Sunday Protist - Diplonemids: Metaboly without a pellicle and the dawn of kDNA?

Took you guys a while to get the past Mystery Micrograph, which gave me ample excuses to procrastinate with last week's Sunday Protist. Of course, no one noticed, so it's all good, right? Opisthokont finally got it after every single other discicristate lineage has been eliminated, and grotesquely revealing hints have been given away. Johan guessed their sister clade, Kinetoplastida. More importantly, we need fresh blood on this blog, and thus far, the Mystery Micrograph winners have been an incestuous group, all linked in the present or past with a single department. Srsly. So half the win goes to Johan for getting sort of close-ish enough, as well as being somebody I don't actually know outside the blogosphere...

So the correct answer for MM#05 was...

Rhynchopus, a diplonemid. (Roy et al. 2007 JEM) Note the odd writhing movement in the timelapse (1-8) as well as absense of flagella in most figures. This group is very obscure - check out Table 1 in the above mentioned publication if you can, and marvel at the sea of question marks regarding the various diplonemids...

That's right, yet another obscure protist, Rhynchopus, a genus of diplonemids. And everyone knows what those are, right? *crickets* Well, hang in there and find out! Feel free to point and laugh any time you see 'Rhyncopus' - apparently, I have serious issues spelling that one... (although it's definitely not as bad as Barleeiidae) Speaking of taxonomy, Diplonema was formerly known as 'Isonema', in case anyone gets the urge to scour ancient protistology literature...

Cell Structure
I assumed that the absense of flagella in the SEM was due to them falling off due to stress or just being generally shitty specimens for EM prep. Turns out, that's actually how they roll - flagella are only visible in swarmer cells produced in hungry (starved) cultures. The flatness is also a characteristic attribute of this cell, not so much a dehydration artefact. (Roy et al. 2007 JEM). The cell contains a large flagellar apparatus and a cytostome (cell mouth), generally oriented parallel to each other, with a lip protruding outside the opening:

Drawings of Rhynchopus cell structure (Roy et al. 2007 JEM). Hopefully this makes the above paragraph make a little more sense...

Not much is really known about the diplonemid nuclear genome organisation, although the chromosomes appear to be permanently condensed, just like in the euglenids. Weird chromosome structure is often correlated with genomes on crack, so one can only wonder what kind of oddities could be concealed in their chromatin. However, the nuclear genome does seem to contain a splice leader gene (Sturm et al. 2001 JEM), a characteristic feature of kinetoplastids, which are famous for their polycistronic primary mRNA transcripts - they have several genes following a single promotor, which are then broken up by trans-splicing 5' cap-containing splice leaders before each gene. Rumour has it, euglenids might also be capable of splice leader trans-splicing, so this may well be a general euglenozoan trait.

Diplonemids are interesting from an evolutionary perspective. While sharing some distinctive features with many euglenids (metaboly, condensed nuclear chromatin, cytopharynx structure), they are actually basal to kinetoplastids, with the euglenids basal to both kinetoplastids AND diplonemids. Together, they form the Euglenozoa*:

(Simpson et al. 2004 Protist; tree of Euglenozoa; note diplonemids branching basal to kinetoplastids, and not to euglenids.)

* By the way, Euglenozoa = large group of excavates containing kinetoplastids (eg. Trypanosomes), euglenids (eg. Euglena) and diplonemids (subject of this post). We like to recycle fragments of Latin lexicon wherever possible.

Metaboly
Many euglenids share a characteristic form of motility called 'metaboly', which is essentially a writhing motion of a cell, where it twists and turns like mad, moving a blog of cytoplasm from one end to the other. Euglenids are covered with protein pellicle strips arranged longitudinally from one end of the cell to the other. Among many euglenids, those strips have a very complex structure, which enables them to slide against each other and allow metaboly to occur. Beneath each stript is a bundle of microtubules. (This page pretty much has nearly everything you ever wanted to know about Euglenids.)

[rant]One anoying thing about Euglenid biologists (that is, people studying euglenids, not euglenids who have chosen a questionable career path...) is their referring to the pellicle strips as 'cytoskeleton', which the rest of us reserve strictly for microtubules, actin filaments and intermediate fibres. As cool as your strips might be, they do not consist of actin, 'tubes OR intermediate fibres, and are therefore NOT cytoskeleton. Otherwise anyone outside 'Euglenology' becomes very confused. Thanks. [/rant]

Anyway, you may wonder why we're rambling about Euglenids all of the sudden. Thing is, diplonemids also have metaboly-like motility. Except that they lack pellicle strips altogether. However, they still have the longitudinal bundles of microtubules lining the cortex of the cell:

TEM across membrane showing the densely packed microtubule bundles just underneath the membrane (Roy et al. 2007 JEM)

The 'function' or significance of euglenoid metaboly is still unknown. Now, it is often said that the euglenid pellicle strips 'cause' the euglenoid motion (metaboly) (eg. Leander et al. 2001 Evol, although they get more careful in later publications). The secondary fusion of pelicle strips, eg. as in Phacus, inhibits metaboly from happening, but that's unrelated to the acquisition of metaboly in the first place. As we've seen in the topmost figure, diplonemids too seem to exhibit metaboly, but no pellicle stripts. This would make them a good model for examining the mechanisms of metaboly, as well as its potential adaptive value, if any exists.

Furthermore, diplonemids are basal to kinetoplastids, and if they share a trait with euglenids, one wonders if kinetoplastids have retained it, as it would be more parsimonious to assume their common ancestor must've had it as well. Kinetoplastids, at least their Trypanosome representatives, also have pellicular microtubules (Woods et al. 1989 J Cell Sci), and some Bodonids have even been observed to excibit metaboly-like motion (Swale 1973 Biol J Linn Soc, and references made to Cryptobia helicis, eg. in Vickerman 1977 J Protozool, although I can't seem to access the original C.helicis account by Kozloff 1948 J Morphol), although it is uncertain if the pellucular 'tubes have anything to do with it.

Some ciliates have also been described to undergo metaboly-like motion, although the similarity between them is uncertain. If more organisms do actually exhibit something similar to euglenoid motion, then perhaps it's not so special after all. It could merely be enhanced by the pellicle strips providing extra rigidity to the longitudinal 'ridges' formed by the microtubules. Thus, there may well be no function of metaboly - it may just be an artefact of having fairly rigid structures lining your cell from head to toe, which in turn would have evolved for entirely different reasons (or lack thereof).

So here's a mini project for someone with lots of time and stray grant money lying about:
- determine the mechanisms of metaboly in both the pellicle-bearing euglenids and 'naked' diplonemids; likely, it has something to do with the organisation and dynamics of pellicular microtubules. Use [MT-disrupting] drugs, they're good for science.
- examine other eukaryotes with some sort of pellicular MT bundles, and see how they roll move about.
- look over older accounts of 'metaboly-like' motion in non-euglenids, and verify them. Check if anything is known about their cytoskeletal organisation.
- my guess would be, the much-noted euglenoid motility might actually be just an epiphenomenon of euglenid cytoskeletal and pellicular structures, which are shaped like that for other reasons. It likely has no function*. Ie, perhaps it's slightly...overrated!

*There have been observations of eukaryovores tending to be more likely to exhibit metaboly than bacteriovores, perhaps due to larger prey sizes, although, as pointed out in the aforementioned link, this correlation is rather fuzzy; and, even in the case of there being such a relationship, the presence or absense of metaboly could be a secondary feature resulting from different requirements of the cytoskeleton itself in eukaryo- and bacteriophagy. That was quite possibly the worst sentence in the history of English.

Mitochondrial Genomes
Diplonemid mitochondria are arranged in a cortical network pattern, and are devoid of a kinetoplast (characteristic of their sister clade)(Roy et al. 2007 JEM). At first glance, the mitochondrial genome seems mundanely circular (and unlinked):

(Marande et al. 2005 Euk Cell; Diplonema circular mitochondrial DNA in TEM)

However, those circles are about ~7Kb in size, which is small for mitochondrial DNA. (Marande et al. 2005). This is starting to get a little weirder. Diplonemids also seem to have flattened, rather than discoidal, mitochondrial cristae - quite unusual for a discicristate. Furthermore, some genes seem to be fragmented and distributed over multiple chromosomes. This is starting to get quite interesting. So why would we expect weird mitochondrial genomes in diplonemids anyway?

Because among the neighbouring kinetoplastids, they happen to be on crack:

(Simpson et al. 2002 MBE; evolution of Trypanosome 'chainmail' genomes and its relatives.)

I won't go into detail here, but let's just say Trypanosome MtDNA organisation is a major piece of supportive evidence for the 7th Day LSD theory of intelligent design, wherein the intelligent designer got fucking high on acid and designed the protists on his day off. I've alluded to it elsewhere, as it is a substantial part of my personal faith, and therefore true. I should go form a church around my revelation...

Basically, the intelligent designer spent too much time watching fantasy movies set in medieval Europe (before humans were even created), and was wondering if it would be possible to replicate various pieces of arms and armour using DNA molecules. You would do it too if you were bored, high and almighty. So this very intelligent designer created 'chainmail' genomes in Trypanosomes. Not only that, but he was also apparenly bored with the canonical DNA -> pre-mRNA -> splicing ->translation pathway, and decided to spice it up with some RNA editing. Why bother producing proper mRNA from the start when you can make shit mRNA and then fix it with a whole bunch of guideRNAs. And then laugh at biologists trying to come up with elaborate adaptive explanation for the evolution of this nonsense. Hahaa!

Basically, big circles code precursor mRNA, which is then fixed up and debugged by 'gRNA' from little circles. This process requires upwards of about 500 proteins to work. Other eukaryotes just use RNA polymerase. Efficiency at its best. Yeah.

Interestingly, in the Euglenids, Petalomonas is suspected of having mitochnodrial RNA editing on crack possibly even exceeding that of Trypanosomes! Unfortunately, the understanding of Euglenid mitochondrial genomes seems to be a mess at the moment, but they do have potential to turn out to be weird. (Roy et al. 2007 Protist).

So what about those fragmented genes hanging out on different chromosomes? Marande et al. (2005) suggest they may be the beginnings of gRNAs. They seem to be significantly larger than Trypanosome gRNAs, and thus may be merely fragments that get trans-spliced together. But often where there is trans-splicing of gene fragments, there may also be RNA editing (eg. dinoflagellate mitochondrial DNA). The diplonemid mitochondrial genome is actually relatively massive (~56 chromosomes, 360Kb total), and turns out that most mitochondrial genomes actually only contain one type of circular plasmid; among the few exceptions are the trypanosomes and Amoebodinium (all from Marande et al. 2005). Hmmm. Thus, while little is still known (or published anyway) about the Diplonemid mitochondrial genomes, they seem to be quite promising for studying the early evolution of kDNA architecture.

If euglenids have similar madness too, one can only wonder why Heterolobosean mitochondrial genomes may reveal. Perhaps the origins of the evolutionary absurdity known as kinetoplast DNA might actually lie earlier on in the discicristates?

There seem to be multitudes of similarities among the euglenozoa, now that I look at it again. And the sister Heterolobosea are too poorly known genetically (or even developmentally) to make any conclusions or generalisations there. But it's quite fascinating to watch the vast and diverse evolutionary history of eukaryotes slowly come together, piece by piece, into a giant tapestry of evolutionary stories. Our intelligent designer on LSD had no idea how much sense he would inadvertently make amid the nonsense...


PS: Please feel free to point out any defects/inaccuracies -- I've been staring at this review post far too long to notice anything!

References:
Leander, B., Witek, R., & Farmer, M. (2001). TRENDS IN THE EVOLUTION OF THE EUGLENID PELLICLE Evolution, 55 (11) DOI: 10.1554/0014-3820(2001)055[2215:TITEOT]2.0.CO;2

Marande, W., Lukes, J., & Burger, G. (2005). Unique Mitochondrial Genome Structure in Diplonemids, the Sister Group of Kinetoplastids Eukaryotic Cell, 4 (6), 1137-1146 DOI: 10.1128/EC.4.6.1137-1146.2005

ROY, J., FAKTOROVÁ, D., BENADA, O., LUKEŠ, J., & BURGER, G. (2007). Description of Rhynchopus euleeides n. sp. (Diplonemea), a Free-Living Marine Euglenozoan The Journal of Eukaryotic Microbiology, 54 (2), 137-145 DOI: 10.1111/j.1550-7408.2007.00244.x

Roy, J., Faktorová, D., Lukeš, J., & Burger, G. (2007). Unusual Mitochondrial Genome Structures throughout the Euglenozoa Protist, 158 (3), 385-396 DOI: 10.1016/j.protis.2007.03.002

Simpson AG, Lukes J, & Roger AJ (2002). The evolutionary history of kinetoplastids and their kinetoplasts. Molecular biology and evolution, 19 (12), 2071-83 PMID: 12446799

SIMPSON, A. (2004). Early Evolution within Kinetoplastids (Euglenozoa), and the Late Emergence of Trypanosomatids Protist, 155 (4), 407-422 DOI: 10.1078/1434461042650389

STURM, N., MASLOV, D., GRISARD, E., & CAMPBELL, D. (2001). Diplonema spp. Possess Spliced Leader RNA Genes Similar to the Kinetoplastida The Journal of Eukaryotic Microbiology, 48 (3), 325-331 DOI: 10.1111/j.1550-7408.2001.tb00321.x

SWALE, E. (1973). A study of the colourless flagellate Rhynchomonas nasuta (Stokes) Klebs Biological Journal of the Linnean Society, 5 (3), 255-264 DOI: 10.1111/j.1095-8312.1973.tb00705.x

VICKERMAN, K. (1977). DNA Throughout the Single Mitochondrion of a Kinetoplastid Flagellate: Observations on the Ultrastructure of Cryptobia vaginalis (Hesse, 1910) The Journal of Eukaryotic Microbiology, 24 (2), 221-233 DOI: 10.1111/j.1550-7408.1977.tb00970.x