Seema Shirolikar

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.

Mixed micelle formation with hydrophobic and hydrophilic Pluronic block copolymers: Implications for controlled and targeted drug delivery

Pluronic block copolymers offer affluent phase behavioral characteristics and are extensively investigated for drug delivery applications. Hydrophobic Pluronics produce larger aggregates whereas hydrophilic Pluronics often generate small-sized micelles in aqueous milieu. To overcome the limitations and combine the advantages of different kinds of Pluronics the mixing of such two types of Pluronics is studied here, especially for hydrophobic Pluronic L81 and relatively hydrophilic Pluronic P123. Critical micelle concentration (CMC) of the developed binary mixtures was 0.032 mg/ml as evidenced from pyrene fluorescence spectroscopy and is located in between that of the individual Pluronics. Dynamic light scattering (DLS) showed very small particle sizes (∼20 nm) and low polydispersity indices for most of the mixed micelles. Transmission electron microscopy (TEM) demonstrated spherical shape of micelles. Based upon the ratio of hydrophobic and hydrophilic Pluronics, dispersions of varied stability were obtained. With 0.1/1.0 wt.% and 0.5/3.0 wt.% of Pluronic L81/P123, stable dispersions were obtained. Stability was assessed from turbidity measurement, size analysis and clarity of dispersion on standing. Micelles were also found to be stable in bovine serum albumin (BSA) solution. Mixed micelles showed fairly high entrapment efficiency, loading capacity and sustained release profile for aceclofenac (Acl), a model hydrophobe. Presence of salt lowered Acl solubilization in micelles. Thermodynamic parameters for Acl solubilization in mixed micelles revealed high partition coefficient values and spontaneity of drug solubilization. Thus, the developed novel mixed micelles hold promise in controlled and targeted drug delivery owing to their very small size, high entrapment efficiency and stability.

F-actin-based extensions of the head cyst cell adhere to the maturing spermatids to maintain them in a tight bundle and prevent their premature release in Drosophila testis

BackgroundIn Drosophila, all the 64 clonally derived spermatocytes differentiate in syncytium inside two somatic-origin cyst cells. They elongate to form slender spermatids, which are individualized and then released into the seminal vesicle. During individualization, differentiating spermatids are organized in a tight bundle inside the cyst, which is expected to play an important role in sperm selection. However, actual significance of this process and its underlying mechanism are unclear.ResultsWe show that dynamic F-actin-based processes extend from the head cyst cell at the start of individualization, filling the interstitial space at the rostral ends of the maturing spermatid bundle. In addition to actin, these structures contained lamin, beta-catenin, dynamin, myosin VI and several other filopodial components. Further, pharmacological and genetic analyses showed that cytoskeletal stability and dynamin function are essential for their maintenance. Disruption of these F-actin based processes was associated with spermatid bundle disassembly and premature sperm release inside the testis.ConclusionAltogether, our data suggests that the head cyst cell adheres to the maturing spermatid heads through F-actin-based extensions, thus maintaining them in a tight bundle. This is likely to regulate mature sperm release into the seminal vesicle. Overall, this process bears resemblance to mammalian spermiation.

Anomalies in the motion dynamics of long-flagella mutants of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii has long been used as a model organism in studies of cell motility and flagellar dynamics. The motility of the well-conserved ‘9+2’ axoneme in its flagella remains a subject of immense curiosity. Using high-speed videography and morphological analyses, we have characterized long-flagella mutants (lf1, lf2-1, lf2-5, lf3-2, and lf4) of C. reinhardtii for biophysical parameters such as swimming velocities, waveforms, beat frequencies, and swimming trajectories. These mutants are aberrant in proteins involved in the regulation of flagellar length and bring about a phenotypic increase in this length. Our results reveal that the flagellar beat frequency and swimming velocity are negatively correlated with the length of the flagella. When compared to the wild-type, any increase in the flagellar length reduces both the swimming velocities (by 26–57%) and beat frequencies (by 8–16%). We demonstrate that with no apparent aberrations/ultrastructural deformities in the mutant axonemes, it is this increased length that has a critical role to play in the motion dynamics of C. reinhardtii cells, and, provided there are no significant changes in their flagellar proteome, any increase in this length compromises the swimming velocity either by reduction of the beat frequency or by an alteration in the waveform of the flagella.

KCl induces a caspase-independent programmed cell death in the unicellular green chlorophyte Chlamydomonas reinhardtii (Chlorophyceae)

Abstract: Programmed cell death (PCD) plays an important role in mediating adaptation responses under adverse conditions such as high salinity. To understand the molecular mechanism of adaptation of algal cells to salt, the freshwater alga Chlamydomonas reinhardtii was challenged with 200 mM KCl. In the present study, vegetative cells of C. reinhardtii undergo cell death when exposed to 200 mM KCl, this death being dose-dependent, with 100–800 mM KCl causing 16–64% cell death. Within half an hour of KCl exposure, a ~1.9-fold rise in the intracellular H2O2 content followed an increase in antioxidant enzyme (superoxide dismutats, catalase, and ascorbate peroxidase) activities and their transcript levels. Furthermore, apoptotic hallmarks such as disruption of mitochondrial membrane potential, DNA nicks, apoptosis-inducing factor (AIF) release into the cytoplasm, and genomic DNA fragmentation were observed. Interestingly, KCl stress did not stimulate caspase-3-like protease activity. Additionally, cells undergoing PCD showed characteristic shrinkage with an accumulation of lipids and vacuoles, along with degraded chloroplast. These results illustrate that KCl induces reactive oxygen species production that leads to AIF release from mitochondria, causing a caspase-independent cell death in C. reinhardtii.

Seedless synthesis of gold nanorods employing isopropyl radicals generated using gamma radiolysis technique

The one step synthesis of short aspect ratio gold nanorods using gamma radiation method by incorporating cetyltrimethyl ammonium bromide (CTAB) as a directing agent is reported in this communication. In present method of synthesis, isopropyl radical is selectively generated in the system by carrying out radiolysis of aqueous solution containing 2.75 × 10−2 mol dm−3 of acetone, 0.2 mol dm−3 isopropanol, 4 × 10−4 mol dm−2 Au1+ and 6 × 10−5 mol dm−3 Ag+. The isopropyl radical brings about the reduction of Au1+ to Au0, which undergoes coalescence with other Au0 resulting in formation of Au nanorods. The formation of nanorods is attributed to slower reaction of isopropyl radical with Au1+. The TEM of as-prepared nanoparticles confirmed the formation of uniform sized nanorods having aspect ratio 3.

Atypical septate junctions maintain the somatic enclosure around maturing spermatids and prevent premature sperm release in Drosophila testis.

Tight junctions prevent the paracellular flow and maintain cell polarity in an epithelium. These are also essential for maintaining the blood-testis-barrier involved in regulating sperm differentiation. Septate junctions are orthologous to the tight junctions in insects. In Drosophila testis, major septate junction components co-localize at the interface of germline and somatic cells initially and then condense between the two somatic cells in a cyst after germline meiosis. Their localization is extensively remodeled in subsequent stages. We find that characteristic septate junctions are formed between the somatic cyst cells at the elongated spermatid stage. Consistent with the previous reports, knockdown of essential junctional components, Discs-large-1 and Neurexin-IV, in the somatic cyst cells, during the early stages, disrupted sperm differentiation beyond the spermatocyte stage. Somatic knockdown of these proteins during the final stages of spermatid maturation caused premature release of spermatids inside the testes, resulting in partial loss of male fertility. These results indicate the importance of maintaining mechanical integrity of the somatic enclosure during spermatid coiling and release in Drosophila testis. It also highlights the functional similarity with the tight junction proteins during spermatogenesis in mammalian testes. Summary statement Dubey et al., showed that septate junctions stitch the somatic enclosure around maturing spermatids in Drosophila testis. Maintaining the integrity of this junction is essential for proper release of spermatids.