Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function

The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP) is caused by mutations in the SPG4 (spastin) gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ) phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, we observed the opposite phenotype: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that we observe in spastin mutants may be relevant to an understanding of human Spastin's role in maintenance of axon terminals in the spinal cord.     

Cold temperature improves mobility and survival in Drosophila models of autosomal-dominant hereditary spastic paraplegia (AD-HSP)

Autosomal-dominant hereditary spastic paraplegia (AD-HSP) is a crippling neurodegenerative disease for which effective treatment or cure remains unknown. Victims experience progressive mobility loss due to degeneration of the longest axons in the spinal cord. Over half of AD-HSP cases arise from loss-of-function mutations in spastin, which encodes a microtubule-severing AAA ATPase. In Drosophila models of AD-HSP, larvae lacking Spastin exhibit abnormal motor neuron morphology and function, and most die as pupae. Adult survivors display impaired mobility, reminiscent of the human disease. Here, we show that rearing pupae or adults at reduced temperature (18°C), compared with the standard temperature of 24°C, improves the survival and mobility of adult spastin mutants but leaves wild-type flies unaffected. Flies expressing human spastin with pathogenic mutations are similarly rescued. Additionally, larval cooling partially rescues the larval synaptic phenotype. Cooling thus alleviates known spastin phenotypes for each developmental stage at which it is administered and, notably, is effective even in mature adults. We find further that cold treatment rescues larval synaptic defects in flies with mutations in Flower (a protein with no known relation to Spastin) and mobility defects in flies lacking Kat60-L1, another microtubule-severing protein enriched in the CNS. Together, these data support the hypothesis that the beneficial effects of cold extend beyond specific alleviation of Spastin dysfunction, to at least a subset of cellular and behavioral neuronal defects. Mild hypothermia, a common neuroprotective technique in clinical treatment of acute anoxia, might thus hold additional promise as a therapeutic approach for AD-HSP and, potentially, for other neurodegenerative diseases.KEY WORDS: Spastin, Hereditary spastic paraplegia, AD-HSP, Microtubule severing, Cold treatment, Therapeutic hypothermia, Drosophila disease model     

Drosophila Atlastin regulates the stability of muscle microtubules and is required for synapse development

Hereditary spastic paraplegia (HSP) is an inherited neurological disorder characterized by progressive spasticity and weakness of the lower extremities. The most common early-onset form of HSP is caused by mutations in the human gene that encodes the dynamin-family GTPase Atlastin-1 (Atl-1). Recently, loss of the Drosophila ortholog of Atl-1 (Atl) has been found to induce locomotor impairments from the earliest adult stages, suggesting the developmental role of atlastin-subfamily GTPases. Here, we provide evidence that Atl is required for normal growth of muscles and synapses at the neuromuscular junction (NMJ). Atl protein is highly expressed in larval body-wall muscles. Loss-of-function mutations in the atl gene reduce the size of muscles and increase the number of synaptic boutons. Rescue of these defects is accomplished by muscular, but not neuronal expression of Atl. Loss of Atl also disrupts ER and Golgi morphogenesis in muscles and reduces the synaptic levels of the scaffold proteins Dlg and α-spectrin. We also provide evidence that Atl functions with the microtubule-severing protein Spastin to disassemble microtubules in muscles. Finally, we demonstrate that the microtubule-destabilizing drug vinblastine alleviates synapse and muscle defects in atl mutants. Together, our results suggest that Atl controls synapse development and ER and Golgi morphogenesis by regulating microtubule stability.     

Role of Spastin in Apical Domain Control along the Rhabdomere Elongation in Drosophila Photoreceptor

Background

Mutations in spastin are the most common cause of hereditary spastin paraplegia, a neurodegenerative disease. In this study, the role of spastin was examined in Drosophila photoreceptor development.

Methodology/Principal Findings

The spastin mutation in developing pupal eyes causes a mild mislocalization of the apical membrane domain at the distal section, but the apical domain was dramatically reduced at the proximal section of the developing pupal eye. Since the rhabdomeres in developing pupal eyes grow from distal to proximal, this phenotype strongly suggests that spastin is required for apical domain maintenance during rhabdomere elongation. This role of spastin in apical domain modulation was further supported by spastin''s gain-of-function phenotype. Spastin overexpression in photoreceptors caused the expansion of the apical membrane domain from apical to basolateral in the developing photoreceptor. Although the localizations of the apical domain and adherens junctions (AJs) were severely expanded, there were no defects in cell polarity.

Conclusions/Significance

These results strongly suggest that spastin is essential for apical domain biogenesis during rhabdomere elongation in Drosophila photoreceptor morphogenesis.     

The hereditary spastic paraplegia gene, spastin, regulates microtubule stability to modulate synaptic structure and function

BACKGROUND: Hereditary Spastic Paraplegia (HSP) is a devastating neurological disease causing spastic weakness of the lower extremities and eventual axonal degeneration. Over 20 genes have been linked to HSP in humans; however, mutations in one gene, spastin (SPG4), are the cause of >40% of all cases. Spastin is a member of the ATPases associated with diverse cellular activities (AAA) protein family, and contains a microtubule interacting and organelle transport (MIT) domain. Previous work in cell culture has proposed a role for Spastin in regulating microtubules. RESULTS: Employing Drosophila transgenic methods for overexpression and RNA interference (RNAi), we have investigated the role of Spastin in vivo. We show that Drosophila Spastin (D-Spastin) is enriched in axons and synaptic connections. At neuromuscular junctions (NMJ), Dspastin RNAi causes morphological undergrowth and reduced synaptic area. Moreover, Dspastin overexpression reduces synaptic strength, whereas Dspastin RNAi elevates synaptic currents. By using antibodies against posttranslationally modified alpha-Tubulin, we find that Dspastin regulates microtubule stability. Functional synaptic defects caused by Dspastin RNAi and overexpression were pharmacologically alleviated by agents that destabilize and stabilize microtubules, respectively. CONCLUSIONS: Loss of Dspastin in Drosophila causes an aberrantly stabilized microtubule cytoskeleton in neurons and defects in synaptic growth and neurotransmission. These in vivo data strongly support previous reports, providing a probable cause for the neuronal dysfunction in spastin-linked HSP disease. The role of Spastin in regulating neuronal microtubule stability suggests therapeutic targets for HSP treatment and may provide insight into neurological disorders linked to microtubule dysfunction.     

Making more microtubules by severing: a common theme of noncentrosomal microtubule arrays?

Two related enzymes, katanin and spastin, use the energy from ATP hydrolysis to sever microtubules. Two new studies (one in this issue; see McNally et al., p. 881) show that microtubule severing by katanin provides a means for increasing microtubule density in meiotic spindles. Interestingly, loss of spastin leads to a sparser microtubule array in axons and synaptic boutons. Together, these studies hint at a wider role for microtubule-severing enzymes in the formation and organization of noncentrosomal microtubule arrays by generating new seeds for microtubule growth.     

The Saccharomyces cerevisiae Kinesin-related Motor Kar3p Acts at Preanaphase Spindle Poles to Limit the Number and Length of Cytoplasmic Microtubules

The Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle–specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential α-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.     

Linking axonal degeneration to microtubule remodeling by Spastin-mediated microtubule severing

Mutations in the AAA adenosine triphosphatase (ATPase) Spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia, which is a retrograde axonopathy primarily characterized pathologically by the degeneration of long spinal neurons in the corticospinal tracts and the dorsal columns. Using recombinant Spastin, we find that six mutant forms of Spastin, including three disease-associated forms, are severely impaired in ATPase activity. In contrast to a mutation designed to prevent adenosine triphosphate (ATP) binding, an ATP hydrolysis-deficient Spastin mutant predicted to remain kinetically trapped on target proteins decorates microtubules in transfected cells. Analysis of disease-associated missense mutations shows that some more closely resemble the canonical hydrolysis mutant, whereas others resemble the ATP-binding mutant. Using real-time imaging, we show that Spastin severs microtubules when added to permeabilized, cytosol-depleted cells stably expressing GFP-tubulin. Using purified components, we also show that Spastin interacts directly with microtubules and is sufficient for severing. These studies suggest that defects in microtubule severing are a cause of axonal degeneration in human disease.     

Clasp-mediated microtubule bundling regulates persistent motility and contact repulsion in Drosophila macrophages in vivo

Drosophila melanogaster macrophages are highly migratory cells that lend themselves beautifully to high resolution in vivo imaging experiments. By expressing fluorescent probes to reveal actin and microtubules, we can observe the dynamic interplay of these two cytoskeletal networks as macrophages migrate and interact with one another within a living organism. We show that before an episode of persistent motility, whether responding to developmental guidance or wound cues, macrophages assemble a polarized array of microtubules that bundle into a compass-like arm that appears to anticipate the direction of migration. Whenever cells collide with one another, their microtubule arms transiently align just before cell–cell repulsion, and we show that forcing depolymerization of microtubules by expression of Spastin leads to their defective polarity and failure to contact inhibit from one another. The same is true in orbit/clasp mutants, indicating a pivotal role for this microtubule-binding protein in the assembly and/or functioning of the microtubule arm during polarized migration and contact repulsion.     

The mbk-2 kinase is required for inactivation of MEI-1/katanin in the one-cell Caenorhabditis elegans embryo

The Caenorhabditis elegans early embryo is widely used to study the regulation of microtubule-related processes. In a screen for mutants affecting the first cell division, we isolated a temperature-sensitive mutation affecting pronuclear migration and spindle positioning, phenotypes typically linked to microtubule or centrosome defects. In the mutant, microtubules are shorter and chromosome segregation is impaired, while centrosome organization appears normal. The mutation corresponds to a strong loss of function in mbk-2, a conserved serine/threonine kinase. The microtubule-related defects are due to the postmeiotic persistence of MEI-1, a homologue of the microtubule-severing protein katanin. In addition, P-granule distribution is abnormal in mbk-2 mutants, consistent with genetic evidence that mbk-2 has other functions and with the requirement of mbk-2 activity at the one-cell stage. We propose that mbk-2 potentiates the degradation of MEI-1 and other proteins, possibly via direct phosphorylation.     

Drosophila S6 Kinase Like Inhibits Neuromuscular Junction Growth by Downregulating the BMP Receptor Thickveins

Synaptic connections must be precisely controlled to ensure proper neural circuit formation. In Drosophila melanogaster, bone morphogenetic protein (BMP) promotes growth of the neuromuscular junction (NMJ) by binding and activating the BMP ligand receptors wishful thinking (Wit) and thickveins (Tkv) expressed in motor neurons. We report here that an evolutionally conserved, previously uncharacterized member of the S6 kinase (S6K) family S6K like (S6KL) acts as a negative regulator of BMP signaling. S6KL null mutants were viable and fertile but exhibited more satellite boutons, fewer and larger synaptic vesicles, larger spontaneous miniature excitatory junctional potential (mEJP) amplitudes, and reduced synaptic endocytosis at the NMJ terminals. Reducing the gene dose by half of tkv in S6KL mutant background reversed the NMJ overgrowth phenotype. The NMJ phenotypes of S6KL mutants were accompanied by an elevated level of Tkv protein and phosphorylated Mad, an effector of the BMP signaling pathway, in the nervous system. In addition, Tkv physically interacted with S6KL in cultured S2 cells. Furthermore, knockdown of S6KL enhanced Tkv expression, while S6KL overexpression downregulated Tkv in cultured S2 cells. This latter effect was blocked by the proteasome inhibitor MG132. Our results together demonstrate for the first time that S6KL regulates synaptic development and function by facilitating proteasomal degradation of the BMP receptor Tkv.     

Katanin maintains meiotic metaphase chromosome alignment and spindle structure in vivo and has multiple effects on microtubules in vitro

Assembly of Caenorhabditis elegans female meiotic spindles requires both MEI-1 and MEI-2 subunits of the microtubule-severing ATPase katanin. Strong loss-of-function mutants assemble apolar intersecting microtubule arrays, whereas weaker mutants assemble bipolar meiotic spindles that are longer than wild type. To determine whether katanin is also required for spindle maintenance, we monitored metaphase I spindles after a fast-acting mei-1(ts) mutant was shifted to a nonpermissive temperature. Within 4 min of temperature shift, bivalents moved off the metaphase plate, and microtubule bundles within the spindle lengthened and developed a high degree of curvature. Spindles eventually lost bipolar structure. Immunofluorescence of embryos fixed at increasing temperature indicated that MEI-1 was lost from spindle microtubules before loss of ASPM-1, indicating that MEI-1 and ASPM-1 act independently at spindle poles. We quantified the microtubule-severing activity of purified MEI-1/MEI-2 complexes corresponding to six different point mutations and found a linear relationship between microtubule disassembly rate and meiotic spindle length. Previous work showed that katanin is required for severing at points where two microtubules intersect in vivo. We show that purified MEI-1/MEI-2 complexes preferentially sever at intersections between two microtubules and directly bundle microtubules in vitro. These activities could promote parallel/antiparallel microtubule organization in meiotic spindles.     

Contribution of Noncentrosomal Microtubules to Spindle Assembly in Drosophila Spermatocytes

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.     

Analysis of the rice mutant dwarf and gladius leaf 1. Aberrant katanin-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling

Molecular genetic studies of plant dwarf mutants have indicated that gibberellin (GA) and brassinosteroid (BR) are two major factors that determine plant height; dwarf mutants that are caused by other defects are relatively rare, especially in monocot species. Here, we report a rice (Oryza sativa) dwarf mutant, dwarf and gladius leaf 1 (dgl1), which exhibits only minimal response to GA and BR. In addition to the dwarf phenotype, dgl1 produces leaves with abnormally rounded tip regions. Positional cloning of DGL1 revealed that it encodes a 60-kD microtubule-severing katanin-like protein. The protein was found to be important in cell elongation and division, based on the observed cell phenotypes. GA biosynthetic genes are up-regulated in dgl1, but the expression of BR biosynthetic genes is not enhanced. The enhanced expression of GA biosynthetic genes in dgl1 is not caused by inappropriate GA signaling because the expression of these genes was repressed by GA3 treatment, and degradation of the rice DELLA protein SLR1 was triggered by GA3 in this mutant. Instead, aberrant microtubule organization caused by the loss of the microtubule-severing function of DGL1 may result in enhanced expression of GA biosynthetic genes in that enhanced expression was also observed in a BR-deficient mutant with aberrant microtubule organization. These results suggest that the function of DGL1 is important for cell and organ elongation in rice, and aberrant DGL1-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling.     

Creative destruction of the microtubule array

Comment on: Mukherjee S, et al. Cell Cycle 2012; 11:2359-66.Typical cells contain a dense array of microtubules that serves as a structural backbone and also provides a substrate against which molecular motor proteins generate force. Cells transitioning through the cell cycle or undergoing significant morphological changes must be able to tear apart the microtubule array and reconstruct it into new configurations, either partially or completely. The microtubule field was revolutionized in the 1980s with the introduction of the dynamic instability model,¹ now broadly recognized as a fundamental mechanism by which microtubule populations are reconfigured.² Dynamic instability involves the catastrophic disassembly of microtubules, generally from their plus ends, as well as the rapid reassembly of microtubules and selective stabilization of particular ones. Microtubules can be stabilized along their length by binding to various proteins and can be attached at their minus ends to structures such as the centrosome and “captured” at their plus ends by proteins in the cell’s cortex.² Given the contribution of these stabilizing and anchoring factors, additional mechanisms beyond dynamic instability are required to tear down previous microtubule structures so that new ones can be constructed. Borrowing from the field of economics, we refer to this as creative destruction.Various proteins such as stathmin³ and kinesin-13⁴ contribute to creative destruction by promoting loss of tubulin subunits from the ends of the microtubules. We find especially interesting a category of AAA enzymes called microtubule-severing proteins that use the energy of ATP hydrolysis to yank at tubulin subunits within the microtubule, thereby causing the lattice to break.⁵ If this occurs along the length of the microtubule, the microtubule will be severed into pieces. If this occurs at either of the two ends of the microtubule, the microtubule will lose subunits from that end. The first discovered and best-studied microtubule-severing proteins are katanin and spastin.Thanks to David Sharp and his colleagues at Albert Einstein College of Medicine, as well as other workers in the field, we now know that cells express at least five other AAA proteins with potential microtubule-severing properties, on the basis of sequence similarity to katanin and spastin in the AAA region.⁵ Two of these, called katanin-like-1 and katanin-like-2, are very similar to katanin. The three others are similar to one another, collectively termed fidgetins (fidgetin, fidgetin-like-1 and fidgetin-like-2). One possibility is that all seven of the microtubule-severing proteins are regulated similarly and are functionally redundant with one another. A more compelling possibility is that, while there is some functional redundancy, there is also a division of labor, with each severing protein displaying distinct properties and carrying out its own duties. Thus far, Sharp’s studies on mitosis support the latter scenario, with katanin, fidgetin and spastin having characteristic distributions within the spindle, resulting in unique phenotypes when depleted.⁶In a new article, Sharp’s group has confirmed that fidgetin has microtubule-severing properties. Interestingly, fidgetin depolymerizes microtubules preferentially from the minus end.⁷ In addition, the new work shows that in human U2OS cells, fidgetin targets to the centrosome, where most minus ends of microtubules are clustered, suggesting a scenario by which fidgetin suppresses microtubule growth from the centrosome as well as attachment to it. Consistent with this scenario, the authors show that experimental depletion of fidgetin reduces that speed of poleward tubulin flux as well as the speed of anaphase A chromatid-to-pole motion and also results in an increase in both the number and length of astral microtubules. Notably, this contrasts with katanin, which favors the plus ends of microtubules, for example, at the chromosome during cell division⁶ and at the leading edge of motile cells.⁸The authors close their article by pointing out that microtubule-severing is important beyond mitosis, for example, in the restructuring of the microtubule array in neurons and migrating cells, and we would point to plants as well.⁹ We previously described a mechanism called “cut and run,” wherein the severing of microtubules is important for motility within the microtubule array, as short microtubules are more mobile than long ones.⁹ Now, inspired by the work of Sharp and colleagues, we envision “creative destruction” as another way of understanding the crucial roles played by a diversity of microtubule-severing proteins in cells.     

Suppression of Patronin deficiency by altered Hippo signaling in Drosophila organ development

The microtubule network is crucial for cell structure and function. Patronin is a conserved protein involved in protecting the minus end of microtubules. Conversely, Klp10A is a kinesin-like microtubule depolymerase. Here we report the role of Drosophila Patronin and Klp10A for cell survival in developing organs. Loss of Patronin reduces the size of organs by activation of a caspase in imaginal discs. Reduced wing by Patronin RNAi is suppressed by knockdown of Spastin (Spas) but not Katanin 60, suggesting that Patronin is inhibitory to the severing function of Spas at the minus end. Patronin RNAi phenotype is also recovered by overexpressing Death-associated inhibitor of apoptosis 1 (Diap1), a Yorkie target gene. Heterozygote mutations in Hippo pathway genes, including hippo and warts (wts), suppress the Patronin RNAi wing phenotypes. Furthermore, Patronin physically interacts with Merlin and Expanded while reducing their function. Patronin and Klp10A antagonistically regulate their levels. Wing phenotypes of Patronin RNAi are rescued by knockdown of Klp10A, consistent with their antagonistic interaction. Klp10A overexpression also causes organ size reduction that is partially suppressed by Diap1 overexpression or wts heterozygote mutation. Taken together, this study suggests that the antagonistic interaction between Patronin and Klp10A is required for controlling cell survival and organ size by modulating microtubule stability and Hippo components.Subject terms: Development, Genetic interaction     

The hangover gene negatively regulates bouton addition at the Drosophila neuromuscular junction

The synaptic growth of neurons during the development and adult life of an animal is a very dynamic and highly regulated process. During larval development in Drosophila new boutons and branches are added at the glutamatergic neuromuscular junction (NMJ) until a balance between neuronal activity and morphological structures is reached. Analysis of several Drosophila mutants suggest that bouton number and size might be regulated by separate signaling processes [Budnik, V., 1996. Synapse maturation and structural plasticity at Drosophila neuromuscular junctions. Curr. Opin. Neurobiol. 6, 858-867.]. Here we show a new role for Hangover as a negative regulator of bouton number at the NMJ. The hangover gene (hang) encodes a nuclear zinc finger protein. It has a function in neuronal plasticity mediating ethanol tolerance, a behavior that develops upon previous experience with ethanol. hang^AE10 mutants have more boutons and an extended synaptic span. Moreover, Hang expression in the motoneuron is required for the regulation of bouton number and the overall length of muscle innervation. However, the increase in bouton number does not correlate with a change in synaptic transmission, suggesting a mechanism independent from neuronal activity leads to the surplus of synaptic boutons. In contrast, we find that expression levels of the cell adhesion molecule Fasciclin II (FASII) are reduced in the hang mutant. This finding suggests that the increase in bouton number in hang mutants is caused by a reduction in FASII expression, thus, linking the regulation of nuclear gene expression with the addition of boutons at the NMJ regulated by cell adhesion molecules.     

CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca(2+)-permeable channels and stomatal closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca²⁺ in guard cell ion channel regulation. However, genetic mutants in Ca²⁺ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca²⁺-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca²⁺ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca²⁺-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca²⁺-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca²⁺ oscillation experiments revealed that Ca²⁺-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca²⁺-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca²⁺-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.