During organismal development, countless cell types integrate extra- and intracellular cues to form functional organs. These cues determine the destination of migrating cells, the morphology and genomic programming of differentiating cells, and the formation of networks, such as the synaptic network of the brain. While nearly all cell types undergo morphogenesis during development, the transformation of neural progenitor cells into polarized, functionally integrated neurons is especially complex, and critical to neural circuit function. This process includes cellular sphere symmetry breaking for neurite initiation, neurite elongation, axon/dendrite polarity specification, and the formation of the axonal growth cone—a cytoskeleton-rich structure that transduces guidance cues to direct the axon to its targets (McCormick and Gupton, 2020; Dorskind and Kolodkin, 2021; Zang et al., 2021). The cytoskeleton, which is comprised of microtubules, intermediate filaments, and actin filaments, undergoes major morphological rearrangements throughout these events, particularly at the cell periphery. Microtubules and actin filaments and their interactions have been a major focus of study in the context of early neuronal morphogenesis (Suter and Forscher, 2000; Dehmelt and Halpain, 2004; Dent et al., 2011; Bearce et al., 2015; Cammarata et al., 2016). During early development neurons respond to attractive and repulsive guidance cues to direct their migration and outgrowth, and cytoskeleton-associated proteins are important signal integrators and regulators of these processes.

Previous studies found that Navigators and their invertebrate homologs have AAA+ nucleotide triphosphatase (NTPase), calponin homology (CH), and coiled-coil (CC) domains, with the exception that NAV1 lacks the CH domain, likely due to secondary loss (Maes et al., 2002; Peeters et al., 2004; Stringham and Schmidt, 2009; Abe et al., 2014; Accogli et al., 2022). However, those differ in the number of reported CC domains (2, 3, or 4), and in the reported presence or absence of conserved microtubule- or cytoskeletal-binding domains. To resolve these discrepancies and further investigate the structure of Navigator family proteins, we here used current protein sequence analysis tools to study human (h) NAV1, hNAV2, and hNAV3; as well as NAV homologs Unc-53 (C. elegans) and Sickie (D. melanogaster). Since each protein has multiple potential isoforms generated from different reported transcript variants, we focused our analysis on well-documented isoforms with NCBI CCDS (consensus cDNA sequence) or Ensembl Canonical designations. The isoforms and details of sequence analysis are indicated in Figure 1, and in Supplementary Excel Workbooks 1–6.

Navigator family proteins are relatively large (>200 kD) and overall basic proteins (pI 8.2–9.5). With the exception of NAV1 (which lacks the CH domain), Navigators contain an N-terminal CH domain (~110 aa), and a C-terminal AAA+ domain (Figure 1; Supplementary Excel Worksheets S1–S5). Navigator CH domains were previously proposed to contain a single actin binding domain (Coy et al., 2002), but more recent studies suggest that CH domains typically contain multiple actin-binding sequences (Yin et al., 2020). The AAA+ domains have a core ATPase region (116–155 aa) identified by sequence analysis, flanked by extended AAA+ domain structures (401–452 total aa) inferred by structure prediction with AlphaFold (Jumper et al., 2021) and conservation analysis with COBALT (NCBI; see below). As determined by DeepCoil2 (Zimmermann et al., 2018), each protein also contains two CC domains (each between 40 and 70 aa), in conserved locations proximal to the AAA+ domain (Figure 1). We also found that Navigator family proteins contain extensive low complexity sequences (LCSs), such as serine-rich regions (SRRs), and numerous predicted intrinsically disordered regions (IDRs), with LCSs often overlapping IDRs (Figure 1).

Additional motifs predicted in Navigator family proteins included monopartite (mNLS) and bipartite (bNLS) nuclear localization sequences (NLSs; Figure 1; Lu et al., 2021), which target proteins for nuclear import (Figure 1). However, only NAV1 contains an NLS in the first 60 amino acids, where NLSs are most effective. NLS predictions do not necessarily indicate functional efficacy, but typically reflect highly basic sequences (Pemberton and Paschal, 2005). Since Navigators (including NAV1) are predominantly associated with cytoskeletal structures outside the nucleus, such as axons and growth cones, the significance of NLS motifs in Navigator family proteins remains uncertain.

Contrary to previous research reporting a “highly conserved” microtubule-binding domain (MBD) between the CH and CC domains (Peeters et al., 2004), the present analysis found no known consensus MBDs in Navigator family proteins (Figure 1). The putative MBD identified in NAV1 by deletion analysis (Martinez-Lopez et al., 2005) showed only low homology to the corresponding regions of NAV2 (36% identity) and NAV3 (38% identity), and no significant homology to Sickie or Unc-53. Likewise, the putative MBD (also termed a cytoskeleton-interacting domain) of NAV2 (Muley et al., 2008) showed only low homology to the corresponding regions of NAV1 (35% identity) and NAV3 (50% identity), and no significant homology to Sickie or Unc-53. Notably, the putative MBD regions contained extensive IDRs. Since IDRs may play a key role in the assembly and disassembly of microtubules and actin filaments (Wright and Dyson, 2015), it seems plausible that the poorly conserved IDRs in Navigator family proteins may mediate interactions with cytoskeletal proteins. Our analysis found that other proposed interaction sequences, including putative actin-binding (LKK, polyproline) and SH3-binding (PXXP) motifs (Coy et al., 2002; Stringham et al., 2002; Abe et al., 2014), were not conserved among Navigator family proteins.

Another mode of interaction with microtubules is suggested by previous studies that identified Navigators as microtubule plus-end tracking proteins (+TIPs) that reorganize the cytoskeleton (Martinez-Lopez et al., 2005; van Haren et al., 2009; Sanchez-Huertas et al., 2020). Many +TIPs bind microtubule end-binding (EB) family proteins through hydrophobic SxIP motifs (Honnappa et al., 2009), which are present in Unc-53 (Figure 1, this paper), Sickie (Abe et al., 2014), NAV1 (Sanchez-Huertas et al., 2020), and NAV2 (Abe et al., 2014). To assess SxIP motifs in Navigators, we used criteria established by proteomic analyses (Jiang et al., 2012). Specifically, SxIP motifs are considered functional if they meet three criteria: (a) they have the canonical sequence [ST]-X-[IL]-P (Ser or Thr may be the first amino acid, and Ile or Leu may be the third amino acid); (b) they meet “SxIP-9AA” contextual criteria requiring the presence of at least one basic amino acid but no acidic amino acids; and (c) are found in an IDR (Jiang et al., 2012). These criteria identified one SxIP motif in Unc-53, two in Sickie, two in hNAV1, four in hNAV2, and three in hNAV3 (Figure 1). The SxIP motifs were invariably found in the N-terminal half of Navigators, although hNAV2 had an additional SxIP motif in the C-terminal half of the protein. A previously reported SxIP motif adjacent to the AAA+ domain (Abe et al., 2014) was not confirmed, because it contained an acidic amino acid and was not in an IDR.

Prediction of three-dimensional Navigator protein structures using AlphaFold (Jumper et al., 2021) confirmed and extended results from sequence analysis. Indeed, AlphaFold identified the CH domain (absent in NAV1), two CC domains, and the AAA+ domain in each human Navigator family protein (Figure 2). Interestingly, the AAA+ domains predicted by AlphaFold were substantially larger (~400–450 aa) than the core AAA+ NTPase domains predicted from sequence analysis (~110–160 aa), consistent with typical properties of AAA+ family proteins (Hanson and Whiteheart, 2005; Seraphim and Houry, 2020). Outside the major domains of Navigators, the predicted positions of amino acids were generally low confidence, consistent with the predominance of LCSs and IDRs. The low confidence of predicted inter-domain sequence positions could result from a lack of experimentally determined structures for similar sequences, although AlphaFold can accurately predict protein structures even when similar structures are unknown (Jumper et al., 2021). Thus, it is likely that putative IDRs in Navigator family proteins do not form stable 3-dimensional structures, but are instead flexible and dynamic.

To examine the conservation of sequences and domains across Navigator family proteins, NCBI COBALT was used to align Unc-53, Sickie, hNAV2, and hNAV3 (Figure 3; hNAV1 was excluded because it lacks the CH domain.). This analysis showed that the CH, CC1, CC2, and AAA+ domains were highly conserved among these proteins, while intervening sequences were less conserved and had frequent insertions or deletions (Figure 3). The cytoskeletal-interacting domain of hNAV2 that was defined in deletion experiments (Muley et al., 2008) was not well conserved in other Navigators. Interestingly, the core AAA+ domain was flanked by highly conserved sequences spanning ~400-450aa, confirming that the extended AAA+ structural domain predicted by AlphaFold is indeed highly conserved across the Navigators.

To determine how different transcript variants and isoforms might affect protein structure and potential interactions, we compared well-documented isoforms for several Navigator family proteins. For hNAV1, we compared the canonical isoform 1 (1877 aa) and the shorter isoform 2 (1,483 aa): alternative exon usage affected IDR and LCS sequences but did not alter CC or AAA+ domains. For hNAV2, we compared isoforms 2 (2,429 aa) and 5 (2,488 aa): the additional sequences in isoform 5 likewise affected LCS and IDR regions, but not CH, CC, or AAA+ domains (Figure 4). For Sickie, we compared isoforms B (2,197 aa) and J (2,707 aa): in this case, isoform J contained additional N-terminal protein sequences (including an IDR), but major domains (CH, CC, AAA+) were again not altered (Figure 4). From these examples, we conclude that different NAV isoforms mainly alter regions outside the major conserved domains, possibly to modulate cytoskeletal binding or other targeting interactions.

Together, the data indicate that Navigator family proteins, including their invertebrate homologs, have conserved AAA+, CC1, and CC2 domains, usually accompanied by an N-terminal region with multiple SxIPs and one CH domain (except in NAV1). These major domains are connected by extensive LCS and IDR sequences, which may serve to diversify interactions with the cytoskeleton or other targets. The AAA+ domain of Navigator family proteins belongs to the McrB family in the helix-2 insert clade of AAA+ proteins (Iyer et al., 2004). Interestingly, the McrB family AAA+ proteins use GTP rather than ATP as cellular energy source (Nirwan et al., 2019), suggesting that Navigator family proteins may be GTPases that function in cellular morphogenesis.

So far, Navigators have been characterized in diverse species of protostome and deuterostome animals such as roundworm, insect, and human. Thus, Navigators were likely present already in the earliest bilaterian animals (~570 million years ago), and possibly earlier. However, it is unknown if any non-bilaterian species produce Navigators.

One putative Navigator-related protein, named DDB0185859 (protein sequence Q54Q34), has been described in a Protist, Dictyostelium discoideum, a species of slime mold (Friedberg and Rivero, 2010). DDB0185859 was characterized as having a single CH domain (CH1-type) related to that in Navigators. Sequence analysis using current algorithms confirmed that DDB0185859 has an N-terminal CH domain with homology to Navigators, but no coiled-coil or AAA+ domains were identified (Supplementary Figure S1). Thus, while the CH domain of DDB0185859 is related to the CH domain in Navigators, DDB0185859 does not itself qualify as a Navigator. Interestingly, an SxIP motif was also identified in DDB0185859, although it is unknown whether Protists utilize such motifs to interact with microtubules. Since Dictyostelium discoideum belongs to the kingdom Protista, it is likely that singlet CH1-type proteins evolved in the earliest eukaryotes (Friedberg and Rivero, 2010), and may have used SxIP motifs to interact with microtubules.

Searches in OrthoDB identified additional putative Navigator-related proteins in two groups of non-bilaterian animals: Hydra vulgaris (freshwater polyp) in the group Cnidaria, and Trichoplax adhaerens and Trichoplax sp. H2 in the group Placozoa. The homologies and gene names were assigned by automated systems and are considered preliminary. In Hydra, the LOC100207311 Neuron Navigator 1 gene encodes multiple isoforms, the longest of which is isoform X1 with 1,514 aa. Sequence analysis of isoforms X1 (XP_047126242.1) and X4 (XP_047126245.1) revealed a C-terminal AAA+ (P-loop NTPase) domain, but no CH or coiled-coil domains. In Trichoplax adhaerens, hypothetical protein TRIADDRAFT_54591 (785 aa) was tagged as “similar to neuron navigator 1.” Sequence analysis of TRIADDRAFT_54591 (XP_002111049.1) identified two coiled-coil domains, but no CH or AAA+ domains. In Trichoplax sp. H2, the Neuron Navigator 2 (NAV2) gene encodes a 1,349 aa protein (RDD46365.1), which on sequence analysis was found to contain two CC domains and a C-terminal AAA+ domain (Supplementary Figure S1). The CC domains each consisted of two subdomains separated by only 4–6 aa (Supplementary Figure S1; Supplementary Excel Worksheet S6). By FASTA Protein Similarity Search, NAV2 of Trichoplax sp. H2 was most closely related to the Navigator family based on sequences from vertebrate and invertebrate Navigators. Interestingly, the relative positions of the coiled-coil and AAA+ domains in NAV2 of Trichoplax sp. H2 are similar as in NAVs of humans and other Bilateria (Figures 4, 5). Thus, Trichoplax sp. H2 NAV2 resembles the C-terminal half of Navigator proteins in bilaterians, but lacks the CH domain present in most other Navigators. Indeed, Trichoplax sp. H2 NAV2 resembles vertebrate NAV1 in lacking the CH domain, although NAV1 is thought to have lost the CH domain secondarily (Maes et al., 2002; Figure 5). Since Trichoplax belong to Placozoa, a basal eumetazoan clade that lacks neurons (Srivastava et al., 2008; Kamm et al., 2018), it appears that the C-terminal region of Navigators (containing two CC and one AAA+ domain) was present in early Metazoa (Figure 5).

In summary, proteins similar to Navigator N-terminal (containing the CH domain) and C-terminal (containing two coiled-coil and one AAA+ domains) regions have been identified in non-bilaterian species. However, no complete Navigator proteins have been recorded outside Bilateria. Interestingly, the McrB clade of AAA+ domains are posited to have undergone trans-kingdom horizontal gene transfer to eukaryotes from the ancestral prokaryotes from which they probably originated (Iyer et al., 2004). We speculate that complete Navigators formed by fusion of N- and C-terminal precursors, as a consequence of genome rearrangement around the time when Bilateria arose (Figures 4, 5).

Both the invertebrate and vertebrate Navigators are associated with regulating the cytoskeleton during cell migration and morphogenesis. This next section will review the current literature to highlight how the Navigators regulate the cytoskeleton, binding partners that have been implicated, and how Navigators may influence cellular behavior, especially migration and morphogenesis. Table 1 summarizes these reports.

Several studies have pointed to a role for the Navigators in various cancers, most of them highlighting differential expression of one of the Navigators in association with cancer cells or cancer progression, including breast cancer, colorectal cancer and uterine sarcoma (Li et al., 2010; Carlsson et al., 2012, 2013; Cohen-Dvashi et al., 2015; Tan et al., 2015; Davidson et al., 2017; Hu et al., 2019; Uboveja et al., 2020). In particular, Nav2 was shown to promote invasion of colorectal cancer cells (Ishiguro et al., 2002; Tan et al., 2015). These studies report that Nav2 is highly expressed in colorectal cancer cells, and that Nav2 promotes migration and invasion of cancer cells, and therefore overexpression of Nav2 likely promotes metastasis (Ishiguro et al., 2002, Tan et al., 2015). These observations are interesting in light of the discovery of Nav1 involvement in macropinocytosis (Powers et al., 2022), as cancer cells use macropinocytosis to obtain nutrients to drive rapid cell proliferation (Recouvreux and Commisso, 2017; Zhang and Commisso, 2019; Xiao et al., 2021). While the Navigators are apparently not uniformly either upregulated or downregulated in cancer, the differential expression of Navigators across different types of cancers and a potential connection to macropinocytosis presents an interesting area for further study.

Many outstanding questions remain regarding the functions, mechanisms, and similarities and differences among the Navigators, as well as their roles across the multiple tissues in which they are expressed (Maes et al., 2002). Investigating the function of all three Navigators in different tissues and comparing functions across tissues and between Navigators will give insight into their specific roles. Tissue-specific single knockouts of the Navigators and comparison of phenotypes will be necessary to assess the similarities and differences.

The conservation of the AAA+ domain from invertebrates through vertebrates, possibly stemming from early metazoans, suggests this is a defining feature of the Navigator family. However, little is known about the relevance of the AAA+ domain to the Navigators’ cellular roles. In vitro experiments investigating the nucleoside triphosphatase activity of this domain are lacking. Identification of substrates for the AAA+ activity, and cellular experiments with mutated AAA+ domains to test the contribution of this domain to Navigator function would begin to answer important questions. Similar structure–function experiments investigating the other functional protein domains in the Navigators will provide additional insights. Curiously, as noted above, amino acid sequence suggests that Navigators belong to the McrB family within the helix-2 insert clade (Iyer et al., 2004), which, unlike the majority of AAA+ proteins, hydrolyze GTP rather than ATP as their nucleoside triphosphatase activity (Nirwan et al., 2019). However, neither ATPase or GTPase activity for the Navigators has yet been experimentally confirmed. Although some eukaryotes contain members of this clade (Iyer et al., 2004), the best characterized member is critical in prokaryotic DNA restriction modification. McrB functions in bacteriophage defense as the GTP hydrolyzing subunit of the E. coli McrBC restriction system (Nirwan et al., 2019). McrB forms hexamers in the presence of GTP and binds to the endonuclease subunit McrC to perform DNA translocation and cleavage (Panne et al., 1999; Nirwan et al., 2019). It is unclear whether and how this ancestral function aligns with the previous identification of Nav2/HELAD1 as a DNA helicase (Ishiguro et al., 2002). According to current knowledge, the Navigators are mostly concentrated within cytoplasmic regions of cells. Nevertheless, it is too soon to rule out a function associated with nucleic acids. Our protein domain analysis (Figure 1) suggests a possible nuclear trafficking of the Navigators due to the presence of one or more nuclear localization signals. A previous study also reported Nav3/POMFIL1 immunoreactivity at nuclear pores (Coy et al., 2002). Investigating the potential nuclear functions of the Navigators could lead to discovery of yet undescribed functions of this protein family.

Most AAA+ proteins form a hexameric ring with a central pore that mediates substrate translocation driven by the motor-like NTPase activity (Nirwan et al., 2019; Puchades et al., 2020; Khan et al., 2022). It is an outstanding question whether the Navigators form hexamers, and whether they might interact to form hetero-oligomers comprised of multiple Navigators. Therefore, determining whether Navigators interact with one another, determining their native structure, and identifying their key substrate(s) would yield critical insights into Navigator function within the cell.

Finally, to date there have been no targeted investigations of the potential redundancy or interaction among the Navigator proteins, despite the fact that they are expressed in many of the same tissues (Maes et al., 2002). While knockout or knockdown experiments on a single Navigator have been performed, experiments using double or triple knockouts would aid in understanding any redundancy or cooperation among the Navigators. This line of inquiry would likely lead to yet unexplored areas of Navigator biology.

From nematodes to humans, the Navigators and their homologs have proven to be essential for proper development of multiple tissues, including the mammalian nervous system. The Navigators likely integrate cellular responses to multiple extracellular guidance cues in different cell types, and coordinate the cytoskeletal response to these cues via both microtubules and actin filaments, in conjunction with membrane trafficking (Figure 7). Based on the discovery that Nav1 promotes macropinocytosis in neurons (Powers et al., 2022) and the similar domain structure of all the Navigators, we propose that uptake of fluid-phase cues and/or downregulation of cell surface receptors could represent general mechanisms that explain the function of the Navigators in cell migration and guidance, as well as cancer. Cancer cells use macropinocytosis to obtain extra nutrients and metabolites (Commisso et al., 2013; Qian et al., 2014; Palm et al., 2015; Recouvreux and Commisso, 2017), suggesting the possibility of a similar mechanism during the energy-demanding process of neuronal outgrowth. The ancient evolutionary origins of fluid-phase uptake mechanisms like macropinocytosis (King and Kay, 2019) aligns with the idea that macropinocytosis may be crucial to multiple biological processes. It is also likely that the Navigators influence morphogenesis and migration via interaction with other signaling molecules that directly or indirectly affect cytoskeletal dynamics (Figure 7). The recent identification of a patient with a neurodevelopmental disorder attributable to the loss of Nav2 (Accogli et al., 2022) underscores the urgency to characterize the molecules and the cellular processes in which the Navigators participate, which will further our understanding of human health and disease.

RP: conceptualization, writing—original draft version, writing—reviewing and editing, figure preparation. RH: conceptualization, writing—original draft version, writing—reviewing and editing, figure preparation. SH: conceptualization, writing—reviewing and editing, figure preparation, supervision. All authors contributed to the article and approved the submitted version.

This work was supported by National Institutes of Health (NIH) grants NS37311 and MH087823 and was a component of the National Cooperative Reprogrammed Cell Research Groups (NCRCRG) to study mental illness which was supported by NIH grant U19 MH107367.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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