Cryptomonads: solar-powered armoured battleships

I've been 'scoping around some pond water lately and came across some relatively big cryptomonads (g. Cryptomonas, I think). Cryptos aren't all that rare, but most of them whirl about rather hyperactively, rendering them as troublesome photo subjects. This specimen, on the other hand, had a convenient habit of pausing every once in a while to have its picture taken. Finally, I have my own cryptomonad shots!

Cryptomonas(?) sp. The cell is about ~30µm long, pretty big for a cryptomonad. On its right side the cryptomonad has a furrow – or, in some species, an tube-like gullet – lined with ejectisomes (particularly visible in the top right image). The vesicle at the anterior tip of the cell is its contractile vacuole. Refractile stuff is the starch granules. 40x objective, DIC

Despite their small size and superficially generic algal appearance, cryptomonads do have quite a few awesome bits about them. From an evolutionary standpoint, they have pretty damn awesome plastids – products of secondary endosymbiosis of red algae, complete with a shrunken relict nucleus ("nucleomorph") of the red algal ex-host! The plastids also have four membranes, complicating the delivery of plastid-targetting proteins from the cryptomonad host nucleus. But I'll save that story for some other time, and instead keep it superficial. Literally: it has ejectile things lining its surface, and who doesn't like the idea of a microscopic solar-powered hyperactive battleship?

Prior to embarking on some battle scenes, lets look around the ship's anatomy a little bit mostly as an excuse to show off a diagram. At its fore we have a pair of flagella, lined with little hairs – also a characteristic of many Alveolates and Stramenopiles, with whom Cryptomonads might share the secondary red algal symbiosis event with. Much of the cell is occupied with a single plastid, making the fucker a bit difficult to diagram. In all his/her/its infinite wisdom, the designer apparently failed to take into consideration the future pains of this student attempting to tame the wild beast that is Illustrator while drawing this cell. Asshole. Besides the plastid, there's also a single mitochondrion and a bunch of other small crap that a eukaryote ought to have. The plastid's outermost (fourth) membrane is contiguous with the endoplasmic reticulum system, presumably homologous to the original digestive vesicle that enveloped the 'enslaved'* red alga. The third membrane derives from the red algal cell membrane, whereas the inner pair are the usual plastid membranes. Pop quiz: where would you expect to find the relict endosymbiont's nucleus (the nucleomorph)? (Answer at the bottom of the post, or in the diagram if you're so inclined to 'cheat' ;p)

*Google [scholar] "Cavalier-Smith" and "enslaved". When he likes certain words, he really likes them.

Back to the surface. The cryptomonad surface is quite complex, consisting of an inner and surface periplast layers separated by the cell membrane. Sometimes the surface layer can be be covered in scales, sometimes fibrous matter. This periplast is perforated with pores for ejectisomes, much like battlements on a warship. Ejectisomes themselves consist of coiled proteinaceous ribbons that extend forcefully upon firing.

Cryptomonad periplast. IPC – inner periplast layer, PM – plasma membrane, S – scales (of the surface periplast layer). On the right is a freeze fracture EM of the plasma membrane, which shows imprints of the surface scales (vaguely hexagonal) and pores for ejectisomes (E). In other words, the surface of an armoured warship with battlements. (Brett & Wetherbee 1986 Protoplasma)

Ejectisomes – more generally, extrusomes – are not all that unusual in the protist world. Many ciliates are loaded with menacing trichocysts and green algae like Pyramimonas are not afraid to fire similar structures either. Some bacterial endo- and episymbionts also bear similar coiled structures, but that's a topic for some other day as well. Extrusomes can also be used more locally to glue prey to the organism – if you, upon finding yourself shrunk to microns, accidentally bump into a frail-looking centrohelid heliozoan, be afraid. Be very afraid. It will smother you in adhesive proteins from the extrusomes lining its fragile-looking axopodia and devour you alive and possibly paralysed.

Ejectisomes in cryptomonads and their non-photosynthetic close relatives, katablepharids. Pyramimonas is only distantly related, and probably evolved its ejectisomes completely independently. (Kugrens et al. 1994 Protoplasma: nice review on protist ejectisomes in general, excluding ciliates)

One of the poor cryptomonads got stuck as my slide was drying out, and in its agony, released an explosion of ejectisomes. As any other biologist excessively attached to their subjects, I hate seeing protists die; at least this one didn't die in vain but gave us a nice demonstration. Extrusome firing often accompanies stress in protists that have them, drying out definitely qualifying. The following images are quite graphic, and not for the faint of heart. At least because the image quality is seriously compromised by a random layer of air between the coverslip and the specimen covered with remnants of water – a total chaos of refraction indexes.

Lysed cryptomonad on a dried out slide, surrounded fired ejectisomes. The fibrils around the cryptomonad remains are the uncoiled ribbons propelling the ejectisomes (refractile granules seen well in phase contrast, bottom images). 40x obj, DIC and PC.

While the cryptomonad may use its ejectisomes for hunting (most photosynthetic unicellular protists tend to be predators as well), perhaps they play a larger role in defense. Partly in stabbing its own predators, but additionally in a way that's quite counterintuitive to large creatures like us – sudden movement.

You might notice there isn't really much projectile action per se happening at the microbial scale. The firing is closer to an extrusion of a structure rather than freely propelling it a far distance. Furthermore, unlike an actual battleship, the cryptomonad can stop and turn almost instantaneously, and doesn't have much inertia. There is a reason for that, and it lies in the physics of fluid dynamics, a topic few of us outside biophysical biology concern ourselves with. Luckily, Purcell took care of that for us in his rather interesting 1977 paper, "Life at low Reynold's Number*" – turns out, the effect of viscosity on the behaviour of an object depends on its size, and water from a microorganism's perspective is a very different substance than what it is to us. In fact, it helps to imagine that microbial creatures live in honey or molasses – while water's viscosity doesn't actually change, it acts on µm-size things in a manner somewhat similar to how highly viscous fluids would act on things of our scale. Biophysics is quite a bit different at that scale, and different strategies are required in dealing with it.

*Reynold's number = proportion between object's velocity*size*[fluid density] and the fluid's viscosity)

In highly viscous fluids, coasting is not really an option. Things stop as soon as the driving force ceases to be applied, as anyone who's paddled a canoe across a lake of molasses would know (Bostonians from the early 1900's, perhaps?). This is why you don't really see stiff fins on bacteria or single-celled eukaryotes, at least not for motility itself. There are many ways to use a flagellum – a topic deserving of its own post – the beating strategy requiring it to be flexible at the right times. More importantly to our topic, you can't realistically give something enough force for it to keep moving like a bullet, so shooting things is out of question. Instead, the projectile must keep being pushed, usually by something unfolding or unraveling – in the case of the cryptomonad, a coiled protein ribbon. Cryptomonad artillery is perhaps more similar to harpoons than cannons.

This means a fired ejectisome can be used to essentially "push off" in the opposite direction, providing the organism with a sudden, drastic movement it wouldn't be able to obtain by flapping its flagella. The armoury of a threatened cryptomonad may be more important in providing it with rapid escape than damaging its pursuers. The microbial art of war is seldom discussed in non-enzymatic terms, but it is too a diverse and fascinating area, peppered with counterintuitive surprises. Life, and war, are indeed very different at low Reynold's numbers.

References
Brett, S., & Wetherbee, R. (1986). A comparative study of periplast structure inCryptomonas cryophila andC. ovata (Cryptophyceae) Protoplasma, 131 (1), 23-31 DOI: 10.1007/BF01281684

Kugrens, P., Lee, R., & Corliss, J. (1994). Ultrastructure, biogenesis, and functions of extrusive organelles in selected non-ciliate protists Protoplasma, 181 (1-4), 164-190 DOI: 10.1007/BF01666394

Purcell, E. (1977). Life at low Reynolds number American Journal of Physics, 45 (1) DOI: 10.1119/1.10903

Answer to the nucleomorph scavenger hunt: between the third (red algal) and second (plastid outer) membranes. The nucleus was originally in the cytoplasm, within the red algal cell membrane and outside the plastid. Oh, and if you want real topological clusterfuck, may I recommend the tertiary endosymbiosis in Kryptoperidinium – also try to count the genomes!

Snippets of the beauty of sliced axonemes

Look what I found in obscure ultrastructure literature!

It's a star! (Ringo 1967 J Cell Biol)

That's a cross section through the transitional zone of the Chlamydomonas flagellum. The geometric intricacy actually comes from the need to transition from nine triplets of the basal body to the flagellum's nine doublets and a central pair (of microtubules). As evident in the figure below, this can get quite tricky:

Cross-sections through a Chlamydomonas flagellum and basal body. (Ringo 1967 J Cell Biol)

This could be inspiration to some fun baking project...

Speaking of sliced flagella, there are some more wonderful patterns hidden away in the axonemes of rather obscure 'heliozoans' (centrohelids and actinophryids):

Left: Spiral arrangement of microtubules in the axoneme of actinophryid(?) Echinosphaerium. (Jones & Taylor 1981 JCS) Right: Cross section through an axopod of Actionosphaerum (actinoprhyid) containing an axoneme in the centre. m - mitochondrion. (Tilney et al. 1961 JCB)

(axonemes are the cytoskeletal support within axopodia, the long spikey protrusions that make the creatures appear like miniature suns)

And here's one so bizarre it takes three additional diagrams to explain it:

Axoneme of Cienkowskya mereschkovskyi*, a centrohelid 'heliozoan' reportedly closely related to Heterophrys. (Febvre-Chevalier + Febvre 1984 Origins of Life)

*To the slavic ear, this sounds painfully like a masculine adjective applied to a feminine genus name...owww.

And back to basal bodies, parabasalian TEMs are a work of art in their own right. Especially from Joeniids:

Rows of basal bodies in the ciliary region of Pachyjoenia howa. (Brugerolle & Bordereau 2004 Eur J Protistol)

This has nothing to do whatsoever with neither protists nor axonemes, but rather a funky-looking reject from my research images. It's cytoplasmic GFP in a partly lysed plant cell (normally the GFP should look diffuse) Not sure why it does that though, but often lysing cells become very bright, and squashed material is lush with autofluorescence.

My favourite colour is GFP.

Real posts to come soon... still mildly overwhelmed by stuff at the moment. Currently working on gathering up literature both new and old on a supergroup that may or may not actually exist, and is a complete and total mess either way (the new 'Hacrobia'). On top of other things.

To give you an idea, the previously incertae sedis centrohelids are in it, apparently, and their older grouping, "heliozoa", has only recently been dissolved (Nikolaev et al 2004 PNAS), so what is now spread out over at least two supergroups (centrohelids in 'Hacrobia' and actinophryids in Stramenopila) used to be lumped under one category and now you might as well rip all your hair out trying to figure anything out in there. It basically means you have to deal with the damn things on a genus-by-genus, taxon-by-taxon basis. (luckily, they're really cute so it's not that bad ^^) Also, a couple extensive papers happen to be written by The One We Fear, and I'm still denying the fact I may eventually have to actually read them. Grrr. Oh, and add to that the horrible and sometimes altogether lacking availability of older literature, requiring you to assemble epic shopping lists for the inter-library loan people...

Should probably also write another sentence of that manuscript to feel as if I've done something this week. Describe a couple more figures that don't actually exist yet... wish all my control pictures didn't totally suck. Redoing experiments again to get better images of...controls. Sigh. Working on projects in two completely unrelated fields (and different buildings) is a bit draining... /rant

Tree of Roots

Flagellar roots, that is. Tree of phylogenetic roots would be another fun project though...

You know when you see a page full of diagrams and get overcome by this urge to map them onto some phylogeny just for the hell of it? Especially when your other option is to actually write up the results and discussion sections your supervisor's sort of waiting for? (wrote two whole paragraphs' worth today, so I can take the rest of the day off, right?) Anyway, here comes Sleigh 1988 BioSystems p279, modern phylogeny edition:

Phylogeny of Sleigh representations of flagellar root structures. Diagrams from Sleigh 1988 BioSystems; phylogeny based on A Tree of Eukaryotes v1.2 (complete references therein).

Sleigh came up with a way to represent the structure of flagellar root apparatuses in order to compare them between various groups. These diagrams are used today by people working with protist cytoskeletons, and are reportedly a pain in the ass to make (rather unkind on one's 3D imagination capabilities). The flagellar root was traditionally considered to be a reliable character for taxonomic work, although it seems to be rather dangerous in some cases, as morphological any traits often tend to be. The flagellar root apparatus is quite complicated, and very often is responsible for the organisation of the rest of the cell. An annoying thing about them is how little is often known about the biochemistry of the various root elements, as materials besides tubulin can be freely used. In fact, older literature is full of descriptions of various fibrillar systems that have yet to be followed up on with modern cell biology techniques.

Luckily, I somehow resisted the temptation to add other people's Sleigh diagrams onto the tree; hopefully won't succumb any time soon as I actually have real work to do. Hopefully fate won't take me to Simpson 2003 anytime soon...

Does anyone else find making diagrams quite...relaxing?

(Sunday Protist on its way...keep on getting distracted while looking stuff up for it)

Reference
SLEIGH, M. (1988). Flagellar root maps allow speculative comparisons of root patterns and of their ontogeny Biosystems, 21 (3-4), 277-282 DOI: 10.1016/0303-2647(88)90023-8

Awesome talks on cellular evolution

While I'm busy and neglecting my blogging duties, have a look at these talks that someone sent me the night before a midterm (please don't do that! T_T): Evolutionary Perspectives on Mechanisms of Cellular Organisation
They are pretty awesome, although I have yet to look through all of them. But I did notice Lynch and just had to watch his talk on cell evolution:

Michael Lynch 2010 "What Would it Take to Make an Evolutionary Cell Biology?"

That talk makes me OMG SO VERY EXCITED!!!

Not only does Lynch practise a healthy dose of neutralism (the 'right' kind of evol biology, in my completely unbiased opinion...), he also seems to be very supportive of cellular evolutionary biology! =D His genome evolution stuff is pretty awesome, and I definitely recommend his 2007 PNAS The fraility of adaptive hypotheses for the origins of organismal complexity as a wonderful intro to proper evolutionary modeling -- that is, one involving drift and constructive neutral phenomena rather than dismissing them for the simplistic storytelling exercise that is adaptationism...

PS: Neutralism vs. Selectionism is a false dichotomy, btw -- selection and drift work in tandem, sometimes one stronger than the other in certain circumstances, but usually not for long. That's obviously more complicated than pure selectionism or pure neutralism (ie. people who deny selection, somehow...), which is why many people prefer to ignore one or the other. Usually drift. It's among the more poorly-taught concepts in intro biol classes, which is kind of scary. I'm an completely sick of people (esp. authors!!!) who apparently insist on subscribing to this false dichotomy, arguing over whether one entity or another is a product of selection or drift. How about BOTH? Grrrrr...

Been reading too much non-biological evolution literature. If it seemed like many biologists don't get evolution, it's about as bad or even worse in evolutionary phsychology, linguistics, etc. Of course, some non-biologists do get it. But not too many...

PPS: Google ad after I published this: "Darwinism is Not Science (And Neither is Intelligent Design) Discover a New Theory of Evolution" *facepalm*

Words that need to disappear forever: "Oncogene"

Srsly, what the fuck is an 'oncogene'? Genes involved in cancer DO NOT FORM ANY SORT OF NATURAL CLASS. You can name a gene family based on phylogeny (best), or function (still ok), but involvement in this massive variety of often-unrelated cell-cycle and growth related defects we lump together as 'cancer'? FUCK NO. And "oncoprotein" makes me hurl even farther.

Furthermore, what the flying fuck is a "proto-oncogene"!? A gene that is just poised to cause/inhibit cancer but evolution hasn't allowed it yet?

'Oncogene', 'oncoprotein' and all derivations thereof must be BANNED. There is no value whatsoever to lumping genes into such categories, especially for cancer research. First of all, we have "cancer-related", which has the vague air about it that is justly deserved ('oncogene' sounds so concrete...); second of all, you'd think that something being vaguely involved in cancer somehow must be about the most useless information ever for cancer researchers. Aren't they more interested in how this gene is involved, not that it can somehow participate in this absurdly complicated process ultimately involving the entire fucking cell?

Anyway, my two cents before I run off to a cell physiol exam... aren't you glad you don't have to teach me? =P

Yet another Woese Tree rant

o_O OMG, I'm getting infected by You-Know-Who's writing style...

I just sent this to my cell physiol prof:

The tree in slide 9 of the first lecture powerpoint is absolutely awful and grotesquely outdated (apparently based on Woese 1990). First of all, it is well-supported that the root is more likely to lie either between Eubacteria and Archaea+Eukarya, or within Eubacteria (I'm partial to the latter). It's rather murky in that particular diagram. Secondly, the eukaryote side of the tree makes one want to cry/punch a giant hole in the wall: Trichomonas and Giardia aren't exceptionaly likely to be the basal eukaryotes, and group reasonably well with Trypanosoma+Euglena, forming what may well be a monophyletic group. In any case, Trichomonas and Giardia seem to form a clade.

Dicty is WAAAY more closely related to Humans+Yeast than either Maize or Paramecium -- the later two are on a fundamentally different branch of eukaryotes -- the bikonts (eg. Cavalier-Smith 2009 J. Euk. Microbiol) or the corticates (Cavalier-Smith 2010 Biol. Lett.) Dicty, Humans and Yeast are members of a quite well-supported group (Unikonts), and this tree erroneously suggests that multicellularity evolved in the common ancestor of Plants, Animals and Fungi, which is completely wrong. Multicellularity evolved in each of those lineages independently, and in quite a few other lineages as well, up to ~16 times in the Eukaryotic kingdom (King 2004 Dev. Cell) and arguably a few times among prokaryotes as well (eg. Streptomyces).

I can't believe this is in a 2008 edition of the textbook! This tree has been outdated for at least a decade! How does one go about formally complaining with things like this? I refuse to let any suggestion of "crown/higher eukaryotes" vs. "stem/lower eukaryotes" go by unnoticed. The very notion of higher/lower organisms is thoroughly wrong and must be abandoned. Even in an introductory diagram. ESPECIALLY in an introductory diagram. Especially when they have the nerve to HIGHLIGHT Maize, Yeast and Humans as if they're some special enlightened life forms.

The era of Woese 1990 is over. We can move on anytime now...

The diagram comes from the "Big Alberts" text, 2008 edition. They should bloody know better by now! >.<

Candidate for the Twisted Tree of Life Award?

Also, their 'interesting' spelling of eukaryotes is in itself a rantworthy topic for some other day...

Heterolobosea II - 'Split Morphology Disorder': amoebo-flagellate transformation in Naegleria

Earlier, in Heterolobosea I, I promised brain-eating amoebae with a split morphology disorder. Having a bit of a morphology fetish, I find the latter topic fascinating, so bear with me as we get into some gory details of cell biology, which I strive to make at least somewhat readable to sane human beings. As always, please let me know if anything is unclear, or *gasp* inaccurate...

Fundamentals of cellular morphology
Most organisms strive to have some semblance of shape (including bacteria). To crudely simplify matters, in the style of biochemistry, cells are sphaerical double membraned lipid vesicles. Thus, by default, a cell 'wants' to be a round blob. That would be it's natural state.

But most cells are not round blobs. In fact, they can have some rather complex shapes like metazoan neurons, forams, parabasalians or the endlessly weird and sophisticated ciliates. The deeper you venture into the realm of protist diversity, the more awe-inspiring the cornucopia of variety that is cell morphology. Luckily, this vast variety also has some order to it, for much of it happens to fall into two fundamental 'genres' of cellular morphology: flagellates and amoebae*. Of course, as with anything in biology, there are exceptions, and things that have a comfortable niche in-between. Unicellular organisms also have this tendency to construct cortical of extracellular structures, which can have a large influence on their shape. But the key determinant of cellular morphology remains the cytoskeleton, and the composition of the cytoskeleton largely determines which of the two categories the cell allies to. The cytoskeleton is an intricate, often highly dynamic, complex system; it is a misconception to see cells as bags of cytoplasm with organelles floating about -- the innards of a cell are strictly regulated and usually connected to various cytoskeletal and membraneous elements. Things don't just float about aimless inside there.

*There's also a third type, the cyst, which is basically a lack of morphology (rounded up cell) usually with external protection of some sort. It often lacks any elaborate cytoskeletal organisation, both actin- and tubulin-based. Cysts are often used for mitosis or meiosis, as well as resting through periods of unfavourable conditions.

There are two main component systems of the cytoskeleton: actin and tubulin, ignoring the plethora of miscellaneous proteins that have been used for various structural jobs. You are likely familiar with microtubules as spindle fibres during mitosis. You may also be familiar with actin as a key player in cell motility and morphology in animal systems. Tubulin is also important for the flagellar apparatus -- we've yet to find one composed of actin (and probably with good reason). Actin is involved heavily in endomembrane trafficking within a cell, as well as endo- and exocytosis. If interested, this week's issue of Science has a nice overview of actin in morphogenesis and cell movement.

Their roles in morphogenesis, the formation of morphology (in this case, cellular), are much less clearly defined. Furthermore, it depends largely on the species -- plants, for example, rely very heavily upon microtubules for morphology, with actin being a minor player. Interestingly, plants also lack centrioles or basal bodies, except for in some gametes if I recall. Thus, the mitotic spindle in plants (and diatoms) forms entirely without centrioles. Yeast cells can also do mitosis without basal bodies if you surgically remove them. It seems like the characteristic centriole arrangement at mitosis is a way to ensure their inheritance in both daughter cells, rather than organising the spindle. More on this later, it will become relevant.

Amoeboid cells, as you may know, are primarily actin-based. In fact, amoebozoans tend to suck at internal microtubules, except for spindle formation at mitosis. They hate tubulin about as much as plants hate actin. Actin-based cells don't have to be amorphous -- they are still able obtain complex morphologies like that of neurons (although there may be some serious tubulin involvement in that. My knowledge of animal cell biol is rather pathetic). But there is a positive correlation between amoeboid-ness and actin-ness ('actinity'?). Turns out, the entire unikont clade, if it exists (ok, opisthokonts and amoebozoans), seems to really prefer actin over tubulin.

In contrast, flagellates are primarily tubulin-based -- of course, they still use actin for some intracellular work, but the shape depends largely on the whims of the 'tubes (microtubules, in the slang of our local plant cytoskeleton lab...). Perhaps not relying much on flagella in the amoeboid case allows them to lose so microtubule organisation pathways, thereby switching to actin; flagellates tend rely heavily on intact tubulin systems, and may thus be less prone to losing their structure. Also, if you're a flagellate, you kind of need shape for a modicum of streamlining -- try swimming around as a flattened reticulate blob of some sort! Keep in mind that life at that scale is very different -- viscosity becomes a crucial factor when considering unicellular motility. Perhaps being hydrodynamic isn't even as important as simply retaining shape. Otherwise you'd be like a blob of molasses trying to swim through a sea of maple syrup. Not gonna get very far.

Whatever the reason, amoeboid cells tend to have a predominantly actin-based cytoskeleton; whereas flagellates have a penchant for tubulin. Of course, not all organisms are decisive enough to make this committment, so we've got amoeboflagellates in the middle:

But your conventional amoeboflagellates are only the beginning -- plenty of cells, especially Cercozoans for some reason, fancy transitioning between being more amoeboid or more flagellate. But few cells actually dispense with flagella and basal bodies altogether, only to form them anew when special conditions arise.

As we've seen earlier, de novo centriole formation was until recent considered fairly impossible (ignoring the organism we're about to prod at). As we all know, there must be a reason for the guaranteed inheritance of centrioles at mitosis, and they must be hard to form. After all, tubulin nucleation doesn't happen randomly very often, and new microtubules seldom start without some sort of 'seed' (gamma-tubulin), as the tubulin has to form a ring prior to growing into a tube, which isn't likely to happen on its own. (Actin, on the other hand, is only two monomers wide, and can form spontaneously relatively easily) Thus, for basal bodies to pop out of nowhere is also rather unlikely, but, as we're about to see (and as several more recent experiments show in yeasts and animals), de novo centriole formation can and does happen.

Naegleria and Tetramitus: Heteroloboseans with 'split morphology disorder'
Several paragraphs into our journey, we've yet to see any Heteroloboseans. Let's change that. Meet Naegleria, famous to medical biologists as a brain-eating opportunist, and to real biologists as the master of de novo flagellar creation:

Naegleria gruberi. (by Jonckheere at ToLWeb)

And before someone accuses me of focusing on biomedically relevant organisms, Tetramitus, another Heterolobosean, does it too, and has more flagella to oogle at:

Dramatic transformation of Tetramitus between amoeboid and flagellate forms. (Outka & Kluss 1967 JCB)

In fact, while I failed to find a nice picture of Naegleria's flagellate form, that of Tetramitus reveals its marvelous complexity. Keep in mind this thing was a 'formless' amoeba a few hours before:

Quite a complex structure to be formed within a 4h period! Note that while the organelle positions in amoeboid cells are fairly flexible, they become fixed in the flagellate form. It even has subpellicular microtubule bundles, a cytostome, and the oral groove characteristic of Excavates. This cell is unquestionably worthy of being called a flagellate, so this really is a complete transformation between two fundamentally different cell morphologies. (Outka & Kluss 1967 JCB)

Of course, heteroloboseans are not the only group to claim amoebo-flagellate transformations; other predominantly flagellate groups like the parabasalia also contain organisms with a bit of a split personality morphology disorder: Gigantomonas herculea, for example, is a termite endosymbiotic Trichomonad that can also switch between amoeboid and thoroughly flagellate forms (Brugerolle 2005 Acta Protozool.). However, unlike Naegleria, Tetramitus et al., they still retain basal bodies during the amoeboid stage.

Of Naegleria's culinary preferences
But because of Naegleria's pesky little habit to occasionally get into human brains and eat them (oops!), it gets a lot more research attention (Which, in the world of Heterolobosean biology, doesn't really mean very much) It should be noted that while there is some waves of 'Naegleraphobia' out there, incidences of human infection are very very VERY rare (small handful of cases a year; some years without any cases whatsoever). Unfortunately, it is almost universally fatal, but so are many much more ubiquitous diseases. But that's enough excuse for the media to put up a scary article on Naegleria every once in a blue moon, often accompanied by an image of... Amoeba proteus or Chaos sp. Hey, at least they're still eukaryotes!

Before we continue on with our hard core cell biology, let's clarify Naegleria is not a parasite, but rather an opportunist that seems to not mind the 37 degree heat of the human body. It gets into the human brain through the nose, generally from swimming in warm water. I'm not sure if it even makes it past the brain - it may eat the brain, kill its host, and end there, as the likelihood of ending up in a suitable environment after invading the brain is pretty low. So Naegleria's gastronomic inclinations may well just be an accident, albeit a rather costly one to the few humans it infects.

Ok, that exceeded my dose of biomed tolerance for the next three months. I often add "-clinical -patient -pathology" when searching for articles online; that's how much I avoid medicine. I'm a cruel, evil person, I know =P

Amoeboid to flagellate transformation
So here's an overview of Naegleria's life cycle. It consists of an eating and mitotically able amoeboid form (interestingly, mitosis happens without basal bodies), a flagellate form induced by addition of water (where swimming is favoured over crawling). Presumably, it can still feed in its flagellate form, considering it bothers to construct an elaborate oral groove and cytostome. The third form is a resting cyst, which it forms and hibernates in when times are bad. Thus, Naegleria's life cycle actually involves all three fundamental cell types, which is why it's such a wonderful system for studying the transformations between them (and cellular differentiation.)

Naegleria's principal morphology is amoeboid, which is the stage for mitosis. Flagellation happens upon addition of water, whereas encystment is often a response to unsuitable living conditions. Note that mitosis happens without basal bodies. (based on this figure, in turn based on Fulton 1970 in Methods in Cell Physiology; also, don't pay any attention to the portrayal of the flagellar root appartus...)

I'm probably trying to get through way too much in one post, so I'll unfortunately have to do an injustice to the gory details of the cell and molecular biology behind this process, and just give a skimpy overview.

The transitiona between the principal cell morphologies involve a regulation of actin and tubulin monomer levels - or the amount and concentration for the 'building blocks' for each morphology. In fact, the reason many organism encyst for mitosis (or at least reduce their flagellar length) is to redirect tubulin from the cytoskeleton and flagellum to form the mitotic spindle. Otherwise, you'd have to synthesise a bunch of new tubulin which would soon become useless. If you look at some images of Naegleria amoebae undergoing mitosis, you'd notice the cells are much rounder than normal.

Naturally, there's also a regulation of proteins involved in cytoskeletal organisation for each system (and the interactions between them), but that doesn't seem to be studied at all in Naegleria. We do know that the commitment to flagellation involves a rapid increase of tubulin production, since the amoeboid form has barely any.

A quick caveat: a common misconception is that flagella are microtubular things that 'stick out' of the cell, and are on the 'outside' -- flagella are bound by the cytoplasmioc plasma membrane, so the tubulin is quite accessible to regulation. To give you an idea of how a flagellum grows, have a look at these TEMs from Naegleria:

Note that it's still surrounded by the plasma membrane. There's even a little knob at the growing end. (Dingle & Fulton 1966 JCB)

Naegleria takes ~120min at 25C to transition from an amoeba to a flagellate. The earliest event in this process is the initiation of tubulin synthesis, followed by basal body assembly 30min later. Cells round up as actin filaments depolymerise, and basal bodies begin to sprout flagella at about the hour mark. In another 20min, the tubulin cytoskeleton is molded into the proper flagellar body shape, with flagella reaching full length at ~110-120min.

See text. Actin vs. tubulin concentrations are relative, and do not imply either reaches zero at either end, although the amoeboid tubulin concentrations are very low, whereas actin levels remain more or less constant. (After Fulton & Walsh 1980 JCB; free access)

The data above was gathered rather painfully via drug experiments -- you can target actin and tubulin for various forms of disruption (as well as protein synthesis), and determine at which timepoints actin or tubulin is the most crucial for a proper differentiation. Now we have sleek antibodies and GFP and genetic tools of all sorts (they had many of the techniques before, but they has become much cheaper, easier and higher quality over the years. Perhaps at an expense of the quality of science itself, since you can cover up sloppy work with sexy pictures quite a bit easier...), so we can actually see the changes in actin and tubulin throughout the transformation process:

Immunostaining of the cytoskeleton during flagellar transformation. red - actin; green - tubulin. Yellow - co-localisation of actin and tubulin. Note that these are different cells, as they had to be fixed (killed) for immunostaining. I would love to see a life cell timelapse of this process!* Note how the flagellate morphology is 'molded' by the longitudinal microtubule bundles. (Walsh 2006 Eur J Cell Biol)

*Doesn't look like Naegleria has had its genome sequenced yet. And yet we're about to sequence 10K vertebrate genomes. Would they mind sparing us a couple slots? But once it happens, transgenic lines and away we go with sexy timelapse. In 4D. *drools* (4D hyperstacks are really hard to analyse, so despite sounding sexy, the scientific value of some of their applications is debatable)

Hopefully I've convinced you at least that Naegleria would make an interesting model for studying cell differentiation and transitions in morphology, as well as cytoskeletal dynamics. While the field is still miniscule, at least its non-clinical component, there is work being done right now on investigating the regulation of cytoskeletal development, such as this paper on the localisation of microtubule-related mRNAs. But not much.

It's actually kind of worrisome: the generation of protist cell biologists from the 60's and 70's is now retiring, while the younger researchers in protistology are predominantly molecular biologists. They're useful, sure, but I insist that there's more to an organism than its genome. I know a few molecular biologists who agree, but they still do genomics work! They say there's wonderful opportunity in investigating protist cell biology in the near future, but if the older cell biologist retire before they can pass on their knowledge and skills and wisdom, are we going to now be set back a good decade or two, wasting our time to make the same mistakes and learn what has already been learned? Fancy equipment cannot replace experience and insight, nor can insight be written into a paper.

But anyway, I'd just like to point out again the humans, yeast and Arabidopsis are not the best organisms to study all biological processes - they're good for some things, and not quite so nice for others. This is where we should use a wide diversity of models to understand processes that are more accessible there - such as flagellar transformation in Heteroloboseans. I'm not aware of any other system that involves such a complete transition between three fundamental cell types, even with de novo basal body formation! Seriously, what more can you ask for? (let's hope it has a 'nice' genome...)

Apologies to the less cell-biology-oriented portion of our audience. But at least we've got pretty pictures! =P

References
Dingle AD, & Fulton C (1966). Development of the flagellar apparatus of Naegleria. The Journal of cell biology, 31 (1), 43-54 PMID: 5971974

Fulton C, & Walsh C (1980). Cell differentiation and flagellar elongation in Naegleria gruberi. Dependence on transcription and translation. The Journal of cell biology, 85 (2), 346-60 PMID: 6154711

González-Robles, A., Cristóbal-Ramos, A., González-Lázaro, M., Omaña-Molina, M., & Martínez-Palomo, A. (2009). Naegleria fowleri: Light and electron microscopy study of mitosis Experimental Parasitology, 122 (3), 212-217 DOI: 10.1016/j.exppara.2009.03.016

Outka DE, & Kluss BC (1967). The ameba-to-flagellate transformation in Tetramitus rostratus. II. Microtubular morphogenesis. The Journal of cell biology, 35 (2), 323-46 PMID: 4861775

WALSH, C. (2007). The role of actin, actomyosin and microtubules in defining cell shape during the differentiation of Naegleria amebae into flagellates European Journal of Cell Biology, 86 (2), 85-98 DOI: 10.1016/j.ejcb.2006.10.003

Sunday Protist - Scenedesmus: Of coenobia and cell cycles

Busy starting working on slides for a lab meeting talk tomorrow, so today's Sunday Protist will be very brief and undetailed. And thoroughly uninformative. For real this time. Seriously, I say this every freaking time and end up writing a minireview on the damn thing... it's just so damn alluring to go and read up random stuff on these organisms!

We've been ignoring Archaeplastids again. Here, have some Scenedesmus:

Scenedesmus dimorphus(?) 40x obj, DIC

Ok, my images are shitty (damn thing is motile!), let's look at a professional one:

Source here; Scenedesmus in DIC

What you see here is a green alga that divides multiple times without splitting off to form a coenobium - a clonal 'colony' of cells sharing an immediate ancestry. The sizes of coenobia depend on environmental conditions, such as availability of nutrients and sunlight, as well as presence of predators (they grow bigger to avoid being eaten). Often the end cells grow spikes (like the ones in the lower image).

Actually, this thing is somewhat relevant to the slides I'm working on - regulation of mitosis (and 'counting' cell divisions) is a very important aspect of multicellular development, since it's not helpful to have cells that divide too much or too little. Actually, of extreme interest to humans is this slight issue that arises when cells divide too much - we call it cancer. As some may sadly know personally, the effects of uncontrolled division can lead to rather dire consequences.

In Arabidopsis, stomata consist of a pair of guard cells surrounding a pore. Those guard cells come from a common precursor, called a guard mother cell (GMC). The stomatal lineage has a few specific genes seemingly responsible for enhancing the regulation of GMC division to ensure it happens once and only once. A double mitosis at that stage would result in guard cell clusters (which poses a problem to their opening and closing). There are some mutants where the regulation of this division gets screwed up - in some lines, you get single guard cells (ie no mitosis, and no pore); in others, you get clusters, sometimes massive ones. The molecular and genetic details behind cell cycle regulation are a fucking mess, and make one's head spin. Which is what makes giving talks on the subject so much [evil] fun! XP bwahahaha!

Now another question regarding those clusters is whether the nuclei go wild first, and then cell division follows to ensure each is separated off (sorta like palintomy), or whether the cells fail to exit the cell cycle upon division, and keep going into mitosis over and over again (with the complete set of mitotic events). When speaking of division, one must be careful to keep track of nuclear ploidy levels (DNA replication), karyokinesis (nuclear division) and cytokinesis (cell division). Now 'mitosis' is one of those words that's used to describe most, some, or all of the above, as if the cell cycle isn't confusing enough as is. Note that each of these proceses interact with a million other things, and have regulatory checkpoints and so on. Which results in wonderful diagrams like this or papers like this or this Annual Review from hell. Seriously, you do NOT want that latter paper as a first intro to cell cycle. You just don't.

If any of the above confuses you, the point is that the cell cycle is bloody complicated.

Back to Scenedesmus. The coenobium produced offspring inside each of its cells, which then vacate their parent cells and go on to explore the great world beyond. The characteristic divisions happen while the daughter colony coenobium is still inside the parent cell. (Pickett-Heaps & Staehelin 1975 J Phycol), and depend on the conditions present during the division. The details are descibed in (Zachleder et al. 2002 Eur. J. Phycol), which I'll try to walk you through (and hopefully not fail too badly). So this is roughly how Scenedesmus regulates its length (in terms of cell number) in response to the length of light periods:

Cell cycle events in daughter coenobia under various conditions.
A) Typical cell cycle. G1 - 'gap' one (main growth phase), S - 'synthesis' (DNA replication), G2 - 'gap' two (post replication growth phase), M- mitosis.
B) Cell cycle of Scenedesmus under high temperature and low irradiance. CP - commitment point, where the cell 'commits' to divide regardless of environmental conditions to follow. Unlike the canonical model (A), where the cell immediately undergoes cytokinesis after karyokinesis, the daughter coenobium undergoes another growth phase (G3) prior to undergoing cell division. Thus, this is a nice model for separating nuclear mitosis and cytokinesis.
C) Placing the synchronised cells into the dark after the first commitment point results in only one round of mitotic divisions. (the bar above represents amount of time spent in light and dark) (presumably, the cells don't divide in the dark)
D) Cells placed in the dark after the second commitment point. This results in two rounds of mitotic divisions, which also overlap (so the S of the second stage hits the nuclei when the first division stage is at M; ie during mitosis, the nuclei undergo replication. It would be so much fun to prod at this at the molecular level! *drools*
E)Darkness after the third commitment point. Three overlapping cell cycles. Note how G3 of the first cycle is elongated in order to undergo cytokinesis after all the nuclear divisions have been carried out. Seriously, how does the organism coordinate its cell cycles carefully enough to allow this madness to proceed smoothly?
(Zachleder et al. 2002 Eur. J. Phycol)

So what happens under constant light? (something that never happens in nature save for the polar regions)

The 4th commitment point is activated, but the G3 of the first cycle fails to proceed long enough to allow for the fourth nuclear mitosis to occur prior to cytokinesis. The cell divides while the nuclea still have twice the proper ploidy. The nucleus then divides after the cells are released from the parent, and you end up with a dikaryotic/binucleate organism. (Zachleder et al. 2002)

And of course, how can you do anything cell cycle related without drugs?

Cell cycle arrest via 3h cyclohexamide treatment (which halts protein synthesis) results in longer cell cycle and increase in the number of commitment points within a single division cycle (from main text). Under constant light, the mitotic phase in each of the cycles was postpones, even in the third and fourth cycles, whose commitment points occured after inhibitor removal. Thus, this drug affected the regulation of all of the overlapping cell cycles within that generation. Furthermore, even though the fourth cycle was initiated after the inhibitor removal, it had a greatly elongated G2 phase and still underwent mitosis after cytokinesis and release of the daughter coenobium! (thereby resulting in binucleate cells yet again). (Zachleder et al. 2002)

Of course, when you have one drug, why not try another? (the gateway drug concept works quite well in cell biology research, perhaps even better than in 'real life'...)

A) Constant treatment with FdUrd (fluorodeoxyuridine), which blocks the S-phase, causes the cells to grow in size, but fail to undergo replication (thereby leaving the nucleus at the normal G1 levels). Interestingly, the pathways regulating cell size remain oblivious to the failure of nuclear replication.
B) The removal of FdUrd after the second commitment point results in a highly unusual (for Scenedesmus) division patterns, where the G3 phase disappears altogether, and cytokinesis happens immediately following nuclear mitosis. While the cell cycles still overlap, they are no longer coordinated to 'wait' for the last nuclear division to be complete. This pattern is quite normal for its relatives, including Chlamydomonas. (Zachleder et al. 2002)

Interestingly, the adult organism still develops properly, suggesting that perhaps this strange mode of division may be a byproduct of other changes in cell cycle regulation. I can't say much more about this, as evolutionary biologists seem to REALLY not care about cell cycle regulation. They already tend to neglect the existence of cells (ie, organisms are more than just their genomes!), and it will be a while before we realise that those cells have clocks, which also evolved through time. Wait, but what about irreducible complexity? Not the clocks!!! (that was a really cool animation of simulation of clock evolution!)

So now I wonder whether the mutant stomatal clusters undergo something weird similar to this, or if something else is going on entirely. Being a systematics-loving cell biologist guarantees that I'll never have any friends, but I'm still surprised and disappointed by my colleague's strict aversion to anything that is not their pet model organism. I think comparative cell biology could do wonders to our understanding of the mainstream systems, as well as the very fundamentals of how the unit of life can function. Actually, Scenedesmus got me thinking just now...the most likely thing would be your usual "cell forgets it shouldn't divide" scenario, ie. a differentiation/cell cycle exit lag. But the overlapping cell cycles and a palintomy-like division remains a competing hypothesis that must be dealt with.

Another interesting detail (and now I'm pulling crap out of my ass) is the presence of spikes on the ends of the Scenedesmus coenobium -- somehow, the end cells 'know' they're at the end. Now what is interesting is whether they sense some intercellular morphogen gradient (unlikely), or can sense some signals from neighbouring cells, and the absense of this signal on the end may promote spike growth. Again, this ties in with the stomatal pathway - stomata are generally spaced out without [ideally] any of them touching (although that may happen once or twice per wild type leaf, by accident). Since they rely on opening and closing to function, being stuck together creates a bit of a hinderence to proper functioning. There are several mechanisms to ensure that when a precursor cell divides to form a stomatal precursor (a meristemoid), the meristemoid is on the opposite side of another stomatal lineage cell. Perhaps Scenedemus terminal cells use a similar method of 'neighbour sensing'.

Would be fun just to assemble a showcase of the diversity of the various cell cycles throughout both eukaryotic and prokaryotic domains of life. The cell cycle is fundamental to anything that divides and propagates (that is, anything biological), and there are some truly fascinating deviations from the canonical models!

WE NEED COMPARATIVE CELL BIOLOGY TO HAPPEN! Come on guys, don't leave it to the idiots like me to dabble in on our spare time! DO SOMETHING. Srsly. People are choking each other in biomed because there's simply no space for everyone to be there, whereas comparative cell biology and cellular evolution seem to be overgrown by weeds, with an occasional ball of tumbleweed rolling by, to the chorus of crickets in the background, and TC-S solitarily strolling by and churning out his Univeral Theories of Everything with no one to argue with. He must feel very lonely there. He may even want company.

This must be the strangest advertisement ever. ^_^

Anyway, here was my very short, completely-devoid-of-scholarly-literature Sunday Protist entry. Is it a problem when you just can't resist the tempation to read random crap about random crap? Damn. It's wrecking my undergrad performance! (Seriously. No one in those classes gives a damn that you've read hundreds of papers more than any other undergrad around. Grrr. Fucking pre-meds and those who cater to their whims. Grrrr.)

Anyway, now to skip over a couple phyla to my plant cycle... must prepare slides to I can pretend I've actually done something lately during the lab meeting. Ummm... lots of background should be able to confuse them long enough to not realise none of that is actually my own work! =P Especially if we stop to discuss the convoluted cell cycle diagrams at every opportunity (and with my boss, we will!) also, I'm armed with a couple (incomplete) graphs at the moment! Graphs are powerful creatures in biology. "I think your theory may be flaw-..." "I QUANTIFIED STUFF!" "Oh...ok! =D"

*Also, a small rant (can a blog post ever be complete without a rant?): could people please take the effort to discriminate between a true colony and a coenobium? If a bunch of organisms of different ancestries congregate together, that would be a colony; if an organism reproduces a bunch of times but the offspring don't split off, that's a coenobium. A coenobium is clonal, and a much more likely form of cooperation. Although coenobium generally applies to unicellular organisms, so it gets murky when similar stuff happens in clonal colonies of multicellular organisms. Perhaps one should make it mandatory to specify whether the 'colony' is clonal or not in those cases? It makes a difference from the evolutionary perspective... also, my understanding is that random stuff congregating together is actually a pretty unlikely occurence compared to clonal stuff not bothering to break apart. Would that be true?

PS: this thing is kinda cool-looking; also unicellular and multinuclear! (ie. coenocytic) Btw, if anyone is confused with regards to multinucleate/coenocytic/plasmodial, they all mean pretty much the same thing, except the former tends to be used by zoologists, the middle by botanists and phycologists, and the latter by mycologists (and to some extent, developmental zoologists). Since we don't really ever talk to each other, there's plenty of opportunity for the language to diversify into multitudes of mutually unintelligible dialects - would this be an example of sympatric or allopatric speciation then? Is a discipline more like a niche or a geographically isolated location? Or both?

References
Pickett-Heaps, J., & Staehelin, L. (1975). THE ULTRASTRUCTURE OF SCENEDESMUS (CHLOROPHYCEAE). II. CELL DIVISION AND COLONY FORMATION1 Journal of Phycology, 11 (2), 186-202 DOI: 10.1111/j.0022-3646.1975.00186.x

ZACHLEDER, V., BI??OV??, K., V??TOV??, M., KUB??N, ?., & HENDRYCHOV??, J. (2002). Variety of cell cycle patterns in the alga Scenedesmus quadricauda (Chlorophyta) as revealed by application of illumination regimes and inhibitors European Journal of Phycology, 37 (3), 361-371 DOI: 10.1017/S0967026202003815