Real-time Cell Adhesion Research Articles

Adhesion of eukaryotic cell lines on the gold surface modified with extracellular matrix proteins monitored by the piezoelectric sensor

The piezoelectric sensor (quartz crystal microbalance, QCM) was used to monitor cell adhesion in real time. Two cell lines, rat epithelial cells (WB F344) and lung melanoma cells (B16F10) were used. The cells were adhered and grown on the gold surface of the sensor pre-coated with adsorbed layer of extracellular matrix proteins as vitronectin and laminin. The process of cell attachment and spreading on the gold surface was continuously monitored and displayed by changes of the resonant frequency Δ f and resistance Δ R values of the piezoelectric resonators. The initial phase of cell attachment and spreading induced a decrease of frequency and increase of resistance relating viscoelastic properties of the cell monolayer on the sensing surface. The steady-state of both shifts was achieved after a few hours. The presence and state of cells on the surface was confirmed by fluorescent microscopy. The obtained results demonstrate that the piezoelectric sensor is suitable for studies of the cell adhesion processes. Thus obtained cell-based biosensor has potential for identification and screening of biologically active drugs and other biomolecules affecting cellular shape and attachment.

Mice Lacking PIP5Kβ or PIP5Kγ Have Unique Cytoskeletal Changes within Their Megakaryocytes & Platelets.

Phosphatidylinositol-4, 5-bisphosphate (PIP2) is vital for the signaling cascades that are critical for actin dynamics. Although all PIP5K isoforms (α , β , and γ ) synthesize PIP2 by phosphorylating PI4P, the isoforms have different primary structures, expression levels in various tissues, and intracellular localization. To test the hypothesis that the functions of these isoforms are also different, we have generated murine lines that contain null mutations in either of two platelet PIP5K isoforms, PIP5Kβ and PIP5Kγ . PIP5Kβ -null mice were born 42% less than anticipated by Mendelian genetics, and heterozygotes were born 18% less than the expected frequency. Adult PIP5Kβ −/− mice had normal platelet counts and exhibited no spontaneous hemorrhage. However, PIP5Kβ −/− platelets had impaired aggregation in response to thrombin and thromboxane analogues. In addition, following agonist stimulation they failed to properly synthesize additional PIP2. Platelets lacking PIP5Kβ also exhibited a marked defect in spreading upon immobilized fibrinogen. This failure to spread was associated with a striking decrease in filopodia formation and extension of actin rich lamellipodia. In contrast to the phenotype of PIP5Kβ -knockout mice, we found that targeted disruption of PIP5Kγ results in early prenatal mortality due to a cardiovascular developmental defect. This early lethality prevented studies of hematopoietic cells derived from the bone marrow or the liver. However, we were able to analyze yolk sac progenitor cells that were treated with thrombopoietin ex vivo, differentiating them into megakaryocytes. Similar to PIP5Kβ knockout megakaryocytes, PIP5Kγ knockout megakaryocytes had normal basal levels of PIP2, but decreased synthesis following stimulation with thrombin. Using spinning disk video confocal microscopy, we examined membrane dynamics during cell adhesion in real time. Wild type megakaryocytes actively formed and contracted lamellipodia, and rapidly spread upon the fibrinogen matrix. In contrast, PIP5Kγ -null megakaryocytes continuously extended and retracted membrane blebs, rather than lamellipodia, but eventually spread as much as wild type cells. This observation is consistent with the previous suggestion that PIP2 contributes to the stable association of the membrane with the cytoskeleton. Using laser tweezers to pull the cell membrane apart from the cytoskeleton, we found that long tethers of the membrane could easily be drawn from PIP5Kγ −/− megakaryocytes, but not from PIP5Kγ +/− or PIP5Kβ −/− megakaryocytes. This implies that PIP5Kγ −/− megakaryocytes have a defect in their ability to anchor the cell membrane to the cytoskeleton. This defect was attributable to the ability of PIP5Kγ to synthesize PIP2 since it could be rescued by adding back wild type PIP5Kγ , but could not be rescued by a catalytically inactive PIP5Kγ mutant. Accordingly, our data are consistent with the hypothesis that different PIP5K isoforms contribute to compartmentalized pools of PIP2 that contribute to different aspect of platelet &megakaryocyte actin dynamics. PIP5Kβ synthesizes PIP2 that contributes to the formation of filopodia and lamellipodia, while PIP5Kγ generates PIP2 that is required for the stable association of the membrane with the cytoskeleton.

PIP5Kγ-Null Mutation Induces Cytoskeletal Changes within Megakaryocytes.

All eukaryotic cells contain the phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2) that serves multiple roles in signaling cascades critical for actin dynamics. Phosphatidylinositol-4-phosphate 5 kinase (PIP5K) catalyzes the synthesis of PIP2 by phosphorylating PI4P. Although the classical PIP5K isoforms (α, β, and γ) generate the same phospholipid product, the isoforms have different primary structures, are expressed at different levels in various tissues, and localize in different intracellular compartments. Therefore, it is likely that the functions of these isoforms are different as well. PIP5Kγ is unique because it associates directly with talin and consequently localizes in focal adhesion. Given the role of PIP2 in actin dynamics, we generated a murine line containing a null mutation in the PIP5Kγ gene. We found that the targeted disruption of PIP5Kγ results in early prenatal mortality that is associated with pleotropic effects involving the cardiovascular, neurologic, and hematopoietic systems. This early lethality prevented studies of hematopoietic cells derived from the bone marrow or the liver. However, we were able to analyze yolk sac progenitor cells that were treated with thrombopoietin ex vivo, differentiating them into megakaryocytes. After 5 days in culture, many of the non-adherent yolk sac progenitor cells were multinucleated, and approximately 70% expressed CD41 (αIIb), a marker of the megakaryocyte lineage. We then examined their cytoskeletal content and dynamics. Absence of PIP5Kγ had no effect on the quantity of F-actin in unstimulated or thrombin-stimulated cells that were adherent to fibrinogen or were maintained in suspension in the absence of stimulation. Next, we examined membrane dynamics during cell adhesion in real time using spinning disk video confocal microscopy. Wild type megakaryocytes actively formed and contracted lamellipodia and rapidly spread upon the fibrinogen matrix. In contrast, PIP5Kγ-null megakaryocytes continuously extended and retracted membrane blebs, rather than lamellipodia, but eventually spread as much as wild type cells. This observation is consistent with the previous suggestion that PIP2 contributes to the stable association of the membrane with the cytoskeleton. It is also noteworthy that a similar phenotype has been described in cells with defective anchoring of the membrane to the cytoskeleton because of a lack of filamin. Accordingly, our data are consistent with the hypothesis that PIP5Kγ generates compartmentalized pools of PIP2 that contribute to the association of the membrane with the cytoskeleton.

Biophysical methods for monitoring cell-substrate interactions in drug discovery.

Cell-substrate interactions are implicated in a number of relevant pathways for drug targets such as angiogenesis, arteriosclerosis, chronic inflammatory diseases, and carcinogenesis. Moreover, cell adhesion and cytoskeletal activity have served as valuable indicators for cytotoxicity, cell density, and cell morphology. This review focuses on impedance, capacitance, resonant frequency, and refractive index measurements for monitoring cell adhesion in real time and without the use of cell labeling. ECIS, QCM, and OWLS deliver information about the cell-substrate interactions, cell-cell contact, and the strength of cell adhesion. Because of high sensitivity of these assays, events down to the single cell level have been observed, and resolutions at the nanometer level of cell-substrate distances have been achieved. The physical principles, including assay sensitivity and selectivity, are discussed in the context of cellular pathways of cell adhesion and migration. With the miniaturization of these types of sensors, cell migration and adhesion measurements in combination with specific fluorescent assays might thus deliver a high-content platform for drug development.

Endothelial cell adhesion in real time. Measurements in vitro by tandem scanning confocal image analysis.

Real time measurements of cell-substratum adhesion in endothelial cells were obtained by tandem scanning confocal microscopy of sites of focal contact (focal adhesions) at the abluminal cell surface. Focal contact sites were sharply defined (low radiance levels) in the living cell such that the images could be enhanced, digitized, and isolated from other cellular detail. Sites of focal contact are the principal determinant of cell-substratum adhesion. Measurements of (a) the focal contact area and (b) the closeness of contact (inverse radiance) were used to nominally define the adhesion of a single cell or field of cells, and to record spontaneous and induced changes of cell adhesion in real time. The topography of focal contacts was estimated by calculating separation distances from radiance values using a calibration technique based on interference ring optics. While slightly closer contact was noted between the cell membrane and substratum at or near the center of each focal contact, separation distances throughout the adhesion regions were always < 50 nm. Subtraction of consecutive images revealed continuous spontaneous remodeling of individual focal adhesions in unperturbed cells during periods of < 1 min. Despite extensive remodeling of focal contact sites, however, cell adhesion calculated for an entire cell over extended periods varied by < 10%. When cytoskeletal stability was impaired by exposure to cytochalasin or when cells were exposed to proteolytic enzyme, endothelial adhesion declined rapidly. Such changes were recorded at the level of single cells, groups of cells, and at single focal adhesions. In both unperturbed and manipulated cells, the dynamics of remodeling and cell adhesion characteristics varied greatly between individual sites within the same cell; disappearance of existing sites and appearance of new ones often occurred within minutes while adjacent sites underwent minimal remodelling. Tandem scanning confocal microscopy image analysis of living cells in real time provides repetitive spatial, temporal, and quantitative information about cell adhesion. Such an approach should allow more precise quantitative analyses to be made of the interactions between extracellular matrix, adhesion proteins, integrins, and the cytoskeleton in the living cell.