Has cellular biology and biochemistry shown us a new form of matter? It’s certainly something I never studied in PChem back in the day. It goes by multiple names, and may be more than one thing.
Start with the nucleolus — it’s been known for years, a visible agglomeration of proteins and RNA in the nucleus, not bound by a membrane. Then there is the processing body (aka stress granule), also made of proteins and RNA (but different ones — transcription factors and mRNAs). Then there is the nuclear pore, made of ‘low complexity sequence tails of proteins surrounding the pore (mostly phenylalanine glycine repeats — aka FG repeats) thought to form a barrier to protein movement through the pore. Then there are RNA granules – said to occur by a phase transition to a hydrogel-like phase (whatever that is). Neurologists have long been interested in FUS/TLS a protein which is mutated in some forms of Amyotrophic Lateral Sclerosis and dementia.
I do think that we’re at the blind men and the elephant stage trying to sort all this out (which, of course, makes it fascinating and a fit subject for scientific work — apologies Wittgenstein — “What we cannot speak about we must pass over in silence”
So in what follows you’ll find a lot of information about these matters, which does not have a neat and tidy explanation. This is what science looks like when it’s being done.
[ Cell vol. 149 pp. 753 – 767, 768 -779 ’12 ] RNA granules don’t just occur in dendrites — they are found in (1) germ cell P granules of C. elegans embryos (2) polar granules of Drosophila embryos (3) stress grnules appearing in cultured yeast and mammalian cells on nutrient deprivation or other forms of metabolic stress (4) neuronal granules transporting mRNAs to dendrites.
Unsurprisingly, the granule contains RNA binding proteins (with KH or RNA Recognition motif (RRM) domains). These domains allow proteins containing them to recognize 3’ untranslated regions of target mRNA in a sequence specific manner (really?).
This work shows that structures resembling RNA granules can be reversibly aggregated and disaggregated in a soluble cellfree system in response to a small molecule (a biotinylated isoxazole ) The proteins in the granules contain low complexity sequences (LC sequences). which show little diversity in their amino acid composition (which is usually repetitive). One example is the leucine rich domain. LC sequences are all you need for aggregation by the isoxazole. The domains undergo a concentration dependent phase transition to a hydrogel-like state with no chemical present?? The hydrogels are made of uniformly polymerized amyloidlike fibers. The fibers form and dissolve and don’t cause trouble (unlike classic amyloid).
LC sequences are particularly enriched in RNA and DNA binding proteins. FUS (FUsed in Sarcoma) is an RNA binding protein containing an LC domain (Gly/Ser Tyr Gly/Ser repeats). Hydrogel droplets formed from the LC sequence of FUS can retain proteins containing either the FUS LC sequence or other LC sequences.
This work finds a potential use for LC sequences — they allow the movement of regulatory proteins into and out of organized subcellular domains, via reversible polymerization into dynamic amyloidlike fibers. It’s possible that something similar occurs in Cajal bodies, nuclear speckles and nuclear factories involved in RNA splicing.
[ Proc. Natl. Acad. Sci. vol. 99 pp. 13583 – 13588 ’02 ] They range in size from 2000 Angstroms to several microns. None of them are bounded by a membrane. It is thought that the same processes leading to the formation of nuclear bodies (e.g. a phase transition) is responsible for similar bodies occuring in the cytoplasm) — e.g. P bodies (Processing bodies), stress granules.
Each type is identified immunologically by antibodies against its components (either signature proteins or ribonucleoproteins or even small nuclear RNAs. They include
l. The Cajal body (the coiled body)
2. The promyelocytic body (PML body, POD)
3. Splicing related bodies
a. SC35 speckles (interchromatin granule cluster)
4. The GEM body
5. The matrix associated deacetylase body
6. HAP body
7. nucleoli associated paraspeckles
8. Nucleoli themselves.
The integrity of a nuclear body can be disrupted after depletion of its normal components — PODs are disrupted in acute PML.
The Cajal body and GEM are colocalized, but otherwise there doesn’t seem to be much association among the different nuclear bodies.
[ Cell vol. 162 pp. 1066 – 1077 ’15 ] FUS forms liquid compartments at sites of DNA damage in the nucleus and in the cytoplasm on stress. With time liquid droplets of FUS convert with time to an aggregated state, a conversion accelerated by mutations (in the prionlike domain) derived from patients.
Why is the compartment called liquidlike? FUS molecules rapidly rearrange within the compartment. The comaprtments formed by FUS are spherical. Two FUS compartments can fuse and relax into one sphere.
FUS compartments belong to a set of RNA protein compartments (P granules, nucleoli) which ‘probably’ form by liquid liquid demixing (phase separation) from cytoplasm.
The conversion between a liquid to a solidlike state is concentration dependent, and mutations blocking nuclear localization sequence (NLS) functgion produce increased concentrations in the cytoplasm with aggregation.
The prionlike domain of FUS is intrinsically disordered.
[ Neuron vol. 88 pp. 678 – 690 ’15 ] Mutations in a bunch of RNA binding proteins (TDP43, FUS, ataxin2, hnRNPA1, hnRNPB2) are associated with ALS/FTD (Amyotrophic Lateral Sclerosis/FrontoTemporal Dementia). Poorly soluble assemblies of the mutant RNA binding protein are found in the nucleus and cytoplasm in the patients.
The assemblies differ from amyloids in the following ways
l. They are soluble in urea
2. They have low beta sheet content
3. They have a mixed granular/fibrillar appearance on EM
4. They don’t bind dyes diagnostic for amyloid (e.g. thioflavin T)
5. When fluorescently labeled, they don’t show the reductions in in vivo fluorescent lifetimes typical of conventional amyloid.
This work shows that the LC domain (Low Complexity domain) of normal FUS undergoes phase transitions, reversibly shifting between dispersed liquid droplets and hydrogel-like phases (defined how). FUS mutants limit the ability to shift between phases, instead increasing the propensity of FUS to condense into poorly soluble stable (e.g. irreversible) fibrillar hydrogel-like assemblies (e.g. a new type of phase. Spontaneous occurrence of this might explain sporadic ALS/FTD with FUS pathology even when no mutations are present. These assemblies selectively entrap other ribonucleoproteins, impair local RNP granule function and decrease new protein synthesis in axon terminals of cultured neurons. The work was done in C. elegans.
“The biophysics of conversion from liquid droplet to reversible hydrogel is not yet clear”. Thw two differ only slightly in viscosity.
The FG repeats (phenylalanine, glycine repeats) of nucleoporins show structural characteristics typical of natively unfolded proteins (e.g. highly flexible proteins lacking ordered secondary structure). They can be quite long (200 – 700 amino acids in yeast). Protease sensitivity shows that most FG repeat containing nucleoporins are disordered in situ within the nuclear pore complexes of purified yeast nuclei. This makes it likely that they form a meshwork of random coils at the pore through which nuclear transport proceeds. Natively unfolded proteins show the following biochemical features
l. multiple domains allowing simultaneous interactions with multiple binding partners
2. nonrigid binding domains that can accomodate a variety of interacting partners
3. fast molecular association and dissociation rates.
Another model has FG domains interacting with each other in the pore to form a protein meshwork which acts as a separate hydrophobic phase. Transport complexes can partititon into this phase because they can bind to the GF repeats. Proteins unable to bind to the FG repeats are excluded from the hydrophobic phase. Molecules below 30 – 40 kiloDaltons get through the water filled holes in the gel.
To get through the pore a midsize protein must recruit a large receptor to pass through a narrow channel. The receptors replace the FG – FG binding of the nups with each other by binding to themselves — they essentially dissolve into the gel.
An alternate view holds that FG repeats form a network of unlinked polymers whose thermally activated undulations create a zone of ‘entropic exclusion’. The entropic penalty in collapsing the chains allows a barrier to form. However by binding to the repeats, carriers can circumvent the exclusion — replacing one type of bond with another.
There are several models for the FG repeats in the nuclear pore. The most convincing (to me) is the ‘selective phase’ model — a sievelike meshwork is formed within the NPC via interactions between FG repeats. The size of the FG mesh determines the upper limits of the diffusion gate (e.g. — the molecules getting through without help — in this case under 30 kiloDaltons). The binding of nuclear transport receptors (NTRs) to the FG repeats is proposed to locally dissolve the FG-FG network, allowing passage of whatever is bound to the NTRs.
‘Sufficiently concentrated’ solutions of cohesive FG domains spontaneously form FG hydrogels (which excludes inert molecules over 50 Angstroms in diameter ). Cargo NTR complexes migrate into such hydrogels ‘up to’ 20,000 times faster than the respective cargoes alone. The intragel diffusion rate of a typical importinBeta:cargo complex predicts a similar NPC passage time (10 milliSeconds) as was actually ssen in living NPCs.
The FG repeat domain of the yeast nucleoporin Nsp1 forms a hydrogel-like structure in vitro which requires hydrophobic interactions between the aromatic rings of the phenylalanines. This work assembled FG hydrogels in vitro, and studied protein entry into them and diffusion through them usingfluorescence microscopy. The influx of various nuclear transport receptors of the importin beta family into the Nsp1 FG hydrogel was 1000 times faster than the entry of a control protein. Access of a model cargo bound to importin beta was accelerated by over 20,000 fold (compared to free cargo). However, not every FG hydrogel shows selectivity. To achieve selective permeability the total FG concentration within the gel had to be raised above 50 milliMolar. This has led the authors to introduce the concept of the saturated hydrogel, in which all the FG repeats must extend completely and undergo a maximum number of interactions. It seems likely (to the authors of the editorial not the authors of the paper) that newly made FG proteins would immediately curl up and form intramolecular FG bridges (rather than intermolecular ones) In vitro gel formation can only be induced from lyophilized proteins under extreme pH and salt. The authors suggest that nuclear transport receptors act as chaperones preventing intramolecuular FG interactions after synthesis. Under more physiologic conditions, the FG domain of Nsp1 formed neither homo nor heterotypic interactions with other FG nucleoporins.
FG repeat domains (they contain a hydrophobic patch, usually FG, FxFG, or GLFG, surrounded by more hydroplic spacers) account for 12 – 20% of the mass of a nuclear pore complex. Up to 50 FG repeat domains may occur in a single protein. FG repeats occur in various flavors — examples are FxFG repeats
So there you have it — quite a mess. Figure it out and get on the boat to Sweden