Biomedical subjects

Lingsong Dai

Publications and source records attributed to Lingsong Dai.

3 recordsLinked to original sources

PubMed2005

A fast and robust quantitative time-lapse assay for cell migration.

We describe a simple and widely applicable method to measure cell migration in time-lapse sequences of fluorescently labeled cells in culture. Briefly, binarized cell images obtained after thresholding were cumulatively projected, and the covered areas were measured. This procedure determines the time course of the track area successively covered by the cell population. Under conditions where cell growth is negligible, a robust index of cell motility is derived from normalized plots for the displacement of cells over time. We applied this method to quantitatively examine the migration of B35 neuroblastoma cells transiently expressing GFP and to C6 glioma cells after staining with Hoechst 33258. This sensitive assay detected the influence of agents which inhibit actin polymerization (cytochalasin B) or interfere with the maintenance of cell polarity (methyl-beta-cyclodextrin) on cell migration. Thus, this assay is a versatile tool to measure quickly the migration of different cell types using different labeling strategies.

Animals↗

PubMed2005

Collapsin response mediator protein-4 regulates F-actin bundling.

Collapsin response mediator proteins (CRMPs) form a family of cytosolic phosphoproteins which are involved in the signal transduction of semaphorin 3A leading to growth cone collapse. These proteins interact with a variety of cytosolic proteins including tubulin heterodimers. Here, we show that CRMP-4 co-localizes with F-actin in regular rib-like structures within lamellipodia of B35 neuroblastoma cells. Furthermore, depolymerization of actin fibers changed the distribution of GFP-CRMP-4 in vivo. In vitro, recombinant CRMP-4 formed homo-oligomers, bound to F-actin and organized F-actin into tight bundles. Both oligomerization and F-actin bundling depended on the C-terminal part of CRMP-4. The stoichiometry of actin and CRMP-4 in bundles was approximately 1:1 and the apparent equilibrium constant of the microfilament-CRMP-4 interaction was estimated from bundling assays as K(app) = 730 mM(-1). CRMP-4 was abundant in the cytosol of B35 neuroblastoma cells and its concentration was measured as approximately 1.7 microM. Overexpression of CRMP-4 inhibited the migration of B35 neuroblastoma cells, while knockdown of CRMP-4 enhanced cell migration and disturbed rib-like actin-structures in lamellipodia. Taken together, our data indicate that CRMP-4 promotes bundling of F-actin in vitro, that it is an important component of rib-like actin bundles in lamellipodia in vivo and that it functionally regulates the actin cytoskeleton in motile cells. These findings suggest a specific regulatory role of CRMP-4 towards the actin cytoskeleton which may by be relevant for growth cone collapse.

Actins↗

PubMed2003

Subcellular localization of collapsin response mediator proteins to lipid rafts.

Collapsin response mediator proteins (CRMPs) are involved in signal transduction after exposure of neural cells to the axon guidance molecule Semaphorin 3A/collapsin. All five known CRMPs are expressed in the developing cerebral cortex and neocortical neurons are responsive to Semaphorin 3A. Here, we examine the expression and subcellular localization of CRMPs in neocortical neurons and in neonatal rat brain. In neocortical neurons CRMP-4 was detected in the perikaryon with a diffuse cytosolic distribution. In neurites and at growth cones punctate staining patterns were observed. Extraction of neuron cultures with methyl-beta-cyclodextrin to deplete cholesterol caused rapid redistribution of the punctate CRMP-4 staining into larger patches and abundant growth cone collapse. Western blotting of brain extracts demonstrated for all CRMPs the existence of soluble, detergent-extractable, and Triton X-100-resistant forms. Furthermore, sucrose density gradient centrifugation after solubilization of brain membranes with Triton X-100 revealed that CRMP-1, -3, -5, and to a lower extent CRMP-4 are associated with a detergent-resistant fraction with low buoyant density, but CRMP-2 was not detectable in this fraction. Thus, we propose that lipid rafts form sites for the compartmentalization of signaling events involving specific CRMPs and that the integrity of these membrane microdomains is essential for the maintenance of growth cones.

Actin Cytoskeleton↗