Blebs, pressure driven protrusions of the cell membrane, facilitate the movement of eukaryotic cells such as the soil amoeba Dictyostelium discoideum, white blood cells and cancer cells. Blebs initiate when the cell membrane separates from the underlying cortex. A local rupture of the cortex, has been suggested as a mechanism by which blebs are initiated. However, much clarity is still needed about how cells inherently regulate rupture of the cortex in locations where blebs are expected to form. In this work, we examine the role of membrane energy and the motor protein myosin II (myosin) in facilitating the cell driven rupture of the cortex. We perform under-agarose chemotaxis experiments, using Dictyostelium discoideum cells, to visualize the dynamics of myosin and calculate changes in membrane energy in the blebbing region. To facilitate a rapid detection of blebs and analysis of the energy and myosin distribution at the cell front, we introduce an autonomous bleb detection algorithm that takes in discrete cell boundaries and returns the coordinate location of blebs with its shape characteristics. We are able to identify by microscopy naturally occurring gaps in the cortex prior to membrane detachment at sites of bleb nucleation. These gaps form at positions calculated to have high membrane energy, and are associated with areas of myosin enrichment. Myosin is also shown to accumulate in the cortex prior to bleb initiation and just before the complete disassembly of the cortex. Together our findings provide direct spatial and temporal evidence to support cortex rupture as an intrinsic bleb initiation mechanism and suggests that myosin clusters are associated with regions of high membrane energy where its contractile activity leads to a rupture of the cortex at points of maximal energy.

Aggregation competent Dictyostelium discoideum cells (Ax2 expressing LifeAct-GFP and Ax2 expressing myosin-GFP/Life-Act RFP) chomotaxing towards 4 micromolar CAMP under a block of agarose gel (0.7%) were imaged for 30 seconds using a spinning disk confocal microscope. Images were adjusted for brightness and contrast using Image J. See the Methods section our manuscript for more details.