Tag Archives: Carl Sagan

Cytocapsular tubes

“Extraordinary claims require extraordinary evidence”  Carl Sagan.  That goes in spades for a recent PNAS article vol. 115 pp. E1137 – E1146 2018 — http://www.pnas.org/content/115/6/E1137.

Start by looking at the following 3 videos

pnas.1717910115.sm01-3.avi

pnas.1717910115.sm02-2.avi

pnas.1717910115.sm03-2.avi

Unfortunately, to get to them, it appears that you must be a PNAS subscriber, so I’ll tell you what they show.

First, some background. Cell culture usually involves putting them into a Petri dish, something inherently two dimensional, but most cells in our body live in a 3 dimensional environment. So this work implanted Human Mammary Epithelial Cells (HMECs) into a 3 dimensional matrix of Matrigel (an extracellular matrix surrogate secreted by mouse sarcoma cells, containing, laminin, nidogen, collagen and heparan sulfate proteoglycans) Matrigel also contains the growth factors Transforming Growth Factor Beta (TGFbeta) and Epidermal Growth Factor (EGF), both of which prevent differentiation and promote proliferation. The 3d Matrigel matrix is softer and spongier than the hard surface of a Petri dish.

The behavior of these cells in the Matrigel is completely different than that usually seen. In fact no one has ever seen anything like it before, which is why, if possible you must look at the 3 movies the paper supplies in the supplementary material (referenced above).

The cells move around, but nowhere to be seen are the lamellipodia or the filopodia seen in 2 dimensional cell culture.

In the first movie a small cell repeatedly generates and retracts multiple membranous protrusions called cytocapsues (some larger than the cell itself) which the cell sometimes enters.

The second has multiple cells within the tubes formed by the cytocapsules (cytocapsular tubes) migrating within them. The cells drag the tubes through the matrix, without breaking the tube or allowing another tube to merge with it.

If replicated, this work has bearing on embryology, normal organ function and cancer metastasis.

No one has ever seen anything like this. Why not? The authors say that ‘only when the polymerization, density and viscoelasticity of the Matrigel is tightly controlled are the tubes seen. Unlike secreted extracellular vesicles, cells get inside the cytocapsular tubes. The tubes themselves interconnect and form networks. The tubes degrade and decompose without cells inside them.

They note that polymerized actin (microfilaments) are present under the tube membranes.

Even though the membranes are mostly lipids, proteins are required, and eIF4E levels are elevated. Inhibiting it suppresses cytocapsular tube generation.

It’s worth repeating Sagan – “Extraordinary claims require extraordinary evidence” Stay tuned

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Maybe there is something to it after all

Nearly 8 years ago I wrote a post (see below) about a rather fantastic paper, that said that in order to turn on gene transcription, the DNA had to be damaged first. This caused all sorts of repair enzymes to rush to the damaged site, opening up the chromatin there and allowing RNA polymerase II (which is large) to get to the DNA and transcription to proceed. I wrote the original author who was Italian, who said he was ill, but nothing further appeared about the idea (as far as I know). Remember what Carl Sagan said “Extraordinary claims require extraordinary evidence.”

Now Science (vol. 351 p. 147 ’16 ) has an abstract of an article in Nat. Commun. 6, 10191 (2015). “Curiously, DNA repair factors have been found associated with tran- scriptionally paused, inducible genes. Bunch et al. show that the activation of paused and inducible genes in human tissue culture cells triggers DNA breaks at the RNA polymerase pause site. The subsequent recruitment and signaling activity of DNA repair factors is critical for DNA repair, release of the RNA polymerase, and the transition to the transcrip- tion elongation phase of gene expression.”

Here’s the relevant portion of the post from 2/08. How about that ! ! ! DNA breaks are even more spectacular than 8 oxo-guanine

An incredible article appeared last month in the journal Science. (see below for the abstract). If it can be verified and if it applies generally, our conception of just how genes coding for protein are turned on will be radically changed (yes, there are many other kinds of genes other than those coding for proteins). If DNA compaction, nucleosomes, histones, lysine methylation and demethylation, the histone code, nuclear hormone receptors (particularly the estrogen receptor), DNA glycosylase and topoisomerase aren’t old friends have a look at the first comment on this post for the background you need (it’s back on the Skeptical Chymist). Don’t worry, there is plenty of chemistry to follow.

Some histone code modifications are reversible, particularly acetylation of the epsilon amino group of lysine. Enzymes acetylating histone lysines are called histone acetylases, those removing it are called histone deacetylatases (HDACs). However, lysine methylation was thought to be permanent until ’04 when several enzymes able to demethylate lysine were found. One such enzyme is called LSD1 (it has nothing to do with the hallucinogen). It removes the two methyl groups from lysine #9 of histone #3 (H3K9me2). If this modification is present on a nucleosome near a gene, the gene is silenced, so the methyls must be removed so the protein it codes for can be made.

The estrogen receptor + estrogen complex bound to the ERE (the estrogen response element – a 15 nucleotide DNA sequence) triggers H3K9me2 removal. The process of demethylation is oxidative (how else would you split a nitrogen to hydrocarbon bond?). Hydrogen peroxide is produced, a loose cannon which oxidizes the juicy electron-rich bases of DNA nearby, forming in particular 8 oxo-guanine, as guanine is the most easily oxidized DNA base. Since 21% of the DNA bases in our genome are guanine, H2O2 doesn’t have far to look. This calls in some fairly heavy artillery (DNA glycosylase to remove the 8 oxo-guanine, topoisomerase IIbeta to unwind the DNA so it can be repaired, the repair enzymes, etc, etc…). Naturally this opens up the compacted DNA structure around the gene allowing RNA polymerase II to do its work transcribing the estrogen responsive gene into mRNA (once the damage is repaired).

So according to this paper, estrogen turns on gene transcription by damaging DNA. This is fantastic (if true). There’s more. The estrogen receptor is but one member of a group of proteins called nuclear hormone receptors. The name comes from the fact that other hormones (progesterone, androgen, thyroid, glucocorticoids, mineralocorticoids) have their own proteins that turn on (or turn off) genes the same way. Subsequently it was found that some vitamin metabolites (vitamin D3, vitamin A) have similar receptors even though they aren’t hormones. The human genome contains 48 such proteins. Less than half of them have known ligands. Those with known ligands have their finger in just about every metabolic pie in the cell.

One final point. It has been estimated that 8-oxoguanine is formed 100,000 times each day in every cell. Perhaps its formation is physiologic rather than pathologic. Where does that leave antioxidant therapy, which has been touted to do everything but cure hemorrhoids? Well, one such trial was done on 29,000 Finnish men at high risk for lung cancer (they were smokers) [New England J. Med. vol. 330 pp. 1029-1035 (1994)] Alpha tocopherol (one antioxidant used in the study) didn’t decrease the incidence of lung cancer, and there was an 18% higher incidence of lung cancer among the men receiving beta carotene (another antioxidant). In medicine, theory is great but data trumps it every time.

Science vol. 301 pp. 202 – 206 ’08, B. Perillo et. al.

Modifications at the N-terminal tails of nucleosomal histones are required for efficient transcription in vivo. We analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine–DNA glycosylase 1 and topoisomeraseIIβ, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription.

The old year goes out with a bang

A huge amount of cellular genomics will have to be redone if the following paper is replicated. Remember “Extraordinary claims require extraordinary evidence.” Carl Sagan.

What’s all the shouting about? Normally when you think about messenger RNA (mRNA) as it exists in the cytoplasm after the initial transcript is significantly massaged in the nucleus, you think about the part that codes for amino acids. This ‘coding region’ is the part that is translated into amino acids by the ribosome. But mRNA is invariably larger having nucleotides at each end (3′ and 5′) which have other uses. These are called the 3′ Untranslated Region (3′ UTR) and 5′ Untranslated Region (5′ UTR).

So if you do single cell RNA sequencing (which we can do now) it shouldn’t matter what nucleotide sequence you search for (5′ UTR, 3′ UTR or the coding region) as all mRNA contains one of each.

Not so says this paper [ Neuron vol. 88 pp. 1149 – 1156 ’15 ].

Given the mRNA for a given protein in a single cell, using a probe for the 3’UTR and a probe for the coding sequence should give you the same abundance for both. That’s not what they found at all for single neurons from the brain. In some cases there was much more RNA coding for the 3’UTR than for the coding segment of a given mRNA for a protein. In others there was much less. Even more impressively is that the 3’UTR/(3’UTR + coding) ratio for a given protein varies between different parts of the brain. Obviously this ratio should be .5 given what we knew about mRNA in the past. The ratio has to be between 0 and 1.

Well they looked at a lot of proteins. The did find around 1,400 genes with a ratio of .5 (as expected), but they found 700 showing a ratio of .2 (lots more 3’UTR than coding sequence), and 1,100 showing a ratio of .8. Overall plotting the ratio vs. number of genes with that ratio gives something looking like a bell curve (Gaussian distribution).

It’s long been known that mRNA levels don’t exactly correlate with the levels of proteins made from them. If there’s lots of 3’UTRs around the authors found that there was relatively little protein made from the gene.

A variety of brain atlases have published mRNA abundances for various regions of the brain. If they just used one probe (as they probably did) this is clearly not enough.

The 3’UTRs may be acting as ceRNAs (competitive endogenous RNAs). These have been known for years — I’ve included a post of 3 years ago on the subject (at the end).

So this work (if replicated) throws everything we thought we knew about mRNA into a cocked hat. It’s why I love science, there’s always something really new to think about. Happy New Year !!!

Chemiotics II
Lotsa stuff, basically scientific — molecular biology, organic chemistry, medicine (neurology), math — and music
Why drug discovery is so hard: reason #20 — competitive endogenous RNAs

The chemist will appreciate le Chatelier’s principle in action in what follows. We are far from knowing all the players controlling cellular behavior. So how in the world will we find drugs to change cellular behavior when we don’t know all the things affecting it. The latest previously unknown cellular player to enter the lists are competitive endogenous RNAs (ceRNAs). For details see Cell vol. 147 pp. 344 – 357, 382 – 395 ’11. The background the pure chemist needs for what follows can all be found in the category “Molecular Biology Survival Guide.

Recall that microRNAs are short (20 something) polynucleotides which bind to the 3′ untranslated region (3′ UTR) of mRNA, and either (1) inhibit its translation into protein (2) cause its degradation. In each case, less of the corresponding protein is made. The microRNA and the appropriate sequence in the 3′ UTR of the mRNA form an RNA-RNA double helix (G on one strand binding to C on the other, etc.). Visualizing such helices is duck soup for a chemist.

Molecular biology is full of such semantic cherry bombs as nonCoding DNA (which meant DNA which didn’t cord for protein), a subset of Junk DNA. Another is the pseudogene — these are genes that look like they should code for protein, except that they don’t because of lack of an initiation codon or a premature termination codon. Except for these differences, they have the nucleotide sequence to code for a known protein. It is estimated that the human genome contains as many pseudogenes (20,000) as it contains true protein coding genes [ Genome Res. vol. 12 pp. 272 – 280 ’02 ]. We now know that well over half the genome is transcribed into mRNA, including the pseudogenes.

PTEN (you don’t want to know what it stands for) is a 403 amino acid protein which is one of the most commonly mutated proteins in human cancer. Our genome also contains a pseudogene for it (called PTENP). Interestingly deletion of PTENP (not PTEN) is found in some cancers. However PTENP deletion is associated with decreased amounts of the PTEN protein itself, something you don’t want as PTEN is a tumor suppressor. How PTEN accomplishes this appears to be fairly well known, but is irrelevant here.

Why should loss of PTENP decrease PTEN itself? The reason is because the mRNA made from PTENP, even though it has a premature termination codon, and can’t be made into protein, is just as long, so it also contains the 3’UTR of PTEN. This means PTENP is sopping up microRNAs which would otherwise decrease the level of PTEN. Think of PTENP mRNA as a sponge.

Subtle isn’t it? But there’s far more. At least PTENP mRNA closely resembles the PTEN mRNA. However other mRNAs coding for completely different proteins, also have binding sites in their 3’UTR for the microRNA which binds to the 3UTR of PTEN, resulting in its destruction. So transcription of a completely different gene (the example of ZEB2 is given) can control the abundance of another protein. Essentially its mRNA is acting as a sponge, sopping up the killer microRNA.

It gets worse. Most microRNAs have binding sites on the mRNAs of many different proteins, and PTEN itself has a 3’UTR which binds to 10 different microRNAs.

So here is a completely unexpected mechanism of control of protein levels in the cell. The general term for this is competitive endogenous RNA (ceRNA). Two years ago the number of human microRNAs was thought to be around 1,000. Unlike protein coding genes, it’s far from obvious how to find them by looking at the sequence of our genome, so there may be quite a few more.

So most microRNAs bind the 3’UTR of more than one protein (the average number is unclear at this point), and most proteins have binding sites for microRNAs in their 3’UTR (again the average number is unclear). What a mess. What subtlety. What an opportunity for the regulation of cellular function. Who is going to be smart enough to figure out a drug which will change this in a way that we want. Absence of evidence of a regulatory mechanism is not evidence of its absence. A little humility is in order.