Elena Sivan-Loukianova

Drosophila KAP Interacts with the Kinesin II Motor Subunit KLP64D to Assemble Chordotonal Sensory Cilia, but Not Sperm Tails

BACKGROUND Kinesin II-mediated anterograde intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia in various cell types. Kinesin associated protein (KAP) is identified as the non-motor accessory subunit of Kinesin II, but its role in the corresponding motor function is not understood. RESULTS We show that mutations in the Drosophila KAP (DmKap) gene could eliminate the sensory cilia as well as the sound-evoked potentials of Johnstons organ (JO) neurons. Ultrastructure analysis of these mutants revealed that the ciliary axonemes are absent. Mutations in Klp64D, which codes for a Kinesin II motor subunit in Drosophila, show similar ciliary defects. All these defects are rescued by exclusive expression of DmKAP and KLP64D/KIF3A in the JO neurons of respective mutants. Furthermore, reduced copy number of the DmKap gene was found to enhance the defects of hypomorphic Klp64D alleles. Unexpectedly, however, both the DmKap and the Klp64D mutant adults produce vigorously motile sperm with normal axonemes. CONCLUSIONS KAP plays an essential role in Kinesin II function, which is required for the axoneme growth and maintenance of the cilia in Drosophila type I sensory neurons. However, the flagellar assembly in Drosophila spermatids does not require Kinesin II and is independent of IFT.

An IFT-A Protein Is Required to Delimit Functionally Distinct Zones in Mechanosensory Cilia

BACKGROUND Conserved intraflagellar transport (IFT) particle proteins and IFT-associated motors are needed to assemble most eukaryotic cilia and flagella. Proteins in an IFT-A subcomplex are generally required for dynein-driven retrograde IFT, from the ciliary tip to the base. We describe novel structural and functional roles for IFT-A proteins in chordotonal organs, insect mechanosensory organs with cilia that are both sensory and motile. RESULTS The reduced mechanoreceptor potential A (rempA) locus of Drosophila encodes the IFT-A component IFT140. Chordotonal cilia are shortened in rempA mutants and an IFT-B protein accumulates in the mutant cilia, consistent with a defect in retrograde IFT. A functional REMPA-YFP fusion protein concentrates at the site of the ciliary dilation (CD), a highly structured axonemal inclusion of hitherto unknown composition and function. The CD is absent in rempA mutants, and REMPA-YFP is undetectable in the absence of another IFT-A protein, IFT122. In a mutant lacking the IFT dynein motor, the CD is disorganized and REMPA-YFP is mislocalized. A TRPV ion channel, required to generate sensory potentials and regulate ciliary motility, is normally localized in the cilia, proximal to the CD. This channel spreads into the distal part of the cilia in dynein mutants and is undetectable in rempA mutants. CONCLUSIONS IFT-A proteins are located at and required by the ciliary dilation, which separates chordotonal cilia into functionally distinct zones. A requirement for IFT140 in stable TRPV channel expression also suggests that IFT-A proteins may mediate preciliary transport of some membrane proteins.

Reevaluation of the primary motor cortex connections with the thalamus in primates.

Six injections (∼1 mm in diameter) of biotinylated dextran amine (BDA) were placed in different locations of the primary motor cortex of the rhesus monkey. Anterograde and retrograde labeling patterns in the thalamus were charted and individual labeled axons traced in continuous serial sections. Both anterograde and retrograde labeling in the thalamus was extensive, spanning several millimeters mediolaterally and including ventral lateral, ventral anterior, centromedian, and centrolateral nuclei. Paracentral, mediodorsal, lateral posterior, and medial pulvinar nuclei were also labeled. Two basic types of corticothalamic axons were identified: small to medium‐width, type 1 axons that formed large terminal fields with small boutons, and thick, type 2 axons that formed small terminal fields with large boutons. Within each group, subtypes were identified based on specific features of the axons and terminals: two subtypes of type 1 axons and four subtypes of type 2 axons. The results revealed multiple modes of corticothalamic connectivity: sparsely distributed type 1 axons, dense plexuses of type 1 axons, type 2 axon terminal fields either singly or in clusters, and mixed plexuses of type 1 and type 2 axons. Only some cells in the plexuses were retrogradely labeled; some plexuses did not contain any labeled neurons, and many retrogradely labeled neurons were in the regions devoid of anterograde labeling. These connectivity patterns differed between thalamic nuclei. The results revealed much more complex relationships between M1 and thalamus than were previously thought to exist. It is suggested that this connectivity is neither of exclusively a feedback nature nor perfectly reciprocal but is subserved by a multitude of channels, most likely originating from different populations of cortical neurons, and feeding into a variety of functionally different neuronal networks, with each processing specific information. J. Comp. Neurol. 457:133–158, 2003.

Dynamic range compression in the honey bee auditory system toward waggle dance sounds.

Honey bee foragers use a “waggle dance” to inform nestmates about direction and distance to locations of attractive food. The sound and air flows generated by dancers wing and abdominal vibrations have been implicated as important cues, but the decoding mechanisms for these dance messages are poorly understood. To understand the neural mechanisms of honey bee dance communication, we analyzed the anatomy of antenna and Johnstons organ (JO) in the pedicel of the antenna, as well as the mechanical and neural response characteristics of antenna and JO to acoustic stimuli, respectively. The honey bee JO consists of about 300–320 scolopidia connected with about 48 cuticular “knobs” around the circumference of the pedicel. Each scolopidium contains bipolar sensory neurons with both type I and II cilia. The mechanical sensitivities of the antennal flagellum are specifically high in response to low but not high intensity stimuli of 265–350 Hz frequencies. The structural characteristics of antenna but not JO neurons seem to be responsible for the non-linear responses of the flagellum in contrast to mosquito and fruit fly. The honey bee flagellum is a sensitive movement detector responding to 20 nm tip displacement, which is comparable to female mosquito. Furthermore, the JO neurons have the ability to preserve both frequency and temporal information of acoustic stimuli including the “waggle dance” sound. Intriguingly, the response of JO neurons was found to be age-dependent, demonstrating that the dance communication is only possible between aged foragers. These results suggest that the matured honey bee antennae and JO neurons are best tuned to detect 250–300 Hz sound generated during “waggle dance” from the distance in a dark hive, and that sufficient responses of the JO neurons are obtained by reducing the mechanical sensitivity of the flagellum in a near-field of dancer. This nonlinear effect brings about dynamic range compression in the honey bee auditory system.

Synaptic ultrastructure of Drosophila Johnston's organ axon terminals as revealed by an enhancer trap.

The role of auditory circuitry is to decipher relevant information from acoustic signals. Acoustic parameters used by different insect species vary widely. All these auditory systems, however, share a common transducer: tympanal organs as well as the Drosophila flagellar ears use chordotonal organs as the auditory mechanoreceptors. We here describe the central neural projections of the Drosophila Johnstons organ (JO). These neurons, which represent the antennal auditory organ, terminate in the antennomechanosensory center. To ensure correct identification of these terminals we made use of a β‐galactosidase‐expressing transgene that labels JO neurons specifically. Analysis of these projection pathways shows that parallel JO fibers display extensive contacts, including putative gap junctions. We find that the synaptic boutons show both chemical synaptic structures as well as putative gap junctions, indicating mixed synapses, and belong largely to the divergent type, with multiple small postsynaptic processes. The ultrastructure of JO fibers and synapses may indicate an ability to process temporally discretized acoustic information. J. Comp. Neurol. 491:46–55, 2005.

Posterior parietal cortex projections to the ventral lateral and some association thalamic nuclei in Macaca mulatta

The study focused on projections from the posterior parietal cortex (PPC) to the ventral lateral thalamic nucleus (VL) and three thalamic association nuclei, mediodorsal (MD), lateral posterior (LP) and pulvinar. For light microscopic analysis small biotinylated dextran amine (BDA) or biocytin injections were placed in midrostral and dorsal portions of the inferior parietal lobule (IPL), respectively. The distribution of anterograde and retrograde labeling was charted, and representative axons and terminal fields were reconstructed in the sagittal plane to examine their features. Two types of fibers were identified--those of thin diameter forming diffuse terminal fields with small boutons, and thick fibers forming focal terminal fields with large boutons. Area PFG injection of BDA resulted in labeling of both types of fibers in LP, MD, and pulvinar, whereas only fibers of the first type were found in VL. Biocytin injection in area Opt resulted in preferential labeling of large fibers terminating in LP and pulvinar. Further electron microscopic analysis of labeled boutons in VL and LP, following a large wheat germ agglutinin conjugated horseradish peroxidase injection in the middle of IPL, confirmed the existence of small and large corticothalamic boutons and their different termination sites: the small boutons formed synapses on distal dendrites while the large boutons were found close to somata of thalamocortical projection neurons, on the dendrites of local circuit neurons and in complex synaptic arrangements, such as glomeruli. The results demonstrate that projections from small loci of the PPC to functionally and connectionally different thalamic nuclei differ anatomically, implying a different functional impact on these diverse targets.

Cell-type–specific roles of Na+/K+ ATPase subunits in Drosophila auditory mechanosensation

Ion homeostasis is a fundamental cellular process particularly important in excitable cell activities such as hearing. It relies on the Na+/K+ ATPase (also referred to as the Na pump), which is composed of a catalytic α subunit and a β subunit required for its transport to the plasma membrane and for regulating its activity. We show that α and β subunits are expressed in Johnstons organ (JO), the Drosophila auditory organ. We knocked down expression of α subunits (ATPα and α-like) and β subunits (nrv1, nrv2, and nrv3) individually in JO with UAS/Gal4-mediated RNAi. ATPα shows elevated expression in the ablumenal membrane of scolopale cells, which enwrap JO neuronal dendrites in endolymph-like compartments. Knocking down ATPα, but not α-like, in the entire JO or only in scolopale cells using specific drivers, resulted in complete deafness. Among β subunits, nrv2 is expressed in scolopale cells and nrv3 in JO neurons. Knocking down nrv2 in scolopale cells blocked Nrv2 expression, reduced ATPα expression in the scolopale cells, and caused almost complete deafness. Furthermore, knockdown of either nrv2 or ATPα specifically in scolopale cells causes abnormal, electron-dense material accumulation in the scolopale space. Similarly, nrv3 functions in JO but not in scolopale cells, suggesting neuron specificity that parallels nrv2 scolopale cell–specific support of the catalytic ATPα. Our studies provide an amenable model to investigate generation of endolymph-like extracellular compartments.

Myosin VIIA, important for human auditory function, is necessary for Drosophila auditory organ development.

Background Myosin VIIA (MyoVIIA) is an unconventional myosin necessary for vertebrate audition [1]–[5]. Human auditory transduction occurs in sensory hair cells with a staircase-like arrangement of apical protrusions called stereocilia. In these hair cells, MyoVIIA maintains stereocilia organization [6]. Severe mutations in the Drosophila MyoVIIA orthologue, crinkled (ck), are semi-lethal [7] and lead to deafness by disrupting antennal auditory organ (Johnstons Organ, JO) organization [8]. ck/MyoVIIA mutations result in apical detachment of auditory transduction units (scolopidia) from the cuticle that transmits antennal vibrations as mechanical stimuli to JO. Principal Findings Using flies expressing GFP-tagged NompA, a protein required for auditory organ organization in Drosophila, we examined the role of ck/MyoVIIA in JO development and maintenance through confocal microscopy and extracellular electrophysiology. Here we show that ck/MyoVIIA is necessary early in the developing antenna for initial apical attachment of the scolopidia to the articulating joint. ck/MyoVIIA is also necessary to maintain scolopidial attachment throughout adulthood. Moreover, in the adult JO, ck/MyoVIIA genetically interacts with the non-muscle myosin II (through its regulatory light chain protein and the myosin binding subunit of myosin II phosphatase). Such genetic interactions have not previously been observed in scolopidia. These factors are therefore candidates for modulating MyoVIIA activity in vertebrates. Conclusions Our findings indicate that MyoVIIA plays evolutionarily conserved roles in auditory organ development and maintenance in invertebrates and vertebrates, enhancing our understanding of auditory organ development and function, as well as providing significant clues for future research.

Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila

Drosophila Rootletin organizes rootlets in sensory neurons, where it transmits multiple sensory inputs and maintains basal body cohesion, yet it is not required for cilium stability.

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila.

Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function.