bring the diverse India related research together in order to facilitate future collaborations as well as dissemination of the research. The conference also offers avenues for development of doctoral researchers and junior faculty and opportunities for research collaborations to enhance research impact.
Managing and Organising in the Emerging Indian Context
Abstracts of papers (max. 500 words) should be submitted by 31st May 2018 to Dr Soumyadeb Chowdhury: firstname.lastname@example.com
Review decision on submissions by 15th June 2018.
Due to the major reforms pursued by the present government (e.g., demonetisation, GST, amongst others), the Indian economy is now predicted to sustain its economic growth and India to remain as one of the most preferred destinations for foreign investors. A significant improvement for India on the global indicators such as the World Bank rankings on 'Ease of doing Business' are also an outcome of such reforms. Along with these, the number of major and exciting initiatives being pursued in the government (such as Swachh Bharat, Make in India, Skills Development for Youth, Digital India, Smart Cities, Start-ups India, amongst others) are helping India to consolidate and unify its market, strengthen its human resources and create a robust policy framework to lead its economy on a long-term and sustainable path of high growth and build an engine of growth for the global economy.
As majority of the above-mentioned reforms and initiatives are recent, their implications on businesses and their management and related stakeholders is now evolving. Expectedly, there is not much research being conducted on the various aspects of these recent developments in India. Hence, it offers abundant opportunities to pursue research on a variety of topics within the broad fields of management and organisation.
The aim of the conference is to provide a platform to bring the diverse India related research together in order to facilitate future collaborations as well as dissemination of the research. It is key to create such common ground given the above presented development and to sustain the interest being created in further interdisciplinary research and collaborations that contribute to the emerging body of knowledge as well as creating links with key practitioners and stakeholders in India and abroad.
During the conference, we intend to organise specific developmental workshops for doctoral scholars and junior faculty focusing on careers, publications, funding, networking, amongst others.
Since this a developmental conference, feedback will be provided to all participants for taking their papers forward for publication.
Delegate: £120.00 (including refreshments, conference pack, lunches and dinner)
Doctoral Student: £90.00
To book your tickets please click here.
The conference will be chaired by Professor Pawan Budhwar from the Department of Work and Organisational Psychology.
A member of the Work and Organisational Psychology Group, Joint Director of the Aston India Centre for Applied Research and Joint Co-Editor-in-Chief of British Journal of Management, Pawan is golbaly renowned for his research into the international aspects of HRM, and has held many visiting professorships worldwide.
Pawan's research interests lie mainly in the linkages between HRM and performance in different international contexts. He conducts the majority of his empirical work in emerging markets, especially in India. Pawan's work is of great significance given the links with developments within the Indian economy and the scarcity of research in the field within this context. His previous work has been extensively used by academics, especially his three-level framework of factors influencing HRM in a given context. He has examined HRM systems in emerging markets and established links with social, cultural, political and legal contexts, and this is increasingly being referenced by other researchers.
He has received funding from a variety of funders such as the ESRC, Society for HRM (USA), British Academy, and ERDF to investigate a variety of HR aspects in different set-ups such as in Indian Call Centres, wherein he highlighted many people management related issues and challenges, and the problems emerging in this sector.
Prof Pawan Budhwar, Prof Prasanta Dey, Prof Robert Berry, Dr Philip Davies, Dr Vidu Badigannavar, Dr Bimal Arora, Dr Sudeshna Bhattacharya, Dr Soumyadeb Chowdhury, Ms Divya Jyoti and Ms Debashree De.
Aston India Centre for Applied ResearchAston Business SchoolAston UniversityB4 7ET BirminghamUnited KingdomTel: +44 (0) 121 204 3252
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Tuesday, October 25, 2005
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Quality cal vet home loans Resources. Full of Mortgage newsletters, articles, links and other resources - ALL FREE - in one easy to navigate site to save time and money.">
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UKGovernmentGrants reviewsIs this your business?
27 Old Gloucester Street
London, LONDON WC1N, GB
Tel: 0800 856 1570
London, LONDON WC1N, GB
Tel: 0800 856 1570
4 Reviews From Our Community
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My small family jewelry store had been suffering in the past year and we wanted to get business back to usual but needed some funding to help cover some expenses and start increasing marketing spending. We didn't want to go through the banks because of the high interest rates, and we thought the government would be a better option for us. We found the Small Business Grants UK database and started applying to different grants and low interest loans to help us keep our business afloat.
Small Business Grants UK was able to help me make my business dream become true. I dreamed of having a small computer repair store which I was never able to start because of a lack of capital and high interest rates at the bank. I found Small Business Grants UK and was able to find government grants that my business would be eligible for. All I had to do was fill out an application online.
If I was asked to be a marketing agent for Small Business Grants UK, I would gladly do so gladly. This is because they recently helped me get a government loan that I needed to sustain my business so I contacted them inquiring about a loan and they gladly gave me a list of government agencies that granted loans and I sought one that I applied a loan for immediately and luckily got the loan
The professionals working with Small Business Grants UK are highly knowledgeable and helped me through with all of my queries. I had been looking to start up a small business in the UK but was really unsure how to go about it. Thankfully, after being directed to the service, things have proceeded on quite smoothly for me. The service is transparent, highly professional and proved to suit my needs perfectly. I am thankful for having been directed to www.ukgovernmentgrants.org and I would recommend it to anybody seeking fund programs in the UK.
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text: Reviewed Bargain Host on Monday, December 3, 2012 I have been with Bargain Host for over 6 years now and I have numerous web sites hosted on their servers. The prices are very reasonable and now they are offering low priced domain registrations for resellers and I honestly do not believe there is anywhere else cheaper in the UK. Did you find this review helpful?


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This week we look at the selection process for choosing a web design agency. As a client, how do you pick the right company for you? Conversely, as a web design agency, how do you make yourself attractive to clients?
Okay, I admit it, the last person you want to ask about choosing a web design company is a web designer!
there are many cowboys around that damage the entire profession
He is bound to think you should choose him and that his company is the best. In addition, to be honest, there are many cowboys around that damage the entire profession. I hope that the other entries on this site prove that I am not one of those but I still encourage you to take what I say with a pinch of salt. After all, I am bound to be a little biased.
After saying all of that, here are my recommendations for those seeking a web design company to work on their site.
Are they a full time web design company?
A strange question to start with I know but it is one worth asking. There are a surprising number of students working out of their parents' back bedroom. If you have a limited budget that might be fine, but if you are talking about a serious web development project these kinds of groups will get out of their depth very quickly.
How many people are in the company?
The size of the company is a good indication of its capabilities but also of its overheads. A large company is often unresponsive and tends to have a higher charge out rate. A one-man band can be much cheaper and keener to please but can sometimes be overwhelmed by larger more complex projects. Look for a company that is of the size that best suits your project.
What is the main revenue of the company?
Some companies offer web sites in addition to their main business. A number of graphic designers and printers have branched out into the web design business. Equally, many technical development houses have moved across into web design. In my opinion, it is better to seek out a company that specialises in web design and are experts in that area.
Do they charge for initial consultancy?
Some companies will try to charge for initial consultancy or avoid engaging in project specifications until the contract is signed and sealed. My advice is to avoid companies like this. A good web design company should be willing to meet with you free of charge to discuss the specifications of your project before drawing up a fully costed proposal.
Can they build what you want?
Make sure that they have the capabilities to deliver what it is that you require. It helps if they have produced something similar, which they can refer you to. Do they have the right resources and technology? Do they offer web hosting and diagnostics for your requirements? What kind of support do they offer?
Are you tied in to them once your site is built?
This is probably the biggest problem faced by website owners. Many web design companies build websites that neither the client nor another web design company can edit. Make sure that you aren't going to have to go back to the web design company every time you want to make a change on the site. Also, make sure they are using industry standard tools to build the site in the first place. That way if you are unhappy with them you can go to another company for future development work.
What is their portfolio like and can you view the live sites?
You must see some published work. Starting with the design company's own web site, and some of their clients'. Do you like them? Are they visually appealing? More importantly, are they easy to use. Also check that they are capable of tailoring their designs to fit the client and that they don't have too strong a house style.
Do they have clients willing to talk to you?
Is their service of a standard that their clients will recommend them? If they are good and competent designers then they should have clients who are prepared to discuss their experiences.
What is their charge out rate?
Don't be afraid to ask them what they charge per day and what the average value of their projects is. However, watch out, some design agencies will have a low daily rate but will make sure that the project lasts long enough to make a profit! A good web design agency shouldn't have a problem telling you their rates. After all, it is a waste of everybody's time if your budget doesn't fit with their daily rates. However, it is also useful if you give them an indication of what your budget is because they should be able to tailor a project to fit your budget.
What is their approach to project management?
Will they give you a full proposal with timescales and milestones? Do they have dedicated project managers who will look after the running of your project?
How will they build your site?
Do they build with the latest web standards? Do they understand accessibility legislation and your obligation to make your site available to the widest possible audience?
Do they have long-term clients?
Not only does the presence of long term clients demonstrate a high level of customer satisfaction, it also shows an understanding by the web design company of the need to build sites for the long-term. All web sites have to grow and evolve over time and agencies that work with clients over the long-term know how to design a site flexible enough to change with the company's requirements.
Hopefully some of the above was helpful and not too biased towards Headscape. Just to show my complete impartiality you might want to check out the independent directory of web designers at the UK web design association. This will provide you with a complete list of web designers in the UK and allows you to search by county and by skill.


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Lately promulgated rules in Massachusetts require that starting Jan. 1, 2014, physicians who certify patients for medical marijuana should first complete two hours of Persevering with Medical Education credit. On Dec. 6, 2012, the state's first dispensary, Arizona Organix, opened in Glendale. The Oregon Medical Marijuana Dispensary Program publishes a directory of 87 accepted dispensaries on its website as of Mar. 30, 2017. As of Jan. 1, 2017, medical marijuana dispensaries might promote solely to OMMP-registered sufferers and caregivers.
for regulating medical marijuana and set the appliance dates for May 14 via May 25. If you happen to stay in a state the place it is legal and your physician has OKAY'd it, you should buy it from a certified vendor referred to as a dispensary. Some folks might legally develop their own medical marijuana, click here to know more about benefits of marijuana for health.
Nabilone is used for a similar reasons, however it has a person-made chemical that is similar to THC. Cannabidiol (CBD) is a naturally-occurring constituent of the industrial hemp plant. It can be tricky to be on the fitting facet of science, however on the flawed side of ideology.
At eDocMMJ.com our Compassionate Medical Solutions are among the most well established and best reputable in Florida. You can rest assure that you're going to be speaking with a Doctor who really cares about you and your family. Upon approval the Doctor will provide you your medical cannabis card recommendation with responsive and accurate speed. We know you need the medical benefits and pain management relief of cannabis, and we're here to help you with all aspects of acquiring your certification. Sign up now to get rolling.
The FDA has authorised two medication that embrace ingredients also found in marijuana. Dronabinol has artificial THC and is used to treat nausea from chemotherapy and extreme weight reduction in AIDs sufferers. Authorizes using marijuana for medical functions and establishes the Medical Marijuana Management Program. For more information, you can see through All Natural Medical Solutions, Inc.
The legislation doesn't allow sufferers or caregivers to grow marijuana at residence, but it surely does allow for the state-regulated, non-profit distribution of medical marijuana by compassion centers. Imposes statewide pointers outlining how a lot medicinal marijuana sufferers might grow and possess.


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This document will walk you
through the process of accessing and configuring your
The very first thing you should do is change your password.
You may send and receive email through any web browser connected to
the Internet. Point your Internet browser to http://email.[your
domain name], enter your email mailbox name
password, and click the Login button.
You can configure Outlook to automatically check your email
account for new mail. To configure Outlook, complete the
following steps:
If you wish to use this email account as the default
account through which your email is sent, follow these steps:
Regardless of which account you have set as you default, you can
always change the account through which an email is sent by
selecting the account in the Account dropdown menu from within a new
Copyright Goeben Design. All rights reserved worldwide. +


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9749 102 Street, Edmonton
Courtesy Of Lance Joumblat Of Century 21 Masters.
Honey Stop The Car!! This breathtaking home has been featured in Avenue Magazine is a must see. NO CONDO FEES! Extensively upgraded residential attached two storey townhouse in sought after Rossdale's River Valley Estates. Red brick adorns the exterior and wrought iron fencing greets you at the front. There are massive- two bedrooms plus den Master Bedroom with walkin closet, 4pc bathroom ensuite with Jacuzzi tub 6feet deep, Total-2-4pc, 1-3pc and 1-2pc baths. Bright open large living room, generous sized dining room, Chef dream kitchen features top end appliances, granite counter tops and cabinets(high gloss lacquer) from France gorgeous kitchen with access to west facing deck and patio(Largest yard in the complex). Lots of gleaming hardwood, a west facing balcony off the master bedroom. Home features, Hammer custom railing, Vanee Exchanger, Gas and electric fireplaces, Central vac, single attached garage. Fully developed basement with Murphy bed to make 3rd bedroom. This home awaits your arrival.
Closet Organizers, Deck, Parking-Extra, Ceiling 10 ft.
Insulated, Single Garage Attached
Dishwasher-Built-In, Dryer, Garage Control, Garage Opener, Hood Fan, Oven-Microwave, Refrigerator, Washer, Window Coverings, Stove-Gas, Vacuum System Attachments
Back Lane, Fenced, Fruit Trees/Shrubs, Golf Nearby, Landscaped, Public Transportation, Schools, Shopping Nearby, View Downtown
Listing information last updated on January 28th, 2020 at 10:01pm MST.


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MIAMI'S BEST FLOATING FESTIVITY Biscayne Bay. Sand Bars. Island Beaches. Miami River. Brickell Downtown. Miami Beach Dinner parties. Tours. Beach parties. Night parties. Take up to 39 friends.
$395 per hour for 25 Passengers for 4 hours + $198 gratuities (Billed Separately).
If you want to add more guests (More than 25), than each Additional Guest is $60 for up to a TOTAL of 39 passengers (Billed Separately).
Times schedule:
11am-3pm Half a Day
3pm-7pm Half a Day
11am-7pm Full Day
Message us for an INSTANT REPLY and EASY BOOKING
MIAMI'S BEST FLOATING FESTIVITY Biscayne Bay. Sand Bars. Island Beaches. Miami River. Brickell Downtown. Miami Beach Dinner parties. Tours. Beach parties. Night parties. Take up to 39 friends.
This Party Pontoon is equipped with a full kitchen, dining table, bath room, barbeque grill, great sound system (your music or ours). Water toys, including giant lily pad float, water trampoline & paddle boards. See the Miami City Lights or Sunset from the bay. Or spend the day on island beaches or cooking out at anchor. Sanctuary is Miami's premier party boat for 25-39 people. Plan your next one with us. Book your party now Best deal on the water..
We reserve the right to switch the Yacht you reserved if it became unavailable for the date and time you reserved. If that case occurs, than we will make sure you board a very similar yacht to the one you reserved, OR will place you and your guests on LARGER vessel that has similar amanaties to the original reserved yacht.
In addition, we will provide you and your guests FREE transportation to the marina where the replacement vessel is located.
In any event, you will be notified at least 24 hours prior to your planned departure.
Change of Vessel IS NOT a ground for cancellation.
Once reserved and approved, all payments are final and non-refundable. We only issue refunds if your trip or other requested services are prohibited by the United State Coast Guard due to UNSAFE CONDITIONS
No Other Exceptions.
Galene and his team were excellent! Professional, responsive, and provided a wonderful experience. Would highly recommend.
Celebrated my 21st birthday with my friends on here and had the funnest time. Captain made sure everything went smooth, and booking was easy. Definitely an experience worth every penny!!
Everything was amazing!
I had my Birthday on the boat and it was absolutely amazing !
Easy to book and quick to respond to emails !
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text: We offer one, short, Designated Mountain Biking Trail with relatively easy ascend and beautiful rhododendron arches. There is much more designated trail work underway, so keep posted! Biking, in general is permitted on all Multiuse Trails on Doe Mountain. Day Biking User permits are just $5 and Annual Biking permits are just $35. Our future plans are to continue to expand our Mountain Biking Trail System with a variety of trails with different difficulty ratings.


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Stenhousemuir have signed midfielders Nathan Blockley and Martin Scott as they continue to build for the 2017/18 campaign.
Boss Brown Ferguson has already brought in Ross Dunlop (Albion Rovers), Michael Dunlop (Albion Rovers) and Mark McGuigan (Stranraer).
Scott was previously with Hibs and Livingston and helped Arbroath win the League 2 title last season while Blockley is an ex-Airdrie player who makes the move to Ochilview from Peterhead.


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REDD Pratley and Alfie Saunders tore through Yarnton & Cowley's as Witney Swifts ran out 107-run winners in Division 1.
Swifts were all out for 157 as Charlie Fisher top-scored with 43 and Muhammad Adil claimed 5-25 for the home side.
But Pratley then claimed 5-20 and Saunders 4-32 as Yarnton were shot out for just 50 in their reply.
Oxenford beat hosts East & West Hendred by 81 runs on the Target Score (T/S) method.
The visitors racked up 246-2 as Bharadwaj Ramanathan hit 106 and Andrew Dean 40.
Rain reduced Hendred's reply to 39 overs and a revised target of 226, but they were bowled out for 145, Habeel Khalid taking 3-9.
Tom Stewart-Liberty's unbeaten century helped Wolvercote to a 175-run win at Marsh Gibbon.
Liberty struck 120 not out in Wolvercote's 247-5, before Toby Shelley took 5-22 as Marsh Gibbon were shot out for 72.
Stonesfield saw off visitors Faringdon by 101 runs.
Having been bowled out for 164, Stonesfield's Steve Litten (4-20) and Dan Farrant (4-35) combined to bowl Faringdon out for only 63.
Billy Webb starred for Wootton & Boars Hill as they won by 24 runs at Westcott.
Webb hit 69 in Wootton's
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In Division 2, Cairns Fudge eased to a seven-wicket win over visitors Chipping Norton.
Jamie Clarke took 5-24 to help restrict Chippy to
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Ryan Markham hit 84 as Middleton Cheney chased down Hanborough's 218-8 to win by eight wickets.
Fine knocks of 64 not out from Nikunj Patel and 52 not out from Shailesh Babu (52) saw Tetsworth to a seven-wicket win at home to Stanton Harcourt.
Watlington eased past visitors Clifton Hampden by eight wickets.
Ali Kushi took 4-8 in Clifton's 120-8, before Steve Strong hit 64 not out to steer Watlington to 121-2.
Duncan Macdonald starred with bat and ball to help Kilkenny to a three-wicket win at Brill in Division 3.
He took 3-30 and Chris May 3-10 to help restrict Brill to 156-9, and then scored 60 as Kilkenny reached 157-7.
Risinghurst's Lokeschwar Bandhu claimed 6-28 to lead his side to a 69-run win at Chadlington.
Bampton ran out 25-run winners at Dorchester.
They set 239-7, Ben King top-scoring with 42 not out and Harry Platt taking 4-52.
Russell King then took 3-56 as Dorchester were restricted to 214-7, Chris Rudling hitting 51.
Kidlington beat hosts Marcham by 41 runs on the TS method.
Andy Oliver (64) and Craig Woods (51) in Kidlington's 261-6.
After the loss of ten overs due to rain, Marcham's target was revised to 241, but they managed only 200-9, Thomas Mooring hitting 62 and Edd Wilmot taking 4-28.
Broughton & North Newington defeated hosts West Ilsley by six wickets.
Three wickets apiece for Stuart and James Morgan helped to dismiss West Ilsley for 149.
Paul Derry (62no) then led Broughton to 151-4.
In Division 4, a haul of 5-22 for Manoj Perera was in vain as Shrivenham lost by three wickets at Steventon.
Shrivenham were all out for 117, Alex Bowles hitting 50 not out and Phil Armstrong taking 4-15.
Steventon then made 118-7, despite Perera's fine haul.
Deddington defeated Sunningwell by seven wickets.
Ramesh Chennagiri was the star of the show with super spell of 7-33 as Sunningwell were dismissed for 81, which proved no problem for the hosts.
Radley ran out thrilling three-run winners at Uffington in a low-scoring clash.
They were bowled out for 105, Tom Fillery taking 3-28.
But, despite 43 not out for Martin Key, Uffington then slumped to 102 all out, Naheem Ahmed claiming 3-12.
SIMON Palmer returned superb figures of 6-11 as Kennington thumped visitors Freeland by 112 runs in Division 5.
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Oxenford 2nd beat Tetsworth 2nd by 37 runs, despite a haul of 5-16 from Haider Zaman for Tetsworth, while Wolvercote 2nd saw off visitors Yarnton & Cowley 2nd by six wickets, as they successfully chased down Yarnton's 159 all out.
In Division 6, Santha Thirupathi took 5-34 as Letcombe edged out visitors Chesterton by one wicket.
Chesterton were bowled out for 109, Jimmy Appleton scoring 64 not out.
Phil Austin then hit 41 as the hosts squeezed home at 110-9.
Middleton Cheney 2nd beat visitors Dorchester 2nd by seven wickets.
Barry White was on song for Cheney, taking 4-14 in Dorchester's 146 all out, Ian Ferris top-scoring with 56.
Cheney then eased in at 147-3, Harry Boynton top-scoring with 37.
Edward Burns hit 64 as Wootton & Boars Hill 2nd beat Wolvercote 3rd by 39 runs.
Wootton set 190-8, and then Simon Steer claimed 5-36 as Wolvercote were all out for 151.
Hook Norton 2nd eased to a 156-run win at Stanton Harcourt 2nd in Division 7 North.
Grant Wallis hit 77 not out in Hooky's 244-9, before Oscar Raisbeck claimed 5-28 as Stanton were shot out for 88.
Kidlington 3rd hammered visitors Deddington 2nd by 164 runs.
After posting 216-7, Kidlington's Jacob Oliver took 4-25 as Deddington were dismissed for 52.
Elsewhere, Chipping Norton 2nd shot Stonesfield 2nd out for 41 in a seven-wicket victory, while Oxenford 3rd saw off hosts Charlbury 3rd by 23 runs.
Faringdon 2nd defeated visitors Steventon 2nd by six wickets in their Division 7 South clash.
Simon Cornish took 4-24 in Steventon's 164 all out, before Alan Johnson (44no) and Jonathan Ritchie 39 not out saw Faringdon comfortably home to 167-4.
Bampton 2nd's Harry Curtis took 5-16 and Thomas Jones 5-27 to help their side to a 72-run win against Kennington 2nd.


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Meet the Candidates
Seems most people these days have strong beliefs about government, politics and the upcoming elections. Educate yourself about the people you'll be voting for by attending a visit with the candidates for the 9th Congressional District seat: Dan Meuser and Denny Wolff. The event will be at D.H.H. Lengel Middle School Auditorium, Pottsville on Tuesday, October 25 at 7 pm. The event is free but please register by contacting the Schuylkill Chamber of Commerce at 570-622-1942 or online.
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January 2020


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Analysis of the Ashbya gossypii Wiskott-Aldrich syndrome-like gene AgWAL1 indicates that it is required for the maintenance of polarized hyphal growth. Growth and organelle dynamics of the wild type and of wal1 and other mutant strains were monitored by in vivo (fluorescence) time-lapse microscopy. Loss of WAL1 led to slow growth and defects in polarized growth that produced swellings in subapical regions, whereas formation of hyphal tips and dichotomous tip branching occurred as in the wild-type. Few actin cables in Agwal1 cells were found to insert into the hyphal tip, but specific clustering of cortical actin patches was observed in subapical regions of hyphal tips instead of at the hyphal apex. Distribution and movement of vacuoles was observed in vivo using FM4-64. In the wild type and in the slowly growing mutant strains bem2 and cla4, which lack a Rho-GTPase-activating protein and a PAK kinase, respectively, early endosomes appeared in the hyphal tip, whereas very few early endosomes and small vacuoles were found in the wal1 mutant hyphal tips, thus linking the cortical patch defect of wal1 hyphae with the distribution of endosomes. Vivid movement of vacuoles seen in the wild type and in the bem2 mutant in subapical regions was largely reduced in the wal1 and cla4 mutants. The tubular structure of mitochondria (as visualized by DIOC6 in vivo) was similar in the wild type and the wal1 mutant, although wal1 mitochondria appeared to be larger. Interestingly, mitochondria were found to insert into the hyphal tips in both strains. Our results indicate a function for Wal1p in filamentous fungi in coordinating actin patch distribution with polarized hyphal tip growth.
The dynamic reorganization of the actin cytoskeleton is essential for cell polarization, morphogenesis, the distribution of organelles and cytokinesis (Drubin and Nelson, 1996). Elaborate mechanisms ensure temporal and spatial control of these events and require a network of proteins that includes Rho-GTPase modules as central regulators of the actin cytoskeleton (Schmidt and Hall, 1998; Wendland, 2001; Etienne-Manneville and Hall, 2002). In the yeast Saccharomyces cerevisiae and the filamentous fungus Ashbya gossypii, homologs of the Rho-GTPase Cdc42p and its guanine-nucleotide exchange factor Cdc24 have been shown to be required for the establishment of a polarized cortical actin cytoskeleton that is a prerequisite for bud emergence in yeast and the formation of new hyphal tips in A. gossypii (Johnson, 1999; Wendland and Philippsen, 2001). Rho-GTPases relay spatial and temporal information to effector proteins and are thus able to integrate multiple signalling inputs with the state of actin cytoskeletal organization (Schmidt and Hall, 1998). Among these effectors are multidomain adaptor proteins, such as members of the Formin, PAK (p21-activated kinase) and WASP (Wiskott-Aldrich syndrome protein) families, that can act as scaffolds (Frazier and Field, 1997; Ramesh et al., 1999; Mullins, 2000; Pollard et al., 2001; Sharpless and Harris, 2002; Wild et al., 2004). The S. cerevisiae formin Bni1p, which is activated by Cdc42p, has recently been shown to play a central role in the nucleation of actin cables, which is achieved in an Arp2/3 independent manner (Evangelista et al., 2002; Pruyne et al., 2002; Sagot et al., 2002; Evangelista et al., 2003).
WASP family members are conserved from fungi to humans and are built in a modular fashion. They bind to components of the Arp2/3 complex and actin monomers via C-terminal WH2/C/A (WASP-homology-2/central/acidic) domains and activate actin nucleation (Machesky and Insall, 1998; Machesky et al., 1999; Madania et al., 1999; Rohatgi et al., 1999; Winter et al., 1999; Prehoda et al., 2000). Mammalian WASPs carry a GTPase-binding domain that allows direct interaction with Cdc42p. Such a domain is missing from members of the SCAR (suppressor of cAMP receptor) subgroup as well as from all known fungal WASP family members. In the N-terminal part, WASP family members share a WH1/pleckstrin-homology domain (lacking in SCAR proteins), which is involved in lipid binding, and a basic region (missing in Wsp1p of Schizosaccharomyces pombe) (Lee et al., 2000). Similar to WASP, which can link lipid (i.e. phosphatidylinositol-4,5-bisphosphate) signalling with the actin cytoskeleton, the PAK Cla4p in yeast can link lipid (in this case, via phosphatidylinositol-4-phosphate) and Cdc42p signalling (Rohatgi et al., 1999; Wild et al., 2004). The PAK-like kinase Cla4p in A. gossypii was found to be required for hyphal development and septation, and loss of CLA4 led to a partial disorganization of apical actin patch clusters (Ayad-Durieux et al., 2000).
Secretion in hyphal-tip cells of filamentous fungi is highly polarized and growth of the hyphae is restricted to the tip. Cortical actin patches are clustered at the hyphal tips during the hyphal growth phase (Wendland, 2001). In the ectomycorrhizal basidiomycete Suillus bovinus and the dimorphic fungus Candida albicans during hyphal growth stages Cdc42p was found to be localized to hyphal tips, indicating that Cdc42p is not only required for hyphal tip formation but might also play a role during polarized hyphal growth (Gorfer et al., 2001; Hazan and Liu, 2002).
Mutations of S. cerevisiae genes belonging to the Arp2/3 complex or deletion of the S. cerevisiae WASP LAS17/BEE1 led to a loss of cortical actin patch organization (Winter et al., 1997; Li, 1997). The yeast Las17/Bee1 protein acts as an activator of the Arp2/3 complex and is involved in endosome/vacuole motility (Winter et al., 1999; Yarar et al., 1999; Chang et al., 2003). N-WASP was also shown to promote the actin-dependent movement of endosomes and lysosomes in Xenopus eggs (Taunton et al., 2000). Although substantial progress has been made in the analysis of individual WASP-family-member domains, the cellular function of WASP homologs for polarized hyphal growth in filamentous fungi has so far not been elucidated. Recently, we could show that the WASP homolog of the dimorphic fungal pathogen C. albicans was required for hyphal growth. Yeast cells of C. albicans induced to form hyphae by serum at 37°C responded with an initial polar growth phase but failed to maintain polarized growth, which resulted in the formation of pseudohyphae, with true hyphae not being formed (Walther and Wendland, 2004). The C. albicans WASP mutant showed similar defects in the yeast stage to those of the S. cerevisiae WASP mutant, including aberrant localization of cortical actin patches in the mother cell during bud growth, endocytosis and the number of vacuoles per cell. Thus, the growth defects of the C. albicans WASP mutant resembled those also observed in a C. albicans myo5 (encoding a type I myosin) mutant strain (Oberholzer et al., 2002).
We could not study defects during hyphal growth stages in the WASP mutant of C. albicans and so we aimed to isolate a WASP homologue and to characterize its mutant phenotypes in a true filamentous fungus. Deletion of AgWAL1 led to severely reduced polar hyphal growth rates, increased isotropic growth and defects in septation. Cortical actin patches that cluster in hyphal tips of the wild type were found to accumulate specifically in subapical regions in the wal1 mutant, whereas few actin cables inserted into the hyphal apex. In the A. gossypii wild type, a redirection of cytoplasmic streaming and vacuolar movement occurred towards newly growing hyphal tips. Loss of WAL1 led to severely decreased vacuolar movement in subapical hyphal sections, which was also observed in a cla4 mutant. Furthermore, wal1 hyphal tips not only showed an exclusion of cortical actin patches but we also observed a lack of endosomal vesicles in the hyphal tips, suggesting a functional link between endocytosis and vacuole formation mediated by Wal1p in A. gossypii. Because mitochondria were positioned in the hyphal tips of both the wild type and the wal1 mutant, specific mechanisms must exist in filamentous fungi to transport mitochondria and vacuoles to support polarized hyphal growth.
Materials and Methods
Strains and media
The A. gossypii wild-type strain ATCC10895 and a derivative thereof (A. gossypii leu2, thr4) were used. The A. gossypii bem2 and cla4 deletion strains have been described previously (Ayad-Durieux et al., 2000; Wendland and Philippsen, 2000). The S. cerevisiae las17/bee1 deletion strains RLY157 (MATa ura3-52 his3-Δ200 leu2-3,112 lys2-801D bee1::LEU2; kindly provided by R. Li, Harvard Medical School, Boston, MA), YMW171K (MATa las17::kanMX4, ade2-101, his3-200, leu2-1, lys2-801, trp1-63, ura3-52), YMW172K (MATα las17::kanMX4, ade2-101, his3-200, leu2-1, lys2-801, trp1-63, ura3-52), and RH4207 (MATa las17::kanMX4, bar1::LYS2, ade2-101, his3-200, leu2-1, trp1-63, ura3-52; kindly provided by B. Winsor, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France) were used for heterologous complementation experiments. Media were prepared as described previously (Wendland and Philippsen, 2001). A. gossypii strains were transformed by electroporation as described (Wendland et al., 2000). G418/geneticin was added to rich media for selection of antibiotic-resistant transformants at a final concentration of 200 μg ml-1. Yeast cells were transformed according to the lithium-acetate procedure (Gietz et al., 1995). Yeast media were prepared as described (Wendland and Philippsen, 2001). The Escherichia coli strain DH5α served as host for plasmids.
Isolation of AgWAL1
AgWAL1 was isolated in a multicopy suppressor screen using the temperature sensitivity of the las17/bee1 mutant yeast strains. Different plasmid libraries based on yeast episomal plasmids were used containing either A. gossypii or S. cerevisiae genomic DNA inserts. Transformed cells were plated on minimal medium plates and incubated for four days at 37°C, which is the restrictive temperature for bee1/las17 cells. Several yeast transformants could be isolated. Total DNA was extracted from these colonies and transformed into E. coli. Plasmids were recovered and retransformed into the yeast strain RLY157 to verify their ability to enable growth of RLY157 at elevated temperature. This resulted in the isolation of five independent plasmids from a CEN-ARS based S. cerevisiae plasmid library and one plasmid from a YEp352-based A. gossypii plasmid library (kindly provided by P. Stahmann, Fachhochschule Lausitz, Senftenberg, Germany), termed pST-WAL1. The complete double strand sequence of the A. gossypii DNA insert as well as the terminal sequences of the yeast DNA inserts were determined (MWG Biotech, Ebersberg, Germany). The GenBank accession number for A. gossypii WAL1 gene is AY144115.
Construction of plasmids
A plasmid clone, pWAL1cc, containing the complete AgWAL1 gene was constructed by ligating a 3.5 kb SacI fragment of pST-WAL1 into the SacI site of pRS415. Plasmid pWAL1-XL was constructed making use of the in vivo recombination machinery of S. cerevisiae (Wendland, 2003). To this end, a polymerase chain reaction (PCR) fragment containing the complete AgWAL1 open reading frame (ORF) was amplified from pST-WAL1 using the primers XL1-WAL1, 5′-TCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGGTCTTCTAACGGCGGAAGAC-3′, and XL2-WAL1, 5′-ATGACAAGTTCTTGAAAACAAGAATCTTTTTATTGTCAGTACTGATCACCAATCATCAGCATTGTCATCG-3′. This PCR fragment contains terminal homology regions to the AgTEF1 promoter and terminator to direct homologous recombination. Plasmid pRS415-kanMX is a yeast CEN-ARS plasmid that carries the kanMX selectable marker, which consists of the kanamycin resistance ORF (kan) under control of the AgTEF1 promoter and terminator sequences. pRS415-kanMX was linearized using NruI, which cleaves a unique site within the kan ORF. This linearized plasmid cannot be propagated in yeast because S. cerevisiae cannot reseal blunt-end DNA breaks. Linearized plasmid and the AgWAL1 PCR product were co-transformed into yeast. By means of homologous recombination between the linearized plasmid and the PCR product, a new plasmid was generated, pAgWAL1-XL, in which the AgWAL1 ORF is fused to the AgTEF1 promoter and terminator. Correct integration was verified by sequencing.
Gene disruption and complementation
Insertion and deletion of AgWAL1 were performed via PCR-generated GEN3 disruption cassettes as described previously (Wendland et al., 2000). The following primers were used for the generation of the disruption cassettes: S1-WAL1 (5′-TTAAGCGGGCAATTCCCAAAGCATCGAACAAGATTATAGATGTTGGCTAGGGATAACAGGGTAATACAG-3′) and S2-WAL1 (5′-CTCTTGCAGGAGGACCGATGGCTTGTCCAATTGAGACTTGTCAACCTAGGCATGCAAGCTTAGATCT-3′). Disruption of AgWAL1 was verified by PCR using primers G1-WAL1 (5′-CGCGGATCCGTGTATACGCATATTACCTGACGCAC-3′), G2 (5′-GTTTAGTCTGACCATC-3′), G3 (5′-TCGCAGACCGATACCA-3′) and G4-WAL1 (5′-CGCGGATCCGAGCGTGGGAAAGATCTGGGCGTCG-3′). Primary transformants of A. gossypii are heterokaryotic (i.e. mycelia with multinucleate hyphal segments carry two kinds of nuclei with either wild-type or mutant alleles). Sporulation yielded homokaryotic null mutants, which allowed phenotypic analyses of the mutant strains. The Agwal1 mutant phenotype was complemented by plasmids carrying AgWAL1.
Cytological techniques
Septa were stained using calcofluor (0.1 μg ml-1), with cells either grown in liquid rich medium or on slides covered with thin layers of rich medium, as described previously (Wendland and Philippsen, 2000). Actin was stained from exponentially growing cultures. Cells were fixed in formaldehyde (3.7%) and PBS, and stained with rhodamine-phalloidin (Molecular Probes, Leiden, The Netherlands) as described elsewhere (Oberholzer et al., 2002). Endocytosis was visualized using the vital dye FM4-64 (Molecular Probes). Strains were grown to exponential phase in liquid full medium, harvested and incubated with 2 μM FM4-64. Mitochondria were stained using DIOC6 (Molecular Probes). Cells were mounted on deep-well microscopy slides and then either used directly for microscopy to allow the analysis of early endocytosis or incubated with the dye for up to 16 hours before microscopic observation. Fluorescence microscopy was done on a fully motorized Zeiss AxioplanII imaging microscope with the appropriate filter combinations for each dye (Chroma Technology, Rockingham, VT). Images were taken using a digital video imaging system (Princeton Instruments (Roper Scientific), Trenton, NJ, USA) as described previously (Wendland and Philippsen, 2002).
Time-lapse microscopy
Strains were grown in either complete or minimal medium. Microscopy slides were prepared as follows. Deep-well slides were used and filled with 100 μl of YPD agar medium each, containing FM4-64 when required. This medium was heavily vortexed to increase the oxygen content. The deep well was then covered with an extra slide to generate an even surface; once the agar had solidified, the cover was removed, cells were applied and the well sealed with a coverslip. Microscopy was carried out at 26°C. Images were acquired in a fully automated fashion (2.5 second or 60 second intervals for FM4-64 or DIOC6 time-lapse, and 180 seconds for differential interference contrast (DIC) or Nomarski time-lapse series), collected into stacks and processed as video clips with a frame rate of 10 images per second using Metamorph Software (Universal Imaging Corporation, Downingtown, PA, USA).
Isolation and protein comparison of fungal WASP homologues
A multicopy suppressor screen was performed on bee1/las17 mutant yeast strains based on their temperature sensitivity and growth arrest at 37°C with the aim of isolating either extragenic suppressors or the WASP homologue of a filamentous fungus. With an S. cerevisiae genomic library, five plasmids were isolated containing different but overlapping inserts bearing in common the S. cerevisiae LAS17/BEE1 gene. No other isolate was obtained. Using an A. gossypii genomic 2μ-ARS plasmid library, a single plasmid was obtained several times. Sequencing of the insert revealed the presence of an A. gossypii WASP homologue, AgWAL1 (Wiskott-Aldrich syndrome like protein) and adjacent genes. Comparison of the A. gossypii WAL1 locus with S. cerevisiae showed syntenic arrangements to two S. cerevisiae loci on chromosomes XII and XV, which is an indication of ancient synteny potentially revealing the gene order in a common ancestor that was altered in yeast after its apparent genome duplication but retained in A. gossypii. Interestingly, homologues of two S. cerevisiae genes located on different chromosomes (XII and XV) that are positioned next to each other in A. gossypii, WAL1 and the A. gossypii homologue of YLR287c, are also neighbours in Candida albicans (see Fig. S1 in supplementary material).
The AgWAL1 locus represents the only WASP homologue of A. gossypii that could be determined by examination of the complete A. gossypii genome sequence. AgWAL1 (AGR285w) encodes a protein of 732 amino acids and is located on chromosome VII (Dietrich et al., 2004). The highest degree of amino acids sequence identity is found with the S. cerevisiae Las17/Bee1 protein (43%). Comparison of the specific WASP domains of Wal1p with WASP homologues from ascomycete fungi revealed conserved N-terminal WH1 (>65% amino acids sequence identity) and C-terminal VCA (43% and 56% amino acid sequence identity between A. gossypii and C. albicans or A. gossypii and S. cerevisiae, respectively) domains with a variable central proline-rich region (for a protein alignment, see Fig. S2 in supplementary material). Other fungal species whose genomes have been sequenced (e.g. Neurospora crassa, Aspergillus nidulans, Magnaporthe grisea and Ustilago maydis) also contain a unique WASP homologue. Although the WASPs of the filamentous fungi N. crassa, A. nidulans and M. grisea group together in phylogenetic tree analyses, the A. gossypii WASP forms a group with the WASPs of S. cerevisiae and C. albicans (not shown).
Disruption of AgWAL1 results in temperature-sensitive growth inhibition
Disruption of AgWAL1 was achieved by PCR-based gene targeting. Deletion resulted in the removal of 87% of the AgWAL1 ORF, thus generating a null allele (see Fig. S3 in supplementary material). Correct construction of the deletion was verified by PCR using standard verification primers. Primary transformants were wild-type-like in morphology owing to the heterokaryotic nature of the multinucleate hyphal compartments. Upon sporulation and clonal selection of mycelia containing only transformed Agwal1 mutant nuclei, a slow-growth phenotype became evident. Furthermore, growth of Agwal1 mutants at elevated temperature (above 35°C) was completely abolished (Fig. 1A). This temperature-sensitive growth inhibition could be rescued by reintroduction of plasmid-borne AgWAL1 (Fig. 1B). Complementation with plasmid pWAL1cc, which contains only the WAL1 gene, demonstrated that the mutant phenotype was due solely to the disruption of the genomic copy of WAL1. Heterologous complementation of the S. cerevisiae bee1/las17 defect with WAL1 was dependent on the high copy number of plasmid pSTWAL1 (containing a 2μ-ARS), because pWAL1cc (containing a yeast centromere and ARSH4) did not restore growth of the bee1/las17 strain at elevated temperature.
Agwal1 mutant reveals defects in the organization of the actin cytoskeleton.
During the polarized hyphal growth phase in A. gossypii wild-type hyphae cortical actin patches are concentrated in the hyphal tips (Fig. 2A,B). Defects in the organization of the actin cytoskeleton can be observed as isotropic growth phases and swollen hyphal morphologies and result in slow growth phenotypes as was shown for Agrho3 and Agbem2 mutants (Wendland and Philippsen, 2000; Wendland and Philippsen, 2001). Therefore, we analysed the distribution of the cortical actin cytoskeleton in the Agwal1 mutant (Fig. 2C,D). Localization of some of the cortical actin patches along the hyphae occurred in both wild-type and wal1 hyphae. Clustering of cortical actin patches in the hyphal tips, as found in the wild type, did not occur in wal1 hyphal tips (n=500 for each strain). Instead, cortical actin patches characteristically accumulated in subapical regions of wal1 hyphal tips, which was never observed in the wild type (Fig. 2). In the wild type, actin cables formed an abundant meshwork of cables that was tip directed in the hyphal apex. In the wal1 mutant, some actin cables were found to insert into the hyphal tip even when the tip did not contain any obvious cortical actin patches (Fig. 2C,D). Rhodamine-phalloidin staining also revealed the absence of actin rings at presumptive septal sites that can readily be found in wild-type hyphae. The presence and correct positioning of actin rings are, however, prerequisites for septation and chitin-ring formation in A. gossypii (Wendland and Philippsen, 2002). To explore this in more detail, we stained wild-type and mutant hyphae with calcofluor to analyse the distribution of chitin in the cell wall.
Agwal1 mutants show defects in septation
Wild-type hyphae are tube-like and form septa in regular intervals. Chitin is accumulated at these septal sites and forms chitin rings. Furthermore, hyphal tips can be stained more brightly by calcofluor than subapical regions, indicating sites of active secretion (Fig. 3A). By contrast, hyphae of the wal1 mutant are wider than wild-type hyphae, irregular in shape and periodically swollen. Mutant hyphal cell walls were stained uniformly by calcofluor, corresponding to delocalized cell surface growth and the bulbous hyphal shape. As was suggested by the absence of actin rings at presumptive septal sites, chitin rings were only rarely observed in the mutant hyphae (n=200), indicating a severe defect in septation (Fig. 3B). This corresponds to observations in the Agcyk1 and Agcla4 mutants, in which the failure to form actin rings at presumptive septal sites has been shown to be accompanied by lack of chitin accumulation (Ayad-Durieux et al., 2000; Wendland and Philippsen, 2002). Formation of sporangia depends on septation. Thus, lack of septa (as in the Agcyk1 mutant) also resulted in the absence of sporulation in the Agwal1 mutant (not shown).
Agwal1 mutants exhibit defects in the maintenance of polarized hyphal growth
To understand the growth defects of the Agwal1 mutant more clearly, we compared growth of the mutant strain with the wild type using in vivo time-lapse microscopy. First, we characterized growth of the wild type. Juvenile mycelia, approximately 10 hours after inoculation of spores in complete medium, grew initially at a slow extension rate (Fig. 4A; see Movie 1 in supplementary material). Lateral branches were formed and strong vacuolarization of the hyphae was initiated about 6 hours later at the centre of the mycelium. At about this time, an increase in growth speed was observed and dichotomous tip branching occurred, which is a hallmark of colony maturation in A. gossypii (Wendland and Philippsen, 2000). Movie 1 also showed that some newly formed hyphal tips (n=7 in this movie) stopped hyphal elongation and became vacuolarized, and that cytoplasmic flow was redirected out of these hyphae to other growing ends (Fig. 4A, arrowheads). Additionally, growth was found to be terminated in some instances upon contact of a hyphal tip with other hyphae that blocked further elongation (n=4 in this movie), which also resulted in vacuolarization and redirection of cytoplasmic streaming (Fig. 4A, asterisks). Spore germination of the Agwal1 mutant, in contrast to wild-type hyphae, was found not to result in the characteristic bipolar branching pattern (n=100), which, in the wild type, leads to the generation of two germ tubes at opposite ends of the germ cell (Fig. 4). Most often a second hyphal tube was produced directly adjacent to the first hypha. Growth of the mutant hyphae was very slow (Fig. 4B; see Movie 2 in supplementary material). Under time-lapse growth conditions mutant hyphae often stopped growing and hyphal cells lysed, which made it difficult to obtain time-lapse movies at this early stage of growth from the wal1 mutant. Germination of heterokaryotic spores under selective conditions that favoured growth of only the wal1 mutant in liquid culture revealed similar defects in the bipolar germination pattern (n=200).
In adult stages, hyphal growth rate of the wild type was accelerated (at least tenfold) upon hyphal maturation and reached on average 170 μm hour-1 (Fig. 5; see Movie 3 in supplementary material). The appearance of many vacuoles in adult wild-type hyphae occurred in subapical regions soon after hyphal tip growth. Using in vivo time-lapse microscopy, we were able to observe vacuolar movement (cytoplasmic streaming) in wild-type hyphae in the direction of the growing hyphal tip (Fig. 5; see Movies 1,3 in supplementary material). The speed of cytoplasmic streaming was dependent on the rate of tip growth. Interestingly, the direction of cytoplasmic streaming could also be influenced by septation. Once a septum had formed vacuoles could not pass across these sites (Fig. 5, asterisks; see Movie 3 in supplementary material). In the hypha shown, three consecutive septation events occurred. The first septum prevented backward flow of vacuoles. The second septum occurred at a dichotomous bifurcation and led to the direction of the vacuolar streaming into just the left branch. Finally, the third septum compartmentalized a hyphal segment and trapped the cytoplasm within. This also inhibited loss of cytoplasm out of this compartment. The septation event was soon followed by the formation of a new hyphal tip in this compartment (Fig. 5; see Movie 3 in supplementary material). Furthermore, retrograde flow was observed out of hyphae that stopped growing owing, for example, to contact inhibition (arrows indicate the direction of streaming in Fig. 5, whereas the arrowhead points to the relative positions of one of the large vacuoles).
In contrast to the wild type, mutant Agwal1 hyphae reached radial growth rates of a maximum of only 15 μm hour-1 and were thus more than ten times slower than adult wild-type hyphae (Fig. 6; see Movie 4 in supplementary material). Nevertheless, growth direction was still highly polarized and dichotomous tip branching occurred in Agwal1 hyphae. Agwal1 mutant hyphae were filled with cytoplasm and only few vacuoles were visible that occurred very late during these in vivo observations (Fig. 7, bottom row). Cytoplasmic streaming can be seen in Movie 5 (see supplementary material) but streaming is not directed and vacuoles remain localized in the same hyphal segments in which they were formed, in contrast to the wild type. Interestingly, at several positions in subapical regions, swellings of hyphae occurred very rapidly (within 3 minutes; i.e. a single interval of image acquisition during the time-lapse analyses) (Fig. 6, boxes, left to right) and shown (top row to bottom row) as enlarged images in Fig. 7 (and see Movie 5 in supplementary material). The positions at which these swellings occurred might represent attempts of the hyphae to produce lateral branches.
Organelle morphology and movement in A. gossypii wild-type and mutant strains
Wild-type hyphae showed rapid cytoplasmic streaming and vacuolar movement, in contrast to wal1 hyphae, during our DIC observations. Because defects in polarized growth might be the result of defects in exo- or endocytosis, we examined and compared the vacuolar dynamics of different strains in vivo using the vital dye FM 4-64. In wild-type hyphal tips, fast-moving small vesicles (endosomes) were found. There were often tubular structures linking these vesicles with each other and resulting in fusion of vesicles (Fig. 8A; see Movie 6 in supplementary material). Staining of the wild-type endosomes was very efficient, resulting in bright fluorescent images. Staining of wal1 hyphae was always more diffuse (Fig. 8B; see Movie 6 in supplementary material). Strikingly, whereas wild-type hyphae contained endosomes in the hyphal tips and, in subapical regions, had already fused these endosomes to smaller vacuoles, the wal1 hyphal tips contained very few visible endosomal vesicles and thus appeared relatively empty (compare Fig. 8A,B; see Movie 6 in supplementary material). Other A. gossypii slow-growth mutants deficient in genes that are also involved in actin cytoskeleton dynamics had already been analysed (Ayad-Durieux et al., 2000; Wendland and Philippsen, 2000). We stained hyphae of these cla4 and bem2 mutants with FM4-64 to analyse whether early endosomes were present in the hyphal tips of these mutants. Both of these mutant strains contained endosomal vesicles in their hyphal tips that showed movement and tubular structures similar to the wild type (Fig. 8C,D; see Movies 8,9 in supplementary material). To compare the morphology of larger vacuoles of the different strains, we performed high resolution in vivo time-lapse analyses of older (non-growing) hyphal segments under the identical conditions, running for approximately 4:30 minutes with recordings every 2.5 seconds (Fig. 8E-H; see Movies 7-9 in supplementary material). In the wild type, large vacuoles showed rapid changes in shape, became elongated and (in two instances) fusion events were observed. Interestingly, not all vacuoles seen in the wild-type panel of Movie 7 (see supplementary material) are in motion at the same time. In the middle of the movie, two vacuoles appeared to be moving at the same time, coming to a stop and thus appeared round in shape, and afterwards simultaneously initiated motion again. By contrast, the vacuoles on both sides (tipward and farther back) exhibit phases of motion and pausing independently of the vacuoles in the centre. In Movie 7 (see supplementary material), a wal1 hypha is shown imaged under the same conditions as the wild type. Here, large vacuoles of the wal1 hypha showed drastically reduced motion compared with the wild type. (Fig. 8E,F; see Movie 7 in supplementary material). Whereas large vacuoles in the bem2 mutant readily changed shape, similar to the wild type, vacuoles of the cla4 mutant were rounder and thus resembled more closely the vacuoles of wal1 hyphae (Fig. 8G,H; see Movies 8,9 in supplementary material). Because these high-resolution recordings did not readily allow the analysis of vacuolar movement, we analysed wild-type and wal1 hyphae in a time-lapse series over 3.0-4.5 hours with images taken at 1-minute intervals (see Movies 10,11 in supplementary material). In these movies, the generation and tipwards movement of large vacuoles was evident in the wild type. Hyphae of the wal1 mutant grew much more slowly, did not contain endosomes in the hyphal tips, did not generate large vacuoles and displayed only oscillating, not tipwards, movement of vacuoles.
Because, in S. cerevisiae, several organelles are dependent on the actin cytoskeleton for movement, we also wanted to elucidate whether, like vacuolar movement, the motility of mitochondria was affected in the wal1 mutant hyphae (Fig. 8I,J; see Movie 12 in supplementary material). To this end, we stained wild-type and wal1 hyphae with the vital dye DIOC6 and recorded in vivo fluorescence time-lapse series. In the wild type, mitochondria showed tubular structures, localized to the hyphal tip and appeared to be very motile. Mitochondria in wal1 hyphae appeared to be bigger than wild-type mitochondria. However, mitochondria in wal1 hyphae showed a tubular structure and wild-type-like motility. Strikingly, in wal1 hyphae mitochondria were localized along the hyphae also including the hyphal tips. This finding is in sharp contrast to the distribution and movement of vacuoles in wal1 hyphae.
Members of the WASP family regulate a range of cellular processes that require dynamic reorganization of the actin cytoskeleton (Caron, 2002). The role of WASPs has been analysed in great detail in various organisms and a functional understanding has been gained towards the role of WASP in activating the Arp2/3 complex, resulting in the generation of branched actin filaments (Higgs and Pollard, 2001; Weaver et al., 2003). WASP is regulated by phosphorylation and autoinhibition by intramolecular contacts that can be relieved through binding to Cdc42p (Cory et al., 2003; Buck et al., 2004). Fungal WASPs are different from mammalian WASPs in this respect, because they lack a G-protein-binding domain. Consequently, the S. cerevisiae WASP Las17p/Bee1p was shown not to be autoinhibited but regulated by SH3-domain-containing proteins (Rodal et al., 2003).
Polarization of the actin cytoskeleton towards the hyphal tips is required for hyphal growth in filamentous fungi (Wendland, 2001). This results in the clustering of cortical actin patches at the hyphal tip and maintenance of polarized hyphal growth and tip-directed secretion in fungal hyphae (Momany, 2002). Previously, we have characterized the role of Rho-protein modules for polarized hyphal growth and the organization of the actin cytoskeleton in A. gossypii (Wendland and Philippsen, 2001). Because Rho-protein function is exerted via effector proteins, we are interested in elucidating the signalling route from Rho proteins to the actin cytoskeleton. In this effort, we recently analysed the C. albicans WASP homologue WAL1 (Walther and Wendland, 2004). We observed similarities between the yeast stages of C. albicans and S. cerevisiae of WASP mutant strains that include a partially delocalized cortical actin cytoskeleton, defects in bud-site selection and endocytosis (Li, 1997; Walther and Wendland, 2004). Particularly, in the C. albicans WASP mutant, an increased number of vacuoles was found, suggesting a role in vacuole formation. C. albicans is a dimorphic fungus that can be induced to form true hyphae by, for example, the presence of serum at 37°C. The C. albicans WASP mutant, however, was not able to form hyphae under all laboratory conditions tested and thus we were not able to analyse hypha-specific defects in this organism. Therefore, we generated a WASP deletion mutant in the constitutively filamentous fungus A. gossypii.
Growth defects of the A. gossypii wal1 mutant
The Agwal1 mutant shows temperature-sensitive growth, which is completely abolished at elevated temperatures (e.g. 37°C). Polarized hyphal growth was drastically reduced in wal1 hyphae, resembling growth rates of bem2 and cla4. Cla4p was shown to be required for a process termed hyphal maturation (Ayad-Durieux et al., 2000). This describes the phenomenon that growth speed during mycelium formation is slow in juvenile mycelia and increases dramatically - in A. gossypii more then tenfold - in mature mycelia. The speed of polarized hyphal growth is dependent on the transport of secretory vesicles to the hyphal tip and the insertion of these vesicles into the membrane (Katz et al., 1972; Watters and Griffith, 2001). We found that fast polarized hyphal growth occurs at a time when large vacuoles are formed in subapical regions of hyphal filaments. With these vacuoles, transport processes in the direction of growing tips could be observed (see Movies 1,3 in supplementary material). Septation was found to block cytoplasmic streaming (corresponding to bulk tipwards organelle movement) resulting in a redirection of transport (see Movie 3 in supplementary material). This was surprising given that nuclear migration through septal pores was frequently observed in A. gossypii (Alberti-Segui et al., 2000). Another surprising observation was retrograde flow of cytoplasm out of hyphae that stopped growing (e.g. owing to contact inhibition). This indicates that, in A. gossypii, polarized hyphal growth can serve as a sink for cellular components of subapical regions not compartmentalized by septation. In the Agwal1 mutant, long-range transport of vacuoles was not evident in the time-lapse analyses. Large vacuoles were generated in wal1 hyphae, although this process appeared to be delayed in comparison to the wild type. The process of vacuolar fusion might be of importance for polarized hyphal growth. In order to maintain rapid polarized growth, large amounts of cytoplasm need to be produced. This requirement could be lowered if rear parts of hyphal compartments were filled with large vacuoles. Therefore, defects in vacuolar fusion per se might affect polarized growth rates. VAM4/YPT7 encodes a S. cerevisiae gene that is required for vacuole fusion (Wichmann et al., 1992). Deletion of the VAM4/YPT7 homologue avaA in A. nidulans, however, was shown not to reduce the hyphal growth rate, although this mutant exhibited highly fragmented vacuoles (Ohsumi et al., 2002).
Septation defect of Agwal1
Deletion of AgWAL1 resulted in a septation defect similar to that observed in Agcla4 mutants: hyphae showed very few actin rings, and chitin accumulation at presumptive sites of septation was very rare. This is in contrast to the S. cerevisiae bee1/las17 and the C. albicans wal1 mutant strains, which did not show defects in cytokinesis and septum formation but rather showed increased rates of random budding (Madania et al., 1999; Walther and Wendland, 2004). The failure to form actin rings might be due to either the direct involvement of Wal1p in the polymerization of actin or the requirement for Wal1p in transport processes that are necessary to achieve polarized secretion to septal sites. Although many of the key players involved in septum formation in S. cerevisiae share homologues in filamentous fungi, our results indicate that there are mechanistic differences leading to septum formation between yeasts and filamentous fungi (Walther and Wendland, 2003). The wal1 mutant strain was also found to be defective in sporulation. This corresponds to the sporulation defect of the aseptate cyk1 strain (Wendland and Philippsen, 2002). In the wild-type strain, sporangia are formed by fragmentation of hyphae at septal sites. The failure to generate septa therefore results in the inability to produce sporangia and thus spores.
Wal1p plays a central role in the localization of cortical actin patches
Loss of WAL1 resulted in the clustering of cortical actin patches in subapical parts of the hyphae, in contrast to the tipwards localization in the wild type. This represents a new actin cytoskeleton defect not previously observed in filamentous fungi. Delocalization of actin patches indicates that their presence in the hyphal tips is supporting polarized secretion and fast hyphal elongation that, in the wal1 mutant, is now converted into more isotropic growth, resulting in widened diameters and swollen hyphae. Surprisingly, we found that a few actin cables were able to extend into hyphal tips even without the presence of actin patches (Fig. 2). This could be of mechanistic importance for the establishment and maintenance of polarized hyphal growth in filamentous fungi. In S. cerevisiae, Las17p/Bee1p is required for the activation of the Arp2/3 complex and actin nucleation (Winter et al., 1999; Higgs and Pollard, 2001; Zalevsky et al., 2001). This activity might, however, be limited to the nucleation of branched filaments (Higgs and Pollard, 2001). By contrast, the formin family of proteins was shown to represent a class of actin nucleators that assemble straight filaments (Pruyne et al., 2002; Sagot et al., 2002). The wal1 mutant phenotype displayed specific defects in the polarized localization of actin patches, whereas actin filaments were found to be tip localized in the absence of cortical patches in the tip. The reduced hyphal growth rate of wal1 hyphae could therefore be the result of the basic activity provided by formin dependent assembly of actin filaments. This might occur in a Cdc42p-dependent manner, because CDC42 was shown to be required for the establishment of cell polarity in A. gossypii (Wendland and Philippsen, 2001). In such a model, the polarity establishment machinery, involving the Cdc42p/Rho-protein module and formin effectors, is required for choosing a site of growth and polarizing the actin cable network to result in an initial growth phase. Subsequently, activation of WASP leads to the coordination of polarized secretion, the polarization of cortical actin patches, and long-range transport to the hyphal tip, which is a prerequisite of fast hyphal elongation. In wal1 hyphae, slow growth might therefore be due to shortcomings in either the sufficient delivery of secretory vesicles to the tip or in defective exo- or endocytosis.
Nevertheless, in the Agwal1 mutant, cortical actin patches were still generated and localization was clearly enriched in subapical parts of the hyphae. Other proteins that can activate actin assembly might be responsible for the generation of actin patches, such as A. gossypii homologues of the S. cerevisiae ABP1 and PAN1 genes. Currently, we do not know the mechanism for the accumulation of cortical actin patches at these specific subapical sites. However, localization of cortical actin patches to the hyphal tips requires Wal1p. Considering the protein domains of fungal WASPs, the N-terminal WH1/pleckstrin-homology domain can provide a link to plasma-membrane-associated lipids, specifically phosphatidylinositol-4,5-bisphosphate (Rohatgi et al., 1999). AgWal1p might act as a scaffold protein to coordinate the positioning of cortical actin patches with polarized morphogenesis by linking Rho-GTPase signalling with the ability to stimulate the Arp2/3 complex in combination with the use of lipids as positional information. Synergistic activation by lipid and Cdc42p signalling was shown for N-WASP and also the S. cerevisiae Cla4p, which thereby function as `coincidence detectors' (Rohatgi et al., 1999; Wild et al., 2004).
Endocytosis and vacuolar fusion in A. gossypii
Loss of WAL1 not only affected the localization of cortical actin patches but also resulted in defects in vacuolar morphology, positioning and movement. Strikingly, in A. gossypii, the tips of wal1 hyphae were not only devoid of cortical actin patches but also lacking in endosomes. Both actin patches and endosomes formed in subapical parts of the hyphae. In S. cerevisiae, a tight association of actin clumps and endocytic vesicles was observed by inactivating Prk1p kinase using chemical genetics (Sekiya-Kawasaki et al., 2003). Transport of vacuoles in yeast is dependent on the actin cytoskeleton and type-V myosin motors (Weisman, 2003). Loss of AgWAL1 might cripple the ability to generate actin filaments to provide tracks for vacuolar movement in the hyphae. A defect in vacuolar motility might therefore be a secondary consequence of the wal1 mutation. In fact, the motility and directed movement of vacuoles in wal1 hyphae was drastically reduced in comparison to the wild type. Whereas wild-type vacuoles displayed deformations and rapid shape changes, wal1 vacuoles remained round and immobile, suggesting that relatively little motor-protein force influences these vacuoles. Our results further show that this phenotype of wal1 hyphae is specific for vacuolar movement and distribution and does not affect the localization and movement of mitochondria.
We thank R. Li and B. Winsor for strains, and D. Schade for excellent technical assistance. This work was funded by grants of the Deutsche Forschungsgemeinschaft to JW (We2634/2-1) and by funds for the Research Group `Fungal Pathogens' by the Friedrich-Schiller University and the Hans-Knöll Institute. GenBank accession number for WAL1 is AY144115.
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/117/21/4947/DC1
- Accepted June 24, 2004.
- © The Company of Biologists Limited 2004


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