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 link: Entrez Gene
Rac1 orthologs: Biolitmine
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.
Cheng, K. C., Chen, Y. H., Wu, C. L., Lee, W. P., Cheung, C. H. A. and Chiang, H. C. (2021). Rac1 and Akt Exhibit Distinct Roles in Mediating Abeta-Induced Memory Damage and Learning Impairment. Mol Neurobiol. PubMed ID: 34273104
Accumulated β-amyloid (Aβ) in the brain is the hallmark of Alzheimer's disease (AD). Despite Aβ accumulation is known to trigger cellular dysfunctions and learning and memory damage, the detailed molecular mechanism remains elusive. Recent studies have shown that the onset of memory impairment and learning damage in the AD animal is different, suggesting that the underlying mechanism of the development of memory impairment and learning damage may not be the same. In the current study, with the use of Aβ42 transgenic flies as models, this study found that Aβ induces memory damage and learning impairment via differential molecular signaling pathways. In early stage, Aβ activates both Ras and PI3K to regulate Rac1 activity, which affects mostly on memory performance. In later stage, PI3K-Akt is strongly activated by Aβ, which leads to learning damage. Moreover, reduced Akt, but not Rac1, activity promotes cell viability in the Aβ42 transgenic flies, indicating that Akt and Rac1 exhibit differential roles in Aβ regulating toxicity. Taken together, different molecular and cellular mechanisms are involved in Aβ-induced learning damage and memory decline; thus, caution should be taken during the development of therapeutic intervention in the future.
Banka, S., Bennington, A., ..., Kazanietz, M. G. and Millard, T. H. (2022). Activating RAC1 variants in the switch II region cause a developmental syndrome and alter neuronal morphology. Brain. PubMed ID: 35139179
RAC1 is a highly conserved Rho GTPase critical for several cellular and developmental processes. De novo missense RAC1 variants cause a highly variable neurodevelopmental disorder. Most previously reported patients with this disorder have either severe microcephaly or severe macrocephaly. This study describes eight patients with pathogenic missense RAC1 variants affecting residues between Q61 and R68 within the switch II region of RAC1. These patients display variable combinations of developmental delay, intellectual disability, brain anomalies such as polymicrogyria, and cardiovascular defects with normocephaly or relatively milder micro- or macrocephaly. Pulldown assays, NIH3T3 fibroblasts spreading assays and staining for activated PAK1/2/3 and WAVE2 suggest that these variants increase RAC1 activity and over-activate downstream signalling targets. Axons of neurons isolated from Drosophila embryos expressing the most common of the activating variants are significantly shorter, with an increased density of filopodial protrusions. In vivo, these embryos exhibit frequent defects in axonal organization. Class IV dendritic arborisation neurons expressing this variant exhibit a significant reduction in the total area of the dendritic arbour, increased branching and failure of self-avoidance. RNAi knock down of the WAVE regulatory complex component Cyfip significantly rescues these morphological defects. These results establish that activating substitutions affecting residues Q61-R68 within the switch II region of RAC1 cause developmental syndrome (Banka, 2022).
Park, H. G., Kim, Y. D., Cho, E., Lu, T. Y., Yao, C. K., Lee, J. and Lee, S. (2022). Vav independently regulates synaptic growth and plasticity through distinct actin-based processes. J Cell Biol 221(10). PubMed ID: 35976098
Modulation of presynaptic actin dynamics is fundamental to synaptic growth and functional plasticity; yet the underlying molecular and cellular mechanisms remain largely unknown. At Drosophila NMJs, the presynaptic Rac1-SCAR pathway mediates BMP-induced receptor macropinocytosis to inhibit BMP growth signaling. This study shows that the Rho-type GEF Vav acts upstream of Rac1 to inhibit synaptic growth through macropinocytosis. Evidence is presented that Vav-Rac1-SCAR signaling has additional roles in tetanus-induced synaptic plasticity. Presynaptic inactivation of Vav signaling pathway components, but not regulators of macropinocytosis, impairs post-tetanic potentiation (PTP) and enhances synaptic depression depending on external Ca2+ concentration. Interfering with the Vav-Rac1-SCAR pathway also impairs mobilization of reserve pool (RP) vesicles required for tetanus-induced synaptic plasticity. Finally, treatment with an F-actin-stabilizing drug completely restores RP mobilization and plasticity defects in Vav mutants. It is proposed that actin-regulatory Vav-Rac1-SCAR signaling independently regulates structural and functional presynaptic plasticity by driving macropinocytosis and RP mobilization, respectively.
Martinez-Cervantes, J., Shah, P., Phan, A. and Cervantes-Sandoval, I. (2022). High order unimodal olfactory sensory preconditioning in Drosophila. Elife 11. PubMed ID: 36129180
Learning and memory storage is a complex process that has proven challenging to tackle. It is likely that, in nature, the instructive value of reinforcing experiences is acquired rather than innate. The association between seemingly neutral stimuli increases the gamut of possibilities to create meaningful associations and the predictive power of moment-by-moment experiences. This study reports physiological and behavioral evidence of olfactory unimodal sensory preconditioning in fruit flies. The presentation of a pair of odors (S1 and S2) before one of them (S1) is associated with electric shocks elicits a conditional response not only to the trained odor (S1) but to the odor previously paired with it (S2). This occurs even if the S2 odor was never presented in contiguity with the aversive stimulus. In addition, this study shows that inhibition of the small G protein Rac1, a known forgetting regulator, facilitates the association between S1/S2 odors. These results indicate that flies can infer value to olfactory stimuli based on the previous associative structure between odors, and that inhibition of Rac1 lengthens the time window of the olfactory 'sensory buffer', allowing the establishment of associations between odors presented in sequence.
Zhou, S., Li, P., Liu, J., Liao, J., Li, H., Chen, L., Li, Z., Guo, Q., Belguise, K., Yi, B. and Wang, X. (2022). Two Rac1 pools integrate the direction and coordination of collective cell migration. Nat Commun 13(1): 6014. PubMed ID: 36224221
Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, this study reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, these studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration.
Nakamura, M., Hui, J., Stjepic, V. and Parkhurst, S. M. (2023). Scar/WAVE has Rac GTPase-independent functions during cell wound repair. Sci Rep 13(1): 4763. PubMed ID: 36959278
Rho family GTPases regulate both linear and branched actin dynamics by activating downstream effectors to facilitate the assembly and function of complex cellular structures such as lamellipodia and contractile actomyosin rings. Wiskott-Aldrich Syndrome (WAS) family proteins are downstream effectors of Rho family GTPases that usually function in a one-to-one correspondence to regulate branched actin nucleation. In particular, the WAS protein Scar/WAVE has been shown to exhibit one-to-one correspondence with Rac GTPase. This study shows that Rac and SCAR are recruited to cell wounds in the Drosophila repair model and are required for the proper formation and maintenance of the dynamic actomyosin ring formed at the wound periphery. Interestingly, it was found that SCAR is recruited to wounds earlier than Rac and is still recruited to the wound periphery in the presence of a potent Rac inhibitor. It was also shown that while Rac is important for actin recruitment to the actomyosin ring, SCAR serves to organize the actomyosin ring and facilitate its anchoring to the overlying plasma membrane. These differing spatiotemporal recruitment patterns and wound repair phenotypes highlight the Rac-independent functions of SCAR and provide an exciting new context in which to investigate these newly uncovered SCAR functions.
Zhao, J., Zhang, X., Zhao, B., Hu, W., Diao, T., Wang, L., Zhong, Y. and Li, Q. (2023). Genetic dissection of mutual interference between two consecutive learning tasks in Drosophila. Elife 12. PubMed ID: 36897069
Animals can continuously learn different tasks to adapt to changing environments and, therefore, have strategies to effectively cope with inter-task interference, including both proactive interference (Pro-I) and retroactive interference (Retro-I). Many biological mechanisms are known to contribute to learning, memory, and forgetting for a single task, however, mechanisms involved only when learning sequential different tasks are relatively poorly understood. This study dissected the respective molecular mechanisms of Pro-I and Retro-I between two consecutive associative learning tasks in Drosophila. Pro-I is more sensitive to an inter-task interval (ITI) than Retro-I. They occur together at short ITI (<0 min), while only Retro-I remains significant at ITI beyond 20 min. Acutely overexpressing Corkscrew (CSW), an evolutionarily conserved protein tyrosine phosphatase SHP2, in mushroom body (MB) neurons reduces Pro-I, whereas acute knockdown of CSW exacerbates Pro-I. Such function of CSW is further found to rely on the γ subset of MB neurons and the downstream Raf/MAPK pathway. In contrast, manipulating CSW does not affect Retro-I as well as a single learning task. Interestingly, manipulation of Rac1, a molecule that regulates Retro-I, does not affect Pro-I. Thus, these findings suggest that learning different tasks consecutively triggers distinct molecular mechanisms to tune proactive and retroactive interference.

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.

Roles for Rac1 and Cdc42 in planar polarization and hair outgrowth in the wing of Drosophila

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).

Rac1 impairs forgetting-induced cellular plasticity in mushroom body output neurons

Active memory forgetting is a well-regulated biological process thought to be adaptive and to allow proper cognitive functions. Recent efforts have elucidated several molecular players involved in the regulation of olfactory forgetting in Drosophila, including the small G protein Rac1, the dopamine receptor DAMB, and the scaffold protein Scribble. Similarly, recent work has reported that dopaminergic neurons mediate both learningand forgetting-induced plasticity in the mushroom body output neuron MBON-β2α'1. Two open questions remain: how does forgetting affect plasticity in other, highly plastic, mushroom body compartments and how do genes that regulate forgetting affect this cellular plasticity? This study shows that forgetting reverses short-term synaptic depression induced by aversive conditioning in the highly plastic mushroom body output neuron MBON-β1pedc>α/β. In addition, the results indicate that genetic tampering with normal forgetting by inhibition of small G protein Rac1 impairs restoration of depressed odor responses to learned odor by intrinsic forgetting through time passing and forgetting induced acutely by shock stimulation after conditioning or reversal learning. These data further indicate that some forms of forgetting truly erase physiological changes generated by memory encoding (Cervantes-Sandoval, 2020).

New insights have demonstrated that associative olfactory learning changes the output weight of KC synapses onto the corresponding MBON, suggesting a model in which dopamine-induced plasticity tilts the overall MBON network to direct appropriate behavior. In fact, recent physiological studies have shown that learning alters odor drive to specific MBONs. As a whole, these changes can be described as memory traces. Interestingly, reward learning appears to reduce the drive to output pathways that direct avoidance, whereas aversive learning increases drive to avoidance pathways while reducing the drive to approach pathways. this study choose to explore how forgetting and its genetic disruption affected memory traces formed in MBON-γ1pedc>α/β. This trace was selected because it can be easily induced after a very short stimulation of odor (1 s) along with optogenetic stimulation of dopaminergic neuron innervating the same MB compartment. In addition, it has been shown, using optogenetics, and behavior, that MB-γ1 compartment is the fastest to encode new memories, the most unstable or susceptible to memory decay and shock interference. Furthermore, it was shown that memories in this compartment are highly vulnerable to retroactive interference induced by a formation of additional olfactory memory. These features increased chances to observe forgetting related changes after memory encoding (Cervantes-Sandoval, 2020).

Using electrophysiology whole-cell recordings of MBON-γ1pedc>α/β, it has been shown that pairing and odor with specific artificial activation of dopaminergic PPL1-γ1pedc induced odor-specific synaptic depression. In addition, it has been shown that training the flies by pairing 1 min of CS+ with 12 shocks followed by 1 min presentation of CS-, induced a decreased response to the CS+ relative to CS- compared to no change in mock trained animals. This study first tried to confirm that this depression is observed when individual flies are imaged before and after learning and is observable using calcium reporter GCaMP6f. For this, the fly was trained under a confocal microscope, and calcium responses to odors was recorded in MBON-γ1pedc>α/β before and after training using split-gal4 driver MB112c. Pairing 20 sec of methylcyclohexanol (MCH) presentation with electric shock delivered to the fly legs by a floating electric grid platform induced a robust depression of calcium response to the learned odor. This depression was specific to the paired odor and was not observed in octanol (OCT), which was used as a non-paired odor. Additionally, this decreased response was not observed when flies were trained by a mock training (no shock) or backwards training (shock presented before odor onset). Finally, training flies with the reciprocal odor (OCT) showed similar results. These results confirm previous results and demonstrated that aversive olfactory conditioning induces under the microscope induced a robust memory trace represented as a depression of MBON-γ1pedc>α/β calcium responses to trained odors (Cervantes-Sandoval, 2020).

Next, it was asked how is this memory trace affected when forgetting occurs either intrinsically (as time passes) or is induced by interfering-electric shock or reversal learning. For this experiment GCaMP6f was expressed in MBON- γ1pedc>α/β using R12G04-lexA driver. Flies were trained as above and post-training responses were recorded 5, 15, or 30 min after conditioning. Similar to prior result, full depression to learned odor was observed 5 min after conditioning. This depression showed increasing recovery and was no longer significant from preconditioning responses 15 or 30 min after training. No significant changes were detected in the non-paired odor (OCT). This data demonstrate that at least for the memory trace observed in MBON-γ1pedc>α/β under these training conditions, intrinsic forgetting restitutes MCH calcium responses to normal levels after 30 min. It is important to indicate that a previous study showed that the decreased response to CS+ observed after 1 min CS+ odor pairing, lasted for at least 3 to 4 h after training. These differences, of course, could be attributed to the fact that the current study used a reduced training protocol (20s pairing) intending to improve chances of detecting rapid changes in the observed plasticity (Cervantes-Sandoval, 2020).

Previous work has shown that mechanical stimulation mediated by dopaminergic neurons can promote forgetting if presented after learning. Similarly, another study showed a decrease in conditioned response as a result of DAN activation after artificially induced aversive learning. These results suggested a model where dopamine bidirectionally regulates connectivity between KC > MBON; this regulation would be contingent on dopamine release in the context of odor presentation or not. This study asked whether the presentation of electric shock pulses presented after learning restored memory trace observed in MBON-γ1pedc>α/β to preconditioning levels. Results indicate that 12, 90 V shocks presented after conditioning was enough to restore responses to the paired MCH odor back to preconditioning levels. Responses to the non-paired odor were not affected by any of the protocols followed. Additionally, presenting the four shocks alone after conditioning was not enough to restore responses to CS+, indicating that the effect of shocks alone and reversal learning are somehow different (Cervantes-Sandoval, 2020).

Inducing acute memory forgetting can also be achieved by retroactive interference. In flies, it has been demonstrated that training with reversal conditioning, where flies are trained by presenting a first odor paired with electric shock followed by a second non-paired odor as a CS- and then immediately trained with the reverse contingency, showed decrease memory performance to the first CS+. This study now shows that retroactive interference induced forgetting by reversal learning also restored MCH responses to preconditioning levels. Responses to OCT after reversal conditioning were scarcely significantly decreased. Additionally, analysis of the CS+/CS- ratio showed that reversal conditioning not only restored responses to initial associated odor but also interfered with the synaptic depression of the newly learned contingency, namely no difference between responses ratios pre and post-conditioning. These results contrast with findings in plasticity induced in MBON-γ2 α'1, where reversal learning restores responses to initial CS+ and simultaneously depresses responses to the new CS+. The current results suggest the presence of not only retroactive interference to initial memory but also forgetting of secondary memory induced by proactive interference, which has been previously reported behaviorally. This difference can be attributed to the fact that different MB domains have different properties (Cervantes-Sandoval, 2020).

A previous study identified one of the central molecular regulators of active forgetting, the small G protein Rac1; overexpression of dominant negative (DN) form of Rac1 (RacN17) was found to impair normal memory forgetting. The current study tested how the memory trace in MBON-γ1pedc>α/β is affected by genetic disruption of this active forgetting regulation. For this flies that express GCaMP6f on MBON-γ1pedc>α/β using lexA driver, R12G04-lexA, were trained while expressing DN form of Rac1 in KC using gal4 driver R13F02-gal4. Expression of RacN17 in KC was further confined to adulthood using target system. Flies expressing RacN17 expression in adulthood showed a normal complete depression to the learned MCH odor. Nevertheless, these flies showed impaired recovery of memory-induced plasticity in MBON-γ1pedc>α/β after 15 and 30 min after conditioning with MCH. Unexpectedly, a mild non-specific depression to the non-paired odor was observed. This non-specific depression might be a result of Rac1 inhibition broadening odors representation and therefore increase in generalization; other explanations might also be possible. Despite this, two-way Anova analysis with Sidak's multiple comparisons test showed that the depression observed to the paired odor is significantly higher that the non-specific depression to the CS- (Cervantes-Sandoval, 2020).

Control flies carrying all genetic insertion but the uas-RacN17 and subjected to the same temperature conditions, showed normal depression to the learned odor and full recovery of odor response 30 min after conditioning. Control flies did not show a depressed odor response to the non-paired odor. Flies kept at 18°C to keep target system at non-permissive temperature showed normal learning-induced odor depression as well as normal recovery of odor calcium responses. These results suggest that, at least partially, RacN17 inhibits forgetting by impairing the bidirectional regulation of KC > MBON plasticity that is to say the restoration of the depressed odor responses to CS+ (MCH) in MBON-γ1pedc>α/β (Cervantes-Sandoval, 2020).

The above results indicate that the recovery of depressed olfactory responses in MBON-γ1pedc>α/β to a learned odor mediated by intrinsic forgetting, or normal memory decay trough time passing, is impaired when the DN form of Rac1 is expressed in KC. Next, attempts were made to investigate if RacN17 also affected memory trace loss when this is induced by interfering-electric shocks presented after learning. For that flies were trained as before and then 12, 90 V electric shocks were delivered to fly legs to induce acute forgetting. Flies expressing DN form of RacN17 in KC showed no recovery in learned-odor calcium responses (MCH) as compared to control flies. These results indicated that genetically interfering with memory forgetting by the expression of DN RacN17 in KC impairs not only intrinsic forgetting but also acute dopamine-mediated forgetting induced by strong electric shock stimulation after learning (Cervantes-Sandoval, 2020).

Finally, the effects were investigated of DN RacN17 expression on retroactive interference forgetting provoked by reversal conditioning. For this, the flies were trained presenting a first odor paired with electric shock (MCH, CS+) followed by air and a second odor not paired with electric shock (OCT, CS-). After learning flies were subjected to reversal training in which previous CS- odor was now paired with electric shock and the former conditioned odor was now presented as CS-. This protocol acutely induced a complete recovery of cellular memory trace in MBON- γ1pedc>α/β in control animals. Surprisingly, once again analysis of CS+/CS- ratio showed that reversal conditioning not only restored responses to initial associated odor but also interfered with the synaptic depression of the newly learned contingency, namely no difference between responses ratios pre and post conditioning. Again, these results reinforce the suggestion of proactive interference. Expression of RacN17 in KC during adulthood not only impaired memory trace restoration of initial contingency but also induced a strong depression to the secondary paired odor. These results indicated that genetically interfering with memory forgetting by expression of DN RacN17 in KC impairs restoration of olfactory responses in MBON-γ1pedc>α/β induced by intrinsic memory loss (time passing), acute forgetting induced by a non-associative stimuli (electric shock), and acute forgetting by new associations or memory updating (reversal learning) (Cervantes-Sandoval, 2020).

This study indicates that forgetting reverses synaptic depression induced by aversive conditioning in MBON-γ1pedc>α/β. This is true for intrinsic memory forgetting through time passing, and acute forgetting by both interfering-electric shock and retroactive interference provoked by reversal learning. The results also show physiological evidence of proactive interference in MBON-γ1pedc>α/β, previously observed behaviorally in Drosophila, where prior learning interferes with the formation of new learning. Results also indicate that genetic tampering with normal forgetting by inhibition of small G protein Rac1 impairs restoration of depressed odor responses to learned odor by the three mechanisms described above. It has been recently reported that Rac1 partially regulates forgetting through time passing as well as forgetting induced by reversal learning but it does not affect forgetting induced by non-associative experiences like heat stress, electric shocks or odor presented alone. The current results indicate that at least at physiological level Rac1 inhibition does affect odor responses restoration induced by electric shock in MBON-γ1pedc>α/β. It is possible that this apparent discrepancy is due to the fact that this study only explored the memory trace of a single MBON whereas, as mentioned before, behavior arises, most likely, as a combinational effect of the whole KC > MBON network. Therefore, a single compartment analysis does not necessarily reflect final behavior. It is also important to indicate that in this study, a reduced training protocol (20 s odor with four shocks) was used when compared to the classical training paradigm used for behavioral studies (1 min odor with 12 shocks). This mild training session was used to increase chances of observing the reversal of synaptic plasticity. The dynamic of these physiological changes when flies are trained with classical 1-min protocol remains to be studied. It was recently reported that training flies with a single training cycle (1 min odor presentation along with 12 shocks) induces an independent contextual memory that resides in the lateral horn. It is very likely that the forgetting described in this study and others have different dynamics and/or rules to this context-dependent memory (Cervantes-Sandoval, 2020).

In memory research, one school of thought holds that nothing is ever lost from storage and that forgetting represents only a temporal failure or inhibition of access to memory. The other school holds that memory is not completely preserved and that forgetting is a true erasure of information from storage. The current findings indicate that normal forgetting reverses plasticity generated by aversive learning in MBON-γ1pedc>α/β suggesting that forgetting, in the case of short-term non-protein-synthesis dependent memories, truly erases at least some of physiological changes caused by memory encoding. This finding does not exclude the possibility that other compartments have different properties nor that the same phenomenon is true for long-term memories. It is possible that memories that had undergone protein-synthesis dependent memory consolidation are more resistant to permanently reverse the physiological changes that form part of the long-term memory trace. In that case, when talking about forgetting, it is not possible to talk about erasure but rather a transient blockage of memory retrieval (Cervantes-Sandoval, 2020).

Loss-of-function variants in TIAM1 are associated with developmental delay, intellectual disability, and seizures

TIAM Rac1-associated GEF 1 (TIAM1) regulates RAC1 signaling pathways that affect the control of neuronal morphogenesis and neurite outgrowth by modulating the actin cytoskeletal network. To date, TIAM1 has not been associated with a Mendelian disorder. This study describes five individuals with bi-allelic TIAM1 missense variants who have developmental delay, intellectual disability, speech delay, and seizures. Bioinformatic analyses demonstrate that these variants are rare and likely pathogenic. The Drosophila ortholog of TIAM1, still life (sif), is expressed in larval and adult central nervous system (CNS) and is mainly expressed in a subset of neurons, but not in glia. Loss of sif reduces the survival rate, and the surviving adults exhibit climbing defects, are prone to severe seizures, and have a short lifespan. The TIAM1 reference (Ref) cDNA partially rescues the sif loss-of-function (LoF) phenotypes. The function associated with three TIAM1 variants carried by two of the probands were assessed and they were compared to the TIAM1 Ref cDNA function in vivo. TIAM1 p.Arg23Cys has reduced rescue ability when compared to TIAM1 Ref, suggesting that it is a partial LoF variant. In ectopic expression studies, both wild-type sif and TIAM1 Ref are toxic, whereas the three variants (p.Leu862Phe, p.Arg23Cys, and p.Gly328Val) show reduced toxicity, suggesting that they are partial LoF variants. In summary, this study provides evidence that sif is important for appropriate neural function and that TIAM1 variants observed in the probands are disruptive, thus implicating loss of TIAM1 in neurological phenotypes in humans (Lu, 2022).

Many patients with rare diseases undergo a long and frustrating journey to obtain an accurate diagnosis, often referred to as the diagnostic odyssey. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) are effective approaches to identify diagnoses, as 80% or more of rare diseases are estimated to be caused by genomic abnormalities. However, these comprehensive sequencing methods also uncover many variants of uncertain significance (VUSs) with unknown clinical impact Drosophila melanogaster allows effective approaches to probe the functional impacts of these variants (Lu, 2022).

It is argued that bi-allelic loss-of-function (LoF) variants of TIAM Rac1-associated GEF 1 (TIAM1 [MIM: 600687]) cause a disease associated with developmental delay, intellectual disability, speech delay, and seizures. TIAM1 is a guanine nucleotide exchange factor (GEF). GEFs are positive regulators of small GTPases that promote their activation. Each individual GEF has a specificity profile, and TIAM1 is a Ras-related C3 botulinum toxin substrate 1 (RAC1)-specific GEF. RAC1 stimulates signaling pathways that regulate actin cytoskeleton organization, cell movement, differentiation, and proliferation (Lu, 2022).

TIAM1 is enriched in the brain. The rodent ortholog, Tiam1, is also expressed in the brain, is present in dendrites and spines, and is required to maintain proper outgrowth during development. When activated by neurotrophins such as brain-derived neurotrophic factor (BDNF), the TRKB receptor binds and activates TIAM1, which in turn activates RAC1, causing morphological changes by increasing neurite outgrowth. Similarly, when glutamate activates N-methyl-D-aspartate (NMDA) receptors, TIAM1 is also activated to control actin remodeling by inducing RAC1-dependent pathways. The localization of TIAM1 to spines is regulated by par-3 family cell polarity regulator and thereby controls proper spine formation. Mice lacking Tiam1 have simplified dendritic arbors, reduced dendritic spine density, and diminished excitatory synaptic transmission in the dentate gyrus. Taken together, TIAM1 controls neuronal morphogenesis and neurite outgrowth by RAC1-dependent actin cytoskeletal remodeling in rodents. However, alterations in TIAM1 have not yet been reported in the Online Mendelian Inheritance in Man (OMIM) database or the literature as causing human disease (Lu, 2022).

still life (sif) encodes the ortholog of TIAM1 in Drosophila. Previous studies showed that loss of sif leads to reduced locomotor activity. Sif was reported to be localized presynaptically and shown to genetically interact with Fasciclin II, a neural cell-adhesion molecule localized pre- and postsynaptially that controls synaptic growth, stabilization, and presynaptic structural plasticity. Its partial loss causes loss of some boutons at neuromuscular junctions. Also, Sif is highly enriched in lens-secreting cells in fly eyes and affects the distribution pattern of E-cadherin in pupal eyes (Lu, 2022).

This study identified bi-allelic TIAM1 missense variants in five individuals from four families with developmental delay, intellectual disability, speech delay, and seizures. Functional studies in Drosophila revealed that the loss of sif reduces the survival rate of flies, and the surviving adult flies have a remarkably reduced lifespan and exhibit severe climbing defects. In addition, sif LoF mutants display severe seizure-like behaviors when stressed. Expression of the human TIAM1 reference (TIAM1 Ref) cDNA partially rescues the lethality of sif LoF mutants, whereas the variants observed in probands behave as partial LoF mutations in different phenotypic assays. The data support the hypothesis that the variants observed in the probands are the cause of the observed phenotypes (Lu, 2022).

Exome or genome sequencing of probands and parents with rare diseases combined with functional investigations in model organisms has led to the discovery of numerous novel Mendelian diseases. This study has identified TIAM1 as a disease-associated gene. Five individuals are presented with compound-heterozygous or homozygous coding variants in TIAM1. The main phenotypes observed in the probands are developmental delays, with severe deficits in speech and language, intellectual disability, and seizures. Using Drosophila, it was found that loss of sif, the ortholog of TIAM1, causes semi-lethality, and flies that eclose exhibit climbing defects and seizure-like behaviors and have a short lifespan. The sifT2A-GAL4 allele allowed determination if the expression pattern of the gene and shows that sif is expressed in neurons but not in glia. It also allowed the 'humanization' of the fly by replacing fly sif with human TIAM1. TIAM1 Ref can partially rescue the survival rate and lifespan but not the climbing defects and seizure-like phenotypes of sif LoF mutants. The lack of rescue is likely due to the toxicity of the overexpression of TIAM1 Ref in flies. Alternatively, the limited rescue ability is due to functional diversity of the human homologs, as there are four other orthologs of sif in humans besides TIAM1 (Lu, 2022).

The variant pathogenicity prediction algorithm CADD indicates that these TIAM1 variants are rare and likely pathogenic. Since the TIAM1 variants carried by the probands reported in this study are bi-allelic, they are predicted to be LoF variants. To test this hypothesis, three of the six variants were assessed in vivo using two-pronged functional assays based on rescue and ectopic-expression experiments. TIAM1 p.Arg23Cys has reduced rescue ability, suggesting that it is a partial LoF variant. TIAM1 p.Leu862Phe and p.Gly328Val show rescue ability comparable to that of TIAM1 Ref and did not allow drawing a conclusion based on this assay, since TIAM1 cDNAs have different levels of toxicity that also affect viability. Therefore, ectopic-expression experiments were used in a WT background to further test the function of the variants, given that WT sif and TIAM1 Ref induce similar phenotypes, including wing defects, in ectopic-expression assays. Since TIAM1 Ref and the variants are inserted in the same genomic site, functional analyses of the variants can be perormed by comparing ectopic-expression-induced phenotypes. The three proband-associated variants exhibit reduced toxicity when compared to TIAM1 Ref. When the Ref causes a toxic phenotype and the variants are less toxic, the variants can typically be classified as LoF variants. Hence, the ectopic-expression data support the hypothesis that TIAM1 p.Arg23Cys, p.Leu862Phe, and p.Gly328Val are partial LoF variants. Taken together, the data suggest that the TIAM1 variants reported in this study result in a loss of function and are associated with neurodevelopmental phenotypes and seizures (Lu, 2022).

The bioinformatic data show that the Protein-Ligand Interaction (pLI) of TIAM1 is high (0.96), and the observed-to-expected ratio of LoF variants for TIAM1 is 0.20. However, there are 46 heterozygous individuals with TIAM1 LoF alleles observed in gnomAD, suggesting that loss of one copy of TIAM1 is tolerated. A possible reason why TIAM1 is associated with a high pLI score is that it is a very large gene (>400 kb). It has also been reported an autosomal-recessive disease associated with bi-allelic LoF variants in OXR1,65 a large gene (>400 kb) for which the observed-to-expected ratio of LoF variants is 0.19 and the pLI is 0.84 very similar to TIAM1 (Lu, 2022).

Besides TIAM1, sif is also the ortholog of other GEF-encoding genes, including TIAM2, DNMBP (MIM: 611282), ARHGEF37, and ARHGEF38.17 TIAM2 activates RAC1 and controls cell migration in neurons. DNMBP is a GEF for cell division control protein 42 that controls the shaping of cell junctions through binding to tight junction protein ZO-1, and knockdown of DNMBP results in a disorganized configuration of cell junctions. LoF variants of DNMBP cause infantile-onset cataracts in humans. Similarly, sif knockdown in the eye alters the distribution of septate junctions in adjacent cone cells and affects the function of the eye in young flies. Finally, ARHGEF37 and ARHGEF38 are Rho GEFs. ARHGEF37 assists dynamin 2 during clathrin-mediated endocytosis, while ARHGEF38 is an uncharacterized protein (Lu, 2022).

Tiam1 knockout mice have decreased spine density, simplified dendritic arbors, and decreased miniature excitatory postsynaptic currents in the hippocampus, but they exhibit only subtle behavior abnormalities, which may be due to redundancy of other GEFs. The related TIAM2 shares 37% overall identity and 71% Dbl homology (DH) domain identity with TIAM1. The expression levels of Tiam2 correlate with the stages of neuronal morphological development, and Tiam2 knockdown in neurons also causes reduced neurite outgrowth. TIAM1 promotes the formation and growth of spines and synapses by activating RAC1 signaling pathways that control the actin cytoskeleton. Dysfunction of the neuronal cytoskeleton has been implicated in a variety of diseases, including neurological developmental disorders as well as neurodegenerative diseases. Moreover, dysregulation of the neuronal cytoskeletal network also contributes to the pathogenesis of epilepsy (Lu, 2022).

Previous studies show that sif LoF mutants are viable and have reduced locomotor activity. This study generated a more severe LoF allele, sifT2A-GAL4, which leads to semi-lethality, a highly reduced lifespan, and a severe sensitivity to seizure-like behaviors, in addition to the climbing defects. It is worth noting that fly mutants with such severe sensitivity to seizures are rarely observed. It was also shown that these phenotypes are mainly caused by neuronal sif loss based on RNAi knockdown assays. Interestingly, the sif RNAi- with ∼12% of the remaining sif transcripts based on real-time PCR show much stronger phenotypes, including semi-lethality, when compared to sif RNAi-2 with ∼24% remaining sif transcripts, indicating a threshold effect of sif expression between these two values, similar to what has been documented for other genes, like flower (Lu, 2022).

Finally, the limitations of this study are pointed out. The number of individuals described is small and some of the phenotypic features differ. Two of the individuals come from consanguineous families, and additional recessive conditions could be contributing to the phenotype. Additionally, the analysis was based on exome sequencing, and noncoding variants were not assessed and could contribute to variation in the phenotype. With additional identified individuals with bi-allelic TIAM1 variants, it should become more obvious which clinical features are core to the condition (Lu, 2022).

In summary, this study found that bi-allelic pathogenic TIAM1 variants are associated with a neurological disorder in humans. Functional analysis is provided in flies that supports an LoF model for TIAM1-associated variants. Further studies of the underlying mechanism will be necessary to provide a better understanding of the pathological mechanisms and may provide therapeutic strategies (Lu, 2022).

Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division

Asymmetric stem cell division (ASCD) is a key mechanism in development, cancer, and stem cell biology. Drosophila neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic mechanisms. This study shows that the extrinsic axon guidance cues Netrins, secreted by a glial niche surrounding larval brain neural stem cell lineages, regulate NB ASCD. Netrin-Frazzled/DCC signaling modulates, through Abelson kinase, Robo1 signaling threshold levels in Drosophila larval brain neural stem and progenitor cells of NBII lineages. Unbalanced Robo1 signaling levels induce ectopic NBs and progenitor cells due to failures in the ASCD process. Mechanistically, Robo1 signaling directly impinges on the intrinsic ASCD machinery, such as aPKC, Canoe/Afadin, and Numb, through the small GTPases Rac1 and Cdc42, which are required for the localization in mitotic NBs of Par-6, a Cdc42 physical partner and a core component of the Par (Par-6-aPKC-Par3/Bazooka) apical complex (de Torres-Jurado, 2022).

A precise regulation of ASCD is critical in development, tissue homeostasis, and tumorigenesis. Extrinsic signals from specialized microenvironments, the niches, importantly contribute to that regulation by promoting the stem cell fate in the daughter cell that receives those signals, whereas the other daughter cell enters a differentiation program. Intriguingly, the ASCD of some stem cells, including the Drosophila CNS stem cells called NBs, the subject of this study, seems to depend exclusively on intrinsic regulatory mechanisms. Then, are those stem cells completely independent of their surrounding environment? Also, how general is the requirement of niches and the signals secreted from them for maintaining the stem cell fate in an ASCD (de Torres-Jurado, 2022)?

This study shows that the ASCD of Drosophila NBs, a traditional paradigm for studying intrinsic ASCD regulatory mechanisms, do also depend on extrinsic cues secreted by a glial niche, which are in close contact with those neural stem cell lineages in the larval brain. However, these extrinsic signals are not required to maintain the stem cell fate. They ultimately impact on the regulation of intrinsic factors to induce differentiation in one daughter cell, repressing the self-renewal 'basal state' in this cell. Different studies have shown the possibility of growing in culture isolated larval NBs, which are able to form crescents and divide asymmetrically without any additional extrinsic signal. However, most of these experiments were performed using central brain type I NB lineage (NBI) NBs, which do not require Fra and Robo1 signaling for their correct development, or included type II NB lineage (NBII,) but only particular markers were analyzed (i.e., Baz and Pon). This is relevant as, for example, it was observed that the localization of some ASCD regulators were not affected without extrinsic signals (i.e., Insc, Mira, and Brat). In the experiments in culture, some of the latter regulators are frequently used. It is also key to properly quantify the cases of crescent formation at metaphase, as the phenotypes are never fully penetrant and can be even totally rescued at telophase. For example, in the system used in this study, aPKC showed about 50% localization failures at metaphase, implying that there are NBIIs that show aPKC crescents. Nevertheless, finding that glia-secreted cues are specifically required in NBII lineages was indeed very intriguing. NBII lineages are larger than NBI, as they undergo an additional proliferation phase through INPs and hence are more prone to induce tumor-like overgrowth when ASCD fails. Thus, additional levels of regulation might have evolved in these lineages to ensure the correct division of the NB and INPs to avoid overgrowths. This issue will be further examined in the future (de Torres-Jurado, 2022).

This work has unveiled a novel function for the axon guidance cues Netrins and their Fra/DCC-like receptor in regulating self-renewal versus differentiation in neural stem and progenitor cells of larval NBII lineages. The cortex glial niche that surrounds those lineages secretes Netrins, which modulate Robo1 signaling threshold levels through Fra and Abl kinase in stem and progenitor cells. Whereas Robo1 signaling is activated by its ligand Slit, also secreted by the glial niche, Abl kinase represses this signaling, and these balanced Robo1 signaling levels appear to be critical for the cell-fate commitment of the daughter cell prone to differentiate. The cortex glia dynamically undergoes remodeling through larval stages; only at late third instar larval stages (L3) the glia chamber enwrapping each NB lineage forms completely. This study already observed the presence of the secreted ligands Slit and NetA in the cortex glia at L2, suggesting that these cues are being secreted from the glia since the reactivation of dormant NBs at early L2 (de Torres-Jurado, 2022).

Slit-Robo signaling regulates progenitor cell proliferation in the mammalian CNS and promotes the terminal asymmetric division of a differentiation-committed cell in the Drosophila CNS. Likewise, in mammary stem cells, Robo1 favors their asymmetric mode of cell division. Slit-Robo signaling is also required in other stem or progenitor cells to regulate their lineage specification, identity, or their adhesion/anchoring to the niche. No role for Netrin-Fra/DCC signaling has been previously described in all those contexts (de Torres-Jurado, 2022).

Ultimately, a transcriptional control has been pointed out as the most common way of action by which Robo signaling regulate the above-mentioned cellular processes. This study shows a novel, transcription-independent mechanism by which Robo1 signaling regulates ASCD. Robo1 signaling would be required to activate the small GTPases Rac1 and Cdc42 by repressing its inhibitor RhoGAP93B as well as by recruiting the Dock-Pak complex, which, through Pak, can also bind activated forms of both Rac1 and Cdc42 Rac1 and Cdc42 downregulation directly impacted on the intrinsic machinery that modulate ASCD in neural stem and progenitor cells. Specifically, compromising those small GTPases led to defects in the localization of the ASCD regulators, Par-6, aPKC, Cno, and Numb, and the concomitant formation of ectopic NBs (eNBs) within brain neural lineages, a phenotype that recapitulates that of Slit-Robo1 signaling impairment. The overexpression of robo1 caused similar defects than the loss of robo1. It was also a similar phenotype than for the loss of fra, the downregulation of Abl, or the expression of a kinase dead form of Abl (unable to repress Robo1). Hence, based on all those experiments, a working model proposes that Netrin-Fra signaling would be modulating, through Abl kinase, the Robo1 signaling threshold levels necessary to regulate in turn the correct activity of the small GTPases Rac1 and Cdc42. In fact, and according to this, the expression of Rac1V12 within NBII lineages caused the formation of eNBs and led to defects in the localization of aPKC in NB and progenitor cells, a similar phenotype than that observed after overexpressing robo1 in these NBII lineages. It would be interesting to determine whether this novel function of Netrin-Fra/DCC signaling regulating ASCD is also conserved in vertebrates, and whether Robo/Rac1-Cdc42 signaling threshold levels in the above-mentioned contexts are also critical for and dependent on Netrin-DCC signaling, as was have found in Drosophila (de Torres-Jurado, 2022).


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: 13 October 2023 

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