The fidgetin family: Shaking things up among the microtubule‐severing enzymes

The microtubule cytoskeleton is required for several crucial cellular processes, including chromosome segregation, cell polarity and orientation, and intracellular transport. These functions rely on microtubule stability and dynamics, which are regulated by microtubule‐binding proteins (MTBPs). One such type of regulator is the microtubule‐severing enzymes (MSEs), which are ATPases Associated with Diverse Cellular Activities (AAA+ ATPases). The most recently identified family are the fidgetins, which contain three members: fidgetin, fidgetin‐like 1 (FL1), and fidgetin‐like 2 (FL2). Of the three known MSE families, the fidgetins have the most diverse range of functions in the cell, spanning mitosis/meiosis, development, cell migration, DNA repair, and neuronal function. Furthermore, they offer intriguing novel therapeutic targets for cancer, cardiovascular disease, and wound healing. In the two decades since their first report, there has been great progress in our understanding of the fidgetins; however, there is still much left unknown about this unusual family. This review aims to consolidate the present body of knowledge of the fidgetin family of MSEs and to inspire deeper exploration into the fidgetins and the MSEs as a whole.

fidgetins in several key cellular processes, which include mitosis and meiosis, development, and tissue function and disease.We then describe the involvement of the fidgetins in cell migration, with a particular focus on fidgetin-like 2. Finally, we address what is known about the regulation of the fidgetins.We conclude with consideration of the various similarities and distinctions among the three fidgetin family members.
2 | THE MICROTUBULE CYTOSKELETON: THE "BACKBONE" OF THE CELL

| Microtubule structure
Microtubules are made up of αand β-tubulin heterodimers that assemble in a head-to-tail fashion into a protofilament (Figure 1a-c) (Goodson & Jonasson, 2018;Matamoros & Baas, 2016).Thirteen protofilaments form lateral attachments to make a hollow tube of about 24-nm diameter (Figure 1a,c).Microtubules are inherently polar due to the assembly of their tubulin dimers.The minus end, or the end with exposed α-tubulins, tends to be located in the cell interior and experiences much slower polymerization than the plus end, or the end with β-tubulins exposed (Figure 1a) (Goodson & Jonasson, 2018;Matamoros & Baas, 2016).Radial microtubule arrays are common, in which the minus ends are localized to microtubule organizing centers (MTOCs), such as those found in fibroblasts, as well as centrosomes and centrioles (Goodson & Jonasson, 2018;Matamoros & Baas, 2016).New microtubules are often formed at minus ends, where a third subtype of tubulin, γ-tubulin, forms a γ-TuRC (γ-tubulin ring complex), and acts as a template on which microtubule nucleation can begin (Figure 1a; Goodson & Jonasson, 2018;Matamoros & Baas, 2016).In radial arrays, the plus ends face the entirety of the cell periphery, though in other microtubule organizations, such as the parallel arrays seen in columnar epithelial cells, they face one side.When cells migrate, microtubules oriented toward the direction of movement help to polarize the cell (Goodson & Jonasson, 2018).
The tubulin dimer adopts different conformations depending on whether it is in solution or within the microtubule lattice.Both the αand β-tubulin subunits of the tubulin heterodimer contain a GTP, though only β-tubulin has a functional GTPase (Bailey et al., 2016;Downing & Nogales, 1998).It is thought that the free tubulin dimer exists in a range of "bent" conformations, with the αand β-subunits at a slight angle to each other (Figure 1b) (Nogales, 2001;L. X. Peng et al., 2014).Both GTP-bound and GDP-bound heterodimers in solution tend to remain in the lower energy "bent" state (L.X. Peng et al., 2014).In the "straight" conformation seen within the microtubule lattice, the subunits are more linear, so as to bind longitudinally to the next dimer in the protofilament (L.X. Peng et al., 2014).This rotation also increases the surface area of the intradimer interface, allowing for lateral contacts with neighboring protofilaments (L.X. Peng et al., 2014).The tubulin dimer is straightened by the longitudinal and lateral contacts that form within the lattice (Bailey et al., 2016;L. X. Peng et al., 2014), which prevent the dimer from returning to its "bent" configuration when the GTP is hydrolyzed.The GDP-tubulin dimer is then held in the higher-energy "straight" conformation, and the resultant potential energy is stored within the wall of the protofilament, known as "spring loading" (Bailey et al., 2016;Matamoros & Baas, 2016; L. X. Peng et al., 2014).This stored energy is what allows microtubules to generate force (Bailey et al., 2016) and primes the microtubule for depolymerization (Matamoros & Baas, 2016; L. X. Peng et al., 2014).The plus ends have a higher concentration of β-tubulin GTPs that have not yet been hydrolyzed, F I G U R E 1 Microtubule structure and dynamic instability.(a) Microtubules are made up of αand β-tubulin heterodimers that attach in a head-to-tail orientation to form a polar protofilament.Generally, 13 protofilaments come together to form a 24 nm-wide hollow tube with a minus (À) end and a plus (+) end.Nucleation starts at the minus end with a complex of a third tubulin isotype, γ-tubulin: 13 γ-tubulins form a ring and then continue to polymerize into the base of one of the protofilaments onto which αβ-tubulin heterodimers can bind.At the plus end, recently attached αβ-tubulin heterodimers that have not hydrolyzed their GTP yet form a stabilizing GTP cap onto which the +TIP protein complex can assemble.(b) Although both αand β-tubulin bind GTP, only the nucleotide in β-tubulin is hydrolyzable.When β-tubulin is bound to GTP (green), the heterodimer exists in a straight conformation.When β-tubulin is bound to GDP (blue), the heterodimer adopts a bent conformation, which is more energetically favorable.While held within the microtubule lattice, the GDP-bound αβ-tubulin dimer is maintained in the straight conformation, and the energy is stored as strain.forming what is known as the GTP cap (Figure 1a), which both provides stability to the microtubule and can bind microtubule-associated proteins (Bailey et al., 2016;Nogales, 2001).
The most well-known functions of microtubules are in mitosis.
Stable centrosomal microtubules are responsible for determining the cortical asymmetry necessary for cytokinesis (Akhshi et al., 2014).
Dynamic microtubules form the mitotic spindle, which is comprised of three microtubule subpopulations, which attach to each other, to chromosomes, and to the cortex, respectively.During anaphase, these microtubules capture chromatids and move them into separate daughter cells (Burute & Kapitein, 2019).
The stable and dynamic microtubules can be distinguished by their post-translational modifications (PTMs), which are accumulated over time by tubulin incorporated into the lattice.These PTMs, which are deposited by a variety of microtubule-binding proteins (MTBPs), are acquired by the C-terminal terminal tails of the tubulin dimer as they remain external to the microtubule lattice (Figure 1c).They code for information involving microtubule stability, often by affecting the binding of other MTBPs along the microtubule lattice (Matamoros & Baas, 2016;Nogales, 2001).Detyrosination, or removal of the terminal tyrosine of the α-tubulin C-terminal tail, generally indicates microtubule stability (MacRae, 1997;Nogales, 2001).Conversely, F I G U R E 2 Microtubule dynamics.Polymerization: GTP-bound αβ-tubulin heterodimers attach in a head-to-tail fashion along protofilaments that then form lateral attachments to curl into a hollow tube.Within the microtubule lattice, the β-tubulin GTP is hydrolyzed to GDP so that most of the microtubule is made up of GDP-bound tubulin held energetically unfavorably in a straight conformation.A stabilizing GTP cap forms at the plus end where the β-tubulin GTP has not yet been hydrolyzed.Depolymerization: When the GTP cap is lost, such as by microtubule severing, the microtubule disassembles easily.The weaker lateral bonds between protofilaments and the propensity for the interior GDP-bound tubulin dimers to bend causes the protofilaments to curl away from each other before individual tubulin dimers dissociate.The GDP in the free tubulin dimers is exchanged for GTP so that they can be reused in another growing microtubule.This behavior, known as dynamic instability, confers many of a microtubule's key functions in the cell, such as regulation of migration and cytokinesis.(Adapted from Akhmanova and Steinmetz (2015)).

| HOW MICROTUBULE-SEVERING ENZYMES WORK: THEY PUT A " RING" ON IT
In order to perform the many different roles it plays in the cell, the microtubule cytoskeleton interacts with a multitude of MTBPs, which fulfill a diverse array of purposes.Many of the MTBPs influence the stability or dynamicity of microtubules, which in turn affects their function (Garcin & Straube, 2019;Goodson & Jonasson, 2018).Some do so by depositing post-translational modifications onto the C-terminal tails of tubulin in the microtubule lattice (Garcin & Straube, 2019;Goodson & Jonasson, 2018).Others stabilize the plus end of a growing microtubule by decreasing catastrophe or anchoring the microtubule tip to other cytoskeletal components or to membrane-associated complexes (Garcin & Straube, 2019;Goodson & Jonasson, 2018;Lansbergen & Akhmanova, 2006;Morrison, 2007).Destabilizers that bind to the microtubule tip work to depolymerize the microtubule from the plus end (Garcin & Straube, 2019;Goodson & Jonasson, 2018;Lansbergen & Akhmanova, 2006;Morrison, 2007).
MSEs are a class of microtubule destabilizers that sever the microtubule lattice rather than induce depolymerization from the end (Khan et al., 2022;Matamoros & Baas, 2016).The MSEs are part of the Classical Clade in the ATPases Associated with diverse cellular Activities (AAA+) superfamily (Khan et al., 2022).The Meiotic AAA+ proteins, which are Class I ATPases within the Classical Clade of the AAA+ superfamily, have four protein families: VPS4, katanin, spastin, and fidgetin (Figure 3a,b).Of these, VPS4 is the only one that is not an MSE (Khan et al., 2022).

| MSE structure
The MSEs all share the same general domain organization: an N-terminal regulatory domain(s), which includes a microtubule interacting and trafficking (MIT) domain, and a C-terminal catalytic AAA+ ATPase domain (Figure 3a) (Bailey et al., 2016;Khan et al., 2022).The N-terminal MIT domain consists of a three-helix bundle (Bailey et al., 2016;Khan et al., 2022;Kuo & Howard, 2021;Roll-Mecak & McNally, 2010), which is likely to be involved in protein-protein or membrane-protein interactions (Bailey et al., 2016).The MIT domain is connected to the AAA+ ATPase domain by a flexible linker, sometimes known as pore loop 1, which contains at least one aromatic residue that interacts with tubulin (Bailey et al., 2016;Khan et al., 2022).
The Walker-A motif binds the nucleotide, the Walker-B motif coordinates the Mg 2+ and H 2 O that drive ATP hydrolysis (Khan et al., 2022;Sharp & Ross, 2012), and the sensor-1 element contains conserved polar residues that support the Walker-B motif (Khan et al., 2022).
The SRH motif contains the arginine finger element, which coordinates the ATP of the neighboring subunit, thus communicating nucleotide status between subunits to coordinate their activity (Khan et al., 2022).The sensor-2 motif interacts with the ATP, in part through a conserved alanine residue-interestingly, this is an arginine residue in most other AAA+ ATPases-and changes conformation when the MSE is bound to ADP (Khan et al., 2022).This motif is believed to transmit the free energy of ATP hydrolysis to the tubulin dimer to pull it out of the microtubule lattice (Khan et al., 2022).
The first family of MSEs to be identified were the katanins, named for the Japanese katana sword.Originally discovered in 1993 in sea urchin embryos, katanin is evolutionarily the oldest of all three MSE families (Figure 3b) (Kuo & Howard, 2021;Lynn et al., 2021;F. J. McNally & Roll-Mecak, 2018).Its domains are split across two proteins, which form the p60 and p80 subunits (Hartman et al., 1998;Hartman & Vale, 1999;F. J. McNally & Vale, 1993;K. P. McNally et al., 2000).The catalytic p60 subunit contains the MIT domain, which binds the p80 subunit, and the AAA+ ATPase domain (Hartman et al., 1998;Hartman & Vale, 1999;K. P. McNally et al., 2000).The catalytic subunit of human katanins also have a C-terminal Vps4 insertion, though its function is not known (Lynn et al., 2021).The regulatory p80 subunit contains a stretch of WD40 repeats, which directs the protein to spindle poles for severing, as well as a C-terminal domain that binds the p60 subunit to regulate its microtubule-severing activity (Hartman et al., 1998;K. P. McNally et al., 2000).
In 1999, spastins became the second family to be identified when a mutation in the gene was determined to be a cause of the neurodegenerative disease hereditary spastic paraplegia (HSP) (Lumb et al., 2012).Spastins are the first MSEs evolutionarily to be a single polypeptide (Lumb et al., 2012).In addition to the characteristic MIT and AAA+ ATPase domains, they contain an intermediate microtubule-binding domain (MTBD) and an N-terminal hydrophobic domain that allows for membrane insertion (Roll-Mecak & Vale, 2008;Taylor et al., 2012;White et al., 2007).It is also the first MSE to have a nuclear localization signal (NLS), though its nuclear function remains unknown (Beetz et al., 2004;Lumb et al., 2012).
Finally, the third and most recently identified MSEs are the fidgetins.Fidgetin was discovered in the 1940s through a spontaneous mutation in mice: the eponymous phenotype included "fidgety" head shaking and circling behavior, as well as cleft palates and defects in skull bone fusions (Sharp & Ross, 2012).It was not until 2000 when the mutated gene was identified to be an MSE and thus named fidgetin.Two other family members were also identified as fidgetin-like 1 (FL1) and fidgetin-like 2 (FL2) (Bailey et al., 2016;Cox et al., 2000).
FL1 structural analysis showed strong similarity among the AAA+ ATPase domains of the fidgetin family members as well as with katanin and spastin; notably, all three fidgetins contained the conserved α-helices required for microtubule-severing (W.Peng et al., 2013) that had been identified in spastin (Taylor et al., 2012).In support of this structural evidence, centrosomal microtubule dynamics increased or decreased with FL1 overexpression or depletion in mammalian cells (Zhao et al., 2016), and Caenorhabditis elegans FL1 binds to microtubules (Luke-Glaser et al., 2007).In mouse oocytes, FL1 localizes to γ-tubulin in the spindle apparatus during meiosis, and its knockdown leads to an aberrant increase in astral microtubule length and density, assumedly due to decrease FL1 severing (Shou et al., 2022).Interestingly, in zebrafish, on the other hand, in vivo and in vitro work suggested that FL1 may impact microtubule dynamics at the leading edge of cells through plus-end depolymerization and ATPase-mediated removal of EB1/3 (which bind to the stabilized plus ends of growing dynamic microtubules), rather than severing (Fassier et al., 2018).Thus, more work is necessary to further elucidate how FL1 influences microtubule dynamics in different contexts.
Around the same time, purified fidgetin was demonstrated to sever microtubules in vitro (Mukherjee et al., 2012).This was (1) Six MSEs bind to form a hexameric ring, when can then bind the C-terminal tail of a tubulin dimer along the microtubule lattice.(2) The arginine finger being contributed in trans allows for stepwise coordination of the ATPases.Inset shows successive ATP hydrolysis steps leading to progressive pulling of the tubulin C-terminal tail through the central pore of the MSE.(3) The lateral and longitudinal bonds of the tubulin dimer to the adjacent dimers are destabilized so that the dimer can be pulled free.(4) When all the ATPs have been hydrolyzed, the subunits dissociate from the microtubule and from each other.(Adapted from Sharp and Ross ( 2012)).
supported by in vivo work showing that fidgetin overexpression decreased tyrosinated tubulin density (Hu et al., 2017;Li et al., 2020), while fidgetin knockdown led to increased tubulin density (Austin et al., 2017;Hu et al., 2017;Leo et al., 2015) and curving of the elongated, unsevered microtubules along the plasma membrane (Hu et al., 2017).Interestingly, the tubulin PTM specificity of fidgetin differs between species, suggesting a shift in cellular function across evolutionary time (Leo et al., 2015).
FL2 microtubule-severing activity has been implied by several in vivo investigations.Across a range of mammalian cell types, microtubule density at the leading edge (where FL2 functions) increases significantly when FL2 is knocked down (Baker et al., 2021;Charafeddine, 2015;Charafeddine et al., 2015;Kramer, 2019;Smart et al., 2022), and this effect is rescued by exogenous FL2 expression (Charafeddine, 2015;Charafeddine et al., 2015).In FL2-depleted cells, the elongated microtubules curve along the interior of the cell membrane, another phenotype rescued by re-introduction of FL2 protein (Charafeddine, 2015).Of the family members, FL2 is the most closely related to fidgetin (Kuo & Howard, 2021;F. J. McNally & Roll-Mecak, 2018), whose microtubule-severing activity has already been illustrated.Moreover, just like fidgetin, FL2 contains the conserved α-helices required for microtubule-severing, as well as the AAA+ ATPase domain shared by fidgetin, katanin, and spastin (W.Peng et al., 2013).Future work addressing the purification of the FL2 protein may offer insights into its crystal structure as well as in vitro demonstration of its microtubule-severing activity.
There is some debate as to whether the subunits in an AAA+ ATPase act successively or in unison.Although this has not been directly addressed in MSEs, the asymmetric spiral of the hexamer indicates that the subunits cannot fire in unison, as the P6 protomer does not have an adjacent arginine finger (W.Peng et al., 2013;E. Zehr et al., 2017).Thus, in a "hand-over-hand" mechanism, the MSE pulls the C-terminal tail through the central pore in approximately 2-amino acid steps (Figure 4) (Khan et al., 2022;Kuo & Howard, 2021;F. J. McNally & Roll-Mecak, 2018;Roll-Mecak & Vale, 2008;White et al., 2007).Eventually, the tubulin dimer is removed from the lattice by the MSE hexamer, though the extent to which it is unfolded in the process remains to be determined (F.J. McNally & Vale, 1993;Roll-Mecak & McNally, 2010).Regardless, the tubulin dimer is unlikely to be completely unfolded as it moves through the central pore of the MSE; rather, as the lateral and longitudinal contacts within the lattice are broken, the intact tubulin dimer is removed (Figure 4).
The most compelling evidence for why the tubulin dimer is not entirely unfolded is that tubulin folding is not spontaneous (Downing & Nogales, 1998;F. J. McNally & Roll-Mecak, 2018).The entire process requires multiple different assisting proteins.The cytosolic chaperonin CCT, which is also responsible for folding actin monomers, binds and releases the individual subunits successively until they are folded (Downing & Nogales, 1998;Nogales, 2001).The αand β-tubulin monomers are highly unstable and must be immediately bound to cofactors B and A, respectively, to remain in solution (Nogales, 2001).Formation of the dimer requires the αand β-tubulin monomers to be transferred to cofactors D and E, respectively.
Cofactor C then binds and forms a pentameric complex to complete dimerization (Downing & Nogales, 1998;Nogales, 2001).Without the cofactors, the individual monomers rapidly denature (Downing & Nogales, 1998;Nogales, 2001).As tubulin dimers remain in solution and can be incorporated back into the microtubule lattice, it is also unlikely the monomers are separated by MSE activity (Bailey et al., 2016;Kuo & Howard, 2021;Nogales, 2001).MSE disengagement from the microtubule is still poorly understood, though, once ADP-bound, the hexamer disassembles since its oligomerization is ATP-dependent (Bailey et al., 2016;Eckert et al., 2012;Hartman & Vale, 1999;W. Peng et al., 2013;Sharp & Ross, 2012;Yakushiji et al., 2006;E. Zehr et al., 2017;E. A. Zehr et al., 2020).Successive iterations of MSE activity in the same region eventually render the microtubule destabilized enough for the severed portion to break off (Figure 4) (Kuo & Howard, 2021;Sharp & Ross, 2012).The resultant effect on the microtubule depends on where along the lattice the severing occurs.If the MSE acts on a dynamic microtubule or a dynamic region therein, the severed portion depolymerizes into free tubulin (Baker et al., 2021;Charafeddine et al., 2015;Fassier et al., 2018;Kramer, 2019;Kuo & Howard, 2021;Leo et al., 2015;Matamoros et al., 2019;Matamoros & Baas, 2016;O'Rourke et al., 2019;Sharp & Ross, 2012;Smart et al., 2022;Tao et al., 2016).This can promote catastrophe in the remaining intact microtubule because the newly exposed plus end often lacks a GTP cap and thus also begins to depolymerize unless rescued (Austin et al., 2017;Kuo & Howard, 2021;Leo et al., 2015;Matamoros et al., 2019).If the MSE severs a stable region, the fragment that breaks off remains intact and can be transported elsewhere to nucleate new microtubule seeds; the rest of the lattice may then continue to polymerize (Kuo et al., 2019;Leo et al., 2015;Lumb et al., 2012;Lynn et al., 2021;Matamoros & Baas, 2016;F. J. McNally & Roll-Mecak, 2018;Mukherjee et al., 2012).As a result, all three MSE families destabilize microtubules through their microtubule-severing activity in vitro; however, in vivo, by localizing to distinct regions of the cell and the microtubule, their severing activities have different, often opposing, effects on cell behavior (Sharp & Ross, 2012).The breadth of MSE influences on microtubule density and dynamics is thus likely due in large part to affinities for distinct microtubule stability levels (Austin et al., 2017;Kuo & Howard, 2021;Leo et al., 2015;Mukherjee et al., 2012;Sharp & Ross, 2012), as determined by tubulin PTM specificities.
Microtubule severing has a range of effects depending on factors such as microtubule PTMs and intracellular location.Therefore, MSEs have distinct tubulin specificities and cellular functions that can vary across species.For example, across organisms, the katanins have been shown to preferentially sever acetylated or polyglutamylated microtubules, which tend to be long-lived and stable (Lynn et al., 2021;Sharp & Ross, 2012).Generally, they function in mitosis and meiosis and in cell migration (Lynn et al., 2021;Roll-Mecak & McNally, 2010;Sharp & Ross, 2012).Similarly to katanin, spastin is an evolutionarily conserved MSE that primarily plays a role in mitosis (Figure 3b) (Kuo & Howard, 2021 The fidgetin family is perhaps the most functionally diverse of the MSEs as well as the least well-understood.They perform crucial roles in cellular processes such as mitosis and migration (Bailey et al., 2016;Cox et al., 2000;Sharp & Ross, 2012), and they target a variety of microtubule subpopulations.The wide range of functions of this family reflects both the expansion of the roles of MSEs in evolution as well as the necessity of severing in regulating microtubule dynamics.

| FUNCTIONS IN MITOSIS AND MEIOSIS
As a group, the MSEs are known as the Meiotic AAA+ ATPases (Khan et al., 2022).Indeed, all three families have roles in mitosis and meiosis.The mitotic and meiotic roles of katanin and spastin have been described in depth elsewhere (Hu et al., 2017).The roles of the fidgetins are less well-studied, however.Both fidgetin and FL1 generally act on the minus ends of microtubules during mitosis or meiosis.It is interesting to note that the fidgetin family contains the only MSE without an identified mitotic function-FL2.
In Drosophila melanogaster mitosis, fidgetin works together with katanin and spastin during anaphase to separate the sister chromatids.
Katanin severs plus ends of microtubules in the Pacman mechanism, which helps bring the chromatid closer to the chromosomal pole (Mukherjee, 2011;Mukherjee et al., 2012;Roll-Mecak & McNally, 2010;Sharp & Ross, 2012;Zhang et al., 2007).Simultaneously, spastin and fidgetin sever the minus ends of microtubules at the chromosomal pole in the Flux mechanism to help reel the chromatids in (Mukherjee, 2011;Mukherjee et al., 2012;Roll-Mecak & McNally, 2010;Sharp & Ross, 2012;Zhang et al., 2007).Similarly, human fidgetin severs the minus ends of microtubules to stimulate Flux during anaphase (Zhang et al., 2007), and its knockdown leads to increased density of astral microtubules oriented away from the centrosome during metaphase (Mukherjee et al., 2012).The severed astral microtubules often depolymerize too rapidly to be detected (Hu et al., 2017), leading to an initial, albeit incorrect, belief that fidgetin had no microtubule-severing ability (Mukherjee et al., 2012).Additionally, fidgetin localizes to the spindle midzone during telophase and to both the cytoplasm and nucleus during interphase (Mukherjee et al., 2012), suggesting mitotic roles outside of anaphase in human cells.Mouse fidgetin may have an additional meiotic function as it is upregulated in ovaries and testes.It is also enriched in the zona pellucida of oocytes to prevent polyspermy, though its role in both cases is not fully elucidated (C.R. Li, Wang, et al., 2022).
FL1 also has both mitotic and meiotic roles across organisms.In C. elegans gonadal development, FL1 functions in the distal end mitotic zone, which is responsible for cell proliferation.In the proximal end meiotic zone, which is responsible for cell differentiation, FL1 is degraded to prevent its activity: CUL-3 binds FL1 via the MEL-26 substrate adaptor and loads it onto the E1/2/3 ubiquitin complex to initiate its eventual degradation (Luke-Glaser et al., 2007).In mouse development, FL1 severs the minus ends of microtubules, creating new nucleation seeds to increase spindle density during meiosis.
Without FL1, the spindle structure in mouse oocytes was too loose for efficient chromatid separation, resulting in improper oocyte maturation and fertilization and abnormal early embryonic development (Shou et al., 2022).In mouse pachytene spermatocytes, meiotic progression is dependent on correctly timed ubiquitination of FL1.When FL1 was mutated so that it could not be ubiquitinated, the spermatocytes did not progress through meiosis and apoptosed instead (L'Hôte et al., 2011).This finding also helped explain the low testis weight of 97 C mice, which have two isoforms of FL1-full-length and truncated, which compete for degradation.Since the full-length isoform is thus not degraded enough for meiosis to progress efficiently, the cells apoptose, resulting in low testis weight (L'Hôte et al., 2011).

| INVOLVEMENT IN DEVELOPMENT
As mitotic and meiotic regulators, the MSEs also have roles in embryonic development.In fact, fidgetin was the first AAA+ ATPase whose depletion was tied to developmental issues (Cox et al., 2000;Sharp & Ross, 2012;Yang et al., 2005): this was exemplified in fidgetin's discovery, as its mutation led to behavioral and structural defects (Cox et al., 2000;Sharp & Ross, 2012).Although the precise function of the fidgetins during development is still being explored, studies with zebrafish and mice both indicate increased expression during certain developmental stages.
In zebrafish, fidgetin and FL2 are both expressed in somites and in the notochord and brain during the first 24 hours post-fertilization (hpf) (Dong et al., 2021;Kramer, 2019).After that, fidgetin is most expressed in the brain, eyes, and inner ear (Dong et al., 2021).FL2, on the other hand, is expressed in the brain and kidney at 30 hpf (Kramer, 2019), then the pharyngeal arch until 48 hpf (Dong et al., 2021).After that, it is found in the lens of the eye, the otoliths of the ear, the apical ectodermal ridge of the pectoral fins, the tegmentum of the brainstem, and the bulbous anteriosus of the heart (a structure found only in fish), the notochord, and the posterior cardinal vein in the kidney (Kramer, 2019).By 72 hpf, FL2 expression has moved to the swim bladder bud and the outside of the brain (Dong et al., 2021), and by 3 days post-fertilization, it is found mainly in the brain and pectoral fin sprouts (Kramer, 2019).Further investigation of FL1 expression across zebrafish developmental stages has yet to be explored (Dong et al., 2021).
Altogether, this suggests that fidgetin and FL2 have functions in skeletal, nervous, and cardiovascular system development.In fact, when FL2 was knocked down, zebrafish exhibited defects in cellular branching as well as in epithelial, vascular, and neuronal development (Dong et al., 2021).Neuronal cells had longer axons and more branching, and the zebrafish had abnormal vascular branching, pericardial edema, and a lower heart rate.Behaviorally, they had lower swimming velocity and a reduced response to light-to-darkness shift, indicating a defective vision circuit (Dong et al., 2021).Surprisingly, neither fidgetin nor FL1 knockdown had visible effects on zebrafish development.may indicate some compensatory mechanisms between these proteins (Dong et al., 2021).This finding was challenged, however, by Fassier et al., who found that almost all zebrafish embryos in which FL1 had been knocked down failed to hatch.Those that did had curved tails and clear locomotor defects (Fassier et al., 2018).Therefore, more exploration into the role of FL1 in nervous system development is necessary.
In mammalian development, fidgetin and FL2 have somewhat similar expression patterns.They are co-expressed in developing tissue of the heart, brain, lung, liver, kidney, and skeletal muscle in mice (Yang et al., 2005).During late gestation, fidgetin has high expression in the eye, inner ear, and skeletal bone development (Yang et al., 2005).It also promotes platogenesis by interacting with AKAP95; fidgetin mutation can lead to cleft palate in mice (Yang, Mahaffey, Berube, & Frankel, 2006).One fidgetin allele (containing the +94762G>C SNP) is associated with upregulation of plasma folate concentration during embryonic heart development (D.Wang, Wang, et al., 2017).Folate is necessary for de novo dNTP synthesis for DNA replication and repair during cardiogenesis, so increased concentrations decrease the risk of issues underlying congenital heart defects, such as abnormal branchial arch development (D.Wang, Wang, et al., 2017).FL2 is present most clearly in somites and in the nervous system during early mouse development up to E10.5 (embryonic day 10.5), and in the vasculature by E13.5 (Kramer, 2019).The developmental functions of FL2 appear to be more critical than those of fidgetin or FL1: FL2 knockout in mice is embryonic lethal (Kramer, 2019).The expression profile of FL1 in mouse development is not well studied, though it has been shown to be present in developing spleen and testes tissues in mice (Yang et al., 2005).
Fidgetin and FL2 are involved in the development of the cardiovascular, skeletal, and nervous systems in both zebrafish and mice, suggesting some evolutionary conservation of their functions.As there is some overlap in their expression patterns, the possibility of protein interaction was investigated; however, it was determined that fidgetin cannot form oligomers with either FL1 or FL2 (Yang et al., 2005) and thus function separately.More work is required to elucidate the role of FL1 in development and how it may be similar to or distinct from those of the other fidgetins.

| ROLES IN TISSUE FUNCTION AND DYSFUNCTION
The fidgetin family proteins, which are involved in cardiovascular and nervous system development, also have specific functions in these adult tissues.Both fidgetin and FL2 are expressed in cardiovascular cells, though they appear to have opposing effects: fidgetin expression and FL2 depletion have been shown to have therapeutic benefits.
Neuronal cell structure is regulated by fidgetin and FL2 microtubulesevering, while FL1 impacts axonal transport.

| Cardiovascular
Fidgetin may play a cardioprotective role through a non-severing function according to recent evidence.Multiple human single nucleotide polymorphisms (SNPs) associated with cardiorespiratory phenotypes and with heritable pulmonary arterial hypertension have been identified in the distal promoter region of the fidgetin gene, leading to less penetrance of the gene mutation responsible for heritable pulmonary arterial hypertension (Puigdevall et al., 2019).Studies with Han Chinese populations indicated three isoforms of fidgetin, one of which-X3-was associated with lower risk of congenital heart defects when it contained a +94762G>C SNP (D. Wang, Chu, et al., 2017).This allele reduces binding to the CREB1 transcription factor, which serves as a negative regulator of fidgetin X3.As a result, fidgetin X3, which is thus expressed at higher levels, inhibits to a greater degree the proteasomal degradation of reduced folate carrier 1 (RFC1) and dihydrofolate reductase (DHFR).RFC1 and DHFR increase cellular folate levels via transport from circulation and intracellular recycling, respectively (D. Wang, Chu, et al., 2017).This supported work in mouse development showing that increased folate concentrations in the cell due to this fidgetin allele decrease the risk of congenital heart defects (D.Wang, Wang, et al., 2017).
In contrast to fidgetin, FL2 depletion has cardiovascular benefits.FL2 siRNA knockdown after myocardial infarction in mice significantly improved ejection fraction (by increasing systolic volume without the need for compensatory increases in diastolic volume) and angiogenic repair.In contrast, FL2 expression was transiently increased in the cardiac tissue of reporter mice after myocardial infarctions (Kramer, 2019).

| Neuronal
In neurons, fidgetin and FL2 both sever dynamic microtubules, though with differing effects.The functions of fidgetin across neuronal cell types are surprisingly diverse.In D. melanogaster neurons, fidgetin is upregulated after injury; by severing microtubules to create new dynamic microtubule seeds, dendritic but not axonal degeneration is promoted (Tao et al., 2016).In rat hippocampal axons, fidgetin severs unacetylated microtubules to suppress excessive formation of synaptic boutons; when depleted, the labile regions of microtubules are significantly extended, though the number of microtubules is not affected (Leo et al., 2015).Knocking down fidgetin in rat spinal cord neurons leads to an increase in labile microtubules as well as distal axonal mTOR activation, which were both important for increased axonal branching and growth cone mobility (C.Ma et al., 2022).
Fidgetin-like 2's neuronal functions are related to motility and axonal guidance and are described in greater detail below (see "Migration").FL1, on the other hand, functions quite differently: in zebrafish secondary motor neurons, it regulates bi-directional transport of cargo necessary for axonal pathfinding.Here, FL1 forms a tripartite complex with Kif1bβ and Bicd1 (a dynein adaptor), so that their oppositelyoriented motion slows dynein movement (Atkins et al., 2019).

| CANCER AND DNA REPAIR: FIDGETIN AND FL1
Although all three fidgetins have a nuclear localization signal (Kramer, 2019;Li et al., 2020;Mukherjee et al., 2012;Onitake et al., 2012), FL1 is the only with an identified nuclear function.In human, mouse, and zebrafish cells, FL1 promotes cilia disassembly by severing microtubules at the centrosome (Zhao et al., 2016); however, during interphase, it was noted that FL1 had a preferential localization to the nucleus (Zhao et al., 2016).In mouse osteoblasts, the growth factor bFGF, which is upregulated in cancer, promoted FL1 nuclear localization.Interestingly, bFGF treatment also decreased FL1 mRNA levels in these cells (Park et al., 2007).
C. elegans FL1 directly interacts with SMO-1 (a C. elegans SUMO homolog) in the nucleus.Although the function of this interaction remains unknown, depletion of FL1 leads to defective gonad function and sterility in the nematodes (Onitake et al., 2012).Human FL1 was later identified to function as an anti-recombinase (Matsuzaki et al., 2019).Once in the nucleus, it is recruited to sites of homologous repair by the γH2AX histone, where it binds to RAD51 and KIAA0146 (Yuan & Chen, 2013).Binding to RAD51 promotes hydrolysis of its ATP so that it is released from its single-stranded DNA (ssDNA); in this way, FL1 prevents improper RAD51-ssDNA assemblies (Matsuzaki et al., 2019).SWSAP1 binds FL1 to prevent its association with RAD51 in order to protect appropriately formed RAD51-ssDNA complexes (Matsuzaki et al., 2019).
Naturally, FL1's DNA repair function raises the question of a potential role in cancer.In fact, both fidgetin and FL1 are being investigated for their involvement in cancer, and, recently, they have been linked to liver and lung cancer.Interestingly, no association with cancer has been identified for FL2 so far (http://www.proteinatlas.org,FIGNL2; NIH NCI GDC, FIGNL2; http://cancer.sanger.ac.uk,FIGNL2; (Grossman et al., 2016;Tate et al., 2019;Uhlén et al., 2015)).Fidgetin mRNA and protein are both overexpressed in hepatocellular carcinoma, and expression levels are correlated with tumor stage and progression and with poor prognosis.This suggests fidgetin may serve as a predictive biomarker and therapeutic target, though it remains unclear whether its mitotic or migratory function is involved (Zhou et al., 2020).FL1 is also a potential biomarker as its expression is higher in several cancers, including hepatocellular carcinoma, where it is associated with poor survival (Zhen et al., 2022).Moreover, FL1 promoter methylation is decreased in epithelial cell carcinoma and in low-grade gliomas, and the resultant higher expression is correlated with higher immune infiltration (Zhen et al., 2022).
FL1 expression is also higher in lung cancer, and its knockdown is an emerging therapeutic target.In non-small cell lung cancer cells, higher FL1 expression is associated with poorer prognosis, particularly in cisplatin-resistant cells, where it promotes resistance by associating with the nuclear homologous repair factors RAD51 and CCDC36 (Meng et al., 2022).Knockdown of FL1 in these cells suppresses its role in chromosomal DNA replication and homologous repair so that cell proliferation and invasiveness decreases while apoptosis and G1 phase arrest increases (M.Li, Rui, et al., 2021).In small cell lung cancer cells, where FL1 expression is also increased, knockdown of FL1 increases the effectiveness of the chemotherapeutics etoposide and cisplatin by decreasing tumor cell homologous repair mechanisms (J.Ma et al., 2017).Although not a neoplastic condition, FL1 expression also increases in bronchial epithelial cells in response to DNA damage due to exposure to CuO nanoparticles, with similar effects as seen in non-small cell lung cancer: promotion of cell proliferation and migration and suppression of G1 phase arrest and apoptosis (Kwon et al., 2022).

| FUNCTIONS IN CELL MIGRATION
Cell migration is a complex process that requires precise control of cortical microtubule dynamics.This is achieved by the concerted activity of many different MTBPs, including the MSEs (F.J. McNally & Roll-Mecak, 2018;Sharp & Ross, 2012).Indeed, every member of the fidgetin family has an identified function in cell migration.
All three fidgetins influence axonal growth, which can be considered a modified form of motility in neuronal cells (Roll-Mecak & McNally, 2010;Sharp & Ross, 2012).D. melanogaster fidgetin severs acetylated microtubules in neuronal cells to maintain parallel arrays within axons (Leo et al., 2015).Interestingly, however, vertebrate fidgetins appear to target dynamic rather than stable microtubules.In rat astrocytes, fidgetin promotes increased migration by trimming the ends of tyrosinated microtubules near the plasma membrane to prevent them overgrowing and curving along the cortex (Hu et al., 2017).
On the other hand, in rat adult dorsal root ganglion axons, fidgetin targets unacetylated microtubules in order to decrease movement into regions with inhibitory substrates, such as glial scars, both in vitro (Austin et al., 2017;Matamoros et al., 2019) and in vivo after spinal cord injury (Matamoros et al., 2019).While FL1 was first described as a nuclear protein, a cortical function has been identified in the motility of both human and zebrafish embryonic neurons.In each case, FL1 promoted labile microtubule depolymerization at the cortex to aid in growth cone guidance during neuronal development (Fassier et al., 2018).Two isoforms were identified-one full-length and one N-terminally truncated-which have complementary functions, neither of which involves microtubule-severing activity.While other MSEs bind the acidic tail of tubulin, full-length FL1 binds instead to the acidic tail of stabilizing EB proteins on growing microtubules and uses its ATPase to remove it.This disrupts the +TIP complex (proteins that bind to growing plus ends), uncaps the microtubule, and initiates depolymerization.The short isoform, which does not bind EB proteins, appears to be localized by actin to the cortex, where it promotes microtubule catastrophe in order to support directional turning during axon navigation (Fassier et al., 2018).

| FL2 in cell migration
If fidgetin's most characteristic role is in mitosis and if FL1's is in DNA repair, then FL2's is in adherent cell migration.After the D. melanogaster p60 subunit was shown to localize to the cortex of migratory cells to sever dynamic microtubules and suppress cell motility, the potential migratory functions of human severing enzymes was assessed (Sharp & Ross, 2012).Knockdown of FL2 increased cell migration in a scratch assay two-fold.FL2 depletion resulted a significant increase in both cell speed and directionality, indicating that FL2 depletion actually enhances the efficiency by which cells close the wound zone.FL2's function in the cell was previously unknown, and this study offered a novel insight into the (at the time putative) MSE's function (Charafeddine et al., 2015).Furthermore, this finding eventually led to the development of FL2 siRNA as a potential wound-healing therapeutic (Charafeddine et al., 2015;O'Rourke et al., 2019;J. Wang et al., 2021).
FL2 localizes to the leading edge of migrating cells and targets tyrosinated microtubules to suppress cell motility.After FL2 siRNA knockdown, microtubule density at the edge increased significantly, which supported the fact that FL2 is an MSE (Charafeddine et al., 2015;Kramer, 2019;Smart, 2023).The average number and length of EB1 comets at the leading edge also increased, indicating greater microtubule dynamicity as EB1 only binds to the plus ends of growing microtubules (Charafeddine et al., 2015).FL2's regulation of cell migration does not appear to involve the actin cytoskeleton; however, it does involve focal adhesions (Charafeddine, 2015;Smart, 2023;Smart et al., 2022).FL2 knockdown led to a significant increase in focal adhesion size at the leading edge (Charafeddine et al., 2015); the increased focal adhesion size, usually expected to impede cell motility, in fact matched the optimal adhesion size for efficient cell movement (Kim & Wirtz, 2013).This is likely due to FL2 shifting the relative activation of the RhoA and Rac1 Rho GTPases, which are master coordinators of the various components of the cell migration machinery.These larger focal adhesions also had increased amounts of activated focal adhesion kinase, which is a key regulator of focal adhesion turnover (Smart, 2023;Smart et al., 2022).FL2 was found to concentrate at regions where microtubules contact the cortex (Charafeddine, 2015), where they are likely being captured by focal adhesions to trigger their disassembly (Smart, 2023).
FL2 also regulates motility in both neuronal and cardiovascular cells, which is being harnessed for its therapeutic potential.In rat adult dorsal ganglion cells, FL2 severs unacetylated microtubules in the axon to prevent growth cone advancement into regions with inhibitory substrates; thus, it may be important in axon guidance after injury (Baker et al., 2021).When FL2 is knocked down, these cells exhibit longer axons and neurites and more growth into inhibitory regions (Baker et al., 2021).When FL2 small interfering RNA (siRNA) was applied to severed cavernous nerves, axonal regeneration increased, leading to improvement in erectile dysfunction and showing promise as a peri-surgical treatment for nerve injury during radical prostatectomies (Baker et al., 2021).FL2 depletion also leads to increased in vitro cardiovascular cell migration.Knockout in cardiac smooth muscle cells showed more sprouts and more migration out of a spheroid (Kramer, 2019).HUVECs (human umbilical vein endothelial cells) treated with FL2 siRNA exhibited faster directional migration, formed more complex angiogenic networks more quickly, and were more likely to migrate to the tips of sprouts in spheroids (Kramer, 2019).
Finally, FL2-depleted cells had higher and lower ratios, respectively, of tyrosinated and acetylated tubulin to total tubulin; this relative increase in dynamic microtubules is necessary for greater migration (Kramer, 2019).When FL2 siRNA was applied intravenously to mice after a myocardial infarction, revascularization and cardiac ejection fraction were both improved (Kramer, 2019).
Just as cardiac and neuronal injury recovery was improved by increasing cell migration via FL2 knockdown, similarly, skin (Charafeddine et al., 2015;O'Rourke et al., 2019) and corneal (J.Wang et al., 2021) repair were also augmented by treatment with nanoparticles containing FL2 siRNA.The quality of the wound healing improved, including the reappearance of normal tissue structures rather than just scar tissue.Excision wounds treated with FL2 siRNA nanoparticles had new hair follicles, and burn wounds showed a reduction in CD45 staining (a marker of inflammation) lower ratios of collagen III/I staining (an indicator of scar tissue formation), and increased PECAM1 staining (a marker of tissue revascularization) (O'Rourke et al., 2019).Corneal burn wounds treated with nanoparticles containing FL2 siRNA had decreased opacity, less neovascularization, less edema, and more nerve clusters, all of which is necessary for return of optical function, as well as fewer inflammatory cells (J.Wang et al., 2021).

| FIDGETIN FAMILY REGULATION: WHO'S HOLDING THE SCISSORS?
One of the largely untouched frontiers of fidgetin biology is their regulation within the cell.Each member of the family has a different affinity for tubulin PTMs, which is not always consistent between species; however, this is not enough to explain the highly precise circumstances in which their activities occur.Furthermore, while overexpression studies do show clear effects on the cell, the global microtubule disruption and resultant cellular toxicity that might be expected are not consistently present.Likely, this is due to further regulation, such as protein localization, activation, and RNA regulation.
Tubulin PTMs indicate which microtubules are targeted; however, how each MSE distinguishes between microtubule subpopulationssuch as how mammalian fidgetin and FL2 sever dynamic microtubules (Austin et al., 2017;Charafeddine, 2015;Charafeddine et al., 2015;Hu et al., 2017;Kramer, 2019;Matamoros et al., 2019;Smart, 2023), while D. melanogaster fidgetin severs stable microtubules (Leo et al., 2015;Yang, Mahaffey, Bérubé, & Frankel, 2006)-remains to be determined.Interestingly, free tubulin C-terminal tail peptides are sufficient to inhibit severing activity of both katanin (Bailey et al., 2015) and spastin (Roll-Mecak & Vale, 2008), raising the possibility that the activity of these MSEs may be restricted to areas with lower levels of microtubule dynamicity, due to the lower local concentration of free tubulin dimers.Further investigation into similar environmental controls of fidgetin activity is necessary and could elucidate how their activities are localized to particular regions.
Another method of spatial restriction is by compartmentalizing to the cytoplasm or to the nucleus.Fidgetin (Li et al., 2020), FL1 (Onitake et al., 2012), and FL2 (Yang et al., 2005) each have an NLS and have been shown to localize to the nucleus when overexpressed (Yang et al., 2005).Fidgetin and FL1 both have binding partners in the nucleus, where they have a function additional to their role in cytoplasmic microtubule-severing (see above) (Matsuzaki et al., 2019;Yang, Mahaffey, Berube, et al., 2006;Yuan & Chen, 2013).A nuclear role for FL2 has not been described, though more work is needed to ascertain the function of its NLS.Anecdotal evidence suggests that overexpression of FL2 in many adult cells may accumulate in the nucleus and lead to cell death, leading to speculation that nuclear compartmentalization may serve to control cytoplasmic levels.Therefore, regulation of fidgetin family function may occur in part through spatial restriction, though more exploration is necessary.
Finally, the nature of the transcriptional and translational regulation that allows for the precise expression of the MSEs is beginning to be elucidated.Katanin and spastin gene expression are regulated by transcription factor binding and gene methylation (Kelle et al., 2019), opening the question of how the expression of the fidgetins are regulated on the transcriptional level.
Fidgetins are also controlled through RNA regulation, such as RNA splicing.Mammalian and zebrafish FL1 each have a long and a short isoform, which influence microtubule dynamics distinctly (Fassier et al., 2018;L'Hôte et al., 2011).Human fidgetin is known to have several isoforms (D.Wang, Chu, et al., 2017;D. Wang, Wang, et al., 2017), though their distinguishing functions are not yet fully clear.This type of regulation is not unique to the fidgetins: mammalian spastin has two isoforms, which are involved in membrane abscission and microtubule-severing, respectively (Claudiani et al., 2005;Sakoe et al., 2021).So far, FL2 isoforms have not been reported.Therefore, a deeper look is necessary to clarify how splicing of fidgetin family isoforms may contribute to regulation of their activity.
Translational regulation is another area that has provided insight into why some MSEs are only expressed under certain conditions.
Our lab has found that wild-type levels of FL2 protein in many adult cells under static conditions is low (R. L. Birnbaum, 2023); however, FL2 protein expression locally increases at the cell edge after injury to a monolayer, which was shown to be mediated by local translation in cells on the wound periphery (R. Birnbaum et al., 2023;R. L. Birnbaum, 2023).FL2 mRNA similarly localized to the leading edge of cells, suggesting the involvement of local protein synthesis in response to the injury.Investigation into the RNA-binding proteins (RBP) that mediated this mRNA localization suggests that the RBPs IMP1 and IMP2 are involved in this process.RBPs bind mRNAs to control their subcellular localization, local translation, and degradation, thereby tightly regulating their local and temporal expression (R. Birnbaum et al., 2023;R. L. Birnbaum, 2023).
Regulation of local FL2 mRNA translation may thus be due to a tight balance between IMP-mediated transport to the leading edge, let-7 miRNA-mediated FL2 mRNA degradation, and Lin28A inhibition of let-7 activity (R. Birnbaum et al., 2023;R. L. Birnbaum, 2023).
Potential binding sites for IMP1 and IMP2 were also found in fidgetin and FL1, as well as katanin and spastin (R. Birnbaum et al., 2023;R. L. Birnbaum, 2023), indicating that spatial and temporal control of protein expression is an unexplored regulatory mechanism across the MSE families.Altogether, these findings warrant further investigation into the role of translational regulation in the activity and localization of the fidgetin family members and indeed even the other MSEs.

| FIDGETINS EXPAND KNOWN MSE ROLES AND FUNCTIONS
The fidgetin family is the most recently identified family of MSEs.In the two decades since their discovery in 2000, it has become clear that the three members have a wide range of roles in key cellular processes.The diversity thereof offers intriguing evidence for a broadening of the functions of MSEs over evolutionary time.
All the fidgetins share involvement in three crucial processes.
(1) The first is in development.Fidgetin and FL2 have similar expression patterns across embryogenesis, particularly in the nervous and cardiovascular system, which may explain their involvement in the corresponding adult tissues.FL1 was also shown to have a developmental role, though the nature of it remains to be explored further.
(2) The second is in axonal guidance: fidgetin and FL2 both sever dynamic microtubules to aid in directional tuning and growth cone mobility, while FL1 regulates axonal cargo transport.(3) The third is in migration.Fidgetin severs labile microtubules to either increase or decrease migration depending on the cell type: in astrocytes, it promotes migration, whereas in dorsal root ganglion neurons, it prevents movement into the inhibitory regions of glial scars.FL1 initiates the depolymerization of dynamic microtubules to support directional turning of growth cones.Finally, FL2 severs tyrosinated microtubules to suppress cell migration after injury in skin, cardiovascular tissue, neurons, and corneas.
Not functions are shared between all three members.While all three are part of the Meiotic Clade of AAA+ ATPases, only fidgetin and FL1 have mitotic or meiotic roles.Fidgetin works together with katanin and spastin during anaphase, and it is the only fidgetin family member so far to be shown to function in conjunction with the other MSE families.FL1 has a mitotic but not a meiotic function in C. elegans, whereas, in mice, its meiotic role is important for fertility.
Fidgetin and FL1 have also been implicated in cancer.Both exhibit higher expression in cancer cells and are associated with poorer prognosis.FL2, on the other hand, does not appear to be involved in cancer, and its expression has not been detected in any cancer genomes.
(c) Tubulin dimers can be post-translationally modified.Acetylation, which indicates stability, is the only known modification to occur within the lumen of the microtubule.(c') Most modifications occur externally on the C-terminal tail, and the most common are detyrosination (removal of the terminal tyrosine, represented by a yellow diamond) and polyglutamylation, both of which indicate microtubule stability.
; E. Zehr et al., 2017; E. A. Zehr et al., 2020), thus ensuring that F I G U R E 3 MSE structure.(a) All known MSEs share generally the same domain structure: N-terminal regulatory domain(s), responsible for localization, microtubule-binding, and/or protein-protein or membrane-protein interactions, and a C-terminal AAA+ ATPase domain, which hydrolyzes ATP in order to perform its cellular function.For the katanins, these domains are split between two different proteins (p80 and p60) that form the two subunits of the katanin complex.The regulatory p80 subunit contains WD40 repeats involved in intracellular localization and a C-terminal domain that regulates severing and binds the p60 subunit.The catalytic p60 subunit contains a microtubule-interacting and trafficking (MIT) domain, which binds to the p80 subunit, the AAA+ ATPase domain, and a vacuolar protein sorting 4 (VPS4_C) insertion of unknown function.The spastins contain the MIT and AAA+ ATPase domains as well as an N-terminal hydrophobic domain for membrane insertion and a central microtubule-binding domain (MTBD), which binds the microtubule lattice.Fidgetin, fidgetin-like 1, and fidgetin-like 2 contain a putative N-terminal nuclear localization signal (NLS), an MIT domain that binds the microtubule lattice, a catalytic AAA+ ATPase domain, and a C-terminal VPS4_C insertion.(b) Katanins are the oldest of the MSEs, conserved in sea urchins to mammals.Spastins are the next oldest, present in most model organisms.The fidgetin family appeared the most recently, and of the three members, FL1 shares the least homology both genetically and in function.FL2 is the only MSE found exclusively in mammals.(Adapted from (Kuo & Howard, 2021) and (F.J. McNally & Roll-Mecak, 2018)).
Finally, each fidgetin family member has a unique distinction not seen with the other MSE families, further indicating a widening of MSE functions over time.In addition to its severing functions, fidgetin has a role in regulation of proteolysis: expression of one cardioprotective fidgetin allele increases cellular folate levels by preventing proteasomal degradation of folate transporters and recyclers in order to decrease risk of congenital heart defects.While all the MSEs have a nuclear localization signal, its function has only been determined for FL1: FL1 uses its ATPase domain to prevent improper assembly of homologous DNA repair complexes.Finally, FL2 is the only MSE thus far to have no identified mitotic or meiotic function; as it is the most evolutionarily recent severing enzyme-and the only one exclusive to vertebrates-this may suggest a loss of the original function of the clade.Gaining a deeper understanding of the different functions and regulatory mechanisms of the fidgetins will offer information not just about this family but about the other MSEs as well.Further investigation will yield greater clarity about control of the expression, activity, and localization of the fidgetins.Recent findings about the role of local translation of FL2 provide an exciting new avenue of regulation that could further explain the precise timing and circumstance of inherent to the activities of many of the MSEs.The uniquely wide range of roles found in the fidgetin family challenges the fairly narrow understanding of severing enzyme functions: the fidgetins are involved in much more than just mitosis and migration, and this may prompt re-examination of the roles of the other families.