Sinclair, R. M. | Search bioRxiv

bioRxiv2023

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

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.

plant biology↗

bioRxiv2020

A Biophysical Model for Plant Cell Plate Development

Plant cytokinesis, a fundamental process of plant life, involves de novo formation of a cell plate that partitions the cytoplasm of the dividing cell. Cell plate formation is directed by orchestrated delivery, fusion of cytokinetic vesicles, and membrane maturation to the form the nascent cell wall by the timely deposition of polysaccharides such as callose, cellulose, and crosslinking glycans. In contrast to the role of endomembrane protein regulators the role of polysaccharides, in cell plate development is poorly understood. Callose, a {beta}-1-3 glucan polymer, is transiently accumulated during cell plate expansion to be replaced by cellulose in mature stages. Based on the severity of cytokinesis defects in the absence of callose, it has been proposed that it stabilizes this membrane network structure. However, there is currently no theory to understand its role in cytokinesis. Here we extend the Helfrich free energy model for membranes including a phenomenological spreading force as an "areal pressure" generated by callose and/or other polysaccharides. Regular cell plate development in the model is possible, with suitable bending modulus, for a two-dimensional late stage spreading force parameter of between 2-6pN/nm, an osmotic pressure difference of 2-10kPa, and spontaneous curvature between 0-0.04nm-1. With these conditions, stable membrane conformation sizes and morphologies emerge in concordance with stages of cell plate development. With no spreading force, the cell plate fails to mature properly, corroborating experimental observations of cytokinesis arrest in the absence of callose. To reach a nearly mature cell plate, our model requires the late stage onset that the spreading force coupled with a concurrent loss of spontaneous curvature. A simple model based upon production of callose as a quasi-two-dimensional self-avoiding polymer produces the correct phenomenological form of the spreading force, which will be further refined, since matching to our numbers requires an exceptionally high callose synthesis rate. Significance StatementPlant cell division features the development of a unique membrane network called the cell plate that matures to a cell wall which separates the two daughter cells. During cell plate development, callose, a {beta}-1-3 glucan polymer, is transiently synthesized at the cell plate only to be replaced by cellulose in mature stages. The role for this transient callose accumulation at the cell plate is unknown. It has been suggested that callose provides mechanical stability, as well as a spreading force that widens and expands tubular and fenestrated cell plate structures to aid the maturation of the cell plate. Chemical inhibition of callose deposition results in the failure of cell plate development supporting this hypothesis. This publication establishes the need for a spreading force in cell plate development using a biophysical model that predicts cell plate development in the presence and the absence of this force. Such models can potentially be used to decipher for the transition/maturation of membrane networks upon the deposition of polysaccharide polymers.

biophysics↗