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