Crazy Postdoc Update (Charges Dropped)

I meant to post this a while back, but the whole postdoc poisoning case reached an anti-climatic conclusion a few months back.  As you might remember,  Ben Chun Liu, a postdoc at UCSF, was charged with adding ethidum bromide to his co-worker’s water.  The DA initially planned to bring poisoning charges against Liu, but his lawyers argued that ethidium is not a poison at such low concentrations.  

Well that argument seems to have won out.  The Chronicle reports that all charges have been dropped and that Dr. Liu has apologized to his coworker (strangely though, the Chronicle continues to insist that the ethidium bromide turned the water blue, despite it having a dark red color).  While one might question how adding a laboratory reagent to your coworkers’ drinks without telling them is legal, it’s pretty clear that not only was  Dr. Liu’s ”victim” never in any serious danger, but also the defense lawyers arguments were scientifically sound.

One last point that this whole ordeal has been useful in illustrating: In research labs we tend to be overly careful about how we dispose of waste and how we handle chemicals.  In fact that’s what makes this whole case so shocking; biology labs are extremely (and unnecessarily) paranoid about their ethidium waste, which is collected and disposed of separately from all other waste.  Given that it appears we’ve decided its ok to drink this stuff, you’d think that it might be time to reexamine the expensive and time consuming manner in which we deal with the waste, right?  Well, I wouldn’t expect changes anytime soon;  lab safety guidelines are not formed from rational risk assessment, but rather from a “are-we-covered-in-all-possible cases-legally” viewpoint.  Just look at In the Pipeline’s recent analysis of the MSDS of sand: it’s listed as a “cancer hazard” and is only to be used in a chemical fume hood (be sure to bring one to the beach with you next time!). Regardless, Dr. Liu is free and this seems to be the end to the case – I suppose we can all just be thankful that he didn’t add any sand to the water…

Inner Life of the Cell Explained, Part II – “Adhesion” & the Organization of the Cell

Since these “Inner Life of the Cell” posts appear to be popular with high school students studying for their exams and serving as fodder for youtube debates about evolution, I’m going to break with my tradition of only posting in November and finish up these series of posts.  So back to it:

When we last left our leukocyte he had just had been stimulated to provide an inflammation response.  While I’m grouping this part of the explanation under “Adhesion”, most of the adhesion part of this video was covered previously in “Rolling”.  This section deals more with the basic organization of the cell, and the transmission of the adhesion signal.  Both of these will be important in activation, but in the interests of matching with the explanation video I’ve just lumped it in with “Adhesion”.  With that caveat, let’s return to the video and our recently stimulated leukocyte.

0:31-0:37:  We’re know crossing from the outer (or “top”) layer of the leukocyte membrane (the part that has adhered to the endothelial cell) and delving inside it.  We’re still at the membrane but we’re now in the inner part which is an entirely different environment. Whereas the top  or outer part of the membrane was important in contacting the skin cell, the inner portion is dedicated to transmitting the fact that the cells have “docked”. Here’s what the video says:

The inner leaflet of the bilayer has a very different composition than that of the outer leaflet. While some proteins traverse the membrane, others are either anchored into the inner leaflet by covalently attached fatty acid chains, or are recruited through non covalent interactions with membrane proteins. The membrane bound protein complexes are critical for transmission of signals across the plasma membrane.

Basically, there are some proteins that stick through the membrane (like a straw through the peel of an orange) while others are just stuck to the inside (i.e. attached to the underside of the orange peel but not visible from outside) by “fatty acid chains”, and lastly there are things that are stuck to these anchored pieces, but aren’t anchored themselves (like if your friend was holding onto the edge of a cliff and you were holding onto him).  All these proteins are important in conveying messages from the surface to the interior of the cell (in this case “Hey, we’ve adhered to an inflammation site”).

0:38-0:42:  We’re now going deeper into the white blood cell, leaving the membrane (the outermost part) and entering the cystol, the gooey interior of the leukocyte.  A structure known as the cytoskeleton, comprised of spectrin tetramers (spectrin is a protein, and tetramer means that four of them come together to form a functional unit) is attached at several anchor points to the membrane proteins and holds the cell together and keeps it organized (so that everything doesn’t just mix together).

Beneath the lipid bilayer, spectrin tetramers arranged into a hexagonal network are anchored by membrane proteins.  This network forms the membrane skeleton that contributes to membrane stability and membrane protein distribution.  The cytoskeleton is comprised of networks of filamentous proteins that are responsible for the special organization of cytosolic components.

0:43-0:52:  Now we’re delving even deeper and looking at actin filaments.  Actin is one of the most abundant proteins in the cell and forms these long filaments which will constitute an important part of the cytoskeleton.  Whereas spectrin provides a basic structure to the cystoskeleton, actin will be more important in cytoskeleton dynamics and will thus be instrumental in helping convey signals (such as the “Hey we’ve adhered” message) around the cell.

Inside microvillae, actin filaments form tight parallel  bundles which are stabilized by cross-linking proteins.  While (deeper?) in the cystol the actin network adopts a gel-like structure, stabilized by a variety of actin binding proteins.

0:53-1:01:  Remember how I was saying actin was important in dynamics?  Well, here you can witness it yourself.  These long filaments are continuously forming and coming apart allowing the cell to convey information to all its parts.  Note that these filaments have a directionality – they only grow in one direction and are capped at the other end.

Filaments, capped at their minus ends by a protein complex, grow away from the plasma membrane by the addition of actin monomers to their plus end. The actin network is a very dynamic structure with a continuous directional polymerization and disassembly.

1:02-1:07:  Here we see regulation of the actin network by other proteins.  Filaments can be cut in order to cause them to come apart or to regrow to other parts of the cell. Cofilin is an example of one of these kinds of actin regulating proteins.

Severing proteins induce kinks in the filament and lead to short fragments that rapidly depolymerize or give rise to new filaments.

 

1:08-1:15:  Ok now we leave actin and come to microtubules:

The cytoskeleton includes a network of microtubules created by the lateral association of protofilaments formed by the polymerization of tubulin dimers.  While the plus ends of some microtubules extend toward the plasma membrane, proteins stabilize the curved conformation of protofilaments from other microtubules, causing their rapid plus end depolymerization.

Microtubules are created by the joining together of proteins called tubulin (dimer means that two tubulins are a single functional unit in contrast to the actin tetramer (4 molecules = 1 functional unit)).  Much like actin they have directionality, they rapidly polymerize and depolymerize as needed and serve as “tracks” on which certain proteins can travel.  The term protofilament just means a short microtubule that can grow into a full filament.

1:16-1:26:  Next up is kinesin, which seems to have become the star of this video. Kinesins are motor proteins that travel along microtubules carrying important cargo.  In this case, the kinesin is carrying a vesicle, which is essentially a bubble full of proteins and other important molecules that are needed at other parts of the cell. This is a routine function in the cell, and not necessarily specific to the inflammation response.

Microtubules provide tracks along which membrane bound vesicles travel to and from the plasma membrane.  The directional movement of these cargo vesicles is due to a family of motor proteins linking vesicles and microtubules.

1:27-1:34:  As we watch the little kinesin dragging his vesicle we zoom out and see some of the larger functional units of the cell.  Normally proteins and other important molecules diffuse to their correct location, but in some cases diffusion is insufficient (i.e. the molecule is too large) and these motor proteins have to drag them to the correct spot.  Up in the right corner you see a mitchondria, often referred to as the power plant of the cell (it’s important in making energy for the cell), an example of a structure that would require transport by kinesin.  The large glob dominating the center is the centrosome from which all the microtubules originate.

Membrane bound organelles like mitochondria are loosely trapped by the cytoskeleton.  Mitochondria change shape continuously and their orientation is partly dictated by their interaction with microtubules.  All the microtubules originate from the centrosome, a discrete fibrous structure containing two orthogonal centrioles and located near the cell nucleus.

Ok so we’ve seen how the cell can communicate messages from the surface to the nucleus and gotten a basic idea of its organization, but how does it respond appropriately to the inflammation signal?  We’ll look at that in the next post “Activation”.

Crazy Postdoc Update

KTVU reports that the Benchum Liu (the accused postdoc) will plead not guilty to poisoning his labmate Mei Cao.  Interestingly, Liu does not deny adding ethidum bromide (EtBr) to Cao’s water (he even participated in a videotaped reenactment for police), but will be arguing that EtBr is not a poison.  My last post talked about EtBr as a carcinogen, and this is certainly what I’ve been taught my whole scientific career. It’s also extremely believable because a) the flat, planar rings make it a great intercalating agent, and b) I know it intercalates because I use EtBr routinely to visualize DNA.  That said, there are some caveats on why it might not be harmful to humans – first off there’s the issue of delivery – merely ingesting a small molecule like EtBr doesn’t guarantee it gets into your cells (in fact propidium iodide, chemically very similar to EtBr, only stains dead cells). Whether EtBr can effect your cells will  be dictated by the ability of EtBr to diffuse across the membrane (or taken up  via protein transporters) and from there it still has to get into the nucleus, all the while remaining soluble.

So what’s known about EtBr in living systems?  Well according to Wikipedia, its used to treat Trypanosomosis (a parasite) in cattle.  That suggests it’s not so bad, as the cattle presumably survive the treatment and (I assume) can be used as food sources for humans.  Well, then the question becomes how does the amount of EtBr that Liu gave to Cao compare to what cattle get?  Rosie Redfield at the RRResearch Blog, writing about common misconceptions about EtBr, did the the following calculation:

“The recommended dose for cattle is 1mg/kg body weight (up to 50mg/kg has been used in mice). Compare this with the 0.25 – 1 microgram/ml used in molecular biology (previous error corrected – thanks, anonymous commenter). A 50kg researcher would need to drink 50 liters of gel-staining solution to get even the non-toxic dose used in cattle.”

For reference, I use about 1 ul (10^-6 L)  of EtBr per experiment (diluted in 50 mL buffer) and have never seen more than 20 mL of EtBr just sitting around (having 50 L of it would be strange to say the least). So does Liu have a case?  I would say that evidence suggests that EtBr WILL cause DNA mutations, but EtBr probably DOESN’T get into living cells at even high concentrations.  What does that mean?  Cao will probably be okay – in fact the National Toxicology Program (NTP) report on EtBr has a puzzling description of it as “Bitter tasting dark red crystals from alcohol (Budavari, 1989)”, suggesting Cao is not the first person to digest EtBr. That still doesn’t change the fact that Liu did expose her to a potential carcinogen that has very stringent disposal guidelines in his lab, and I’m betting his lab is going to get fined for improper disposal at the very least.  It’s also important to note that the evidence in cattle suggests EtBr won’t harm humans, BUT there hasn’t been a carefully controlled study to show that.  Furthermore, there IS evidence that it can act as a mutagen once inside a cell, so given a choice between ingesting it and not ingesting it a reasonable person would choose the latter. I don’t know what this means legally, but it looks like the case has taken yet another unexpected turn, and this time I was able to learn something along the way.

When Postdocs Attack: The UC Poisioning Case and How EtBr Works

We interrupt our scheduled series of posts to bring you the case of the crazy postdoc (for those of you outside the scientific research community, postdoc = postdoctoral researcher, which is the status you attain after receiving your PhD but before anyone wants to pay you a real salary).  The San Francisco Chronicle first reported this case on Nov 12.  The basic summary of the case is that a UCSF postdoc (Benchun Liu) decided, for no apparent reason, to poison his labmate’s (Mei Cao’s) drinking water.  There does not appear to be a motive, but the case is still unfolding and there still seems to be a lot of confusion about the facts.  For instance there’s the mysterious identity of the poison.  Initially the Chronicle quoted UCSF police Capt. Paul Berlin:

“He told investigators that in both attempted poisonings, he had used a buffer agent designed to control acid in lab solutions, Berlin said.

The agent turns water blue, but Cao drank it anyway, Berlin said.”

Well, pretty much anyone with any chemistry or biology lab experience can make an educated guess that the blue pH buffer is most likely bromothymol blue. Bromothymol blue is used in a lot of loading buffers (think those agarose gels with bright blue bands you might see on CSI when someone is running a DNA test) – it’s yellow when pH is low, blue when pH is high.  At first glance bromothymol blue doesn’t seem like the worst thing to digest – after all its use as a pH indicator suggests it’s relatively inert (as in we add it to our solutions because we don’t expect it to do anything but inform us about our solution pH and who cares if your stomach turns yellow if it’s not doing anything bad?).  On the other hand we also use bromothymol blue at very low concentrations (i.e. 0.001%), and pretty much anything is toxic at high concentrations.   We can check the MSDS (material safety data sheets: the papers that contain all the basic information about the chemicals you work with in lab) to see what’s known about bromothymol blue. This MSDS suggests that ingestion of large doses can cause upset stomachs, this one suggests inducing vomiting immediately after any accidental ingestion.  My guess is that no one has really explored what happens when you swallow bromothymol blue because a) it’s a horrible drug candidate so no one’s done toxicity tests and b) no one has ever accidentally swallowed the contents of the very conspicuous, very blue, tube on their bench.  Still all the evidence would lead me to believe that, while it’s not good for you, swallowing bromothymol blue is also not the worst thing in the world.

However, this story took a completely different turn on Nov 25, when the Chronicle reported this:

“Liu is accused of trying to poison co-worker Mei Cao, 44, by putting a laboratory chemical, ethidium bromide, into a cup near her work station.”

Ethidium bromide (EtBr) is vastly different than bromothymol blue.  For one thing, it’s red not blue (although it dissolves colorlessly into large volumes).  It’s also a carcinogen (cancer causing chemical) and teratogen (causes birth defects).  In a lab the most common use of EtBr is to visualize DNA.  Remember those CSI gels?  Well, when you load DNA into them you don’t actually see it running through the gel. You need to put it under a UV light so that it will fluoresce.  But DNA by itself is not easily visualizable so we treat the gels with EtBr and visualize the DNA indirectly through EtBr.   We can do this because EtBr is an intercalating agent – that means it inserts periodically into DNA strands.  That’s great for looking at DNA on a gel.  It kind of sucks when it’s intercalating in the DNA in your cell.  These insertions will cause a lot of DNA damage (breaks, mutations, etc) that can cause cancer or cell death.  In fact when we handle EtBr in the lab we have special disposal for anything that touches it (gloves, pipette tips, gels), because we don’t want it sitting around in landfills.

As an aside and general note of interest, a lot of chemotherapeutic agents are also intercalating agents.  Take a look at the structure of EtBr and a doxorubicin, an anticancer drug:

EtBr

Doxorubicin

See those rings in the middle of both structures?  Those will give the molecules a nice, flat, shape that allow them to slip into a pocket that is inherent in the famous double helix structure of DNA, causing it to unwind.  When dealing with cancer this is an acceptable but risky treatment, because the cancer cells have a greater chance to be effected (since they’re the cells undergoing rapid division and therefore are making lots of DNA).  However, any cell is susceptible to this mechanism of action, so in cases where there’s no cancer, all you’re doing is hurting normal cells and possibly introducing cancer causing mutations (I may give cancer treatments their own post in the future as there’s plenty to say about them, independent of this case).  But, it’s an interesting point that most cancer drugs are also carcinogens.

Anyway, back to the crazy postdoc case: EtBr is considerably worse than bromothymol blue, and looks nothing like it, making me wonder if it’s confusion by the reporter about the science, or if the postdoc is just that crazy and keeps making up different stories (or used both!). In an even weirder twist on the case, the postdoc has been released while awaiting trial – that seems odd to me in an attempted murder case.  Anyway, the whole case is extremely crazy, and we’ll be following it here as updates come.  So far, I think the best line (pointed out to me by a friend) comes from the most recommended comment on the sfgate story:

“It’s the urology department, so his motive is obvious. He was pissed off.”

Update 11/30/08: Liu has pleaded not guilty, claiming EtBr is not a poison.  He might have a case.  See my update post on why EtBr, even with its intercalating properties, is not as bad as you might believe.

Inner Life of the Cell Explained, Part I – Rolling

So for these next series of posts I’m going to attempt to explain what’s happening in the video.  I should warn you I’m not exactly an expert in inflammation, so if you disagree with an explanation please make yourself known in the comments.    I’m going to use time references from this version of the video, and I’ll split the series into four posts corresponding to the four steps the narrator of the explanation video mentioned: Rolling, Adhesion, Activation, and Transendothelial Migration.

First up is Rolling, but before we get to that, let’s talk about what this video is about.  Although parts of the video are showing general features and processes of any cell, the story here focuses on a process known as leukocyte extravasation.  What does that mean?  Well a leukocyte is a white blood cell, essentially the foot soldier of your immune system.   Extravasation  refers to fluid leaking out of a container.  Leukocyte extravasation is literally white blood cells leaking out of your blood vessels so that they can reach damaged tissue.  Essentially your circulatory system is a highway, allowing your white blood cells to travel around your whole body.  Extravasation is analagous to taking an exit, which is necessarry when you need your immune cells to reach damaged tissue.  This whole video is essentially about the mechanics of taking an exit.

While most of the video does talk about taking an exit, first we have to see how this leukocyte car drives along the circulatory system.  This is the rolling step.  Let’s go frame-by-frame:

 

 

0:10-0:14: We’re inside a blood vessel (essentially standing in the middle of the highway).  Note that we’re not actually inside the cell yet.  A bunch of red blood cells are rushing past us at a fast rate, driven by blood flow.  A few white blood cells creep along the side of the vessel.

0:14-0:16: Now we zoom onto one of those white blood cells and look at the interface between it and the endothelial cell that makes up the blood vessel.  Notice all the bumps on the cell surfaces – those will be key in understanding why the cells “stick” to one another.

0:17-0:22:  An even closer look at this interface.  We’ve zoomed in on the surfaces and can now see individual proteins (those bumps we saw before) making contacts. The camera pans back to let us see that the surfaces of both cells are littered with many of these proteins each making contacts.

Here’s what the explanation video has to say about this portion:

While red blood cells are carried away at high velocity by strong blood flow , leukocytes roll slowly on endothelial cells.  P-selectins on endothelial cells interact with PSGL1 a glycoprotein on leukocyte microvillae.  Leukocytes pushed by the blood flow adhere and roll on endothelial cells because existing interactions are broken while new ones are formed. These interactions are possible because the extended extracellular domains of both proteins emerge from the extracellular matrix which covers the surface of both cell types.

The first sentence should be self explanatory as long as you remember white blood cell = leukocyte and blood vessel cell = endothelial cell.  So what exactly is a P-selectin and what’s PSGL1?  They’re both proteins, P-selectin (yellow chain) is found on the surface of the endothelial cell and PSGL1 (purple chain) is on the surface of the leukocyte.  These two proteins have a certain stickiness with each other (think opposite parts of a velcro band) that lets the leukocyte adhere to the vessel wall.  The blood flow then gently pushes the white blood cell along the surface of the vessel causing the PSGL1 molecules to disconnect and then reconnect to downstream P-selectins.  This also provides a possible reason why the red blood cells don’t also stick to the walls.  Assuming they don’t express PSGL1 (something I don’t know for certain but seems likely), they don’t have any stickiness to the blood vessel.

Ok back to the frame-by-frame:

0:23-0:28:  Now we’re looking at an individual cell – we’re probably supposed to be in the white blood cell but the endothelial cell would have the same basic foundation.  That foundation is the cell membrane which is a lipid bilayer.  Lipids are a class of molecules which stack together to form the membrane which is vizualized here as a sea on which things are floating. The rafts floating on this sea are actually called “lipid rafts” (although some biologists don’t believe these things exist, or that there function is overstated) and they’re ferrying around important receptors and such.  Here’s the explanation video:

The outer leaflet of the lipid bilayer is enriched in sphingolipids and phosphatidylcholine.  Sphingolipid-rich rafts raised above the rest of the leaflet recruit specific membrane proteins. Rafts rigidity is caused by the tight packing of cholesterol molecules against the straight sphingolipids hydrocarbon chains.  Outside the rafts, kinks in unsaturated hydrocarbon chains and lower cholesterol concentration result in increased fluidity.

Pretty much what I said above. Sphingolipids and phosphatidylcholine are both types of lipids.  The membrane varies in rigidity and flexibility by the presence of cholesterol (unevenly distributed throughout the membrane) and some of these lipids have hydrocarbon tails that are not saturated (and therefore more flexible).

0:29-0:36: Ok now back to our story which is an inflammation story.  Our endothelial cell is at an inflammation site so it produces a signal to say “Hey there’s inflammation over here”.  That’s visualized by the orange and green structures at the top.  These proteins from the endothelial cell recognize a receptor on the leukocyte (purple thing on the bottom).  Activating the purple receptor triggers a whole set of pathways within the leukocyte to start the inflammation response (so we leave the cell-cell interface and finally go within the leukocyte).  The explanation video says this:

At sites of inflammation, secreted chemokines, bound to heparin sulfate proteoglycan on endothelial cells are presented to leukocyte 7-transmembrane receptors.  The binding stimulates leukocytes and triggers an intracellular cascade of signaling reactions.

The heparin sulfate proteoglycan is just another protein (made by the endothelial cell) that with the the chemokine activates the surface receptor on the leukocyte (the 7 in 7-transmembrane refers to the number of loops in the receptor).  Activating a receptor in this manner triggers a signaling cascade within the leukocyte.  But that’ll be covered in the activation post.  Next up is the adhesion post – we’ve touched on the basics here, but in the next post we’ll look within the cell and see how it’s organized and ultimately how we’re going to have to change that organization to get the leukocyte out of the circulatory system.

Update 5/25/09: Part II is up!

More “Inner Life of the Cell” Explanations

I’ve already posted twice on this widely seen video, but I haven’t really had a chance to go into explaining it in depth.  The makers of the video already provided an explanation video, and Wayne at the Niches blog did some frame-by-frame analysis, but, judging from the google searches to my blog, people are hoping for a more detailed explanation.  I’ve produced a transcript of the explanation video which I’ll take section by section in the next series of posts.  I’ve transcribed this myself so I can’t attest that it’s 100% accurate – if you notice any errors, let me know.  The basic summary is that this video provides a glimpse of one specific cellular process: the activation of a white blood cell in an inflammation response.  The not-so-basic summary will start with the next post.

Update 11/29/08: Part I- Rolling is up

Update 05/25/09: Part II – “Adhesion” & the Organization of the Cell is up.

While red blood cells are carried away at high velocity by strong blood flow , leukocytes roll slowly on endothelial cells.  P-selectins on endothelial cells interact with PSGL1 a glycoprotein on leukocyte microvillae.  Leukocytes pushed by the blood flow adhere and roll on endothelial cells because existing interactions are broken while new ones are formed.

These interactions are possible because the extended extracellular domains of both proteins emerge from the extracellular matrix which covers the surface of both cell types.The outer leaflet of the lipid bilayer is enriched in sphingolipids and phosphatidylcholine.  Sphingolipid-rich rafts raised above the rest of the leaflet recruit specific membrane proteins. Rafts rigidity is caused by the tight packing of cholesterol molecules against the straight sphingolipids hydrocarbon chains.  Outside the rafts, kinks in unsaturated hydrocarbon chains and lower cholesterol concentration result in increased fluidity. At sites of inflammation, secreted chemokines, bound to heparin-sulfate proteoglycan on endothelial cells are presented to leukocyte 7 transmembrane receptors.  The binding stimulates leukocytes and triggers an intracellular cascade of signaling reactions.

The inner leaflet of the bilayer has a very different composition than that of the outer leaflet. While some proteins traverse the membrane, others are either anchored into the inner leaflet by covalently attached fatty acid chains, or are recruited through non covalent interactions with membrane proteins. The membrane bound protein complexes are critical for transmission of signals across the plasma membrane.

Beneath the lipid bilayer, spectrin tetramers arranged into a hexagonal network are anchored by membrane proteins.  This network forms the membrane skeleton that contributes to membrane stability and membrane protein distribution.  The cytoskeleton is comprised of networks of filamentous proteins that are responsible for the special organization of cytosolic components.   Inside microvillae, actin filaments form tight parallel  bundles which are stabilized by cross-linking proteins.  While (deeper?) in the cystol the actin network adopts a gel-like structure, stabilized by a variety of actin binding proteins.  Filaments, capped at their minus ends by a protein complex, grow away from the plasma membrane by the addition of actin monomers to their plus end. The actin network is a very dynamic structure with a continuous directional polymerization and disassembly.  Severing proteins induce kinks in the filament and lead to short fragments that rapidly depolymerize or give rise to new filaments.

The cytoskeleton includes a network of microtubules created by the lateral association of protofilaments formed by the polymerization of tubulin dimers.  While the plus ends of some microtubules extend toward the plasma membrane, proteins stabilize the curved conformation of protofilaments from other microtubules, causing their rapid plus end depolymerization.  Microtubules provide tracks along which membrane bound vesicles travel to and from the plasma membrane.  The directional movement of these cargo vesicles is due to a family of motor proteins linking vesicles and microtubules.   Membrane bound organelles like mitochondria are loosely trapped by the cytoskeleton.  Mitochondria change shape continuously and their orientation is partly dictated by their interaction with microtubules.  All the microtubules originate from the centrosome, a discrete fibrous structure containing two orthogonal centrioles and located near the cell nucleus.

Pores in the nuclear envelope allow the import of particles containing mRNA and proteins into the cytosol. Here free ribosomes translate the mRNA molecules into proteins.  Some of these proteins will reside in the cytosol.  Others will associate with specialized cytosolic proteins and be imported into mitochondria or other organelles.  The synthesis of cell secreted and integral membrane proteins is initiated by free ribosomes which then dock to protein translocators at the surface of the endoplasmic reticulum.  Nascent proteins pass through an aqueous pore in the translocator.  Cell secreted proteins accumulate in the lumen of the endoplasmic reticulum, while integral membrane proteins become embedded in the endoplasmic reticulum membrane.

Proteins are transported from the endoplasmic reticulum to the Golgi apparatus by vesicles traveling along the microtubules.  Protein glycosylation initiated in the endoplasmic reticulum is completed inside the lumen of the Golgi apparatus.  Fully glycoslated proteins are transported from the Golgi apparatus to the plasma membrane.  When a vesicle fuses with the plasma membrane, proteins contained in the vesicle’s lumen are secreted and proteins embedded in the vesicle’s membrane diffuse in the cell membrane.

At sites of inflammation, chemokines secreted by endothelial cells bind to the extracellular domains of G protein coupled membrane receptors.  This binding causes a conformational change in the cytosolic portion of the receptor and the consequent activation of a subunit of the G protein.  The activation of the G protein subunit triggers a cascade of protein activation, which in turn leads to the activation and clustering of integrins inside lipid rafts.  A dramatic conformational change occurs in the extracellular domain of the activated integrins.  This now allows for their interactions with I-cam proteins, displayed at the surface of endothelial cells.  These strong interactions immobilize the rolling leukocyte at the site of inflammation.  Additional signaling events cause a profound reorganization of the cytoskeleton, resulting in the spreading of one edge of the leukocyte.  The leading edge of the leukocyte inserts itself between endothelial cells and the leukocyte migrates through the blood vessel wall into the inflamed tissue.  Rolling, activation, adhesion, and transendothelial migration are the four steps of a process called leukocyte extravasation

Making “The Inner Life of the Cell”

Even while I’ve neglected this blog, my previous post on The Inner Life of the Cell remains quite popular. David Bolinksy, one of the creators of the video, gave a TED talk on making the movie. His talk is not particularly interesting from a scientific perspective, but one thing that he emphasizes that deserves restatement is the view of the cell as a series of micromachines.  A lot of current biologocial research focuses on understanding how proteins, nucleic acids, small molecules, and all the other contents of a cell,  assemble together to form higher order structures that perform complex reactions.  One of the challenges that has arisen in modern biology is taking large scale datasets that we’ve generated (i.e. the human genome, gene expresssion studies, etc), and using them to understand how all the different parts of the cell talk to one another.  This global approach to biology is relatively young, so understanding, visualizing, and analyzing these data is a huge, new challenge. Videos like this one, while perhaps not the most detailed representations of biology, are helpful in communicating this global view of the cell.  Plus it looks pretty cool.

Human Growth Hormone, UCSF, Genentech and a Whole Lotta Money

So the Mitchell Report is out, casting steroids and human growth hormone (HGH) into day-to-day discourse. Although it would be interesting to talk about what HGH does and how it can enhance athletic performance, I’m willing to bet there are others out there who are already doing that. Instead I thought I’d talk a little bit about another dark side of HGH that you probably haven’t heard about – the patent dispute between the University of California, San Francisco (UCSF), and Genentech, Inc.

Before I begin, I should disclose that I am a graduate student at UCSF and have heard various versions of this story in different amounts of detail from faculty and other potentially biased sources, but I have attempted to research the issue as best I can, and everything here is based entirely on reporting in Science, The Washington Post, and publications of equivalent believability. This is, to the best of my knowledge, the history of HGH.

Growth hormone is essentially a protein which acts as a signaling molecule – HGH is the human version of this hormone. When the protein binds its receptor, signaling pathways are activated that stimulate the growth of the cell (this is, of course, a vast oversimplification, but should serve for the purposes of this story) Because of its ability to trigger these pathways, growth hormone has many therapeutic uses including treatment of multiple sclerosis.

Recall from my earlier posts that the central dogma of biology is that protein is encoded by RNA which is in turn encoded by DNA. Thus there is a DNA sequence which tells a cell to build growth hormone. One common technique in molecular biology is to “copy” a specific piece of DNA (in this case the DNA coding for HGH) and place it into bacteria. The rationale for this is that you can grow lots and lots of bacteria and then extract and purify the protein, cheaply and quickly.

In 1977, Dr. Peter Seeburg, then in a postdoctoral position in Dr. Howard Goodman’s lab at UCSF successfully copied the DNA that coded for HGH and UCSF was awarded the patent for the gene. Incidentally, UCSF’s version of the copied DNA included the DNA sequence that encodes for HGH but with an additional 48 nucleotides (i.e. 48 more characters) added on at the end (these nucleotides are present in the human genome, but don’t actually serve any purpose in producing the protein) – an unimportant scientific distinction, but one which would be important legally years later.

In 1987, Seeburg left UCSF for Genentech where his job would focus on expressing that DNA in bacteria, so that Genentech could grow lots and lots of the bacteria, which would then make lots and lots of HGH, which Genentech would then use to make lots and lots of money. In fact Genentech eventually did just that, producing the drug Protropin which went on to produce over $2 billion in sales.

In 1990, UCSF sued Genentech for $400 million for infringing their HGH patent. Genentech’s response was that they had developed their HGH from DNA that was independent from the original UCSF DNA. This could have actually been entirely possible, except that, at trial, Seeburg would testify that he actually HAD copied the UCSF DNA.

When Seeburg left UCSF, Robert Swanson, then president of Genentech, sent a letter to Seeburg’s old boss, Dr. Goodman asking for the DNA that Seeburg had worked on. Goodman apparently refused so Seeburg, fresh out of his postdoc, visited his old lab on midnight of New Year’s Eve, and took copies of the DNA with him. Seeburg justified this “midnight raid” claiming that it was customary for scientists to take work they had produced with them to their next positions (it is actually quite common, if not quite proper, to do so within academia), and that he had gone at the late hour merely to avoid Dr. Goodman with whom he was no longer on friendly terms. UC later found out about this incident and in 1980 settled for $2 million with Genentech, but still retained full patent rights over their DNA construct.

Genentech, not wishing to use any of UC’s intellectual property, decided to duplicate the old work and isolate the DNA for HGH themselves and then introduce it into bacteria. Except that they couldn’t get it to work. So Seeburg, with, allegedly, the knowledge of his Genentech coworker Dr. David Goeddel, decided to just use the UC construct to make their bacteria. They even published a Nature paper with results from their supposedly novel construct. At trial, Seeburg, no longer with Genentech and testifying on behalf of UC, stated that much of the data from the Nature paper with all new “UC free construct” was fudged. Goeddel denied knowing that Seeburg had cheated, and Seeburg’s coauthors on the Nature paper denied Seeburg’s account of falsified data (although the “midnight raid” and lifting of UC property was corroborated).

In the legal battle, Genentech argued that because the UC sequence contained those extra base pairs, the Genentech DNA, which contained only the HGH DNA, was substantially different, and an entirely new invention (many patent lawyers actually thought Genentech had a decent case, and that Seeburg’s testimony, although indicative of intent to copy, had no bearing on the final legal issue of whether the Genentech construct was sufficiently different). In 1999, a nine member jury ruled 8 to 1 in favor of UC, however the split decision saved Genentech from damages that could have been as high as $1.2 billion. Rather than risk losing on appeal, and with UC having already invested $20 million in legal fees, the two sides settled for $200 million. $50 million went to building my home away from home, Genentech Hall, – the first building at the new UCSF Mission Bay campus:

Picture taken from (http://www.pbase.com/klaorman/image/11306539)

$30 million went to the UC general fund, $35 million went to research at UCSF and the remaining $85 million was split amongst the original inventors and collaborators, including Dr. Seedburg, whose individual share ended up as $17 million.

I’ve glossed over a lot of the science and legal issues involved here, partly because I’m no lawyer and I don’t know enough to say what really happened here, but it’s clear that HGH has had a colorful history that continues today.

Folding 2.0?

I got to listen to a talk today given by Dave Baker, one of the big names in the protein folding field, and I was not disappointed. There’s a lot of potential material from today’s talk that would be good subject matter for this blog, and I’ll no doubt post more about folding in the future, but the highlight of the talk was FoldIt!

Before I get into too much detail about FoldIt specifically there are a couple things about protein folding I glossed over in the last post. In computer modeling of protein folding, there are usually two major problem areas – sampling and scoring.

For a given protein, we know the fundamental building blocks (amino acids) that compose it and the order in which they are connected – the whole problem of protein folding is how that linear chain “curls up” (i.e. folds) in 3D space. If we were to take every potential position and assign it a score (sometimes called the energy score) we could build a gigantic scoring landscape. Imagine the Grand Canyon: every potential spot you could stand represents a particular conformation (i.e amino acid A is a certain distance and certain angle relative to B) and your elevation represents how good that conformation’s score is (let’s say the lower you are the better). Any step you took in one direction would represent slightly changing the conformation (maybe you pull a certain bond a little further apart) and if you had to walk uphill to that new position your changes would be bad, while walking downhill would be good. A good folding algorithm will try and walk down the canyon as far as possible until it finds a position where every possible next step would take it uphill (once again I’m oversimplyfing for clarity, but this is the general idea).

What are sampling and scoring in this analogy? Scoring is basically your ability to recapitulate the Grand Canyon. That is, if you built a computer model of the canyon, your map is going to be of limited resolution (depending on the manner in which it was built) and will not quite recapitulate the real Grand Canyon. I’m not really going to talk much about scoring this post so even if it doesn’t make sense, read on. Sampling is easier to think about – it’s basically how much of the Grand Canyon you are able to visit. If you walk across the whole canyon you can be sure that your lowest point is the actual lowest point in the entire canyon since you walked across the whole damn canyon. Had you walked only 50% of it, you may have found a place in the canyon that’s pretty low , but that unwalked portion contains an even lower point. Sampling is a typically computational intense process – as fast as computers are today, it takes a long time calculate the score for every position. Furthermore you can always take smaller steps between points (think of someone with a large stride as compared to someone with a small stride…the large strider may “stride” past and miss a pathway the small strider will see), so there’s really an infinite amount of points to sample.

Now one way to approach the problem of sampling is by increasing computing power. The most famous example of this is SETI@home, or Folding@home mentioned yesterday. There’s a similar program for called rosetta@home, which is developed by the Baker lab. “rosetta” refers to the program which makes the calculation to assign a score for a given conformation. Simply put, rosetta@home is a screen saver which uses sophisticated algorithms to move around amino acid side chains and try to pack the 3D protein structure into its lowest energy (i.e. most stable) state. By installing rosetta@home you donate your computer’s idle time to performing these calculations which are then communicated back to the Baker lab.

This all well and good, but the Baker lab went even one step further and developed FoldIt! – a computer game similar to rosetta@home. I like to think of FoldIt as the Web 2.0 approach to protein folding (Web 2.0 is a poorly defined keyword first introduced by Tim O’Reilly but which generally refers to harnessing the power of the collective to accomplish tasks – think of wikipedia, digg, or youtube). FoldIt! is a computer game that allows you to change the 3d structure of the protein by moving parts of it around, while rosetta scores your conformation on the fly. Basically instead of having the computer decide which path to take down the canyon, you’re able to run haphazardly around and try to find the lowest point on your own. The great thing is you don’t need to understand anything about protein folding, all you need to do is understand that you need to move thing around and watch your score go up. You’ll quickly realize certain obvious things – having parts of the protein overlap is bad (steric clash in scientific terms) and fitting things into empty space is good, but all you honestly need to do is look at the score and try to make it go up by whatever means necessary. Your scores are submitted back to the Baker lab website and compared against all other players’ scores. Whereas rosetta@home uses the idle time of thousands of computers, FoldIt! uses the idle time of thousands of people and their computers, making it potentially even more powerful.

In an email to students and faculty before his talk, Dave described FoldIt like this:

“We are developing a multiplayer interactive protein folding and design game for both education and research-our hope is that large groups of people interacting with computers and with each other through the multiplayer game may be able to solve hard optimization problems that neither computers nor people can solve alone. Please help us to test and improve the game! “

If we go back to the canyon analogy, rosetta@home is like having thousands of people walking the canyon so you can accomplish the task faster, but the manner in which they walk is still pretty systematic and similar. That is if there are valleys that look like a lot like the bottom of the canyon but aren’t actually (local minima), the similar nature of everyone’s walking manner will cause people to still end up there. In FoldIt! people are running all over the canyon – some are flapping their arms, some are walking backwards – a lot of energy and effort is wasted, but the naivety of the approach frees it from harmful biases and allows hidden paths that were never known before to be discovered. It’s entirely possible that analysis of human generated structures will reveal a few key rules that algorithms were missing entirely – these rules could be folded into new algorithms and would hopefully allow rosetta to generate a better model in the same amount of sampling.

I think FoldIt! is a great idea, and it’ll be interesting to see if it leads to any new ways to thinking about the folding problem. I think this sort of approach is good for areas where we’re still fundamentally unsure of the best ways to approach the problem – the utility might be more limited in fields where we have a pretty good idea of what we’re doing.

I should note that Dave did not present this as a 2.0 approach and was much more bullish on its education prospects (although he was hopeful for its research utility), and that this “naive 2.0 approach” is my own interpretation of his project, but I think the idea itself is something that might have real application in other fields.

To download FoldIt! go here, the game is not yet live (they’re aiming for early next year), but there are playable puzzles and a real time leaderboard.

What exactly is Folding@home doing? The Protein Folding Problem

As this blog reflects an intersection of my interests in technology and general science, I figured an ideal topic to start off with would be the Protein Folding Problem.

You’ve probably heard about the protein folding problem, although you may not have realized it. The most common references online to protein folding usually involve Folding@home (a distributed computing approach to the problem similar to SETI@home). Usually these references are entitled “Use your PS3 to cure cancer” or some other such overstatement. While there are no doubt real implications to understanding protein folding (including possible cancer therapeutics), such overstatements aren’t helpful in understanding exactly what you’re making your PS3 do.

So what is the protein folding problem? In the simplest terms it’s the manner in which a protein adopts its 3D conformation. If you can remember back to basic high school biology you might recall that the central dogma of biology is this:

DNA->RNA->protein

DNA is the set of instructions for making everything in a cell. RNA is an intermediate, essentially a specific subset of the instructions in your DNA that is then assembled into a protein (the biologists amongst you may take exception from this oversimplification, but for now we’ll stick with it) . You may know of proteins as they pertain to your diet, but in actuality proteins are much more- they are the molecular workhorses of the cell. Most of the chemistry and processes carried out in a cell are done by proteins. These include things like breaking down your food into energy, or recognizing viral particles. In the cell, proteins get stuff done.

Ok so what’s this whole folding business? Well proteins are composed of 20 basic building blocks called amino acids. String these amino acids together in a linear chain and you get a protein. In biology, these amino acids are represented by single letters; for instance DYKDDDDK represents an 8 amino acid protein starting with D and ending with K. Because amino acids are (essentially) the only components of proteins every protein can be represented by sequence of letters. Some proteins are very large (hundreds of letters) while others are small (like the 8 letter protein above) – given that there are 20 naturally occurring amino acid possibilities at each point and the unbounded size of the sequence there is an infinte number of potential protein sequences.

Now of course the human genome is finite, and you might recall we’ve gone ahead and sequenced the whole thing - and we’ve gotten pretty good at determining what parts of the DNA are actually turned into protein. So essentially we know what almost all the proteins in your cells are – at least on the level of these string-like representations. However your proteins do stuff based on their actual 3D structure. That is, when I write DYKDDDDK that doesn’t mean there’s a happy trail of letters in your body wandering about the cell and carrying out their business. Rather each letter represents a specific chemical structure. D for instance is Aspartic acid which looks like this:

Y is Tyrosine which looks like this:

You can string them together and see what DY looks like in 2D but of course the world is actually in 3D, and each of those oxygens, carbons, etc occupy a position in space relative to one another.

And therein lies the protein folding problem. How do you translate an amino acid sequence (i.e. DYDDDDK) into a three dimensional structure? Well it’s not an easy problem, as there aren’t a clear set of rules – the position of each part of each amino acid can be influenced by a whole host of factors including the amino acids neighboring it, the presence of water, and many other things. You can start to see that the problem begins to get very complex, much too complex for a mere human to think about.

Enter the computers. Looking at 3D structures of proteins that people have determined experimentally, there’s no clear set of rules (although some trends become evident) – however this is science and there are theoretically some set of axioms we started with (yes, even in biology). Things like the electric charge of these amino acids (things that are positive will be attracted to things that are negative, neutral things will want to pack in the interior and hide from charged water molecules) are known and can be used to calculate energy maps for specific conformations. You can do multiple iterations and find the most stable (i.e. lowest energy) conformation and use it as your predicted protein structure. This is essentially what Folding@home is doing (at least this is one approach to the problem, and the one I believe that Folding@home is taking). You can even do this for known structures and see how good your algorithm is at returning the known structure.

So why does anyone care what a protein looks like in 3D? Well the 3D structure is important for understanding the function of the protein and that’s a very important thing to understand. If you protein is a drug target (say an HIV protein) knowing its 3D structure could help you design a drug that binds a specific structure on the protein and thereby inhibit its function (i.e. viral replication). A lot of diseases are the results of misfolded proteins (cystic fibrosis, Mad Cow Disease) so understanding the folding process itself is an interesting thing.

So there you have it a basic introduction to the protein folding problem and one computational approach to it. There are other approaches as well, and I’ve definitely oversimplified things. If you’re curious I suggest the Folding@home website, wikipedia, or a simple google search to explore further.