experimental-screening-protocols-immunocytochemistry-microscopy-based

Experimental Screening Protocols, Immunocytochemistry and Microscopy-based Imaging Techniques for <em>Penium margaritaceum</em>

The cell wall is the first component of signal reception/transduction for a plant cell during development and when responding to environmental abiotic and ...

Skidmore College

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.

Endosidin 5 disruption of the Golgi apparatus and extracellular matrix secretion in the unicellular charophyte Penium margaritaceum.

Green algae display a wide range of extracellular matrix (ECM) components that include various types of cell walls (CW), scales, crystalline glycoprotein coverings, hydrophobic compounds, and complex gels or mucilage. Recently, new information derived from genomic/transcriptomic screening, advanced biochemical analyses, immunocytochemical studies, and ecophysiology has significantly enhanced and refined our understanding of the green algal ECM. In the later diverging charophyte group of green algae, the CW and other ECM components provide insight into the evolution of plants and the ways the ECM modulates during environmental stress. Chlorophytes produce diverse ECM components, many of which have been exploited for various uses in medicine, food, and biofuel production. This review highlights major advances in ECM studies of green algae.

The extracellular matrix of green algae.

Phenotypic plasticity allows a plant cell to alter its structure and function in response to external pressure. This adaptive phenomenon has also been important in the evolution of plants including the emergence of land plants from a streptophyte alga. Penium margaritaceum is a unicellular zygnematophyte (i.e., the group of streptophyte algae that is sister to land plants) that was employed in order to study phenotypic plasticity with a focus on the role of subcellular expansion centers and the cell wall in this process. Live cell fluorescence labeling, immunofluorescence labeling, transmission electron microscopy, and scanning electron microscopy showed significant subcellular changes and alterations to the cell wall. When treated with the actin-perturbing agent, cytochalasin E, cytokinesis is arrested and cells are transformed into pseudo-filaments made of up to eight or more cellular units. When treated with the cyclin-dependent kinase (CDK) inhibitor, roscovitine, cells converted to a unique phenotype with a narrow isthmus zone.

Chemically induced phenotype plasticity in the unicellular zygnematophyte, Penium margaritaceum.

Penium margaritaceum, a unicellular zygnematophyte (Streptophyta), was employed to elucidate changes in cell expansion when cells were challenged with the fungal pectinolytic enzyme, pectate lyase, and/or the microtubule-disrupting agent, amiprophos-methyl (APM). Microtubule disruption by APM resulted in significant swelling at expansion zones. These swollen zones provided an easy marker for the location of expansion zones, particularly in cells with altered cell wall pectin. Short-term treatment with pectate lyase showed pectin degradation primarily at the isthmus expansion zone and two satellite bands, corresponding to the location of future expansion in daughter cells. When the homogalacturonan lattice of the cell wall was removed by treatment with pectate lyase during long treatments, cell division was maintained, but daughter cell products were considerably smaller. Treatment of cells with a mixture of both pectate lyase and APM resulted in a distinct phenotype, consisting of 'dumbbell'-shaped cells, as APM-induced swelling occurs at the novel expansion centers exposed by pectate lyase treatment. These cells also presented other curious alterations, including an extensive, chloroplast-free cytoplasmic zone at the center of the cell, a septum containing β-glycan, arabinogalactan and homogalacturonan epitopes, unique stacks of endoplasmic reticulum, displaced Golgi bodies, and an extensive network of vacuoles. These results provide insight into the importance of cell wall integrity in defining the location of cell growth and division in P. margaritaceum. Understanding these processes in a unicellular zygnematophyte may provide insights into steps involved in the evolution of land plants.

Aberrant growth and expansion in Penium margaritaceum triggered by disruption of microtubules and the cell wall.

Adhesion and consequent adoption of a sessile habit is a common feature of many green algae and was likely a key mechanism in terrestrialization by an ancient zygnematophyte (i.e., the Zygnematophyceae, the group of algae ancestral to land plants). Penium margaritaceum is a unicellular zygnematophyte that exhibits a multistep adhesion mechanism, which leads to the establishment of the sessile habit. Based on microscopic and immunological data, a dense aggregate of fibrils containing arabinogalactan-protein (AGP)-like components covers the cell surface and is responsible for initial adhesion. The AGP-like fibrils are 20 μm in diameter and possess chemical profiles similar to land plant AGPs. The fibrils attach to the inner cell wall layers and are very likely connected to the plasma membrane as glycophosphatidylinositol (GPI) lipid-anchored proteins, as they are susceptible to phospholipase C treatment. The presence of GPI-anchored AGPs in Penium is further supported by the identification of putative Penium homologs of land plant AGP genes responsible for GPI-anchor synthesis. After adhesion, cells secrete a complex heteropolysaccharide-containing extracellular polymeric substance (EPS) that facilitates gliding motility and the formation of cell aggregates. Fucoidan-like polymers, major components of brown algal CWs, are a major constituent of both the EPS and the adhesive layer of the CW and their role in the adhesion process is still to be examined.

Josephine LoRicco

Department of Biology and Skidmore Microscopy Imaging Center

Skidmore College

Experimental Screening Protocols, Immunocytochemistry and Microscopy-based Imaging Techniques for Penium margaritaceum

Josephine LoRicco

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology and Skidmore Microscopy Imaging Center

Skidmore College

Experimental Screening Protocols, Immunocytochemistry and Microscopy-based Imaging Techniques for Penium margaritaceum

Department of Biology and Skidmore Microscopy Imaging Center