ABSTRACT
BACKGROUND AND AIMS: Endosidins are a group of low-molecular-weight compounds, first identified by 'chemical biology' screening assays, that have been used to target specific components of the endomembrane system. In this study, we employed multiple microscopy-based screening techniques to elucidate the effects of endosidin 5 (ES5) on the Golgi apparatus and the secretion of extracellular matrix (ECM) components in Penium margaritaceum. These effects were compared with those caused by treatments with brefeldin A and concanamycin A. Penium margaritaceum's extensive Golgi apparatus and endomembrane system make it an outstanding model organism for screening changes to the endomembrane system. Here we detail changes to the Golgi apparatus and secretion of ECM material caused by ES5. METHODS: Changes to extracellular polymeric substance (EPS) secretion and cell wall expansion were screened using fluorescence microscopy. Confocal laser scanning microscopy and transmission electron microscopy were used to assess changes to the Golgi apparatus, the cell wall and the vesicular network. Electron tomography was also performed to detail the changes to the Golgi apparatus. KEY RESULTS: While other endosidins were able to impact EPS secretion and cell wall expansion, only ES5 completely inhibited EPS secretion and cell wall expansion over 24 h. Short treatments of ES5 resulted in displacement of the Golgi bodies from their typical linear alignment. The number of cisternae decreased per Golgi stack and trans face cisternae in-curled to form distinct elongate circular profiles. Longer treatment resulted in a transformation of the Golgi body to an irregular aggregate of cisternae. These alterations could be reversed by removal of ES5 and returning cells to culture. CONCLUSIONS: ES5 alters secretion of ECM material in Penium by affecting the Golgi apparatus and does so in a markedly different way from other endomembrane inhibitors such as brefeldin A and concanamycin A.
Subject(s)
Charophyceae , Brefeldin A/pharmacology , Extracellular Polymeric Substance Matrix , Golgi Apparatus , Extracellular MatrixABSTRACT
Penium margaritaceum is a unicellular zygnematophyte (basal Streptophyteor Charophyte) that has been used as a model organism for the study of cell walls of Streptophytes and for elucidating organismal adaptations that were key in the evolution of land plants.. When Penium is incubated in sorbitol-enhance medium, i.e., hyperosmotic medium, 1000-1500 Hechtian strands form within minutes and connect the plasma membrane to the cell wall. As cells acclimate to this osmotic stress over time, further significant changes occur at the cell wall and plasma membrane domains. The homogalacturonan lattice of the outer cell wall layer is significantly reduced and is accompanied by the formation of a highly elongate, "filamentous" phenotype. Distinct peripheral thickenings appear between the CW and plasma membrane and contain membranous components and a branched granular matrix. Monoclonal antibody labeling of these thickenings indicates the presence of rhamnogalacturonan-I epitopes. Acclimatization also results in the proliferation of the cell's vacuolar networks and macroautophagy. Penium's ability to acclimatize to osmotic stress offers insight into the transition of ancient zygnematophytes from an aquatic to terrestrial existence.