Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-mediated adhesion we have investigated homotypic ALCAM-ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesion because inhibition of actin polymerization by cytochalasin D (CytD) strongly induces homotypic ALCAM-ALCAM interactions. This induction of cell adhesion is likely due to clustering of ALCAM at the cell surface, which is observed after CytD treatment. Single-particle tracking demonstrated that the lateral mobility of ALCAM in the cell membrane is increased 30-fold after CytD treatment. In contrast, both surface distribution and adhesion of a glycosylphosphatidylinositol (GPI)-anchored ALCAM mutant are insensitive to CytD, despite the increase in lateral mobility of GPI-ALCAM upon CytD treatment. This demonstrates that clustering of ALCAM is essential for cell adhesion, whereas enhanced diffusion of ALCAM alone is not sufficient for cluster formation. In addition, upon ligand binding, both free diffusion and the freely dragged distance of wild-type ALCAM, but not of GPI-ALCAM, are reduced over time, suggesting strengthening of the cytoskeleton linkage. From these findings we conclude that activation of ALCAM-mediated adhesion is dynamically regulated through actin cytoskeleton-dependent clustering.

We have developed a method for three dimensional (3D) tracking of polystyrene spheres with nanometer resolution. The detection technique is based on measuring the displacement of a polystyrene sphere positioned in the center of a laser beam just behind the focus. A change in the lateral position of the sphere causes a deflection of the beam which can be measured using a position sensitive detector. A change in the axial position of the sphere causes a shift in the axial position of the focus behind another lens, which can be measured using an overfilled photodiode. A feedback system is used to keep the sphere in the center of the laser beam to avoid the influence of lateral displacements on the detection of the axial position. Spatial resolution for a 0.92 μm polystyrene sphere was better than 1 nm in three dimensions using a sampling rate of 1 kHz. This method was applied to track spheres bound to adhesion molecules LFA-1 expressed at the surface of living cells. It turned out to be a useful method to accurately measure the 3D trajectory of biological molecules on cells in real time.

A three‐dimensional single‐particle tracking system was combined with an optical trap to investigate the behavior of transmembrane adhesion proteins. We exploited this setup to investigate which part of the cell adhesion protein LFA‐1 forms a connection to the cytoskeleton after binding to its ligand ICAM‐1. LFA‐1 is an integrin consisting of an α and a β chain. Thus far, only the cytoplasmic tail of the β chain is known to form a connection to the cytoskeleton. We investigated cells that express a mutant form of LFA‐1 that lacks the complete β cytoplasmic tail and therefore is not thought to bind to the cytoskeleton. Interestingly, single‐particle tracking measurements using beads coated with the ligand ICAM‐1 indicate that this mutant form of LFA‐1 does not move freely within the cell membrane, suggesting that LFA‐1 is still connected to the cytoskeleton network. This finding is strongly supported by the observation that LFA‐1 exhibits a more diffusive motion when the cytoskeleton network is disrupted and confirmed by the optical trap measurements used to force the proteins to move through the membrane. Collectively, our findings suggest that the interaction of LFA‐1 with the cytoskeleton cannot solely be attributed to the cytoplasmic part of the β chain. Cytometry 36:189–194, 1999. © 1999 Wiley‐Liss, Inc.

Abstract.We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

A three-dimensional single-particle tracking system was combined with an optical trap to investigate the behavior of transmembrane adhesion proteins. We exploited this setup to investigate which part of the cell adhesion protein LFA-1 forms a connection to the cytoskeleton after binding to its ligand ICAM-1. LFA-1 is an integrin consisting of an and a chain. Thus far, only the cytoplasmic tail of the chain is known to form a connection to the cytoskeleton. We investigated cells that express a mutant form of LFA-1 that lacks the complete cytoplasmic tail and therefore is not thought to bind to the cytoskeleton. Interestingly, single-particle tracking measurements using beads coated with the ligand ICAM-1 indicate that this mutant form of LFA-1 does not move freely within the cell membrane, suggesting that LFA-1 is still connected to the cytoskeleton network. This finding is strongly supported by the observation that LFA-1 exhibits a more diffusive motion when the cytoskeleton network is disrupted and confirmed by the optical trap measurements used to force the proteins to move through the membrane. Collectively, our findings suggest that the interaction of LFA-1 with the cytoskeleton cannot solely be attributed to the cytoplasmic part of the chain.