Quantification of Cell-to-Cell Connectivity Using Particle Bombardment.

Abstract

Plant cells are connected by cytoplasmic bridges called plasmodesmata. Plasmodesmata are lined by the plasma membrane, essentially forming tunnels that directly connect the cytoplasm of adjacent cells through which soluble molecules can move from cell to cell. This cell-to-cell mobility is underpinned by cytoplasmic advection and diffusion in a manner dependent on molecular size. This movement of molecules is regulated by the aperture of plasmodesmata. GREEN FLUORESCENT PROTEIN (GFP) is a 27kDa soluble protein that can move passively between cells via plasmodesmata. Thus, it serves as an ideal probe to assess plasmodesmal aperture. GFP can be transgenically produced in single cells by microprojectile bombardment-mediated transformation, and its cell-to-cell mobility can be measured by live-cell imaging and counting the number of cells (or cell layers) to which it has moved. Thus, the number of cells in which GFP is visible serves as a measure of plasmodesmal aperture and functional cell-to-cell connectivity. Here we present methods for microprojectile bombardment of GFP into leaf epidermal cells and statistical analysis of resulting data.