Rho-kinase: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Rho-kinase

Synonyms -

Cytological map position - 15A

Function - signaling

Keywords - planar cell polarity, cytoskeleton, germband extension

Symbol - rok

FlyBase ID: FBgn0026181

Genetic map position -

Classification - Rho-kinase

Cellular location - cytoplasmic

NCBI link: Entrez Gene
rok orthologs: Biolitmine
Recent literature
Munjal, A., Philippe, J. M., Munro, E. and Lecuit, T. (2015). A self-organized biomechanical network drives shape changes during tissue morphogenesis. Nature 524: 351-355. PubMed ID: 26214737
Tissue morphogenesis is orchestrated by cell shape changes. Forces required to power these changes are generated by non-muscle myosin II (MyoII) motor proteins pulling filamentous actin (F-actin). Actomyosin networks undergo cycles of assembly and disassembly (pulses) to cause cell deformations alternating with steps of stabilization to result in irreversible shape changes. Although this ratchet-like behaviour operates in a variety of contexts, the underlying mechanisms remain unclear. This study investigated the role of MyoII regulation through the conserved Rho1-Rok pathway during Drosophila melanogaster germband extension. This morphogenetic process is powered by cell intercalation, which involves the shrinkage of junctions in the dorsal-ventral axis (vertical junctions) followed by junction extension in the anterior-posterior axis. While polarized flows of medial-apical MyoII pulses deform vertical junctions, MyoII enrichment on these junctions (planar polarity) stabilizes them. Two critical properties of MyoII dynamics were identified that underlie stability and pulsatility: exchange kinetics governed by phosphorylation-dephosphorylation cycles of the MyoII regulatory light chain; and advection due to contraction of the motors on F-actin networks. Spatial control over MyoII exchange kinetics establishes two stable regimes of high and low dissociation rates, resulting in MyoII planar polarity. Pulsatility emerges at intermediate dissociation rates, enabling convergent advection of MyoII and its upstream regulators Rho1 GTP, Rok and MyoII phosphatase. Notably, pulsatility is not an outcome of an upstream Rho1 pacemaker. Rather, it is a self-organized system that involves positive and negative biomechanical feedback between MyoII advection and dissociation rates.

Deng, H., Wang, W., Yu, J., Zheng, Y., Qing, Y. and Pan, D. (2015). Spectrin regulates Hippo signaling by modulating cortical actomyosin activity. Elife 4: e06567. PubMed ID: 25826608
The Hippo pathway controls tissue growth through a core kinase cascade that impinges on the transcription of growth-regulatory genes. Understanding how this pathway is regulated in development remains a major challenge. Recent studies suggested that Hippo signaling can be modulated by cytoskeletal tension through a Rok-myosin II pathway. How cytoskeletal tension is regulated or its relationship to the other known upstream regulators of the Hippo pathway remains poorly defined. This study identifies the spectrins, α-spec, β-spec, or βH-spec contractile proteins at the cytoskeleton-membrane interface, as an upstream regulator of the Hippo signaling pathway. In contrast to canonical upstream regulators such as Crumbs, Kibra, Expanded, and Merlin, spectrin regulates Hippo signaling in a distinct way by modulating cortical actomyosin activity through non-muscle myosin II. These results uncover an essential mediator of Hippo signaling by cytoskeleton tension, providing a new entry point to dissecting how mechanical signals regulate Hippo signaling in living tissues.
Vega-Macaya, F., Manieu, C., Valdivia, M., Mlodzik, M. and Olguin, P. (2016). Establishment of the muscle-tendon junction during thorax morphogenesis in Drosophila requires the Rho-kinase. Genetics [Epub ahead of print]. PubMed ID: 27585845
The assembly of the musculoskeletal system in Drosophila relies on the integration of chemical and mechanical signaling between the developing muscles with ectodermal cells specialized as "tendon cells". Mechanical tension generated at the junction of flight muscles and tendon cells of the notum epithelium is required for muscle morphogenesis and is balanced by the epithelium in order to not deform. Drosophila Rho kinase (Drok) is necessary in tendon cells to assemble stable myotendinous junctions (MTJ), which are required for muscle morphogenesis and survival. In addition, Drok is required in tendon cells to maintain epithelial shape and cell orientation in the notum, independently of chascon (chas). Loss of Drok function in tendon cells results in miss-orientation of tendon cell extensions and abnormal accumulation of Thrombospondin and βPS-integrin, which may cause abnormal myotendinous junction formation and muscle morphogenesis. This role does not depend exclusively on non-muscular Myosin-II activation (Myo-II), indicating that other DRok targets are key in this process. It is proposed that DRok function in tendon cells is key to promote the establishment of MTJ attachment and to balance mechanical tension generated at the MTJ by muscle compaction.
Kotoula, V., Moressis, A., Semelidou, O. and Skoulakis, E. M. C. (2017). Drk-mediated signaling to Rho kinase is required for anesthesia-resistant memory in Drosophila. Proc Natl Acad Sci U S A 114(41): 10984-10989. PubMed ID: 28973902
Anesthesia-resistant memory (ARM) was described decades ago, but the mechanisms that underlie this protein synthesis-independent form of consolidated memory in Drosophila remain poorly understood. Whether the several signaling molecules, receptors, and synaptic proteins currently implicated in ARM operate in one or more pathways and how they function in the process remain unclear. This study presents evidence that Drk, the Drosophila ortholog of the adaptor protein Grb2, is essential for ARM within adult mushroom body neurons. Significantly, Drk signals engage the Rho kinase Drok, implicating dynamic cytoskeletal changes in ARM, and this is supported by reduced F-actin in the mutants and after pharmacological inhibition of Drok. Interestingly, Drk-Drok signaling appears independent of the function of Radish (Rsh), a protein long implicated in ARM, suggesting that the process involves at least two distinct molecular pathways. Based on these results, it is proposed that signaling pathways involved in structural plasticity likely underlie this form of translation-independent memory.
Benhra, N., Barrio, L., Muzzopappa, M. and Milan, M. (2018). Chromosomal instability induces cellular invasion in epithelial tissues. Dev Cell. PubMed ID: 30245154
Most sporadic carcinomas with high metastatic activity show an increased rate of changes in chromosome structure and number, known as chromosomal instability (CIN). However, the role of CIN in driving invasiveness remains unclear. Using an epithelial model in Drosophila, evidence is presented that CIN promotes a rapid and general invasive behavior. Cells with an abnormal number of chromosomes delaminate from the epithelium, extend actin-based cellular protrusions, form membrane blebs, and invade neighboring tissues. This behavior is governed by the activation of non-muscle Myosin II by Rho kinase and by the expression of the secreted EGF/Spitz ligand. This study has unraveled fundamental roles of the mitogen-activated protein kinase pathways mediated by the Fos proto-oncogene and the Capicua tumor suppressor gene in the invasive behavior of CIN-induced aneuploid cells. These results support the proposal that the simple production of unbalanced karyotypes contributes to CIN-induced metastatic progression.
Kirkland, N. J., Yuen, A. C., Tozluoglu, M., Hui, N., Paluch, E. K. and Mao, Y. (2020). Tissue Mechanics Regulate Mitotic Nuclear Dynamics during Epithelial Development. Curr Biol. PubMed ID: 32413305
Cell divisions are essential for tissue growth. In pseudostratified epithelia, where nuclei are staggered across the tissue, each nucleus migrates apically before undergoing mitosis. Successful apical nuclear migration is critical for planar-orientated cell divisions in densely packed epithelia. Most previous investigations have focused on the local cellular mechanisms controlling nuclear migration. Inter-species and inter-organ comparisons of different pseudostratified epithelia suggest global tissue architecture may influence nuclear dynamics, but the underlying mechanisms remain elusive. This study used the developing Drosophila wing disc to systematically investigate, in a single epithelial type, how changes in tissue architecture during growth influence mitotic nuclear migration. Distinct nuclear dynamics were observed at discrete developmental stages, as epithelial morphology changes. Genetic and physical perturbations were used to show a direct effect of cell density on mitotic nuclear positioning. Rho kinase and Diaphanous, which facilitate mitotic cell rounding in confined cell conditions, are essential for efficient apical nuclear movement. Perturbation of Diaphanous causes increasing defects in apical nuclear migration as the tissue grows and cell density increases, and these defects can be reversed by acute physical reduction of cell density. These findings reveal how the mechanical environment imposed on cells within a tissue alters the molecular and cellular mechanisms adopted by single cells for mitosis.
Espinoza, C. Y. and Berg, C. A. (2020). Detecting New Allies: Modifier Screen Identifies a Genetic Interaction Between Imaginal disc growth factor 3 and combover, a Rho-kinase Substrate, During Dorsal Appendage Tube Formation in Drosophila. G3 (Bethesda). PubMed ID: 32855169
Biological tube formation underlies organ development, and when disrupted, can cause severe birth defects. To investigate the genetic basis of tubulogenesis, the formation was studied of Drosophila melanogaster eggshell structures, called dorsal appendages, which are produced by epithelial tubes. Previously it was found that precise levels of Drosophila Chitinase-like proteins (CLPs), encoded by the Imaginal disc growth factor (Idgf) gene family, are needed to regulate dorsal-appendage tube closure and tube migration. To identify factors that act in the Idgf pathway, a genetic modifier screen was developed based on the finding that overexpressing idgf3 causes dorsal appendage defects with ~50% frequency. Importantly, mutant alleles identified combover (cmb), a substrate of Rho-kinase (Rok) and a component of the Planar Cell Polarity (PCP) pathway, as an idgf3-interacting gene: loss of function enhanced while gain of function suppressed the dorsal appendage defects. Since PCP drives cell intercalation in other systems, it was asked if cmb/+ affected cell intercalation in this model, but no evidence was found of its involvement in this step. Instead, it was found that loss of cmb dominantly enhanced tube defects associated with idgf3 overexpression by expanding the apical area of dorsal appendage cells. Apical surface area determines tube volume and shape; in this way, Idgf3 and cmb regulate tube morphology.

Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. It remains unknown how this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology. Drosophila Rho-kinase (Rok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Rok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain (Mizuno, 1999 and Winter, 2001), and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton (Winter, 2001).

The rok transcript is ubiquitous in all stages of embryogenesis and in imaginal discs. Drok2 mutants die before developing into wandering 3rd instar larvae. To investigate the function of rok in the morphogenesis of adult tissues, somatic Drok2 clones generated by mitotic recombination were examined. Homozygous Drok2 clones in the eye are of similar size as their wild-type siblings (twin spots), indicating no gross defects in proliferation or survival of Drok2 mutant cells. However, about 50% of ommatidia exhibit an increase or decrease in photoreceptor numbers. A change of photoreceptor number has also been reported in eye clones homozygous for a hypomorphic RhoA allele. Of those ommatidia with the correct number of photoreceptors, 60% were misrotated, whereas in control clones all ommatidia were properly rotated. These phenotypes resemble those of planar cell polarity (PCP) mutants such as frizzled, dishevelled, and RhoA, suggesting that rok may also participate in the signaling cascade controlling PCP. Unlike fz and dsh, however, very few ommatidia showed aberrant chirality (Winter, 2001).

The function of rok in the wing was determined, where PCP is exhibited at a single cell level. In wild-type wings, each cell produces a single, distally oriented wing hair, while in Drok2 adult wing clones, greater than 70% of the cells produce multiple hairs. In contrast to most known PCP genes, including fz and dsh, which exhibit both stereotypical orientation and multiple hair defects, most hairs within Drok2 clones maintain a largely distal orientation (Winter, 2001).

To investigate the nature of the rok phenotype during the development of wing cell polarity, phalloidin was used to examine the F-actin distribution in Drok2 clones during prehair initiation. Mutant clones were positively marked using the MARCM strategy. In control clones or in heterozygous tissue neighboring Drok2 mutant clones, each wing cell generates a single F-actin bundle from its distal vertex, extending toward its distal neighbor. However, in Drok2 clones, a majority of the cells generate more than one F-actin bundle. The F-actin-based prehairs always initiate at the cell periphery. Notably, the majority of the prehairs maintain a roughly distal orientation (in the case of multiple hairs, the vector average remains distal). The similarities between the pupal and adult clonal phenotypes indicate that the multiple hair phenotype of adult Drok2 clones is likely the result of failure to restrict F-actin bundle assembly to a single site during prehair formation. The multiple hair/actin phenotype is seen only in mutant cells and never in neighboring heterozygous or wild-type cells, indicating that rok acts in a cell-autonomous manner (Winter, 2001).

The similarity of the Drok2 clonal phenotype in the wing to aspects of the phenotypes of fz, dsh, and RhoA led to the hypothesis that Rok may act downstream of Fz/Dsh. To assess the genetic interactions among these genes, the multiple hair phenotype was quantitated in a defined region: the ventral surface of the proximal-anterior region of the wing. Use was made of the dsh1 allele, which is defective for PCP function without affecting Wg signaling. In dsh1 hemizygous males, an average of 16.8 cells with multiple hairs (12% of the cells) are present in this region. When Rok is overexpressed via a tubP-Drok transgene in the dsh1 hemizygous background, the average number of cells exhibiting multiple hairs is reduced by more than 7-fold to 2.3 per wing region. tubP-Drok expression in a wild-type genetic background does not give rise to any obvious phenotypes. Suppression of the dsh1 multiple hair phenotype by tubP-Drok expression could also be seen when F-actin-based prehairs were visualized in the pupal wing. In contrast to the suppression of the dsh1 phenotype by overexpression of Rok, reduction of rok dosage by 50% (assuming that Drok2 is null) results in a 2.5-fold increase in the number of multiple hair cells (Winter, 2001).

Genetic interactions between rok and fz were examined in a different assay. The proper level of Fz/Dsh signaling is critical for the generation of wild-type PCP, since both overexpression and loss of function of these genes result in polarity defects in the eye and the wing. Overexpression of Fz 30 hr after puparium formation (APF) produces primarily a multiple hair phenotype that is suppressed by dsh1 heterozygosity. Similarly, reducing the wild-type copy number of RhoA and rok by half suppresses the phenotype by 2- to 2.5-fold. Taken together, these experiments suggest that Rok functions downstream of Fz/Dsh in restricting the number of F-actin-based prehairs (Winter, 2001).

Rok signaling regulates the phosphorylation of nonmuscle myosin regulatory light chain (MRLC), and hence the activity of myosin II. Does the phosphorylation state of MRLC modify the multiple hair phenotype of dishevelled mutants? Use was made of a series of mutant spaghetti squash (sqh) transgenes (sqh codes for the Drosophila MRLC) with point mutations in the primary (Ser-21) and secondary (Thr-20) phosphorylation sites, changing them either to glutamic acid (phosphomimetic), or to nonphosphorylatable alanine. Can the phosphorylation state of MRLC also modulate Fz/Dsh signaling? An examination was made to determine whether the phosphomimetic and nonphosphorylatable forms of MRLC could directly modify the dsh1 multiple hair phenotype. Introducing one copy of sqhE20E21 reduces the number of multiple hair cells in dsh1 mutants by 5-fold. sqhE21, or sqhA20E21, also suppresses the dsh1 phenotype by more than 2-fold. In contrast, introduction of sqhA21 into the dsh1 background enhances the multiple hair phenotype. The involvement of MRLC in the Fz/Dsh pathway was also examined using the Fz-overexpression assay. Reducing the wild-type sqh gene dosage from two to one, by introducing a single copy of the sqhAX3 null allele, results in a 2-fold suppression of the multiple hair phenotype caused by Fz overexpression. These results support the notion that MRLC functions in the PCP pathway to restrict F-actin bundle assembly to a single site (Winter, 2001).

MRLC phosphorylation in response to Rok activation would be predicted to modify the conformation and elevate the catalytic activity of its associated heavy chain, Zipper (Zip). Does Zip also participate in regulating actin distribution/wing hair number in response to Fz/Dsh? Loss of one copy of the zip gene enhances the dsh1 phenotype by 4.5-fold, consistent with the genetic interaction data between fz/dsh and sqh. These results suggest that myosin II functions positively downstream of Fz/Dsh in regulating actin prehair development (Winter, 2001).

The localization of Zip protein in wing cells further supports its role downstream of Fz/Dsh. At the apical surface of the pupal wing cell, Zip is asymmetrically localized to the distal portion of the cell, where prehair growth initiates. This distal localization appears to coincide, temporally, with prehair initiation. To test whether Zip localization could be modified by Fz/Dsh signaling, Zip distribution at the apical surface was examined in dsh1 mutants. Instead of being concentrated in the distal region of the cell, Zip is concentrated in the center of the cell, where prehairs form in dsh1 mutants (Winter, 2001).

Does reduction in myosin II/Zip activity also result in the multihair phenotype? Use was made of the hypomorphic zip02957, since zip and sqh null mutations appear to be cell lethal in the wing. As is the case with rok, some homozygous zip02957 wing cells possess multiple F-actin prehairs (Winter, 2001).

Tests were performed to see if the gene crinkled (ck) is involved in the Fz/Dsh signaling pathway regulating wing hair number because (1) ck mutant cells in the wing lead to multiple hair and split hair phenotypes, and (2) ck encodes the Drosophila myosin VIIA protein. Mutations in mouse myosinVIIA lead to stereocilia disorganization and the formation of multiple bundles of stereocilia (Winter, 2001 and references therein).

Reduction of ck activity potently suppresses the dsh1 multiple hair phenotype. This result contrasts with the result that zip1 enhances the dsh1 multiple hair phenotype, and suggests that the two myosin heavy chains have opposing effects in regulating prehair assembly (Winter, 2001).

Both myosin heavy chain genes were tested for their ability to interact with the hs-fz induced multiple hair phenotype, and again it was found that they have opposing effects. Surprisingly, loss of one copy of zip slightly but significantly enhances the late hs-fz multiple hair phenotype, while loss of one copy of ck markedly suppresses this phenotype. These results are the reverse of what one would expect based on their interactions with dsh1, and suggests the possibility that there is a signal from Fz to Ck that is independent of Dsh, or that the multiple hair phenotypes resulting from hypo- or hyper-activity of the Fz/Dsh pathway arise via distinct biochemical mechanisms (Winter, 2001).

To further assess the nature of the relationship between the two myosins, the effect of raising or lowering the activity of MRLC on the ck phenotype was tested. The multiple hair phenotype in animals homozygous for a weak ck mutation is enhanced by one copy of the sqhE20E21 transgene (and hence, a probable increase in myosin II activity), but not by a sqhA20A21 transgene. Taken together, these experiments suggest that a balance between the activities of myosin II and myosin VIIA is important in regulating wing hair number (Winter, 2001).

Unlike other characterized PCP mutants that affect both orientation and number of wing hairs, the primary defect in Drok2 clones appears to be the presence of multiple hairs per cell, with little or no wing hair orientation defect. This suggested that Rok and what lies downstream are involved in transmitting a subset of the Fz/Dsh signal. Supporting this idea, it was found that tubP-Drok and sqhE20E21 suppress the multiple hair phenotype of dsh1, but not the hair misorientation phenotype. Additional data supporting this conclusion comes from observing the site of prehair initiation. Prehairs emerge aberrantly from the center of dsh1 mutant cells, rather than from the distal vertex as seen in wild type cells. Such mispositioning of prehair initiation correlates with the failure to acquire the proper distal orientation. While tubP-Drok expression suppresses multiple prehair formation, it does not affect the site of F-actin initiation in dsh1. Finally, the hair orientation defect resulting from Fz overexpression (via hs-fz) at 24 hours is suppressed by reducing dsh gene dosage but not that of RhoA, rok, sqh or ck. Taken together, these observations suggest that separate mechanisms allow Fz/Dsh to independently regulate the number and the orientation of prehairs, and that only the former involves Rok signaling (Winter, 2001).

Mammalian and C. elegans Rho-kinase/ROCK have been implicated in regulating actin stress fiber formation in fibroblasts (Leung, 1996; Amano, 1997; Ishizaki, 1997) or changes in hypodermal cell shape during embryonic development (Wissmann, 1997). How Rho-kinase/ROCK is regulated by an extracellular signal in a physiological process is unknown. Drosophila Rho-kinase has been shown in this study to be required for restricting the number of sites of F-actin bundle assembly in response to activation of the Fz/Dsh pathway. Together with the previously established genetic link between Fz/Dsh and RhoA, a molecular pathway from Fz/Dsh to the regulation of actin structure has been established. Proteins that physically mediate the interaction between Fz and Dsh, and between Dsh and RhoA, remain to be identified (Winter, 2001 and references therein).

The data presented in this study suggest that the Rok/myosin II pathway is involved in regulating the number -- but not orientation -- of the wing hair. What then are the components that regulate wing hair orientation? One possibility is that a bifurcation of the pathway occurs at the level of RhoA, with a separate effector pathway regulating wing hair orientation. In the eye, the JNK pathway has been implicated in functioning downstream of RhoA in regulating ommatidial polarity. However, the function of the JNK pathway in the wing has not been described, and a signaling pathway that regulates transcription is unlikely to encode the requisite spatial information necessary for selection of the site of prehair initiation. Therefore, it is likely that a separate signal from or upstream of RhoA may control the selection of the F-actin assembly site, and therefore the orientation of the wing hair (Winter, 2001 and references therein).

Several other genes have been implicated in regulating wing hair number based on their loss-of-function phenotypes, including inturned (in), fuzzy(fy), and multiple wing hair (mwh). These gene products are thought to participate in a distinct branch of the polarity signaling pathway for the following reasons: (1) they each have significant effects on both hair orientation and hair number, they are unlikely to function downstream of Rok; (2) expression of the phosphomimetic Sqh-E20E21, or the nonactivatable Sqh-A20A21, in mwh or in mutant backgrounds does not modify their phenotypes (Winter, 2001); (3) while Rok functions in the eye as well as in the wing, In, Fy, and Mwh have no apparent role in PCP signaling in the eye (Winter, 2001).

The misorientation of ommatidia in Drok2 eye clones is also consistent with its functioning in the Fz/Dsh/RhoA pathway. It is conceivable that Rok may act as an effector of RhoA in regulating the process of ommatidial rotation. Alternatively, loss of Rok function could lead to a disruption of the actin cytoskeleton and a consequent loss of the appropriate cell -- cell contacts that support intercellular signaling essential for ommatidial rotation. Although rok mutants exhibit misrotation of ommatidia, they acquire the proper chiral form. Determination of ommatidial rotation may reflect a Rok-dependent subset of PCP signaling in the eye that is, in some way, molecularly analogous to the subset of PCP signaling in the wing, as reflected in the regulation of hair number (Winter, 2001).

Biochemical and cell culture studies have identified many potential substrates for Rho-kinase/ROCK (reviewed in Amano, 2000) The relative contribution of phosphorylation of these different substrates in mediating various functions of Rho-kinase/ROCK is unclear. Strong genetic evidence has been provided that phosphorylation of MRLC at key residues (Ser-21 in Drosophila, equivalent to Ser-19 in mammals) is a critical output for Rok signaling. Phosphomimetic MRLC expression not only suppresses the multiple wing hair phenotype, but also lethality, of Drok2 mutants. Biochemical and immunostaining experiments further demonstrate that Rok modulates MRLC phosphorylation in vivo. Previous work has suggested that Rok regulates phosphorylation of MRLC, both by direct phosphorylation and also by phosphorylation and inhibition of the myosin binding subunit of myosin phosphatase (MBS: see Drosophila Myosin binding subunit) (Amano, 2000). However, at present the relative contribution of direct phosphorylation by Rok of MRLC, versus phosphorylation of MBS that indirectly affects MRLC phosphorylation is not known (Winter, 2001).

The direct genetic interactions between dsh1 and various sqh transgenes further substantiates the notion that regulation of MRLC phosphorylation is not only a major output of Rok signaling, but also of Fz/Dsh signaling to the actin cytoskeleton in regulating hair number (Winter, 2001).

By what mechanism do myosins restrict F-actin bundle formation? In light of the finding that myosin II is concentrated at the site of prehair formation, it seems plausible that myosin II is actively involved in either the recruitment of F-actin to the prehair site, or that it directly participates in the assembly of actin bundles, or both. Studies of mammalian myosin II provide a precedent for a role in the formation of F-actin bundles. Phosphorylation of MRLC promotes a conformational change in myosin II from a folded to an extended state that readily forms multivalent bipolar filaments capable of binding multiple actin filaments. This is thought to result in F-actin bundling and stress fiber formation (Winter, 2001 and references therein).

It appears that in the developing wing, the level of MRLC phosphorylation/myosin II activity must be within an optimal range to establish the formation of a single hair. It is possible that the efficiency of F-actin bundle formation is regulated by MRLC phosphorylation in a manner similar to the control of stress fiber formation. If one further assumes that there are certain bundling substrates present only at limiting concentrations (e.g., F-actin itself), then one would predict that the assembly of one F-actin bundle would reduce the probability of forming a second bundle. When MRLC phosphorylation falls below some threshold level (e.g., in rok mutant cells), the efficiency of primary bundle formation is reduced, and thus the concentration of the limiting substrate remains at sufficient levels to support the assembly of secondary bundles/prehairs. Conversely, if MRLC is hyperphosphorylated (e.g., in Fz-overexpressing cells), the bundling efficiency may increase such that the threshold concentration for bundle formation would be reduced, thereby increasing the probability of assembling multiple bundles/prehairs. Future studies will be required to determine the detailed mechanisms involved (Winter, 2001).

In addition to nonmuscle myosin II, which resembles the myosin II from skeletal muscle, there exists a large class of unconventional myosins that have different properties and potential functions in nonmuscle cells. For instance, several different classes of unconventional myosins are expressed in inner ear epithelium with different subcellular localization. Mutations in three of the unconventional myosins, myosin VI, VIIA, and XV, cause hearing/balancing defects in mice, two of which when mutated in humans result in deafness. Of particular interest in the context of this study is myosin VIIA, mutations of which are responsible for mouse shaker-1 and human Usher's syndrome 1B. Loss-of-function ck (Drosophila Myosin VIIA) mutants exhibit a multiple hair and split wing hair phenotype. ck exhibits strong genetic interactions with components of the signal transduction pathway defined in this study, and has the opposite effects as that of myosin II. The seemingly antagonistic relationship between myosin II and myosinVIIA may suggest a mechanism in which the balance of the activities or stoichiometry of these two myosins is critical for the common process they regulate. For example, myosin II and myosin VIIA may share some common, limiting component(s) required for their activity. Thus, by reducing the myosin VIIA dose, myosin II has a larger share of the common component(s) and thus its activity is upregulated (Winter, 2001 and references therein).

Recent evidence suggests that vertebrate homologs of Dsh and Fz regulate cell morphogenesis using a pathway(s) analogous to that of the Drosophila PCP pathway. The findings described in this study of Rok raise the possibility that the Fz/Dsh cytoskeletal pathway may also be at work in regulating cell polarity in vertebrates, including the stereocilia formation in the inner ear (Winter, 2001).

Asymmetrically deployed actomyosin-based contractility generates a boundary between developing leg segments in Drosophila

Classically, it has been assumed that adhesive differences are a primary means of sorting cells to their respective territories. Yet it is becoming clear that no single, simple mechanism is at play. In the few instances studied, an emergent theme along developmental boundaries is the generation of asymmetry in cell mechanical properties. The repertoire of ways in which cells might establish and then put mechanical asymmetry to work is not fully appreciated since only a few boundaries have been molecularly studied. This study characterize one such boundary in the develop leg epithelium of Drosophila. The region of the pretarsus / tarsus is a known gene expression boundary that also exhibits a lineage restriction. This study show that the interface comprising this boundary is strikingly aligned compared to other cell interfaces across the disk. The boundary also exhibits an asymmetry for both Myosin II accumulation as well as one of its activators, Rho Kinase. Furthermore, the enrichment correlates with increased mechanical tension across that interface, and that tension is Rho Kinase-dependent. Lastly, interfering with actomyosin contractility, either by depletion of myosin heavy chain or expression of a phosphomimetic variant of regulatory light chain causes defects in alignment of the interfaces. These data suggest strongly that mechanical asymmetries are key in establishing and maintaining this developmental boundary (Ly, 2017).

A key component comprising a developmental boundary is the special mechanical properties imposed to its interfaces. Insights into these properties have been gained from the few tissues that have been studied, such as rhombomere boundaries in the vertebrate, but especially the study of several boundaries in Drosophila. The latter studies in Drosophila have afforded much higher resolution so far than study of rhombomeres. Still, relatively few boundaries overall have been studied, and that makes it difficult to draw any generalizations for how the underlying mechanics makes the boundary. This paper reports initial studies on the late-arising developmental boundary necessary for leg segmentation. The pretarsal / tarsal boundary was more aligned than the canonical AP compartment boundary. The rail exhibits an asymmetry in actomyosin accumulation as well as one of its activators, Rho Kinase. This is shown to result in increased tension along the boundary, which is important in aligning its interfaces (Ly, 2017).

Polarized actomyosin enrichment leads to increased cell bond tension along the pretarsal / tarsal interfaces. The fold increase of tension compared with the orthogonal rung interfaces is in line with differences observed in several other tissues, such as the Antero-posterior and dorso-ventral compartment boundaries. Here, along the pretarsal / tarsal interface, actomyosin contractility generates a very smooth, arcing boundary. The alignment is significant, as it is even more aligned than the well-studied AP compartment boundary. In itself, this fact strongly suggests that study of the pretarsal / tarsal boundary will complement the information obtained though study of other developmental boundaries (Ly, 2017).

The data revealing enrichment of the Myosin II regulatory light chain as well as Rho Kinase along rail interfaces strongly implicates contractility in alignment, and the degree of mis-alignment observed in zip mutants supports this contention. Furthermore, treatment with a Rho Kinase inhibitor reduced actomyosin enrichment and released tension along the rail, rapidly generating a less aligned state. In addition, since removal of the Rho Kinase inhibitor led to the rapid re-establishment of alignment, the data collectively argue that asymmetric contractility can drive this alignment event. Still, Rho Kinase inhibitors can affect other protein kinases, such as Atypical Protein Kinase (aPKC). Thus, even though a quite selective Rho Kinase inhibitor was used, it is still possible that another kinase also contributes to alignment, perhaps targeting a factor in addition to the myosin regulatory light chain (Ly, 2017).

The expression of a phosphomimetic form of the Myosin II regulatory light chain generated defects along the rail. The precise mechanism involved awaits live-imaging the formation of the aligned interface. Without that capability in this epithelium, it cannot be determined whether the phosphomimetic form of the Myosin II generated defects due to decreased cycling between on and off states along interfaces normally enriched for myosin, or to increased activity along the normally depleted (rung) interfaces. Nevertheless, regulated contractility is certainly important to alignment (Ly, 2017).

Actomyosin enrichment and the resultant increased tension is a theme observed repeatedly along cell interfaces. Interestingly, the outcome of that increase in tension can be quite different in different circumstances. In some cases, tension stabilizes cell interfaces, as has been observed along the parasegment boundary of the embryonic epithelium, as well as the AP and DV compartment boundaries in developing imaginal disk epithelia. While actomyosin enrichment leads to stabilization in those cases, in other instances, enrichment and the associated increased tension drives interface shrinkage. Those shrinkage outcomes are crucial to the directed junctional remodeling events necessary for convergence extension. Similar shrinkage events are also observed in tissues at steady-state. For example, across the epithelial field in the developing wing, junctional shrinkage events maintain the proper geometry of cell packing. Just how actomoysin enrichment and junctional tension can be directed toward two quite diametrically opposed outcomes, shrinkage or stabilization, is unclear at present. This issue will only be resolved by examining more boundaries of each class, and by identifying more components that act along those interfaces (Ly, 2017).

In fact the pretarsal / tarsal boundary described in this study has several features in common with another interface described previously. In the late embryonic epidermis, well-after convergence and extension, a select set of cells within each parasegment organizes into aligned columns. Those aligning cell columns exhibit enrichments similar to those described here along the smooth, arcing pretarsal / tarsal rail. In addition, in both cases the cells that constitute the boundary assume elongate, rectilinear shapes. A comparison of the mechanics underlying these two alignment events could potentially reveal how actomoysin enrichment and junctional tension can be directed toward stabilization (Ly, 2017).

Besides exhibiting alignment, some boundary interfaces, such as the AP and DV compartment boundaries, are resilient to challenges from neighboring cells, whether from cell division or intercalation. The mechanical basis for this is becoming more clear. The pretarsal / tarsal boundary develops a late-acting lineage-restriction, so it is interesting to consider the degree to which increased tension contributes to the restriction. Interestingly, in depleting or manipulating Myosin II activity the pretarsal/tarsal boundary became very irregular. Yet, no evidence was found for 'invasions' from one territory to the other, at least not in these fixed preparations. This suggests that tension is not sufficient for this restriction in the leg. Perhaps like the DV compartment in the wing a combination of mechanical tension, as seen here, plus oriented divisions and cell elongation contribute to boundary integrity. Alternatively, the affinity properties of the pretarsal versus tarsal cells may well contribute to the lineage restriction (Ly, 2017).

Finally, it is noted that the interfaces flanking the rail are also aligned to a significant degree. This differs from the situation observed along the AP compartment boundary where the adjacent interfaces were used as examples of relatively unaligned interfaces. That raises the interesting question of whether the interfaces flanking the rail are actively aligned. For instance, machinery similar to that deployed along the rail might align the -1 and + interfaces. Alternatively, the flanking interfaces might be aligned only passively, as a consequence of the geometry enforced by the rail interface on the other cell interfaces. If there is an active process aligning the flanking interfaces, MyoII would appear to be minimally involved. No significant enrichment of MyoII was observed along the -1 interface compared to the adjacent rung, and although these interfaces retained some tension, the level was much reduced along the -1 and + interfaces compared to the rails (Ly, 2017).

It is not yet known how the polarized enrichments are first established along the pretarsal / tarsal boundary. There is a fairly well-understood gene regulatory hierarchy that establishes the pretarsal and tarsal territories during the mid third instar period of development. The initially rough borders between the two territories are subsequently refined by further cross-regulatory interactions. Thus, it is no surprise that interfering with the transcriptional regulator, C15, can cause defects along the boundary. In addition, among the factors that are genetically regulated by this transcriptional circuitry are Fasciclin II and the leucine rich proteins, Capricious and Tartan (Caps; Trn). However, it is not known how direct that regulation might be. Moreover, neither removing Fasciclin II, nor both Caps and Trn, generated phenotypes that seemed clarifying. This suggests that key factors remain to be defined. A similar limitation extends to the parasegmental, AP and DV boundaries. While the Wingless, Hedgehog and Notch pathways, respectively, have been implicated at those boundaries, the analyses still leave open the possibility that control by each of those pathways is indirect. Unraveling the direct links from cell signaling to the mechanics of tissue boundaries remains an important goal in studying morphogenesis (Ly, 2017).

Adherens junction remodelling during mitotic rounding of pseudostratified epithelial cells

Epithelial cells undergo cortical rounding at the onset of mitosis to enable spindle orientation in the plane of the epithelium. In cuboidal epithelia in culture, the adherens junction protein E-cadherin recruits Pins/LGN/GPSM2 and Mud/NuMA to orient the mitotic spindle. In the pseudostratified columnar epithelial cells of Drosophila, septate junctions recruit Mud/NuMA to orient the spindle, while Pins/LGN/GPSM2 is surprisingly dispensable. This study shows that these pseudostratified epithelial cells downregulate E-cadherin as they round up for mitosis. Preventing cortical rounding by inhibiting Rho-kinase-mediated actomyosin contractility blocks downregulation of E-cadherin during mitosis. Mitotic activation of Rho-kinase depends on the RhoGEF ECT2/Pebble and its binding partners RacGAP1/MgcRacGAP/CYK4/Tum and MKLP1/KIF23/ZEN4/Pav. Cell cycle control of these Rho activators is mediated by the Aurora A and B kinases, which act redundantly during mitotic rounding. Thus, in Drosophila pseudostratified epithelia, disruption of adherens junctions during mitosis necessitates planar spindle orientation by septate junctions to maintain epithelial integrity (Aguilar-Aragon, 2020).

Adherens junctions have long been thought to be continuously essential for maintaining epithelial form and function. The current findings demonstrate transient loss of adherens junctions during division of pseudostratified epithelial cells, an event that involves adherens junction remodelling during the extensive rounding up of cell shape in mitosis. This study has furthermore shown that loss of adherens junctions is a direct consequence of the increased Rho activity and actomyosin contractility that drives mitotic rounding, which is both necessary and sufficient to regulate the level of junctional E-cadherin in the pseudostratified wing imaginal disc epithelium of Drosophila. These findings are consistent with previous observations that adherens junctions can be removed via E-cadherin endocytosis upon planar polarised Rho activation and actomyosin-driven junctional shrinkage generated during cell-cell rearrangements in the Drosophila embryo and recent optogenetic experiments in human cells. However, the global loss of E-cadherin observed during mitosis of pseudostratified epithelial cells is unprecedented and may be uniquely required by the rapid transformation of these cells from their highly columnar shape to a rounded sphere at mitosis, which involves a rapid increase in apical area and junctional length. This change in cell chape, driven by global actomyosin contractility during mitotic rounding, may both spread out junctions and disrupt cadherin-cadherin contacts between neighbouring cells to favour endocytosis (Aguilar-Aragon, 2020).

Notably, loss of E-cadherin does not seem to occur during mitosis in cuboidal epithelial cells, which undergo a much milder cell shape change during mitotic rounding, such as the Drosophila follicle cell epithelium or in many human cultured epithelial cell lines. Indeed, E-cadherin was reported to play essential roles in planar spindle orientation in cultured human cells. Thus, the pseudostratified epithelia of Drosophila face a unique challenge of orienting the mitotic spindle in the plane of the epithelium without the use of adherens junctions as a cue, which may explain why these cells instead rely on septate junctions, while planar spindle orientation can occur normally in the cuboidal follicle cell epithelium before septate junctions form. In the absence of septate junctions, spindle-orienting factors such as Pins, Mud, Dlg and Scrib localise to lateral membranes, overlapping with E-cadherin, which can directly interact with Scrib in both Drosophila and human cells (Aguilar-Aragon, 2020).

The findings of this study also shed light on the molecular mechanisms linking the cell cycle with control of Rho activation during mitotic rounding. Downstream of the master mitotic kinase Cdk1, a key role for both Aurora A and B kinases (acting redundantly) was identified in initiating mitosis and maintaining cortical rounding, with Aurora B then acting alone to drive furrow formation during cytokinesis. Aurora kinases are known to activate the key cell cycle kinase Plk1/Polo, which can then activate RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl, possibly via the kinesin-like protein MKLP1/KIF23/ZEN4/Pav. One report suggested that Aurora B also acts to directly phosphorylate RacGAP1/MgcRacGAP/CYK4/Tum on S387, but this study found that a CRISPR-knockin mutation of this site (tumS387A) is homozygous viable and fertile in Drosophila. In addition, Aurora B can also directly phosphorylate MKLP1/KIF23/ZEN4/Pav to oligomerise and activate RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl during cytokinesis, via a mechanism involving plasma membrane association of clustered C1 domains from the RacGAP1/MgcRacGAP/CYK4/Tum protein. Accordingly, it was found that a CRISPR-knockin mutation of this site (pavS734A/S735A) is homozygous lethal in Drosophila, suggesting that Aurora A/B could act via this complex during mitotic rounding. Indeed, MKLP1/KIF23/ZEN4/Pav undergoes cell cycle-dependent re-localisation from the nucleus (in interphase) to the cytoplasm (in mitosis), including a clear localisation to the entire plasma membrane during mitosis and then to the cleavage furrow during cytokinesis. The results show that mitotic activation of the MKLP1/KIF23/ZEN4/Pav binding partner RacGAP1/MgcRacGAP/CYK4/Tum also involves translocation from the nucleus to the cytoplasm, similar to ECT2/Pbl, where all three proteins are then available to bind the entire plasma membrane and generate global Rho activity to drive cortical contractility and loss of adherens junctions during mitotic rounding (Aguilar-Aragon, 2020).

The complex of MKLP1/KIF23/ZEN4/Pav and RacGAP1/MgcRacGAP/CYK4/Tum is often referred to as the 'centralspindlin' complex, due to its association with the spindle midzone at anaphase. However, the use of this term was avoided because association with the spindle midzone is not required for the function of this complex in activating ECT2/Pbl and Rho at the plasma membrane during cytokinesis. Furthermore, the results show that the same complex also functions prior to anaphase or cytokinesis in activating ECT2/Pbl and Rho for cortical rounding and downregulation of adherens junctions from the very onset of mitosis (Aguilar-Aragon, 2020).

Importantly, the function of the three cell cycle-regulated Rho activators discussed in this study - MKLP1/KIF23/ZEN4/Pav, RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl - on the regulation of adherens junctions appears to be dose-dependent and cell shape change-dependent. The sudden re-localisation of these proteins to the cytoplasm at mitosis drives a global increase in Rho activation, cell shape change and loss of junctions. In contrast, the relatively low level of these proteins in the cytoplasm during interphase appears to contribute to the normal maintenance of Rho activity at the junctional actomyosin ring to maintain adherens junctions-at least in mammalian cells. In Drosophila, an interphase role of ECT2/Pbl in contributing to Rho activation and maintenance of adherens junctions is also plausible and should be most easily distinguished from the mitotic role in non-dividing epithelial cells. However, in the ovarian follicle cell epithelium, silencing of ECT2/Pbl by RNAi affected cytokinesis (leading to larger cells) but did not affect overall epithelial architecture after cells arrest their proliferation, suggesting that any interphase function may be obscured by redundancy with other RhoGEFs in this tissue. Note also that, in this cuboidal epithelium, mitotic rounding itself is more subtle and ECT2/Pbl is dispensable for planar spindle orientation and epithelial integrity (Aguilar-Aragon, 2020).

Finally, it is noted that ECT2/Pbl was initially reported to be capable of acting as GEF for another GTPase, Cdc42, in addition to Rho 38. This activity suggested a possible role in regulating the apical Cdc42-Par6-aPKC complex in epithelial polarity and in mitosis. In Drosophila, ECT2/Pbl was found to drive transient apical spreading of the Cdc42-Par6-aPKC complex during mitotic rounding of pupal notum epithelial cells. This study found evidence for a similar function of ECT2/Pbl in activating Cdc42 during interphase in the post-mitotic ovarian follicle cell epithelium, although the loss-of-function phenotype of ECT2/Pbl is obscured due to redundancy with other Cdc42 GEFs such as beta-PIX. Loss of both ECT2/Pbl and beta-PIX reduced the level of the Cdc42-Par6-aPKC complex localising to the apical domain of post-mitotic follicle cells and also reduced apical ZO-1 localisation in human Caco2 intestinal epithelial cells in culture. In contrast, the gain-of-function phenotype caused by ECT2/Pbl overexpression is a neoplastic tumour-like phenotype and clearly involves ectopic spreading of the apical Cdc42-Par6-aPKC complex, in addition to persistent cell rounding in both cuboidal follicle cells and pseudostratified wing epithelial cells (Aguilar-Aragon, 2020).

In conclusion, the findings provide new insights into the cell biology of mitotic rounding, identifying remodelling of adherens junctions as a key event in pseudostratified epithelia, where rounding is extensive, but not cuboidal epithelia, where rounding is more subtle. These results are consistent with the hypothesis that Rho activation and actomyosin contractility can stabilise adherens junctions in the absence of mechanical strain, but that Rho activation can induce E-cadherin endocytosis above a critical strain threshold, be it either junctional shrinkage or expansion, both of which may alter the geometry of the junctional actomyosin ring and disrupt cadherin-cadherin contacts between neighbouring cells to favour endocytic internalisation of E-cadherin. The results also clarify the molecular mechanisms linking cell cycle control machinery, particularly the Aurora A and B kinases, with Rho activation and mitotic rounding. Lastly, this work may have direct relevance to certain human epithelial cancers, such as lung cancer or glioma, where overexpression of ECT2/Pbl has been reported to correlate for poor prognosis, and where the findings suggest it could drive not only disruption of epithelial polarity via activation of Cdc42 or Rac but also loss of adherens junctions via sustained Rho activation to promote tumour progression (Aguilar-Aragon, 2020).

Ras acts as a molecular switch between two forms of consolidated memory in Drosophila

Long-lasting, consolidated memories require not only positive biological processes that facilitate long-term memories (LTM) but also the suppression of inhibitory processes that prevent them. The mushroom body neurons (MBn) in Drosophila melanogaster store protein synthesis-dependent LTM (PSD-LTM) as well as protein synthesis-independent, anesthesia-resistant memory (ARM). The formation of ARM inhibits PSD-LTM but the underlying molecular processes that mediate this interaction remain unknown. This study demonstrates that the Ras-->Raf-->rho kinase (ROCK) pathway in MBn suppresses ARM consolidation, allowing the formation of PSD-LTM. The initial results revealed that the effects of Ras on memory are due to postacquisition processes. Ras knockdown enhanced memory expression but had no effect on acquisition. Additionally, increasing Ras activity optogenetically after, but not before, acquisition impaired memory performance. The elevated memory produced by Ras knockdown is a result of increased ARM. While Ras knockdown enhanced the consolidation of ARM, it eliminated PSD-LTM. These effects are mediated by the downstream kinase Raf. Similar to Ras, knockdown of Raf enhanced ARM consolidation and impaired PSD-LTM. Surprisingly, knockdown of the canonical downstream extracellular signal-regulated kinase did not reproduce the phenotypes observed with Ras and Raf knockdown. Rather, Ras/Raf inhibition of ROCK was found to be responsible for suppressing ARM. Constitutively active ROCK enhanced ARM and impaired PSD-LTM, while decreasing ROCK activity rescued the enhanced ARM produced by Ras knockdown. It is concluded that MBn Ras/Raf inhibition of ROCK suppresses the consolidation of ARM, which permits the formation of PSD-LTM (Noyes, 2020).

Consolidation is a process required for the formation of long-lasting memories. This process of converting memories that are initially sensitive to disruption from a variety of insults to more resilient ones is well conserved and many of its characteristics are shared across species. For example, memory in invertebrates and vertebrates lasts longer following multiple spaced training sessions, undergoes both molecular/cellular and systems consolidation, and can be disrupted by inhibition of protein synthesis (Noyes, 2020).

The fruit fly Drosophila melanogaster forms two distinguishable types of consolidated aversive olfactory memory: 1) anesthesia-resistant memory (ARM), which reportedly decays to negligible levels by 4 d after conditioning, can be generated by a single training session; 2) protein synthesis-dependent long-term memory (PSD-LTM), which shows limited decay, requires spaced training. These two types of consolidated memory are not independent from one another. The formation of ARM impairs either the formation or expression of PSD-LTM. Although circuit mechanisms possibly responsible for this relationship are beginning to be dissected, the molecular requirements in the mushroom body (MB), a brain region critical for the storage and retrieval of PSD-LTM and ARM remain unknown (Noyes, 2020).

The small GTPase Ras85D (Ras) is a Drosophila homolog of the mammalian Ras family genes KRAS, NRAS, and HRAS. Activated Ras proteins act as signaling switches, initiating signaling cascades through multiple downstream effector proteins. Precise induction and regulation of Ras activity is essential for mammalian synaptic plasticity and memory. Although upstream regulators of Ras, like NF1 and DRK, have been explored for their roles in Drosophila learning and memory Ras itself has not been thoroughly examined. A large RNA interference (RNAi) screen identified Ras85D as a memory suppressor gene but did not detail its specific role in memory suppression (Noyes, 2020).

This study reports that Ras activity in the MB acts as a switch between the two forms of consolidated memory, required both for PSD-LTM and inhibition of ARM. Increasing Ras activity dramatically reduced memory expression. This effect was determined to be due to Ras regulation of ARM. Knockdown of Ras enhanced the consolidation of ARM, leading to an overall increase in memory, while Ras knockdown eliminated PSD-LTM following spaced training. Although the effect of Ras on both ARM and PSD-LTM was found to be mediated by Raf, it is independent from the canonical downstream extracellular signal-regulated kinase (ERK). Instead, Ras/Raf-mediated inhibition of rho kinase (ROCK) suppresses ARM and is required for PSD-LTM (Noyes, 2020).

Based on the results, a model in which ARM consolidation is suppressed by a training-induced increase in Ras activity. Raf activity is increased in γ MBn following training, presumably through Ras, but the receptor(s) initiating this signaling are not known. Ras can be regulated through G-coupled protein receptors. It is possible that dopamine or an unknown coneurotransmitter released from dopaminergic neurons (DAn) during training initiates Ras signaling. This would provide a link between MP1 DAn, which are proposed to gate LTM, and Ras. The participation of ROCK in consolidation suggests that PSD-LTM and ARM are modulated by changes in the actin cytoskeleton (36) but does not directly indicate whether these changes occur in the pre- or post-synaptic compartments. Of the several genes known to be required for ARM, Bruchpilot (Brp) is the only one with a well-established, specific subcellular compartmentalization. Brp is localized to presynaptic active zones and is required for normal presynaptic morphology and synaptic transmission, indicating that ARM may result from a form of presynaptic plasticity in the MB. Additionally, the DAn that are required for memory formation innervate MB axons and modulate synaptic strength between MBn and downstream MB output neurons. The results demonstrating that artificial activation of Ras increases axonal pERK in γ MBn is evidence that Ras/Raf signaling participates in axonal signal transduction and is consistent with a previous report highlighting a role for presynaptic Raf activity in γ MBn. ROCK activity in mammalian axons is critical for a number of processes; however, it has not been tested whether ROCK signaling occurs in γ MBn axons (Noyes, 2020).

The hypothesis that ARM inhibits the formation PSD-LTM was based on the observation that spaced training, which generates PSD-LTM, eliminates or precludes ARM. Subsequent research at the systems neuroscience level revealed that two sets of neurons, MP1 DAn and serotonergic projection neurons (SPn), appear to be responsible for the promotion of PSD-LTM through the suppression of ARM. The activity of these neurons is increased during spaced training. This activity reduces ARM, while inhibiting their activity enhances ARM. Blocking the activity of either set of neurons during spaced training does not prevent memory formation but prevents the formation of PSD-LTM. This suggests that without SPn and MP1 DAn activity, ARM occurs by default and is preferentially expressed at the expense of PSD-LTM. Ras fulfills the requirements as the intracellular and molecular switch regulating the inverse relationship between ARM and PSD-LTM. The suppression of ARM and formation of PSD-LTM both require Ras in γ MBn, which are downstream in the circuit from the ARM/PSD-LTM-gating MP1 DAn that synapse directly on to γ MBn (Noyes, 2020).

The mammalian counterpart for ARM, if one exists, is unknown. Protein synthesis-independent ARM has been reported to be measurable up to 4 d after conditioning, while mammalian protein synthesis-independent memory lasts only hours. Despite the lack of a clear and direct mammalian counterpart to ARM, it is becoming apparent that many of the same genes that are involved in ARM also play a role in mammalian memory and plasticity. Ras, Raf, and CDC42 negatively regulate ARM but in mammals are positive regulators of LTM. Conversely, reduced ROCK or dunce, the latter purported to function through the SPn, impair ARM. In mammals, inhibition of ROCK or a mammalian ortholog of dnc (48), PDE4, enhances memory. It seems likely that discovering more genetic regulators of ARM will reveal previously unknown genetic regulators of mammalian memory. Based on the genes and their functions discussed in this paper, it is possible that factors that promote ARM in Drosophila function in memory suppression in mammals (Noyes, 2020).

The effect of ROCK on memory is not restricted to γ MBn. ROCK is also required in α/β MBn for ARM. In this neuron type, the effects of ROCK are not mediated by Ras but through Drk, the Drosophila homolog of Grb2. It is interesting to consider whether the ROCK substrate(s) mediating enhanced ARM in α/β and γ MBn are the same even though the upstream signaling components are distinct. Several ROCK targets have been established as important for normal memory, including cofilin and nonmuscle myosin II (Noyes, 2020).

A recent report indicates that ERK activity in γ MBn slows forgetting. The current results revealing that ERK knockdown reduces memory support this conclusion. However, the former report finds that Raf RNAi expression in γ MBn reduces memory, which is at odds with the finding that Raf RNAi enhances memory. The most likely explanation for this discrepancy is the use of different gal4/UAS-RNAi combinations that produce different levels of gene knockdown. It is interesting to consider that Raf signaling in γ MBn might regulate three forms of memory: consolidated ARM and PSD-LTM through ROCK and labile memory through ERK (Noyes, 2020).

Assembly of a persistent apical actin network by the formin Frl/Fmnl tunes epithelial cell deformability

Tissue remodelling during Drosophila embryogenesis is notably driven by epithelial cell contractility. This behaviour arises from the Rho1-Rok-induced pulsatile accumulation of non-muscle myosin II pulling on actin filaments of the medioapical cortex. While recent studies have highlighted the mechanisms governing the emergence of Rho1-Rok-myosin II pulsatility, little is known about how F-actin organization influences this process. This study shows that the medioapical cortex consists of two entangled F-actin subpopulations. One exhibits pulsatile dynamics of actin polymerization in a Rho1-dependent manner. The other forms a persistent and homogeneous network independent of Rho1. The formin Frl (also known as Fmnl) has been identified as a critical nucleator of the persistent network, since modulating its level in mutants or by overexpression decreases or increases the network density. Absence of this network yields sparse connectivity affecting the homogeneous force transmission to the cell boundaries. This reduces the propagation range of contractile forces and results in tissue-scale morphogenetic defects (Dehapiot, 2020).

Animal cells can modify their shape to complete complex processes such as cell migration, division or tissue morphogenesis. These behaviours arise from the contractile properties of the actomyosin cortex and its ability to build up tension. Recent advances have shown that cortical contractility can occur in a pulsatile manner by taking the form of local and transient accumulations of myosin II (MyoII). These MyoII pulses underlie a variety of morphogenetic processes, ranging from single-cell polarization to tissue-scale remodelling. Although recent evidence suggest that MyoII pulsatility can spontaneously emerge6, the spatiotemporal pattern of cortical contractility must be controlled to produce reproducible morphogenetic outcomes. In most studied systems, this control is achieved through the conserved RhoA GTPase signalling pathway, which activates MyoII through the regulation of Rho-associated kinase (ROCK; Rok in Drosophila) and MyoII light-chain phosphatase (Dehapiot, 2020).

In addition to MyoII regulation, another key parameter that influences cortical contractility resides in the organization and dynamics of the F-actin network. Typically, the cortex assembles as a thin layer of actin filaments bound to the plasma membrane. The cortical network is both highly plastic and mechanically rigid, conferring to the cells the ability to adapt and exert forces on their environment. These remarkable properties stem from the action of actin-binding proteins (ABPs) regulating the organization and turnover of the network. Actin nucleators, such as the Arp2/3 complex or formins, promote filament polymerization that leads, respectively, to the assembly of highly branched or sparse F-actin networks. These networks can be remodelled by actin bundlers (fascin and plastin) or cross-linkers (filamin and α-actinin), and their filament turnover regulated by profilin, capping proteins or members of the actin-depolymerizing factor and cofilin families. Modulating the dynamic organization of F-actin networks through ABPs can significantly modify how MyoII contractility gives rise to cortical tension (Dehapiot, 2020).

In embryonic Drosophila epithelial cells, MyoII pulses appear in the medioapical (medial) part of the cell and produce sustained or repeated cycles of apical contraction and relaxation. MyoII pulsatility, together with adherens junction (AJ) remodelling, gives rise to a variety of tissue morphogenetic events such as mesoderm-endoderm invagination, convergent extension or tissue dorsal closure (DC). While the mechanisms underlying the emergence of MyoII pulsatility have been widely studied, little is known about how medioapical F-actin supports pulsatile contractility. It has been shown that the spatiotemporal organization of F-actin modifies the viscoelastic properties of the cortex and its ability to propagate tension. Cortical F-actin can also influence MyoII activation by serving as a scaffold for the motor-driven advection of regulators such as Rho1, Rok or Rho GTPase-activating proteins (RhoGAPs), which are required for pulse assembly and disassembly. In this study, focusing on two highly pulsatile tissues, namely the ectodermal cells during germband extension (GBE) and amnioserosa cells during DC, the regulation of the medioapical F-actin network was study and attempts were made to understand how it supports the propagation of contractile forces to the surrounding tissue (Dehapiot, 2020).

While most studies of cortical pulsed contractility have focused on the emergence of MyoII pulsatility, this study examined how cortical F-actin influences this process in embryonic Drosophila epithelial cells. In both ectodermal (GBE) and amnioserosa cells (DC) this study showed that the medioapical cortex consists of two differentially regulated but entangled subpopulations of actin filaments. These two populations share the same subcellular localization but undergo distinct spatiotemporal dynamics. The Rho1-induced pulsatile F-actin, together with MyoII, promotes local cell deformations, while the persistent network ensures homogeneous connectivity between pulses and AJs and hence spatial propagation of deformation (Dehapiot, 2020).

The formin Frl was identified as a critical nucleator that promotes the assembly of the persistent network. This constitutes a previously unappreciated role for this formin, since it has been so far mainly described as participating in lamellipodia and filopodia formation. It would be interesting to know whether, like in other systems, Frl is regulated by Cdc42 or Rac1 to promote the persistent network assembly. Furthermore, it is probable that other formins are involved in this process, since the lack of Frl only partially reduced the network density in ectodermal cells. The formin DAAM would constitute a good candidate, since it cooperates with Frl during axon growth in Drosophila (Dehapiot, 2020).

This study also showed that Frl antagonizes apical cell contractility by impairing Rho1 signalling in amnioserosa cells. While this antagonism may depend on cell context, it will be interesting to identify the crosstalk mechanisms operating between Frl and the GTPase. To this end, previous studies report that F-actin can negatively feedback on Rho1 activation. Indeed, it is possible that, like in the Caenorhabditis elegans zygote, some Rho1 inhibitors (for example, RhoGAPs) bind to cortical F-actin in the systems examined in this study. Consequently, modifying the persistent network density, through Frl loss or gain of function, could in turn modulate the levels of apical Rho1 activation. It has also been shown that advection acts as a positive feedback for pulsatility by increasing the local concentration of upstream regulators (for example, Rho1 and Rok) (Dehapiot, 2020).

It will therefore be interesting to study how the persistent network influences this feedback mechanism. The current data also revealed that modulating Frl levels affects epithelial dynamics at the cellular and tissue scales. Although this is probably influenced by the effect of Frl on medial actomyosin pulsatility and/or MyoII junctional density, this study designed a series of analyses to understand how the persistent network may influence pulsed contractility in mechanical terms. It has been suggested that medioapical F-actin acts as a scaffold to transmit contractile forces to AJs and, by extension, to the surrounding tissue. The results revealed that the persistent network does indeed play a key role in this process by promoting the uniform and long-range propagation of contractile forces. A numerical model was desigened to qualitatively recapitulate the experimental measurements and to provide solid evidence that Frl influences epithelial dynamics through the persistent network (Dehapiot, 2020).

Tissue morphogenesis requires interactions between cellular-and tissue-scale deformations, the propagation of which in space and time are little understood. This study showed that differentially regulated subpopulations of actin filaments play a key role in this process by promoting distinctly the emergence and the spatial propagation of cortical deformations. The findings echo previous experimental and theoretical studies demonstrating that the F-actin network, through its cross-linking state, the length of its filaments or its turnover, can mediate the amplitude and the length scale at which cortical stresses propagate. It will be important to unravel how cells tune these properties in different tissues and developmental stages to further understand how mechano-chemical information drives embryo morphogenesis (Dehapiot, 2020).


cDNA clone length - 6259

Bases in 5' UTR - 1022

Exons - 10

Bases in 3' UTR - 1064


Amino Acids - 1390

Structural Domains

The Drosophila homolog of Rho-kinase/ROK alpha, Rok, which has conserved the basic structural feature of Rho-kinase/ROK alpha consisting of the N-terminal kinase, central coiled-coil and C-terminal pleckstrin homology (PH) domains, has been identified (Mizuno, 1999).

Rok structure has been compared with that of rat ROK-beta. Both proteins are composed of a conserved N-terminal domain, a serine/threonine kinase domain, a large coiled-coil domain with an embedded Rho-binding domain, and a C-terminal Plekstrin-homology (PH) domain split by a Cys-rich domain (Winter, 2001).

Rho-kinase: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 17 August 2020

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.