Four-dimensional quantitative analysis of cell plate development using lattice light sheet microscopy identifies robust transition points between growth phases

Abstract

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodeling, and timely polysaccharide deposition, such as callose. Such a dynamic process requires dissection in time and space; hence this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM) we studied cell plate development in four dimensions, monitored by the behavior of the cytokinesis specific GTPase RABA2a. We monitored the entire length of cell plate development, from its first emergence, with the aid of RABA2a, both in the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis, we identified distinct behavioral patterns allowing for the identification of three easily trackable, cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited phase transition, establishing quantitatively the critical role and timing of the polysaccharide in cell plate expansion and maturation. This study exemplifies the power of LLSM, combined with quantitative analysis to decode and untangle such a complex process. HighlightWe employed lattice light sheet 4D microscopy in plants to dissect cytokinesis, a multistage process involving orchestrating delivery of membranes and timely polysaccharide deposition. Robust quantitative analysis revealed distinct phase shifts, while inhibition of callose deposition abolished the phase transition.