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Structural Model of Bax in the Membrane

Bax is a 21 kDa molecule, consisting of 9 alpha helices – one of these helices is hydrophobic (a9), and two are amphiphatic (a5 and a6) [1]. Early structural topology studies of membrane-inserted Bax molecule hypothesize the insertion of 3 of these helices (a5, a6 and a9) [2]. This model was termed as the umbrella model, where a hairpin turn was believed to exist between a5 and a6. The other helices were located on the surface of the membrane, although no role in oligomerization or pore-formation was associated to these helices.

In 2013, an Australian group published crystal structures of Bax fragments with the BH3 domain of Bid [3]. This publication, while not showing the structure of Bax in the membrane, introduced a key piece to the puzzle of Bax oligomerization. They showed that the first few helices of Bax (a2 to a4 and a small part of a5) form what is essentially a dimerization domain. They do so by swapping helices between each subunit (swap dimer) to create interaction interfaces between the two subunits. This did not necessarily resolve a debate on how Bax oligomers assemble, but it gave our group a springboard to pattern our structural studies of Bax structure in the membrane.

In cooperation with Enrica Bordignon and Gunnar Jeschke at ETH Zürich, our postdoctoral researcher, Stephanie Bleicken elucidated the structure of Bax in the presence or absence of liposomes using double electron-electron resonance (DEER) [4]. In this study, we generated single- and double-cysteine mutants of Bax and labeled them with organic molecules that contain unpaired electrons (spin labels).

DEER was used to measure the distance between two spin-labels (Figure 1). Using single-cysteine mutants, we were able to see the intra-dimer (or even inter-dimer) distances between two spin labels at the same position of Bax. Using double-cysteine mutants, we were able to measure the distance between spin labels within a single Bax molecule in a dimer. To remove background from the distances that would’ve been acquired from Bax dimers, we added unlabeled Bax to the system. The distances were used as constraints to generate a model of the structure of Bax.

deer-with-text

Figure 1. Use of single- and double-cysteine mutants in elucidating Bax structure

We were able to show that between solution and membrane inserted form, not a lot of changes occur in the dimerization domain, in line with the swap dimer model.

However, our distance constraints do not support a hairpin in between a5 and a6, where we saw the distance to be farther away and made us question the umbrella model of Bax.

Interestingly, the a9 distances cannot be properly resolved indicating a flexible and mobile helix in the membrane. Note that a9 is a hydrophobic helix and therefore probably transmembrane. Even more interesting is that the distance between the a6 of two monomeric units in a dimer is 3 nm, which is similar to the thickness of a membrane.

Structural Model of Active Bax at the Membrane

Figure 2. Strucural Model of Bax in the Membrane. (A) Bax model showing flexibility of label at position 149 (end of a6). (B) Bax model showing flexibility of labels at positions 169 (N-terminal of a9) and 193 (C-terminal of a9). (C) Clamp model of Bax in the membrane. (D) Hypothetical arrangement of Bax dimers around a membrane pore. Figure from [4].

These evidences led us to propose a clamp model of Bax in the membrane (Figure 2). The clamp model still agrees with the topologies measured with the umbrella model. Furthermore, the flexibility of a9 suggests that while it is needed for the initial steps of membrane targeting, its direct role in pore formation may be expendable.

References:

[1] M. Suzuki, R. J. Youle, and N. Tjandra. Structure of bax: Coregulation of dimer formation and intracellular localization. Cell, 103:645–654, 2000.

[2] M. G. Annis, E. L. Soucie, P. J. Dlugosz, J. A. Cruz-Aguado, L. Z. Penn, B. Leber, and
D. W. Andrews. Bax forms multispanning monomers that oligomerize to permeabilize
membranes during apoptosis. EMBO Journal, 24:2096–2103, 2005.

[3] P. E. Czabotar, D. Westphal, G. Dawson, S. Ma, C. Hockings, W. D. Fairlie, E. F. Lee,
S. Yao, A. Y. Robin, B. J. Smith, D. C. S. Huang, R. M. Kluck, J. M. Adams, and P. M.
Colman. Bax crystal structures reveal how bh3 domains activate bax and nucleate its
oligomerization to induce apoptosis. Cell, 152:519–531, 2013.

[4] S. Bleicken, G. Jeschke, C. Stegmueller, R. Salvador-Gallego, A. J. Garc´ıa-S´aez, and
E. Bordignon. Structural model of active bax at the membrane. Molecular Cell, 2014.

Note: This is a five-part piece on our group’s recent contributions to Bax’s structure and function in apoptosis.

Quick links:

Part 1. Insights on Bax pore-forming activity

Part 2. Structural Model of Bax in the Membrane

Part 3. Counting Bax Molecules in the Membrane – A single molecule approach (to be released)

Part 4. Bax forms Rings, Arcs and Lines in Apoptotic Mitochondria (to be released)

Part 5. Membrane effects of Bax (to be released)

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Insights on Bax pore-forming activity

The Bcl-2 family of proteins regulates apoptosis by mitochondrial outer membrane permeabilization (MOMP). For years, it was believed that effector proteins Bax and Bak do so by making pores in the membrane. However, direct evidence of a pore in the mitochondria outer membrane has never been shown. In the mean time, the interplay of different family members and the mechanism of action that regulate and control MOMP are still not well-understood.

Up until the mid 2000s, the nature of the apoptotic pore has not been characterized. But with the advent of better technologies, and our improved ability to recombinantly produce Bax and Bak, various biophysical studies that characterize the mechanism of pore formation emerged.

In 2013, our lab produced two papers to help understand the mechanism of pore-formation of Bcl-2 proteins.

The first paper [1] distinguished the mechanism of action of Bax and Bcl-xL on model membrane systems. In this paper, we used single-vesicle permeabilization studies to show that Bax and Bcl-xL both permeabilize model membranes. However pores induced by Bcl-xL eventually close in time, while Bax remains open. In short, Bax makes stable pores.

This is quite intriguing in some sense because if one looks at the 3D structures of Bax and Bcl-xL in solution, they are very similar with great overlap. However, Bax acts as a pro-apoptotic affector in apoptosis, while Bcl-xL is anti-apoptotic.

We rationalized this mechanistic difference by Bax’s ability to self-interact and oligomerize in the membrane (as shown by Fluorescence Correlation Spectroscopy). During the oligomerization, Bax undergoes a conformational change, which is able to stabilize the open pore. On the other hand, Bcl-xL does not oligomerize in the membrane, thereby only producing transient pores.

To understand this better, one needs to look at the energetics of pore formation in lipid membranes [2]. An asymmetric insertion of material into a lipid bilayer introduces membrane tension. The reaction of the bilayer is then to rearrange the lipids and in doing so, may stochastically open up a pore. The opening of the pore would expose the hydrophobic tails of the lipids to water, and this is energetically not favored. To prevent exposure of the hydrophobic tails, lipids at the edge of the pore will bend and introduce packing defects in the membrane. And while this is less energetically costly than exposing hydrophobic regions, it is unfavorable. The energetic cost of keeping a pore open is often called line tension. In the absence of agents to stabilize the edge of the pore, the pore will simply close.

In the case of Bax and Bcl-xL, this is what we hypothesized to be happening. The insertion of proteins introduces enough membrane tension to open pores in the membrane. However only Bax is able to stabilize the edge of the pore, supposedly through oligomerization.

In the 2nd paper, Bleicken and Landeta, et al. [3] showed that Bax and Bak pores are not of a particular size. Using similar single vesicle permeabilization studies, we demonstrated that changing the concentration of the proteins changes the total permeabilized area in the membrane. This is akin to saying that the size of the pore is dependent on how many proteins are in the membrane.

While this may be generally be intuitive, pore size mainly depends on the nature of the pore. Pores may either be protein-lined (or proteinaceous) or toroidal (both protein and lipids line the pore)[4]. In protein-lined pores, the proteins serve as a scaffold to stabilize the pore; as such the protein structure defines the size of the pore. In these pores, proteins often form an oligomer with a defined number of units, and therefore the pore size is also definite. In toroidal pores, both protein and lipids line the pore, as such protein size is more flexible. They do not necessarily form a scaffold that holds the pore strongly together, but rather serve as a support so that lipids intercalate around the pore rim. The protein oligomers are believed to alleviate curvature stress of the lipid bending around the pore.

Unfortunately, there is no membrane-inserted structure of Bax to show how Bax stabilizes the pore edge. Putative membrane-inserted structures show that three helices in Bax are membrane-associated [5] , but no real 3D structure for Bax in the membrane has ever been solved to support this.

References

[1] S. Bleicken, C. Wagner, Ana J. García-Sáez, Mechanistic Differences in the Membrane Activity of Bax and Bcl-xL Correlate with Their Opposing Roles in Apoptosis, Biophys. J., 104 (2013) 421-431.

[2] M.-T. Lee, F.-Y. Chen, H.W. Huang, Energetics of Pore Formation Induced by Membrane Active Peptides, Biochemistry-US, 43 (2004) 3590-3599.

[3] S. Bleicken, O. Landeta, A. Landajuela, G. Basanez, A.J. Garcia-Saez, Proapoptotic Bax and Bak Proteins Form Stable Protein-permeable Pores of Tunable Size, J. Biol. Chem., 288 (2013) 33241-33252

[4] K. Cosentino, U. Ros, A.J. Garcia-Saez, Assembling the puzzle: Oligomerization of alpha-pore forming proteins in membranes, Biochim Biophys Acta, 1858 (2016) 457-466

[5] M. G. Annis, E. L. Soucie, P. J. Dlugosz, J. A. Cruz-Aguado, L. Z. Penn, B. Leber, and
D. W. Andrews. Bax forms multispanning monomers that oligomerize to permeabilize
membranes during apoptosis. EMBO J., 24 (2005) 2096–2103 .

Note: This is a five-part piece on our group’s recent contributions to Bax’s structure and function in apoptosis.

Quick links:

Part 1. Insights on Bax pore-forming activity

Part 2. Structural Model of Bax in the Membrane

Part 3. Counting Bax Molecules in the Membrane – A single molecule approach (to be released)

Part 4. Bax forms Rings, Arcs and Lines in Apoptotic Mitochondria (to be released)

Part 5. Membrane effects of Bax (to be released)

Introduction

In 2011, I started this blog to chronicle my journey as a PhD Student in the lab of Ana Garcia-Saez. As a chemist dipping his feet into biophysics, I was at times overwhelmed by the challenges that my project presented to me. But due to the crazy, albeit amazing colleagues, I was able to get through all these challenges.

Bax is a pro-apoptotic protein of the Bcl-2 family that serves as an effector of mitochondrial outer membrane permeabilization (MOMP). MOMP is considered the point of no return in apoptosis as this process releases factors like cytochrome c that trigger downstream processes. It is believed that Bax oligomerizes in the membrane to form pores that allow the release of the apoptotic factors. However, nobody has seen the pores in relation to Bax activity.

In 2012, my supervisor pooled all the researchers in our lab working with characterizing the pore-forming activity of Bax. To put it simply all the researchers aimed to find out how does Bax form pores in the membrane, and what is the structure of this Bax pore.  The main difference is that we used different techniques for this common goal. As such, we dubbed this umbrella project, the “Bax Project”. My part in the project revolved around the use of Atomic Force Microscopy to characterize Bax in the membrane.

Bax Project employed our expertise in working with Bax in model membrane systems and use this to collaborate with structural biology experts. Some these collaborators included Enrica Bordignon and Gunnar Jeschke. They were our electroparamagnetic resonance spectroscopy collaborators from ETH Zürich. Enrica has since then went to the Freie Universität in Berlin. We also have collaborations with Markus Axmann and Prof Joachim Spatz who helped us with single molecule microscopy experiments (as well as teaching us enough microscopy to be able to set up our own microscopes in the lab). We also have to include Enrico Klotzsch and Jonas Ries in this front also for their expertise in microscopy. Both Enrico and Markus have also worked with Gerhard Schütz at the Technische Universität Wien, whom a colleague was able to visit for further consultation with single molecule microscopy. For super resolution microscopy, we worked with Markus Mund and Jonas Ries at the EMBL as well as with Jale Schneider and Johann Engelhardt (at Stefan Hell’s group) at the DKFZ.

Fast forward to 2016 and we have mostly finished the work we set out four years ago. Of course, as we answer some questions, this leads us to new exciting and questions. In the next few entries, I will be giving a few summaries and stories of the various papers we have published for the Bax Project, and maybe give you a glimpse of where our group is heading to.

Quick links:

Part 1. Insights on Bax pore-forming activity

Part 2. Structural Model of Bax in the Membrane

Part 3. Counting Bax Molecules in the Membrane – A single molecule approach (to be released)

Part 4. Bax forms Rings, Arcs and Lines in Apoptotic Mitochondria (to be released)

Part 5. Membrane effects of Bax (to be released)

 

Note: This is a five-part piece on our group’s recent contributions to Bax’s structure and function in apoptosis.

CRISPR/Cas 9 challenge

I’ve been on some sort of blogging hiatus lately, mostly because I have been on a conference and writing spree for the majority of 2015.

I’ve already attended 3 conferences this year and there’s one more in October. Such is the fun world of scientists. (PS. I’m an introvert so I’m always scared of meeting new people, but conferences could also be very fun! I’ll expound more on my next entry.)

Furthermore, I recently submitted a feature article to Science in School in EMBL, which is now under review. I am also in the midst of revisions of a research article that I plan to submit by the end of the month. There’s also the thesis that I hope to submit in October. That’s a lot of writing assignment for the next few weeks.

But aside from that, I am swamped with learning new biology techniques, specifically knocking out genes using the CRISPR/Cas9.

Here’s a nice introduction about the CRISPR/Cas9 system.

And if you’re interested in the specific protocol, here’s one from Nature Protocols

http://www.nature.com/nprot/journal/v8/n11/full/nprot.2013.143.html

On my part I was using the pX458 plasmid which has a GFP reporter. This plasmid codes for the sgRNA sequence, as well as the SpCas9 and GFP. GFP positive cells can then be sorted from the population to help isolate single clones. Unfortunately, it hasn’t worked yet for me and I’m still trouble shooting the process. (rant warning!!!) I sort of wasted three months of my life and I’m not feeling so happy about it (end rant).

Anyway, to keep me sane, I took some pictures of the cultures from the failed single clones.

The leaning tower of culture dishes Plates for each isolated single clone

A recent study measured the effectiveness of peer review as a way of scientific gate keeping[1] . This article was summarized in phys.org, which was where I found it [2].

I found it very interesting as a scientist who works with quite a number of data sets and computation, how the methods of data mining and cataloguing can help evaluate the current practice of science. In this fast-paced, internet-crazy generation, the publishing data is indeed within our fingertips. I like how it is being used for studies like this to improve science.

With this in mind, the conclusion of the paper is something that is not necessarily “new” knowledge to scientists. But it does give us a confirmation of what most people believe about the peer review process. To summarize, I think most scientists believe in the peer review process. On the average, the peer review process has predicted the impact of articles well. They state that peer review scores (explained as 3 for immediate acceptance, 2 for minor revisions, 1 for major revisions and 0 for rejection) tend to correlate with impact (measured as no. of citations). However, the authors did say that peer review is not perfect. There are other possible biases that could lead to rejection of eventual impactful papers.

The authors discuss the different limitations of the peer review process including the nature of publishing as an enterprise (with limited resources), the Matthew effect (a published author in a high-ranking journal will tend to be able to publish in other high-ranking journal or simply put “the rich get richer, and the poor get poorer”), or even their use of citations as a measure of impact. They also discussed scientific gatekeeping in the context of high-risk, high-reward situations. They say that while some unconventional, controversial contributions may be impactful and even lead to breakthroughs, most of them are actually bad ideas.

Studies like this should be able to springboard discussions and eventual reform of the peer review process. Because more and more data about biases and limitations are gathered, improvement in the system should naturally come next. And indeed, there has been some effort to do so including debates on open access, impact factor as a measure of journal quality/scientist’s ability, reviewer vs. author anonymity, gender biases in science,work-life balance in academia, etc — all of which could potentially help the practice of science in the digital age.

Finally, they conclude that peer review weeds out the bad from the good, but is not able to distinguish the good from the great. In the end, the scientific community will be able to decide what is excellent science from the rest — regardless of where it is published.

Now, who has not heard of that before?

———–

[1] Siler, K., Lee, K., and Bero, L. “Measuring the effectiveness of scientific gatekeeping“. Proceedings of the National Academy of Sciences (PNAS), 112, 360-365 (2014)

[2] Malory M., “Peer review could reject breakthrough manuscripts, study shows“. phys.org, Published: December 2014, Date acessed: March 2015.

Season’s Greetings

Christmas Greeting

Science Break!

For the past few months, I have been on a constant writing spree — having one writing assignment after another.

I had to write (1) a few parts for a journal article with a collaborator, (2) a protocol paper, (3) a book chapter, and finally, (4) my own journal article. I’m currently on 3 and returning the edits/answers for review for 2, and then I still have to start 4. All of this on top of doing experiments. I still have no idea how to fit my thesis in there.

This past few weeks were quite mentally tough, so it was a welcome reprieve to come to the DKFZ graduation ceremony. One of my colleagues, Corinna Wagner also graduated, so I came as moral support!

Just like last year, I was again part of the student band, “The Wild Types”. We are a group of current (and former) PhD Students in Heidelberg and we come from different graduate programs and institutes: DKFZ, HBIGS, and EMBL. We performed as the opening act, intermission and ending number during the ceremonies, and then outside during the reception.

What I love most about this experience is getting to perform in a semi-relaxed environment that you shared with your peers in science. It is also a chance to remember that there is a light at the end of the tunnel, and to put the cliché, “we’re all in this together”.

The Wild Types for me has not only been fellow artists, who I deeply admire and respect, but also a family. It has grown over the years, but I really learned to love them.

The string that holds us together is Foivos Tsokanos, working on Signal Transduction of Cancer at the DKFZ by day and our manager/musical director. He also plays all kinds of instruments: guitar, bass, drums and percussions, piano combined, with singing.

Milene Costa da Silva, working on Iron Homeostasis at the University Hospital in Heidelberg, who is also our amazing lead vocalist turned bass guitar player.

Markus Graf, formerly working at the Radiology Department and now a Professor in Heilbronn University, also one our bass guitar players,

Reinhard Liebers, working on Epigenetics at the DKFZ while being our Percussionist/part of Sailing Conductors.

David Peralta, formerly working on Redox Regulation at the DKFZ and now an assistant editor at Wiley-VCH in Weinheim, another vocalist

Joel Perez-Perri, a postdoctoral fellow at the EMBL working on RNA biology and one of our main guitarist

Elena Senís Herrero, working on Viral Vectors and Viral Host Interactions at the BioQuant in Heidelberg, who also sings and plays guitar.

Laura Wiehle, also working on Epigenetics with Reinhard at the DKFZ, is our violinist

and then there’s me, the Biophysicist from Tübingen who sings (and sometimes plays the piano)

I have nothing but admiration and praise to these group of people I now consider part of my family in Heidelberg.

They are one of the reasons I have not yet gone crazy doing my PhD. =)

A few weeks ago, I attended a software carpentry workshop in EMBL

It introduces a few important tools for those who would like to utilize computer programming to better their research life.

Some of the tools we learned about were:

1. Unix Shell

2. Python/Numpy

3. Git (Version Control System) — I’m super impressed by Git!!

4. Cluster computing

5. Web tools

As someone who had only dabbled on programming, it was a nice refresher. I liked learning Git, Shell and Clusters. The best thing for me about the course was the opportunity to go through these tools in a guided environment. I tend to be a little apprehensive of learning new software because I usually end up deleting something important or making my system very unstable. With the course, I was able to go over this fear.

As one of the lecturers said, they didn’t aim for it to be detailed and comprehensive enough so that we will be experts at the end, but to get us over that first steep step in the learning curve so that we would hopefully be able to go beyond and create something useful for us in the future. My immediate response to this is that I think it did help me with the first step of the learning curve. Only time will tell if it will bear fruit, but I am eager to try in the next few weeks/months.

For more information about software carpentry, Visit their website at http://software-carpentry.org/

They have most of the resources for helping scientist be better users of technology to aid in their research. It is also a portal for more experienced people to share their knowledge and expertise. Maybe someday, I’d be able to give back to the community and teach a course or two.

—-

As a little side story, I met a guy named Aidan Budd during the workshop. I already heard about him around the scientific twitter-verse in Heidelberg when we organized the Communication Career Day at the DKFZ, but I never got to directly interact with him. He is a scientist at EMBL but is doing more project management work. However, I realized his main passion was helping scientists become better people, by not only engaging their scientific needs but voicing out their needs/concerns as a person.

As an educator, this has always been my qualms with science, in that it gets too results-driven that we forget that without the person there is no scientist. I am often reflecting the lack of cura personalis, a term I love to borrow from my Jesuit formation, in scientific practice. I am not saying that working conditions in science is extremely bad, but it could be improved. And people like Aidan who speak up about it, help us challenge the status quo: scientists should just “man up” and simply “get the job done without complaints”.

I also talk to several colleagues about their woes in the lab and often times, our chat boxes would end up being flooded by rants of dealing with supervisors, bosses, or colleagues. Some of it are quite harmless but might still get one in a foul mood. Some of them are just wrong in so many levels that it definitely gets one in a foul mood, not just for the day, but for the whole week or month. I also saw an article about mental health in academia, showing this culture of simply accepting it as “the way it is” is not healthy and should be addressed systematically. The running joke between some of my friends is that we should write a book about it considering the length of conversations we already had. I bet some other scientists have their own stories — both less tragic, or more vibrant than ours.

To this end, there is one thing we know for sure. That scientists need a lot of support from peers and family to overcome this. It is not simply a fight for the scientists but also a fight for the practice of science — which in truth, should involve everyone (but this is a topic for another blog post!!).

Scientists are first and foremost, people.

This recent feature from Nature shows an interesting take on some education paradigm changes implemented from some Universities across the globe.
1. The first one is the Technical University Munich, which was dubbed innovative university. It was the first university to implement organizational changes to promote quality and become globally-competitive. One of the talking points was structured graduate school. As the BSc and MSc in Germany are well-structured, I shall comment only about the Doktorand or PhD. The Doktorarbeit or PhD work is very research-oriented, so you don’t need a definitive course requirement. This often times lead to horror stories at most universities, where there are no checks and balances to mentorship and graduate formation. The excellence initiative of German universities sponsored by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) provided funding for Universities to be more competitive.  This created funding for programs such as some that I am familiar with like the Cell Networks in Heidelberg (which sort of led to the construction of BioQuant, my former research building), and the HBIGS (a graduate school at the faculty of biosciences). If you look at the website TUM also garnered a lot of these Excellence Initiative funding.

For international graduate students like me, one good entry point to Germany is through the graduate schools offered by research institutes like the Helmholtz Graduate School at Helmholtz centers (like the one at DKFZ) and the International Max Planck Research Schools (IMPRS) at various Max Planck Institutes. Most of these graduate schools are both loosely structured, allowing proper mentorship and guidance to researchers, while maintaining some freedom to choose one’s endeavors. In the DKFZ, for example, we are required to take 3 semesters of “progress in cancer research” lectures to update us on current trends, topics and researches about cancer (since we are a cancer center, after all) and several scientific and science-related courses and seminars. While the PICR is a strict requirement, the others are up to us to fulfill. Scientific courses include those that help us learn techniques, summer schools, etc., while science-related courses can be soft-skills like presenting in english, science writing, or even German language courses. On top of this, we attend several networking activities like conferences and retreats. But the best part for me is the thesis advisory committee (TAC) and its consequent annual meetings. In the TAC meetings two other senior scientists aside from your PI evaluate your progress and plans. In this way, proper guidance is given to the student’s research, while still having some freedom for self-learning.

After the PhD, Universities with the Excellence Initiative funding, have centers (like the aforementioned CellNetworks in Heidelberg) for Postdocs and junior professorships/privatdozent.

As the article points out, the main challenge for Germany is how to keep this sustainable as the funding for these excellence clusters will run out soon, unless this becomes enacted into federal or state laws. (and from a personal standpoint, there’s this 12-year rule to worry about).

2. KAIST’s flipped classroom or Education 3.0 is a prime example for student-centered learning within the confines of a university. The idea of self-learning and cooperative-learning is very intriguing on the one hand, but also something not so new. The self-learning part is taking responsibility to know the material before class, something that traditional lectures fail to do. I also love the cooperative-learning component, where the students are placed in a situation that they can help each other and voice out their concerns in a smaller group (vs. say 30 people in a class.)

The main drawback, I see here is the need to hire/train more people to supervise these group discussions. Teaching assistants are good candidates for this. However, facilitation is very different to standing in front telling people what they should know. It would also be interesting to know whether this style is better for more basic classes than for more difficult courses, or if the course material difficulty would even matter. While I don’t think there should be any difference, this in turn lies on how well facilitation works.

3. Massive online open courses are gaining popularity in the recent years. MOOCs are a distant cousin to this flipped classroom environment. While both promote the idea of self-learning, the cooperative-learning is less structured. The fora can be lively or dull depending on the participants, and because the audience is more heterogeneous, it is harder to plan and consider all possible questions or situations.

I have tried a few of them myself, and the learning part really depends on one’s motivation to finish. I started quite a lot of them but failed to finish most of them as more pressing life matters arrived.  Furthermore, sometimes the peer evaluation is not that helpful, specially if your peers didn’t take time to really comment and criticize your work.

Akin to a social network, MOOCs also promote connectivity, but can also result in particularly awkward forum threads. And just like facebook or twitter, MOOCs need to evolve in such a way that too much  or too less anonymity would not be detrimental to learning. In the end, they say, humans could only hold up to a few real connections

4. The last paradigm example from South Africa is perhaps the most familiar to me, as we tried to promote programs within the Ateneo de Manila University to be more inclusive. In the Philippines, the main problem is that public basic education is not as good as private school curriculum. So when university applications are considered, more often than not, the richer people are able to afford university and qualify through the exams. In the Ateneo, groups like Pathways to Higher Education and Ateneo Center for Educational Development help bridge the gap and give public school children a fighting chance. Furthermore, Ateneo supports deserving students who wouldn’t be able to go through university without the help through full or partial scholarships. Within the student body, camaraderie between scholars is very strong. Aside from the financial and academic support offered, students also have avenues for social and emotional support.

I like this paradigm of inclusivity because it gives people an equal footing in an otherwise unfair world. By making university more accessible, we make it possible for those who are otherwise unable to in the first place to learn.

Giant unilamellar vesicles (GUVs) are free-standing lipid bilayers that organize to diameters between 20 and 300 µm, approximating the size a eukaryotic cell. One way to make them is through electroformation where lipids dissolved in chloroform are deposited and dried on an electrode surface (usually platinum) and then submerged in an aqueous solution and subjected to an electrical current1, 2. Over the years several papers have studied the effects of different types of currents, strength of electrical fields on vesicle formation3-5.

In our lab, we mainly use GUVs to look at the permeabilizing properties of membrane-active agents. Among others, we have characterized differences in pore-activity of Bcl-2 proteins6, 7. As this method has become more and more popular, the need for proper statistics and fast analysis prompted the lab to develop a software to analyze GUV permeabilization8.

We monitor vesicle permeabilization by looking at the amount of dye that has penetrated the GUVs. GUVs are usually marked with a lipidic dye (a dye that loves hydrophobic mixtures, and will not solubilize in water) to be able to detect it in the image. An outer dye is added to the GUV-containing solution. Non-permeabilized GUVs will have no fluorescence signal within it, while permeabilized GUVs will have varying fluorescent intensities (Figure 1A). The degree of filling is the intensity inside with respect to the intensity outside. The effect of different proteins and lipid compositions can be assessed by the amount of permeabilization (Figure 1B). Furthermore a distribution of filling degrees per condition can be plotted to see whether vesicles are filled in an all-or-none manner (The GUVs are either filled or not-filled, which may indicate presence of stable pores) or graded (GUVs have varying filling degrees) (Figure 1C).

Figure 1. Permeabilization Analysis of Single GUVs. GUVs are placed in a solution with an external dye (green, Alexa 488) and the flux of the dye into the GUV results in increase of fluorescence. (B) After Incubation, the intensity is monitored for all GUVs and all conditions to determine differences in biological function. (C) Furthermore, the distribution of filling degree can be assessed to show whether the filling characteristic is all-or-none or graded.

Figure 1. Permeabilization Analysis of Single GUVs. GUVs are placed in a solution with an external dye (green, Alexa 488) and the flux of the dye into the GUV results in increase of fluorescence. (B) After Incubation, the intensity is monitored for all GUVs and all conditions to determine differences in biological function. (C) Furthermore, the distribution of filling degree can be assessed to show whether the filling characteristic is all-or-none or graded.

One can also monitor pore stability by adding different colored dyes at different time points. In Figure 2, Alexa 488 was added to the GUVs at the initial point and Alexa 633 was added after 1 hour (or any other incubation time of choice). Depending on the stability of the pores, only one dye or both will permeabilize the GUV (Figure 2A-B). The filling degree for both dyes for each GUV can then be monitored for all conditions (FIGURE 2C-E) and good statistics can be established (FIGURE 2F).

Pore stability measurements uses two dyes placed at two time points. Stable pores remain open (A), where as unstable pores close and hinder the entrance of the 2nd dye (B). The degree of filling of both dyes is monitored for all GUVs and conditions (C,D,E) to give better statistics and infer differences in the pore stability (F).

Figure 2. Pore stability measurements uses two dyes (Alexa 488, green and Alexa 633, red) placed at two time points. Stable pores remain open (A), where as unstable pores close and hinder the entrance of the 2nd dye (B). The degree of filling of both dyes is monitored for all GUVs and conditions (C,D,E) to give better statistics and infer differences in the pore stability (F).

One can also monitor the pore size by adding dyes attached to different size markers. For example cytochrome c-Alexa 488 (12 kDa) and APC-Alexa 633 (105 kDa) will have different filling degrees and kinetics depending on the pore size. One can monitor the filling over time and several equations to find the total permeabilized area have been established before7.

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