Rac1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Rac1

Synonyms -

Cytological map position - 61F5

Function - GTPase - signaling protein

Keyword(s) - cytoskeleton

Symbol - Rac1

FlyBase ID: FBgn0010333

Genetic map position - 3-

Classification - GTP-binding protein (rho-subfamily).

Cellular location - cytoplasmic

NCBI links: | Entrez Gene | | HomoloGene
Recent literature
Ojelade, S. A., et al. (2015). Rsu1 regulates ethanol consumption in Drosophila and humans. Proc Natl Acad Sci U S A 112: E4085-4093. PubMed ID: 26170296
Alcohol abuse is highly prevalent, but little is understood about the molecular causes. This study reports that Ras suppressor 1 (Rsu1) affects ethanol consumption in flies and humans. Drosophila lacking Rsu1 show reduced sensitivity to ethanol-induced sedation. Rsu1 is required in the adult nervous system for normal sensitivity;t it acts downstream of the integrin cell adhesion molecule and upstream of the Rac1 GTPase to regulate the actin cytoskeleton. In an ethanol preference assay, global loss of Rsu1 causes high naive preference. In contrast, flies lacking Rsu1 only in the mushroom bodies of the brain show normal naive preference but then fail to acquire ethanol preference like normal flies. Rsu1 is, thus, required in distinct neurons to modulate naive and acquired ethanol preference. In humans, polymorphisms in RSU1 are associated with brain activation in the ventral striatum during reward anticipation in adolescents and alcohol consumption in both adolescents and adults. Together, these data suggest a conserved role for integrin/Rsu1/Rac1/actin signaling in modulating reward-related phenotypes, including ethanol consumption, across phyla.

Gombos, R., Migh, E., Antal, O., Mukherjee, A., Jenny, A. and Mihaly, J. (2015). The formin DAAM functions as molecular effector of the planar cell polarity pathway during axonal development in Drosophila. J Neurosci 35: 10154-10167. PubMed ID: 26180192
Recent studies established that the planar cell polarity (PCP) pathway is critical for various aspects of nervous system development and function, including axonal guidance. Although it seems clear that PCP signaling regulates actin dynamics, the mechanisms through which this occurs remain elusive. This study established a functional link between the PCP system and one specific actin regulator, the formin DAAM, which has previously been shown to be required for embryonic axonal morphogenesis and filopodia formation in the growth cone. dDAAM also plays a pivotal role during axonal growth and guidance in the adult Drosophila mushroom body, a brain center for learning and memory. By using a combination of genetic and biochemical assays, it was demonstrated that Wnt5 and the PCP signaling proteins Frizzled, Strabismus, and Dishevelled act in concert with the small GTPase Rac1 to activate the actin assembly functions of dDAAM essential for correct targeting of mushroom body axons. Collectively, these data suggest that dDAAM is used as a major molecular effector of the PCP guidance pathway. By uncovering a signaling system from the Wnt5 guidance cue to an actin assembly factor, it is proposed that the Wnt5/PCP navigation system is linked by dDAAM to the regulation of the growth cone actin cytoskeleton, and thereby growth cone behavior, in a direct way.

Sollier, K., Gaude, H. M., Chartier, F. J. and Laprise, P. (2015). Rac1 controls epithelial tube length through the apical secretion and polarity pathways. Biol Open 5: 49-54. PubMed ID: 26700724
The morphometric parameters of epithelial tubes are critical to the physiology and homeostasis of most organs. In addition, many human diseases are associated with tube-size defects. This study shows that Rac1 limits epithelial tube elongation in the developing fly trachea by promoting Rab5-dependent endocytosis of the apical determinant Crumbs. Rac1 is also involved in a positive feedback loop with the septate junction protein Coracle. Thereby, Rac1 precludes paracellular diffusion and contributes to the septate junction-dependent secretion of the chitin-modifying enzymes Vermiform and Serpentine, which restrict epithelial tube length independently of Crumbs. Thus, Rac1 is a critical component of two important pathways controlling epithelial tube morphogenesis (Sollier, 2015).

Cervantes-Sandoval, I., Chakraborty, M., MacMullen, C. and Davis, R.L. (2016). Scribble scaffolds a signalosome for active forgetting. Neuron [Epub ahead of print]. PubMed ID: 27263975
Forgetting, one part of the brain's memory management system, provides balance to the encoding and consolidation of new information by removing unused or unwanted memories or by suppressing their expression. Recent studies have identified the small G protein, Rac1, as a key player in the Drosophila mushroom bodies neurons (MBn) for active forgetting. It has also been shown that a few dopaminergic neurons (DAn) that innervate the MBn mediate forgetting. This study shows that Scribble, a scaffolding protein known primarily for its role as a cell polarity determinant, orchestrates the intracellular signaling for normal forgetting. Knocking down scribble expression in either MBn or DAn impairs normal memory loss. Scribble interacts physically and genetically with Rac1, Pak3, and Cofilin within MBn, nucleating a forgetting signalosome that is downstream of dopaminergic inputs that regulate forgetting. These results bind disparate molecular players in active forgetting into a single signaling pathway: Dopamine→ Dopamine Receptor→ Scribble→ Rac→ Cofilin.

Spinner, M. A., Walla, D. A. and Herman, T. G. (2018). Drosophila Syd-1 has RhoGAP activity that is required for presynaptic clustering of Bruchpilot/ELKS but not Neurexin-1. Genetics 208(2): 705-716. PubMed ID: 29217522
Syd-1 proteins are required for presynaptic development in worm, fly, and mouse. Syd-1 proteins in all three species contain a Rho GTPase activating protein (GAP)-like domain of unclear significance: invertebrate Syd-1s are thought to lack GAP activity, and mouse mSYD1A has GAP activity that is thought to be dispensable for its function. This study shows that Drosophila melanogaster Syd-1 can interact with all six fly Rhos and has GAP activity toward Rac1 and Cdc42. During development, fly Syd-1 clusters multiple presynaptic proteins at the neuromuscular junction (NMJ), including the cell adhesion molecule Neurexin (Nrx-1) and the active zone (AZ) component Bruchpilot (Brp), both of which Syd-1 binds directly. A mutant form of Syd-1 that specifically lacks GAP activity localizes normally to presynaptic sites and is sufficient to recruit Nrx-1 but fails to cluster Brp normally. Evidence is provided that Syd-1 participates with Rac1 in two separate functions: (1) together with the Rac guanine exchange factor (RacGEF) Trio, GAP-active Syd-1 is required to regulate the nucleotide-bound state of Rac1, thereby promoting Brp clustering; and (2) Syd-1, independent of its GAP activity, is required for the recruitment of Nrx-1 to boutons, including the recruitment of Nrx-1 that is promoted by GTP-bound Rac1. It is concluded that, contrary to current models, the GAP domain of fly Syd-1 is active and required for presynaptic development; it is suggested that the same may be true of vertebrate Syd-1 proteins. In addition, the data provide new molecular insight into the ability of Rac1 to promote presynaptic development.
Zhang, X., Li, Q., Wang, L., Liu, Z. J. and Zhong, Y. (2018). Active Protection: Learning-Activated Raf/MAPK Activity Protects Labile Memory from Rac1-Independent Forgetting. Neuron 98(1):142-155. PubMed ID: 29551489
Active forgetting explains the intrinsic instability of a labile memory lasting for hours. However, how such memory maintains stability against unwanted disruption is not completely understood. This study reports a learning-activated active protection mechanism that enables labile memory to resist disruptive sensory experiences in Drosophila. Aversive olfactory conditioning activates mitogen-activated protein kinase (MAPK) transiently in the mushroom-body gamma lobe, where labile-aversive memory is stored. This increased MAPK activity significantly prolongs labile memory retention and enhances its resistance to disruption induced by heat shock, electric shock, or odor reactivation. Such experience-induced forgetting cannot be prevented by inhibition of Rac1 activity. Instead, protection of Rac1-independent forgetting correlates with non-muscle myosin II activity and persistence of learning-induced presynaptic structural changes. Increased Raf/MAPK activity, together with suppressed Rac1 activity, completely blocks labile memory decay. Thus, learning not only leads to memory formation, but also activates active protection and active forgetting to regulate the formed memory.

Rac, Rho (see Drosophila Rho1) and Cdc42 are members of the Rho family of small guanosine triphosphatases (GTPases) are all implicated in the temporal and spatial control of actin filament organization in the cytoskeleton. In mammalian fibroblasts actin filaments exist principally in three types of structures: the cortical actin network, actin stress fibers, and cell surface protursions including membrane ruffles and microspikes. Stress fibers emanate from distinct areas of the plasma membrane known as focal adhesions, where clusters of integrin receptors bind to extracellular matrix proteins such as fibronectin and collagen. A number of proteins are found associated with focal adhesions at the intracellular face of the plasma membrane, including vinculin, talin, tensin and alpha-actinin (Ridley, 1992a and references). Ruffling, in contrast to stress fibers which emanate throughout the cell, is a phenomenon of the actin reorganization at the membrane. It is thought that ruffling is a prelude to the accumulation of multiple large intracellular vesicles by pinocytosis. It is likely that these two processes are linked, as there is a close association between ruffling and pinocytosis (Ridley 1992b).

Although structurally related, the three proteins serve different functions, to judge by their diverse roles in cultured cells. Rho controls the assembly of actin stress fibers and focal adhesion complexes (Ridley, 1992a). Rac proteins, approximately 60% identical to Rho, regulate actin filament accumulation at the plasma membrane to produce lamellipodia (cellular protrusions involved in cell motility) and membrane ruffles. Rac also induces stress fiber formation but this function is dependent on the presence of Rho (Ridley, 1992b). Cdc42 stimulates the formation of filopodia (finger like projections that form around actin bundles whose barbed ends are oriented in the direction of outgrowth, involved in motility and axon growth cone spreading) (Nobes, 1995a).

Rac stimulates the assembly of multimolecular focal complexes at the plasma membrane. These complexes, which are associated with lamellipodia and filopodia, contain vinculin, paxillin, and focal adhesion kinase, but are formed independently of rho-induced focal adhesions. These Rac induced focal complexes at the leading edge of motile cells are distinct from focal adhesions in three ways: (1) they are much smaller, (2) they do not have the characteristic elongated, arrowhead shape of Rho-regulated focal adhesions and (3) they are not arranged within the cell in the same fashion as focal adhesions. Activation of Cdc42 in cultured cells leads to the sequential activation of Rac and then Rho, suggesting a molecular model for the coordinated control of cell motility by members of the Rho family of GTPases (Nobes, 1995a).

In Drosophila, Rac and Rho are both involved in dorsal closure. This involvement requires the cytoskeleton and downstream gene function, in particular the newly discovered serine/threonine kinase PAK, that binds and is activated by Rac and CDC42 (Harden, 1996). Hemipterous and Jun N-terminal kinase are downstream targets of Rac and CDC42 in the PAK initiated phosphorylation cascade. The dorsal closure phenotype is discussed more fully in the Hemipterous and JNK sites.

Rac and Cdc42 are GTP binding proteins that serve a function similar to that of Ras; all three are molecular switches that can activate protein kinases. A kinase cascade has been characterized in vertebrates that is homologous to that involving Hemipterous in Drosophila. Another direct target of Rac characterized in vertebrates is phosphatidylinositol (PI) 3-kinase (see Drosophila Pi3K92E). This interaction depends on Rac being in a GTP-bound state and requires an intact Rac effector domain (Bokoch, 1996). The function of PI3K is required for the generation of phospholipids, which leads to formation of diacylglycerol and the activation of protein kinase C.

Rac1 and CDC42 control actin-dependent processes in the fly's wing imaginal disc epithelium, and plays a role in the formation of the polarized outgrowth of wing hairs. At approximately 35 hours after puparium formation each wing epithelial cell forms a hair by extending a single process from its apical membrane. The emergence of a wing hair is presaged by the accumulation of actin on the distal side of the cell. Outgrowth initiates from this site and is oriented distally. At this stage, the outgrowth is termed a prehair. Subsequently, the prehair elongates and tilts up out of the plain of the epithelium so that its base comes to lie in the center of the apical membrane. Tissue polarity mutants such as frizzled, dishevelled, and prickle interfere with the choice of the site at which actin begins to accumulate before wing hair formation. Because these genes act early in the polarization pathway, they are likely candidates to act as molecules that transmit the polarization signal. Frizzled is unlikely to be involved in structural aspects of hair formation; rather it must polarize some feature of the cell that can later be used to determine either the site of hair outgrowth or cellular orientation. The tissue polarity genes inturned, fuzzy, and multiple wing hair act downstream of frizzled and are required only for hair polarity. These genes may help translate the cellular polarity generated by the Frizzled pathway into polarized hair outgrowth (Wong, 1993 and Eaton, 1996).

Rac and the other Ras family GTPases can be mutated to dominant negative forms that interfere with the functioning of wild type GTPases. When a dominant negative Rac1 is expressed in the wing, a polarity phenotype similar to that of inturned and fuzzy is produced. Cells that express a dominant negative Cdc42 often make no hair at all. Expression of dominant negative Cdc42 interferes with actin polymerization in wing hairs. In wild-type cells prior to hair formation, actin is distributed around the periphery in the apical junction region. Actin fibers are observed running across the cells in many directions, often with a radial arrangement. The first sign of hair formation is the accumulatin of actin on the distal side of the cell. At the level of the intercellular junction, actin filaments are often observed extending from the distal vertex into the center of the cell. As the hairs extend, actin disappears from the junctional region. Dominant negative Cdc42 causes dramatic defects in the actin organization that correlate with prehair outgrowth. By the time their wild-type neighbors have extended actin-filled prehairs, cells expressing dominant negative Cdc42 have not polarized the distribution of actin filaments distally, and no sign of outgrowth is evident. These data suggest that Cdc42 is specifically required for actin polymerization in developing wing hairs and that actin polymerization is required for outgrowth (Eaton, 1996).

While Cdc42 is critical for the outgrowth but not the placement of wing hairs, Rac1 is involved in the placement but not outgrowth. Dominant negative Rac1 causes duplication or triplication of wing hairs, but no defects in the appearance of the hairs themselves. Normally there is a continuous band of junctional actin around the perimeter of the apical aspect of wing epithelia. Upon expression of dominant negative Rac1 the normal continuous band of junctional actin is reduced in amount and fraught with gaps. The process by which duplicate hairs are filled with normal amounts of actin is delayed. During formation of hairs the majority of microtubules (see ß1 tubulin for related information) span the cell at the level of apical junctions, while 30 hours later, most microtubules comprise a longitudinal array that runs from the apex to the base of each cell. This suggests that microtubule organization is developmentally regulated during wing formation. Dominant negative Rac1 expression disorganizes the apical microtubule web. It is concluded that Rac1 is needed to maintain the proper organization of the apical microtubule web before prehair formation, at the time planar polarization is thought to occur. It is likely that Rac1 acts at the sites of cell-cell contact where junctions are located, and that dominant negative Rac1 disrupts planar polarization because of the disorganization of junctional actin and microtubules (Eaton, 1996).

Besides being involved in dorsal closure and hair polarity, the Rho family of GTPases are also involved in oogenesis. Rac is involved specifically in border cell migration. Analysis of heat shock regulated Rac1 indicates a requirement both for the initiation and continuation of migration. For more information about border cell migration see Slow border cells (Slbo). Expression of constitutively active Rac is unable to rescue the border cell migration defect in mutant slbo, suggesting that the two function in different pathways. Rac, Rho and Cdc42 are also required in the germ line for proper nurse cell cytoplamic transport. At stage 11 of oogenesis, a network of actin filaments polymerizes to form a cage around nurse cell nuclei, apparently preventing their movement during the transfer of nurse cell cytoplasm to the oocyte. This transfer process, known as "dumping," appears to result from the myosin-based contraction of subcortical actin. Limiting the amounts of each of the Rho family members results in the absence of the actin cage that normally surrounds stage 11 nurse cell nuclei. This results in the obstruction of ring canals and the failure of complete transfer, results similar to those occurring in other mutants that affect the cytoskeleton, such as chickadee, singed and quail (Murphy, 1996 and references).

The exact protein interactions required for Rac1 involvement in hair cell placement, follicle cell migration, and formation of the nurse cell actin cage are not yet known. Does Rac signal through Hemipterous in these instances as it does in dorsal closure, or are the effects of Rac confined to cytoskeletal organization independent of nuclear signaling? The involvement of Rac in two signaling cascades, one involving the serine/threonine kinase PAK and another potentially involving protein kinase C, leaves no shortage of pathways to be examined.

A mutational analysis of three Rac genes reveals different functions of these genes in developmental processes

Rac GTPases regulate the actin cytoskeleton to control changes in cell shape. To date, the analysis of Rac function during development has relied heavily on the use of dominant mutant isoforms. Here, loss-of-function mutations have been used to show that the three Drosophila Rac genes, Rac1, Rac2 and Mtl, have overlapping functions in the control of epithelial morphogenesis, myoblast fusion, and axon growth and guidance. They are not required for the establishment of planar cell polarity, as had been suggested on the basis of studies using dominant mutant isoforms. The guanine nucleotide exchange factor, Trio, is essential for Rac function in axon growth and guidance, but not for epithelial morphogenesis or myoblast fusion. Different Rac activators thus act in different developmental processes. The specific cellular response to Rac activation may be determined more by the upstream activator than the specific Rac protein involved (Hakeda-Suzuki, 2002).

In Drosophila, studies using constitutively active and dominant negative mutants have implicated Rac1 in closure of the dorsal epidermis, myoblast fusion, the establishment of planar cell polarity, and the control of axon growth and guidance. Each of these processes requires dynamic remodelling of the actin cytoskeleton, although the extracellular signals and the cellular responses involved seem to be different in each case. Given the ability of dominant mutant Rac proteins to interfere with cytoskeletal dynamics, it is not surprising to find that they perturb each of these processes. But are endogenous Rac proteins actually required for these processes, and if so, which proteins are involved, and how are they regulated? These questions cannot be answered using dominant mutant proteins. They require the phenotypic analysis of loss-of-function mutations in each of the endogenous Rac genes (Hakeda-Suzuki, 2002).

The Drosophila genome contains two highly similar Rac genes, Rac1 and Rac2. A third gene, Mtl, encodes a closely related GTPase that is structurally similar to both Rac and Cdc42 GTPases, but functionally behaves like Rac1 and Rac2. Rac1, Rac2 and Mtl are therefore referred to collectively as the Drosophila Rac genes. All three genes are ubiquitously expressed during development. A loss-of-function mutation in the Mtl gene was generated by imprecise excision of a P-element inserted in the first non-coding exon. A 2,068-base pair (bp) deletion was recovered that removes the entire Mtl open reading frame, but no part of any other predicted gene. Animals homozygous for this deletion, MtlDelta, as well as both Rac1 and Rac2 single mutants, are viable and fertile. The Rac2 Mtl double mutant is also viable and fertile. All other combinations are homozygous lethal. These loss-of-function mutations have been used to assess the contribution of each Rac protein to a set of distinct cell-shape changes that occur during Drosophila development. Embryos were examined lacking both the maternal and zygotic contributions of one or more Rac gene, and also pupae and adults that were homozygous mutant either entirely or in large clones of cells. For pupae and adults, both the strong hypomorph Rac1J10 and the null allele Rac1J11 were used, together with the null deletion alleles for Rac2 and Mtl (Rac2Delta and MtlDelta). Analyses in the embryo were restricted to the use of the Rac1J10 allele, since triple mutant embryos could not be recovered using the null allele Rac1J11. Evidently, Rac proteins also have important but still uncharacterized functions during oogenesis and early embryogenesis (Hakeda-Suzuki, 2002).

During Drosophila embryogenesis, opposing lateral epidermal sheets move towards one another, meeting and fusing seamlessly at the dorsal midline. This process of dorsal closure resembles ventral enclosure in Caenorhabditis elegans; and wound healing in vertebrates. It is believed to be driven, at least in part, by an actomyosin contractile ring that assembles at the leading edge, with lamellipodial and filopodial protrusions facilitating adhesion and alignment as the epidermis is sealed. Expression of dominant negative Rac1 in epidermal cells prevents formation of the acto-myosin cable and completion of dorsal closure, suggesting that at least one endogenous Rac protein might be involved. This study has determined that all three Rac proteins contribute to dorsal closure. Triple mutant Rac embryos fail to complete dorsal closure. Little or no actin accumulation is seen at the leading epidermal edge, and both lamellipodia and filopodia are lacking. The underlying amnioserosa cells appear normal (Hakeda-Suzuki, 2002).

Weaker and less frequent defects are also seen in Rac1;Rac2 and Rac1;Mtl double mutant embryos. All remaining single and double mutant embryos successfully complete dorsal closure. Dorsal closure thus relies more on Rac1 than either Rac2 or Mtl, although any one of the three is largely sufficient. Quite different cell-shape changes occur during cell fusion, a striking example of which is the fusion of myoblasts to form multinucleate muscle fibers. The role of the actin cytoskeleton in myoblast fusion remains unclear. Most likely, it is involved in the formation of a vesicular prefusion complex that assembles at the apposed plasma membranes. Expression of either dominant negative or dominant active Rac1 in Drosophila myoblasts blocks their fusion, but here too the precise roles and contributions of individual Rac genes are unknown. Little or no myoblast fusion occurs in either Rac1 Rac2 double mutant or Rac1;Rac2;Mtl triple mutant embryos. In contrast, myoblast fusion appears to be complete in Rac1 and Mtl single and double mutants, whereas only a few isolated myoblasts fail to fuse in Rac2 single mutants and Rac2;Mtl double mutants. Myoblast fusion thus requires either Rac1 or Rac2, but not Mtl (Hakeda-Suzuki, 2002).

Actin rearrangements also underlie the establishment of planar cell polarity (PCP) within an epithelium. In Drosophila, PCP has been studied most extenstively in the context of eye and wing development. Photoreceptors in the eye are arranged in a trapezoidal fashion, giving each ommatidium a specific chirality and orientation. In the wing, each epithelial cell forms a single distally oriented hair, preceded by the assembly of an actin-based 'pre-hair' at the distal vertex of the cell. The involvement of Rac proteins in PCP has been suggested by the finding that both dominant negative and constitutively active forms of Rac1 disrupt ommatidial orientation in the eye, and that dominant negative Rac1 also induces the formation of multiple hairs per cell in the wing. To test more critically a requirement for Rac proteins in PCP, clones of cells in the eye and the wing were examined that were triply mutant for null alleles of Rac1, Rac2 and Mtl. No PCP defects could be detected within these clones in either tissue. Presumably, the PCP defects previously reported are due to cross-inhibition or cross-activation of other pathways by the dominant mutant Rac proteins used (Hakeda-Suzuki, 2002).

The most complex changes in cell shape that occur during development take place in the nervous system, as differentiating neurons extend axons and dendrites towards their specific target cells. Dominant mutant Rac proteins are crude tools with which to address the complexities and subtleties of neuronal differentiation, and it is perhaps not surprising that their use has sometimes led to conflicting results. To begin to tease apart the diverse functions of Rac GTPases in nervous system development, the embryonic central nervous system (CNS) and peripheral nervous system (PNS), and the adult visual system, were examined in single, double, and triple mutants for Rac1, Rac2 and Mtl. Embryonic CNS axon pathways were examined using anti-Fasciclin II (FasII) monoclonal antibody 1D4. FasII labels axons in three longitudinal fascicles on each side of the midline, and is thus a sensitive marker to detect any misrouting of longitudinal axons across the midline. Such midline guidance errors occur in 33% of segments in Mtl mutant embryos, but only at very low frequency (less than 2%) in embryos lacking one or both of Rac1 and Rac2. Mutations in Rac1, and to a lesser extent Rac2, enhance the frequency of the midline guidance errors in Mtl mutant embryos (to 75% and 42% of segments, respectively. Axon guidance defects also occur in the visual system of whole-eye Rac mosaics generated using eyFLP. In wild-type adults and control mosaics, photoreceptor axons establish precise topographic connections in the lamina and medulla of the optic lobe. These projection patterns are largely normal in each of the single mutants, and only mild defects occur in Rac1 Rac2 and Rac2 Mtl double mutants. Projection defects are more pronounced in Rac1 Mtl double mutants, and are severe in the triple mutant. These defects include local disruptions in topographic mapping, and a frequent misrouting of photoreceptor axons around and beyond the medulla. Specification of photoreceptor cell fate appears to be normal, even in triple mutant clones. The projection defects observed in the triple mutant can be rescued by reintroducing either Rac1 or Mtl specifically in the eye using a GMR transgene (Hakeda-Suzuki, 2002).

Together, these data establish a critical role for endogenous Rac proteins in axon guidance. The three Rac proteins have overlapping functions in axon guidance in both the CNS and visual system, just as they do in the mushroom bodies. Nevertheless, some degree of specialization can be discerned. For example, axon guidance at the CNS midline depends more on Mtl than Rac1, whereas Rac1 is more important than Mtl in the mushroom bodies (Hakeda-Suzuki, 2002).

Whereas single and double mutant embryos reveal a role for Rac proteins in axon guidance, triple mutant embryos demonstrate the essential function of Rac proteins in axon growth. Severe growth defects occur in Rac1;Rac2;Mtl homozygous mutant embryos. In the CNS, FasII-positive axons rarely extend from one segment into the next, and very few sensory axons from the PNS reach the CNS. Specification of neuronal and glial cell fate appears relatively normal, as does dendritic growth and morphology. These axon growth defects were quantified by determining the frequency with which axons from the dorsal cluster of sensory neurons reach the lateral cluster on their path towards the CNS. This analysis of the PNS confirmed the general impression gained from the CNS: axon stalling is severe in Rac1;Rac2;Mtl triple mutants, occurs occasionally in Rac1;Mtl double mutants, and is rare in all other combinations (Hakeda-Suzuki, 2002).

These data demonstrate that axon growth also requires Rac activity, but only at a very low level. This activity can be provided by any one of the three Rac proteins alone, although Rac2 is less effective than either Rac1 or Mtl. Axon growth is thus maintained at levels of Rac activity that are insufficient for accurate guidance, consistent with the idea that a low level of Rac activity is essential to drive the growth cone forward, while spatially restricted bursts of high activity may be needed to turn it (Hakeda-Suzuki, 2002).

Endogenous Rac GTPases thus function in morphogenesis of the epidermis, mesoderm, and nervous system. Are they regulated by the same or different upstream activators in each of these tissues? The guanine nucleotide exchange factor Trio activates Rac1, Rac2 and Mtl in vitro, and loss of trio function in the visual system results in projection errors of photoreceptor axons similar to those observed in Rac triple mutants. Axon guidance errors and occasional stalling defects also occur in embryos lacking zygotic trio function. Axon stalling becomes severe in both the CNS and PNS if the maternal trio function is also eliminated. As with the Rac proteins, low levels of Trio activity are sufficient but essential for axon growth. This critical requirement for Trio in axon growth is particularly striking, given that the Drosophila genome encodes at least 22 other Rho family GTPase exchange factors, several of which are also expressed in the developing nervous system (Hakeda-Suzuki, 2002).

In the embryonic nervous system and adult visual system, loss of trio function thus results in defects remarkably similar to those observed upon loss of Rac function, consistent with the idea that Trio and Rac proteins act in a common pathway in vivo. An epistasis experiment was performed to test this. Overexpression of the Trio GEF1 domain using the eye-specific GMR promoter results in a severely disrupted eye morphology and highly aberrant photoreceptor axon projections. If Trio signals through Rac proteins in vivo, then these defects should be dependent on Rac function. This is indeed the case. Both the eye morphology and axon projection defects are almost completely suppressed in animals homozygous for loss-of-function mutations in either Rac1 or Rac2. Mtl alone does not suppress this trio gain-of-function phenotype. The Rac1;Rac2;Mtl triple mutant phenotype is completely epistatic to the trio gain-of-function phenotype. These data demonstrate that Trio GEF1 does indeed act through Rac proteins in vivo, and further suggest that Rac1 and Rac2 are its preferred substrates. The trio loss-of-function phenotype is however much more severe than the Rac1;Rac2 double mutant phenotype, suggesting that endogenous Trio may also activate Mtl, at least when Rac1 and Rac2 are lacking (Hakeda-Suzuki, 2002).

Having identified Trio as the primary activator of Rac proteins during axon growth, whether Trio is required for any of the other Rac functions was investigated. Dorsal closure occurs normally in embryos lacking both maternal and zygotic trio function. Myoblast fusion also appears complete in these embryos, but myotubes often fail to attach themselves correctly to the epidermis. Thus, although it is expressed in both the epidermis and mesoderm, Trio is not required for either dorsal closure or myoblast fusion (Hakeda-Suzuki, 2002).

Thus endogenous Rac proteins control cell-sheet spreading, cell fusion, and axon growth and guidance, and they also regulate axon branching. Each of these processes involves its own characteristic restructuring of the cytoskeleton, and hence is likely to be mediated by a different set of Rac effectors. What determines which of these effector pathways will be stimulated when Rac proteins are activated? One possibility would be that distinct Rac proteins have distinct effectors. This may well be the case for myoblast fusion, which can be mediated by Rac1 or Rac2, but not Mtl. However, in most cases Rac1, Rac2 and Mtl have largely overlapping functions, indicating that they also share a common set of effectors. A similar pattern of overlapping functions in diverse processes has also recently been reported for the three C. elegans Rac genes. In general, the cellular response is therefore unlikely to be dictated by the specific Rac protein involved. These results suggest an alternative possibility. Trio, despite its widespread expression, is required for only a limited subset of Rac functions. This suggests that the set of effectors a Rac protein engages, and hence the cellular response it induces, might also depend on how or where it has been activated. Trio, for example, might activate Rac proteins to a level, for a duration, or in a subcellular location, that allows it to stimulate only those effector pathways that control motility and guidance. Exploring the basis for specificity in Rac function is an important task for the future (Hakeda-Suzuki, 2002).

Two components of aversive memory in Drosophila, anesthesia-sensitive and anesthesia-resistant memory, require distinct domains within the Rgk1 small GTPase

For aversive olfactory memory in Drosophila, multiple components have been identified that exhibit different stabilities. Intermediate-term memory generated after single cycle conditioning is divided into anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM), with the latter being more stable. This study determined that the ASM and ARM pathways converged on the Rgk1 small GTPase and that the N-terminal domain-deleted Rgk1 was sufficient for ASM formation, whereas the full-length form was required for ARM formation. Rgk1 is specifically accumulated at the synaptic site of the Kenyon cells (KCs), the intrinsic neurons of the mushroom bodies (MBs), which play a pivotal role in olfactory memory formation. A higher than normal Rgk1 level enhanced memory retention, which is consistent with the result that Rgk1 suppressed Rac-dependent memory decay; these findings suggest that rgk1 bolsters ASM via the suppression of forgetting. It is proposed that Rgk1 plays a pivotal role in the regulation of memory stabilization by serving as a molecular node that resides at KC synapses, where the ASM and ARM pathway may interact (Murakami, 2017).

Drosophila olfactory learning and memory, in which an odor is associated with stimuli that induce innate responses such as aversion, has served as a useful model with which to elucidate the molecular basis of memory. Olfactory memory is divided into several temporal components and the intermediate-term memory (ITM) generated after single cycle conditioning is further classified into two distinct phases, anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM). Evidence has suggested that ASM and ARM are distinctly regulated at the neuronal level and at the molecular level (Murakami, 2017).

Mushroom bodies (MBs) represent the principal mediator of olfactory memory. Kenyon cells (KCs) are the intrinsic neurons of MBs, which are bilaterally located clusters of neurons that project anteriorly to form characteristic lobe structures and are a platform of MB-extrinsic neurons that project onto or out of the MBs. To elucidate the molecular mechanisms that underlie olfactory memory, screenings for MB-expressing genes have been a useful strategy. A technique used to examine gene expression in a small amount of tissue samples has enabled the investigation of the expression profile in MBs with a substantial dynamic range of expression levels and high sensitivity, thereby representing a promising approach with which to identify novel genes responsible for memory. This study deep sequenced RNA isolated from adult MBs and identified rgk1 as a KC-specific gene (Murakami, 2017).

The RGK protein family, for which Drosophila Rgk1 exhibits significant protein homology, belongs to the Ras-related small GTPase subfamily, which is composed of Kir/Gem, Rad, Rem, and Rem2. Their roles include the regulation of Ca2+ channel activity and the reorganization of cytoskeleton. Notably, mammalian REM2 is expressed in the brain and has been shown to be important for synaptogenesis, as well as activity-dependent dendritic complexity. These findings raise the possibility that RGK proteins may have a role in the synaptic plasticity that underlies memory formation. Drosophila has several genes that encode proteins homologous to the RGK family, including rgk1. Therefore, based on the ample resources available in Drosophila for the investigation of neuronal morphology and functions, Drosophila Rgk proteins will provide a good opportunity to elucidate the function of RGK family proteins (Murakami, 2017).

This study describes the analysis of Drosophila rgk1, which exhibited specific expression in KCs. Rgk1 accumulated at synaptic sites and was required for olfactory aversive memory, making the current study the first to demonstrate the role of an RGK family protein in behavioral plasticity. These data suggest that Rgk1 supports ASM via the suppression of Rac-dependent memory decay, whereas the N-terminal domain has a specific role in ARM formation. Together, these findings indicated that Rgk1 functions as a critical synaptic component that modulates the stability of olfactory memory (Murakami, 2017).

It is proposed that the ITM is genetically divided into three components: the rut-, dnc-, and rgk1-dependent pathways. The rut and dnc pathway act specifically for ASM and ARM, respectively, whereas rgk1 acts for both ASM and ARM, albeit partially. Consistent with this notion, it is noteworthy that the ASM and ARM pathways converge on Rgk1, yet the functional domains may be dissected; the full-length form of Rgk1 is required for ARM, whereas the molecule that lacks the N-terminal domain is capable of generating ASM, which suggests that the protein(s) required for ARM formation may interact with the N-terminal domain of Rgk1 (Murakami, 2017).

The data suggested that Rgk1 acts for both ASM and ARM, whereas the rgk1 deletion mutant, which was shown to be a protein null, exhibited only a partial reduction in ITM; these findings imply that Rgk1 regulates an aspect of each memory component. This idea may be explained by the expression pattern of Rgk1. Rgk1 exhibited exclusive expression and cell-type specificity in the KCs, whereas the memory components have been shown to be regulated by the neuronal network spread outside of the MBs and are encoded by multiple neuronal populations. For example, two parallel pathways exist for ARM and ASM is modulated, not only by MB-extrinsic neurons, but also by the ellipsoid body that localizes outside of the MBs. dnc-dependent ARM requires antennal lobe local neurons and octopamine-dependent ARM requires α'/β' KCs, in neither of which was Rgk1 detected. Therefore, Rgk1 may support a specific part of memory components that exists in a subset of KCs (Murakami, 2017).

The specific expression of Rgk1 in KCs suggests its dedicated role in MB function. Rgk1 exhibited cell-type specificity in KCs from anatomical and functional points of view. Rgk1 is strongly expressed in α/β and γ KCs and weakly expressed in α'/β' KCs and the expression of the rgk1-sh transgene in α/β and γ KCs was sufficient to disrupt memory. Several genes required for memory formation have been shown to be expressed preferentially in the KCs and the notable genes include dunce, rutabaga, and DC0. Although a recent study in KC dendrites showed that the modulation of neurotransmission into the KCs affects memory strength, KC synapses are thought to be the site in which memory is formed and stored. The current analyses with immunostaining and GFP fusion transgenes indicated that Rgk1 is localized to synaptic sites of the KC axons, which raises the possibility that Rgk1 may regulate the synaptic plasticity that underlies olfactory memory. Among the RGK family proteins, Rem2 is highly expressed in the CNS and regulates synapse development through interactions with 14-3-3 proteins, which have been shown to be localized to synapses and are required for hippocampal long-term potentiation and associative learning and memory. In Drosophila, 14-3-3Ζ is enriched in the MBs and is required for olfactory memory. In addition, the C-terminal region of Drosophila Rgk1 contains serine and threonine residues that exhibit homology to binding sites for 14-3-3 proteins in mammalian RGK proteins. Therefore, Rgk1 and 14-3-3Ζ may act together in the synaptic plasticity that underlies olfactory memory (Murakami, 2017).

The roles of RGK family proteins in neuronal functions have been investigated extensively. The current data, when combined with the accumulated data on the function of RGK family proteins, provide novel insights into the mechanism that governs two distinct intermediate-term memories, ASM and ARM. Regarding the regulation of ASM, the data showed that Rgk1 suppressed the forgetting that was facilitated by Rac. Rac is a major regulator of cytoskeletal remodeling. Similarly, mammalian RGK proteins participate in the regulation of cell shape through the regulation of actin and microtubule remodeling. Rgk1 may affect Rac activity indirectly by sharing an event in which Rac also participates because there have been no reports showing that RGK proteins regulate Rac activity directly; further, it was determined that rgk1 transgene expression did not affect the projection defect of KC axons caused by RacV12 induction during development. Therefore, it is suggested that Rgk1 signaling and Rac signaling may merge at the level of downstream effectors in the regulation of forgetting. A member of the mammalian RGK1 proteins, Gem, has been shown to regulate Rho GTPase signaling through interactions with Ezrin, Gimp, and Rho kinase. Rho kinase is a central effector for Rho GTPases and has been shown to phosphorylate LIM-kinase. In Drosophila, the Rho-kinase ortholog DRok has been shown to interact with LIM-kinase. Furthermore, Rac regulates actin reorganization through LIM kinase and cofilin and the PAK/LIM-kinase/cofilin pathway has been postulated to be critical in the regulation of memory decay by Rac. It was shown recently that Scribble scaffolds a signalosome consisting of Rac, Pak3, and Cofilin, which also regulates memory decay. Therefore, Rgk1 may counteract the consequence of Rac activity (i.e., memory decay) through the suppression of the Rho-kinase/LIM-kinase pathway. DRok is a potential candidate for further investigation of the molecular mechanism in which Rgk1 acts to regulate memory decay (Murakami, 2017).

The data indicated that Rgk1 is required for ARM in addition to ASM. It has been shown that Synapsin and Brp specifically regulate ASM and ARM, respectively. The functions of Synapsin and Brp may be differentiated in a synapse by regulating distinct modes of neurotransmission. The exact mechanism has not been identified for this hypothesis; however, the regulation of voltage-gated calcium channels may be one of the key factors that modulate the neurotransmission. Voltage-gated calcium channels are activated by membrane depolarization and the subsequent Ca2+ increase triggers synaptic vesicle release. The regulation of voltage-gated calcium channels has been shown to be important in memory; a β-subunit of voltage-dependent Ca2+ channels, Cavβ3, negatively regulates memory in rodents. Importantly, Brp regulates the clustering of Ca2+ channels at the active zone. Moreover, it has been demonstrated extensively that mammalian RGK family proteins regulate voltage-gated calcium channels. Kir/Gem and Rem2 interact with the Ca2+ channel β-subunit and regulate Ca2+ channel activity. In addition, the ability to regulate Ca2+ channels has been shown to be conserved in Drosophila Rgk1. Therefore, both Brp and Rgk1 may regulate ARM through the regulation of calcium channels, the former through the regulation of their assembly and the latter through the direct regulation of their activity. The finding that Rgk1 localized to the synaptic site and colocalized with Brp lends plausibility to the scenario that Rgk1 regulates voltage-gated calcium channels at the active zone (Murakami, 2017).

Several memory genes identified in Drosophila, including rutabaga, PKA-R, and CREB, have homologous genes that have been shown to regulate behavioral plasticity in other species. The identification of Drosophila rgk1 as a novel memory gene raises the possibility for another conserved mechanism that governs memory. There is limited research regarding the role of RGK proteins at the behavioral level in other species; however, the extensively documented functions of RGK proteins with respect to the regulation of neuronal functions, combined with the data presented in this study regarding Drosophila Rgk1, raise the possibility of an evolutionally conserved function for RGK family proteins in memory (Murakami, 2017).

Genetic dissection of active forgetting in labile and consolidated memories in Drosophila

Different memory components are forgotten through distinct molecular mechanisms. In Drosophila, the activation of 2 Rho GTPases (Rac1 and Cdc42), respectively, underlies the forgetting of an early labile memory (anesthesia-sensitive memory, ASM) and a form of consolidated memory (anesthesia-resistant memory, ARM). This study dissected the molecular mechanisms that tie Rac1 and Cdc42 to the different types of memory forgetting. Two WASP family proteins, SCAR/WAVE and WASp, act downstream of Rac1 and Cdc42 separately to regulate ASM and ARM forgetting in mushroom body neurons. Arp2/3 complex, which organizes branched actin polymerization, is a canonical downstream effector of WASP family proteins. However, this study found that Arp2/3 complex is required in Cdc42/WASp-mediated ARM forgetting but not in Rac1/SCAR-mediated ASM forgetting. Instead, Rac1/SCAR may function with formin Diaphanous (Dia), a nucleator that facilitates linear actin polymerization, in ASM forgetting. The present study, complementing the previously identified Rac1/cofilin pathway that regulates actin depolymerization, suggests that Rho GTPases regulate forgetting by recruiting both actin polymerization and depolymerization pathways. Moreover, Rac1 and Cdc42 may regulate different types of memory forgetting by tapping into different actin polymerization mechanisms (Gao, 2019).

There are 3 major findings. First, 2 WASP family proteins, SCAR/WAVE and WASp, act as downstream effectors of Rac1-mediated ASM forgetting and Cdc42-mediated ARM forgetting, respectively. Second, although the Arp2/3 complex is a well-established effector that links activation of WASP family proteins to actin polymerization, it is only required in Cdc42/WASp-mediated ARM forgetting. Instead, formin Dia functions together with Rac1/SCAR in ASM forgetting. Third, feeding inhibitors of the Arp2/3 complex and Dia to fruit flies led to rather specific effects on ASM and ARM forgetting, raising the possibility of developing drugs on these molecular targets to treat memory-related diseases (Gao, 2019).

The effect of Rac1 on ASM forgetting has been tied to the activation of an actin depolymerization regulator cofilin presumably through a PAK/LIMK phosphorylation cascade. However, actin dynamics is a balanced play that requires continuous turnover between polymerization and depolymerization. It is not known whether signaling pathways regulating actin polymerization also play a role. There are 3 families of proteins that nucleate and promote actin polymerization, Arp2/3 complex, WH2-domain proteins, and formin. The finding that Arp2/3 complex and formin Dia function in ARM and ASM forgetting suggests that both actin polymerization and depolymerization pathways contribute to forgetting. How Arp2/3 complex and Dia separately contribute to ARM and ASM forgetting remains an open question. It is yet to be determined whether these proteins have different expression or subcellular locations in the MB neurons. However, it is interesting that Arp2/3 complex and formins are specialized in different types of actin polymerization (Gao, 2019).

In a working model, Cdc42 activates Arp2/3 complex via a canonical pathway (Cdc42/WASp/Arp2/3 complex), while Rac1-mediated ASM forgetting depends on SCAR/WAVE complex. This complex, in addition to SCAR/WAVE, includes at least 4 other members: Sra-1, Abi, HSPC300, and Kette. These additional members are thought to hold SCAR/WAVE in the complex in an inactive state, until GTP-bound Rac1 binds to Sra-1 and relieves the inhibition. On the other hand, the intact complex is essential for the stability of the SCAR/WAVE protein as well (i.e., failure to keep the intact complex can lead to SCAR degradation). This latter effect may explain the observation that RNAi knockdown of SCAR complex members has the same effect on inhibiting forgetting as the knockdown of SCAR. As a WASP family protein, SCAR/WAVE is able to associate with and activate Arp2/3 complex through its C-terminal region. However, RNAi knockdown of Arp2 and Arp3 and pharmacological inhibition of Arp2/3 complex specifically affects ARM forgetting, while no effects on ASM retention were observed. It is therefore proposed that Rac1/SCAR may function through Arp2/3 complex-independent mechanisms. SCAR/WAVE complex is reported to physically associates with Dia through one of its members, Abi, to regulate actin dynamics. Behavioral characterization of Dia knockdown and overexpression, as well as the genetic epistasis experiment, support the idea that Dia could be downstream of Rac1/SCAR in ASM forgetting. Details about the functional coordination between SCAR/WAVE and Dia therefore await further clarification (Gao, 2019).


cDNA clone length - 1733

Bases in 5' UTR - 265

Bases in 3' UTR - 923


Amino Acids - 192

Structural Domains

Rac1 is 92% identical to human Rac1 (Luo, 1994). Rac1 and Rac2 of Drosophila are are 93% identical (Harden, 1995).

Rac, a small GTPase in the Ras superfamily, regulates at least two biological processes in animal cells: (1) the polymerization of actin and the assembly of integrin complexes to produce lamellipodia and ruffles; and (2) the activity of an NADPH oxidase in phagocytic cells. NADPH oxidase activation is mediated through a Rac effector protein, p67phox. Using chimeras made between Rac and the closely related GTPase, Rho, two distinct effector sites have been identified in Rac, one N-terminal and one C-terminal, both of which are required for activation of p67phox. The same two effector sites are essential for Rac-induced actin polymerization in fibroblasts. p65PAK, a ubiquitous serine/threonine kinase, interacts with Rac at both the N- and C-terminal effector sites, but the GTPase-activating protein, bcr interacts with Rac in a different region. This makes p65PAK a candidate effector of Rac-induced lamellipodium formation (Diekmann, 1995).

Rac1: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 2 June 2002 

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