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 Johnston's 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.     

Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport

This paper describes the molecular and biochemical properties of KLP68D, a new kinesin-like motor protein in Drosophila melanogaster. Sequence analysis of a full-length cDNA encoding KLP68D demonstrates that this protein has a domain that shares significant sequence identity with the entire 340-amin acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled- coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different than those moved by kinesin heavy chain or other plus-end directed motors.     

Kinesin-II is required for axonal transport of choline acetyltransferase in Drosophila.

KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport.     

Kinesin II is required for cell survival and adherens junction positioning in Drosophila photoreceptors

Photoreceptor morphogenesis requires specific and coordinated localization of junctional markers at different stages of development. Here, we provide evidence that Drosophila Klp64D, a homolog of Kif3A motor subunit of the heterotrimeric Kinesin II complex, is essential for viability of developing photoreceptors and localization of junctional proteins. Genetic analysis of mutant clones shows that absence of Klp64D protein in early larval eye disc does not affect initial differentiation, but results in abnormal nuclear position in differentiating photoreceptors. These cells eventually die in the pupal stage, indicating klp64D's role in cell viability. The function of Klp64D protein is cell type specific because the p35 cell death inhibitor can rescue cell death in cone cells but not photoreceptors. In contrast to early induction of mutant clones, late induction during third instar larval stage just prior to pupation allows survival of single‐ or few‐celled clones of klp64D mutant cells. Analysis of these lately induced clones shows that Klp64D function is essential for Bazooka (Par‐3 homolog) and Armadillo localization to the adherens junction (AJ) in pupal photoreceptors. These findings suggest that Kinesin II complex plays a cell type‐specific function in the localization of AJ and cell polarity proteins in the developing retina, thereby contributing to photoreceptor morphogenesis. genesis 48:522–530, 2010. © 2010 Wiley‐Liss, Inc.     

A new kinesin-like protein (Klp1) localized to a single microtubule of the Chlamydomonas flagellum

The kinesin superfamily of mechanochemical proteins has been implicated in a wide variety of cellular processes. We have begun studies of kinesins in the unicellular biflagellate alga, Chlamydomonas reinhardtii. A full-length cDNA, KLP1, has been cloned and sequenced, and found to encode a new member of the kinesin superfamily. An antibody was raised against the nonconserved tail region of the Klp1 protein, and it was used to probe for Klp1 in extracts of isolated flagella and in situ. Immunofluorescence of whole cells indicated that Klp1 was present in both the flagella and cell bodies. In wild-type flagella, Klp1 was found tightly to the axoneme; immunogold labeling of wild-type axonemal whole mounts showed that Klp1 was restricted to one of the two central pair microtubules at the core of the axoneme. Klp1 was absent from the flagella of mutants lacking the central pair microtubules, but was present in mutant flagella from pf16 cells, which contain an unstable C1 microtubule, indicating that Klp1 was bound to the C2 central pair microtubule. Localization of Klp1 to the C2 microtubule was confirmed by immunogold labeling of negatively stained and thin-sectioned axonemes. These findings suggest that Klp1 may play a role in rotation or twisting of the central pair microtubules.     

Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse

Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin-based movement of large protein-aggregates aids this process. Choline acetyltransferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates toward the synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. Here, we show that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution.     

Biochemical and Molecular Dynamic Simulation Analysis of a Weak Coiled Coil Association between Kinesin-II Stalks

Definition

Kinesin-2 refers to the family of motor proteins represented by conserved, heterotrimeric kinesin-II and homodimeric Osm3/Kif17 class of motors.

Background

Kinesin-II, a microtubule-based anterograde motor, is composed of three different conserved subunits, named KLP64D, KLP68D and DmKAP in Drosophila. Although previous reports indicated that coiled coil interaction between the middle segments of two dissimilar motor subunits established the heterodimer, the molecular basis of the association is still unknown.

Methodology/Principal Findings

Here, we present a detailed heterodimeric association model of the KLP64D/68D stalk supported by extensive experimental analysis and molecular dynamic simulations. We find that KLP64D stalk is unstable, but forms a weak coiled coil heteroduplex with the KLP68D stalk when coexpressed in bacteria. Local instabilities, relative affinities between the C-terminal stalk segments, and dynamic long-range interactions along the stalks specify the heterodimerization. Thermal unfolding studies and independent simulations further suggest that interactions between the C-terminal stalk fragments are comparatively stable, whereas the N-terminal stalk reversibly unfolds at ambient temperature.

Conclusions/Significance

Results obtained in this study suggest that coiled coil interaction between the C-terminal stalks of kinesin-II motor subunits is held together through a few hydrophobic and charged interactions. The N-terminal stalk segments are flexible and could uncoil reversibly during a motor walk. This supports the requirement for a flexible coiled coil association between the motor subunits, and its role in motor function needs to be elucidated.     

Kinesin‐2 differentially regulates the anterograde axonal transports of acetylcholinesterase and choline acetyltransferase in Drosophila

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are involved in acetylcholine synthesis and degradation at pre‐ and postsynaptic compartments, respectively. Here we show that their anterograde transport in Drosophila larval ganglion is microtubule‐dependent and occurs in two different time profiles. AChE transport is constitutive while that of ChAT occurs in a brief pulse during third instar larva stage. Mutations in the kinesin‐2 motor subunit Klp64D and separate siRNA‐mediated knock‐outs of all the three kinesin‐2 subunits disrupt the ChAT and AChE transports, and these antigens accumulate in discrete nonoverlapping punctae in neuronal cell bodies and axons. Quantification analysis further showed that mutations in Klp64D could independently affect the anterograde transport of AChE even before that of ChAT. Finally, ChAT and AChE were coimmunoprecipitated with the kinesin‐2 subunits but not with each other. Altogether, these suggest that kinesin‐2 independently transports AChE and ChAT within the same axon. It also implies that cargo availability could regulate the rate and frequency of transports by kinesin motors. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Hormonal modulation of glycogen reserves In the fat body of castor semilooperAchaea janata Linn (Lepidoptera,Noctuidea)

Histochemical details of the fat body in the fifth instar larval stage, pupa and adult moth of the castor semilooperAchaea janata were elucidated in detail using light and electron microscopy in conjunction with glycogen storage patterns using polyacrylamide gel electrophoresis. The periodic-acid Schiff staining for glycogen in fat body was maximum in the spinning stage of the larva, when compared to the feeding stage and prepupal stages, and higher in the pupa than in the larva and the adult moth. In insulin injected and juvenile hormone treated fat body, glycogen deposition was more than in glucagon injected tissues. The periodic-acid Schiff stained bands in PAGE had electrophoretic mobility similar to the corresponding protein band numbers, indicating their glycoprotein nature.     

Development of the adult leg epidermis in <Emphasis Type="Italic">Manduca sexta</Emphasis>: contribution of different larval cell populations

During metamorphosis of the tobacco hornworm Manduca sexta, the simple thoracic legs of the larva are remodeled into the more complex adult legs. Most of the adult leg epidermis derives from the adult primordia, small sets of epidermal cells located in specific regions of the larval leg, which proliferate rapidly in the final larval instar. In contrast, the contribution of the epidermal cells outside the primordia is unknown. In this study we have determined their contribution to the adult leg by labeling them with 5-bromodeoxyuridine (BUdR) and following their fate. Although the labeled cells diminished drastically in number, small groups of these cells persisted into the midpupal stage suggesting that they do contribute to the adult leg epidermis. We also found that during the wandering stage the adult primordia went through active proliferation and very little cell death, while the cells outside the primordia went through extensive cell death accounting for the decrease in their number. Our results indicate that two distinct cell populations exist outside the adult primordia. Most cells belong to the first population, which is larval-specific and disappears through apoptosis early in metamorphosis. The second population consists of polymorphic cells that contribute to the larval, pupal and adult leg epidermis.Edited by D. Tautz     

Kinesin-2 differentially regulates the anterograde axonal transports of acetylcholinesterase and choline acetyltransferase in Drosophila

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are involved in acetylcholine synthesis and degradation at pre- and postsynaptic compartments, respectively. Here we show that their anterograde transport in Drosophila larval ganglion is microtubule-dependent and occurs in two different time profiles. AChE transport is constitutive while that of ChAT occurs in a brief pulse during third instar larva stage. Mutations in the kinesin-2 motor subunit Klp64D and separate siRNA-mediated knock-outs of all the three kinesin-2 subunits disrupt the ChAT and AChE transports, and these antigens accumulate in discrete nonoverlapping punctae in neuronal cell bodies and axons. Quantification analysis further showed that mutations in Klp64D could independently affect the anterograde transport of AChE even before that of ChAT. Finally, ChAT and AChE were coimmunoprecipitated with the kinesin-2 subunits but not with each other. Altogether, these suggest that kinesin-2 independently transports AChE and ChAT within the same axon. It also implies that cargo availability could regulate the rate and frequency of transports by kinesin motors.     

pkl1+and klp2+: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis

We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast. The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function. Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase. During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots. In metaphase-arrested cells these migrate back and forth across the nucleus. During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone. Neither klp2(+) nor pkl1(+) is essential, and the double deletion is also wild type for both vegetative and sexual reproduction. Each deletion rescues different alleles of cut7(ts), a KLP that contributes to spindle formation and elongation. When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2 Delta,dhc1-d1 in karyogamy, pkl1 Delta,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both. Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1 Delta shortens it. The anaphase spindle of klp2 Delta becomes longer than the cell, leading it to curl around the cell's ends. Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.     

Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

Developmental expression of tryptophan hydroxylase gene in <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

To describe the serotonergic system in a tunicate larva, we cloned a gene encoding for tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis, in the ascidian Ciona intestinalis and studied its expression pattern during development. Ci-TPH expression was found from tailbud stage in the precursor cells of the visceral ganglion and in the tail. In the larva, TPH-expressing neurons formed two clusters in the anterior central nervous system at the level of the visceral ganglion. Moreover, we found Ci-TPH expression at the level of the muscle cells of the tail and suggested that this localisation might be at the level of neuro-muscolar junctions. Moreover, we discussed the involvement of serotonin in the control of larval locomotory activity.     

Babesia gibsoni: Detection in blood smears and formalin-fixed, paraffin-embedded tissues using deoxyribonucleic acid in situ hybridization analysis

In this study, we attempted to detect Babesia gibsoni in blood smears and formalin-fixed, paraffin-embedded tissues obtained from B. gibsoni-infected dogs using in situ hybridization. Using a digoxigenin-conjugated deoxyribonucleic acid (DNA) probe, both intraerythrocytic and exoerythrocytic parasites in the culture could be specifically stained in blood smears fixed with 4% phosphate-buffered paraformaldehyde. This indicated that genomic DNA extracted from the parasites could be detected using in situ hybridization. Moreover, the parasite could be specifically stained in paraffin-embedded spleen, lymph node, and kidney sections using in situ hybridization. Infected erythrocytes in blood vessels in the spleen and kidney, hemosiderin-laden macrophages in the spleen, and phagocytized erythrocytes, which seemed to be infected with the parasites, in lymph nodes were also specifically stained. This suggests that in situ hybridization can be utilized to investigate both the life cycle of B. gibsoni and the pathological condition of canine babesiosis.     

Detection of mRNA and hnRNA using a digoxigenin labelled cDNA probe byin situ hybridization on frozen tissue sections

Summary A full-length cDNA clone encoding the constant region of T cell receptor chain was labelled by random priming DNA with digoxigenin-dUTP. The probe was used to estimate the relative amount of the receptor chain mRNA byin situ hybridization on frozen sections from human thymus and lymph nodes. The hybridization was visualized in blue using an anti-digoxigenin antibody conjugated with alkaline phosphatase and a subsequent enzyme-catalysed colour reaction. The distributions of the signal in tissue sections were as expected. Moreover, labelled cells showed hybrids both in the cytoplasm and in the nucleus, and strongly and weakly stained cells were clearly distinguishable. The results indicate that this method ofin situ hybridization should be useful in the detection of specific mRNA in frozen sections.     

人单拷贝及重复DNA序列的原位PCR

在培养的人小肠癌转移腹水细胞系细胞中进行了Y染色体特异的重复序列及单拷贝序列的原位扩增与检测.结果显示原位PCR法的灵敏度比直接的原位杂交法明显提高.     

Adjacent chromosomal regions can evolve at very different rates: Evolution of theDrosophila 68C glue gene cluster

Summary The 68C puff is a highly transcribed region of theDrosophila melanogaster salivary gland polytene chromosomes. Three different classes of messenger RNA originate in a 5000-bp region in the puff; each class is translated to one of the salivary gland glue proteins sgs-3, sgs-7, or sgs-8. These messenger RNA classes are coordinately controlled, with each RNA appearing in the third larval instar and disappearing at the time of puparium formation. Their disappearance is initiated by the action of the steroid hormone ecdysterone. In the work reported here, we studied evolution of this hormone-regulated gene cluster in themelanogaster species subgroup ofDrosophila. Genome blot hybridization experiments showed that five other species of this subgroup have DNA sequences that hybridize toD. melanogaster 68C sequences, and that these sequences are divided into a highly conserved region, which does not contain the glue genes, and an extraordinarily diverged region, which does. Molecular cloning of this DNA fromD. simulans, D. erecta, D. yakuba, andD. teissieri confirmed the division of the region into a slowly and a rapidly evolving protion, and also showed that the rapidly evolving region of each species codes for third instar larval salivary gland RNAs homologous to theD. melanogaster glue mRNAs. The highly conserved region is at least 13,000 bp long, and is not known to code for any RNAs.     

原钙黏附蛋白18b基因抑制对斑马鱼神经系统发育的影响

原钙黏附蛋白18b(Protocadherin18b,Pcdh18b)属于钙黏附蛋白家族成员．为了研究pcdh18b基因抑制对斑马鱼神经系统发育的影响,针对pcdh18b的翻译起始位点设计一个吗啡啉修饰的反义寡核苷酸抑制其表达,在斑马鱼受精卵一到二细胞期注射并且验证其有效性．注射后用原位杂交和吖啶橙染色检测神经系统的表型和标志基因的表达．pcdh18b下调使神经前体细胞的标志基因neurog1、神经元标志基因elavl3和神经胶质细胞标志基因gfap的表达均出现下调,中后脑边界的标志基因pax2a和wnt1表达减弱并出现神经管分叉现象,同时与后脑分节相关的基因krox20表达减少．吖啶橙染色显示pcdh18b下调后斑马鱼中脑、后脑及中后脑边界细胞凋亡增多．这些结果表明pcdh18b抑制导致了斑马鱼神经系统发育的异常．     

Photoperiod- and temperature-dependent induction of larval diapause in a multivoltine bruchid, Bruchidius dorsalis

The wild bruchid beetle, Bruchidius dorsalis Fahraeus (Coleoptera: Bruchidae), has a multivoltine life cycle and overwinters in several developmental stages in the middle part of Japan. We investigated the incidence of diapause under different conditions of photoperiod (from L8:D16 to L16:D8) and temperature (at 20 °C and 24 °C). Our experiments revealed the following results: (1) B. dorsalis entered diapause at the final (late fourth) instar larva under short photoperiods, (2) the larval diapause incidence was dependent on temperature (critical photoperiods were 12.5 h at 20 °C and 12 h at 24 °C), (3) some individuals did not enter diapause under short-photoperiod conditions at 24 °C, and (4) the sensitive stages to the photoperiod were from the late egg stage to the early first instar larva. Based on these results, we discuss not only the evolution of a complex overwintering strategy inB. dorsalis but also the domestication process of stored-bean pests.