InteractiveFly: GeneBrief

Rootletin: Biological Overview | References

Cilia are essential for cell signaling and sensory perception. In many cell types, a cytoskeletal structure called the ciliary rootlet links the cilium to the cell body. Previous studies indicated that rootlets support the long-term stability of some cilia. This study reports that Drosophila melanogaster Rootletin (Root), the sole orthologue of the mammalian paralogs Rootletin and C-Nap1, assembles into rootlets of diverse lengths among sensory neuron subtypes. Root mutant neurons lack rootlets and have dramatically impaired sensory function, resulting in behavior defects associated with mechanosensation and chemosensation. Root is required for cohesion of basal bodies, but the cilium structure appears normal in Root mutant neurons. Normal rootlet assembly requires centrioles. The N terminus of Root contains a conserved domain and is essential for Root function in vivo. Ectopically expressed Root resides at the base of mother centrioles in spermatocytes and localizes asymmetrically to mother centrosomes in neuroblasts, both requiring Bld10, a basal body protein with varied functions (Chen, 2015).

As the major microtubule (MT)-organizing center in animal cells, the centrosome consists of a pair of MT-based centrioles that organizes a protein matrix called the pericentriolar material to regulate MT assembly. In specific cell types, the mother centriole can mature into a basal body to organize a cilium, a slender protrusion that contains an MT-based axoneme assembled from the distal tip of the basal body. Cilia generally fall into two classes: motile cilia and primary (nonmotile) cilia. Motile cilia are often present in specialized epithelia, where they beat in coordinated waves, whereas most vertebrate cells can produce a primary cilium to sense diverse extracellular signals and transduce them into important cellular responses. Disruption of cilium assembly or function causes a spectrum of diseases named ciliopathies (Chen, 2015).

In many cell types, a fibrous cytoskeletal structure called the ciliary rootlet links the base of the cilium to the cell body. Across species, the rootlet ultrastructure consists of cross-striations appearing at intervals of 50-70 nm along its length. The size of rootlets varies among cell types, with prominent ones, for example, in mammalian photoreceptors. In mammals, Rootletin (Root, also known as ciliary rootlet coiled-coil protein) is the primary constituent of ciliary rootlets, and endogenous Root is expressed in photoreceptors and all major ciliated epithelia but absent from the spermatozoa. In mammalian cilia, Root resides only in the rootlet and does not extend into the basal body or cilium (Yang, 2002). However, the Caenorhabditis elegans Root orthologue, CHE-10, localizes at the proximal end of the basal body and extends into the transition zone, the most proximal region of the cilium (Mohan, 2013). In proliferating mammalian cells when cilia are not assembled, Root forms fibrous linkers between the centriole pairs and interacts with its paralog C-Nap1 (also known as CEP250) to promote centrosome cohesion in the cell cycle (Chen, 2015).

Over decades, biologists have been intrigued by what the in vivo function of the rootlet may be. In green algae, the rootlet fibers appear to anchor the flagella and to help absorb the mechanical stress generated by flagellar beating. Root mutant mice lack rootlets yet do not show overt defects in development, reproductive performance, or overall health, and Root is not required for normal ciliary functions during development (Yang, 2005a). However, Root is important for the long-term stability of the cilium, particularly in specialized cells, such as photoreceptors. Studies in C. elegans showed that CHE-10 (Root orthologue) maintains cilium structure through preserving intraflagellar transport and the integrity of the transition zone and the basal body (Mohan, 2013). However, the role of CHE-10 may have diverged somewhat from Root in other organisms as it localizes to the basal body and transition zone of cilia and is required in neurons that lack rootlets (Chen, 2015).

This study has identified Drosophila Root as the sole orthologue of mammalian Root and C-Nap1, and has shown that it localizes to the ciliary rootlet in sensory neurons and, upon ectopic expression, at the proximal end of mother centrioles in spermatocytes. Root is required for neuron sensory perception, affecting various behaviors related to mechanosensation and chemosensation. Root is essential for basal body cohesion and for organizing the ciliary rootlet, and its N terminus containing the evolutionarily conserved Rootletin domain is critical for Root function and rootlet assembly in vivo. This study shows that Drosophila Root organizes rootlets at the base of primary cilia in sensory neurons and is essential for sensory neuron functions, including negative geotaxis, taste, touch response, and hearing (Chen, 2015).

A recent study of Root loss of function using RNAi knockdown in Drosophila also showed the essential role for Root in sensory perception of Ch neurons (Styczynska-Soczka, 2015). This study shows that Root is not required for normal cilium assembly, and it is likely that the required neuronal function of Root is at the rootlets, as rescue constructs that express tagged versions of Root rescued phenotypes completely or partially, and partial rescue coincided with assembly of smaller rootlets. Root was required for cohesion of the basal body pair in ciliated neurons, and centrioles, but not cilia, were required for rootlet assembly. Furthermore, the conserved Root domain is required for rootlet formation and for Root function, but not for localization to basal bodies (Chen, 2015).

Bld10, a presumptive Root partner, was not required for Root assembly into rootlets in sensory neurons but was required for ectopic Root localization to the proximal base of the centriole at the threshold of the lumen. In addition, ectopic Root localized asymmetrically in NBs, accumulating much more at the mother centrosome (Chen, 2015).

How do rootlets affect sensory neuron function? Because rootlets appear to always be associated with cilia, it is likely that rootlets support the structure and/or functions of cilia, enabling their role as sensors of environmental cues. However, Root mutant mice, which lack rootlets, develop normally, and during development Root is not essential for normal cilium functions, including environmental perception and cilium beating (Yang, 2005a); however, the long-term stability of cilia requires Root (Yang, 2005a; Chen, 2015 and references therein).

One important consideration for the mouse phenotypes is that the paralog, C-Nap1, may have redundant functions with Root. Indeed, in this study, it was found that even very small rootlets, resembling the localization of C-Nap1 at the base of centrioles, could rescue Root⁶⁶. How can the rootlet, and especially a short rootlet, support mechanosensation? It has been proposed that a cytoskeletal structure (e.g., possibly the rootlet cytoskeleton) links mechanosensation from extracellular forces via the dendrite to the axon or synapse. Because the rootlet does not span across the neuron from the basal body to the axon, perhaps it links to another cytoskeleton like MTs. The conserved Root domain, which this study shows is essential for Root function but not localization to basal bodies, interacts with several kinesin light chains (Yang, 2005b), supporting the idea of a possible linkage between the rootlet and the MT cytoskeleton (Chen, 2015).

In C. elegans, che-10 (Root orthologue) mutants show much more severe defects, with cilium, transition zone, and basal body degeneration during development due to severe defects in intraflagellar transport and preciliary membrane disruption that affects delivery of basal body and ciliary components (Mohan, 2013). But these defects may not necessarily be attributed to the rootlet structure because unlike in mammalian cells, CHE-10 also localizes within the basal body and the transition zone (a 'nonfilament form' of CHE-10) in neurons both with and without rootlets (Mohan, 2013). Moreover, in che-10 mutants, cilium degeneration also occurs in neurons without rootlets. Thus, in C. elegans, CHE-10, which is required for sensory neuron function, may have acquired new functions that have deviated from its function in mammals and Drosophila where Root is restricted to the Rootlet and the proximal base of centrioles. This study found that in Drosophila the loss of rootlets impairs sensory neuron functions (Chen, 2015).

Interestingly, the size of rootlets appears to affect neuronal function, particularly in ChOs that normally have long rootlets, because it was observed that shortened rootlets, resulting serendipitously from GFP-Root expression in the Root⁶⁶ mutant background, only partially restored the JO hearing impairment. The morphologically normal appearance and stability of cilia in Root⁶⁶ neurons indicate that rootlets may mediate signal transduction from cilia to the cell body, perhaps as a key structural element of the mechanoreceptor. Shorter rootlets may transduce signal less efficiently than longer ones in the JO, explaining why GFP-Root did not completely rescue the Root66 phenotype (Chen, 2015).

Alternatively, rootlets may be important for ciliary protein trafficking at the base of the cilium and between the dendrite and the cilium. In this scenario, long rootlets may support trafficking along the dendrite more efficiently than short ones. If this is the case, defective trafficking must be limited because loss of intraflagellar transport trafficking would result in failure to maintain the cilium structure and produce a more severe uncoordination phenotype (Chen, 2015).

With ectopic Root expression, this study showed that in a Drosophila cell line without cilia or rootlets, Root organized rootlet-like structures extending from the centrioles. However, ectopically expressed Root in cells such as NBs, spermatocytes, and spermatids localized to a smaller focus in the centrioles/centrosomes. In Ch neurons, Root assembles into longer rootlets than in Es neurons. It will be interesting to know what determines the forms of Root protein (centrosomal form vs. rootlet form), and in the case of rootlets, what defines their length. How Root is targeted to basal bodies and how the Root domain regulates rootlet assembly remain important questions (Chen, 2015).

Root, like its mammalian orthologue C-Nap1, specifically associates with mother centrioles upon ectopic expression in testes or NBs. Centriolar localization of Root in NBs and testes was shown to require the proximal centriolar protein Bld10, yet Bld10 is not required for Root localization to rootlets in ciliated neurons. Therefore, different mechanisms may regulate the recruitment of Root to centrioles in proliferating cells versus rootlet assembly at basal bodies in ciliated neurons. Overall, this study shows that Drosophila Root is a key structural component of ciliary rootlets that assembles in a centriole-dependent manner, and ciliary rootlets are necessary for neuronal sensory functions (Chen, 2015).

The Drosophila homologue of Rootletin is required for mechanosensory function and ciliary rootlet formation in chordotonal sensory neurons

In vertebrates, rootletin is the major structural component of the ciliary rootlet and is also part of the tether linking the centrioles of the centrosome. Various functions have been ascribed to the rootlet, including maintenance of ciliary integrity through anchoring and facilitation of transport to the cilium or at the base of the cilium. In Drosophila, Rootletin function has not been explored. In the Drosophila embryo, Rootletin is expressed exclusively in cell lineages of type I sensory neurons, the only somatic cells bearing a cilium. Expression is strongest in mechanosensory chordotonal neurons. Knock-down of Rootletin results in loss of ciliary rootlet in these neurons and severe disruption of their sensory function. However, the sensory cilium appears largely normal in structure and in localisation of proteins suggesting no strong defect in ciliogenesis. No evidence was found for a defect in cell division. It is concluded that the role of Rootletin as a component of the ciliary rootlet is conserved in Drosophila. In contrast, lack of a general role in cell division is consistent with lack of centriole tethering during the centrosome cycle in Drosophila. Although the evidence is consistent with an anchoring role for the rootlet, severe loss of mechanosensory function of chordotonal (Ch) neurons upon Rootletin knock-down may suggest a direct role for the rootlet in the mechanotransduction mechanism itself (Styczynska-Soczka, 2015).

The ciliary rootlet has long been known from transmission electron microscopy studies as the striated fibrous structure extending from the cilium basal body towards the cell nucleus . A rootlet is present at the base of most cilia, but it is particularly robust in cells with large or motile cilia. For instance, mammalian photoreceptors have a large rootlet at the base of a connecting cilium that links to the large photoreceptive outer segment. The rootlet has been speculated to have various functions, including contraction, association with organelles, transport/trafficking and anchoring of the basal body and axoneme (Styczynska-Soczka, 2015).

Knowledge of the rootlet was advanced by the discovery of its major constituent protein, a coiled-coil protein known as rootletin (encoded by the CROCC [ciliary rootlet coiled-coil] gene in humans). Mouse rootletin is a large 2009 amino acid residue protein with a globular head domain and a tail domain consisting of extended coiled-coil structures. The tail domain mediates polymerisation, whilst the head domain interacts with kinesin light chain 3 (KLC-3) (Yang, 2005b). It seems likely that rootletin is the only structural constituent of the ciliary rootlet, and its depletion causes loss of the rootlet (Yang, 2005a; Yang, 2006). Hence, rootletin-deficient mice have been used to assess the function of the rootlet. Interestingly, mice lacking rootletin only exhibit a prominent ciliary phenotype in photoreceptors (Yang, 2005a), which are cells with high rootletin expression levels and a particularly robust rootlet. Rootletin-depleted mouse photoreceptors show signs of degeneration at 18 months, reflected by shortening, disorganisation and loss of the photoreceptor outer segments (Yang, 2005a). The requirement for the rootlet was interpreted as the need for the small connecting cilium to hold in place the large outer segment. It is notable that cells with less prominent rootlets did not show this phenotype (Styczynska-Soczka, 2015).

Thus, the hypothesis has emerged that the rootlet is required in the stability and function of cilia that are subjected to mechanical stress. Other functions for the rootlet in cilium biology, particularly in cells other than photoreceptors, are unclear. An association with KLC-3 led to suggestions that it might be involved in transport to the cilium or in facilitating intraflagellar transport (IFT) at the base of the cilium, but no transport defect was noted in rootletin mutant mice. However, in Caenorhabditis elegans the rootletin orthologue, Che-10, was shown to indirectly influence IFT by modulating the preassembly/localisation of various IFT proteins to the periciliary membrane compartment (Mohan, 2013). Interestingly, in Che-10 mutants, cilia are initially formed normally but start to degenerate in late larvae (Styczynska-Soczka, 2015).

Aside from cilia, rootletin has also been shown to have a role in centriole cohesion during the centrosome duplication cycle (Yang, 2006; Bahe, 2005; Nam, 2015). In metaphase cells, the centrosome consists of two tightly associated centrioles. After mitotic (M) phase exit, but before cell division, these become separated but loosely attached via linker proteins, known as a G1-G2 tether (Nam, 2015). Rootletin has been shown to be one of these linker proteins and so is required for centriole cohesion in G1 and S phases. It associates with the related C-Nap1, which is itself associated with the ends of centrioles, thereby forming filaments that maintain a loose connection between the centrioles (Yang, 2006; Bahe, 2005). Phosphorylation of C-Nap1/rootletin by Nek2 kinase allows separation of the centrioles in late G2 before proceeding to M phase (Bahe, 2005; Styczynska-Soczka, 2015 and references therein).

This study explores the function of the presumed Drosophila homologue of rootletin, which is encoded by the gene CG6129 (hereafter referred to as Rootletin). Drosophila displays several distinctive features relevant to Rootletin function. First, the centrosome duplication cycle is modified in Drosophila such that there is no G1-G2 tether either in the early embryo or larval neuroblasts. Instead, the centrosomes split immediately after mitosis. It is therefore of interest to ask whether Rootletin is required for Drosophila centrosome duplication (Styczynska-Soczka, 2015).

A second feature of Drosophila is that it has very few ciliated cell types. The only somatic cells bearing cilia are the type I sensory neurons, in which olfactory, gustatory or mechanosensory reception are performed via a specialised terminal cilium. While the cilia in these classes of neuron all have an associated rootlet, the most robust and prominent rootlets are found in the chordotonal (Ch) neurons. Ch neurons are auditory and proprioceptive mechanosensors and may be presumed to be under mechanical stress. Although nothing has been described of Rootletin function, it is highly represented in the transcriptome of Ch neurons (Cachero, 2011). This study investigated the expression and function of Drosophila Rootletin with particular focus on Ch neuron structure and function (Styczynska-Soczka, 2015).

Drosophila Rootletin (CG6129) is required for the formation of the ciliary rootlet of Ch neurons. This strongly supports the conclusion that it performs a conserved function as a structural component of the rootlet. The expression pattern of Rootletin also highlights this function: it is restricted to ciliated cells, i.e., the type I sensory neurons, and among these cells, it is most abundantly and persistently expressed in Ch neurons, whose cilia have very robust rootlets. In contrast, Rootletin does not seem to be required for non-ciliated cells (Styczynska-Soczka, 2015).

In Ch neurons, loss of Rootletin and the rootlet results in functionally defective sensory responses. Despite this, the Ch neuron cilium itself does not appear to be strongly defective structurally. This may suggest a defective mechanotransduction process rather than defective development of the cilium. Based on observations in mouse photoreceptors, it was proposed that a rootlet is required for mechanical stability of large or motile ciliary structures. Clearly, the Ch neurons could be described in this category as these mechanosensory cells must be under mechanical stress in their function. Moreover, the Ch neuron cilium has the molecular machinery for ciliary motility, and there is biophysical evidence that cell or ciliary motility might be important for mechanotransduction. The rootlet can therefore be seen to provide a solid anchor for this in order to maintain dendritic integrity. Age-related decline in fly proprioceptive function may be consistent with the stress/anchor hypothesis. However, at the light microscope level, no sign of collapse or shortening of the cilium with age was found in Rootletin knock-down flies (Styczynska-Soczka, 2015).

An alternative explanation for Ch neuron dysfunction and age-related decline is a requirement for rootlet/Rootletin in transport of components to the base of the cilium or within the cilium, as has been proposed for the orthologue in C. elegans. In Drosophila Ch neurons, it is clear that transport and IFT are not strongly defective as ciliogenesis appears to occur largely normally. Whilst subtle changes in the channel NompC localisation suggest that Rootletin might be indirectly involved in some aspects of IFT, no change in the localisation of IFT protein, RempA, suggests a lack of general disruption of IFT. It is possible, however, that a subtle impairment of IFT disrupts transport necessary for long-term ciliary homeostasis rather than ciliogenesis. Given the severe loss in neuronal function, even in young flies, an alternative explanation is that the rootlet directly participates in mechanotransduction, such as being required to maintain or transmit tension during cilium stimulation (Styczynska-Soczka, 2015).

In other organisms, rootletin is required for centriole cohesion or tethering after centriole duplication (Yang, 2006; Fry, 1998). In vertebrates, rootletin forms the tether in association with the related C-Nap1 (CEP250) protein (Yang, 2006). It seems unlikely that Rootletin is the centrosome linker protein in Drosophila because it is not expressed generally. Moreover, there is no separate C-Nap1 orthologue in Drosophila. Lack of this function would be consistent with observations that Drosophila cells seem to lack centriole tethering. Instead, centrioles separate immediately upon disengagement during the centrosome cycle. In mammalian cells, centrosome separation upon entering mitosis is achieved by Nek2 phosphorylation of rootletin and C-Nap1 (Lee, 2008). Interestingly, Drosophila retains an orthologue of Nek2, and in cultured Drosophila cells Nek2 knockdown causes mitotic spindle defects (Prigent, 2005). In the absence of a role for Rootletin or C-Nap1, the role of Nek2 in this process is unclear (Styczynska-Soczka, 2015).

Despite the lack of a role in centriole tethering in centrosomes, it seems that Rootletin plays a 'tethering-like' role in the basal body of the Ch neuron cilium since the proximal centriole is lost upon Rootletin knock-down. Indeed, on TEM of wild-type neurons, the proximal centriole appears to be held in place by strands of the rootlet that pass around it before joining with the distal centriole (Styczynska-Soczka, 2015).

It is concluded that the role of Rootletin as a component of the ciliary rootlet is conserved in Drosophila. In contrast, lack of a general role in cell division is consistent with lack of centriole tethering during the centrosome cycle in Drosophila. Although the evidence is consistent with an anchoring role for the rootlet, severe loss of mechanosensory function of Ch neurons upon Rootletin knock-down may suggest a direct role for the rootlet in the mechanotransduction mechanism itself. In contrast, any effect on ciliary transport appears to be subtle (Styczynska-Soczka, 2015).

The gene regulatory cascade linking proneural specification with differentiation in Drosophila sensory neurons

In neurogenesis, neural cell fate specification is generally triggered by proneural transcription factors. While the role of proneural factors in fate specification is well studied, the link between neural specification and the cellular pathways that ultimately must be activated to construct specialised neurons is usually obscure. High-resolution temporal profiling of gene expression reveals the events downstream of atonal proneural gene function during the development of Drosophila chordotonal (mechanosensory) neurons. Among other findings, this reveals the onset of expression of genes required for construction of the ciliary dendrite, a key specialisation of mechanosensory neurons. It was determined that atonal activates this cellular differentiation pathway in several ways. Firstly, atonal directly regulates Rfx, a well-known highly conserved ciliogenesis transcriptional regulator. Unexpectedly, differences in Rfx regulation by proneural factors may underlie variations in ciliary dendrite specialisation in different sensory neuronal lineages. In contrast, fd3F encodes a novel forkhead family transcription factor that is exclusively expressed in differentiating chordotonal neurons. fd3F regulates genes required for specialized aspects of chordotonal dendrite physiology. In addition to these intermediate transcriptional regulators, it was shown that atonal directly regulates a novel gene, dilatory, that is directly associated with ciliogenesis during neuronal differentiation. This analysis demonstrates how early cell fate specification factors can regulate structural and physiological differentiation of neuronal cell types. It also suggests a model for how subtype differentiation in different neuronal lineages may be regulated by different proneural factors. In addition, it provides a paradigm for how transcriptional regulation may modulate the ciliogenesis pathway to give rise to structurally and functionally specialised ciliary dendrites (Cachero, 2011).

Numerous genetic and misexpression analyses in a range of organisms have shown that proneural factors influence a neuron's ultimate phenotype (including its subtype identity) at an early stage in its development. However, the nature of this influence on the cell biological processes of neuronal differentiation has remained obscure. This study bridges the gap between early specification by the proneural factor, ato, and the differentiation of Ch neurons. The current model in both Drosophila and vertebrates is that proneural factors activate two types of target gene during neural precursor specification: a common target set for shared neuronal properties and a unique target set for subtype-specific properties. The data suggest that such neuronal subtype differences are ultimately controlled by proneural factors in several ways: by the differential regulation of both specific and common intermediate transcription factors, which in turn regulate genes for aspects of neuronal structural and functional differentiation, and by direct regulation of potential differentiation genes (Cachero, 2011).

The proneural factors ato and sc commit cells to similar but distinct neural precursor fates: Ch and ES neurons are evolutionarily related cell types with similar but distinct structural and physiological properties. Notably, both are characterised by the possession of specialised ciliary-based dendrites. Thus, ciliogenesis is a key pathway that must ultimately be activated in sensory neurons subsequent to proneural factor function. However, there are important differences between the dendrites of Ch and ES neurons. Ch dendrites have a more prototypically organised axonemal structure and possess a characteristic ciliary dilation - a specialisation that separates the Ch ciliary dendrite into functionally distinct zones. Moreover, there is evidence for an active 'beat' of Ch cilia during sensory transduction. In general, ES dendrites appear reduced in structure: although a basal body and short axoneme are present, the tip of the dendrite consists of a 'tubular body' of irregularly packed microtubules. Thus the basic ciliogenesis pathway must be modulated differently in Ch and ES differentiation, and ultimately this must reflect a difference in function between ato and sc proneural factors. The ciliogenic regulator Rfx is expressed and required for both ES and Ch lineages, but it is more strongly and more persistently expressed in Ch lineages (the Ch-enriched pattern). This modulation of Rfx expression is at least partly due to differences in its regulation by proneural factors, since it appears to be a direct target of ato but not sc. This study hypothesises that differences in Rfx regulation by the proneural factors lead to differences in implementation of a core cilia biogenesis program, thereby directly linking early proneural factor function with key differences of neuronal morphology. Consistent with this idea, the data show that several known or predicted ciliogenesis genes also exhibit this Ch-enriched pattern, and some of these are predicted or known Rfx targets (Cachero, 2011).

In this view, the subtype differences between Ch and ES neurons are partly produced by quantitative differences in timing or level of expression of a common differentiation process, which ultimately depends on a qualitative difference in Rfx regulation by the proneural factors. A possible example of this is CG6129. This gene is a predicted Rfx target gene and is expressed in a Ch-enriched pattern (Laurenon, 2007). The homologous mouse protein (Rootletin) localises to the ciliary rootlet and is required for its formation. Thus Ch-enriched expression of CG6129 explains the presence of the ciliary rootlet in Ch neurons but not ES neurons. One prediction of this hypothesis is that overexpression of Rfx in ES neurons will upregulate Ch-enriched genes, and this is borne out by preliminary experiments that show an increase in CG6129 expression in ES neurons upon Rfx overexpression. It is notable that differences in IFT activity are proposed to underlie differences in ciliary morphology while RFX class factors have been associated with regulating genes for IFT in a variety of organisms. This work suggests that variations in Rfx expression level and timing should be explored as a possible factor in cilium diversity (Cachero, 2011).

fd3F fits the more conventional view of a proneural target gene that implements a subtype-specific program of differentiation. It is expressed downstream of ato uniquely in Ch neurons and regulates genes required for functional specialisation of the Ch ciliary dendrite. It is likely that Forkhead factors regulate specialisation of ciliogenesis in other organisms. In C. elegans, FKH-2 is expressed widely early in development but is also required specifically for ciliary specialisation of one type of sensory neuron. Foxj1 in mice, Xenopus, and zebrafish appears to be required for the motile cilia of the lung airway and embryonic node, but not for primary cilia. It remains to be determined whether fd3F regulates the machinery for the active beat that occurs in Ch dendrites as part of sensory transduction. Together, these studies of Rfx and fd3F extend the previously limited knowledge of the gene regulatory network underlying ciliogenesis and provide insight into how the core program may be modified to produce the highly specialised and diverse morphologies that cilia adopt for different functions (Cachero, 2011).

Previous to this study, little was known about how ato/sc proneural genes control the acquisition of Ch/ES subtype identity, except that regulation of the Cut homeodomain transcription factor is involved. Mutant and misexpression analyses show that cut is a fate selector switch for ES identity downstream of sc, but nothing is known of its mode of action or targets. Whereas Rfx and fd3F functions are likely to be confined to neuronal morphology, cut affects the identity of support cells too. As a fate switch in the entire lineage, it appears likely that cut is involved in high-level fate specification (like proneural genes) rather than regulating aspects of differentiation directly. However, it is also possible that cut may repress ciliogenesis genes in ES neurons, either directly or by repressing Rfx expression. It will be important to integrate cut into the Ch/ES gene regulatory network in the future (Cachero, 2011).

In the temporal expression profiling data, there is a steady increase in the number of known or suspected differentiation genes expressed in developing Ch cells. Many more are not expressed until after the analysis ends. Ciliogenesis is a highly intricate cellular process requiring the coordination of perhaps hundreds of genes and differences in expression onset may indicate prerequisite steps in the process of differentiation and ciliogenesis. A surprising observation was the significant number of ciliogenesis and differentiation genes that are expressed even at the earliest profiling time point. This is unexpected, since the earliest time point is predicted to be not only before differentiation but also even before cell divisions have generated the neurons. It is suggested that further analysis of expression timing may lead to insights into the cell biology of ciliogenesis. The early activation of differentiation genes may reflect the rapid pace of development in the Drosophila embryo. Thus, early expression of ciliogenesis genes may provide components that prime cells for rapid cilium assembly later once differentiation has been triggered. Along these lines, the findings mirror striking observations of retinal ganglion cells, whose rapid differentiation within 15 minutes of the exit from mitosis has been taken to imply that genes required in postmitotic cells must be transcribed before cell division. A more intriguing possibility is that early expression reflects an orderly time course for ciliogenesis that begins many hours before the final cell division. For example, unc is thought to be required for the conversion of the mitotic centriole to ciliogenic basal body, but this stuyd found that the mRNA and fusion protein are expressed even in SOPs, several cell divisions before terminal differentiation. Interestingly, in mammals newly replicated centrioles mature over two cell cycles. It is conceivable that the sensory neuron basal body might similarly need time to mature (Cachero, 2011).

Since Rfx and some ciliogenesis genes are expressed in SOPs, what prevents ciliogenesis from being activated in the non-neuronal support cells? One possibility would be an extension of model recently proposed for the generation of support cell differences, in which Notch signalling between daughter cells confines the function of genes to one branch of the lineage. This would predict that ciliogenesis genes and/or Rfx are Notch target genes. Another possibility is that some of the gene products are asymmetrically segregated. Thirdly, ciliogenesis may not be triggered until one or more key gene products are produced in the neuronal cell (Cachero, 2011).

As a corollary, it will be important to explore further the gene regulatory network underlying the temporal and cell-type differences in ciliogenesis genes. Some early expressed differentiation genes are known or predicted Rfx targets (Laurenon, 2007). This gives a rationale for the early regulation of Rfx by ato in Ch lineages. However, in both C. elegans and D. melanogaster, Rfx regulates only a subset of ciliogenesis genes (notably, it does not regulate IFT-A genes). Further studies on ato target genes and the ciliogenesis regulatory network in sensory neurons will identify other important regulators. It remains to be determined how many differentiation genes are, like dila, direct targets of ato. Interestingly, vertebrate proneural factors are hypothesised to regulate directly the transition from cycling neural progenitor (or neural stem cell) to postmitotic differentiating neuron. Perhaps ato has retained some part of an ancestral proneural factor function in direct regulation of terminal differentiation despite the subsequent evolution of SOPs that must undergo several divisions before differentiating (Cachero, 2011).

β-Catenin is a Nek2 substrate involved in centrosome separation

β-Catenin plays important roles in cell adhesion and gene transcription, and has been shown recently to be essential for the establishment of a bipolar mitotic spindle. This study shows that β-catenin is a component of interphase centrosomes and that stabilization of β-catenin, mimicking mutations found in cancers, induces centrosome splitting. Centrosomes are held together by a dynamic linker regulated by Nek2 kinase and its substrates C-Nap1 (centrosomal Nek2-associated protein 1) and Rootletin. β-catenin binds to and is phosphorylated by Nek2, and is in a complex with Rootletin. In interphase, β-catenin colocalizes with Rootletin between C-Nap1 puncta at the proximal end of centrioles, and this localization is dependent on C-Nap1 and Rootletin. In mitosis, when Nek2 activity increases, β-catenin localizes to centrosomes at spindle poles independent of Rootletin. Increased Nek2 activity disrupts the interaction of Rootletin with centrosomes and results in binding of β-catenin to Rootletin-independent sites on centrosomes, an event that is required for centrosome separation. These results identify β-catenin as a component of the intercentrosomal linker and define a new function for β-catenin as a key regulator of mitotic centrosome separation (Bahmanyar, 2008).

Search PubMed for articles about Drosophila Rootletin

Bahe, S., Stierhof, Y. D., Wilkinson, C. J., Leiss, F. and Nigg, E. A. (2005). Rootletin forms centriole-associated filaments and functions in centrosome cohesion. J Cell Biol 171: 27-33. PubMed ID: 16203858

Bahmanyar, S., Kaplan, D. D., Deluca, J. G., Giddings, T. H., Jr., O'Toole, E. T., Winey, M., Salmon, E. D., Casey, P. J., Nelson, W. J. and Barth, A. I. (2008). beta-Catenin is a Nek2 substrate involved in centrosome separation. Genes Dev 22: 91-105. PubMed ID: 18086858

Cachero, S., Simpson, T. I., Zur Lage, P. I., Ma, L., Newton, F. G., Holohan, E. E., Armstrong, J. D. and Jarman, A. P. (2011). The gene regulatory cascade linking proneural specification with differentiation in Drosophila sensory neurons. PLoS Biol 9: e1000568. PubMed ID: 21283833

Chen, J. V., Kao, L. R., Jana, S. C., Sivan-Loukianova, E., Mendonca, S., Cabrera, O. A., Singh, P., Cabernard, C., Eberl, D. F., Bettencourt-Dias, M. and Megraw, T. L. (2015). Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila. J Cell Biol 211: 435-453. PubMed ID: 26483560

Fry, A. M., Mayor, T., Meraldi, P., Stierhof, Y. D., Tanaka, K. and Nigg, E. A. (1998). C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2. J Cell Biol 141: 1563-1574. PubMed ID: 9647649

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date revised: 22 January 2016

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