Given the evidence summarized above, the last common eukaryotic ancestor had a motile 9+2 flagellum, was anchored on a basal body of triplet microtubules, and required IFT for its assembly. How did such a complex system evolve? Clearly it must have been preceded by a microtubule cytoskeleton with dynein and kinesin motors. Strong arguments have been made that the driving force for the evolution of a microtubule cytoskeleton and its associated motor proteins was the ability to accurately segregate a large, nuclear membrane-enclosed genome by mitosis. Although the complicated checks and balances used to assure mitotic fidelity vary widely in extant eukaryotes, some aspects of mitosis have been sufficiently conserved and are so central to the process that they must have been present at an early stage. Other aspects of mitosis, assumed to be ancient because of their simplicity, reveal a minimal apparatus but may not reflect the ancestral condition. Although model organisms such as fungi have provided many details of such minimal systems, the tremendous variety of extant mitotic mechanisms (as reviewed in ref. 41
) should not be forgotten. Mitosis requires a microtubule organizing center (MTOC) that duplicates once per cell cycle, a connection between each chromatid and one of the duplicated MTOCs, and separation of the MTOCs with their associated chromatids. These MTOCs vary structurally from the simple nuclear membrane-embedded spindle pole bodies of some unicellular organisms to the complex centriole-containing centrosomes of many metazoan cells. Most organisms form two microtubule arrays during mitosis, one oriented toward the chromosomes to link each chromatid to its MTOC, and a second that assembles between the two MTOCs to form a scaffold for MTOC separation. In G1
phase of the cell cycle, prior to DNA and MTOC duplication, the primitive cell would have had a single MTOC, with a single array of microtubules directed away from the nucleus that defined the polarity of the cell. It is this cytoplasmic microtubule array that most likely provided the raw material for evolution of the flagellar axoneme42–44
A polarized array of microtubules projecting from one side of the nucleus, as seen in most cells today, need only become linked into a bundle to provide an organelle that could distort the cell membrane and form a protoflagellum (). Microtubules radiating from the MTOC that were not incorporated into this protoflagellar bundle would continue to provide a cytoskeleton for general cytoplasmic transport and organization of the endomembrane system, and the interaction of motors such as kinesin and dynein with this microtubule cytoskeleton would provide directed motility of associated vesicles. Such movement along the protoflagellar bundle could direct exocytosis and endocytosis to a specific region of the cell membrane, creating a new membrane domain. The similarity of IFT proteins to proteins involved in vesicle trafficking18
and the similarity of IFT kinesin and dynein to cytoplasmic versions of these motors, argues that axonemes evolved from proteins that were already in use in microtubule-based vesicular transport systems.
Figure 3 Proposed steps in the transition from an early eukaryote, with a polarized morphology based on asymmetric placement of a microtubule organizing center, but lacking flagella (left), through an intermediate with a protoflagellum that supported gliding and (more ...)
Early eukaryotes, lacking any other means of locomotion, were presumably benthic amoeboid cells and could not yet swim. Microtubule-based motors moving along a parallel bundle of microtubules in the protoflagellum would have provided at least two specific advantages to these organisms. Simple coupling of retrograde movement to transmembrane proteins would convert the protoflagellum to a feeding organelle, bringing particles that adhered to its surface back toward the cell body by retrograde IFT for subsequent phagocytosis. Alternatively, if substrate adhesion through IFT-associated protoflagellar transmembrane proteins was strong, and cell body adhesion proportionately weak, retrograde IFT could support gliding motility. Flagellar gliding as a means of locomotion is common in many pelagic, benthic and soil flagellates today, and such surface motility has also been described for metazoan cilia, suggesting that it was either an early adaptation of the IFT system, or one that has happened repeatedly during subsequent evolution44
. As the coupling mechanisms between flagellar adhesion molecules and gliding motors have not been widely studied, their evolutionary history remains unknown.
Movement of secretory vesicles along a polarized microtubule bundle would also provide a polarized distribution of cell surface molecules, including receptors. While the localization of receptors to ciliary and flagellar surfaces has been documented in both protists and in metazoan sensory cilia, including chemosensory cilia in C. elegans
, kidney cilia in vertebrates, and the highly modified cilia in sensory neurons of the vertebrate retina, evidence that specific receptors have been localized to this membrane domain since before eukaryotic divergence is only fragmentary (reviewed in ref. 44
). Receptor localization does not require the complex structure of a flagellum, but the ability to define a polarized cell surface domain for sensory signaling may have been one of the driving forces in early flagellar evolution.
Flagella are first and foremost organelles that beat, and their complex structure would not have evolved without strong selection for motility, as witnessed by the rapid loss of much of this complexity in non-motile sensory cilia, and the complete loss of flagella in non-motile organisms such as yeasts. However strong this advantage of a beating flagellum might have been to early eukaryotes, intermediate stages must have existed that provided intermediate levels of motion; a sudden jump from a benthic, amoeboid or gliding organism to one that can swim by flagellar beating is not plausible. To understand the advantages of less vigorous bending motility, one need only look at flagellar function among existing flagellates. Many organisms use flagella to generate feeding currents that increase the frequency with which food particles (bacteria, other eukaryotes, or detritus) can be ingested, while others use flagellar movements to aid in trapping food particles45,46
. Feeding currents are common in organisms such as choanoflagellates, which attach to the substrate with stalks and use flagellar beating to create currents past the stationary cell, in mastigamoebae, which create currents while continuing to move by amoeboid activity, and in many biflagellates such as bodonids, which create currents with an anterior flagellum and glide on a posterior flagellum. Even a modest ability to vibrate or wave a protoflagellum could have provided the initial selective advantage that drove further development of single microtubules into doublet microtubules, and favored diversification of dyneins that could take advantage of this new doublet microtubule track ().
Along with increased motility came the increased need to anchor the axoneme, which may have driven both centriole/basal body evolution and the development of links between the flagellar base and the cell membrane. These links would segregate the flagellar compartment from the rest of the cytoplasm, requiring further refinements in the IFT sorting/trafficking mechanism so that standard vesicle fusion occurred at the flagellar base, and IFT movement transported both membrane and non-membrane components to and from the flagellar compartment. In addition, such links create a boundary between the flagellar membrane and the rest of the plasma membrane, sequestering receptors and adhesion molecules into a unique membrane domain.
The ability to bend does not require an axoneme with 9-fold symmetry, and many axonemes have been discovered that depart from this pattern, yet most of these departures appear to be more recent modifications of an ancestral 9+2 pattern. As argued in more detail elsewhere, I propose that motility regulation by a central apparatus provided a strong selective advantage to the organism in which it evolved, and that the most successful regulatory mechanism was based on an apparatus built on a scaffold of two central microtubules, with regulatory signals transmitted through radial spokes of a defined length44,47
. The geometry of this regulatory mechanism presumably favors an outer cylinder of precisely nine doublet microtubules, with the distance between doublets determined by the reach of dyneins that must span each interdoublet gap. Whether central pair regulation was based on a fixed central pair orientation, as found today in metazoans such as bivalves48
, sea urchins49
, and ctenophores50
and possibly in excavates such as euglenids51
, or a rotating central pair52
, as commonly found in green algae such as Chlamydomonas53
, and chromalveolates such as Paramecium55
, remains to be determined. Central pair rotation may allow regulation of bends in different beat planes, and therefore be a more flexible regulatory system for organisms whose survival is most highly dependent on rapid changes in flagellar beat parameters47,53
. The origin of radial spokes remains, at this time, one of the greater mysteries of flagellar evolution, as related proteins have not been identified in other microtubule-associated regulatory complexes.