Label-Free Rapid Separation and Enrichment of Bone Marrow-Derived Mesenchymal Stem Cells from a Heterogeneous Cell Mixture Using a Dielectrophoresis Device.

Bone marrow-derived mesenchymal stem cells (BMSCs) are an important cell resource for stem cell-based therapy, which are generally isolated and enriched by the density-gradient method based on cell size and density after collection of tissue samples. Since this method has limitations with regards to purity and repeatability, development of alternative label-free methods for BMSC separation is desired. In the present study, rapid label-free separation and enrichment of BMSCs from a heterogeneous cell mixture with bone marrow-derived promyelocytes was successfully achieved using a dielectrophoresis (DEP) device comprising saw-shaped electrodes. Upon application of an electric field, HL-60 cells as models of promyelocytes aggregated and floated between the saw-shaped electrodes, while UE7T-13 cells as models of BMSCs were effectively captured on the tips of the saw-shaped electrodes. After washing out the HL-60 cells from the device selectively, the purity of the UE7T-13 cells was increased from 33% to 83.5% within 5 min. Although further experiments and optimization are required, these results show the potential of the DEP device as a label-free rapid cell isolation system yielding high purity for rare and precious cells such as BMSCs.

A modified FCCS procedure applied to Ly49A-MHC class I cis-interaction studies in cell membranes.

The activity of natural killer (NK) cells is regulated by a fine-tuned balance between activating and inhibitory receptors. Dual-color fluorescence cross-correlation spectroscopy (FCCS) was used to directly demonstrate a so-called cis-interaction between a member of the inhibitory NK cell receptor family Ly49 (Ly49A), and its ligand, the major histocompatibility complex (MHC) class I, within the plasma membrane of the same cell. By a refined FCCS model, calibrated by positive and negative control experiments on cells from the same lymphoid cell line, concentrations and diffusion coefficients of free and interacting proteins could be determined on a collection of cells. Using the intrinsic intercellular variation of their expression levels for titration, it was found that the fraction of Ly49A receptors bound in cis increase with increasing amounts of MHC class I ligand. This increase shows a tendency to be more abrupt than for a diffusion limited - three dimensional bimolecular reaction, which most likely reflects the two-dimensional confinement of the reaction. For the Ly49A- MHC class I interaction it indicates that within a critical concentration range the local concentration level of MHC class I can provide a distinct regulation mechanism of the NK cell activity.

Multipoint fluorescence correlation spectroscopy with total internal reflection fluorescence microscope.

We report simultaneous determination of diffusion coefficients at different points of a cell membrane using a multipoint fluorescence correlation spectroscopy (FCS) system. A system carrying seven detection areas in the evanescent field is achieved by using seven optical fibers on the image plane in the detection port of an objective-type total internal reflection FCS (TIR-FCS) system. Fluctuation of fluorescence intensity is monitored and evaluated using seven photomultiplier tubes (PMTs) and a newly constructed multichannel correlator. We demonstrate simultaneous-multipoint FCS, with a 3-mus time resolution, to investigate heterogeneous structures such as cell membranes and membrane-binding molecular dynamics near glass surfaces in live cells.

The novel cargo Alcadein induces vesicle association of kinesin-1 motor components and activates axonal transport.

Alcadeinalpha (Alcalpha) is an evolutionarily conserved type I membrane protein expressed in neurons. We show here that Alcalpha strongly associates with kinesin light chain (K(D) approximately 4-8x10(-9) M) through a novel tryptophan- and aspartic acid-containing sequence. Alcalpha can induce kinesin-1 association with vesicles and functions as a novel cargo in axonal anterograde transport. JNK-interacting protein 1 (JIP1), an adaptor protein for kinesin-1, perturbs the transport of Alcalpha, and the kinesin-1 motor complex dissociates from Alcalpha-containing vesicles in a JIP1 concentration-dependent manner. Alcalpha-containing vesicles were transported with a velocity different from that of amyloid beta-protein precursor (APP)-containing vesicles, which are transported by the same kinesin-1 motor. Alcalpha- and APP-containing vesicles comprised mostly separate populations in axons in vivo. Interactions of Alcalpha with kinesin-1 blocked transport of APP-containing vesicles and increased beta-amyloid generation. Inappropriate interactions of Alc- and APP-containing vesicles with kinesin-1 may promote aberrant APP metabolism in Alzheimer's disease.

Lateral mobility of membrane-binding proteins in living cells measured by total internal reflection fluorescence correlation spectroscopy.

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) allows us to measure diffusion constants and the number of fluorescent molecules in a small area of an evanescent field generated on the objective of a microscope. The application of TIR-FCS makes possible the characterization of reversible association and dissociation rates between fluorescent ligands and their receptors in supported phospholipid bilayers. Here, for the first time, we extend TIR-FCS to a cellular application for measuring the lateral diffusion of a membrane-binding fluorescent protein, farnesylated EGFP, on the plasma membranes of cultured HeLa and COS7 cells. We detected two kinds of diffusional motion-fast three-dimensional diffusion (D(1)) and much slower two-dimensional diffusion (D(2)), simultaneously. Conventional FCS and single-molecule tracking confirmed that D(1) was free diffusion of farnesylated EGFP close to the plasma membrane in cytosol and D(2) was lateral diffusion in the plasma membrane. These results suggest that TIR-FCS is a powerful technique to monitor movement of membrane-localized molecules and membrane dynamics in living cells.