Altered membrane dynamics of quantum dot-conjugated integrins during osteogenic differentiation of human bone marrow derived progenitor cells

Abstract

Functionalized quantum dots offer several advantages for tracking the motion of individual molecules on the cell surface, including selective binding, precise optical identification of cell surface molecules, and detailed examination of the molecular motion without photobleaching. We have used quantum dots conjugated with integrin antibodies and performed studies to quantitatively demonstrate changes in the integrin dynamics during osteogenic differentiation of human bone marrow derived progenitor cells (BMPCs). Consistent with the unusually strong BMPC adhesion previously observed, integrins on the surface of undifferentiated BMPC were found in clusters and the lateral diffusion was slow (e.g., approximately 10(-11) cm2/s). At times as early as those after a 3-day incubation in the osteogenic differentiation media, the integrin diffusion coefficients increased by an order of magnitude, and the integrin dynamics became indistinguishable from that measured on the surface of terminally differentiated human osteoblasts. Furthermore, microfilaments in BMPCs consisted of atypically thick bundles of stress fibers that were responsible for restricting the integrin lateral mobility. Studies using laser optical tweezers showed that, unlike fully differentiated osteoblasts, the BMPC cytoskeleton is weakly associated with its cell membrane. Based on these findings, it appears likely that the altered integrin dynamics is correlated with BMPC differentiation and that the integrin lateral mobility is restricted by direct links to microfilaments.

FIGURE 1

Confocal fluorescence images of integrin distribution on the surface of human osteoblast (left panel) and BMPC (right panel). Integrins were visualized using FITC-conjugated anti-CD49d antibodies. Integrins on the osteoblast were uniformly distributed. In contrast, integrins on the BMPC were found to cluster in punctates. Images were recorded using a 60×/NA = 1.4 microscope objective.

FIGURE 2

Composite overlay of DIC and fluorescence images. A typical DIC image of the BMPC was superimposed with a fluorescence image of integrins visualized using conjugated quantum dots (red) indicated by arrows. Optimization of antibody dilution (10⁻³ mg/ml) and quantum dot concentration (0.1 nM) resulted in labeling <20 integrin molecules per cell. Images were recorded using a 100×/NA = 1.4 microscope objective.

FIGURE 3

Typical integrin tracking experiment. Quantum dot-conjugated integrins were tracked for 30 s at 150-ms intervals on the cell surface. Representative trajectories of integrins monitored on the surface of human osteoblast (A) and BMPC (B) show that the integrin executed a confined movement in the terminally differentiated osteoblast (C, diamonds) but was found laterally immobile in BMPC (C, triangles). The first four data points in the integrin-confined movement were used to calculate the microdiffusion coefficient reported in this work (solid line).

FIGURE 4

Histograms of integrin diffusion coefficients. After incubation of osteoblasts in the normal growth media for 1, 3, 7, and 14 days, the integrin diffusion coefficients were measured and histograms were constructed. To construct a histogram, at least 20 quantum dot-conjugated integrins from 3–5 osteoblasts and at least 50 integrins from 30 BMPCs were monitored, and their diffusion coefficients were determined as described in the Materials and Methods section. Histograms of integrin diffusions on human osteoblasts or BMPCs were constructed at early differentiation stages (e.g., <3 days; (A)) and at later stages (e.g., >7 days; (B)). For osteoblasts, the integrin diffusion coefficients did not statistically differ between the values determined at day 1 with those at day 3 (p = 0.59), day 7 (p = 0.85), and day 14 (p = 0.38). For BMPCs, the integrin diffusion coefficients became significantly greater after a 3-day incubation in the osteogenic media (p < 0.01) but did not further increase upon longer incubation (e.g., 7 or 14 days).

FIGURE 5

Effect of osteogenic factors on integrin diffusion. Human osteoblasts were incubated for 3 days in the osteogenic media, and the integrin diffusion coefficients were measured (A). Similarly, BMPCs were incubated for 3 days in the normal growth media, and the integrin diffusion coefficients were measured (B). These treatments did not affect or alter the integrin diffusion characteristics. The osteogenic factors do not have direct effects on the integrin diffusion. Each histogram represents data for 20–50 individual quantum dots tracked from five osteoblasts and 10 BMPCs.

FIGURE 6

Actin cytoskeleton organization in osteoblasts (A) and BMPCs (B). BMPCs are typically bigger in size than osteoblasts and contain thicker actin stress fibers with no noticeable actin network that can be seen in osteoblasts. When microfilaments were disrupted using cytochalasin D, the integrins were found to diffuse more rapidly both in osteoblasts (C) and BMPCs (D). These two histograms of the integrin diffusion coefficients are statistically indistinguishable (p = 0.83). However, when compared to the control, the diffusion coefficient distributions were statistically different (p = 0.02 for osteoblasts and p < 0.01 for BMPCs). Each histogram represents data for at least 20 individual quantum dots tracked from 3–5 cells at day 1.

FIGURE 7

Membrane tethers extracted from osteoblasts (A) and BMPCs (B). Fluorescent beads (0.5-μm diameter) were attached to the cell membrane and pulled away from the cell by LOT. The thin membrane tethers extending from the beads to the cell body (white arrows) appear as faint shadows in the superimposed brightfield/fluorescence images.

FIGURE 8

Tether extraction experiment. The motion of the laser tweezers with the trapped bead continued at constant speed 1.5 μm/s until the bead escaped and quickly retracted to the cell surface. The tether length was determined from images recorded at 0.1-s intervals. At day 1, the tether lengths in osteoblasts and BMPCs were determined. The optical forces applied to produce these tethers were estimated to be between 3 and 10 pN. The tether length distributions, constructed from measurements of 35–40 tethers (n = 20–25 cells), show that longer tethers could be produced more frequently in undifferentiated BMPCs, indicating a weaker cell membrane-cytoskeleton association.