Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles ¹. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.Building on the classic inverted motility assay ², the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo ³. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA⁴, quantum dots ⁵ or a wide variety of antigens via biotinylated antibodies ^4-6.Download video file.^{(57M, mov)}     

Structural characterization of the large soluble oligomers of the GTPase effector domain of dynamin

Dynamin, a protein playing crucial roles in endocytosis, oligomerizes to form spirals around the necks of incipient vesicles and helps their scission from membranes. This oligomerization is known to be mediated by the GTPase effector domain (GED). Here we have characterized the structural features of recombinant GED using a variety of biophysical methods. Gel filtration and dynamic light scattering experiments indicate that in solution, the GED has an intrinsic tendency to oligomerize. It forms large soluble oligomers (molecular mass > 600 kDa). Interestingly, they exist in equilibrium with the monomer, the equilibrium being largely in favour of the oligomers. This equilibrium, observed for the first time for GED, may have regulatory implications for dynamin function. From the circular dichroism measurements the multimers are seen to have a high helical content. From multidimensional NMR analysis we have determined that about 30 residues in the monomeric units constituting the oligomers are flexible, and these include a 17 residue stretch near the N-terminal. This contains two short segments with helical propensities in an otherwise dynamic structure. Negatively charged SDS micelles cause dissociation of the oligomers into monomers, and interestingly, the helical characteristics of the oligomer are completely retained in the individual monomers. The segments along the chain that are likely to form helices have been predicted from five different algorithms, all of which identify two long stretches. Surface electrostatic potential calculation for these helices reveals that there is a distribution of neutral, positive and negative potentials, suggesting that both electrostatic and hydrophobic interactions could be playing important roles in the oligomer core formation. A single point mutation, I697A, in one of the helices inhibited oligomerization quite substantially, indicating firstly, a special role of this residue, and secondly, a decisive, though localized, contribution of hydrophobic interaction in the association process.     

Biodiversity acts as insurance of productivity of bacterial communities under abiotic perturbations

Anthropogenic disturbances are detrimental to the functioning and stability of natural ecosystems. Critical ecosystem processes driven by microbial communities are subjected to these disturbances. Here, we examine the stabilizing role of bacterial diversity on community biomass in the presence of abiotic perturbations such as addition of heavy metals, NaCl and warming. Bacterial communities with a diversity gradient of 1–12 species were subjected to the different treatments, and community biomass (OD₆₀₀) was measured after 24 h. We found that initial species richness and phylogenetic structure impact the biomass of communities. Under abiotic perturbations, the presence of tolerant species in community largely contributed in community biomass production. Bacterial diversity stabilized the biomass across the treatments, and differential response of bacterial species to different perturbations was the key reason behind these effects. The results suggest that biodiversity is crucial for maintaining the stability of ecosystem functioning and acts as ecological insurance under abiotic perturbations. Biodiversity in natural ecosystems may also uphold the ecosystem functioning under anthropogenic disturbance.     

Combinatorial determination of sequence specificity for nanomolar DNA-binding hairpin polyamides

Development of sequence-specific DNA-binding drugs is an important pharmacological goal, given the fact that numerous existing DNA-directed chemotherapeutic drugs rely on the strength and selectivity of their DNA interactions for therapeutic activity. Among the DNA-binding antibiotics, hairpin polyamides represent the only class of small molecules that can practically bind any predetermined DNA sequence. DNA recognition by these ligands depends on their side-by-side amino acid pairings in the DNA minor groove. Extensive studies have revealed that these molecules show extremely high affinity for sequence-directed, minor groove interaction. However, the specificity of such interactions in the presence of a large selection of sequences such as the human genome is not known. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA binding specificity of two hairpin polyamides, ImPyPyPy-gamma-PyPyPyPy-beta-Dp and ImPyPyPy-gamma-ImPyPyPy-beta-Dp, in the presence of more than 134 million different sequences. These were verified by restriction endonuclease protection assays and DNase I footprinting analysis. Our data showed that both hairpin polyamides preferentially selected DNA sequences having consensus recognition sites as defined by the Dervan pairing rules. These consensus sequences were rather degenerate, as expected, given that the stacked pyrrole-pyrrole amino acid pairs present in both polyamides are unable to discriminate between A.T and T.A base pairs. However, no individual sequence within these degenerate consensus sequences was preferentially selected by REPSA, indicating that these hairpin polyamides are truly consensus-specific DNA-binding ligands. We also discovered a preference for overlapping consensus binding sites among the sequences selected by the hairpin polyamide ImPyPyPy-gamma-PyPyPyPy-beta-Dp, and confirmed by DNase I footprinting that these complex sites provide higher binding affinity. These data suggest that multiple hairpin polyamides can cooperatively bind to their highest-affinity sites.     

Modern and ancestral genotypes of Mycobacterium tuberculosis from Andhra Pradesh, India

Traditionally, the distribution of the Mycobacterium tuberculosis genotypes in India has been characterized by widespread prevalence of ancestral lineages (TbD1+ strains and variants) in the south and the modern forms (TbD1(-) CAS and variants) predominating in the north of India. The pattern was, however, not clearly known in the south-central region such as Hyderabad and the rest of the state of Andhra Pradesh where the prevalence of both tuberculosis (TB) and human immunodeficiency virus (HIV) infection is one of the highest in the country; this area has been the hotspot of TB vaccine trials. Spoligotyping of 101 clinical isolates obtained from Hyderabad and rural Andhra Pradesh confirmed the occurrence of major genogroups such as the ancestral (or the TbD1+ type or the East African Indian (EAI) type), the Central Asian (CAS) or Delhi type and the Beijing lineage in Andhra Pradesh. Sixty five different spoligotype patterns were observed for the isolates included in this study; these were further analyzed based on specific genetic signatures/mutations. It was found that the major genogroups, CAS and "ancestral," were almost equally prevalent in our collection but followed a north-south compartmentalization as was also reported previously. However, we observed a significant presence of MANU lineage in south Andhra Pradesh, which was earlier reported to be overwhelmingly present in Mumbai. This study portrays genotypic diversity of M. tuberculosis from the Indian state of Andhra Pradesh and provides a much needed snapshot of the strain diversity that will be helpful in devising effective TB control programs in this part of the world.     

Somatic embryogenesis in Leptadenia reticulata (Retz.) Wight and Arn along with assessment of shoot and callus cultures for HPTLC fingerprint and quantification of p-coumaric acid

Plant Cell, Tissue and Organ Culture (PCTOC) - Leptadenia reticulata (Retz.) Wight and Arn is an important medicinal plant of Asclepiadaceae family. In the present study regeneration was attempted...     

Screening Preeclamptic Cord Plasma for Proteins Associated with Decreased Breast Cancer Susceptibility

Preeclampsia, a complication of pregnancy characterized by hypertension and proteinuria, has been found to reduce the subsequent risk for breast cancer in female offspring. As this pro- tective effect could be due to exposure to preeclampsia-specific proteins during intrauterine life, the proteomic profiles of umbilical cord blood plasma between preeclamptic and normotensive pregnancies were compared. Umbilical cord plasma samples, depleted of 14 abundant proteins, were subjected to proteomic analysis using the quantitative method of nanoACQUITY ultra performance liquid chromatography-mass spectrometry with elevated energy mode of acquisitionE （NanoUPLC-MSE）. Sixty-nine differentially expressed proteins were identified, of which 15 and 6 proteins were only detected in preeclamptic and normotensive pregnancies, respectively.Additionally, expression of 8 proteins （gelsolin, complement C5, keratin type I cytoskeletal 10, pigment epithelium-derived factor, complement factor B, complement component C7, hemoglobin subunit gamma-2 and alpha-fetoprotein） were up-regulated in preeclampsia with a fold change of 1〉 2.0 when compared to normotensive pregnancies. The identification of alpha-fetoprotein in pre- eclamptic umbilical cord blood plasma supported the validity of this screen as alpha-fetoprotein has anti-estrogenic properties and has previously been linked to preeclampsia as well as a reduced breast cancer risk. The findings of this pilot study may provide new insights into the mechanistic link between preeclampsia and potentially reduced breast cancer susceptibility in adult life.     

Mlh1 is required for female fertility in Drosophila melanogaster: An outcome of effects on meiotic crossing over,ovarian follicles and egg activation

Mismatch repair (MMR) system, a conserved DNA repair pathway, plays crucial role in DNA recombination and is involved in gametogenesis. The impact of alterations in MMR family of proteins (bacterial MutS and MutL homologues) on mammalian fertility is well documented. However, an insight to the role of MMR in reproduction of non-mammalian organisms is limited. Hence, in the present study, we analysed the impact of mlh1 (a MutL homologue) on meiotic crossing over/recombination and fertility in a genetically tractable model, Drosophila melanogaster. Using mlh1^e00130 hypomorphic allele, we report female specific adverse reproductive outcome for reduced mlh1 in Drosophila: mlh1^e00130 homozygous females had severely reduced fertility while males were fertile. Further, mlh1^e00130 females contained small ovaries with large number of early stages as well as significantly reduced mature oocytes, and laid fewer eggs, indicating discrepancies in egg production and ovulation. These observations contrast the sex independent and/or male specific sterility and normal follicular development as well as ovulation reported so far for MMR family proteins in mammals. However, analogous to the role(s) of mlh1 in meiotic crossing over and DNA repair processes underlying mammalian fertility, ovarian follicles from mlh1^e00130 females contained significantly increased DNA double strand breaks (DSBs) and reduced synaptonemal complex foci. In addition, large proportion of fertilized eggs display discrepancies in egg activation and fail to proceed beyond stage 5 of embryogenesis. Hence, reduction of the Mlh1 protein level leads to defective oocytes that fail to complete embryogenesis after fertilization thereby reducing female fertility.