SURFACE-MORPHOLOGY AND MECHANICAL-PROPERTIES OF MDCK MONOLAYERS BY ATOMIC-FORCE MICROSCOPY

We describe the morphology and mechanical stability of the apical surf ace of MDCK monolayers by atomic force microscopy (AFM). Living cells could be imaged in physiological solution for several hours without no ticeable deterioration. Cell boundaries appear as ridges that clearly demarcate neighboring cells. In some cases the nucleus of individual c ells could,be seen, though apparently only in very thin areas of the m onolayer. Two types of protrusions on the surface could be visualized. Smooth bulges that varied in width from a few hundred nanometers to s everal micrometers, which appear to represent relatively rigid subapic al structures. Another type of protrusion extended well above the memb rane and was swept back and forth during the imaging. However, the mic rovilli that are typically present on the apical surface could not be resolved. For comparison, a transformed MDCK cell line expressing the K-ras oncogene was also examined. When cultured on solid substrata at low density, the R5 cells spread out and are less than 100 nm thick ov er large areas with both extensive processes and rounded edges. Many i ntracellular structures such as the nucleus, cytoskeletal elements and vesicles could be visualized. None of the intracellular structures se en in the AFM images could be seen by scanning electron microscopy. Bo th R5 cells and MDCK monolayers required imaging forces of > 2 nN for good image contrast. Force measurements on the MDCK monolayers show th at they are very soft, with an effective spring constant of similar to 0.002 N/m for the apical plasma membrane, over the first micrometer o f deformation, resulting in a height deformation of approximately 500 nm per nanoNewton of applied force. The mechanical properties of the c ells could be manipulated by addition of glutaraldehyde. These changes were monitored in real time by collecting force curves during the fix ation reaction. The curves show a stiffening of the apical plasma memb rane that was completed in similar to 1 minute. On the basis of these measurements and the imaging forces required, we conclude that deforma tion of the plasma membrane is an important component of the contrast mechanism, in effect 'staining' structures based on their relative rig idity.