Friday Feature: The Virtual Fish

Your Friday treat is a movie from Sean Megason’s lab of the development of a zebrafish ear.  Sean has a plan to provide a complete (“in toto”) image set describing the entire development of a vertebrate, using methods described here (Megason SG (2009). In toto imaging of embryogenesis with confocal time-lapse microscopy. Methods in molecular biology (Clifton, N.J.), 546, 317-32 PMID: 19378112).  When the project is complete — which will not be tomorrow — there will be a movie recording every cell division, and every morphological rearrangement, that happens as a zebrafish egg turns into a functioning fish.  And then you will be able to sit at your computer and analyze vertebrate development without needing to get so much as a single finger wet.

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This is from a zebrafish embryo in which both the nucleus (green) and the membrane (red) of every cell has been fluorescently labeled.  If you watch carefully, you can see individual cells divide (one green blob becomes two) and move into new positions, creating (for example) the circle of cells that then opens up into the tube of the inner ear.

Amazing.

Friday Feature: Frustrating neutrophils

Neutrophils have a special place in the study of cell motility.  It seems that about one in 5 talks on cell motility starts with this classic video of neutrophil crawling, from David Rogers (Vanderbilt) circa 1950 (a YouTube version set to music, just for a little variety):

(credits for original here).

I am not saying this is a bad thing.  It’s a great movie, and a wonderful way to introduce a semi-naive audience to the topic.  And it’s fascinating to see the neutrophil change direction in response to the movement of the bacterium.  How does the neutrophil know where the bacterium has gone?

Friday Feature: Inflammatory behavior

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Today’s movies were generously sent to me by Markus Covert (Stanford University).  This is a sampling of a very comprehensive and impressive study of the behavior of NF-κB in single cells, just published in Nature (Tay et al.  2010.  Single-cell NF-kappaB dynamics reveal digital activation and analogue information processing. Nature 466 267-71. PMID: 20581820).  It’s increasingly clear that a number of signaling pathways — calcium, p53, NF-κB  — use spatial and temporal dynamics to deliver complex, nuanced information on the single cell level.  Trying to understand how these pathways are working, or how to manipulate them using drugs, without appreciating that fact is like trying to understand what happened at the World Cup by watching a slice of the action that starts above waist level: you might be able to see when a goal or a near miss happens, but you’d have no idea about what led up to it.

What we’re looking at here is a fluorescent fusion protein of NF-κB being transported into the nucleus in response to the addition of TNF-α, then coming out again.  NF-κB is a transcription factor, so it needs to be in the nucleus to do its job; moving the pre-made protein in and out of the nuclear compartment is a quick way to switch NF-κB-induced genes on and off. NF-κB is usually sequestered in the cytoplasm by a protein called IκB.  When an activating signal, such as TNF-α, comes along, a kinase, IKK, is turned on (I am sweeping a good deal of complexity under the rug here) that phosphorylates IκB, causing it to be degraded by the ubiquitin pathway.  The degradation of IκB releases NF-κB, which then goes into the nucleus and turns on various genes including the one for IκB.

The NF-κB—IκB interaction is thus a negative feedback loop with a fast arm (protein degradation) and a slow arm (protein synthesis), which can lead to oscillations.  (Think about tweaking the taps on your shower when the temperature isn’t right (fast) — and the water taking its sweet time to make its way up the pipe to the shower head and fall onto your alternately shivering and scalded body (slow).  Oscillations, and much swearing, result).  The behavior of these oscillations can determine which downstream genes get activated.  Since NF-κB is a remarkably versatile and important transcription factor — it’s central to the immune response, and implicated in cancer, inflammation, autoimmune disease, learning and memory — the question of exactly how the activation of downstream genes is controlled is really quite important.

Controls are cool

Many of you know that as a post-doc in Uri Alon’s lab, Galit Lahav caused a small revolution in our understanding of how the p53 network responds to DNA damage.  By looking at single cells instead of populations, she showed that individual cells responding to the damage caused by gamma-irradiation show a series of stereotyped pulses (shown in this movie); different cells show different numbers of pulses, and as you increase the amount of damage, the number of pulses per cell increases.  Now the Lahav lab has identified another previously unsuspected feature of the p53 response (Loewer A, Batchelor E, Gaglia G, Lahav G.  2010.  Basal Dynamics of p53 Reveal Transcriptionally Attenuated Pulses in Cycling Cells Cell 142 89-100. PMID: 20598361). It turns out that p53 is being activated in normal growing cells all the time.  Because the cell cycle of cells in culture is unsynchronized, this activation can only be seen by looking at single cells. Since p53 may be the most studied protein on the planet, discovering something completely new and unexpected about its activities isn’t an everyday event.

The story started with an experiment that was originally intended as a control, looking at unstressed cells.  Unexpectedly, in these unstressed, undamaged cells they found p53 pulses that are indistinguishable in shape from the pulses seen in gamma-irradiated cells. The first clue to where these pulses come from was the observation that they’re correlated with specific stages of the cell cycle, primarily happening right after mitosis.  Loewer et al. used a Cdk inhibitor to show that when the cell cycle is stopped, the pulses go away.  And the pulses were also selectively stopped when the ATM/DNA-PK pathway, which monitors double-stranded DNA breaks, was inhibited. It appears that these pulses are triggered by transient DNA damage that is a routine part of the cell cycle.

Friday Feature: Calling for help

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This amazing movie, from Niethammer P, Grabher C, Look AT, Mitchison TJ. 2009 A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature 459 996-9 PMCID: PMC2803098, shows leukocytes (the white blobs) rushing to the site of a wound in response to a hydrogen peroxide signal (fluorescence in upper panel).

We’ve known for a while that leukocytes rapidly (within minutes) home to the sites of wounds.  What hasn’t been clear is what signal attracts them.  We’ve also known for a while that hydrogen peroxide is generated in wound sites: but until now, the general belief has been that it comes from the leukocytes that are attracted into the wound.  Hydrogen peroxide has a role in killing bacteria, at least under some conditions, and so this all seemed to make sense.  Until this movie.  You can’t really tell by eye, but the quantitative analysis clearly shows that the hydrogen peroxide production starts before the first leukocyte arrives.  In fact, the timing is such that it seems very plausible that it is the hydrogen peroxide that calls in the leukocytes: soon after the hydrogen peroxide reaches the nearest blood vessel, you start seeing purposeful leukocytes making tracks towards the wound.

Is the hydrogen peroxide gradient we see causal, or is it a byproduct of something else? There are five enzymes in the zebrafish genome that can produce hydrogen peroxide, directly or indirectly (four NADPH oxidases (Nox-1, -2, -4 and -5), and Dual Oxidase, abbreviated Duox).  Niethammer et al. showed that small molecule inhibitors that inhibit all 5 enzymes prevented the hydrogen peroxide gradient from forming in response to a wound, and also strongly reduced leukocyte recruitment to the wound.  Using antisense morpholinos and quantitative PCR, they then narrowed down which of the five possible enzymes could be responsible: none of the Nox enzymes seems to be involved, but knocking out Duox blocked both gradient formation and leukocyte recruitment.  Problem solved: the leukocyte recruitment signal has been discovered!

Well — there may still be more to the story.

Happy 4th!

These are not fireworks.  Martin Wuehr (Mitchison lab) kindly put together some of his pictures of asters in early frog and zebrafish embryos to give you a firework-like experience that doesn’t depend on good weather, trekking down to Boston Common, or indeed being in the United States.  Enjoy!

Friday Feature: Variable death

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It’s Friday, and what better way to get into the mood for beer hour than to talk about death?  To make sure that the mood doesn’t get too lugubrious, let’s stick to the death of entities that are unlikely to remind you of anyone you know, such as single cells.  This video shows HeLa cells undergoing apoptosis, from work described in Spencer SL, Gaudet S, Albeck JG, Burke JM, Sorger PK. 2009.  Non-genetic origins of cell-to-cell variability in TRAIL-induced apoptosis. Nature 459 428-32.  PMC2858974.  You’ll see that the greenest cells round up and bleb and die very fast, whereas the less green cells die much more slowly.  And thereby hangs a tale.

Apoptosis — programmed cell death — is one of those processes that you would think would be entirely predictable.  If you’ve triggered a cell to die, you’d think that would be the end of it.  But this turns out not to be true.  If a clonal population of cells is exposed to TRAIL (TNF-related apoptosis-inducing ligand), typically some survive; and the time to death is also very variable.   On the face of it, this variability in genetically identical cells is quite mystifying.  What makes it more than a mere curiosity is that this same variable response might be important in clinical settings, where it’s typical for cancer therapeutics to kill some cancer cells and spare others (a phenomenon called “fractional kill”).

Sabrina Spencer (who has now left the Sorger lab for post-doctoral training in the Meyer lab), set out to ask what kind of mechanism might explain this variable death.  The most traditional and obvious candidate, perhaps, is cell cycle phase; another obvious (though less traditional) hypothesis is that some yet-to-be-identified key factor is present at very small numbers per cell, so that chance fluctuations in its level create significant differences in the functioning of the death pathway.

But there is a third possible explanation.

Friday Feature: Watching the immune system work

I can’t resist it — another movie.  This one is from Ralph Weissleder‘s lab, and it’s a doozy.  We are looking inside the spleen of a live mouse, and we are watching a monocyte (blue track) sitting peacefully in the spleen wake up in response to an injury and get out into the blood to help heal the wound.

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Swirski FK, Nahrendorf M, Etzrodt M, Wildgruber M, Cortez-Retamozo V, Panizzi P, Figueiredo JL, Kohler RH, Chudnovskiy A, Waterman P, Aikawa E, Mempel TR, Libby P, Weissleder R, Pittet MJ. 2009. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science 325 612-6. PMCID: PMC2803111

I know not everyone adores immunology, but I do.  Where else do you get such frequent, visible cell fate decisions, and so many hugely consequential checks and balances?  It’s a systems biologist’s playground.  (Those who feel that their own corner of biology is even better, feel free to comment.  Neurobiology, anyone?)

Monitoring mothers

Roy Kishony pointed out this cool paper by Suckjoon Jun, one of the Fellows at the FAS Center for Systems Biology.  (Wang P, Robert L, Pelletier J, Dang WL, Taddei F, Wright A, and Jun S. 2010.  Robust growth of Escherichia coli.  Current Biology 20 1-5.  PMID: 20537537).  There are two things that are cool here.  The first is the technology: a microfluidic device called the “mother machine” that traps a single “mother” cell at the bottom of a growth channel where it is immobilized for study.  As the mother grows, a chain of daughter cells get pushed up to the top of the channel and carried away by the flow of growth medium, which also provides sufficient resupply of nutrients to diffuse to the bottom of the channel and keep the mother cell happy (they checked).  The whole thing looks like a tiny sausage machine.

Friday Feature: Carboxysome spacing

I promise that not every Friday Feature will be a movie, but since I mentioned the Silver lab’s ambitions to control how cells use light energy in the last post, here is a lovely movie of cyanobacteria growing and dividing, in which you can see the remarkably regular spacing of labeled carboxysomes.  Carboxysomes are where much of the magic of carbon fixation happens: they’re said to be responsible for about 40% of all the carbon fixation on the planet.  Dave Savage got interested in how they work, and, not unnaturally, wanted to see what they look like.  In this movie they are labeled with YFP.

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Savage DF, Afonso B, Chen AH, Silver PA.  2010. Spatially ordered dynamics of the bacterial carbon fixation machinery. Science.  327 1258-61. PMID: 20203050

The remarkable regularity you see is not just an illusion: carboxysomes are evenly spaced along the length of the cell, and their position adjusts as the cell grows (or as new carboxysomes appear) so that the spacing remains regular.  They “wiggle” due to diffusion far less than you would expect them to.  The spacing seems to be determined by the cytoskeleton, using mechanisms that were originally described in connection with the regular spacing of certain plasmids.