Sohail Ahmed - Academia.edu (original) (raw)

Papers by Sohail Ahmed

Communicative & Integrative Biology, 2010

Journal of Molecular Signaling, 2007

We have identified human ArhGAP9 as a novel MAP kinase docking protein that interacts with Erk2 a... more We have identified human ArhGAP9 as a novel MAP kinase docking protein that interacts with Erk2 and p38α through complementarily charged residues in the WW domain of ArhGAP9 and the CD domains of Erk2 and p38α. This interaction sequesters the MAP kinases in their inactive states through displacement of MAP kinase kinases targeting the same sites. While over-expression of wild type ArhGAP9 caused MAP kinase activation by the epidermal growth factor receptor (EGFR) to be suppressed and preserved the actin stress fibres in quiescent Swiss 3T3 fibroblasts, over-expression of an ArhGAP9 mutant defective in MAP kinase binding restored EGFR-induced MAP kinase activation and resulted in significant disruption of the stress fibres, consistent with the role of Erk activation in disassembly of actin stress fibres. The interaction between ArhGAP9 and the MAP kinases represents a novel mechanism of cross-talk between Rho GTPase and MAP kinase signaling.

Single Molecule Spectroscopy and Superresolution Imaging VII, 2014

ABSTRACT IRSp53 is a Cdc42 effector and a member of the Inverse-Bin-Amphiphysins-Rvs (I-BAR) doma... more ABSTRACT IRSp53 is a Cdc42 effector and a member of the Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain family which can induce negative membrane curvature. IRSp53 generates filopodia by coupling membrane protrusion (I-BAR domain) with actin dynamics through its SH3 domain binding partners. Dynamin 1 (Dyn1), a large GTPase associated with endocytosis, is a novel interacting partner of IRSp53 that localises to filopodia. Using rapid time-lapse TIRF microscopy we have shown that Dyn1 localized to a subcellular region just behind Mena at the leading edge, or in filopodial tip complexes when co-expressed with IRSp53. Dyn1-GFP was strongly localized in the filopodial shaft during the early phase of elongation, after which it moved rearward, suggestive of a role in early filopodia assembly. Mena and Eps8, accumulate at the tip complex in sequence and are involved in filopodial extension and retraction, respectively (Chou at al, 2014 submitted). Here we describe the use of dSTORM to investigate the molecular architecture of filopodia and in particular the size of the F-actin bundle in these structures. The data suggest that direct Stochastic Optical Reconstruction Microscopy (dSTORM) in combination with other techniques will allow the molecular architecture of

Stem Cells and Development, 2012

Neurospheres are widely used to propagate and investigate neural stem cells (NSCs) and neural pro... more Neurospheres are widely used to propagate and investigate neural stem cells (NSCs) and neural progenitors (NPs). However, the exact cell types present within neurospheres are still unknown. To identify cell types, we used single-cell mRNA profiling of 48 genes in 187 neurosphere cells. Using a clustering algorithm, we identified 3 discrete cell populations within neurospheres. One cell population [cluster unsorted (US) 1] expresses high Bmi1 and Hes5 and low Myc and Klf12. Cluster US2 shows intermediate expression of most of the genes analyzed. Cluster US3 expresses low Bmi1 and Hes5 and high Myc and Klf12. The mRNA profiles of these 3 cell populations correlate with a developmental timeline of early, intermediate, and late NPs, as seen in vivo from the mouse brain. We enriched the cell population for neurosphere-forming cells (NFCs) using morphological criteria of forward scatter (FSC) and side scatter (SSC). FSC/ SSC high cells generated 2.29-fold more neurospheres than FSC/SSC low cells at clonal density. FSC/SSC high cells were enriched for NSCs and Lewis-X + ve cells, possessed higher phosphacan levels, and were of a larger cell size. Clustering of both FSC/SSC high and FSC/SSC low cells identified an NFC cluster. Significantly, the mRNA profile of the NFC cluster drew close resemblance to that of early NPs. Taken together, data suggest that the neurosphere culture system can be used to model central nervous system development, and that early NPs are the cell population that gives rise to neurospheres. In future work, it may be possible to further dissect the NFCs and reveal the molecular signature for NSCs.

Journal of Biological Chemistry, 2009

The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downs... more The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downstream of membranebound receptors through distinct effector pathways. Cdc42-effector protein interactions represent important elements of cell signaling pathways that regulate cell biology in systems as diverse as yeast and humans. To derive mechanistic insights into cell signaling pathways, it is vital that we generate quantitative data from in vivo systems. We need to be able to measure parameters such as protein concentrations, rates of diffusion, and dissociation constants (K D) of protein-protein interactions in vivo. Here we show how single wavelength fluorescence cross-correlation spectroscopy in combination with Förster resonance energy transfer analysis can be used to determine K D of Cdc42effector interactions in live mammalian cells. Constructs encoding green fluorescent protein or monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain (CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP) and insulin receptor substrate protein (IRSp53), were expressed as pairs in Chinese hamster ovary cells, and concentrations of free protein as well as complexed protein were determined. The measured K D for Cdc42V12-N-WASP, Cdc42V12-CRIB, and Cdc42V12-IRSp53 was 27, 250, and 391 nM, respectively. The determination of K D for Cdc42-effector interactions opens the way to describe cell signaling pathways quantitatively in vivo in mammalian cells.

Journal of Biological Chemistry, 2009

The transducer of Cdc42-dependent actin assembly (Toca-1)-N-WASP complex was isolated as an essen... more The transducer of Cdc42-dependent actin assembly (Toca-1)-N-WASP complex was isolated as an essential cofactor for Cdc42-driven actin polymerization in vitro. Toca-1 consists of an N-terminal F-BAR domain, followed by a Cdc42 binding site (HR1 domain) and an SH3 domain, (the N-WASP interacting site). N-WASP is an activator of actin nucleation through the Arp2/3 complex. The aim of the present study was to investigate the cellular function of the Toca-1-N-WASP complex. We report that Toca-1 induces filopodia and neurites as does N-WASP in N1E115 neuroblastoma cells. Toca-1 requires the F-BAR domain, Cdc42 binding site, and SH3 domain to induce filopodia. Toca-1 and N-WASP both require each other to induce filopodia. The expression of Toca-1 and N-WASP affects the distribution, size, and number of Rab5 positive membranes. Toca-1 interacts directly with N-WASP in filopodia and Rab5 membrane as seen by Forster resonance energy transfer. Thus the Toca-1-N-WASP complex localizes to and induces the formation of filopodia and endocytic vesicles. Last, three inhibitors of endocytosis, Dynamin-K44A, Eps15⌬95/295, and clathrin heavy chain RNA interference, block Toca-1-induced filopodial formation. Taken together, these data suggest that the Toca-1-N-WASP complex can link filopodial formation to endocytosis. Cell migration and axon guidance rely on morphological actin-based structures such as filopodia and lamellipodia/ membrane ruffles (1). Cdc42 is a member of the Rho family of GTPases and plays a prominent role in cell signaling pathways that control cell migration as well as cell shape and polarity. Cdc42 was first identified as a yeast protein involved in cell division that affects budding of daughter cells (2). In mammalian cells, one specific function of Cdc42 is to activate filopodial formation (3, 4). Filopodia are dynamic actin-based structures that protrude from the plasma membrane and are thought to play a role in a diverse number of cellular and developmental processes such as synaptogenesis, dorsal closure in embryos, and viral uptake (5-7). Cdc42-mediated filopodial formation involves a number of proteins including IRSp53, Mena, Eps8, and N-WASP (8-11). A potential mechanism by which Cdc42 generates filopodia is by recruiting IRSp53, which then recruits Mena, Eps8, and N-WASP (11). IRSp53 can induce membrane deformation via its I-BAR 2,3 domain (12) and its SH3 domain

IEEE Transactions on Biomedical Engineering, 2012

Neural stem cells/neural progenitors (NSCs/NPs) are cells that give rise to the main cell types o... more Neural stem cells/neural progenitors (NSCs/NPs) are cells that give rise to the main cell types of the nervous system: oligodendrocytes, neurons, and astrocytes. Studying NSCs/NPs with time-lapse microscopy is critical to the understanding of the biology of these cells. However, NSCs/NPs are very sensitive to phototoxic damage, and therefore, fluorescent dyes cannot be used to follow these cells. Also, since in most of NSC/NP-related experiments, a large number of cells neesd to be monitored. Consequently, the acquisition of a huge amount of images is required. An additional difficulty is related to our original suspension living, tracking objective, behavior much closer to the natural, in vivo, way of development of the cells. Indeed, unlike adherent cells, suspension cells float freely in a liquid solution, thus, making their dynamics very different from that of adherent cells. As a result, existing visual tracking algorithms that have primarily been developed to track adherent cells are no longer adequate to tackle living cells in suspension. This paper presents a novel automated 3-D visual tracking of suspension living cells for time-lapse image acquisition using phase-contrast microscopy. This new tracking method can potentially strongly impact on current 3-D video microscopy methods, paving the way for innovative analysis of NSCs/NPs and as a result, on the study of neurodegenerative diseases.

FEBS Letters, 1984

The phosphorylation potential (AG,) in mitochondria has been found to be relatively insensitive t... more The phosphorylation potential (AG,) in mitochondria has been found to be relatively insensitive to changes in the protonmotive force (A@. Similar observations have been made for lactose transport in ~s~herjchia co& Localised chemiosmosis has been invoked to explain these data. Here a postulate of localised chemiosmosis was put to experimental test and found to be incorrect. Alternative explanations for the Apdependence of lactose transport are presented.

Cytometry Part A, 2009

The study of neuronal morphology and neurite outgrowth has been enhanced by the combination of im... more The study of neuronal morphology and neurite outgrowth has been enhanced by the combination of imaging informatics and high content screening, in which thousands of images are acquired using robotic fluorescent microscopy. To understand the process of neurite outgrowth in the context of neuroregeneration, we used mouse neuroblastoma N1E115 as our model neuronal cell. Six-thousand cellular images of four different culture conditions were acquired with two-channel widefield fluorescent microscopy. We developed a software package called NeuronCyto. It is a fully automatic solution for neurite length measurement and complexity analysis. A novel approach based on topological analysis is presented to segment cells. The detected nuclei were used as references to initialize the level set function. Merging and splitting of cells segments were prevented using dynamic watershed lines based on the constraint of topological dependence. A tracing algorithm was developed to automatically trace neurites and measure their lengths quantitatively on a cell-by-cell basis. NeuronCyto analyzes three important biologically relevant features, which are the length, branching complexity, and number of neurites. The application of NeuronCyto on the experiments of Toca-1 and serum starvation show that the transfection of Toca-1 cDNA induces longer neurites with more complexities than serum starvation.

Cancer Research, 2008

The failure of current glioma therapies is mainly due to the ability of the tumor cells to invade... more The failure of current glioma therapies is mainly due to the ability of the tumor cells to invade extensively the surrounding healthy brain tissue, hence escaping localized treatments. Neural stem cells (NSC) are able to home in on tumor foci at sites distant from the main tumor mass, possibly enabling treatment of scattered glioma clusters. To make the strategy more effective, we performed a cDNA expression library screening to identify the candidate genes that once overexpressed would enhance the tropism of NSCs for gliomas. Here, we show that a previously unannotated gene, the one encoding transmembrane protein 18 (TMEM18), is one such gene. Overexpression of TMEM18 was seen in the current study to provide NSCs and neural precursors an increased migration capacity toward glioblastoma cells in vitro and in the rat brain. Functional inactivation of the TMEM18 gene resulted in almost complete loss of the migration activity of these cells. Thus, TMEM18 is a novel cell migration modul...

Biophysical Journal, 2011

Biophysical Journal, 2009

The quantification of biological interactions is very important in life sciences. Here we report ... more The quantification of biological interactions is very important in life sciences. Here we report for the first time, to our knowledge, the determination of a biomolecular dissociation constant (K D) in living zebrafish embryos at physiological protein expression levels. For that purpose, we extend the application of single wavelength fluorescence cross-correlation spectroscopy into small organisms and measure the interaction of Cdc42, a small Rho-GTPase, and IQGAP1, an actin-binding scaffolding protein. Cdc42 and IQGAP1 were labeled with monomeric red fluorescent protein and enhanced green fluorescent protein, respectively. Both fluorophores were excited at a single wavelength of 514 nm, simplifying the fluorescence spectroscopy measurements and allowing quantification. For the determination of the interaction, we used two Cdc42 mutants, the constitutively active Cdc42 G12V which is in a predominantly GTP-bound form and the dominant-negative GDP-bound Cdc42 T17N. While Cdc42 G12V binds to IQGAP1 with an apparent K D of~100 nM, Cdc42 T17N has at least a one-order-of-magnitude lower affinity for the same protein. As a comparison, we measure the same protein-protein interactions in Chinese hamster ovary cell cultures but observe significant differences in protein mobility and K D from the zebrafish measurements, supporting the notion that bimolecular interactions depend on the biological system under investigation and are best performed under physiologically relevant conditions.

Biophysical Journal, 2012

Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissocia... more Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissociation constants (K d s) of biomolecules. The determination of a K d depends on the accurate measurement of the auto-and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of~0.5 or less for different FCCS schemes. We use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and determine the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation volumes for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Fö rster (or fluorescence) resonance energy transfer between the labels. We deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation volumes. We use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the K d .

pakentomol.com

In order to elaborate the feeding preference of Mythimna separata (Walk.), fifteen plants viz., c... more In order to elaborate the feeding preference of Mythimna separata (Walk.), fifteen plants viz., chulai (Amaranthus viridis), dib (Typha angustata), garlic (Allium sativum), gram (Cicer arientinum), khabbal (Cynodon dactylon), Korbooti (Euphorbia belioscopia), okra ...

Int. J. Agric. Biol, 2005

The LC50 values for malathion-resistant (PAK) and organophosphate-susceptible (FSS-II) strains of... more The LC50 values for malathion-resistant (PAK) and organophosphate-susceptible (FSS-II) strains of red flour beetle, Tribolium castaneum larvae were determined through residual film/filter paper impregnated method against six insecticides with novel modes of action such as abamectin (Sure 1.8 EC), spinosad (Tracer 240 SC), indoxacarb (Steward 150 SC), azadirachtin (Nimbokil 60 EC), buprofezin, and polychlorinated petroleum hydrocarbon (Tenekil 100 EC). The

Pak. Entomol, 2005

The efficacy of Khar Booti (Haloxylon recurvum), neem (Azadiracta indica) as first application on... more The efficacy of Khar Booti (Haloxylon recurvum), neem (Azadiracta indica) as first application on setts in furrow against termites in sugarcane was compared with chlorpyrifos (40 EC) and control. The botanicals were used in 1:20 in water. Overall mean of termites count ( ...

Communicative & Integrative Biology, 2010

Journal of Molecular Signaling, 2007

We have identified human ArhGAP9 as a novel MAP kinase docking protein that interacts with Erk2 a... more We have identified human ArhGAP9 as a novel MAP kinase docking protein that interacts with Erk2 and p38α through complementarily charged residues in the WW domain of ArhGAP9 and the CD domains of Erk2 and p38α. This interaction sequesters the MAP kinases in their inactive states through displacement of MAP kinase kinases targeting the same sites. While over-expression of wild type ArhGAP9 caused MAP kinase activation by the epidermal growth factor receptor (EGFR) to be suppressed and preserved the actin stress fibres in quiescent Swiss 3T3 fibroblasts, over-expression of an ArhGAP9 mutant defective in MAP kinase binding restored EGFR-induced MAP kinase activation and resulted in significant disruption of the stress fibres, consistent with the role of Erk activation in disassembly of actin stress fibres. The interaction between ArhGAP9 and the MAP kinases represents a novel mechanism of cross-talk between Rho GTPase and MAP kinase signaling.

Single Molecule Spectroscopy and Superresolution Imaging VII, 2014

ABSTRACT IRSp53 is a Cdc42 effector and a member of the Inverse-Bin-Amphiphysins-Rvs (I-BAR) doma... more ABSTRACT IRSp53 is a Cdc42 effector and a member of the Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain family which can induce negative membrane curvature. IRSp53 generates filopodia by coupling membrane protrusion (I-BAR domain) with actin dynamics through its SH3 domain binding partners. Dynamin 1 (Dyn1), a large GTPase associated with endocytosis, is a novel interacting partner of IRSp53 that localises to filopodia. Using rapid time-lapse TIRF microscopy we have shown that Dyn1 localized to a subcellular region just behind Mena at the leading edge, or in filopodial tip complexes when co-expressed with IRSp53. Dyn1-GFP was strongly localized in the filopodial shaft during the early phase of elongation, after which it moved rearward, suggestive of a role in early filopodia assembly. Mena and Eps8, accumulate at the tip complex in sequence and are involved in filopodial extension and retraction, respectively (Chou at al, 2014 submitted). Here we describe the use of dSTORM to investigate the molecular architecture of filopodia and in particular the size of the F-actin bundle in these structures. The data suggest that direct Stochastic Optical Reconstruction Microscopy (dSTORM) in combination with other techniques will allow the molecular architecture of

Stem Cells and Development, 2012

Neurospheres are widely used to propagate and investigate neural stem cells (NSCs) and neural pro... more Neurospheres are widely used to propagate and investigate neural stem cells (NSCs) and neural progenitors (NPs). However, the exact cell types present within neurospheres are still unknown. To identify cell types, we used single-cell mRNA profiling of 48 genes in 187 neurosphere cells. Using a clustering algorithm, we identified 3 discrete cell populations within neurospheres. One cell population [cluster unsorted (US) 1] expresses high Bmi1 and Hes5 and low Myc and Klf12. Cluster US2 shows intermediate expression of most of the genes analyzed. Cluster US3 expresses low Bmi1 and Hes5 and high Myc and Klf12. The mRNA profiles of these 3 cell populations correlate with a developmental timeline of early, intermediate, and late NPs, as seen in vivo from the mouse brain. We enriched the cell population for neurosphere-forming cells (NFCs) using morphological criteria of forward scatter (FSC) and side scatter (SSC). FSC/ SSC high cells generated 2.29-fold more neurospheres than FSC/SSC low cells at clonal density. FSC/SSC high cells were enriched for NSCs and Lewis-X + ve cells, possessed higher phosphacan levels, and were of a larger cell size. Clustering of both FSC/SSC high and FSC/SSC low cells identified an NFC cluster. Significantly, the mRNA profile of the NFC cluster drew close resemblance to that of early NPs. Taken together, data suggest that the neurosphere culture system can be used to model central nervous system development, and that early NPs are the cell population that gives rise to neurospheres. In future work, it may be possible to further dissect the NFCs and reveal the molecular signature for NSCs.

Journal of Biological Chemistry, 2009

The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downs... more The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downstream of membranebound receptors through distinct effector pathways. Cdc42-effector protein interactions represent important elements of cell signaling pathways that regulate cell biology in systems as diverse as yeast and humans. To derive mechanistic insights into cell signaling pathways, it is vital that we generate quantitative data from in vivo systems. We need to be able to measure parameters such as protein concentrations, rates of diffusion, and dissociation constants (K D) of protein-protein interactions in vivo. Here we show how single wavelength fluorescence cross-correlation spectroscopy in combination with Förster resonance energy transfer analysis can be used to determine K D of Cdc42effector interactions in live mammalian cells. Constructs encoding green fluorescent protein or monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain (CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP) and insulin receptor substrate protein (IRSp53), were expressed as pairs in Chinese hamster ovary cells, and concentrations of free protein as well as complexed protein were determined. The measured K D for Cdc42V12-N-WASP, Cdc42V12-CRIB, and Cdc42V12-IRSp53 was 27, 250, and 391 nM, respectively. The determination of K D for Cdc42-effector interactions opens the way to describe cell signaling pathways quantitatively in vivo in mammalian cells.

Journal of Biological Chemistry, 2009

The transducer of Cdc42-dependent actin assembly (Toca-1)-N-WASP complex was isolated as an essen... more The transducer of Cdc42-dependent actin assembly (Toca-1)-N-WASP complex was isolated as an essential cofactor for Cdc42-driven actin polymerization in vitro. Toca-1 consists of an N-terminal F-BAR domain, followed by a Cdc42 binding site (HR1 domain) and an SH3 domain, (the N-WASP interacting site). N-WASP is an activator of actin nucleation through the Arp2/3 complex. The aim of the present study was to investigate the cellular function of the Toca-1-N-WASP complex. We report that Toca-1 induces filopodia and neurites as does N-WASP in N1E115 neuroblastoma cells. Toca-1 requires the F-BAR domain, Cdc42 binding site, and SH3 domain to induce filopodia. Toca-1 and N-WASP both require each other to induce filopodia. The expression of Toca-1 and N-WASP affects the distribution, size, and number of Rab5 positive membranes. Toca-1 interacts directly with N-WASP in filopodia and Rab5 membrane as seen by Forster resonance energy transfer. Thus the Toca-1-N-WASP complex localizes to and induces the formation of filopodia and endocytic vesicles. Last, three inhibitors of endocytosis, Dynamin-K44A, Eps15⌬95/295, and clathrin heavy chain RNA interference, block Toca-1-induced filopodial formation. Taken together, these data suggest that the Toca-1-N-WASP complex can link filopodial formation to endocytosis. Cell migration and axon guidance rely on morphological actin-based structures such as filopodia and lamellipodia/ membrane ruffles (1). Cdc42 is a member of the Rho family of GTPases and plays a prominent role in cell signaling pathways that control cell migration as well as cell shape and polarity. Cdc42 was first identified as a yeast protein involved in cell division that affects budding of daughter cells (2). In mammalian cells, one specific function of Cdc42 is to activate filopodial formation (3, 4). Filopodia are dynamic actin-based structures that protrude from the plasma membrane and are thought to play a role in a diverse number of cellular and developmental processes such as synaptogenesis, dorsal closure in embryos, and viral uptake (5-7). Cdc42-mediated filopodial formation involves a number of proteins including IRSp53, Mena, Eps8, and N-WASP (8-11). A potential mechanism by which Cdc42 generates filopodia is by recruiting IRSp53, which then recruits Mena, Eps8, and N-WASP (11). IRSp53 can induce membrane deformation via its I-BAR 2,3 domain (12) and its SH3 domain

IEEE Transactions on Biomedical Engineering, 2012

Neural stem cells/neural progenitors (NSCs/NPs) are cells that give rise to the main cell types o... more Neural stem cells/neural progenitors (NSCs/NPs) are cells that give rise to the main cell types of the nervous system: oligodendrocytes, neurons, and astrocytes. Studying NSCs/NPs with time-lapse microscopy is critical to the understanding of the biology of these cells. However, NSCs/NPs are very sensitive to phototoxic damage, and therefore, fluorescent dyes cannot be used to follow these cells. Also, since in most of NSC/NP-related experiments, a large number of cells neesd to be monitored. Consequently, the acquisition of a huge amount of images is required. An additional difficulty is related to our original suspension living, tracking objective, behavior much closer to the natural, in vivo, way of development of the cells. Indeed, unlike adherent cells, suspension cells float freely in a liquid solution, thus, making their dynamics very different from that of adherent cells. As a result, existing visual tracking algorithms that have primarily been developed to track adherent cells are no longer adequate to tackle living cells in suspension. This paper presents a novel automated 3-D visual tracking of suspension living cells for time-lapse image acquisition using phase-contrast microscopy. This new tracking method can potentially strongly impact on current 3-D video microscopy methods, paving the way for innovative analysis of NSCs/NPs and as a result, on the study of neurodegenerative diseases.

FEBS Letters, 1984

The phosphorylation potential (AG,) in mitochondria has been found to be relatively insensitive t... more The phosphorylation potential (AG,) in mitochondria has been found to be relatively insensitive to changes in the protonmotive force (A@. Similar observations have been made for lactose transport in ~s~herjchia co& Localised chemiosmosis has been invoked to explain these data. Here a postulate of localised chemiosmosis was put to experimental test and found to be incorrect. Alternative explanations for the Apdependence of lactose transport are presented.

Cytometry Part A, 2009

The study of neuronal morphology and neurite outgrowth has been enhanced by the combination of im... more The study of neuronal morphology and neurite outgrowth has been enhanced by the combination of imaging informatics and high content screening, in which thousands of images are acquired using robotic fluorescent microscopy. To understand the process of neurite outgrowth in the context of neuroregeneration, we used mouse neuroblastoma N1E115 as our model neuronal cell. Six-thousand cellular images of four different culture conditions were acquired with two-channel widefield fluorescent microscopy. We developed a software package called NeuronCyto. It is a fully automatic solution for neurite length measurement and complexity analysis. A novel approach based on topological analysis is presented to segment cells. The detected nuclei were used as references to initialize the level set function. Merging and splitting of cells segments were prevented using dynamic watershed lines based on the constraint of topological dependence. A tracing algorithm was developed to automatically trace neurites and measure their lengths quantitatively on a cell-by-cell basis. NeuronCyto analyzes three important biologically relevant features, which are the length, branching complexity, and number of neurites. The application of NeuronCyto on the experiments of Toca-1 and serum starvation show that the transfection of Toca-1 cDNA induces longer neurites with more complexities than serum starvation.

Cancer Research, 2008

The failure of current glioma therapies is mainly due to the ability of the tumor cells to invade... more The failure of current glioma therapies is mainly due to the ability of the tumor cells to invade extensively the surrounding healthy brain tissue, hence escaping localized treatments. Neural stem cells (NSC) are able to home in on tumor foci at sites distant from the main tumor mass, possibly enabling treatment of scattered glioma clusters. To make the strategy more effective, we performed a cDNA expression library screening to identify the candidate genes that once overexpressed would enhance the tropism of NSCs for gliomas. Here, we show that a previously unannotated gene, the one encoding transmembrane protein 18 (TMEM18), is one such gene. Overexpression of TMEM18 was seen in the current study to provide NSCs and neural precursors an increased migration capacity toward glioblastoma cells in vitro and in the rat brain. Functional inactivation of the TMEM18 gene resulted in almost complete loss of the migration activity of these cells. Thus, TMEM18 is a novel cell migration modul...

Biophysical Journal, 2011

Biophysical Journal, 2009

The quantification of biological interactions is very important in life sciences. Here we report ... more The quantification of biological interactions is very important in life sciences. Here we report for the first time, to our knowledge, the determination of a biomolecular dissociation constant (K D) in living zebrafish embryos at physiological protein expression levels. For that purpose, we extend the application of single wavelength fluorescence cross-correlation spectroscopy into small organisms and measure the interaction of Cdc42, a small Rho-GTPase, and IQGAP1, an actin-binding scaffolding protein. Cdc42 and IQGAP1 were labeled with monomeric red fluorescent protein and enhanced green fluorescent protein, respectively. Both fluorophores were excited at a single wavelength of 514 nm, simplifying the fluorescence spectroscopy measurements and allowing quantification. For the determination of the interaction, we used two Cdc42 mutants, the constitutively active Cdc42 G12V which is in a predominantly GTP-bound form and the dominant-negative GDP-bound Cdc42 T17N. While Cdc42 G12V binds to IQGAP1 with an apparent K D of~100 nM, Cdc42 T17N has at least a one-order-of-magnitude lower affinity for the same protein. As a comparison, we measure the same protein-protein interactions in Chinese hamster ovary cell cultures but observe significant differences in protein mobility and K D from the zebrafish measurements, supporting the notion that bimolecular interactions depend on the biological system under investigation and are best performed under physiologically relevant conditions.

Biophysical Journal, 2012

Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissocia... more Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissociation constants (K d s) of biomolecules. The determination of a K d depends on the accurate measurement of the auto-and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of~0.5 or less for different FCCS schemes. We use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and determine the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation volumes for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Fö rster (or fluorescence) resonance energy transfer between the labels. We deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation volumes. We use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the K d .

pakentomol.com

In order to elaborate the feeding preference of Mythimna separata (Walk.), fifteen plants viz., c... more In order to elaborate the feeding preference of Mythimna separata (Walk.), fifteen plants viz., chulai (Amaranthus viridis), dib (Typha angustata), garlic (Allium sativum), gram (Cicer arientinum), khabbal (Cynodon dactylon), Korbooti (Euphorbia belioscopia), okra ...

Int. J. Agric. Biol, 2005

The LC50 values for malathion-resistant (PAK) and organophosphate-susceptible (FSS-II) strains of... more The LC50 values for malathion-resistant (PAK) and organophosphate-susceptible (FSS-II) strains of red flour beetle, Tribolium castaneum larvae were determined through residual film/filter paper impregnated method against six insecticides with novel modes of action such as abamectin (Sure 1.8 EC), spinosad (Tracer 240 SC), indoxacarb (Steward 150 SC), azadirachtin (Nimbokil 60 EC), buprofezin, and polychlorinated petroleum hydrocarbon (Tenekil 100 EC). The

Pak. Entomol, 2005

The efficacy of Khar Booti (Haloxylon recurvum), neem (Azadiracta indica) as first application on... more The efficacy of Khar Booti (Haloxylon recurvum), neem (Azadiracta indica) as first application on setts in furrow against termites in sugarcane was compared with chlorpyrifos (40 EC) and control. The botanicals were used in 1:20 in water. Overall mean of termites count ( ...