Optineurin mediates negative regulation of Rab8 function by TBC1D17, a GTPase activating protein

Advance Online Publication August 1, 2012 doi: 10.1242/?jcs.102327 Rab GTPases regulate various membrane trafficking pathways but the mechanisms by which GTPase activating proteins recognize specific Rabs are not clear. Rab8 is involved in controlling several functions including the trafficking of transferrin receptor from early endosome to recycling endosome. Here we provide evidence to show that TBC1D17, a Rab GTPase activating protein, through its catalytic activity, regulates Rab8-mediated endocytic trafficking of transferrin receptor. Optineurin, a Rab8-binding effector protein, mediates interaction and colocalisation of TBC1D17 with Rab8. A non-catalytic region of TBC1D17 is required for direct interaction with optineurin. Co-expression of Rab8, but not other Rabs tested, rescues the inhibition of transferrin receptor trafficking by TBC1D17. Activated GTP-bound form of Rab8 is localized to the tubules emanating from the endocytic recycling compartment. Through its catalytic activity, TBC1D17 inhibits recruitment of Rab8 to the tubules and reduces colocalisation between transferrin receptor and Rab8. Knockdown of optineurin or TBC1D17 resulted in enhanced recruitment of Rab8 to the tubules. A glaucoma-associated mutant of optineurin, E50K causes enhanced inhibition of Rab8 by TBC1D17 resulting in defective endocytic recycling of transferrin receptor. Our results show that TBC1D17, through its interaction with optineurin, regulates Rab8-mediated endocytic recycling of transferrin receptor and recruitment of Rab8 to the tubules. We describe a mechanism of regulating a Rab GTPase by an effector protein (optineurin) that acts as an adaptor to bring together a Rab (Rab8) and its GTPase activating protein (TBC1D17).

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Modeling the self-organized phosphatidylinositol lipids signaling system in chemotactic cells based on quantitative image analysis

Advance Online Publication August 16, 2012 doi: 10.1242/?jcs.108373 The phosphatidylinositol (PtdIns) lipids reaction is a key signaling event; this signal is responsible for gradient sensing in eukaryotic cell chemotaxis. The self-organization activity of the PtdIns lipids reaction induces an inherent polarity even in the absence of an external chemoattractant gradient by producing a localized PtdIns 3,4,5-trisphosphate [PtdIns(3,4,5)P3]-enriched domain on the membrane. Experimentally, we found that such a domain could exhibit two types of behavior: 1) persistent domain formation that travels on the membrane and 2) stochastic formation of transient domains. Taking advantage of the simultaneous visualization of PtdIns(3,4,5)P3 and its enzyme, phosphatase and a tensin homolog (PTEN), the inter-dependence of their spatiotemporal dynamics was demonstrated statistically. Based on this statistical analysis, we developed a theoretical model for the self-organization of PtdIns lipids signaling that can accurately reproduce both persistent and transient domain formation; these types of formations can be explained by the oscillatory and excitability properties of the system, respectively.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), which permits unrestricted non-commercial use, distribution and reproduction in any medium provided that the original work is properly cited and all further distributions of the work or adaptation are subject to the same Creative Commons License terms.

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Functional involvement of Rab1A in microtubule-dependent anterograde melanosome transport in melanocytes

Advance Online Publication August 1, 2012 doi: 10.1242/?jcs.109314 Morié Ishida, Norihiko Ohbayashi, Yuto Maruta, Yuka Ebata and Mitsunori Fukuda*?*Correspondence: Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan. Tel.: +81-22-795-7731; Fax: +81-22-795-7733; E-mail: nori{at}m.tohoku.ac.jp. Melanosomes are transported to the cell periphery of melanocytes by coordination between bidirectional microtubule-dependent movements and unidirectional actin-dependent movement. Although both the mechanism of the actin-dependent melanosome transport and the mechanism of the microtubule-dependent retrograde melanosome transport in mammalian skin melanocytes have already been determined, almost nothing is known about the mechanism of the microtubule-dependent anterograde melanosome transport. Small GTPase Rab proteins are common regulators of membrane traffic in all eukaryotes, and in this study we performed genome-wide screening for Rab proteins that are involved in anterograde melanosome transport by expressing 60 different constitutive active (and negative) mutants and succeeded in identifying Rab1A, originally described as a Golgi-resident Rab, as a prime candidate. Endogenous Rab1A protein was found to be localized on mature melanosomes in melanocytes, and its functional ablation either by siRNA-mediated knockdown or by overexpression of a cytosolic form of Rab1A-GTPase-activating protein/TBC1D20 induced perinuclear melanosome aggregation. The results of time-lapse imaging further revealed that long-range anterograde melanosome movements were specifically suppressed in Rab1A-deficient melanocytes, whereas retrograde melanosome transport occurred normally. Taken together, these findings indicate that Rab1A is the first crucial component of the anterograde melanosome transport machinery to be identified in mammalian skin melanocytes.

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Diverse, but not yet pluralistic

The question of pluralism in Egypt is becoming more relevant with every passing day, writes Ayman Abdel-Wahab

Egypt is not a pluralistic society -- not yet. But it is an open society, one in which cultural and social diversity have always been a source of vitality and societal prowess.

What we need to do is manage the diversity to make it an asset, not a liability. In the heat of political rivalry, the line may be blurred between diversity as a natural phenomenon and the political and social framework this diversity needs in order to survive.

To put it in a nutshell, diversity has to be institutionalised, ensconced into our legal system, defended and channelled into productive courses. Things like the status of women, social inequities, as well as urgent regional matters need to be addressed in a systematic manner. The demands of Upper Egypt for development cannot be ignored, nor can the rights of the Bedouins of Sinai and the inhabitants of Nubia be overlooked. Sectarian strife between the Muslims and Christians and discrimination against certain sections of society must also be confronted.

Our society may be diverse, but the diversity needs to be managed, or it will backfire. Egypt has always been a melting pot, and we need to work harder to keep it so.

Political groups are apt to exploit factional issues for their own benefit. This will happen, no doubt. But we need to keep an eye on what's going on. We need to work double time to make sure that diversity becomes a source of inspiration, not of frustration.

For one thing, we have to boost civil society so it may act as a magnet for the immense energy of our younger generations. The youth deserves to have its say in the future of its country. And it needs to learn how to appreciate civil rights and respect the special needs of certain parts of society. The key to diversity is civil society. If it is fully engaged in development and democratisation, Egypt will remain the melting pot it always was.

The writer is director of the Civil Society Unit at Al-Ahram Centre for Political and Strategic Studies.

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PA vows to hold elections

Local elections are on the cards, but Hamas continues to protest that it has no freedom to campaign in the West Bank, reports Khaled Amayreh in Ramallah

The Palestinian Authority (PA) is planning to hold local elections in the West Bank in mid-October despite Hamas's refusal to do likewise in the Gaza Strip.

The Fatah-dominated PA views elections as the ultimate arbiter between itself and Islamist Hamas, which is in control of the Gaza Strip. Hamas views elections as the would-be fruit of national reconciliation with Fatah.

Despite several agreements and understandings reached through active Arab -- especially Egyptian -- mediation, the two main Palestinian political groups have failed to reach a solid final agreement that would end more than five years of tension, starting soon after Hamas defeated Fatah in the 2006 general elections.

With a supposedly large chunk of eligible Palestinian voters set to boycott the upcoming elections, it is expected that the turnout will be modest, which would allow Hamas to further question the credibility of the elections under current circumstances.

Most of the municipal and local councils in the West Bank are already controlled by Fatah following the sacking of pro-Hamas councils and mayors in the context of the oft-bitter hostility between the two groups.

In many cities and localities, the PA has been refusing to organise elections, fearing Islamists would win the polls.

However, with Hamas substantially weakened in the West Bank due to a combined repression campaign by the PA security agencies and the Israeli occupation army, which saw most Hamas's community leaders and members of parliament thrown behind bars, the PA seems fairly encouraged to hold the elections.

Hamas has been demanding, but to no avail, that the PA introduce preparations for genuine elections, including allowing freedom of speech and other civil liberties that would allow Islamists to campaign freely.

Israel, for its part, has kept up its vehement hostility towards Hamas, vowing to detain Islamist candidates for their "affiliation with a terrorist organisation".

The PA admits it can do nothing to prevent Israel from targeting Hamas, apart from appealing to the international community to pressure Tel Aviv.

But international pressure on Israel may not be good enough for Hamas to risk delivering its leaders and candidates into the jaws of the lion -- risking having a new generation of Islamist leaders languishing in open-ended detention in Israeli jails and detention camps without charge or trial.

The holding of local elections in the West Bank, excluding East Jerusalem, comes in the midst of severe political and economic crises facing the PA and threatening its very survival.

Earlier this week, the extremist Israeli foreign minister, Avigdor Lieberman, called for the elimination of President Mahmoud Abbas, calling him a terrorist.

Lieberman is the second most powerful politician in Israel following Prime Minister Binyamin Netanyahu, and his remarks shouldn't be taken lightly.

Abbas has vowed to once again seek United Nations recognition of a Palestinian state. However, it seems that behind-the-scenes pressure by the United States and its allies may have succeeded in convincing the Palestinian leadership to at least postpone its UN recognition bid for the time being.

Another postponement at the UN is guaranteed to weaken Abbas in the view of his people. The aging Palestinian leader has been severely castigated of late after saying during a meeting with a visiting Israeli delegation, which included rabbis, in Ramallah last week that "Israel was created in order to stay forever."

Several Palestinian leaders and intellectuals scoffed at the remarks, calling them "treasonous and catastrophic".

Abdel-Sattar Qassem, a former presidential candidate and university professor, said Abbas had no right whatsoever to sell out the Palestinian cause.

"Palestine is not the property of the Abbas family; he has no right to cede what doesn't belong to him and his family," he said.

Nonetheless, most if not all Fatah leaders have remained mum, refusing to comment on Abbas's remarks, which most Palestinians view as an embarrassing taboo.

The main reason for Fatah's refusal to publicly distance itself from the remarks has to do with the fact that Abbas tightly controls Fatah's coffers and any severe criticisms directed at the chairman may cost the critics their financial survival.

Meanwhile, a severe economic crisis, whose harshness has no parallel since the start of the Israeli occupation 1967, is hitting hard in the occupied territories, causing families to adopt unprecedented austerity measures.

With its foreign aid and revenue dwindling, the PA government is barely able to pay salaries for its estimated 160,000 civil servants.

As a result, the PA has failed to pay electricity and water bills to Israel, prompting the Israeli government to threaten to cut off electricity to the West Bank, including Abbas's own office in Ramallah.

On Monday, 4 September, the state-run Israeli TV disclosed the content of a letter sent to Netanyahu by Energy Minister Uzi Landaou in which the latter threatened to cut electricity off to the West Bank.

Landaou, an extreme right winger, was quoted as saying that the PA was indebted to Israel for huge sums of money which would force him to cut off power to the Fayyad government.

The suffocating economic crisis is forcing most Palestinians to prioritise their spending, with many families unable to send their grown up sons and daughters back to college due to poverty.

Demonstrations protesting widespread poverty have been taking place in some areas such as Ramallah and Bethlehem. However, the scope of the protests doesn't seem to pose an immediate threat to the survival of the PA regime in Ramallah.

The PA is not a sovereign entity and is not free to carry out normal economic activities such as free import and export, due to Israeli restrictions and control over border crossings connecting the West Bank to the outside world.

This is the reason most economic experts argue that it is nearly impossible for the PA to successfully overcome its recurrent economic and financial crises as long as Israel remains in control of the Palestinian territories.

Others go as far as arguing that it too late for the Palestinians to establish a viable and territorially contiguous Palestinian state on the West Bank, given the phenomenal expansion of Jewish colonies throughout the occupied territory.

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Mechanosensitive mechanisms in transcriptional regulation

Advance Online Publication July 13, 2012 doi: 10.1242/?jcs.093005 July 1, 2012 J Cell Sci 125, 3061-3073. Akiko Mammoto1,*, Tadanori Mammoto1,* and Donald E. Ingber1,2,3,‡

1Vascular Biology Program, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA

2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA 02115, USA

3Harvard School of Engineering and Applied Sciences, Cambridge, MA 02139, USA ?‡Author for correspondence (don.ingber{at}wyss.harvard.edu) ?* These authors contributed equally to this work

Transcriptional regulation contributes to the maintenance of pluripotency, self-renewal and differentiation in embryonic cells and in stem cells. Therefore, control of gene expression at the level of transcription is crucial for embryonic development, as well as for organogenesis, functional adaptation, and regeneration in adult tissues and organs. In the past, most work has focused on how transcriptional regulation results from the complex interplay between chemical cues, adhesion signals, transcription factors and their co-regulators during development. However, chemical signaling alone is not sufficient to explain how three-dimensional (3D) tissues and organs are constructed and maintained through the spatiotemporal control of transcriptional activities. Accumulated evidence indicates that mechanical cues, which include physical forces (e.g. tension, compression or shear stress), alterations in extracellular matrix (ECM) mechanics and changes in cell shape, are transmitted to the nucleus directly or indirectly to orchestrate transcriptional activities that are crucial for embryogenesis and organogenesis. In this Commentary, we review how the mechanical control of gene transcription contributes to the maintenance of pluripotency, determination of cell fate, pattern formation and organogenesis, as well as how it is involved in the control of cell and tissue function throughout embryogenesis and adult life. A deeper understanding of these mechanosensitive transcriptional control mechanisms should lead to new approaches to tissue engineering and regenerative medicine.

Key words Funding

This work was supported by the National Institutes of Health [grant numbers CA45548 and DE019023], the Department of Defense [grant number BC074986], AHA, the Hearst Foundation and ABTA. Deposited in PMC for release after 12 months.

This article is part of a Minifocus on Mechanotransduction. For further reading, please see related articles: ‘Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues’ by Brendon M. Baker and Christopher S. Chen (J. Cell Sci. 125, 3015-3024). ‘Finding the weakest link – exploring integrin-mediated mechanical molecular pathways’ by Pere Roca-Cusachs et al. (J. Cell Sci. 125, 3025-3038). ‘Signalling through mechanical inputs – a coordinated process’ by Huimin Zhang and Michel Labouesse (J. Cell Sci. 125, 3039-3049). ‘United we stand – integrating the actin cytoskeleton and cell–matrix adhesions in cellular mechanotransduction’ by Ulrich S. Schwarz and Margaret L. Gardel (J. Cell Sci. 125, 3051-3060). ‘Molecular force transduction by ion channels – diversity and unifying principles’ by Sergei Sukharev and Frederick Sachs (J. Cell Sci. 125, 3075-3083).

© 2012. Published by The Company of Biologists Ltd

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Features: Zewail versus students

After an 18-month delay, promises and counter-promises, the Nile University versus Zewail City of Science and Technology saga continues, writes Sarah Eissa Clockwise from top: the Nile University premises; the sit-in; a student cleaning up; during the construction phase; students say the NU name is all over the place even on sewer covers

Students at Egypt's private Nile University (NU) began an open-ended sit-in last Saturday, saying that they were prepared to continue their protest on the university's campus, which they are officially not allowed to enter, until their demands are met. The students said the sit-in, started when university authorities did not respond to them, was an act of last resort. They face an uncertain future since the land on which the Nile University sits was granted last year to Nobel Prize-winning Egyptian scientist Ahmed Zewail to establish the Zewail City of Science and Technology (ZCST).

Nile University is a non-profit, privately-owned and autonomously-managed institution of higher learning, founded in 2006. Students at the institution have been waiting to move in for some time to begin their studies, but have now been told that the ZCST will be using the university's buildings instead. According to the students, the decision on the university's future and to award the land and buildings to the ZCST instead was political motivated. Many parents, professors, clerks, other private and public university students, and some political forces support the sit-in, the students said.

In a statement at last weekend's press conference, the NU students said that despite the fact that they were engaged in a peaceful sit-in, thugs had been sent to attack them. When one of the students and a staff member had gone to the police to ask them to protect the sit-in, they were themselves detained, the students said, with the authorities demanding that the students either end the sit-in or risk continued detention. Six Central Security Forces (CSF) trucks had been seen outside university premises. An officer had asked for the students' names, in order to protect them, he claimed, but the students said that they were surprised to find that the officer reported to retired general Salah Azazi, security director of the ZCST.

At the press conference, the NU students said they were not against the Zewail project, but they did not want to see it take the place of NU. If they were allowed to keep the NU buildings, they would be willing to press the administration to host the Zewail project until the ZCST was able to find buildings of its own. In the meantime, they would not end the sit-in, the students said. After a year-and-a-half of broken promises from previous governments, they said they hoped the new government would support them, allowing NU to use the campus permanently.

During the press conference, students Mohamed Osama and Mustafa Shamaa, the latter a previous NU student union president, announced that they would be beginning a 48-hour hunger strike on Monday if no steps were taken. Shamaa said that the hunger strike was the only way that the students could find to make the government and public opinion pay attention to their plight.

In visits to the sit-in, Al-Ahram Weekly found the students apparently exhausted though enthusiastic about speaking about NU's problem. Ahmed Nassar, present NU Student Union president and a fourth-year computer-engineering student, said that for a year-and-a-half they had wanted to enter the university buildings, which had been built and equipped using university money and donations. However, they had not been able to do so because of decisions taken by former prime ministers Essam Sharaf and Ahmed Shafik, which had allowed the Zewail project to acquire the land, according to Nassar without due legal title.

Over the previous year-and-a-half, the students had protested the decision in front of the People's Assembly (PA), the prime minister's office and the Ministry of Higher Education, he said. With each new minister and prime minister they had made a new stand, also sending petitions to the former ruling Supreme Council of the Armed Forces (SCAF) and the prime minister in person. But all they had received were more promises, he said.

Ahmed Ismail, a research assistant in the software engineering track in the computer and information technology department at NU, said that Masters students would not be able to graduate because there was nowhere for them to put their equipment while the buildings were under legal guard. The equipment had to be kept in a Smart Village garage lab for the time being, he said, where students had been attending classes.

According to Mohamed Ghoneim, a member of the ZCST board of trustees, speaking to the Al-Hayat Al-Youm television programme, the NU students had never entered the university's premises. Ismail disagreed, saying that a graduation ceremony, science fair, and conferences had been held there in conjunction with the Menoufiya and Ain Shams universities. "There are 27 PDF files of photos proving that we have used the buildings," the students said, adding that there was even a scale model of the university on the NU campus. They said that not only had the ZCST tried to take the buildings from them, but it had also taken equipment, computers and books found inside the disputed buildings.

Ghoneim said that the NU board of trustees had surrendered the university's land and buildings to the Ministry of Higher Education and Ministry of Communication on 17 February 2011. In October, the land and buildings had been transferred to the ZCST. To avoid any problems, members of the ZCST administration had met with academic representatives of both universities and had decided to transfer all NU students, researchers and staff to the ZCST. Current students could choose to graduate from either NU or ZCST, he said, adding that it was not possible, however, for the two institutions formally to merge.

NU students Nassar and Ismail said that the intention was to kill NU slowly, inevitably bringing about its closure. Ismail was also concerned about NU workers, "there are more than 200 employees, and we do not want to see them lose their jobs. Transferring the students does not mean that the problem is solved," he said.

"It's an 'acquisition' not a merger," said Dina El-Agami, a fourth-year student in business. El-Agami is one of many students who will be travelling abroad on an exchange programme, and she was worried at the possibility of closure faced by NU. Anyone choosing to work or study at NU believes in the university's mission, she said, and they could not simply abandon the institution in favour of ZCST. Besides, she said, the ZCST project was vague and the institution's specialties were different from those at NU. Ismail and Nassar agreed, adding that Egypt needed both institutions equally.

NU president professor Tareq Khalil said that he had not surrendered the NU's land and buildings to ZCST. "Can anyone give up a university," he asked. "The university has an independent status and was established by presidential decree. No one can simply give it up," he said. In a previous interview with the Weekly, Khalil had explained that NU was established in 2006 as the only non-profit university in Egypt. Following the passage of Law 12/2009 allowing for the establishment of civil universities, the NU was approved in January 2011 by the Supreme Council for Civil and Private Universities. However, due to the 25 January Revolution, the presidential decree was never signed, he said.

This has left the university in the present legal problem, with Menna Syam, a research assistant in the Centre for Informatics, saying that the students were using two buildings in the Smart Village, for which the rent had not been paid. A new campus could not be built as long as the money spent on the present one had not been recouped, she said. Ismail added that over the previous five years, NU had accomplished many achievements, including student Haitham Desouki's winning first prize in the Stars of Science competition. It also had many more ambitious plans, including the development of pilotless airplanes and the application that had won the Google Ibdaa competition. The university also has joint projects with Microsoft and Google, he said.

According to students, there are around 300 post-graduate students at NU. More than 500 former students have graduated, and there are around 88 undergraduates, "Many students have applied to join NU, but have found no places available," El-Agami said. The university offers some 100 per cent scholarships, and Ismail said his scholarship paid him a salary as a researcher, showing that the university was not just for those able to afford its fees. Other scholarships are worth around 50 per cent of fees, he said, with all Masters students having scholarships.

According to Zewail, many professors have come to Egypt specifically to take part in the ZCST project, but Nassar said that professor Yehia Ismail, claimed by Zewail, had in fact been hired by NU. Ismail himself has said that he was employed by both institutions. For the students, Zewail has not shown that he has the right to the land for the ZCST, and they complain of his claiming the 127 acres used by NU. "I don't mind him taking the land, but he should give the buildings back to us," Ismail said. "He is just insisting because of the buildings, and it's not his right to do so," he added.

Ahmed Khalil, a fourth-year business student, commented that the university's server room can only be used by NU professors, as it uses fingerprint recognition. The NU logo was everywhere on campus, he said, even on the sewer covers. "Even the sewage manhole covers bear the university logo," said one of the university security guards, who supported the students' demands. The students had been peaceful in their protests, he said, and had not tried to storm the university.

University president Tareq Khalil said that there were now proposals to look into the issue again by committees of presidential advisors. Meanwhile, the students criticised the information on the controversy carried by some media outlets, showing the Weekly an issue of one state newspaper that they said had published lies about them.

For their part, supports of Zewail last Friday formed a human chain to support the ZCST on the Qasr Al-Nile bridge in Cairo. One of the participants said that they were dreaming of being able to register at Zewail's ZCST. "At first, we were not sure what we could do to support the project, but now we have decided to take active steps. If we do not learn in the ZCST, the next generations will be able to do so," she said.

She said that she did not believe that the NU students had the right to use the buildings. She did not oppose NU, she said, but she wanted to see the completion of ZCST. Why didn't people look towards a bigger dream, she asked, "a complete science city, not a university set up under the previous regime?"

Zewail had not taken the NU buildings, she added. The land had originally belonged to him, and after the Revolution he had been able to enforce his right to it. Ramy Isaac, Cairo coordinator of the Friends of Zewail group, refused to comment on the NU case and its relation to the Zewail project. The projects should not be discussed together, he said.

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