We present a technical platform that allows us to monitor and measure cortex and membrane dynamics during bleb-based chemotaxis. Using D. discoideum cells expressing LifeAct-GFP and crawling under agarose containing RITC-dextran, we were able to simultaneously visualize the actin cortex and the cell membrane throughout bleb formation. Using these images, we then applied edge detect to generate points on the cell boundary with coordinates in a coordinate plane. Then we fitted these points to a curve with known x and y coordinate functions. The result was to parameterize the cell outline. With the parameterization, we demonstrate how to compute data for geometric features such as cell area, bleb area and edge curvature. This allows us to collect vital data for the analysis of blebbing.

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.

Blebs, pressure driven protrusions of the plasma membrane, facilitate the movement of cells such as the soil amoeba Dictyostelium discoideum and other eukaryotes such as white blood cells and cancer cells. Blebs initiate or nucleate when proteins connecting the membrane to the cortex detach, either as a result of a rupture of the cortex or as a direct consequence of a build up in hydrostatic pressure. While linker detachment resulting from excess hydrostatic pressure is well understood, the mechanism by which cells rupture their cortex in locations of bleb formation is not so clear. Consequently, existing predictive models of bleb site selection do not account for it. To resolve this, we propose a model for bleb initiation which combines the geometric forces on the cell cortex/membrane complex with the underlying activity of actin binding proteins. In our model gaps, resulting from a rupture of the cortex, form at locations of high membrane energy where an accumulation of myosin II helps to weaken the cortex. We validate this model in part through a membrane energy functional which combines stresses on the cell boundary from membrane tension, curvature, membrane-cortex linker tension with hydrostatic pressure. Application of this functional to microscopy images of chemotaxing Dictyostelium discoideum cells identifies bleb nucleation sites at the highest energy locations 96.7% of the time. Sensitivity analysis of the model components points to membrane tension and hydrostatic pressure, all of which are regulated by myosin II, as critical to model predictability. Furthermore, microscopy reveals discrete clusters of myosin II along the leading edge of the cell, with blebs emerging from 80% of these sites. Together, our findings suggest a critical role for myosin II in bleb initiation through the formation of gaps and provides a predictive mathematical model for quantitative studies of blebbing. Author summaryEukaryotic cells such as white blood cells and cancer cells have been observed to move by making spherical herniation of their plasma membrane, referred to as blebs. The precise mechanism by which cells select locations around their boundary to initiate blebs is unclear. We hypothesize that blebs initiate at locations of high membrane energy where an accumulation of myosin II helps to rupture the cortex and/or detach linker proteins. We test this hypothesis by formulating a free energy functional representation of membrane energy to predict where blebs will initiate. The functional March 16, 2020 1/19 accounts for geometric forces due to membrane tension, curvature and membrane-cortex linker tension as well as hydrostatic pressure. Application of the functional to data from the soil amoeba, Dictyostelium disodium, identifies blebs at the highest energy locations over 90% of the time. Sensitivity analysis of model components points to membrane tension and hydrostatic pressure, all influenced by myosin II, as major forces driving bleb initiation. Additionally, we observe clusters of myosin II at locations of bleb ...

Blebs, pressure driven protrusions of the plasma membrane, facilitate the movement of cell such as the soil amoeba Dictyostelium discoideum in a three dimensional environment. The goal of the article is to develop a means to predict nucleation sites.We accomplish this through an energy functional that includes the influence of cell membrane geometry (membrane curvature and tension), membrane-cortex linking protein lengths as well as local pressure differentials. We apply the resulting functional to the parameterized microscopy images of chemotaxing Dictyostelium cells. By restricting the functional to the cell boundary influenced by the cyclic AMP (cAMP) chemo-attractant (the cell anterior), we find that the next nucleation site ranks high in the top 10 energy values. More specifically, if we look only at the boundary segment defined by the extent of the expected bleb, then 96.8% of the highest energy sites identify the nucleation. February 27, 20191/20This work concerns the prediction of nucleation sites in the soil amoeba-like 1 Dictyostelium discoideum. We define a real valued functional combining input from 2 cortex and membrane geometry such as membrane curvature and tension, cortex to 3 membrane separation and local pressure differences. We show that the functional may 4 be used to predict the location of bleb nucleation. In the region influenced by the 5 cAMP gradient (the cell anterior), the next blebbing site lies in the ten highest energy 6 functional values 70% of the time. The correctness increases to 96.8% provided we 7 restrict attention to the segment in the general location of the next bleb. We verify 8 these claims through the observation of microscopy images. The images are sequential 9 at 1.66 and 0.8 seconds per image. We first identify the earliest sign of the bleb. We 10 then use several observational factors to identify the nucleation site and estimate the 11 corresponding location in the prior image.12 21 D. discoideum is a species of soil-living amoeba-like organism. It is a eukaryotic 22 organism whose motility and shape is controlled predominantly by intricate actin based 23 structures in the cytoplasm. In turn, the cell cytoplasm is encased by a plasma 24 membrane, a 4-5nm thick semi-permeable lipid bilayer [38]. Beneath this membrane is 25 the cell cortex, an assembly of thin cross-linked actin filaments held together firmly by 26 cross linking proteins such as Filamin, α-Actinin, Fimbrin and Fascin. The cortex 27 thickness is several hundred nanometers [1]. The cortex plays a major role in 28 February 27, 2019 2/20 maintaining cell shape and the formation of motility structures. Its contractile 29 capabilities are due to the presence of Myosin II in the network. The membrane is 30 attached to the cortex by trans membrane proteins such as Talin [9]. In addition to the 31 cortex, the cytoplasm includes distinct actin-based structures referred to as the 32 cytoskeleton. The fluid part of the cytoplasm (cytosol) contains water, ions and 33 dissolved molecules. 34 Blebs and blebbing have ...