Membrane order in pollen tubes-Methodology and unexpected findings.

Abstract

The plasma membrane of mammalian cells is made up of two co-existing liquid phases, liquid ordered (Lo) phase and liquid disordered (Ld) phase. In pollen tubes that grow 3-20 μm/min upon germination, little is known about the membrane order and the existence of ordered membrane domains. Accordingly, we stained pollen tubes with the solvatochromic probe di-4-ANEPPDHQ, which undergoes a red emission shift in Lo-phase compared to Ld-phase membranes, because the latter are more hydrated. Growing pollen tubes were imaged live using widefield fluorescence microscopy with the emission split into two channels recorded simultaneously. Z-series were captured, deconvolved and the relative abundance of Lo- versus Ld-phase was quantified by generalised polarisation (GP). The plasma membrane was first outlined by marking sequential points, which were used as a basis for finding the most intense pixels performing searches along short line segments perpendicular to the local orientation of the plasma membrane. Additional points were then interpolated and similarly optimized. An area outside the pollen tube was automatically defined and used for background subtraction. The mapped plasma membrane was used to automatically identify the tip of the pollen tube and three distance maps were generated (i) from the tip along the plasma membrane, (ii) from tip inside the pollen tube and (iii) inwards from the plasma membrane. These maps were combined to delineate specific regions of interest. The membrane order of apical vesicles (AV) was compared to that of three biologically relevant zones along the plasma membrane. Interestingly, the highest membrane order was observed in the AV and the tip zone of the plasma membrane, whereas the order of the plasma membrane along the tube was lower. Pollen tubes thus differ from other eukaryotic cells, whose plasma membranes consistently has the highest order.