Naegleria: a classic model for de novo basal body assembly

As has been discussed above, Naegleria was one of the first reported cases of de novo basal body assembly [4], and for decades remained the best-studied example. It was also by studying Naegleria differentiation, in particular the induction of ?- and ?-tubulin isoforms specific to flagellates, that led to the origin of the multitubulin hypothesis, which predicted the existence of multiple types of tubulin that would be used to build different cellular structures [5]. Both flagellar ?- and ?-tubulins, which are incorporated into basal bodies, flagella, and cortical microtubules, undergo highly regulated synthesis during differentiation [3, 5, 22, 35, 36]. Evidence has been presented that another, very divergent, ?-tubulin is used for mitosis in Naegleria [37].

An area of great promise for future research in Naegleria is how the majority of differentiating Naegleria cells assemble exactly two basal bodies and two flagella. There are already some provocative observations in the literature that hint at an interesting counting mechanism.

Naegleria strain NEG is normally diploid (2n) [11], but in culture it often becomes tetraploid (4n), presumably due to failure of mitotic nuclei to separate [2] (p. 459). While the diploid strains tend to have two flagella (2n-2f), the tetraploids initially tend to have four flagella (4n-4f). This configuration is metastable, however, and after some growth in culture tetraploid cells tend to revert to forming two flagella upon differentiation (i.e., 4n-2f). In this state, they look very similar to strain NB-1, which is a stable tetraploid that typically makes two flagella (i.e., 4n-2f). In both cases, 4n-2f cells seem to have looser control over their counting, with around 20 % flagellates having 3–4 flagella, compared to only 2 % of 2n-2f NEG flagellates [2] (p. 413). These simple observations are easily reproduced [2, 25], but perhaps more challenging to understand. While ideas of possible precursors that divide along with cell division are appealing [38] (p. 199), they do not seem necessary since known proteins seem sufficient to nucleate the formation of a new basal body independent of any precursor structure (e.g., [39, 40]).

Strikingly, sublethal temperature shocks at appropriate times during differentiation can dramatically increase the number of basal bodies and flagella that Naegleria assembles [41, 42]. For example, on average strain NB-1 normally assembles 2.2 flagella. However, after a 38° temperature shock, this average rises to 4.5, with a range of up to 18 flagella on a single cell [41]. These multiflagellate cells display disorganized swimming and tumbling. When these flagellates revert to amoebae in the same nonnutrient environment, they immediately redifferentiate without division, but with only the normal number of flagella (average of 2.1) [41]. Why heat-shock temporarily alters flagellar number, as well as the nature of the normal control mechanism, remain interesting challenges for future investigation.

In three published reports from JooHun Lee’s laboratory, it has been suggested that a novel entity regulates Naegleria basal body assembly in an unprecedented manner [43–45]. Their work presents evidence that Naegleria amoebae maintain a novel protein complex through numerous generations. This complex, containing a Naegleria transacetylase protein, is reported to accumulate ?-tubulin, pericentrin, and myosin II. The resulting “GPM” complex, present in amoebae, moves to the site of basal body assembly, and provides the focus where two basal bodies form de novo. Then the complex (including ?-tubulin) leaves the site of basal body assembly, travels to the other end of the cell, and disassembles, leaving the basal bodies behind. In this study, the presence of ?-tubulin is used to build the hypothesis that the complex might transiently nucleate the start of basal body assembly. Although provocative, the reliance on mammalian antibodies without properly defined epitopes in Naegleria to trace the movement and fate of the GPM complex leaves room for serious disagreement with these findings. In the experience of our laboratories, Naegleria proteins are sufficiently divergent from other species that the immunofluorescence signal when using heterologous antibodies (if there is any) is almost always to unknown antigens, or proteins trapped at the posterior end of amoebae (e.g., [8]). Specifically, both our labs have tried heterologous antibodies to ?-tubulin, without success. This is in stark contrast to results obtained by using affinity-purified antibodies raised to the single Naegleria ?-tubulin gene product. These antibodies reveal that ?-tubulin is localized to the basal bodies during their assembly, and remains stably localized there—parallel to the result observed for ?-tubulin in other species [8]. In addition, our results indicate that ?-tubulin, like other basal body proteins, is not present in amoebae: the mRNA for ?-tubulin is induced early in differentiation [9], and ?-tubulin antigen accumulates as the basal bodies are assembled [8]. The fact that Lee’s results show the heterologous antibody epitopes are already present in amoebae, and go on to dissociate from the basal bodies, make it seems likely to us that the recognized epitope is not ?-tubulin. In their most recent paper [44], Lee et al. used a new antibody to a Naegleria ?-tubulin peptide, but in immunogold electron microscopy found that this antibody did not colocalize with the structure recognized by the heterologous ?-tubulin antibody they had used to define the GPM complex. (Similar objections apply to the heterologous pericentrin antibody they used; in this case it is also unknown what epitope is staining, and no pericentrin gene has been curated in the Naegleria genome). While the Lee laboratory’s ideas are provocative and interesting, resolving the issues caused by heterologous antibodies as well as more precise colocalization studies are essential to understanding their results. We hope these issues can be resolved in the near future.

Given the current interest in control of centriole formation, we would love to be able to discuss the role of individual genes in the control of Naegleria basal body assembly. For example, in animal cells there have been a series of key papers dissecting the role of polo-like kinase 4 (PLK4) in the control of centriole assembly and number (e.g., [46, 47]). In these animal cells, PLK4 localizes to existing centrioles and there becomes activated and appears to regulate the normal assembly of a single new centriole. In addition, overexpression of PLK4 can induce de novo centriole formation. One can imagine such roles for PLK4 in the rapid formation of basal bodies during Naegleria differentiation, but so far no Plk4 gene has been recognized in the Naegleria genome. This could be due to genetic divergence, but a comparative study indicates that orthologs of Plk4 may be limited to Ophisthokonts (animals and fungi) [48]. While Naegleria
Plk1 might play the role of Plk4 in the amoeboflagellate, any role of polo-like kinases in this system remains a challenge for future research, particularly given the current lack of tools for gene manipulation in Naegleria cells.