Day 2- Single Cell Modeling

After the coffee break and some illuminating discussions, we came back to the next session: single cell modeling!

Philip Nelson- Single-photon sensitivity in human vision

Philip started this session by giving us a really nice background on the “evolvement” of science. If we have nice night/dim light vision, we would have a better chance of survival-not falling to be a prey! However, there is this notion that the emission of light is lumpy and random, and so is the capture/absorption of light. This leads us to wonder, what is the sensitivity of human vision (perception threshold)?

He goes on to discuss the landmark experiment done by Hecht on determining the minimum number of photons required for perception of light dating back to 1942, and some considerations involved in the experiment’s design. Barlow later pointed out there could be discrepancy due to false positive reports. He also discovered the role of spontaneous isomerization in vision perception. Later on, Baylor’s single cell data showed that there are not only spontaneous isomerizations, but also some lower signal rumble. Barlow also has the insight that ” the very first synapse must discarded some genuine signals”, which Rob Smith claimed later that it is “discarding half of the real signals”.

By using the “high-tech” instruments available now, scientists are able to confirm directly that rod cells impose no threshold, and test out the models proposed. Take home message here is that even with challenging technical details, we could still try to have the model which in Philip’s words: “The best fitting model is the one that maximizes a likelihood function defined by the rather limited experimental dataset.”

 

Po-Yi Ho- Interrogating the Bacterial Cell Cycle by Cell Dimension Perturbations and Stochastic Modeling

Po-Yi is testing the growth law of bacteria by perturbing the cell dimensions and looking at the bacteria cell cycle, using E.coli as a model system.

Single bacteria cells grow exponentially. Particularly, average E. coli cell volume scales exponentially with growth rate, with a scaling exponent equal to the time from initiation of a round of DNA replication to the cell division at which the corresponding sister chromosomes segregate, this is known as the Schaechter’s growth law. So how are cell division and DNA replication coupled and regulated? They found that cells initiate replication at a constant size delta per origin of replication. So they proposed this “adder-per-origin” model, which states that cells add a constant volume delta per origin between initiations, then divide after a constant C+D minutes.

When they try to perturb the bacterial cell dimensions by varying the expression levels of mreB and ftsZ (bacterial homologue of actin and tubulin, respectively), they found that decreased mreB levels resulted in increased cell width (with little change in cell length), whereas decreased ftsZ levels resulted in increased cell length. Moreover, they also found that the growth law remained valid over a range of growth conditions and dimension perturbations, which is kind of impressive! In conclusion, the timing of replication initiation governs that of cell division, and cell volume is the key.

 

Rosanna Smith- Gene reporters and the problem of measurement in live cells

Rosanna showed that even though fluorescence reporters are a convenient tool for us to measure protein expression in live cells, there could be some “side-effects” (that are not so loved obviously) that could perturb the results and affect the interpretation of data.

They showed that some reporters that are used to assess gene expression may have noise that impacts the single cell reporting. As an example, they used the pluripotency factor Nanog, and looked at the stochastic transcription from two alleles. They illustrated that different reporters can have different Nanog expression patterns, meaning that the reporters will perturb the dynamics that we are originally trying to measure.

This talk prompts me to think more about the experimental design and the intrinsic technical difficulties associated!

***

After a delicious and satiating lunch along with nice relaxing small talks, we are ready for the cultural tour to National Palace Museum, and the highly-anticipated Taipei 101 tour! The National Palace Museum has all the art treasures dating back to thousands of years ago! Next, we went up the observatory deck of Taipei 101. The elevator is pretty impressive (even for people afraid of height), it only took us 37 sec to get to floor 89 (382 meters above the ground)! The view of the whole Taipei city is amazing, although there was some fog and the visibility was not the best. The wind damper used to stabilize this skyscraper is also pretty impressive, it helped this tall building survive through different typhoons that hit Taiwan!

-Ivy

Mechanobiology

Taekjip Ha kicks off the mechanobiology section with tension

He’s going to talk about his now pretty famous (if you are into that kind of thing) tension sensors. This is important because all cells sense their outside environment and respond to it through mechanosensitive membrane proteins, which transmit this important information to the cytoplasm of the cell.

He uses short lengths of DNA with a fluorophore on each end. FRET occurs at low tension, and at high tension FRET is abated as the molecule is stretched.

The tension sensors can be programmed to lose their FRET at pN scales, on a range from 4 pN to 60 pN. So it’s a pretty direct way to measure how much force cells need.

Amazingly to me (I study integrin nanoclustering) Takejip shows that only one or two strongly binding integrins are required to induce successful binding of receptors to much less strong integrin adhesions and induce cell spreading.

[continuing our fairy tale theme, Takejip compares this phenomenon to the princess and the pea – tenuous but I’ll take it]

Next, he shows that Notch, another membrane protein, is depended on between 4 pN and 12 pN – a really small amount of force, in order to drop Gal 4 and allow it to go to the nucleus. At the end, Jagged1 is mentioned, which has a profile similar to that of notch – it needs 4 to 12 pN of force to activate, and seems to follow the ‘catch bond’ schema.

Pakorn Kanchanawong unfolds vinculin with 3D super resolution

 

Pakorn is very well known by now, but I’ve never seen him talk. He uses iPALM to get  sub 15 nm isotropic resolution in x y and z, and brought out some pretty memorable papers on the ultrastructure of focal adhesions.

Here he has done the same in cadherin based adhesions, in adherens junctions between cells. These are  super complex adhesions, and iPALM only currently works in the TIRF zone…so Pakorn and his team created a fake cell like substrate on the coverslip, so that the cell makes a junction that can be imaged.

Next he showcases a whole host of information about the cadherin adhesions, which appear fairly clearly segregated into three layers. The middle one, the interface zone, contains vinculin and its on this that he focuses.

By tagging both the N and C terminals of vinculin, Pakorn find that it stretches from 5 nm to 30 nm in length! To stretch it must be phosphorylated by Abl kinase, as well as having mechanical force applied across it. In addition, it turns out that Zyxin and VASP are taken with the vinculin, reaching nearly the height of the cortical actin.

A complex and rather elegant use of drugs and precise measurements (the best combination) from Pakorn once again, showing that mechanics and phosphorylation couple to produce vinculin stretch and subsequent molecular clutch engagement.

Ashley Nord describes the mechanosensitivity of flagellar machinery in bacteria

Ashley Nord for the final talk in this section. She describes this minimalist machinery that operates the bacterial flagellar – the alien like tendrils that drive bacterial swimming.

She describes mechanosensitivity in the ‘stators’ and does some pretty clever measurements on them. These are ion channels, which lend energy to the engine that is the flagellar machinery. The first thing she does is prove that more stators translate to a faster motor.

She also finds that the stators turn over dynamically – after a little while, they leave and are replaced by new stators. Crucially, in viscous solutions, the number of stators goes up to the maximum amount available. In less viscous solutions, the number of these stators goes down to about 4.

Okay and that’s me out for now. I will let the others take over!

Michael Shannon

 

Structured light, deep imaging and automated super resolution

Morning everyone. Everyone is looking pretty fresh and ready for the day, and appear to have entirely dodged jetlag. I myself am dangerously full of caffeine and ready to go.

The lecture hall is filling up, with little biophysical interactions (ha) going on all around…we await the introduction by Jung-Chi Liao.

Jung-Chi asks out thanks for bringing all of the rain from across Asia – funny as it has been raining here for 4 days straight now [note – I ask someone later and this is not normal in Taipei!]. He reminds us of the upcoming Shilin night market and cultural tours plus the banquet at the end. A big thanks to the other organisers, particularly Wei-Chun.

Suliana Manley – Aesop’s fable by SIM

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Achilles Kapanidis and Suliana Manley 

And up first is Suliana Manley : she uses Super Resolution live microscopy to reveal that cell wall manipulating proteins are also responsible for changing cell size after division.

Suliana opens with the popular Aesop’s story the ‘tortoise and the hare’. She uses the story to describe the ability of individual bacteria to decide how big they should be, a bit like the hare, choosing his/her moment to race through the cell cycle.

The model is Caulobacter crescentus – and she shows us that population of such bacterial cells plateaus to become all the same size – the larger ones get smaller and the smaller get larger. With this in mind, she uses Structured Illumination Microscopy to address the question of how this happens.

Its known, says Suliana, that the divisome, the group of proteins that manage the construction and destruction of the peptidoglycan cell wall are the ones that also form a contractile ring when cells are ready to divide in two. By using SIM, which is really gentle technique, the group can watch several cell cycles and collect ample data while tagging these two proteins.

What’s interesting is that by treating the bacteria with a pedtidoglycan inhibitor (fosfomycin), the cells get longer (by decreasing the rate of pedtidoglycan synthesis). It seems therefore that the availability of peptidoglycan intermediates might be a way to control the size of such cells.

Bi-Chang Chen – lattice light sheet extends to localisation

A swift change then, after some questions, to Bi-Chang chen – well known for his work on the lattice light sheet in Eric Betzig’s lab.

As usual, the lattice images never cease to amaze. Using multiple Bessel beams and a spatial light modulator, the team have been able to image whole cells, and even whole brains – like that of the fly – 185 um maps. That’s because beam energy is spread, photodamage is reduced and signal to noise is maintained. The sample is moved up and down to achieve magnificent images.

Of most interest to me is the single molecular side of things. In particular, swept z stacks of sox2 transcription factors in live cells are impressive. I quite fancied that I could see ‘hop’ diffusion, certainly there were regions where the Sox2 molecules were clustering together in a very interesting consistent looking way. Bi-Chang also describes some new self blinking dyes suitable for super resolution localisation microscopy. This type of dye is probably going to be invaluable for live cell localisation: especially for nanocluster tracking.

Shean-Jen Chen: Adaptive optics in a new guise

Next up Shean-Jen Chen who is going to talk to us about the availability of his multi-photon, which has recently been revamped and outfitter with some sweet new adaptive optics that he calls ‘temporal focusing’.

It’s a multi-photon ‘scope but not as we know it. He’s made it fully biotissue compatible, in all of its messy, undulating glory.

First, he has used a grating to create multiphoton at the sample plane where the now separate wavelength beams converge. Second, he uses a Digital Micromirror Device (DMD) basically a plate with 1000s of tiny controllable mirrors, with which he can structure the light going onto the sample, to get to that high spatial frequency information: only one lens as this is non -linear. The effective resolution laterally is 168 nm and axially is 1.3 um.

Finally, the juicy bit – he uses a deformable mirror to compensate for temporal distortion. He basically measures how the light should look and corrects for deformation in the image. Biotissue has diverse refractive indices, so correcting for them with adaptive optics like this is desirable.

The result of all of that is high speed (30 cell volumes per second) and much nicer resolution, which will be invaluable in years to come, as the biologists dream up new avenues, some of which might be 6 cells deep!

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Takeharu Nagai and Yemima Riani

 

Masahiro Ueda: super resolution x 1000

OK so the final talk of this session comes from Masahiro Ueda. This one really stands out for me, something that has been in my mind for some time!

Masahiro has some very impressive statistics. That’s because he’s found a way to automate super resolution microscopy, as well as drug treatments of such cells. With this technique, we are looking at 1000s of cells per day – incredible: my record on the dSTORM at King’s is around 80 cells in one (long day). So this really is a big deal.

The set up involves a two 96 well plates, one full of chemicals or drugs, the other full of cells, which both go into a chamber with the microscope at 37 degrees, with 5 % CO2. The drugs are added to the cells at time points with a robot (and extensive washing – the video of this robot washing itself was strangely amusing to us, the jetlagged), while the cells are located using machine learning software, autofocusing (on the side of the aperture) and a quick fluorescence check to avoid unnecessary time wasting.

The example given here is the clear relationship between EGFR trimerization and an activation marker, the adaptor protein Grb2. The Grb2 proteins cluster and hover around the areas of no longer diffusing EGFR trimers. Very promising stuff.

Oh, and yes, they did also engineer an objective oil supply device for all of those stage shifts!

Phooph! That’s the end of that section *runs into the rain to reinvigorate*

Next up, mechanobiology!­

A Single-Cell State of Mind in Taipei

Ni Hao! As I eagerly await my arrival to Taipei, Taiwan, for the Biophysical Society thematic meeting, Single-Cell Biophysics: Measurement, Modulation, and Modeling (June 17 – June 20), I thought that I would try to be productive with my time on this 12-hour flight to write this brief introductory post rather than spend my time dreaming of beef noodle soup, fried egg pancakes, potstickers, bubble tea, stinky tofu, shaved ice desserts, etc. (I think you get the idea; I really enjoy food). This is my first international conference, and I am very excited and honored to be able to share my research and immerse myself in this highly collaborative environment – to hear the inspiring works of researchers from around the world as well as engage in a fluid exchange of scientific ideas. I would also like to express my appreciation and thank the Biophysical Society for the opportunity to blog over these next few days. In addition to stuffing myself fueling myself with all of the street food that Taiwan has to offer, my colleagues and I will be covering this thematic meeting, providing our unique prospective and sharing with all of you readers the new experimental, computational, and theoretical advances that are emerging within the field to help provide a more complete understanding of the intricate and dynamic biophysical forces that instruct single-cell behavior.

 

A little about me, I am a Bioengineering PhD student conducting stem cell research at the University of California – Berkeley. What particularly attracted me to this international thematic meeting – beyond the intimate relevance to my doctoral research of developing new engineering methods to study how extrinsic niche cues guide stem cell fate decisions at the single-cell level – is the opportunity to interact with a community comprised of such diverse perspectives: biologists, chemists, physicists, engineers. I think about single cells every day – single adult neural stem cells to be exact – as I’m sure that the other researchers attending this meeting do as well. And just as each cell that comprises a tissue is distinct, defined by its own gene expression signature and shaped by different forces and experiences, each scientist/engineer here provides his/her own unique perspective to this meeting. We are all driven by different motivations, bring with us different skillsets and goals. Just as I aim to understand the heterogeneity of adult neural stem cell behavior, I look forward to learning from each of the attendees and immersing myself in the rich diversity of experiences brought here to Taipei.

 

Speaking of which, this is not my first time to Taiwan nor is this my second or third. Being half Taiwanese, a significant portion of my family still lives on this island, and I hold dear the beautiful culture that has been and always will be a part of my life. But this trip is different, and I’m excited. I’m excited to experience Taiwan with a scientific mindset. I’m looking forward to attending all of the talks and poster sessions, perhaps with a red bean bun (or two or five) in hand or a heaping serving of Taro ice cream.

 

Olivia J. Scheideler (University of California – Berkeley)

Biophysicists Finding Balance: Father’s Day 2017

June 18 is Father’s Day in the US. In honor of the occasion, we spoke with Biophysical Society member Seth Weinberg, Virginia Commonwealth University, about what it is like to be a biophysicist and a parent, and how the two roles impact each other.


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How many children do you have? What are their ages?

My wife Gabrielle and I have 3-year old twin girls, Hannah and Meredith.

At what stage of your career did you have your children? 

My daughters were born about three months before I started my first faculty position.  It was a very hectic time, finishing up the last few months of a post-doc, moving, and then starting up my own research group.

 Has your career been influenced or changed by your role as a parent? How?

I like to think that my experiences raising twins has made me more patient with my own students. Having become a parent and starting my own lab at roughly the same time, I have thought more about my role in preparing the next generation.  Especially being the father of two daughters, I have become more aware of how important it is to promote opportunities for women in science.

How has your career been influenced by your own father?

My own scientific career has stemmed from my desire to understand how complicated systems work, and much of that desire originates from my father.  My father was a science and engineering teacher, and from a very young age, he encouraged me to make learning and being curious into activities that were fun.

What has been the most challenging aspect of being a biophysicist and a parent?

Being away from my family to attend meetings and conferences is definitely challenging.  As much as I enjoy seeing and catching up with colleagues, it is still difficult to be away from my family during trips.

Have there been any benefits to being both a father and a scientist?

In perhaps a subtle way, my daughters have inspired my interests as a scientist.  My daughters are fraternal twins, and since their birth, I’ve become more interested in ‘nature vs. nurture’ types of questions.  I’m constantly amazed at how different my daughters are, despite their nearly identical childhoods (so far at least).  As a scientist, I’ve become interested in understanding how important randomness is in our biology and trying to understand how one system can generate different behaviors in response to the same input.

Would you encourage your children to be scientists?

I don’t plan to encourage them to be scientists any more or less than other specific careers.  I hope to encourage them to pursue careers that they will enjoy and find fulfilling.  Although, with my own career and my wife as a nurse, I wouldn’t be surprised if one or both pursued some career in the biomedical sciences.

How would your children describe your work?

I asked my daughters what they think I do at work. One said, “Monkey!” and the other laughed.

Any advice for other fathers or prospective fathers pursuing science careers?

Pursuing a science career is challenging – constantly being pulled in multiple directions and never knowing for sure if you are doing things right.  Being a parent is pretty much the exact same thing, so as a scientist, you are as prepared as you can be (which is to say, you are never really prepared)!

Pre-Meeting Introduction: Single-Cell biophysics: measurement, modulation and modelling

Hi everyone. I’m Michael and I’ll be one of three people covering the meeting. I’m a single cell biophysicist from London, currently working in Dylan Owen’s lab at King’s College (KCL).

The lab and I work on ways to quantitate biological phenomena on the nanoscale, particularly molecular clustering. I personally have been looking into the nanoscale goings on of integrin adhesions in migrating T cells. It’s a fun system to work in, because the adhesions are so much smaller than what are found in most other cells. What I’ve found appears to be a specific system of membrane nanoclustering, that is altered to tune the speed of a migrating T cell.

We think that this might be pretty important, especially considering a speed change experienced by cells with a mutation in PTPN22 – an integrin signal modulating phosphatase that is associated with autoimmune disease predisposition.

As well as this, we are very interesting in the dynamics and ultrastructure of the actin cytoskeleton, and mechanisms of membrane protein nanoclustering – how this might relate to the picket-fence model, lipid rafts and scaffold type protein regulation.

Some very relevant topics await: on the first day I’m very interested to hear Suliana Manley for live super resolution microscopy and Pakorn Kanchanawong on his cadherin adhesome work. I hope you are too!

As light rain continues to fall over Taipei, I think I can hear some kind of horn (of Gondor?), which I’m going to take it as my signal to start exploring the city. I always enjoy the sounds you get in such a place – city sounds are my favourite as they are so complex and multi-layered – familiar somehow and yet so different to what I’m used to in London.

So, with that, I’ll see you all tomorrow, for an engaging first day full of imaging techniques, mechanobiology, nanotechnology and the cell cycle!

Michael Shannon (Dylan Owen lab, KCL)

Getting ready for the Biophysical Society Thematic Meeting on Single-Cell Biophysics!

(IAMS building, photo credit: IAMS)

I am very grateful to have the opportunity to attend my first Biophysical Society Meeting! This year, the meeting “Single-Cell Biophysics: Measurement, Modulation, and Modeling” will be held June 17-20 at Dr. Poe Lecture Hall Foyer of the Institute of Atomic and Molecular Science (IAMS) building, National Taiwan University (NTU). As its name implies, the meeting this year is focused on single-cell biophysics, and it is bringing together people from different fields around the world! I can’t wait to have nice discussions on exciting sciences, meeting new friends, and becoming part of this wonderful community!

(National Taiwan University Library, photo credit: Office of International Affairs, NTU)

As a local here in Taiwan, I have pursued my undergrad and master degree at National Taiwan University (NTU). I remember those good old days of working till midnight in the lab, and then go across the street from our lab to grab some delicious Taiwanese street snacks (chicken fillet/popcorn chicken & bubble milk tea …and so many more)! (Good science and delicious food go together really well! Guess I am lucky to be schooled here in Taipei!)

(Taipei, photo credit: Travel Taipei)

We will be covering this thematic meeting over these days, hope you enjoy reading our articles! I believe it would be a rewarding experience!

🌸Ivy