Recently I've written a few articles about the benefits of leasing a car. I've gotten a lot of feedback on it and I stand by the fact that almost nobody should buy a car.
You are paying 5K over 10 years to have a 30K vehicle.
You are paying 30K over 5 years for a 30K vehicle that you could have leased for 18K.
Option 2, leasing for 18K, saves you 12K of cash over the first 5 years. Cash is king. The average person thinks about what they can "save" over the final 5 years. The rich think about how they can invest in what they save the first 5 years. Let's say you invest 12K into something that will bring you income. The question becomes how much income can you produce with that 12K investment over the next five years.
Look at it from a 10-year perspective. It's true that by leasing I will pay 15K more over the final 5 years than if I had bought, assuming only minor maintenance for the buyer. But the person who leases can turn every dollar that they save in the first few years of that time period into something they can invest in that will produce income for them over the next five years.
Is it better to have 12K earlier to invest or have 18K later of savings? Not considering inflation, it depends on what you can do with that 12K up front.
The middle class doesn't add in the cost of owning with money that they could have saved by leasing in the beginning and investing it into something that brings them other streams of income. The question is not just calculating what the monthly cost to own is over a given time period versus leasing. This leaves out all income producing scenarios.
This is not counting all the other things I already went over in detail in my previous article on the benefits of leasing. It's not about building equity, it's about what will make you the most money.
Leasing is more favorable for cash flow.
The middle class looks at the cost of things. It is impossible to create real wealth just by saving money on a middle class income. When the wealthy throw money around on ridiculous things — cars, boats, planes, vacation homes, it no longer matters that the things are poor investments, because the money they are spending is minuscule compared to the abundance they've created.
In reality, anyone worried about spending $300 a month to use a car and not have it build "equity" will never create big boy money because their focus is not on creating more income.
The middle class is concerned about a couple of hundred dollars when they should have their mind on a couple of million dollars.
I encourage everyone in the middle class to get on Cardone University and get themselves out. There is no freedom in the middle class, it will continue to be squeezed, and buying a car instead of leasing one will not get anyone rich.


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ROSEMONT, IL (June 2, 2017) – As Stacy May-Johnson walked off the field after a 9-4 victory, the first one to greet her, with a huge smile, was none other than her daughter, Corrie. "I didn't know if she'd be here, or if she'd be at home in bed, but here she is," May-Johnson said.
As Johnson spoke with the media after the game, teammate Emily Allard came up to Corrie with an even bigger smile.
"Did you see your mommy win?" Allard said with a smile as Corrie giggles.
"It was a special thing, but the girls made it really special and really fun," May-Johnson added.
It all started in the bottom of the first, with two outs on a 2-0 count, when Natalie Hernandez saw a pitch she liked and took a swing. Her single to center made way for designated player Emily Allard to score the Bandits' first run of the game after she reached with a base hit of her own.
The Racers responded to Hernandez's RBI single in the first with an edge. It only took one turn through the lineup for the Racers' leadoff batter, Taylor Schlopy, to crush a ball to left, scoring two after Griffin Joiner reached first with a single.
Hernandez later added her second base hit and RBI of the game, allowing the tying run to score after Akron had pulled ahead.
The following at bat, Stacy May-Johnson, who came out of retirement for a second time to join the Bandits for the 2017 season, saw her first regular season at-bat since 2011 as a pinch-hitter. Facing a 1-0 count, May-Johnson hit a hard fly ball to right, which quickly dropped to send Allard home and Carosone to second.
After the Bandits scored six runs from three straight scoring plays, Allard then was brought to the plate for the second time in the inning, this time driving in final run to seal an explosive eight-run inning.
Following the rally, Shelby Turnier relieved Bandits starter Haylie Wagner (W, 2-0) in the top of the sixth. Wagner left after tossing five innings while surrendering six hits and two runs. Turnier struck out the final batter of the game, Akron's Sam Fischer, to seal the 9-4 win.
The Bandits are off to a 2-0 start, marking their third consecutive season with a pair of wins to kick off the campaign. The finale of the three-game set against the Racers is scheduled for Saturday night at 7:35 p.m. and the game will be broadcast on NPF TV.


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Infection and Infertility
Part of book or chapter of book English OPEN
Wu, Wei; Tang, Qiuqin; Gu, Hao; Chen, Yiqiu; Xia, Yankai; Wang, Jiahao Sha and Xinru;
Publisher: InTech
[1] Kanda, T. et al. Case of mumps orchitis after vaccination. International Journal of Urology 21, 426-428 (2014). doi:10.1111/iju.12305.
[2] Whyte, D. et al. Mumps epidemiology in the mid-west of Ireland 2004-2008: increasing disease burden in the university/college setting. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin 14, 11-15, (2009). pii:19182.
[5] Zhou, X. L. et al. Effects of hepatitis B virus S protein on human sperm function. Human Reproduction 24, 1575-1583 (2009). doi:10.1093/humrep/dep050.
[6] Huang, J. M. et al. Effects of hepatitis B virus infection on human sperm chromosomes. World Journal of Gastroenterology 9, 736-740 (2003).
[7] Huang, J. M. et al. Studies on the integration of hepatitis B virus DNA sequence in human sperm chromosomes. Asian Journal of Andrology 4, 209-212 (2002).
[8] Bu, Z. et al. Effect of male hepatitis B virus infection on outcomes of in vitro fertilization and embryo transfer treatment: insights from couples undergoing oocyte donation. International Journal of Clinical and experimental Medicine 7, 1860-1866 (2014).
[9] Hadchouel, M. et al. Presence of HBV DNA in spermatozoa: a possible vertical transmission of HBV via the germ line. Journal of Medical Virology 16, 61-66 (1985).
[10] Englert, Y. et al. Medically assisted reproduction in the presence of chronic viral diseases. Human Reproduction Update 10, 149-162 (2004). doi:10.1093/humupd/dmh013.
[11] Paavonen, J. & Eggert-Kruse, W. Chlamydia trachomatis: impact on human reproduction. Human Reproduction Update 5, 433-447 (1999).
[12] Joki-Korpela, P. et al. The role of Chlamydia trachomatis infection in male infertility. Fertility and sterility 91, 1448-1450 (2009). doi:10.1016/j.fertnstert.2008.06.051.
Association of the IL1RN Gene VNTR Polymorphism with Human Male Infertility (2012)
Toxoplasma gondii Decreases the Reproductive Fitness in Mice (2014)
InTech via InTech (Part of book or chapter of book, 2016)

redpajama_set_name: RedPajamaCommonCrawl


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MANGA.TOKYO > Anime & Otaku News > This Week's Top 10 Most Popular Anime News (11-17 October 2019)
This Week's Top 10 Most Popular Anime News (11-17 October 2019)
by manga.tokyo
©2017 川原 礫/KADOKAWA アスキー・メディアワークス/SAO-A Project
Hello MANGA.TOKYO readers!
A new week has brought new anime news so we are back to bring you a brief recap of the top 10 most popular anime news this week! There have been more announcements for the January 2020 season and our anime info pages for Winter 2020 have started to fill up with new information on all the anime that will start next season! Keep up to date by checking the page from time to time, and if you are still wondering what to watch from this season, check out our Fall 2019 anime list:
Fall 2019 Anime: Official Twitter Hashtags & Pages
Now let's see what the most popular anime news for the week 11-17 October 2019 were!
Most Popular Anime News of the Week
10. Merch! Merch! Merch!
Merchandise of the Day
9. Science can be very romantic! Especially if the topic of research is Love itself.
RikeKoi Reveals Trailer and Key Visual
8. An exciting story seems to wait for us in In/Spectre!
In/Spectre Introduces Additional Characters
7. If it exists, there's an anime for it, right? This anime follows girls while they go fishing!
Hokago Teibo Nisshi Anime Announced for Spring 2020
6. Darwin's Game is something for everybody who enjoys the gamble-to-survive kind of genre!
Darwin's Game Announces Main Cast for Winter 2020 Start
5. Did you already put Appare-Ranman! on your plan-to-watch list? We can't wait for this story!
Appare-Ranman! Anime Takes Viewers Across America
4. These girls are fighting out in the open sea and continue to look good doing it!
High School Fleet Movie Reveals New Visual and Characters
3. Last week we found Ghost in the Shell ranking at number three, and this week it's the review of Paprika!
Paprika Movie Review
2. The biggest gaming event in Japan was of course reviewed by us!
Tokyo Game Show 2019 Event Report
1. Everybody was excited for the continuation of Sword Art Online Alicization, as were we!
Sword Art Online Alicization WoU Episode 1 Preview Stills and Synopsis
Until Next Week!
Check our reviews section for our Summer 2019 weekly anime reviews and for more anime movie and anime series reviews. MANGA. TOKYO will soon be back with another weekly digest.
Until then, don't forget to follow us on Facebook, Twitter, Instagram, Reddit, and Pinterest, and bookmark MANGA.TOKYO for all the latest news in the otaku world!
The latest anime, manga, games, and live action news on MANGA.TOKYO
5 Centimeters Per Second Movie Review
This Week's Top 10 Most Popular Anime News (13-19 April 2018)
This Week's Top 10 Most Popular Anime News (6-12 April 2018)
This Week's Top 10 Most Popular Anime News (16-22 March 2018)
This Week's Top 10 Most Popular Anime News (16-22 February 2018)
This Week's Top 10 Most Popular Anime News (11-17 May 2018)
This Week's Top 10 Most Popular Anime News (9-15 February 2018)


This year sees the 50th anniversary of one of the world's most beloved, iconic and desirable cars: the Porsche 911. This motoring great is equally fashionable bought as a classic or new as a status symbol. It is the ultimate in auto style and performance.
On the outside, the modern 911 has changed little from the 1963 original. The diagonally sloping roof line, angled windscreen and funky circular headlamps prove the adage 'if it ain't broke, don't fix it.' While new 911s have had their edges softened, the basic shape has largely been maintained through various facelifts and special editions. The other enduring element is the rear-sited engine, giving an at the time controversial rear-wheel drive, that made the Porsche stand out from rivals.
However, on the road there has been some serious updating. The earliest 911s were powered by a 2.0 litre, 130bhp engine for a 0-60mph speed of just over 9 seconds – good for its time but considered tortoise-like these days. The 2013 anniversary edition 911 comes with a 3.4 litre engine, returning 350bhp and will get you to 60mph in 4.8 seconds, almost half the original 911's time.
The cost new in 1963 was £4,095, which equates to about £68,000 of today's money, which is reassuringly consistent, as the latest 911 will lighten your wallet by about £73,000. Bearing in mind the weight of history behind these cars, it's not a bad price.
While the Porsche 911 represents success in the same way as a Rolex watch or a bespoke suit, it grew from the fairly banal origins of the VW Beetle, designed by none other than Ferdinand Porsche for pre-war Germany. The 911 was unveiled to the world after years of development at the 1963 Frankfurt Motor Show and at the time was named the 901. The 911 designation came about after Peugeot claimed exclusive use of three-digit model numbers using a '0' as the centre number.
Since then, the 911 has become a worldwide motoring icon, with more than 800,000 examples sold. It is beloved of drivers everywhere and is a particular celebrity favourite, with famous owners including US talk show host, Jay Leno, pop-star and well-known Ferrari-fanatic, Jay Kay, Arnold Schwarzenegger and David Beckham. American comedian and actor, Jerry Seinfeld, has a collection of Porsches numbering more than 50, of which many are 911s.
Porsche fans may be interested in a new book celebrating the car's anniversary, 'The Porsche 911 Book, 50th Anniversary Edition' by publisher TeNeues.


Always encrypt confidential data – This step requires your company to put in a bit of extra work, but it serves as a great security defense by making it very difficult for unauthorized individuals to read your customers' sensitive records. Therefore, it's well worth the effort.
Destroy paper documents when they are no longer in use – This one isn't just a suggestion – it's a law! Under the Fair and Accurate Credit Transaction Act (FACTA) Disposal Rule, organizations are required to shred or otherwise destroy customer information printed on paper once they are done using it. Taking this measure limits the amount of customer data that will be at risk if someone breaks into your company.
Keep your devices updated – Staying up-to-date in terms of your operating system, web browser and security software will give your company maximum protection against cyberthreats that have the potential to steal customer data, such as viruses and malware.
Limit access to customer information – As a rule of thumb, you should only allow those at your company who absolutely need to access customer data to do so. The fewer people who can view the data, the lower the chance that someone will make a mistake that compromises it.
Use a network monitoring service – When threats to your customers' data arise, it's important to catch and address them quickly. Hiring an East Norriton network monitoring service will help you do so, thereby giving your company peace of mind that customer information isn't being compromised under the radar.
When you make use of these methods, you'll greatly decrease the risk of your customers' information falling into the wrong hands and, in turn, keep your business running smoothly.
Let's Talk Solutions – Contact Alura Today!
If you'd like some assistance with monitoring and maintaining your company's network, Alura Business Solutions is the place to contact! Since 2005, our East Norriton network monitoring service has been helping organizations of all sizes and types keep their data highly secure by identifying and eradicating threats using NetAlert and NetAudit. If you're interested in joining our client family, just give us a call at 844-558-7200. We look forward to showing you why we've gained a reputation for being a network monitoring service East Norriton organizations can rely on!


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Karla kalendrisaba
Kanada hääl
Teised arvamuslood
The far-left and the Kremlin (1)
Estonian Life No. 1 2022 - Laas Leivat
This writer has on occasion pointed to the mutually beneficial relationship between many European rightwing fringe movements and aggressive anti-Western Russian politics.
While the far-right's visibility in Europe has dramatically grown in recent years, especially with their anti-vaxxer exhortations, several years ago the influence of the far-right and the far-left in the EU elections was about equal. Both received about 8-10% of the vote. In other words, political extremes in the EU parliament represented about 20% of its members. Their combined influence has grown since.
The far-right's affinity to the Kremlin defies historic precedent.
Not so with much of the far-left. It still adheres to the time-worn tradition of being Soviet (now Russian) apologists, even useful idiots. They're true to their legacy from three generations ago, solidly rooted by well-known fellow travelers. One recalls Walter Duranty, the British-born, Moscow-based, New York Times journalist who ridiculed and lied in press reports about the 1930's Ukrainian famine while simultaneously confiding to the British embassy that ten million had died from starvation in the Soviet Union. In fact, nearly four million Ukrainians alone perished from a deliberately managed starvation program.
(One must acknowledge a rudimentary difference between the far right and the far left in their support of Russia's policies. The left fringe knowingly and deliberately fosters pro-Kremlin positions, some with ideological or intellectual motives, others out of nostalgia for the 1917 revolution, etc. The ultra-right presents a more strident, visceral posture, not out of ideological zeal. Putin is their poster hero who protects cultural conservatism and battles the permissive, decadent liberalism of the West.)
In the 1940's Bernard J. Stern, a US scholar and recognized expert on Soviet nationalities authored an article for the American Sociological Review praising the virtues of the Communist policy towards minorities and captive republics, that the latter supposedly enjoyed the right to self-determination and the right to secede from the USSR. He praised Josef Stalin as a skillful statesman. The academic piece was typically unfettered Soviet propaganda published in a recognized academic journal.
Under Putin, Russia is adopting Soviet era propaganda as historical fact. The stark facts that were exposed in the early 1990's, the brutality, and failures of the of the USSR are have been rejected and replaced by a myth, one that the Western left fringe honours, has taken their place.
Perhaps one of the most distinct examples of this is the observation of Jean-Luc Mélanchon, leader of France's Left Party, made after Russia had invaded and annexed Crimea, who insisted that Crimea belonged to Russia: "The Crimean ports are vital for the security of Russia. It is absolutely predictable that the Russians would not give in…….The Russian nation cannot allow North Americans and NATO moved to their doors."
The European far-left's relationship with the Kremlin was built on "old comrade" networks, developed by the Soviet KGB, conatcts, according to intelligence sources, are still alive. An appropriate example is Germany's Die Linke party, the direct descendant of the Marxist-Leninist ruling party of the former East Germany, the Socialist Unity Party. It has been calling for an end to NATO and the creation of a military organization including Russia "from Lisbon to Vladivostok". This coincides perfectly with Putin's current demand – the dismantling of the North Atlantic Alliance.
Ukrainian researchers have identified numerous far left groups, all of whom have substantial followers at their websites, at least two of which are Canada based. Canadian Dimension and Globalresearch, both support the Kremlin's claims of Ukraine residing within Russian space and its domination, and justify the suppression of domestic political dissent.
How many of these Kremlin proxies are funded by Russia is unknown. But often they cite Russian official media or even Russian neo-Nazi sources. Their authors/contributors have visited Russian occupied territories such as Crimea and the Donbass region of Ukraine and signed declarations backing the aggressive international behavior benefitting the Russian ruling elite.
While many dismissed the ultra-left of the West as dupes of Moscow, before the collapse of the Soviet Union, they still espoused some Marxist doctrine. One party rule, state ownership and Communists in power jibed with their ideals. But Russia today gives capitalism a bad name. The most affluent capitalists are those closest to the Kremlin leadership, whose corrupt relationship with these government-favoured oligarchs has made Putin one of the wealthiest men in the country.
In spite of this the far-left still supports this non-Marxist ruling elite, who do not adhere to any socialist principles domestically or internationally. While not accusing the Russian regime of capitalism, the far-left calls it "progressive" for its anti-American stance, and its use of some oil and gas revenues for social programs. The left certainly denies any Russian imperialistic ambitions.
Regardless of which one has more influence, the far-left and the ultra-right together help to expose a large number of people to pro-Moscow propaganda. Both are remorseless Kremlin sycophants.
Laas Leivat, Toronto
0 #1 ... 2022-01-11 15:44
https://nationalinterest.org/feature/ukraine-washington%E2%80%99s-bellicose-security-client-192772
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Q: How to draw STRAIGHT lines in manim? I want to create a video with manim, which includes some straight lines.
Manim's class Line is actually line segments. Changing the buff attribute to an negative value doesn't work. I tried to calculate the intersect of the line with the screen border, but it's too complicated. Then I expand the line like this:
def expand_line(line: Line, ratio=100) -> None:
    ends = line.get_start_and_end()
    delta = ends[1] - ends[0]
    line.put_start_and_end_on(ends[0] - ratio * delta, ends[1] + ratio * delta)

It works well; but the animations like Create goes too fast.
Are there any classes or other better ways to draw straight lines?

A:
I tried to calculate the intersect of the line with the screen border, but it's too complicated.

You had the right idea here. Here's my solution:
class Test(Scene):
    def extend_line(self, line: Sequence[np.ndarray], **kwargs):
        sr_points = ScreenRectangle(height=config.frame_height).get_vertices()
        inters = []
        for i in adjacent_pairs(sr_points):
            inters += [line_intersection(i, line)]
        inters.sort(key=np.linalg.norm)  # we want the nearest intersections
        return Line(*inters[:2], **kwargs)

    def construct(self):
        a = self.extend_line([LEFT * 2, UP])
        self.wait()
        self.play(Create(a))

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In order to graduate high-school you must have a minimum of 40 service hours. All forms must be turned into 1SGT. Navarra (Room 505) before school or during first or second lunch. All forms must be filled out according to the guidelines posted on the school's website otherwise they will not be counted.


"Holding on to anger is like grasping a hot coal with the intent of throwing it at someone else; you are the one who gets burnt".
Anger is an emotion we all experience almost every-day. It is a strong negative emotion that has the ability to ruin the state of mind. Anger makes one emotionally disturbed, intellectually confused, physically violent and spiritually lost. The overall effect of anger is deadly.
"Anger is an enemy with the face of a friend. Anger is like a very sharp sword. Anger destroys everything".
So, when does one become angry? Or more importantly, one must ask themselves, why am I angry? Or why should I become angry. It is a question that has different answers as different people have different trigger points, temper and reason.
In Sanskrit, anger has many synonyms but in English, they mean the same. For example, some of the synonyms of anger are Cope(कोप), Krodh(क्रोध), Dwesh(द्वेष) etc however the cause of Cope, Krodh and Dwesh are not the same. The cause of Cope is issue based. It is temporary. Cope has to be shown at times in the interest of work. It is necessary to survive in this world. Krodh on the other hand is person based. In other words it is grudge. This should not be there. If this exists, slowly this grows into Dwesh (literally translates to Dvi+visha which is double poison). And Dwesh kills you every single day. They say poison kills you once and for all but Dwesh exists till death. The feeling of Dwesh is a nasty one. The very presence of the other person physically and mentally causes you to breed hatred and resentment towards the other individual. It eats you from within and you lose your state of mind. The peace which you actually deserve is lost for the simple cause of breeding hatred, anger and all such negative thoughts in your head.
A very good example to illustrate what damage anger does to oneself can be learnt from the Ramayana. Manthara, the personal maid of Queen Kaikeyi was obsessed with Bharat occupying the throne. She hated the fact that the whole kingdom loved Rama more than Bharat. This hatred that she bore flared up when she came to know that Rama was going to be coronated as King of Ayodhya. No sooner did she hear this than she ran to Kaikeyi to give her the "bad" news. Kaikeyi was joyful upon hearing the news but then Manthara manipulated her into thinking that Rama would enslave her son and rule like a tyrant. The seed of hatred has been planted. Now the course of action. Years ago, Kaikeyi received two vows from her husband, King Dasharatha. She decided to use them – First ask for Bharat (her son) to be coronated as King and second- Banish Rama to vanvas (to live in the forest) for 14 years.
You might think what is the big deal? Now Rama was not only loved by his father and mother but was loved by all. The whole kingdom in fact had started making preparations for his coronation. No sooner did the news break out to the people they were at a loss of words. And what was the news- Dasharatha fell unconscious (because he couldn't imagine surviving without his favourite son), Rama has given up the throne and will go to the forest for 14 years (to fulfil his duty to abide by the promise given by his father to his mother), Bharat will be King, Kaikeyi asked such boons and Manthara was the root cause. Just observe. One person bore a seed of hatred and see how catastrophic the results turned out. The events later on turned to be worse but the point is look how dangerous how anger is.
"You will not be punished for your anger, you will be punished by your anger".
"From anger, complete delusion arises, and from delusion bewilderment of memory. When the memory is bewildered, intelligence is lost and when intelligence is lost one falls down into the material pool".
In conclusion, Anger is something we must avoid as much as possible. Unless and until is required to show it, it must be kept at bay. Anger can't be avoided entirely because it is a part of our system but it can always be kept under control. As mentioned earlier, Cope must be used but never let it transform into Krodh and dwesh therein. Anger can truly disturb our state of mind. We may not be able to control our feelings but we surely have the power to control the shape of the mind. Its ok to feel angry at times but when it turns the state of mind into that of depression or resentment, beware!! You might have taken the wrong turn.
2019 started off in a crazy way!! An egg went on to bag the highest number of likes on instagram and shortly came the "#10 year challenge" . It required an individual to post a picture of him/her in the present and 10 years ago i.e., A picture that was clicked back in 2007-2009 and a current picture of the guy/girl would have to be posted with the script : #10 year challenge to show the person's past and present appearance. It is also called #HowHardDidAgingHityou.
In 10 years, How much have we appreciably evolved in the mental platform?
In 10 years, How mature have we become?
In 10 years, How has your thought process evolved ? Is it actually evolving ? Are we actually involving in something useful ?
I mean, think over it! Is it really necessary? We all age but don't you think that if we are actually mature we would focus on improving our understanding on the grand scale rather focusing on how our body has changed. The body has to age and die out one day or the other and this is the law of nature. No amount of external make-up can change the fact. Even the external make-up is temporary. We cannot control this change but what is under our control is the directionality of our mind. It takes the slightest tap to set the mind on the wrong path. Whether we like it or not, we must see the truth as it is- External beauty is temporary. It is the character that matters.
" Knowledge will give you power, but character respect".
"As a person puts on new garments, giving up old ones, the soul similarly accepts new material bodies, giving up old ones".
" As the embodied soul continuously passes, in this body, from boyhood to youth to old age, the soul similarly passes into another body at death. A sober person is not bewildered by such a change".
Hence let us not worry how we looked 10 or 50 years ago- sure it will be nostalgic but it is more important to develop and control our minds. Let us strive to ameliorate our notions about the world and our psyche and see things the way they are.
The year has come to an end. Pretty much, everyone has had their share of ups and downs this year. A multitude of people would have already listed down "New Year Resolutions". These include to improve physical and mental well being, improve finances, improve career, education, get over grudges and so on.
35% of participants who failed their New Year's Resolutions admitted they had unrealistic goals, 33% of participants didn't keep track of their progress and a further 23% forgot about it. About one in 10 respondents claimed they made too many resolutions.. There can be several reasons such as – Lost track of it, Lack of will power, too many distractions and incapable of handling them, "Ahh leave it, will try it from next year". Just as one plans ahead of any journey, similarly in the journey of life also, every individual must chalk out his/her steps. If one consults their own experience, they will understand that it is better to lay out short term goals which lead to a long term one. Many a times, people aim at the target but forget to reconsider the path. An aimless arrow is the most dangerous one.
As the year ends, I thought of putting across a few thoughtful quotes which would surely lighten the hearts of knowledge within us and take a step closer to realizing one's self.
"Be a good person, but don't waste time to prove it".
"You're the driver of your own life, don't let anyone steal your seat".
"Never apologize to others for their misunderstanding of who you are".
"Be the same person, privately and publicly".
"If you want to fly, give up everything that weighs you down".
"The less you respond to negative people, the more peaceful your life becomes".
"If you want to be happy, do not dwell in the past, do not worry about the future, focus on living in the present".
"Talking about our problems is our greatest addiction. Break the habit. Talk about your joys".
"Never get too attached to anyone, because attachments is the root of expectations and expectations lead to suffering".
"Overthinking is the biggest cause of unhappiness".
"Greatness in man lies in simplicity. Seeking attention only leads to disappointment".
"Man acts according to his intelligence".
"Remain humble no matter what your status is".
"Never make a decision when you are in extremes of moods. Always make decisions with a calm mind".
"Righteousness is useless unless paired with foresight".
Hope these quotes from the ancient times bring upon the light of wisdom in our life to carry on even in life's darkest times.


text:
Q: Generating interesting random TMs To get a more intuitive understanding of the halting problem I want to generate some random TMs and see how they behave, what some heuristics can tell about them, etc.
The problem is that, if I correctly recall a paper I once saw, the probability for random TMs, from some reasonable distribution, to quickly halt or enter a trivial infinite loop approaches 1 as the number of states grows. That makes for boring research.
I want to know if there is a distribution of TMs in which this is not true. That is, for most TMs, it's not obvious if they halt or not. Although I'm not particular about TMs - any model of computation will do.


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Trip from Alipurduar to Pratapgarh
Weather: POSSIBLE LIGHT RAIN OVERNIGHT AND IN THE EVENING.
Pratapgarh, also called Bela, Bela Pratapgarh, Partapgarh or Partabgarh, is the birthplace of the national poet Harivansh Rai Bachchan.
The district is named after its headquarters town Bela Pratapgarh, commonly known as Pratapgarh. Raja Pratap Singh, a local raja between 1628–1682, located his headquarters at Rampur near the old town of Aror. There he built a garh (fort) and called it Pratapgarh after himself.
Subsequently, the area around the fort became known as Pratapgarh. When the district was constituted in 1858, its headquarters was established at Bela, which came to be known as Bela Pratapgarh, the name Bela presumably being derived from the temple of Bela Bhawani on the bank of river Sai. It is popularly known as "Bela Mai" – meaning mother goddess Bela.
Sai and the Ganges are the main rivers flowing through Pratapgarh district. Pratapgarh It is bounded on the north by the district of Sultanpur, on the south by Allahabad, on the east by Jaunpur, on the west by Fatehpur and north-west by Rae Bareli.
Attraction in Pratapgarh
Route Map from Alipurduar to Pratapgarh
Bandhavgarh
Manendragarh
Mainpat
Jashpur Nagar
Chirimiri
Katghora
Ratanpur


On a recent jaunt to the south-west we ventured into the pages of the Cornwall Guide by Weekend Journals and found ourselves near Penzance at a rugged and surprisingly quiet (for a hot day in the middle of the summer holidays) Tremenheere Sculpture Gardens.
Our first encounter of the site was to spend 15 minutes in the near-complete darkness of a small converted water tank. Although that sounds a little bizarre it becomes more normal once we explain that it was all part of an artwork by light/vision legend, James Turrell. The tank has a small hole in it's roof with a mirror and becomes a camera obscura; we waited in the dark long enough our eyes to adjust and the view from directly above the tank was projected onto the wall in front of us. As there isn't quite enough light to wake up all of the cells in our eyes we see the tree canopy in black and white. We took the picture below from on top of the tank to give an idea of what we saw but it's really one of those things that is best appreciated in person.
Exiting the tank and re-entering the daylight made the colours outside seem much more vivid; the orange of the crocosmia and various fungi popped against a verdant green background.
The artwork displayed at Tremenheere all seems to stem from ideas of the earth and has a real connection to nature which is so fitting given where the sculptures sit. Each piece we had to discover rather than having it offered to us and we found that all the more engaging. A particular favourite was Turrell's Skyspace and the line of grasses that Richard Long uses to point to the sea whilst referencing the waves with movement.
Most of all we enjoyed the way that the artwork made us see texture and form in things that grew in the grounds and appreciate the cracks in the dry earth for their sculptural qualities, or the graphic lines of bamboo.
We absolutely recommend a visit to Tremenheere Sculpture Gardens.
When we found ourselves in Melbourne Australia a couple of months ago we couldn't help but check out their botanic garden.


601. Azathioprine for ocular inflammatory diseases. Pasadhika S, Kempen JH, Newcomb CW, Liesegang TL, Pujari SS, Rosenbaum JT, Thorne JE, Foster CS, Jabs DA, Levy-Clarke GA, Nussenblatt RB, Suhler EB. Am J Ophthalmol. 2009 Oct;148(4):500-509.e2. Epub 2009 Jul 1.
602. Local treatment for lymphoid malignancies of the eye. Giuliari GP, Hinkle DM, Foster CS. Anticancer Agents Med Chem. 2009 Dec;9(10):1123-8. Review.
603.Current trends in the management of ocular symptoms in Adamantiades-Behçet's disease. Zakka FR, Chang PY, Giuliari GP, Foster CS. Clin Ophthalmol. 2009;3:567-79. Epub 2009 Oct 19.
604. Clinical review: treatment of vitreoretinal lymphoma. Pe'er J, Hochberg FH, Foster CS. Ocul Immunol Inflamm. 2009 Sep-Oct;17(5):299-306. Review.
605. Infliximab for the treatment of refractory scleritis. Doctor P, Sultan A, Syed S, Christen W, Bhat P, Foster CS. Br J Ophthalmol. 2010 May;94(5):579-83. Epub 2009 Dec 2.
606. Serum soluble interleukin-2 receptor levels do not correlate with an associated systemic disease in patients with uveitis. Papadaki T, Iaccheri B, Fiore T, Zacharopoulos I, Thadani S, Christen W, Androudi S, Foster CS. Acta Ophthalmol. 2010 Mar;88(2):e28-9. Epub 2009 Jun 2.
607. Cataracts and uveitis. Jancevski M, Foster CS. Discov Med. 2010 Jan;9(44):51-4.
608. Cyclosporine for ocular inflammatory diseases. Kaçmaz RO, Kempen JH, Newcomb C, Daniel E, Gangaputra S, Nussenblatt RB, Rosenbaum JT, Suhler EB, Thorne JE, Jabs DA, Levy-Clarke GA, Foster CS. Ophthalmology. 2010 Mar;117(3):576-84. Epub 2010 Jan 19.
610. Mycophenolate mofetil for ocular inflammation. Daniel E, Thorne JE, Newcomb CW, Pujari SS, Kaçmaz RO, Levy-Clarke GA, Nussenblatt RB, Rosenbaum JT, Suhler EB, Foster CS, Jabs DA, Kempen JH. Am J Ophthalmol. 2010 Mar;149(3):423-32.e1-2. Epub 2009 Dec 30.
611. Ocular disease in patients with ANCA-positive vasculitis. Watkins AS, Kempen JH, Choi D, Liesegang TL, Pujari SS, Newcomb C, Nussenblatt RB, Rosenbaum JT, Thorne JE, Foster CS, Jabs DA, Levy-Clarke GA, Suhler EB, Smith JR. J Ocul Biol Dis Infor. 2009 Dec 12;3(1):12-19.
612. Cataracts and uveitis. Jancevski M, Foster CS. Discov Med. 2010 Jan;9(44):51-4.
613. Ceron OM, Hinkle DM, Foster CS. Intraocular Inflammatory Disorders Update. Retinal Physician. 2009 Feb:24-8.
614. Combination of rituximab and intravenous immunoglobulin for recalcitrant ocular cicatricial pemphigoid: a preliminary report. Foster CS, Chang PY, Ahmed AR. Ophthalmology. 2010 May;117(5):861-9. Epub 2010 Jan 4.
615. The spectrum of fundus autofluorescence findings in birdshot chorioretinopathy. Giuliari G, Hinkle DM, Foster CS. J Ophthalmol. 2009;2009:567693. Epub 2010 Feb 8.
616. Amorese L, Foster CS. Parasitic Keratitis. Contemporary Ophthalmology. 2010 Jan 15.
617. Multicenter Uveitis Steroid Treatment Trial Research Group. The Multicenter Uveitis Steroid Treatment (MUST) Trial: Rationale, Design and Baseline Characteristics. Am J Ophthalmol 2010;149:550-561. e10. Epub 2010 Jan 25.
618. Cyclophosphamide for ocular inflammatory diseases. Pujari SS, Kempen JH, Newcomb CW, Gangaputra S, Daniel E, Suhler EB, Thorne JE, Jabs DA, Levy-Clarke GA, Nussenblatt RB, Rosenbaum JT, Foster CS. Ophthalmology. 2010 Feb;117(2):356-65. Epub 2009 Dec 6.
619. Hypopyon in patients with uveitis. Zaidi AA, Ying GS, Daniel E, Gangaputra S, Rosenbaum JT, Suhler EB, Thorne JE, Foster CS, Jabs DA, Levy-Clarke GA, Nussenblatt RB, Kempen JH; Systemic Immunosuppressive Therapy for Eye Diseases Cohort Study. Ophthalmology. 2010 Feb;117(2):366-72. Epub 2009 Dec 14.
620. Tumalad M, Mansoor A, Foster CS. Ocular Manifestations of Herpes Simplex Virus Infection. Contemporary Ophthalmology. 2010 Feb 28.
622. Nonsteroidal anti-inflammatory therapy and recurrent acute anterior uveitis. Fiorelli VM, Bhat P, Foster CS. Ocul Immunol Inflamm. 2010 Apr;18(2):116-20.
623. Durrani K, Foster CS. Management of Uveitis: Current concepts and modalitites. Contemporary Ophthalmology. 2010 Apr 30.
624. Lee F, Foster CS. Pharmacotherapy of Uveitis. Expert Opinion on Pharmacotherapy. 2010 May;11(7):1135-46.
625. Masood K, Foster CS. Birdshot Retinochoroidopathy: An Updated Review of Diagnosis, Treatment, and Management Options. Contemporary Ophthalmology. 2010 May 15.
626. Siddique SS, Foster CS. Central Serous Chorioretinopathy. Contemporary Ophthalmology. 2010 Jun 15.
627. Adverse effects of smoking on patients with ocular inflammation. Galor A, Feuer W, Kempen JH, Kaçmaz RO, Liesegang TL, Suhler EB, Foster CS, Jabs DA, Levy-Clarke GA, Nussenblatt RB, Rosenbaum JT, Thorne JE. Br J Ophthalmol. 2010 Jul;94(7):848-53.
628. Portero-Benito A, Foster CS. Ocular Albinism. Contemporary Ophthalmology. 2010 Jul 15.
629. Mehta M, Foster CS. Histopathologic Differential Diagnosis of Common Eyelid Lesions. Contemporary Ophthalmology. 2010 Jul 30.
630. Microperimetry findings in patients with birdshot chorioretinopathy. Giuliari GP, Pujari S, Shaikh M, Marvell D, Foster CS. Can J Ophthalmol. 2010 Aug;45(4):399-403.
631. Keratoprosthesis in autoimmune disease. Ciralsky J, Papaliodis GN, Foster CS, Dohlman CH, Chodosh J. Ocul Immunol Inflamm. 2010 Aug;18(4):275-80.
632. Villaneuva- Palafox SK, Shaikh M, Foster CS. Neuromyelitis Optica (Devic's Disease) : An entity distinct from multiple sclerosis. Contemporary Ophthalmology. 2010 Aug 15.
633. Durezol (Difluprednate Ophthalmic Emulsion 0.05%) compared with Pred Forte 1% ophthalmic suspension in the treatment of endogenous anterior uveitis. Foster CS, Davanzo R, Flynn TE, McLeod K, Vogel R, Crockett RS. J Ocul Pharmacol Ther. 2010 Oct;26(5):475-83.
634. Recurrent conjunctivitis and scleritis secondary to coexistent conjunctival pemiphigus vulgaris and cryptic herpes simplex infection: a case report. Mehta M, Dacey M, Stephen Foster C. Ocul Immunol Inflamm. 2010 Dec;18(6):454-6. Epub 2010 Sep 2.
635. Scleral necrosis in a patient with congenital erythropoietic porphyria. Siddique SS, Gonzalez-Gonzalez LA, Thakuria P, Chang PY, Foster CS. Cornea. 2011 Jan;30(1):97-9.
636. Pars plana vitrectomy versus immunomodulatory therapy for intermediate uveitis: a prospective, randomized pilot study. Quinones K, Choi JY, Yilmaz T, Kafkala C, Letko E, Foster CS. Ocul Immunol Inflamm. 2010 Oct;18(5):411-7.
637. Effects of AIN457, a fully human antibody to interleukin-17A, on psoriasis, rheumatoid arthritis, and uveitis. Hueber W, Patel DD, Dryja T, Wright AM, Koroleva I, Bruin G, Antoni C, Draelos Z, Gold MH; Psoriasis Study Group, Durez P, Tak PP, Gomez-Reino JJ; Rheumatoid Arthritis Study Group, Foster CS, Kim RY, Samson CM, Falk NS, Chu DS, Callanan D, Nguyen QD; Uveitis Study Group, Rose K, Haider A, Di Padova F. Sci Transl Med. 2010 Oct 6.
638. Krader CG, Foster CS. Dexamethasone Implant an Option For Posterior Segment Uveitis. Ophthalmology Times. 2010 Oct 15; 35 (20): 1, 11.
639. Herpes keratitis in a patient undergoing treatment with topical mitomycin C. Siddique SS, Gonzalez-Gonzalez LA, Amorese L, Shaikh M, Foster S. Cornea. 2011 Apr;30(4):466-7.
640. Anterior segment optical coherence tomography. Jancevski M, Foster CS. Semin Ophthalmol. 2010 Sep-Nov;25.
641. Foster CS, Anesi S. The Successful Approach to Ocular Rosacea. Drugs and Disease, Refractive Eyecare. Dec 2010: 11-12, 19.
642. Dexamethasone intravitreal implant for noninfectious intermediate or posterior uveitis. Lowder C, Belfort R Jr, Lightman S, Foster CS, Robinson MR, Schiffman RM, Li XY, Cui H, Whitcup SM; Ozurdex HURON Study Group. Arch Ophthalmol. 2011 May;129(5):545-53. Epub 2011 Jan 10.
643. Palafox SK, Smitha J, Tauber A, Foster CS. Ophthalmia Neonatorum. Journal of Clinical & Experimental Ophthalmology 2:119 (1-5).
644. Peter Y. Chang, Sana S. Siddique, Mehrine Shaikh and C. Stephen Foster. 2010, Topical corticosteroids in ocular diseases, Current Topics in Steroid Research, Vol. 7, 1-10.
645. Foster CS. Drug Delivery Mechanisms for the Treatment of Uveitis. Retina Today. 2011 March.
646. Mycophenolate mofetil monotherapy in the management of paediatric uveitis. Chang PY, Giuliari GP, Shaikh M, Thakuria P, Makhoul D, Foster CS. Eye (Lond). 2011 Apr;25(4):427-35. Epub 2011 Mar 18.
649. Difluprednate for inflammatory eye disorders. Mulki L, Foster CS. Drugs Today (Barc). 2011 May;47(5):327-33.
650. Gonzalez-Gonzalez LA, Foster CS. Secukinumab. Drugs of the Future. 2011 36(6): 441-445.


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November 20, 2019 - Extreme Makeover: Home Edition—the world's biggest, most transformative, inspirational and aspirational home show ever—is coming back to television with 10 new episodes on HGTV. And, to give fans an early look at the highly anticipated reboot, HGTV has released a nearly three-minute sneak peek video on HGTV.com, HGTV Instagram and HGTV Facebook. In 2020, viewers will witness the full power of massive, community-led home transformations each week, beginning with the premiere episode of Extreme Makeover: Home Edition on Sunday, Feb. 16, at 9 p.m. ET/PT.
a deserving family gets the renovation help they need to change their lives. Working alongside Jesse, neighbors, local building experts and celebrity guests, the show's regularly appearing designers—Breegan Jane, Carrie Locklyn and Darren Keefe—create customized homes that perfectly accommodate the needs of each inspiring family.
"Today's short teaser video is just a taste of what everyone can expect from Extreme Makeover: Home Edition," said Jane Latman, president, HGTV. "We reimagined television's most iconic home renovation show. We cast Jesse as host, reviewed hundreds of deeply emotional family stories, added more special guests who have a passion to help others, and executed truly unbelievable, life-changing, whole-home renovations. With help from more than 10,000 volunteers—families, friends and neighbors—sharing more than 560,000 hours of their own time, we improved the lives of 10 special families and created another can't miss television series on HGTV."
HGTV's spin on the iconic home renovation series Extreme Makeover: Home Edition will showcase great stories, inspired volunteers and gorgeous home renovations for families who give back to their communities. The whole-home overhauls will include interior, exterior and landscaping—all completed in record time while the family is sent away.
The new episodes will be produced by Endemol Shine North America with Sharon Levy, DJ Nurre, Michael Heyerman, Brady Connell and Jesse Tyler Ferguson serving as executive producers. The company also produced the original version of Extreme Makeover: Home Edition for ABC.
Fans can follow HGTV on Facebook and Twitter and follow Extreme Makeover: Home Edition on Facebook and Twitter for the latest news and updates. Online users can join the conversation using #HGTVEXTREME.


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Teaching Tools in Plant Biology
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Research ArticleResearch Article
Two Arabidopsis Phragmoplast-Associated Kinesins Play a Critical Role in Cytokinesis during Male Gametogenesis
Yuh-Ru Julie Lee, Yan Li, Bo Liu
Yuh-Ru Julie Lee
Section of Plant Biology, University of California, Davis, California 95616
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China
Bo Liu
Published August 2007. DOI: https://doi.org/10.1105/tpc.107.050716
American Society of Plant Biologists
In plant cells, cytokinesis is brought about by the phragmoplast. The phragmoplast has a dynamic microtubule array of two mirrored sets of microtubules, which are aligned perpendicularly to the division plane with their plus ends located at the division site. It is not well understood how the phragmoplast microtubule array is organized. In Arabidopsis thaliana, two homologous microtubule motor kinesins, PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B, localize exclusively at the juxtaposing plus ends of the antiparallel microtubules in the middle region of the phragmoplast. When either kinesin was knocked out by T-DNA insertions, mutant plants did not show a noticeable defect. However, in the absence of both kinesins, postmeiotic development of the male gametophyte was severely inhibited. In dividing microspores of the double mutant, microtubules often became disorganized following chromatid segregation and failed to form an antiparallel microtubule array between reforming nuclei. Consequently, the first postmeiotic cytokinesis was abolished without the formation of a cell plate, which led to failures in the birth of the generative cell and, subsequently, the sperm. Thus, our results indicate that Kinesin-12A and Kinesin-12B jointly play a critical role in the organization of phragmoplast microtubules during cytokinesis in the microspore that is essential for cell plate formation. Furthermore, we conclude that Kinesin-12 members serve as dynamic linkers of the plus ends of antiparallel microtubules in the phragmoplast.
In angiosperms, cytokinesis is brought about by the phragmoplast, an apparatus containing a framework of microtubules, together with actin microfilaments and membranous organelles (Staehelin and Hepler, 1996). In the phragmoplast, microtubules are organized in two mirrored sets: their plus ends are juxtaposed at the division site and their minus ends face the reforming nuclei. These antiparallel microtubules serve as tracks along which Golgi-derived vesicles are transported toward microtubule plus ends. Vesicle fusion gives rise to the cell plate, the physical barrier dividing the cytoplasm of the mother cell. Thus, the arrangement of this antiparallel array and the positional maintenance of microtubule plus ends allow vesicles to be unidirectionally delivered toward microtubule plus ends, to the division site.
Phragmoplast microtubules are derived from those of the spindle midzone and are highly dynamic (Zhang et al., 1990). The establishment of the antiparallel phragmoplast array involves microtubule–microtubule sliding driven by motors in the Kinesin-5 family (Asada et al., 1997). After the antiparallel pattern is established, however, tubulin dimers are continuously polymerized onto the plus end of the phragmoplast microtubules (Vantard et al., 1990; Asada et al., 1991). Newly added microtubule segments at the plus ends would tend to overlap in an antiparallel fashion. But a recent tomographic investigation of samples prepared by rapid freezing and freeze substitution indicates that microtubule plus ends do not overlap in the middle of the phragmoplast in meristematic cells (Austin et al., 2005). Thus, the possibility is excluded for microtubules from opposite sets to slide against each other once the phragmoplast microtubule array is established. Newly added microtubule segments, therefore, have to be continuously translocated away from the middle line of the phragmoplast in order to have the plus ends remain at the division site. A plausible scenario is that one or more plus end–directed kinesins may act at the plus ends of oppositely oriented phragmoplast microtubules to continuously generate the outward force.
In the model plant Arabidopsis thaliana, there are 61 genes encoding microtubule-based motor kinesins in >10 subfamilies (Reddy and Day, 2001). Among them, two homologous genes, PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B, exhibit a cell cycle–dependent localization pattern and specifically decorate the plus ends of phragmoplast microtubules (Lee and Liu, 2000, 2004; Pan et al., 2004). Kinesin-12 members in both plants and animals bear a signature neck sequence of plus end–directed kinesins (Lee and Liu, 2000; Miki et al., 2005). Indeed, the Xenopus Kinesin-12 Xl KLP2 exhibits plus end–directed motility in vitro (Boleti et al., 1996). It has been hypothesized that Kinesin-12s serve as dynamic linkers between two mirrored sets of microtubules in the phragmoplast (Lee and Liu, 2000; Liu and Lee, 2001).
In this report, we used mutants in which genes encoding PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B were inactivated by T-DNA insertions for functional studies of these two kinesins. The data presented here indicate that Kinesin-12s play a critical role in organizing phragmoplast microtubules and, consequently, in cytokinesis.
The function of Kinesin-12A/B seemed to be different from those of reported proteins acting on phragmoplast microtubules. For example, TKRP125 of the BIMC/Kinesin-5 subfamily functions in the sliding of interdigitated microtubules (Asada et al., 1997). The At MAP65-3/PLE protein in the MAP65/PRC/Ase1p family plays a critical role in phragmoplast microtubule organization by maintaining the dimension of the microtubule array, and mutations at the corresponding locus lead to the formation of multinucleated root cells (Müller et al., 2004). Conversely, microtubule bundling activity conferred by MAP65-1 has to be downregulated via its phosphorylation by a mitogen-activated protein kinase cascade in the phragmoplast (Sasabe et al., 2006). Phragmoplast localization of the kinase is dependent on two novel plant kinesins that are essential for cytokinesis (Nishihama et al., 2002; Strompen et al., 2002). Two homologous kinesins, POK1 and POK2, play a role in the orientation of the phragmoplast and, consequently, in the orientation of the cell plate, but not in the organization of phragmoplast microtubules (Müller et al., 2006). Interestingly, loss of function of these proteins does not alter the overall organization of the antiparallel pattern of phragmoplast microtubules. This study indicates that PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B play a fundamental role in the organization of the phragmoplast microtubule array.
Loss-of-Function Mutations at the Kinesin-12A/B Loci
In order to test whether Kinesin-12A/B are indeed critical for the organization of phragmoplast microtubules, T-DNA insertional mutations were recovered (Figure 1A ). Mutations at either the Kinesin-12A or Kinesin-12B locus do not cause a noticeable defect in the growth and reproduction of the plant (Pan et al., 2004). We reasoned that these genes might function redundantly during cytokinesis. Homozygous double mutants were generated using different alleles of T-DNA insertional mutations (Figure 1A). The kinesin-12a-1 and kinesin-12a-2 mutations had the T-DNA sequence inserted in the 2nd and 12th exons, respectively, and kinesin-12b-1 and kinesin-12b-2 had T-DNA inserted in the 1st and 15th exons, respectively. In contrast with single mutants, the kinesin-12a-1 kinesin-12b-2 and kinesin-12a-2 kinesin-12b-2 double mutants consistently produced significantly fewer seeds in their fruits. The result shown in Figure 1B, as well as other results shown here, were from the kinesin-12a-1 kinesin-12b-2 mutant, unless noted otherwise. Mature siliques of wild-type plants had ∼80% mature seeds. Mature siliques of the double mutants, however, had <40%. The mutant siliques often contained small white structures, which could be either unfertilized ovules or fertilized ovules containing early aborted embryos. While the double mutants produced ∼50% fewer seeds than the wild type, the single mutants produced seeds comparable to those of wild-type plants (Figure 1C). Because the F1 plants resembled their homozygous mutant parent (Figure 1C), we concluded that the incomplete penetrance of the low-fertility phenotype was inheritable.
Mutations at the Kinesin-12A and Kinesin-12B Loci.
(A) Diagrammatical representation of the Kinesin-12A and Kinesin-12B gene structures. Introns are shown as lines, and exons are shown as boxes. Positions of the T-DNA mutational insertions of kinesin-12a-1, kinesin-12a-2, kinesin-12b-1, and kinesin-12b-2 are shown at top of the diagrams.
(B) The homozygous double mutant of kinesin-12a-1 and kinesin-12b-2 produced significantly fewer seeds in siliques. Red arrows point to ovule positions where no seeds were found.
(C) Quantification of seed production in wild-type and mutant siliques. The y axis represents the percentage of ovule positions with seeds. Plants bearing homozygous single mutations at either locus produced similar numbers of seeds as their wild-type counterparts. The homozygous double mutant and its F1 progeny produced ∼50% fewer seeds. Genotypes of the plants are as follows: 12A 12B for plants with wild-type alleles at both Kinesin-12A and Kinesin-12B loci; 12a 12B for plants with a homozygous mutation at the Kinesin-12A locus; 12A 12b for plants with a homozygous mutation at the Kinesin-12B locus; 12a 12b for the homozygous double mutant; and 12a 12b (F1) for the F1 progeny of 12a 12b.
Mutations in the Kinesin-12A/B Genes Resulted in Defective Pollen Grains
Because Arabidopsis is a self-pollinating plant, reduction of seed formation could be due to defects in either the male or female gametophyte, or both, or to defects in embryogenesis. At first, prefertilization and postfertilization ovules of the mutant were cleared for microscopic examination using Normaski optics. No observable defect was detected in either the developing embryo sacs or developing embryos (data not shown). In order to reveal whether gametophytes were responsible for the phenotype, we then performed reciprocal pollination experiments by applying mutant pollen grains onto wild-type stigma, and vice versa. Our results indicated that pollen grains of double mutant plants caused reductions of seed formation in wild-type siliques (average seed number/silique = 7; n = 7) and wild-type pollen grains restored the seed production level in mutant siliques (average seed number/silique = 43; n = 7).
Furthermore, reciprocal crosses were performed to analyze genetic transmission via gametophytes. When pollen grains of the Kinesin-12A/kinesin-12a-1; kinesin-12b-2/kinesin-12b-2 or kinesin-12a-1/kinesin-12a-1; Kinesin-12B/kinesin-12b-2 mutant were used to pollinate the stigma of a wild-type female parent, transmission of the double mutant allele (kinesin-12a-1; kinesin-12b-2) was severely reduced (male transmission efficiency = 12.9 and 20.7%, respectively) (Table 1 ). When these mutants were used as female receivers of the wild-type pollen grains, transmission of the double mutant allele through the female gamete was moderately reduced (female transmission efficiency = 64.3 and 61.7%, respectively) (Table 1). Thus, in the double mutants, the phenotype of reduction of seed formation was mainly caused by defective pollen grains. Because the transmission through the female gamete was not perfect, it was also not ruled out that these kinesins might play a role in multiple phragmoplasts associated with the cellularization of the female gametophyte.
Transmission Efficiency of kinesin-12a (12a) and kinesin-12b (12b) Mutations in Reciprocal Crosses between Mutant and Wild-Type Plants
We further analyzed male gametophytes of the double mutant by fluorescence and electron microscopy. The pollen grain is a young male gametophyte in angiosperms. The mature male gametophyte of flowering plants is composed of three haploid cells, of which two sperm cells are suspended in the cytoplasm of the vegetative (pollen tube) cell (McCormick, 2004). Pollen grains collected from open flowers of both wild-type and double mutant plants were then examined by staining with the DNA-specific dye 4′,6-diamidino-2-phenylindole (DAPI). When wild-type flowers were open, pollen grains were already mature, and they had two brightly stained sperm nuclei and a faintly stained vegetative nucleus (Figure 2A , a and b). Pollen grains isolated from mutant flowers often contained two loosely packed DNA masses (Figure 2B, a and b), and they resembled the DNA mass of the vegetative nucleus in wild-type pollen grains. To test whether the abnormal mutant pollen grains produced a generative cell or sperm, we examined them by transmission electron microscopy. In wild-type pollen grains, the vegetative nucleus and sperm (only one revealed in this section) were found in the pollen cytoplasm (Figure 2A, c). The sperm nucleus and cytoplasm were completely isolated from the vegetative cytoplasm by the sperm cell wall (Figure 2A, d). Defective mutant pollen grains, however, lacked sperm. Instead, two identical nuclei were found in single sections (Figure 2B, c). The two nuclei were suspended in the vegetative cytoplasm, and no cell wall–like structure was detected between them (Figure 2B, d).
The Double Mutant Failed to Produce Male Gametes.
(A) In the wild type (a), a mature pollen grain contains two sperm and one vegetative nucleus. The sperm nuclei (arrows) and the vegetative nucleus (arrowhead) were revealed by DAPI staining. In (b), a differential interference contrast image shows the pollen appearance. Transmission electron microscopy images ([c] and [d]) show the vegetative nucleus (VN) and one sperm cell (arrow) in the pollen cytoplasm. Note that the sperm cytoplasm was physically separated from the pollen cytoplasm by a barrier (arrows in [d]). The other sperm cell was not included in this section. Bars = 10 μm in (b) for (a) and (b), 4 μm in (c), and 1 μm in (d).
(B) In the double mutant, a defective pollen grain failed to produce sperm. DAPI staining (a) showed two loosely packed DNA masses (arrowheads) resembling the vegetative nucleus. A differential interference contrast image of this pollen is shown in (b). Transmission electron microscopy images ([c] and [d]) show two similar nuclei (N) suspended in the pollen cytoplasm. Note that between the nuclear envelopes of the two nuclei (arrowheads), there was no barrier as seen in ([A], [d]). Bars = 10 μm in (b) for (a) and (b), 4 μm in (c), and 1 μm in (d).
(C) Quantification of pollen grains in the categories of two sperm nuclei plus one vegetative nucleus (2+1), two identical nuclei (1+1) or one DNA mass (1), and shrunken appearance (s). The y axis represents the proportion of pollen grains in each category. Pollen grains in the three categories were quantified in the wild type (12A/12A; 12B/12B), single mutants (12a/12a; 12B/12B and 12A/12A; 12b/12b), various heterozygous double mutants (12A/12a; 12B/12b, 12a/12a; 12B/12b, and 12A/12a; 12b/12b), and the homozygous double mutant (12a/12a; 12b/12b).
While many defective pollen grains were consistently detected in the double mutants, very few such pollen grains were observed in the wild type and single mutants. The difference among pollen grains of these different genetic backgrounds was striking when they were classified into three categories: those with two sperm nuclei and one vegetative nucleus (2+1); those with two similar nuclei (1+1) or those with one large DNA mass (1); and those aborted ones with a shrunken appearance (s) (Figure 2C). In the pollen grains with one large DNA mass, the DNA mass likely resulted from the overlap of two indiscernible nuclei by fluorescence microscopy, as sister chromatids segregated normally in the mutants (data not shown). Alternatively, it was also not ruled out that two nuclei might fuse to become one. Nevertheless, both scenarios reflected the failure of spermatogenesis. Ninety-seven percent of wild-type pollen grains contained three nuclei, 3% contained one or two nuclei, and 0% were aborted (n = 103). In the kinesin-12a-1 single mutant, the distribution of pollen grains in these three categories was 96, 3, and 1%, respectively (n = 529). The homozygous kinesin-12b-1 mutant had very similar distribution of different pollen grains, as did the heterozygous Kinesin-12A/kinesin-12a-1; Kinesin-12B/kinesin-12b-2 mutant. In mutants bearing one copy of either Kinesin-12A or Kinesin-12B, defective pollen grains were detected more frequently than in the aforementioned mutants (Figure 2C). In the kinesin-12a-1 kinesin-12b-2 homozygous double mutant, however, only 42% of pollen grains contained three nuclei; 41% of pollen grains were either binucleate or uninucleate, and 17% of pollen grains were shrunken (n = 1825). These data, therefore, suggested that the increased loss of functional Kinesin-12A/B genes was accompanied by the increase of defective pollen grains. It was noticed that mutants bearing one functional copy of either Kinesin-12A or Kinesin-12B produced significantly more than half of normal pollen grains, which could result from possible inheritance of the wild-type product through meiosis. Thus, our data suggested that in the double mutant, the failure of gametogenesis frequently took place due to the failure in cytokinesis, resulting in the absence of the male gamete sperm.
Pollen grains isolated from open flowers of the homozygous double mutant were subject to germination in vitro. It was found that defective pollen grains with two similar nuclei were able to produce pollen tubes as trinucleate pollen grains (data not shown). Thus, a defective pollen grain and resulting tube would fail to reach the ovule, which would reduce the fertility in the double mutant.
Kinesin-12A/B Play a Role in the Organization of Phragmoplast Microtubules
It was hoped that the T-DNA insertions would inactivate Kinesin-12 gene expression in homozygous mutants. The Kinesin-12A and/or Kinesin-12B transcripts were detected in the wild type (12A/12A; 12B/12B) by RT-PCR (Figure 3A ). Moreover, a corresponding transcript was detected in mutants bearing either one or two copies of the wild-type Kinesin-12A or Kinesin-12B gene (12a/12a; 12B/12B, 12A/12A; 12b/12b, 12a/12a; 12B/12b, and 12A/12a; 12b/12b). While the At1g13320 transcript encoding protein phosphatase 2A, as a positive control, was detected in the wild type and all mutants, corresponding transcripts were not detected in homozygous mutants (Figure 3A). Thus, the insertions inactivated the expression of the Kinesin-12A/B genes.
Alteration of At Kinesin-12 Expression by T-DNA Insertional Mutations.
(A) Absence of the Kinesin-12A and/or Kinesin-12B transcripts in single and double homozygous mutants by RT-PCR. The transcripts were detected in the wild type (12A/12A; 12B/12B), and either transcript was detected in mutants bearing either one or two copies of the wild-type Kinesin-12A or Kinesin-12B gene (12a/12a; 12B/12B, 12A/12A; 12b/12b, 12a/12a; 12B/12b, and 12A/12a; 12b/12b). The At1g13320 transcript encoding protein phosphatase 2A (PP2A) was used as a positive control.
(B) Localization of At Kinesin-12A in dividing microspores of the wild type (12A 12B) and the double mutant (12a 12b). The At Kinesin-12A signal is pseudocolored green, and DNA is pseudocolored red. While in the wild type, microspore-specific signals (white arrowheads) were detected between the vegetative nucleus (blue arrows) and the generative nucleus (purple arrows), no such signal was detected in the microspore of the double mutant. The peripheral signal was due to the autofluorescence of the pollen coat. Bars = 5 μm.
To determine the activity of Kinesin-12A/B in the dividing microspores, antibodies raised against the C-terminal domain of Kinesin-12A were used. The antibodies recognize both Kinesin-12A and Kinesin-12B (Pan et al., 2004). In the wild-type microspores, Kinesin-12A/B appeared as discrete signals between the forming vegetative nucleus and the generative nucleus (Figure 3B). No detectable signal was revealed in double mutant microspores at identical stages, indicating that Kinesin-12A/B were absent from the phragmoplast in these mutant cells (Figure 3B).
To further elucidate what had caused the frequent failure of cell division, antitubulin immunofluorescence was performed in microspores of both the wild type and the double mutant. In wild-type microspores, upon the completion of mitosis, microtubules were gradually organized into the typical phragmoplast array between two reforming daughter nuclei located toward one end of the microspore (Figure 4A , a to c). At first, microtubules were organized into an antiparallel array, and a clear dark line was revealed in the middle (Figure 4A, a). Such a dark line marks the plus end of phragmoplast microtubules. The progression of cytokinesis was accompanied by the expansion of the phragmoplast microtubule array toward the periphery and the shortening of microtubules at their ends (Figure 4A, b). Once the cell plate was laid down centrifugally, the phragmoplast microtubule array appeared in a hollow cylinder-like configuration (Figure 4A, c). The cell plate eventually met the plasma membrane of the microspore to render a convex lens–shaped generative cell and a larger vegetative cell (McCormick, 2004).
Comparisons of Microtubule Organization and Cell Plate Development during the First Mitotic Cell Division in the Microspore.
Microtubules are pseudocolored green, and DNA is pseudocolored blue.
(A) In wild-type microspores (a), upon the completion of mitosis, microtubules were organized into an antiparallel array between two DNA masses. A typical phragmoplast microtubule array (b) had a dark line in the middle. The phragmoplast microtubule array (c) appeared in a barrel-like shape at this late stage of cytokinesis.
(B) In defective microspores of the homozygous double mutant, microtubules failed to be organized into an antiparallel phragmoplast array. Microtubules (a) polymerized into bundles between two DNA masses after mitosis. More microtubules were formed between two reforming nuclei (b), and they did not appear to have a dark line by tubulin immunofluorescence in the middle (c). Aggregates/bundles of microtubules ([d] and [e]) remained to be associated with one nucleus toward the periphery. Bar = 5 μm.
In dividing microspores of the double mutant, however, microtubules were frequently disorganized after mitosis was complete (Figure 4B, a to e). In all microspores upon completion of anaphase, coalesced microtubule bundles were found between the two sister chromatid masses (Figure 4B, a). At later stages, microtubule bundles remained unshortened, and their distal ends toward the reforming daughter (son) nuclei extended at or near the nuclear envelope (Figure 4B, b and c). The antitubulin immunofluorescence often did not reveal a dark line in the center of the microtubule mass (Figure 4B, c). This result suggested that these microtubules had not had their plus ends organized in the middle of the phragmoplast, as was seen in the wild-type cells. In pollen grains collected from open flowers of the homozygous double mutant, microtubule bundles were still detected, and they were associated with the nucleus toward the periphery of the cytoplasm (Figure 4B, d). Microtubule bundles often became randomly organized, with no reminiscence of the phragmoplast array (Figure 4B, e).
Among wild-type microspores, 95% demonstrated normal phragmoplast arrays with mirrored microtubule sets during their first cytokinesis (n = 56). Only 26% of mutant microspores, however, showed similar phragmoplast arrays at comparable cytokinesis stages as wild-type microspores (n = 118).
Mutant Microspores Failed to Form the Cell Plate
Earlier reports on cytokinesis mutants revealed that fragments of cell plate–like structures were still formed even though cytokinesis failed (Lauber et al., 1997). In those mutants, cells undergoing cytokinesis have microtubules organized into the mirrored phragmoplast array. Here, we report that in kinesin-12a kinesin-12b homozygous double mutants, microtubules no longer bear the typical phragmoplast array. Thus, we wanted to examine whether the defective mutant microspores assembled the cell plate when microtubules were disorganized. Immunolocalization of KNOLLE, a syntaxin-like protein localized at the cell plate (Lauber et al., 1997), was performed in both wild-type and mutant microspores. In the wild-type microspore bearing a mature phragmoplast, KNOLLE was densely accumulated at the division site (Figure 5 ). In the mutant microspore bearing aberrantly organized microtubules, KNOLLE had a diffuse localization pattern among microtubule bundles (Figure 5). This result suggests that the accumulation of KNOLLE-bearing vesicles at the division site is dependent on the antiparallel microtubule array of the phragmoplast, in which the plus ends of microtubules face each other in the middle. Hence, in the defective mutant microspores, disorganized microtubules, when their plus ends were not placed in the middle of the phragmoplast, prevented KNOLLE from accumulating at the cell division site.
The Cell Plate Failed To Be Formed in Defective Microspores.
(A) Localization of the cell plate marker KNOLLE in developing pollen grains of the wild type and the double mutant. In a wild type (12A 12B) microspore, the developing cell plate marked by the syntaxin-like protein KNOLLE (arrowheads) was formed in the middle region of the phragmoplast. A dark midline was clearly seen by tubulin immunofluorescence (arrowheads). In defective microspores of a homozygous double mutant (12a 12b), microtubules failed to be organized into a phragmoplast array with a dark midline. KNOLLE accumulated around microtubules in a diffuse fashion. The peripheral signal was due to the autofluorescence of the pollen coat. Bars = 10 μm.
(B) Callose accumulation in the wild type and the double mutant. In the wild type, callose (small arrowheads), labeled by aniline blue, appeared between the reforming vegetative nucleus (large arrowhead) and the generative nucleus (arrow) stained by DAPI. The completion of cytokinesis left a callose-rich cell plate (arrowheads) separating the cytoplasms of the generative cell and the vegetative cell (top right). In defective pollen grains in the double mutant, callose accumulated as a large aggregate at the cell cortex (small arrowhead), while two identical nuclei (large arrowheads) were positioned away from the aggregate. Such a callose-rich aggregate (arrowhead) was not organized in a cell plate–like configuration at the cell cortex (bottom right). Bar = 10 μm.
Callose deposition in developing pollen grains of the double mutant was also compared with that in wild-type pollen. After mitosis was completed in wild-type microspores, callose was deposited between two reforming nuclei (Figure 5B). Eventually, a complete callose-rich cell plate was formed (Figure 5A). In the double mutant, defective pollen grains bearing two identical nuclei lacked callose deposition between the nuclei (Figure 5B). Instead, callose was detected at the cell cortex as a large aggregate that did not resemble the cell plate (Figure 5B).
Our results demonstrate that two highly homologous kinesins, Kinesin-12A and Kinesin-12B, serve as dynamic integrators of antiparallel microtubules in the phragmoplast in Arabidopsis. Simultaneous inactivation of both kinesins by genetic means often led to loss of the antiparallel pattern of the phragmoplast microtubule array, which consequently caused the failure of cell plate formation and cytokinesis in dividing microspores. Thus, by acting at the plus end of phragmoplast microtubules, the Kinesin-12A/B motors allow the microtubules without direct contact to remain in two mirrored sets so that transport from both sides of the dividing cell takes place in a unidirectional manner toward the division plane.
The Organization of Phragmoplast Microtubules
Upon the completion of sister chromatid segregation, newly polymerized microtubules coalesce in the spindle midzone and later form the phragmoplast microtubule array (Zhang et al., 1993). Microtubule-associated proteins, known as MAPs, play a regulatory role in microtubule organization (Lloyd and Hussey, 2001; Jürgens, 2005). A group of evolutionarily conserved MAPs with molecular masses of ∼65 kD, known as MAP65/Ase1p/PRC1, have been shown to decorate phragmoplast microtubules (Jiang and Sonobe, 1993; Hussey et al., 2002). Particular isoforms of MAP65s show discrete patterns of localization in the phragmoplast (e.g., preferentially toward the plus or the minus end of phragmoplast microtubules) (Smertenko et al., 2000; Müller et al., 2004; Van Damme et al., 2004). By exclusively decorating the spindle midzone at late anaphase and the phragmoplast midzone, At MAP65-3/PLE contributes to limiting the dimension of the phragmoplast microtubule array, as loss-of-function mutations lead to expansion of the phragmoplast microtubule midzone (Müller et al., 2004). Separately, tobacco (Nicotiana tabacum) MAP65-1 was shown to be a substrate of a phragmoplast-specific mitogen-activated protein kinase cascade, and its phosphorylation is required for the timely depolymerization of phragmoplast microtubules (Sasabe et al., 2006).
The other likely organizing factor of phragmoplast microtubules is the MOR1/GEM1 protein in the XMAP215/Dis1/TOG family (Hussey et al., 2002). The gem1-2 and mor1 mutations at a locus encoding an evolutionarily conserved XMAP125-like MAP also leads to a failure of cytokinesis in the microspore and abnormal cell plate formation in somatic cells (Whittington et al., 2001; Twell et al., 2002; Eleftheriou et al., 2005; Kawamura et al., 2006). But the antiparallel pattern of phragmoplast microtubules is not altered in vegetative cells of the mor1 mutant (Eleftheriou et al., 2005; Kawamura et al., 2006).
While the aforementioned MAPs likely contribute to the general operation of phragmoplast microtubules, their functions may be limited to regulation of the stability and/or dynamics of the microtubules. In other words, unlike Kinesin-12A/B, they are unlikely to contribute to establishing the fundamental pattern of the phragmoplast microtubule array.
Specialized Roles of Distinct Kinesins in Plant Cytokinesis
Besides Kinesin-12A/B, other kinesins have also been found to actively participate in cytokinesis in Arabidopsis and other plants (Lee and Liu, 2004). Those in the Kinesin-5/BIMC subfamily are among the most well characterized. Kinesin-5 in both tobacco and carrot (Daucus carota) cells decorates phragmoplast microtubules with an emphasis toward the plus end and plays a role in microtubule–microtubule sliding (Asada et al., 1997; Barroso et al., 2000). Functions of similar kinesins have yet to be tested in Arabidopsis by genetic means.
Conversely, members in the Kinesin-7 subfamily exhibit spatially and temporally specific localization in the middle region of the phragmoplast. Among them, the NACK1/HIK kinesin interacts physically with a cytokinesis-important mitogen-activated protein kinase cascade to allow a mitogen-activated protein kinase kinase kinase to be targeted to the division site and is required for cytokinesis (Nishihama et al., 2002; Strompen et al., 2002). A similar kinesin, NACK2/TES, is required exclusively for male meiotic cytokinesis (Yang et al., 2003). An identified function of this kinesin–mitogen-activated protein kinase alliance is to downregulate Nt MAP65-1's microtubule-bundling activity by phosphorylation, which is required for the timely execution of cytokinesis in tobacco cells (Sasabe et al., 2006). But the inactivation of the NACK1/HIK kinesin does not alter the pattern of the phragmoplast microtubule array (Nishihama et al., 2002).
Other kinesins have been implicated in the spatial regulation of cytokinesis in Arabidopsis. A novel kinesin, as a cyclin-dependent kinase substrate, localizes to the division site and cortex except for the site once occupied by the preprophase band (Vanstraelen et al., 2006). How this intriguing localization pattern might be linked to phragmoplast operation awaits further examination. In addition, two novel extra-large kinesins, POK1 and POK2, play a redundant role in guiding phragmoplast and cell plate orientation during cytokinesis (Müller et al., 2006). However, unlike At Kinesin-12A/B, these kinesins again do not affect the overall antiparallel organization of phragmoplast microtubules.
Kinesin-12 as a Microtubule Plus End–Associated Motor in the Cell Plate Assembly Matrix
As demonstrated by electron microscopic tomography, antiparallel phragmoplast microtubules do not interdigitate, and plus ends of one group of these microtubules are inserted in an amorphous structure termed the cell plate assembly matrix at the cell division site in dividing somatic cells (Austin et al., 2005). Because a dark line was revealed by antitubulin immunofluorescence in the phragmoplast of dividing microspores (Figure 4A, b and c), the microtubules were probably organized as in somatic cells. It has been postulated that microtubule-stabilizing factors like EB1 could have contributed to capturing the blunt, metastable plus ends of these microtubules within the matrix (Austin et al., 2005). Unfortunately, the composition of the proposed microtubule plus end–capturing complex has not been determined.
Our results have provided evidence that by acting directly at the microtubule plus end, Kinesin-12A/B play a critical role in allowing phragmoplast microtubules to be organized in two mirrored sets with a gap between them (Figure 6A ). We suggest that these motors likely are part of the microtubule plus end–capturing complex in the phragmoplast. However, the mechanism that regulates the temporally specific association of Kinesin-12 with the plus end of phragmoplast microtubules is not clear. The kinesin may be targeted there by interacting with certain anchoring factor(s) residing in the cell plate assembly matrix. Such a targeting mechanism has been reported for the XKLP2 kinesin in Xenopus (Wittmann et al., 2000). Together with such a putative anchoring factor, Kinesin-12A/B can stabilize the plus ends of the phragmoplast microtubules while still allowing tubulin dimers to be added to the plus ends. Kinesin-12 would then drive newly assembled microtubule segments to be translocated in an outward manner by acting as a plus end–directed motor.
Models of the Function of Kinesin-12.
(A) Kinesin-12A/B and their putative anchoring factor(s) form a protein complex that interacts with the plus ends of phragmoplast microtubules located in the middle region. They function in translocating newly polymerized microtubule segments and allow the plus ends to be stably located in the middle region.
(B) The presence of Kinesin-12A/B allows the formation and maintenance of the antiparallel phragmoplast microtubule array. Consequently, successful cytokinesis brings about the cell plate (red), which separates the generative cell from the vegetative cytoplasm. The generative cell undergoes mitosis to produce two sperm cells. The absence of Kinesin-12A/B causes microtubules to be bundled together with mixed polarities. Consequently, materials for cell plate formation do not accumulate in the middle region. Ultimately, two nuclei are suspended in the vegetative cytoplasm. Microtubules are shown in green, and nuclei are shown in blue.
During male gametogenesis, the cytoplasm of the generative cell is physically separated from that of the vegetative cell, as a result of cell plate formation via the phragmoplast (Figure 6B). To date, mutations in genes including TIO, encoding a member of the FUSED kinase family, which acts at the phragmoplast midzone, lead to the failure of cytokinesis after microspore mitosis (Oh et al., 2005). However, it has not been observed that the reported mutations cause a loss of the bipolarity of the phragmoplast microtubule array. Because mutants such as those bearing null tio mutations produce a partial cell plate, one would predict that at least an initial phragmoplast array is established, which might fail to expand. In the absence of Kinesin-12A/B, microtubules frequently fail to be organized into a mirrored phragmoplast array. Consequently, defective microspores failed to produce the generative cell because of the failure of cell plate formation (Figure 6B).
The fact that some microspores still divide normally in the double mutant suggests that there are other factor(s) that play a redundant role like Kinesin-12A/B. In addition, although both kinesins also decorate the plus end of phragmoplast microtubules in somatic cells (Pan et al., 2004), we did not observe any defect in cytokinesis during vegetative growth in the homozygous double mutant. Thus, it further suggests that one or more of the remaining 59 kinesins encoded by the Arabidopsis genome (Reddy and Day, 2001) may play a redundant role like Kinesin-12A/B. We suggest that each of these functionally related kinesins contributes to establishing the phragmoplast microtubule array quantitatively. A qualitative effect is generated when quantitative functions of individual kinesins are combined. In the homozygous double mutant reported here, a significant number of pollen grains were defective despite the fact that no noticeable defect in diploid cells was detected.
In summary, our results demonstrate that the failure of cytokinesis caused by the inactivation of Kinesin-12 resulted directly from the disorganization of phragmoplast microtubules. Such a direct connection between kinesin motors and cell plate formation establishes the significance of proper microtubule organization in plant cytokinesis.
Plant Materials and Growth Conditions
Arabidopsis thaliana plants bearing T-DNA insertion mutations were either the Wassilewskija ecotype (kinesin-12a-1 and kinesin-12a-2) or Columbia (kinesin-12b-1 and kinesin-12b-2). The kinesin-12a-1 and kinesin-12b-1 lines were reported previously (Pan et al., 2004). The kinesin-12a-2 line was recovered from the collection at the Arabidopsis Knockout Facility of the University of Wisconsin Biotechnology Center. The kinesin-12b-2 (SALK_027020) line was recovered from the Sequence-Indexed Library of Insertion Mutations in the Arabidopsis Genome at the Salk Institute Genome Analysis Laboratory. Seedlings were grown under 24 h of light at 22°C and 70% RH. Standard genetic crosses were performed between mutant lines. Progeny from crosses between wild-type Wassilewskija and Columbia plants were used as controls.
PCR-Based Screening
Positive T-DNA insertions were confirmed by a PCR-based method (Krysan et al., 1996). Primers for kinesin-12a-2 were the gene-specific primer YT3 (5′-TACATGTCAGTAAAAGGGTAATGCAATCA-3′) and the T-DNA border–specific primer JL202 (5′-CATTTTATAATAACGCTGCGGACATCTAC-3′) for testing T-DNA insertion and the gene-specific primers 3726F2 (5′-GATGTTTACCACAAGATGAAATTATCAAC-3′) and 3726R (5′-GCTTCTGTAACTAAATTTTCTCCTTCAC-3′) for testing homozygosity. Primers for kinesin-12b-2 were YBTA1 (5′-CTATGGGATTTTGTGGCTCTGC-3′) and the T-DNA border–specific primer LBa1 (5′-ATGGTTCACGTAGTGGGCCATC-3′) for detecting the T-DNA insertion and the gene-specific primers YBTA1 and JPF (5′-TTAGAAGTTTATTGAATCAATGCAGATATG-3′) for testing homozygosity.
RNA Extraction and RT-PCR
Total RNA was isolated from flower buds using PureLink Plant RNA reagent (Invitrogen) as described by the manufacturer. The expression of RNA was detected using PCR amplification of reverse transcription products. Potential genomic DNA contaminants of RNA samples were eliminated by digesting with DNase I before the reverse transcription step. The primers used for RT-PCR were PA1-5 (5′-GCTGGAGAGTTACTTGTTCGG-3′) and PA1-3 (5′-TCCATTGCTGCTCACTACTTG-3′) for Kinesin-12A and PA1L-5 (5′-TGTTCAAGCAGCAGGAGAGTTAC-3′) and PA1L-3 (5′-GCCATAGCATCGTCATTACAAGAAG-3′) for Kinesin-12B. RT-PCR of At1g13320, which encodes a subunit of Ser/Thr protein phosphatase 2A, served as a positive control (Czechowski et al., 2005). After 40 amplification cycles, PCR products were analyzed by gel electrophoresis.
Nuclei in microspores were stained with the dye DAPI according to a published protocol (Park et al., 1998). Immunolocalization experiments were performed according to a published study (Terasaka and Niitsu, 1990). Briefly, developing pollen grains were mechanically released from anthers and fixed with 4% formaldehyde for 1 h at room temperature. Fixed pollen grains were collected by centrifugation at 3000 rpm for 5 min and then digested with 1% Cellulase RS and 1% Pectolyase Y-23 (both from Yakult Honsha) in 50 mM PIPES buffer, pH 5.3, for 2 h with gentle rocking. The pollen grains were then immobilized on poly-l-Lys–coated slides prior to incubation with antibodies. Microtubules were labeled by the DM1A anti-α-tubulin antibody diluted at 1:400 (Sigma-Aldrich); At Kinesin-12A/B were stained by anti-PAKRP1-C at 1:400 (Lee and Liu, 2000); and KNOLLE was stained with the anti-KNOLLE antibodies at 1:100 (Rose Biotechnology). Wide-field fluorescence images were acquired with a CCD camera (Hamamatsu Photonics) using the ImageProPlus 4.0 software package (Media Cybernetics) on an Eclipse E600 microscope equipped with epifluorescence optics (Nikon). Confocal images were collected with a TCS-SP laser scanning confocal microscope (Leica) using argon and krypton lasers. Images were assembled in the Adobe Photoshop 7.0 software package.
Developing pollen grains from wild-type and mutant flowers were stained for callose with aniline blue solution (0.05% [w/v] in 100 mM potassium phosphate buffer, pH 8.5) for 5 min and observed by fluorescence microscopy as described elsewhere (Park and Twell, 2001). Conventional transmission electron microscopy was performed according to a published protocol (Park and Twell, 2001) using the low-viscosity Spurr mini kit (Ted Pella). Samples were observed with a JEM-100S transmission electron microscope (JEOL).
The Arabidopsis Genome Initiative locus identifiers for the major genes mentioned in this study are as follows: Kinesin-12A (At4g14150), Kinesin-12B (At3g23670), and protein phosphatase 2A (At1g13320).
We are grateful to Haihong Liu for her assistance in transmission electron microscopy and to Sally Assmann for her insightful suggestions on the manuscript. We thank the ABRC, the University of Wisconsin Biotechnology Center, the Salk Institute, and the Syngenta Torrey Mesa Research Institute for providing mutant screening services. Critical comments made by anonymous reviewers were greatly appreciated. This work was supported in part by the National Research Initiative of the USDA Cooperative State Research, Education, and Extension Service (Grant 2005-35304-16031 to Y.-R.J.L.) and the Energy Biosciences Program of the U.S. Department of Energy (Grant DE-FG02-04ER15554 to B.L.).
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Bo Liu (bliu{at}ucdavis.edu).
www.plantcell.org/cgi/doi/10.1105/tpc.107.050716
Revised July 29, 2007.
Accepted August 6, 2007.
Published August 24, 2007.
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The Plant Cell Aug 2007, 19 (8) 2595-2605; DOI: 10.1105/tpc.107.050716
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