Brilliant structural work on the Arp2/3 complex with actin filaments and why it makes me depressed

The Arp2/3 complex of 5 proteins forms side branches on existing actin filaments.  The following paper shows its beautiful structure along with movies.  Have a look — it’s open access. https://www.pnas.org/doi/10.1073/pnas.2202723119.

Why should it make me depressed? Because I could spend the next week studying all the ins and outs of the structure and how it works without looking at anything else.  Similar cryoEM studies of other multiprotein machines are coming out which will take similar amounts of time.  Understanding how single enzymes work is much simpler, although similarly elegant — see Cozzarelli’s early work on topoisomerase.

So I’m depressed because I’ll never understand them to the depth I understand enzymes, DNA, RNA etc. etc.

Also the complexity and elegance of these machines brings back my old worries about how they could possibly have arisen simply by chance with selection acting on them.  So I plan to republish a series of old posts about the improbability of our existence, and the possibility of a creator, which was enough to me get thrown off Nature Chemistry as a blogger.

Enough whining.

Here is why the Arp2/3 complex is interesting.  Actin filaments are long (1,000 – 20,000 Angstroms and thin (70 Angstroms).  It you want to move a cell forward by having them grow toward its leading edge, growing actin filaments would puncture the membrane like a bunch of needles, hence the need for side branches, making actin filaments a brush-like mesh which could push the membrane forward as it grows.

The Arp2/3 complex has a molecular mass of 225 kiloDaltons, or probably 2,250 amino acids or 16 thousand atoms.

Arp2 stands for actin related protein 2, something quite similar to the normal actin monomer so it can sneak into the filament. So can Arp3.  The other 5 proteins grab actin monomers and start them polymerizing as a branch.

But even this isn’t enough, as Arp2/3 is intrinsically inactive and multiple classes of nucleation promoting factors (NPFs) are needed to stimulate it.  One such NPF family is the WASP proteins (for Wiskott Aldrich Syndrome Protein) mutations of which cause the syndrome characterized by hereditary thrombocytopenia, eczema and frequent infections.

The paper’s pictures do not include WASP, just the 7 proteins of the complex snuggling up to an actin filament.

In the complex the Arps are in a twisted conformation, in which they resemble actin monomers rather than filamentous actin subunits which have a flattened conformation.  After activation arp2 and arp3 mimic the arrangement of two consecutive subunits along the short pitch helical axis of an actin filament and each arp transitions from a twisted (monomerLike) to a flattened (filamentLike) conformation.

So look at the pictures and the movies and enjoy the elegance of the work of the Blind Watchmaker (if such a thing exists).

The twists and turns of topoisomerase (pun intended)

It is very sad that my late friend Nick Cozzarelli isn’t around to enjoy the latest exploits of the enzyme class he did so much great work on — the topoisomerases. For a social note about him see the end of the post.

We tend to be quite glib about just what goes on inside a nucleus when DNA is opened up and transcribed into mRNA by RNA polymerase II (Pol II). We think of DNA has a linear sequence of 4 different elements (which it is) and stop there. But DNA is a double helix, and the two strands of the helix wind around each other every 10 elements (nucleotides), meaning that within the confines of our nuclei this happens 320,000,000 times.

I’ve written a series of six posts on what we would see if our nuclei were enlarged  by a factor of 100,000 (which is the amount of compaction our DNA must undergo to fit inside the 10 micron (10 millionths of a meter) in diameter nucleus (since if fully extended our DNA would be 1 meter long. So if you compacted the distance from New York to Seattle (2840 miles or 14,995,200 feet) down by this factor you’d get a sphere 150 feet in diameter or half the length of a football (US) field. Now imagine blowing up the diameter and length of the DNA helix by 100,000 and you’d get something looking like a 2,840 mil long strand of linguini which twists on itself  320,000,000 times. The two strands are 3/8th of an inch thick. They twist around each other every 9/16ths of an inch.

For the gory details start at https://luysii.wordpress.com/2010/03/22/the-cell-nucleus-and-its-dna-on-a-human-scale-i/ and follow the links.

Well, we know that for DNA to be copied into mRNA it must be untwisted, the strands separated so RNA polymerase II (Pol II) can get to it.  Pol II is enormous — a mass of 500 kiloDaltons and 7 times thicker at 140 Angstroms than the DNA helix of 20 Angstrom thickness.

Consider the fos gene (which we’ll be talking about later). It contains 380 amino acids (meaning that the gene contains at least 1140 nucleotides ). The actual gene is longer because of introns (3,461 nucleotides), which means that the gene contains 346 complete turns of the double helix, all of which must be unwound to transcribe it into mRNA.

So it’s time for an experiment. Get about 3 feet of cord roughly 3/8 of an inch thick. Tie the ends together, loop one end around a hook in your closet, put a pencil in the other end and rotate it about 100 times (or until you get tired). Keeping everything the same, have a friend put another pencil between the two strands in the middle, separating them. Now pull on the strands to make the separation wider and move the middle pencil toward one end. In the direction of motion the stands will coil even tighter (supercoiling) and behind they’ll unwind.

This should make it harder for Pol II to do its work (or for enzymes which copy DNA to more DNA). This is where the various topoisomerase come in. They cut DNA allowing supercoils to unwind. They remain attached to the DNA they cut so that the DNA can be put back together. There are basically two classes of topoisomerase — Type I topoisomerase cuts one strand, leaving the other intact, type II cuts both.

Who would have thought that type II topoisomerase would be involved in the day to day function of our brain.

Neurons are extended things, with information flowing from dendrites on one side of the cell body to much longer axons on the other. The flow involves depolarization of the cell body as impulses travel toward the axon. We know that certain genes are turned on by this activity (e.g. the DNA coding for the protein is transcribed into mRNA which is translated into protein by the ribosome). They are called activity dependent genes.

This is where [ Cell vol. 1496 – 1498, 1592 – 1605 ’15 ] comes in. Prior to neuronal activity, when activity dependent genes are expressed at low levels, the genes still show the hallmarks of highly expressed genes (e.g. binding by transcription factors and RNA polymerase II, Histone H3 trimethylation of lysine #4 {H3K4Me3 } at promoters).

This work shows that such genes are highly negatively supercoiled (see above) preventing RNA polymerase II (Pol II) from extending into the gene body. On depolarization of the cell body in some way Topoisomerase IIB is activated, leading to double strand breaks (dsbs) within promoters allowing the DNA to unwind and Pol II to productively elongate through gene bodies.

There is evidence that neuronal stimulation leads to dsbs ( Nature NeuroScience vol. 16 pp. 613 – 621 ’13 ) throughout the transcription of immediate early genes (e.g. genes turned on by neural activity). The evidence is that there is phosphorylation of serine #139 on histone variant H2AX (gammaH2AX) which is a chromatin mark deposited on adjacent histones by the DNA damage response pathway immediately after DSBs are found.

Etoposide (a topoisomerase inhibitor) traps the enzyme in a state where it remains bound to the DNA of the dsb. On etoposide Rx, there is an increase in activity dependent genes (Fos, FosB, Npas4). Inhibition of topiosomerase IIB (the most prevalent topoisomerase in neurons) by RNA interference (RNAi) leads to blunted activity dependent induction of these genes. This implies that DNA cutting by topoisomerase IIB is required for gene activation in response to neuronal activity.  Other evidence is that knocking down topoisomerase  using RNA interference (RNAi) stops activity dependent gene transcription.

Further supporting this idea, the authors induced dsbs at promoters of activity dependent genes (Fos, fosB, Npas4) using the CRISPR system. A significant increase in transcription was found when the Fos promoter was targeted.

I frankly find this incredible. Double strand breaks are considered bad things for good reason and the cell mounts huge redundant machines to repair them, yet apparently neurons, the longest lived cells in our bodies are doing this day in and day out. The work is so fantastic that it needs to be replicated.

Social Note: Nick Cozzarelli is one of the reasons Princeton was such a great institution back in the 50s (and hopefully still is). Nick’s father was an immigrant shoemaker living in Jersey City, N. J. Princeton recognized his talent, took him in, allowing him to work his way through on scholarship, waiting tables in commons, etc. etc. He obtained a PhD in biochemistry from Harvard and later became a prof at Berkeley, where he edited the Proceedings of the National Academy of Sciences USA for 10 years. He passed away far too soon of Burkitt’s lymphoma in his late 60s. We were friends as undergraduates and in grad school.

I can only wonder what Nick would say about the latest twists of the topoisomerase story