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Paraflagellar rod (PFR), or sometimes called paraxial rod, is an accessory proteinaceous filament found inside the flagella of some parasitic protozoans belonging to the phylum Euglenozoa, specifically in the species of Crithidia, Euglena, Herpetomonas, Leishmania, Phytomonas and Trypanosoma. As an additional filament to the usual (9+2) axonome, it is an indispensable component of the flagella that makes up the flagellum uniquely in euglenozoa, but not in other organisms. The structural organisations and functions of the PFR are fundamentally similar in different euglenid and kinetoplastid species, but are highly variable in size. A PFR is typically a three-layered mesh of over 40 different proteins that form a trilaminar lattice-like filament running alongside the microtubule. Euglenozoans exhibit unique flagellar movement during locomotion with characteristic non-planar helical motion unlike the flagella of other animals. The PFR helps in maintaining the structural integrity of the flagellum during such movements by imparting an elastic resistance during the undulation of the axoneme. It also serves as a conduit for Ca2+ and cAMP signalling pathway inside the flagellar complex, thereby controlling the mechanics of flagellar motion. It further plays a critical role in the parasite-host interaction that it may provide a target site for antiparasitic drugs.

History
PFR was first identified by Keith Vickerman in 1962.

The proteins of the PFR were first identified by Jeremy S. Hyams of the University College London in 1982. Hyams compared the protein components of the flagella between two euglenids euglenid Euglena gracilis and Chlamydomonas reinhardtii, and noticed that the former species had two proteins absent in the latter. As C. reinhardtii was known to lack a PFR, the proteins having molecular weights of 80 kDa and 69 kDa were found to be that of the PFR in E. gracilis; and were subsequently designated as PFR1 and PFR2 respectively. The next year, the two proteins were further confirmed in Crithidia fasciculata as having molecular weights of 76 kDa and 68 kDa. In 1989, Kathrin Schlaeppi, Judith Deflorin, and Thomas Seebeck from the Institut für Allgemeine Mikrobiologie, Bern, Switzerland, idenfied the PFR gene from Trypanosoma brucei. However, they reported that "two tandemly linked genes of identical nucleotide sequence" were the sources of the PFR1 and PFR2, genome sequences of T. brucei, Trypanosoma cruzi and Leishmania major simultaneously reported in 2005 revealed that the genes for PFR1 and PFR2 are not identical but related and arranged in separate tandem arrays.

The flagellar complex
In all organisms and individual cells possessing flagella, the only internal component is the axoneme, which in turn is composed of microtubules and dyneins. Only in the euglenozoans, such as kinetoplastids (Crithidia, Herpetomonas, Leishmania, Phytomonas and Trypanosoma species) and euglenid (Euglena species), are the flagellum composed of two distinct parts, the axoneme and PFR.

In the trypomastigote and epimastigote forms of kinetoplastids, the flagellum arises from the posterior or middle portion of the main body so that the flagellar membrane and the cell membrane forms a continuous structure known as undulating membrane that creates wavy motions during locomotion. The adhesion of the flagellar membrane to the body surface along the undulating membrane is mediated by the flagellum attachment zone, which is a complex of cytoskeletal filaments. The PFR is attached to the inner surface of the undulating membrane on one side and to the flagellum attachment zone on the other.

As in a typical eukaryote, the axoneme of euglenozoans contains nine doublet (each doublet is a pair of two, A and B tubules) microtubules arranged in a circle that surrounds two central singlet (unpaired) microtubules giving a structural configuration of (9+2) filament. This is in contrast with prokaryotes, where there are no central filaments, giving the flagellum a characteristic (9+0) filament. Again unlike in prokaryotes in which the flagella are a rotor machineries, the beat of the eukaryotic axoneme is powered by the sliding of the doublet microtubules against each other, the sliding of which is generated by the ATPase motor proteins, the dyneins, that are attached to the A tubule. The relative sliding and twisting are maintained by the radial spokes, a complex of over 20 proteins, that link the doublet microtubules to the central pair.

However, the euglenozoan axoneme is functionally different from those of other eukaryotes in that it generates motion as a series of beats consisting of bihelical waves, and each beat starts from the terminal tip and ends at the base. The bihelical waves are alternating left-handed and right-handed forces and separated by relatively relaxed states or kinks after each wave along the axoneme. This creates a drill-like motion on the entire length of the flagellum. As a result, the body is propelled in the direction of the tip of the flagellum.

Molecular architecture
As in typical eukaryotes, the flagella in protozoans are characteristically (9+2) cytoskeletal filaments consisting of nine circular doublet microtubules enclosing two central singlet microtubules. The interlinked microtubules together form the axoneme, the functional structure of the flagellum. A PFR appears under a transmission electron microscope as a three-layered lattice-like filament that runs along the entire length of the flagella, except at the base called flagellar pocket. The microtubules and the PFR are together enclosed entirely by a flagellar membrane. It is present in all the life cycle stages, including trypomastigote, epimastigote and promastigote, of kinetoplastids but not in the amastigote. In an amastigote stage, such as found in Leishmania species and Trypanosoma cruzi, there is no PFR and external flagellum, but only a highly reduced axoneme, or a residual flagellum, that is confined to the flagellar pocket inside the cytoplasm. Thus, this kinetoplastid stage is also referred to as "aflagellate amastigote form" or the individuals as "aflagellated amastigotes."

The PFR has a molecular architecture that is almost similar to intermediate filaments found in invertebrates and vertebrates as one of the cytoskeletons; the others being microtubules and microfilaments. Like intermediate filament, it has high solubility in trypsin, resistance to detergent treatment, similar amino acid composition, and a helical orientation. However, its association with flagellum and specific configuration indicate that it is not a typical cytoskeleton. The general overall thickness of the PFR is 150 nm throughout the entire length. It has three well-defined regions relative to the positions within the axoneme, namely the proximal, the intermediate and the distal zones (or domains). The proximal domain is highly conserved in that the structure is basically same across different species. However, the intermediate and the distal domains exhibit specific organisations of the thick and thin filaments giving rise to variations in the overall size in different species.

The fine structural organisation of the proximal and distal zones have common features. They are both dense plates composed of thin filaments measuring 7–10 nm in width and thick double filaments of 25 nm wide. The proximal domain lies closest to the microtubule and form a peptide link with the outer doublets using four to seven electron dense filaments, while the distal zones are farthest away from the axonome and form the thickest portion. The intermediate domain consists only of thin filaments of 5 nm wide that connect the proximal and distal domains on either sides. The proximal zone is connected to the axoneme by a set of proteins called PFR-axoneme connectors (PACs).

In T. brucei, where the PFR is most extensively studied, the filaments are stacked in bundles. The bundles, called "scissors densities," are arranged 54 nm from each other and oriented at 45° to the axoneme axis. They are connected by thin filaments called "wire densities." Such arrangement is essential for structural integrity while being flexible. The repeating densities give rise to an appearance of a comb teeth along the longitudinal axis. There can be ~370 scissor density planes with each plane containing ~27 pairs of scissors for a 20-µm-long PFR.

Composition
A PFR is composed of more than 40 different proteins that collectively form a trilaminar filament. The total number of proteins and their precise roles are yet incompletely elucidated. Depending on their roles, the PFR proteins can be categorised into four groups: those forming the lattice structure; those involved in calcium signalling; those involved in adenine nucleotide signaling and metabolism; and those which are of unknown function. Most of them are common in all euglenozoa, but at least a dozen of them are uniquely diverse among the species and are present in one or few species. The two most dominant, in terms of number and function, are PFR1 and PFR2, while the rest are recognised as minor components, which are relatively poorly understood on their structure and function. The two proteins are structurally distinct in all species and their molecular size ranges from 70 to 80 kDa for PFR1 and 68 to 82 kDa for PFR2. The proteins are highly conserved between related species. The amino acid and nucleotide sequences are  identical by more than 80% among the kinetoplastids. The two proteins themselves also share high molecular homology, with about 70% identity in their amino acid sequences. Their major difference is that although they share similar antigenic properties, they do not bind to the same epitope of antibodies. The proteins have been variously referred to by different names in different species, such as PFR1 and PFR2 in C. fasciculata, T. evansi, L. mexicana and L. major, PFRC and PFRA in T. brucei and PAR3 and PAR2 in T. cruzi respectively. However, PFR1 and PFR2 are most universally employed.

Assembly
The flagellum with its axoneme and PFR undergoes routine changes during reproductions in kinetoplastids. As the protozoans start to divide, replication of nuclear DNA is followed by nuclear division and then by division of the kinetoplast. This is a signal for the formation of new flagellum. The microtubules and the PFR are always synthesised in a coordinated process. Each cell division results in a production of a daughter flagellum which develops the microtubules and PFR concertedly. The microtubules and PFR originates from the basal body which lies towards the posterior region of the cell in trypomastigote and epimastigote forms, but at the anterior region in promastigote forms.

PFR1 and PFR2 are equally required for complete assembly of the PFR. Euglenozoans that are mutants either of PFR1 or PFR2 can not produce PFR. In experimental conditions, the PFR1 alone can form a rudimentary proximal zone, but not the PFR2. It is estimated that about 80% of the proteins form helices. The N terminal region of the proteins have short helices, but the rest are long helices. In the longest helices there are bout 258 amino acids in PFR1 and 222 amino acids in PFR2, creating a filament length of 41.3 nm and 35.5 nm, respectively.

Functions
In the flagellum, the axoneme is the main locomotory structure, while PFR performs diverse functional roles as an accessory filament. The PFR helps in maintaining the structural integrity of the flagellum during movements by imparting an elastic resistance during the undulation of the axoneme. It acts as a mechanical spring by absorbing, storing and relaying the forces generated by axonemal beating. Therefore, it adds to the efficiency of the flagellar movement which is crucial in euglenozoans as they spend much of their lifetime moving in the host's body fluids. Particularly in T. brucei, the only species which spends its entire lifecycle in continuous motion, except during cell divisions for reproduction, an extra efficient flagellar movement is required.

The PFR also serves as a conduit for Ca2+ and cAMP signalling pathways inside the flagellar complex, thereby controlling the mechanics of flagellar motion. It further plays a critical role in the parasite-host interaction that it may provide a target site for antiparasitic drugs.