In JoVE (1)
Other Publications (9)
- Molecular and Cellular Neurosciences
- Cell Motility and the Cytoskeleton
- Developmental Biology
- American Journal of Physiology. Cell Physiology
- American Journal of Physiology. Cell Physiology
- Tissue Engineering. Part A
- Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry
- Frontiers in Oncology
Articles by Powrnima Joshi in JoVE
Other articles by Powrnima Joshi on PubMed
Amyloid Beta Peptide Adversely Affects Spine Number and Motility in Hippocampal Neurons Molecular and Cellular Neurosciences. Nov, 2006 | Pubmed ID: 16962789 Elevated levels of amyloid-beta peptide (Abeta) are found in Down's syndrome patients and alter synaptic function during the early stages of Alzheimer's disease. Dendritic spines, sites of most excitatory synaptic contacts, are considered to be an important locus for encoding synaptic plasticity. We used time-lapse two-photon imaging of hippocampal pyramidal neurons in organotypic slices to study the effects of Abeta on the development of dendritic spines. We report that exposure of hippocampal neurons to sub-lethal levels of Abeta decreased spine density, increased spine length and subdued spine motility. The effect of Abeta on spine density was reversible. Moreover, Abeta's effect on dendritic spine density was blocked by rolipram, a phosphodiesterase type IV inhibitor, suggesting the involvement of a cAMP dependent pathway. These findings raise the possibility that Abeta-induced spine alterations could underlie the cognitive defects in Alzheimer's disease and Down syndrome.
Changes in Cholesterol Levels in the Plasma Membrane Modulate Cell Signaling and Regulate Cell Adhesion and Migration on Fibronectin Cell Motility and the Cytoskeleton. Mar, 2007 | Pubmed ID: 17238130 The number and distribution of lipid molecules, including cholesterol in particular, in the plasma membrane, may play a key role in regulating several physiological processes in cells. We investigated the role of membrane cholesterol in regulating cell shape, adhesion and motility. The acute depletion of cholesterol from the plasma membrane of cells that were well spread and motile on fibronectin caused the rounding of these cells and decreased their adhesion to and motility on fibronectin. These modifications were less pronounced in cells plated on laminin, vitronectin or plastic, indicating that cholesterol-mediated changes in adhesion and motility are more specific for adhesion mediated by fibronectin-specific integrins, such as alpha5beta1. These changes were accompanied by remodeling of the actin cytoskeleton, the spatial reorganization of paxillin in the membrane, and changes to the dynamics of alpha5 integrin and paxillin-rich focal adhesions. Levels of tyrosine phosphorylation at position 576/577 of FAK and Erk1/Erk2 MAP-kinase activity levels were both lower in cholesterol-depleted than in control cells. These levels normalized only on fibronectin when cholesterol was reincorporated into the cell membrane. Thus, membrane cholesterol content has a specific effect on certain signaling pathways specifically involved in regulating cell motility on fibronectin and organization of the actin cytoskeleton.
Amyloid Precursor Protein is Required for Convergent-extension Movements During Zebrafish Development Developmental Biology. Nov, 2009 | Pubmed ID: 19664615 Amyloid precursor protein (APP) has been a focus of intense investigation because of its role in Alzheimer's disease (AD), however, its biological function remains uncertain. Loss of APP and APP-like proteins results in postnatal lethality in mice, suggesting a role during embryogenesis. Here we show that in a zebrafish model system, knock down of APP results in the generation of fish with dramatically reduced body length and a short, curly tail. In situ examination of gene expression suggests that the APP morphant embryos have defective convergent-extension movements. We also show that wild-type human APP rescues the morphant phenotype, but the Swedish mutant APP, which causes familial AD (fAD), does not rescue the developmental defects. Collectively, this work demonstrates that the zebrafish model is a powerful system to define the role of APP during embryonic development and to evaluate the functional activity of fAD mutant APP.
CDK5 Interacts with Slo and Affects Its Surface Expression and Kinetics Through Direct Phosphorylation American Journal of Physiology. Cell Physiology. Nov, 2011 | Pubmed ID: 22094329 Large conductance calcium activated potassium (BK) channels are ubiquitous and play an important role in a number of diseases. In hair cells of the ear they play a critical role in electrical tuning, a mechanism of frequency discrimination. These channels show variable kinetics and expression along the tonotopic axis. Although the molecular underpinnings to its function in hair cells are poorly understood it is established that BK channels consist of a pore forming alpha subunit (Slo) and a number of accessory subunits. Here we identify CDK5, a member of the cyclin dependent kinase family as an interacting partner of Slo. We show CDK5 to be present in hair cells, and expressed in high concentrations in the cuticular plate and in the circumferential zone. In HEK cells we show that CDK5 inhibits surface expression of Slo by direct phosphorylation of Slo. Similarly, we note that CDK5 affects Slo voltage activation and deactivation kinetics, by a direct phosphorylation of T847. Taken together with its increasing expression along the tonotopic axis, these data suggest that CDK5 likely plays a critical role in electrical tuning and surface expression of Slo in hair cells. Key words: BK channels, CDK5, surface expression and kinetics.
Hair Cell BK Channels Interact with RACK1, and PKC Increases Its Expression on the Cell Surface by Indirect Phosphorylation American Journal of Physiology. Cell Physiology. Jul, 2012 | Pubmed ID: 22538239 Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.
In Vivo Transplantation of Autogenous Marrow-derived Cells Following Rapid Intraoperative Magnetic Separation Based on Hyaluronan to Augment Bone Regeneration Tissue Engineering. Part A. Jan, 2013 | Pubmed ID: 23082937 This project was designed to test the hypothesis that rapid intraoperative processing of bone marrow based on hyaluronan (HA) could be used to improve the outcome of local bone regeneration if the concentration and prevalence of marrow-derived connective tissue progenitors (CTPs) could be increased and nonprogenitors depleted before implantation.
A Quantitative Determination of Magnetic Nanoparticle Separation Using On-off Field Operation of Quadrupole Magnetic Field-flow Fractionation (QMgFFF) Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry. 2013 | Pubmed ID: 23842422 Quadrupole Magnetic Field-Flow Fractionation (QMgFFF) is a technique for characterization of sub-micrometer magnetic particles based on their retention in the magnetic field from flowing suspensions. Different magnetic field strengths and volumetric flow rates were tested using on-off field application and two commercial nanoparticle preparations that significantly differed in their retention parameter, λ (by nearly 8-fold). The fractograms showed a regular pattern of higher retention (98.6% v. 53.3%) for the larger particle (200 nm v. 90 nm) at the higher flow rate (0.05 mL/min v. 0.01 mL/min) at the highest magnetic field (0.52 T), as expected because of its lower retention parameter. The significance of this approach is a demonstration of a system that is simpler in operation than a programmed field QMgFFF in applications to particle mixtures consisting of two distinct particle fractions. This approach could be useful for detection of unwanted particulate contaminants, especially important in industrial and biomedical applications.
Enrichment of Circulating Melanoma Cells (CMCs) Using Negative Selection from Patients with Metastatic Melanoma Oncotarget. May, 2014 | Pubmed ID: 24811334 Circulating tumor cells have emerged as prognostic biomarkers in the treatment of metastatic cancers of epithelial origins viz., breast, colorectal and prostate. These tumors express Epithelial Cell Adhesion Molecule (EpCAM) on their cell surface which is used as an antigen for immunoaffinity capture. However, EpCAM capture technologies are of limited utility for non-epithelial cancers such as melanoma. We report a method to enrich Circulating Melanoma Cells (CMCs) that does not presuppose malignant cell characteristics. CMCs were enriched by centrifugation of blood samples from healthy (N = 10) and patient (N = 11) donors, followed by RBC lysis and immunomagnetic depletion of CD45-positive leukocytes in a specialized magnetic separator. CMCs were identified by immunocytochemistry using Melan-A or S100B as melanoma markers and enumerated using automated microscopy image analyses. Separation was optimized for maximum sensitivity and recovery of CMCs. Our results indicate large number of CMCs in Stage IV melanoma patients. Analysis of survival suggested a trend toward decreased survival with increased number of CMCs. Moreover, melanoma-associated miRs were found to be higher in CMC-enriched fractions in two patients when compared with the unseparated samples, validating this method as applicable for molecular analyses. Negative selection is a promising approach for isolation of CMCs and other EpCAM -negative CTCs, and is amenable to molecular analysis of CMCs. Further studies are required to validate its efficacy at capturing specific circulating cells for genomic analysis, and xenograft studies.