Many cell phenomena involve major morphological changes, particularly in mitosis and the process of cell migration. For cells or neuronal growth cones to migrate, they must extend the leading edge of the plasma membrane as a lamellipodium or filopodium. During extension of filopodia, membrane must move across the surface creating shear and flow. Intracellular biochemical processes driving extension must work against the membrane mechanical properties, but the forces required to extend growth cones have not been measured. In this paper, laser optical tweezers and a nanometer-level analysis system were used to measure the neuronal growth cone membrane mechanical properties through the extension of filopodia-like tethers with IgG-coated beads. Although the probability of a bead attaching to the membrane was constant irrespective of treatment; the probability of forming a tether with a constant force increased dramatically with cytochalasin B or D and dimethylsulfoxide (DMSO). These are treatments that alter the organization of the actin cytoskeleton. The force required to hold a tether at zero velocity (F0) was greater than forces generated by single molecular motors, kinesin and myosin; and F0 decreased with cytochalasin B or D and DMSO in correlation with the changes in the probability of tether formation. The force of the tether on the bead increased linearly with the velocity of tether elongation. From the dependency of tether force on velocity of tether formation, we calculated a parameter related to membrane viscosity, which decreased with cytochalasin B or D, ATP depletion, nocodazole, and DMSO. These results indicate that the actin cytoskeleton affects the membrane mechanical properties, including the force required for membrane extension and the viscoelastic behavior.

The small GTPase racE is essential for cytokinesis in Dictyostelium. We found that this require- ment is restricted to cells grown in suspension. When attached to a substrate, racE null cells form an actomyosin contractile ring and complete cytokinesis normally. Nonetheless, racE null cells fail completely in cytokinesis when in suspension. To understand this conditional requirement for racE, we developed a method to observe cytokinesis in suspension. Using this approach, we found that racE null cells attempt cytokinesis in suspension by forming a contractile ring and cleavage furrow. However, the cells form multiple blebs and fail incytokinesis by regression of the cleavage furrow. We believe this phenotype is caused by the extremely low level of cortical tension found in racE null cells com- pared to wild-type cells. The reduced cortical tension of racE null cells is not caused by a decrease in their content of F-actin. Instead, mitotic racE null cells contain abnormal F-actin aggregates. These results suggest that racE is essential for the organization of the cortical cytoskeleton to maintain proper cortical integrity. This function of racE is independent of attachment to a sub- strate, but can be bypassed by other signaling pathways induced by adhesion to a substrate.

Stimulated secretion in endocrine cells and neuronal synapses causes a rise in endocytosis rates to recover the added membrane. The endocytic process involves the mechanical deformation of the membrane to produce an invagination. Studies of osmotic swelling effects on endocytosis indicate that the increased surface tension is tightly correlated to a significant decrease of endocytosis. When rat basophilic leukemia (RBL) cells are stimulated to secrete, there is a dramatic drop in the membrane tension and only small changes in membrane bending stiffness. Neither the shape change that normally accompanies secretion nor the binding of ligand without secretion causes a drop in tension. Further, tension decreases within 6 s, preceding shape change and measurable changes in endocytosis. After secretion stops, tension recovers. On the basis of these results we suggest that the physical parameter of membrane tension is a major regulator of endocytic rate in RBL cells. Low tensions would stimulate endocytosis and high tensions would stall the endocytic machinery.

Development of the nervous system requires that neuronal growth cones, in coordination with growing axons, migrate along precise paths defined by specific extracellular matrix cues until they ncounter their targets. Laminin promotes growth cone migration through receptors such as the integrins, but the underlying physical mechanism is poorly understood. We have investigated the cytoskeletal associations and surface dynamics of endogenous β1 integrins in chick dorsal root ganglion growth cones migrating on laminin. A single-beam optical gradient trap was used to place 0.5-µm-diameter polystyrene beads conjugated with anti-β1 integrin monoclonal antibodies at desired locations on the growth cone surface. We found a substantial increase in the stable attachment of these beads, with subsequent slow rearward motion, on the front periphery of the growth cone compared to the ase. The surface dynamics of smaller aggregates of integrin were explored by monitoring the temporal and spatial displacements of 40-nm-diameter gold particles coated with anti-β1 integrin antibodies. The small particles were transported preferen tially to the growth cone periphery by brief directed xcursions interspersed with periods of diffusion. In addition, the leading edge of the growth cone was supported to a greater extent by an actin-dependent cytoskeleton that resisted mechanical tether formation. Such a regional differentiation of the growth cone has not been documented previously and has implications for the mechanism of growth cone migration and guidance.

The plasma membrane of most cells is drawn tightly over the cytoskeleton of the cell, resulting in a significant tension being developed in the membrane. The tension in the membrane can be calculated fi-om the force required to separate it ffom the cytoskeleton; and the force itself can be measured rapidly by using laser tweezers. Recent observations indicate that decreasing membrane tension stimulates endocytosis and increasing tension stimulates secretion. Thus, membrane tension provides a simple physical mechanism to control the area of the plasma membrane. Here, we speculate that tension is a global parameter that the cell uses to control physically plasma membrane dynamics, cell shape and cell motility.