Projections of the plasma membrane
Polaric also showed a similar kinetics of internalization of cell membrane to CellMask Orange data not shown. The directions of cell migration are indicated by black arrows.
Both photobleached regions did not move relative to the substratum, indicating that the cell membrane moves in a manner consistent with the fountain flow model.
Plasma membrane function
Each cell image is cropped to identically sized squares, and thus does not show the actual displacement of the cell. Graphs E and F show typical time courses of the fluorescence intensities of the cell membrane and inside the cells, respectively. Figure 2 The total cell surface area does not change during cell migration. These pieces of evidence suggest that many cell membrane components, including the cortical actin cytoskeleton, do not move relative to the substratum, supporting retrograde flow. Interestingly, the beads did not move relative to the substratum as the cell advanced, and the attached beads finally accumulated at the rear of the cells as the cells advanced forward. In feeding, pseudopodia flow around the particle and engulf it, forming a food vacuole. Microscopy For agar-overlay, a thin agar block 0. An agar block was removed to observe the cell surface after fixation see Materials and Methods. The cell membrane was stained with Polaric, a similar fluorescent lipid analogue to CellMask Orange. Note that the bleached regions did not move relative to the substratum, whereas the cell advanced. The cytoskeleton has been well established to contribute to cell migration. One minute after staining, the excess dye was removed by washing with BSS.
The cells under agar-overlay did not extend thin extensions such as filopods and microvilli as far as we observed under a light microscope. Thus, half of the total lipids of the cell membrane were internalized and newly exchanged within approximately 5 min.
Plasma membrane structure
Photobleaching Photobleaching was performed using a confocal microscope as previously described A Typical fluorescence images when a small region of the cell membrane was photobleached 0 sec. Therefore, we concluded that a reservoir of unfolded membrane is dispensable for cell migration, although we cannot exclude the possibility that it contributes to the cell shape change during cell migration in the absence of agar-overlay. Under this condition, it may be practically difficult to explain the circulation of cell membrane by the caterpillar model. How migrating cells maintain a constant cell membrane surface area by coordinating exocytosis and endocytosis and how these membrane dynamics are coordinated with the cytoskeletal systems to drive cell migration remain to be investigated. Clathrin-containing pits and vesicles also mainly appear at the rear of migrating leukocytes and epithelial cells 31 , However, due to a technical limitation at that time, the photobleaching was simultaneously performed on both the dorsal and ventral cell membrane in single cells, and the individual movements could not be distinguished. Note that the probe was already internalized and found in many intracellular vesicles about 5 min after the agar-overly. Dictyostelium is a suitable candidate for studying migrating cells with such a high velocity. Results The total area of the cell surface is constant during cell migration Migrating Dictyostelium cells always change their shape by extending pseudopods and retracting their rear. To clarify which model is suitable, photobleaching experiments were performed separately on the dorsal and ventral cell membranes. In the fastest cell, the half-life was approximately 60 s. Six z-axis images with an interval of 0. Internalization of isotope-labeled surface proteins indicated a time of 45 min for total cell membrane exchange The cytoskeleton has been well established to contribute to cell migration.
Interestingly, the beads did not move relative to the substratum as the cell advanced, and the attached beads finally accumulated at the rear of the cells as the cells advanced forward. The folded surface appears as projections and wrinkles on the cell surface and is utilized as a membrane reservoir.
Figure 1 Three models for the behavior of the cell membrane during cell migration. Pseudopodia 3. The cell without the agar-overlay had many wrinkles, as well as small and long projections, on its surface.
Note that the fluorescence recovered by a simple lateral diffusion of the lipids.
based on 33 review